Nusantara Bioscience vol. 5, no. 2, November 2013 by Nusantara Bioscience

Caledula officinalis photo by Piotr Jasinski (Karlgoro)

| Nus Biosci | vol. 5 | no. 2 | pp. 51-107 | November 2013 | | ISSN 2087-3948 | E-ISSN 2087-3956 |

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EDITORIAL BOARD: Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia (sugiyarto_ys@yahoo.com) Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia (jrwitono@yahoo.com) Editorial Advisory Boards: Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia (janung4@yahoo.com.au) Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia (pattiselannofreddy@yahoo.com) Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India (pmkrai@hotmail.com) Biomedical Sciences, Afiono Agung Prasetyo, Sebelas Maret University, Surakarta, Indonesia (afieagp@yahoo.com) Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia (iyahya@uns.ac.id) Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia (cecep_kusmana@ipb.ac.id) Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia (lufehakim@yahoo.com) Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia (nnsutarno@yahoo.com) Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia (gadis@limnologi.lipi.go.id) Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt (shokry_1@yahoo.com) Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia (charis@ukdw.ac.id) Microbiology, Kateryna Kon, Kharkiv National Medical University, Kharkiv, Ukraine (katerynakon@gmail.com) Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia (ajamsari@yahoo.com) Plant Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China (xiuyunzh@yahoo.com.cn) Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia (pudjiwi@yahoo.com) Management Boards: Managing Editor, Ahmad D. Setyawan, Sebelas Maret University, Surakarta, Indonesia (unsjournals@gmail.com) Associated Editor (English Editor), Wiryono, State University of Bengkulu, Indonesia (wiryonogood@yahoo.com) Associated Editor (English Editor), Suranto, Sebelas Maret University, Surakarta, Indonesia Technical Editor, Ari Pitoyo, Sebelas Maret University, Surakarta, Indonesia (aripitoyo@yahoo.co.id) Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok, Indonesia (nusbiosci@gmail.com)

PUBLISHER: Society for Indonesian Biodiversity CO-PUBLISHER: School of Graduates, Sebelas Maret University Surakarta

FIRST PUBLISHED: 2009

ADDRESS: Bioscience Program, School of Graduates, Sebelas Maret University Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: nusbiosci@gmail.com ONLINE: biosains.mipa.uns.ac.id/nusbioscience

Society for Indonesia Biodiversity

Sebelas Maret University Surakarta

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 5, No. 2, pp. 51-56 November 2013

Effects of autoclaving on the proximate composition of stored castor (Ricinus communis) seeds ANTHONY NEGEDU1,â&#x2122;Ľ, JOSEPH B. AMEH2, VERONICA J. UMOH3, SUNDY E. ATAWODI3, MAHENDRA K. RAI4 1

Raw Materials Research and Development Council, P.M.B. 232, Garki, Abuja, Nigeria. Tel.: +.234-9-4137416-7, Fax.:+234-9-4136034, â&#x2122;Ľ email: tonyneg2000@yahoo.com 2 Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria 3 Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria 4 Department of Biotechnology, SGB Amravati University, Maharashtra, India. Manuscript received: 12 February 2013. Revision accepted: 24 May 2013.

Abstract. Negedu A, Ameh JB, Umoh VJ, Atawodi SE, Rai MK. 2013. Effects of autoclaving on the proximate composition of stored castor (Ricinus communis) seeds. Nusantara Bioscience 5: 51-56. The effect of autoclaving on the proximate composition, free fatty acids and peroxide value of castor (Ricinus communis L.) seeds in storage were studied. Seeds of castor were surface sterilized, dried and divided into two equal sets of 300g each. One set was autoclaved at 15 1b pressure for 30 minutes at 121 oC and the other set served as control. Each set was prepared in triplicates and both sets were stored under same room temperature conditions for a period of 180 days and agitated intermittently. Analysis of the proximate composition showed that autoclaving treatment caused an increased total fat content, reduced moisture, protein, nitrogen free extract (soluble sugar) and ash contents of the seeds in storage, as well as a nonsignificant increase in crude fiber (non-soluble sugar) content. It increased the free fatty acid content and decreased the peroxide value of seed oil. Key words: autoclaving, castor seeds, free fatty acids, peroxide value, proximate composition

Abstrak. Negedu A, Ameh JB, Umoh VJ, Atawodi SE, Rai MK. 2013. Pengaruh perlakuan autoklaf terhadap komposisi proksimat dari benih jarak kepyar yang disimpan (Ricinus communis). Nusantara Bioscience 5: 51-56. Pengaruh perlakuan autoklaf terhadap komposisi proksimat, asam lemak bebas dan nilai peroksida benih jarak kepyar (Ricinus communis L.) dalam penyimpanan dipelajari. Biji jarak kepyar disterilkan permukaannya, dikeringkan dan dibagi menjadi dua kelompok yang sama, masing-masing sebanyak 300g. Salah satu kelompok itu diautoklaf pada tekanan 15 lb selama 30 menit pada suhu 121oC, sedangkan kelompok lainnya digunakan sebagai kontrol. Setiap kelompok diperlakukan dalam tiga ulangan dan kedua kelompok disimpan dalam kondisi suhu ruangan yang sama dalam jangka waktu 180 hari serta beberapa kali dibalik-balik. Analisis komposisi proksimat menunjukkan bahwa perlakuan autoklaf menyebabkan peningkatan kadar lemak total, mengurangi kadar air, protein, ekstrak nitrogen bebas (gula larut) dan kandungan abu dari biji dalam penyimpanan, serta peningkatan secara tidak signifikan kandungan serat kasar (non larut gula). Perlakuan ini meningkatkan kadar asam lemak bebas dan menurunkan nilai peroksida minyak biji. Kata kunci: perlakuan autoklaf, biji jarak kepyar, asam lemak bebas, angka peroksida, komposisi proksimat

INTRODUCTION Vegetation belt influences dietary pattern in West Africa. For instance, in the Southern Nigeria, legumes, nuts, seeds, starchy roots or tubers dominate, while cereals dominate the northern part (Ajayi et al. 2005). In the south eastern Nigeria, popular among the oil seeds used in soups for emulsification and stabilization are Irvingia gabonensis (Ataga and Ota-Ibe 2006); Brachystegia eurycoma and Detarium microcarpum (Ohegbu et al. 2002). But, of particular interest in this study is the castor (Ricinus communis) because of versatile industrial applications. Castor (Ricinus communis L.) (Figure 1) bean plant is a dicotyledonous and monoecious herb of the family Euphorbiaceae and it is considered by most authorities to be native to tropical Africa and may have originated in Abyssinia/Ethiopia (CSIR 1976). The castor is cultivated

for its seeds which yield versatile oil known as castor oil. The seed contains 45-50.6% oil, 12-16% protein, 23-27% fibre, 3-7% NFE, 5% moisture and 2% ash (CSIR 1976). The annual worldwide production stands at 1,311, 669 metric tonnes. The demand for the castor oil is about 453,590 metric tonnes and valued at more than US $500 million (FAO 2005). The oil has about one thousand patented industrial applications and has been used in the production of over four hundred industrial products such as paints, dyes, soaps, cosmetics, polishes, lubricants, plastics, paper, hydraulic fluids, inks, lacquers, machining oils, pigments, sealants, electrical liquids etc. (Roetheli et al. 1991; Gobin et al. 2001). Castor oil enjoys tremendous world demand in the pharmaceutical, cosmetic, textile, paint, leather, lubricant, chemical, plastic, synthetic fibre, automobile and engineering industries (Roetheli et al. 1991; Anjani et al. 2004).

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Figure 1. Ricinus communis. A. Habit, B. Development of flowers into fruit.

After the extraction of castor oil, the high nitrogen containing pomace (meal) is suitable for fertilizer and when detoxified, it can be employed in livestock feed formulation (Uzogara et al. 1990; Joshua 2000). Castor oil is used as an ingredient in the folk medicine for arthritis, cancer, cholera, convulsion, dogbite, guinea worm, oesteomelitis, rheumatism, veneral diseases, tuberculosis and considered an antidote, bactericide, emetic, emollient, insecticide, larvicidal, laxative, purgative etc (Roetheli et al. 1991). It has been reported that the proximate composition of the seeds of some used as soup thickners, such as Mucuna flagellipes (Udensi et al. 2010); Mucuna utilis (Ukachukwu and Obioha 2000; Udensi et al. 2008) have been improved by autoclaving. However, there appears to be dearth of information on the effect that autoclaving would have on the proximate composition of castor seeds. This study was therefore, undertaken to evaluate the effect that autoclaving treatment will have on the proximate composition of castor seeds with a view to recommending it as a pre-storage treatment for the preservation of the nutritional values of seeds.

MATERIALS AND METHODS Collection of seed samples Shortly after harvesting and sun-drying of castor seeds by farmers, seed samples were purchased from local farmers at Ankpa, Kogi State, Nigeria. Visibly mouldy as

well as necrotic lesioned seeds were handpicked and whole seeds that failed to pass through Âž x Âž inch mesh were used for treatments. Sample preparation The approximately uniform and clean seeds were surface sterilized using 1% sodium hypochlorite solution (NaOCl) and rinsed consecutively in sterile de-mineralized water. The surface sterilized seeds were divided into two sets of 300g each and placed in 1 litre autoclavable plastic jars. Each set was prepared in triplicates and one set was autoclaved at 15 1b pressure for 30 minutes at 121oC and cooled, while the second set served as control (raw seed). Both sets were stored under same normal room temperature of 27Âą1oC for a period of 180 days (6 months). At intervals of sixty days (2 months), samples were taken from each set (autoclaved and control) and analyzed for proximate composition using standard methods of AOAC (1995). The biochemical changes that occurred in both sets of seed samples were compared. Analysis of proximate composition Moisture content of the samples was determined by drying to a constant weight of 105oC in a forced draught oven. Crude protein content was determined using the micro Kjeldahl digestion method described by AOAC (1995). The total ash content was determined using the method of Kirk and Sawyer (1991). The total ash present in 5g of the sample was determined by incinerating the sample in a muffle furnace at 550 oC for 3 hours. The

NEGEDU et al. – Effects of autoclaving on Ricinus communis seeds

method described by Kirk and Swayer (1991) was used to determine the crude fibre content of the samples. The protocol for the crude fibre content is briefly given. Two grammes of defatted sample were boiled in 200cm3 of 0.1275 M sulphuric acid solution for 30 minutes with constant agitation. The boiling mixture was poured into a Buckner funnel and washed with boiling water twice. Then the residue was boiled in a 0.313 M sodium hydroxide solution for 30 minutes with constant stirring. The residue was then washed twice with boiling water followed by 1% HCl, then washed with boiling water until free from acid. It was then dried in an oven to a constant weight. Carbohydrate was determined by difference (100-(protein + fat + moisture + ash). The nitrogen value, which is the precursor for protein of a substance, was determined by micro-Kjeldahl method (Guebel et al. 1991). The nitrogen value was converted to protein by multiplying with a factor of 6.25. The crude lipid content of the sample was determined using Soxhlet type of the direct solvent extraction method. The solvent used was petroleum ether (boiling range 40-60 oC). All proximate values were reported in percentage (AOCS 2000; Okwu and Morah 2004). Data analysis Data were expressed as mean±standard error of M (SEM). The data were subjected to one-way analysis of variance (ANOVA). SPSS soft ware was used to analyse the data and P< 0.05 was considered statistically significant.

RESULTS AND DISCUSSION Effects of autoclaving on moisture content of castor seeds after 180 days of storage In the control (un-autoclaved) and autoclaved, there was a similar trend in moisture content levels (Figure 2.). From an initial level of 7.9±0.21%, the moisture declined through 60 days to lower values (5.57±0.16% and 6.45±0.16%) in the control and autoclaved respectively. Following this point, the seed moisture content increased to 8.71±0.19% in the un-autoclaved. At the end of storage period of 180 days, the level of moisture in the control (unautoclaved) was higher than in the autoclaved seeds. Statistical analysis shows that the difference in the values of moisture between the control and autoclaved was significant (P≥0.05). The significant decrease in moisture content of the autoclaved seeds (Figure 2.) agreed with Ward and Diener (1961) on steamed peanuts and Ohegbu et al. (2009) on Brachystegia eurycoma . The decrease could have been as a result of physical damage to the structural integrity of the seeds and denaturation of protein structure leading to reduced water holding capacity of the seeds during storage. Effect of autoclaving on crude protein (cp) content of castor seeds after 180 days of storage Figure 3 presents the trend in the level of crude protein content of the castor seeds after 180 days storage. Within

0-60 days, in the control (un-autoclaved), there was a similar trend in crude protein values. A rise in the initial value (21.28±0.03%) to a higher value (27.07±1.76%) was observed. A slight decline from the initial value (21.28±0.03%) to a lower level (20.80±1.76%) was observed in the autoclaved seeds. Between 120-180 days, a decline in the crude protein value was observed in the unautoclaved and autoclaved (from 26.36±0.40% to 24.47±0.55% and from 20.26±0.40% to 17.78±0.55% respectively). At the end of storage period, the level of crude protein in the control (un-autoclaved) was higher than in the autoclaved seeds). Statistical analysis shows that, the difference in the crude protein values between the un-autoclaved and the control was significantly different (P ≤ 0.05). The decline in the level of crude protein in the autoclaved seeds (Figure 3.) agreed with Ward and Diener (1961) who reported similar result on steamed peanuts and Udeni et al. (2004) who reported similar observation on autoclaved seeds of Mucuna utilis. The increase in protein content could have been due to reduction/destruction of certain protease inhibitors and anti-nutrients like phytic acid and tannins which form complexes with protein and make it unavailable during hydrolysis. Effect of autoclaving on total fat content of castor seeds after 180 days of storage Figure 4 presents the mean values of the total fat content with respect to autoclaving treatment. In the unautoclaved seeds, it was observed that the total fat declined from the initial value (47.55±0.42%) to a lower value (24.93±2.23%) at 60 days, 21.77±0.15% at 120 days and 13.19±1.44% at 180 days. But in the autoclaved, after the decline from the initial value (47.55±0.42%) to a lower value (32.84±2.23%),at 60 days, the value remained almost unchanged till end of storage (180 days). However, at the end of storage, the level of fat content in the autoclaved was higher than in the control (un-autoclaved) and the difference in the level, between the control and the autoclaved was statistically significant (P ≤ 0.05). The higher total fat content of the autoclaved seeds (Figure 4.) agreed with the findings of Ezeokonkwo (2005) who observed increased total fat content of steamed seeds of an oil seed crop (African almond-Terminalia catappa). The significantly higher total fat value in the autoclaved seeds as compared to the control could be as a result of the preservation of the total fat by the autoclaving treatment which might have inactivated the endogenous enzymes (lipoxygenases) of the seeds. This reasoning is supported by the findings of Majunder (2007) and Sreerama et al. (2008) who reported that post harvest practices accelerate moisture migration, together with thermogenesis leading to enhanced deterioration of seed constituents such as fat, but, because endogenous enzymes were inactivated in the autoclaved seeds, there was lesser deterioration of the total fat compared to the raw seeds in which the endogenous enzymes might have been reduced the total fat content.

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Figure 2. Effects autoclaving on the moisture content of castor seeds after 180 days of storage Figure 3. Effect of autoclaving on the crude protein content of castor seeds after 180 days of storage Figure 4. Effects of autoclaving on total fat content of castor seeds after 180 days of storage Figure 5. Effects of autoclaving on crude fiber content of castor seeds after 180 days of storage Figure 6. Effects of autoclaving on the ash content of castor seeds after 180 days of storage Figure 7. Effects of autoclaving on the nitrogen free extract content of castor seeds after 180 days of storage Figure 8. Effects of autoclaving on free fatty acid content of castor seeds after 180 days of storage Figure 9. Effects of autoclaving on the peroxide value of castor seeds after 180 days of storage

Effects of autoclaving on crude fiber content of castor seeds after 180 days of storage The trend in the crude fiber content from 0-180 days of storage is presented in Figure 5. In both the un-autoclaved and autoclaved), a similar trend occurred. From the initial value (10.68±2.11%), the level of crude fiber content rose steadily to a higher value (35.60±30.18%), while a gradual rise was recorded in the autoclaved till the end of storage (36.80a±30.18). However, at the end of storage, the difference in the level of crude fiber between the control and the autoclaved was not statistically significant (P≥ 0.05). The non-significantly higher level of crude fiber content (Figure 5.) observed in the autoclaved seeds during this study agreed with the report of Apata (2008) who observed that autoclaving did not cause appreciable changes in the

crude fiber content of groundnut meal. The non-significant difference between autoclaved and un-autoclaved seeds with respect to crude fiber content could have been due to the presence of some heat-stable factors in the seeds causing less hydrolysis of the structural carbohydrates. The presence of heat-stable factors in other oilseeds such as Jatropha curcas has been reported (Martinez-Herrera et al. (2005). Effects of autoclaving on the total ash content of castor seeds after 180 days of storage The trend in the values of ash content from 0-180 days of storage is presented in Figure 6. In un-autoclaved and autoclaved seeds, a similar trend was observed. A gradual rise from the initial value (3.61±1.01%) to higher values

NEGEDU et al. – Effects of autoclaving on Ricinus communis seeds

after 180 days (6.20±0.25% and 5.24±0.25% respectively) was observed. After 180 days, the value of the ash content in the un-autoclaved was higher than that in the seeds. However, in both (control and autoclaved), at 180 days of storage, the difference in values between them, were statistically significant (P ≤ 0.05) with respect to ash content. The significantly higher level of ash content in the autoclaved seeds than that of un-autoclaved seeds (Figure5.) agreed with the findings of Ezeokonkwo (2005) who obtained similar result from steamed seeds of another oil seed, African almond (Terminalia catappa). Salunkhe and Desai (1986) had also reported increased ash content of steamed groundnuts seeds. The higher level of ash in the un-autoclaved seeds could be attributed to non-leaching of the minerals from the seeds which might have occurred in the autoclaved seeds during the autoclaving process. This reasoning is supported by Ataga and Ota-Ibe (2006) who reported leaching of minerals from steamed seeds of Wild Mango (Irvingia gabonensis) leading to decreased total ash content in the autoclaved seeds than in the control. Effects of autoclaving on the nitrogen free extract (NFE) content of castor seeds after 180 days of storage From the trend in the values of the NFE content of castor seed presented in Figure 5.24, the un-autoclaved showed a rise from the initial value (11.79±1.11%) to a higher value (21.31±1.28%) at 60 days and followed by a gradual decline to a lower level (16.29±1.82%). The value gradually rose to a level higher (19.17±0.07%). In the autoclaved seeds, after the initial rise from the pre-storage value (11.79±1.11%) to a higher level (22.44±1.28%), a steady decline followed till end of storage to a lower value (12.55±0.70%). Statistical analysis reveals that at 180 days, the mean values in the level of the NFE, between the autoclaved and controls (un-autoclaved) were significantly different (P ≤ 0.05). The significant decrease in the soluble sugar (NFEnitrogen free extract) of the autoclaved seeds (Figure 7.) disagreed with the findings of Ezeokonkwo (2005) who reported that roasting or steaming increased the level of soluble carbohydrates (NFE) in another oil seed (Terminalia catappa). In addition, Apata (2008) reported that autoclaving did not induce appreciable changes in the composition of cellulose, non-cellulosic polysaccharides and lignin of processed groundnut meal. It has been reported that some seeds may possess heat– stable factors such as lectins and typsin inhibitors (Martinez–Herrera et al. 2005) which make such seeds more resistant to hydrolysis by heat. The decrease in the level of NFE could be as a result of more stability of some structural carbohydrates of the castor seeds that allowed less hydrolysis of the insoluble sugars (crude fiber) into soluble sugars (NFE).

From the initial value (9.21±0.02%) of the free fatty acid, a rise to higher values occurred (31.74±2.34% in the autoclaved and 62.90±2.34% in the control seeds). This was followed by a decline to lower values (21.41±2.98% and 32.94±2.98% in the autoclaved and un-autoclaved respectively) at the end of storage. At the end of the storage period of 180 days, the level of free fatty acid in the autoclaved seeds was lower compared to the un-autoclaved control). Statistical analysis showed that difference in the levels of free fatty acids between the control and the autoclaved was statistically significant (P ≤ 0.05). The increase in the level of free fatty acid in the autoclaved seeds (Figure 8.) agreed with Oso (1974), Manji et al. (2006) who reported increased free fatty acid in steamed oil palm fruits. The increase could be due to greater liberation of free fatty acid by the heat process or conversion of the oil into their constituent fatty acids. This is supported by Onyeka et al. (2005) on heated fruits of another oil seed, Black pear (Dacryodes edulis). The decline could be as a result of the transformation of the free fatty acid into fatty acid hydroxy peroxides at a rate faster than they were formed, since the peroxides themselves are unstable and decomposed into stable compounds such as aldehydes, ketones, epoxides (Sowunmi 1981). Effects of autoclaving on the peroxide value of castor seeds after 180 days of storage Figure 9, presents the trend in the levels of peroxide value of castor seed during storage. An initial rise in the level of peroxide was observed in the autoclaved and unautoclaved seeds. After 120 days of storage, a decline in the level of peroxide value occurred in the autoclaved and un-autoclaved. The level of peroxide was lower in the autoclaved compared to the un-autoclaved seeds after 180 days of storage period. Statistical analysis showed that between the autoclaved and un-autoclaved seeds, the difference in the level of peroxide value was statistically significant (p≤0.05). The significantly higher level of peroxide value in the un-autoclaved seeds (Figure 9) compared to the autoclaved disagreed with Bankole et al. (2005) who reported higher peroxide in the steamed melon seeds. This variance could have been due to the decrease in the peroxide value resulting from the faster rate of decomposition of the hydroperoxy fatty acids (which are themselves unstable) into secondary products such as ketones, aldehydes, epoxides which are more stable and are largely responsible for the off flavours and objectionable odours in deteriorated seeds or oily products. This is supported by the findings of (Going 1968; Gaillard 1975; Arumughan et al. 1984; Amoo and Asoore 2006), they reported faster rate of decomposition of hydroperoxyfatty acids into secondary products.

CONCLUSION Effects of autoclaving on free fatty acid (FFA) content of castor seeds after 180 days of storage The trend in the levels of free fatty acid content of castor seed due to autoclaving is presented in Figure 8. In both autoclaved and control, a similar trend was observed.

The study has shown that when castor seeds are autoclaved and stored, the total fat content of the seeds increased, with increased free fatty acid level of the seed oil. The increase in the free fatty acid level of an oilseed is

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not healthy for the economic values of the seed, because increased free fatty acid level will cause rise in cost of processing and also attract reduction in seed price as penalty. In addition, the decreased protein and soluble sugars may not be economically advantageous for those interested in using the seed protein and soluble sugars. Therefore, it is not advisable to autoclave seeds before storage. If storage of seeds is for the purpose of economic end products, such as the oil, protein and soluble sugars, then, autoclaving may not be recommended.

REFERENCES Ajayi IA, Oderinde RA, Kajogbola DO, Uponi JI. 2005. Oil content and fatty acid composition of some underutilized legumes from Nigeria. Food Chem 99 (1): 115-120. Amoo LA, Asoore, FP. 2006. Effect of processing on the nutrient composition and oil of peanut (Arachis hypogea) seed flour. J Chem Soc Nigeria 31: 1-5. Anjani K, Raoof MA, Ashoka P, Reddy V, Rao CH. 2004. Sources of resistance to major castor (Ricinus communis L.) diseases. Pl Genet Res Newslett 137: 46-48. AOAC. 1995. Official Methods of Analysis. 13th Edition. Association of Analytical Chemists, Washington DC. USA. AOCS. 2000. American Oil Chemical Society. Official Methods of Analysis. 5th Edition. Association of Official Analytical Chemists. Washington, DC, USA. Apata FD. 2008. Effects of cooking methods on available and unavailable carbohydrates of some tropical grain legumes. African J Biotechnol 7 (16): 2940-2945. Arumughan C, Bhat KK, Sen DP. 1984. Evaluation of some chemical methods of deterioration in edible oils. J Food Sci Technol 21: 395399. Ataga AE, Ota-Ibe G. 2006. Seed-borne fungi of the wild mango (ogbono) [Irvingia gabonensis (Aubry-Leconte ex. Rorke) Bail] and their effects on food composition. Nigerian J Bot 19: 54-60. Bankole SA, Osho A, Joda AO, Enikuomehin OA. 2005. Effect of drying method on the quality and storability of ‘egusi’ melon seed (Colocynthis citrulus L). African J Biotechnol 4: 799-803. CSIR [Council of Scientific and Industrial Research]. 1946-1976. The Wealth of India. CSIR, New Delhi. Ezeokonkwo CA. 2005. Effect of roasting on the nutrient composition of Terminalia catappa L. seeds. Nigerian J Nat Sci 26 (1):19-24. FAO. 2005. World area production and productivity during 2005. Retrieved from http.//ikisan.com/links/ap-castor History.shtml on 7/9/2007 Gobin AMI, Uguru MI, Deckers I. 2001. Oil crops. In: Raemackers RH (ed). Crop production in tropical Africa. CIP Royal Library, Brussels. Going LH. 1968. Oxidative deterioration of partially processed soyabean oil. J Assoc Oil Chem Soc (JAOCS) 53: 632-636. Guelbel DV, Nudel BC, Giulietti F. 1991. A simple and rapid micro Kjeldah method for total nitrogen analysis. Biotech Technol 5 (6): 427-430. Joshua OO. 2005. Some physical properties of castor nut relevant to the design of processing equipment. J Agric Eng Res 77: 113-118

Kirk RS, Sawyer R. 1991. Fats and oils. In: Pearson’s composition and analysis of foods, 9th ed. Longman Group limited. UK. Majunder SK. 2007. Nutritional implication of recently developed techniques of storage and pest control. Central Food Technological Research Institute, Mysore, India. Manji AJ, Aliyu BA, Kafamiya II. 2006. Degradation of groundnut oil used for shallow frying. J Chem Soc Nigeria 31: 22-26. Martinez-Herrera J, Siddhuraju P, Francis G, Davila-Ortiz G, Becker K. 2005. Chemical composition, toxic/antimetabolic constituents and effects of different treatments on their levels in four provenances of Jatropha curcas L. from Mexico. Food Chem 96: 80-89. Ohegbu FO, Iweala JEE, Kanu I. 2011. Studies on the chemical and antinutritional content of some Nigerian spices. Intl J Nutr Metabol 3 (6): 72-76. Ohegbu FO, Onwuchekwa CC, Iweala JEE, Kanu I. 2009. Effect of processing methods on nutritive and antinutritive properties of seeds of Brachystegia eurycoma and Detarium mycrocarpum from Nigeria. Parkistan J Nutr 8 (4): 316-320. Okwu DE, Mor FN. 2004. Mineral and nutritive value of Dennettia tripetala fruits. Fruits 59 (6): 437-442. Onyeka EU, Onuegbu N, Onuoha NU, Ochonogor F. 2005. Effect of extraction pretreatment on the composition and characteristics of seed and pulp oil of African black pear (Dacryodes edulis). Nigeria Food J 23: 13-20. Oso BA. 1978. The lipase activity of Talaromyces emersoni. Canadian J Bot 56: 1840-1843. Roetheli JC, Glaser LK, Brigham RD. 1991. Castor: Assessing the feasibility of U.S. Production. Workshop Summary, Plain view Tx, Sept. 18-19, 1990. Salunkhe DK, Desai BB. 1986. Postharvest Biotechnology of Oil Seeds CRC Press. Florida. Sowunmi OE. 1981. Biochemical changes and nutritional changes in maize (Zea mays L.) and cowpea (Vigna unguiculata L.) during storage. [Ph.D. Dissertation] Faulty of Science, University of Ibadan, Nigeria. Sreerama YN, Sasikala VB, Pratape VM. 2008. Nutritional implications of recently developed techniques of storage and pest control. Food Chem 108 (3): 891-899. St. Angelo AJ, Kuck JC, Ory RL. 1979. Role of Lipoxygenase and Lipid Oxidation in quality of oil seeds. J Agric Food Chem 27 (2): 229-335 Udensi EA, Arisa NU, and Ike E. 2010. Effect of soaking and boiling and autoclaving on the nutritional quality of Mucuna flagellipes (“Ukpo”). African J Biochem Res 4 (2): 47-50. Udensi EA, Arisa NU, Maduka M. 2008. Effect of processing method on the level of antinutritional factors on Mucuna flagellipes. Nigeria Food J 26 (2): 53-59. Udensi EA, Onwuka GI, Okoli EG. 2004. Effect of processing on the levels of some antinutritional factors of Mucuna utilis palnt. Pl Prod J 8 (1): 1-6. Ukachukwu SN, Obioha FC. 2000. Effect of time duration of thermal treatments on the nutritive value of Mucuna cochinchinensis. Global J Pure Appl Sci 9: 11-15. Uzogora SG, Agu LN, Uzogora EO. 1990. A review of traditional fermented foods condiments and beverages in Nigeria: their benefits and possible problems. Ecol Food Nutr 24: 267-288. Ward HS, Diener UL. 1961. Biochemical changes in shelled peanuts caused by storage fungi. 1. Effect of Aspergillus tamarii, four species of Aspergillus glaucus group and Penicillium citrinum. Phyptopathol 51: 244-250.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 5, No. 2, pp. 57-64 November 2013

Changes in growth, hormones levels and essential oil content of Ammi visnaga plants treated with some bioregulators IMAN M. TALAAT1, HEMMAT I. KHATTAB2, AISHA M. AHMED1,♥ 1

Botany Department, National Research Centre, Cairo, Egypt. Tel.: +20-1140355848, Fax.: +20-233370931, ♥e-mail: aishazyat@yahoo.com 2 Botany Department, Faculty of Science, Ain-Shams University, Cairo, Egypt. Manuscript received: 6 August 2013. Revision accepted: 10 September 2013.

Abstract. Talaat IM, Khattab HI, Ahmed AM. 2013. Changes in growth, hormones levels and essential oil content of Ammi visnaga plants treated with some bioregulators. Nusantara Bioscience 5: 57-64. The effects of foliar application of different concentrations of amino acids (tyrosine and phenylalanine) and phenolic acids (trans-cinnamic acid, benzoic acid and salicylic acid) on growth, pigment content, hormones levels and essential oil content of Ammi visnaga L were carried out during two successive seasons. It is clear that foliar application of either amino acids or phenolics significantly promoted the growth parameters in terms of shoot height, fresh and dry biomass, number of branches and number of umbels per plant. The increment of growth parameter was associated with elevated levels of growth promoters (IAA, GA3, total cytokinins) and low level of ABA. The greatest increase in the previously mentioned parameters was measured in plants exposed to different concentrations of phenols particularly in benzoic acid-treated plants. Such effect was concentration dependent. All treatments led to significant increments in seed yield and essential oil content. Moreover, Gas Liquid Chromatographic analysis revealed that the main identified components of essential oil were 2,2-dimethyl butanoic acid, isobutyl isobutyrate,α-isophorone, thymol, fenchyl acetate and linalool. Phenolics and amino acids treatments resulted in qualitative differences in these components of oil. Key words: Ammi visnaga, phenolic compounds, amino acids, hormones, growth criteria, essential oil

Abstrak. Talaat IM, Khattab HI, Ahmed AM. 2013. Perubahan dalam pertumbuhan, kadar hormon dan kandungan minyak atsiri tanaman Ammi visnaga yang diperlakukan dengan beberapa bioregulator. Nusantara Bioscience 5: 57-64. Pengaruh aplikasi daun berbagai konsentrasi asam amino (tirosin dan fenilalanin) dan asam fenolat (asam trans-sinamat, asam benzoat dan asam salisilat) terhadap pertumbuhan, kandungan pigmen, kadar hormon dan kandungan minyak atsiri Ammi visnaga L. telah dilakukan selama dua musim berturut-turut. Hasilnya secara jelas menunjukkan bahwa aplikasi daun dari salah satu asam amino atau asam fenolat secara signifikan meningkatkan parameter pertumbuhan dalam hal tinggi tunas, biomassa segar dan kering, jumlah cabang dan jumlah tangkai bunga per tanaman. Kenaikan parameter pertumbuhan terkait dengan meningkatnya kadar hormon promotor pertumbuhan (IAA, GA3, total sitokinin) dan rendahnya kadar ABA. Peningkatan terbesar parameter tersebut terukur pada tanaman yang terkena berbagai kadar fenol terutama tanaman yang diperlakukan dengan asam benzoat. Efek seperti itu tergantung kadarnya. Semua perlakuan menyebabkan kenaikan signifikan dalam jumlah biji dan kandungan minyak atsiri. Selain itu, analisis Kromatografi Gas Cair mengungkapkan bahwa komponen utama yang teridentifikasi dari minyak atsiri adalah asam 2,2-dimetil butanoat, isobutil isobutirat, α-isoforon, timol, fensil asetat dan linalool. Perlakuan fenolat dan asam amino mengakibatkan perbedaan kualitatif komponen minyak atsiri ini. Kata kunci: Ammi visnaga, senyawa fenolat, asam amino, hormon, kriteria pertumbuhan, minyak atsiri

INTRODUCTION Ammi visnaga, known as Khella, is an annual or perennial herb belongs to family Apiaceae (Umbelliferae). Khella is native to the Mediterranean and is cultivated in Egypt. Ammi visnaga is antiasthmatic, diuretic, lithontripic and vasodilator. It is an effective muscle relaxant and has been used for centuries to alleviate the excruciating pain of kidney stones (Chevallier 1996). The seeds used as a folk medicine for diuretic and lithontripic (Uphof 1959). Visnaga seeds contain oil that includes the substance 'khellin', which is used in the treatment of asthma. They have antispasmodic action on the smaller bronchial muscles, dilate the bronchial, urinary and blood vessels without affecting blood pressure (Bown 1995). Essential oil

of A. visnaga is known for its proprieties against coronary diseases and bronchial asthma (Rose and Hulburd 1992; Satrani et al. 2004). The major components were linalool, isoamyl 2-methyl butyrate, and isopentyl isovalerate (Khadhri et al. 2011). Furthermore, phenolics are low molecular compounds ubiquitous in all tissues of higher plants with great significance in plant development. Phenolic compounds are some of the most widespread molecules among plant secondary metabolites, and are of great significance in plant development (Curir et al. 1990). However, their biological, ecological and agronomical significance in the rhizosphere is much less clear. Furthermore these biomolecules may contribute in soil and water conservation, weed management, mineral element nutrition, as well as

5 (2): 57-64, November 2013

they impact as signal molecule in certain symbiotic relationships, and act as defense molecules against soil pests and pathogens (Makoi1 and Ndakidemi 2007). Additionally, they serve as flower pigments, act as constitutive protection agents against biotic and abiotic stress (Delalonde et al 1996), function as signal molecules, act as allelopathic compounds, and affect cell and plant growth (Dakora 1995; Dakora and Phillips 1996; Ndakidemi and Dakora 2003), are important natural animal toxicants (Adams 1989) and some may function as pesticides (Vidhyasekaran 1988; Waterman and Mole 1989; Beier 1990). They are also functional components of the rhizosphere and its soil organic matter (Haider et al. 1975; Martin 1977). They have long been recognized as allelochemicals for weed control (Rice 1984; Putnam and Tang 1986) phytoestrogens in animals (Adams 1989) and plant defense molecules (Vidhyasekaran 1988). In the rhizosphere, they act as important precursors for the synthesis of soil humic substances (Haider et al. 1975). Salicylic acid participates in the regulation of several physiological processes in plant such as stomatal closure, nutrient uptake, chlorophyll synthesis, protein synthesis, inhibition of ethylene biosynthesis, transpiration and photosynthesis (Khan et al. 2003; Shakirova et al. 2003). SA increase cell metabolic rate (Amin et al. 2008). The biosynthesis of salicylic acid in plants starts from phenylalanine and follows one of two known paths of synthesis which involves trans-cinnamic acid then hydroxylation of benzoic acid which is a direct precursor of salicylic acid (Rask 1995). Moreover, amino acids as organic nitrogenous compounds are the building blocks in the synthesis of proteins (Davies 1982). Amino acids are particularly important for cell growth stimulation. They act as buffers which help to maintain favorable pH value within the plant cell. They protect the plants from ammonia toxicity. They can serve as a source of carbon and energy, as well as protect the plants against pathogens. Amino acids also function in the synthesis of other organic compounds, such as protein, amines, purines and pyrimidines, alkaloids, vitamins, enzymes, terpenoids and others (Goss 1973; Abdel-Aziz and Balbaa 2007). Furthermore, Hass (1975) stated that the biosyntheses of cinnamic acids (which are the starting materials for the synthesis of phenols are derived from phenylalanine and tyrosine. The aim of this study is to investigate the role of some phenolic substances (salicylic acid, t-cinnamic acid and benzoic acid) and amino acids (tyrosine and phenylalanine) on the growth, endogenous hormones, photosynthetic pigments, total, soluble and insoluble carbohydrates of A. visnaga plants as well as the essential oil content of the seeds. MATERIALS AND METHODS Experimental Two pot experiments were conducted in the greenhouse of National Research Centre (NRC), Dokki, Cairo, Egypt, during two successive seasons of 2009/2010 and 2010/2011. Ammi visnaga seeds were obtained from the Department of Medicinal and Aromatic Plants, Ministry of

Agriculture, Giza, Egypt. Ten sterilized seeds were sown in each pot (30cm diameter) in the third week of October. Each pot was filled with 10 kg of air-dried clay soil. Physical and chemical properties of the soil used in this study were determined according to Jackson (1973) and Cottenie et al. (1982) and are presented in Table (1). Eight weeks after sowing, the seedlings were thinned and three plants per pot were left. Pots were divided into three main groups. The first group was exposed to different levels of phenolic compounds (salicylic acid, trans-cinnamic acid and benzoic acid) at concentrations 5, 10 and 20 mg L-1. The second group was sprayed with different levels of amino acids (phenylalanine and tyrosine) at concentrations 50,100 and 200 mg L-1. Phenolic compounds and amino acids were applied after 30 days from the sowing date. The third group was sprayed with H2O to serve as control. The experiments conducted under natural day conditions, with photoperiod 11hr ± 2 and temperature about 27oC ± 2. Table 1. Physical and chemical properties of the soil used Soil texture

pH EC*

Organi Organic cC matter

Total N Total P Total K (%)

Sandy 7.2 0.6 0.9 1.9 0.3 loam Note: EC * = Electric conductivity (salinity)

0.1

All agricultural practices were conducted according to the recommendations by the Egyptian Ministry of Agriculture as follows: fertilizers were added to all pots as follows: cattle manure (2g pot−1), phosphorus (2g pot −1) as calcium super phosphate (15.5% P2O5), nitrogen (2g pot −1) as ammonium sulphate (20.5% N) and potassium (1.5 g pot −1 ) as potassium sulphate (48% K2O). Weeds were removed by hand and only natural pesticides were used for any plant diseases. The growth parameters of differently treated Ammi plants were measured after 75, 119, 180 and 210 days from sowing (stages A, B, C and D respectively). Stage A was at the vegetative growth while stage B at the beginning of flowering and stages C and D were at early fruiting and harvest time. Vegetative growth characters Plant height (cm), fresh and dry weights of shoot (g plant-1) were recorded during the vegetative stage. Plant height (cm), number of branches and umbels (plant-1), fresh and dry weights of shoots (plant-1) were recorded at flowering, early fruiting and fruiting stages. Endogenous hormones The endogenous hormone levels were determined using the method described by Wasfy and Orrin (1975). Chlorophyll (chl) a, chl b and total carotenoids content was measured according to the method o f Association of Official Agricultural Chemists (AOAC 1970). Total and soluble carbohydrate Total and soluble carbohydrate contents were determined according to the method described by Dubois et al. (1956). Then, the insoluble carbohydrates were calculated.

TALAAT et al. – Growth, hormones levels and essential oil content of Ammi visnaga

Figure 1. Ammi visnaga L. (Khella, bisnaga or toothpickweed)

Essential oil isolation The ripening fruits of A. visnaga were collected air dried and weighed for extraction of the essential oil. Five grams of dry fruits were crushed into smaller pieces and reduced to fine powder with the aid of a mechanical grinder. The powder sample was extracted with petroleum ether (PE 4060ºC) for 48h at room temperature. The extract was evaporated to dryness using a rotary evaporation at reduced pressure. The essential oil was passed over dark anhydrous sodium sulfate to remove moisture. The fraction obtained was stored in a refrigerator at 4ºC in dark to identify the chemical constituents of oil (Adams, 2007). GC-MS analysis were carried out on a Varina 3400 system equipped with a DB-5 fused silica column (30 m x 0.25 mm i.d.); Oven temperature was 40 to 240°C at a rate of 4°C min-1, transfer line temperature 260°C, injector temperature 250°C, carrier gas helium with a linear velocity of 31.5 cm s-1, split ratio 1/60, flow rate 1.1 mL min-1, Ionization energy 70 eV; scan time 1 s ; mass range 40-350 amu. Identification of components The components of the oil were identified by comparison of their mass-spectra with those of a computer library or with authentic compounds and confirmed by comparison of their retention Indices with those of authentic compounds. Kovats, Indices (Kováts 1958) were determined by coinjection of the sample with a solution containing a homologous series of n-hydrocarbons, at a temperature run identical to that described above.

Statistical analysis In this experiment, one factor was considered: different concentrations of amino acids (50, 100 and 200 mg L-1), phenolic compounds treatments (5, 10 and 20 mg L-1) and control. The experimental design followed a complete random block design. According to Sendecor and Cochran (1990), the average of data were statistically analyzed using 1-way analysis of variance (ANOVA-1). Significant values determined according to the Least Significant Difference (LSD at 0.05 and at 0.01 p) by using the STATITCF program (1982). RESULTS AND DISCUSSION Effect of amino acids and phenolic compounds on growth parameters Foliar application of different concentrations of either phenols or amino acids stimulate a gradual increases in growth parameters in terms of plant height, number of branches, number of umbels fresh and dry weights and water content of A. visnaga shoots throughout the experimental periods. Results also, investigated that phenols stimulate all the previous morphological parameters particularly at 20 mg L-1 compared with those of amino acids (tyrosine and phenylalanine) throughout the experimental period (Figures 1-6). The greatest increases in all investigated morphological criteria was measured in A. visnaga plants exposed to 20 mg L-1 benzoic acid at all stages. Similar results were obtained by

5 (2): 57-64, November 2013

Balbaa and Talaat (2007) who concluded that phenylalanine treatments significantly promoted plant height, number of branches, fresh and dry weights of rosemary plants. Abd El-Aziz et al. (2007) indicated also that foliar application of tyrosine significantly promoted plant height, number of leaves and branches, fresh and dry weights of branches and shoots and stem diameter in both cuttings of Salvia farinacea plants. It was recorded that application of certain amino acids significantly increased the vegetative growth of Chrysanthemum (El-Fawakhry and El-Tayeb 2003), peppermint (Refaat and Naguib 1998), datura (Youssef et al. 2004) and Pelargonium graveolens (Mahgoub and Talaat 2005). Furthermore, salicylic acid caused significant increases in most growth parameters of different plant species (Abd El-Wahed et al. 2006; El-Khallal et al. 2009; Delavari et al. 2010; Dawoode et al. 2012). The promotive effect of salicylic acid could be attributed to its bioregulator effects on physiological and biochemical processes in plants such as ion uptake, cell elongation, cell division, cell differentiation, sink/source regulation, enzymatic activities, protein synthesis and photosynthetic activity as well as increase the antioxidant capacity of plants (Raskin 1992; Blokhina et al. 2003; El-Tayeb 2005). Effect of amino acids and phenolic compounds on chemical composition The changes of chlorophylls a and b as well as carotenoids content in response to amino acids and phenolics treatments are shown in Figure 7. High pigments levels (chl a, b, carotenoids) were measured in A. visnaga leaves treated with phenols compared with those of amino acids. The maximum increase in chlorophylls and carotenoids are recorded in leaves treated with 20 mg L-1 benzoic acid. The increments in pigment level were attributed to the promotion in its synthesis and/or retardation of pigment degradation. These results are similar to those obtained by Sharma et al. (1995) who found that excised leaves of Tropaeolum majus, treated with t-cinnamic acid, retained more chlorophyll (60% higher at 10-3 M) compared to control. Moreover, the potent effects of particularly salicylic acid might be ascribed firstly to the reduction in chlorophyll loss due to its ability to increase the antioxidant capacity of the plants (Kuorzer et al. 1999) or inducing the synthesis of stabilizing substances (Nemeth et al. 2002). Salicylic acid caused significant increases in photosynthetic pigments (Figure 8). These results corrborate with those of Khodary (2004) and Gunes et al. (2005) on maize, El-Tayeb (2005) on barley, and Dawood et al. ( 2012) on sunflower. The enhancing effects of SA on photosynthetic capacity could be attributed to its stimulatory effects on Rubisco activity and pigment contents (Khodary 2004) as well as increased CO2 assimilation, photosynthetic rate and increased mineral uptake by the plant (Szepesi et al. 2005). In addition, Arfan et al. (2007), pointed that application of salicylic acid improved the photosynthetic capacity and retain pigment content through increasing IAA and Cytokinins therefore inhibits their senescence. Similar results were obtained by Hassanein (2003) on Foeniculum vulgare plants and Abou Dahab (2006) on Philodendron erubescens plant. They reported that foliar application of

the amino acid (tryptophan) caused an increase in photosynthetic pigments contents. The increments of the photosynthetic pigments in the treated A. visnaga leaves were concomitant with a gradual increase in total, soluble and insoluble carbohydrates (Figure 8). The maximum increases in soluble and insoluble carbohydrates were measured in the plants exposed to foliar application of phenolic compounds compared to those treated with amino acids. Moreover, such increments in the levels of total, soluble and insoluble carbohydrates were recorded in leaves exposed to 20 mg L1 benzoic acid. These results are in agreement with those obtained by Goss (1973), who indicated that amino acids can serve as a source of carbon and energy when carbohydrates become deficient in the plant; amino acids are determinate, releasing the ammonia and organic acid from which the amino acid was originally formed. The organic acids then enter the Kreb's cycle, to be broken down to release energy through respiration. These results could also be explained by the findings obtained by Gamal El-Din et al. (1997) who found that treatment of lemon grass plants with 100 ppm phenylalanine in the first cut and ornithine in the second cut recorded the highest level of carbohydrate percentage compared with control. Refaat and Naguib (1998) reported that application of all amino acids (alanine, cytosine, guanine, thiamine and L-tyrosine) increased the total carbohydrates percentage in peppermint leaves. The effect of the amino acids on the total carbohydrates content may be due to their important role on the biosynthesis of chlorophyll molecules which in turn affected carbohydrate metabolism. In this respect, Talaat and Balbaa (2010) reported that chemical analysis of the leaves of sweet basil indicated that the contents of total soluble and total carbohydrates were significantly increased as a result of foliar application of trans-cinnamic acid. Tari et al. (2002) and Dawood et al. (2012) reported that salicylic acid application resulted in a significant increase in total soluble carbohydrates content in leaves of tomato and sunflower, thus maintaining the carbohydrates pool in the chloroplasts at a high level. Plant hormones play an important role in development processes; some of them have a key in the most plant mechanisms. Data represented in Figure 9 showed increments in gibberellins (GA3), indole acetic acid (IAA) and cytokinins (Z & ZR) in plants treated with amino acids and phenolic compounds. High concentrations of gibberellins (GA3), Indole acetic acid (IAA) and cytokinins (Z & ZR) were measured in A. visnaga leaves treated with phenolic compounds compared with amino acids. The highest values of GA3, IAA and cytokinins were recorded in plants exposed to 20 mg L-1 benzoic acid. A reduction in abscisic acid (ABA) level was concomitant with such increments in growth promoters estimated in plants exposed to either phenolic compounds or amino acids. The increases in the levels of endogenous growth promoters could be attributed to the increase in their biosynthesis and/or decrease in their degradation and conjugation. On the other hand, the reduction in ABA level could be due to the shift of the common precursor isopentenyl pyrophosphate to biosynthesis of cytokinins and/or gibberellins

TALAAT et al. â&#x20AC;&#x201C; Growth, hormones levels and essential oil content of Ammi visnaga

150

Branch number

Plant height (cm/ plant)

100

Stages

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

8 6 4 Stages

Fruiting Fruiting Early Earlyfruiting fruiting Flowering Flowering Vegetative Vegetative

2 0

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Treatments

Shoot fresh wt. (g/plant)

Umble number

Fruiting Fruiting Early Earlyfruiting fruiting Flowering Flowering

30 20 Stages

10 0

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Fruiting Fruiting Early Earlyfruiting fruiting Flowering Flowering

70 60 50 40 30 Stages 20 10 0

Treatments

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Fruiting Fruiting Early Earlyfruiting fruiting Flowering Flowering Vegetative Vegetative

Treatments

100 90

40 30 20

Stages

10 0

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Fruiting Fruiting Earlyfruiting fruiting Early Flowering Flowering Vegetative Vegetative

Watercontant content (%) Water (%)

Shoot dry wt. (g/plant)

70 60 50 40

Stages

30 20 10 0

Treatments

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Treatments

2000

Carbohydrates (mg/100gdrywt)

Photosynthetic pigments (mg L-1)

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Chl a+Chl b/Carotenoids Chla+Chlb/Carotinoids Chl Chla/Chl a/Chlb b Chl Chla+Chl a+Chlb b Carotenoids Caretnoids Chl Chlbb Chl Chlaa

1500

1000

500

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

700 600 500 400 300 200 100 A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Cytokinins CYTOKININS ZR ZR ZZ ABA ABA IAA IAA GA 3 GA3

Fruit (g), oil(%),oil yelid(ml/plant)

800

Hormones (ug /g)

Totalcarbohydrate carbohydrates Total Insoluble carbohydrates Insoluble carbohydrate Solublecarbohydrate carbohydrates Soluble

Treatments

Fruiting Fruiting Early Earlyfruiting fruiting Flowering Flowering Vegetative Vegetative

10 8 6 4 2 0

A B1 B2 B3 C1 C2 C3 D1 D2 D3 E1 E2 E3 F1 F2 F3

Treatments

Oil Oil yield yelid Oil Oil % % Fruit Fruityield yelid

5 (2): 57-64, November 2013

Figure 1. Changes in the values of plant height of shoot system of A. visnaga plants (cm plant-1) treated with different concentrations of amino acids and phenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of ten replicates ± SD Figure 2. Changes in the values of branch number of shoot system of A. visnaga plants treated with different concentrations of amino acids and phenolic compounds during the flowering, early fruiting and fruiting stages, each value is mean of ten replicates ± SD Figure 3. Changes in the values of umbels number of shoot system of A. visnaga plants treated with different concentrations of amino acids and phenolic compounds during the flowering , early fruiting and fruiting stages, each value is mean of ten replicates ± SD Figure 4. Changes in the values of fresh weight of shoot system of A. visnaga plants (g plant-1) treated with different concentrations of amino acids and phenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of ten replicates ± SD Figure 5. Changes in the values of dry weight of shoot system of A. visnaga plants (g plant-1) treated with different concentrations of amino acids and phenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of ten replicates ± SD Figure 6. Changes in the percentage of water content of A. visnaga shoots treated with different concentrations of amino acids and phenolic compounds during the vegetative, flowering, early fruiting and fruiting stages, each value is mean of ten replicates ± SD Figure 7. Changes in the values photosynthetic pigments of A. visnaga plants (mg L-1) treated with different concentrations of amino acids and phenolic compounds during the vegetative stage, each value is mean of ten replicates ± SD Figure 8. Changes in the percentage of total, soluble and insoluble carbohydrates of A. visnaga plants (%) treated with different concentrations of amino acids and phenolic compounds during the vegetative stage; each value is mean of ten replicates ± SD Figure 9. Changes in the values of phytohormone contents of A. visnaga Plants (μg g-1) treated with different concentrations of amino acids and phenolic compounds during the vegetative stage Figure 10. Changes in the values of fruit yield (g), oil percentage (%) and oil yield (ml plant-1) of A. visnaga plants treated with different concentrations of amino acids and phenolic compounds, each value is mean of ten replicates ± SD Table 2. The constituents of essential oil of A. visnaga plants

No. Components (%)

1 α-Thujene 2 Myrcene 3 Isobutyl isobutyrate 4 Linalool 5 2,2-Dimethylbutanoic acid 6 α-Isophorone 7 Fenchyl acetate 8 Bornyl acetate 9 Thymol 10 Geranyl acetate 11 Lavandulyl acetate 12 Citronellyl propionate 13 Croweacin 14 α-Damascone 15 (Z,E)-farnesal Total identified Monoterpene compounds Sesquiterpene compounds

931 991 1004 1029 1108 1121 1220 1289 1290 1381 1439 1446 1460 1689 1701

0 22.9 5.7 28.9 13.4 6.3 13.2 9.6 100 100 -

Tyrosine 50 100 200 2.5 1.3 1.0 2.0 0.4 8.0 20.6 35.3 15.9 2.9 0.6 1.3 35.4 55.4 30.4 17.9 0.9 3.0 3.8 0.3 2.5 1.7 0.4 7.8 8.5 1.8 13.1 0.3 1.4 0.2 0.7 0.6 5.6 4.7 1.5 6.7 0.4 1.5 0.6 1.1 100 100 82.6 100 99.4 98.9 0.6 1.1

Phenylalanine 50 100 200 1.2 1.9 3.6 3.6 18.9 18.6 24.1 3.3 1.3 20.6 38.8 50.5 2.7 1.2 9.2 7.8 5.0 2.6 5.1 9.3 2.8 4.9 2.6 9.1 7.6 3.0 7.9 3.3 8.1 11.0 7.1 2.1 1.0 1.4 0.8 100 100 100 98.6 99.2 100 1.4 0.8 -

instead of ABA (Hopkins and Huner 2004). These results are in accordance with those obtained by Shehata et al. (2000), Shehata et al. (2001) and Zaghlool (2002). The increases in IAA and GA3 in shoot tissues of sunflower plant concurrently with the increase in growth rate due to the role of these endogenous hormones in stimulating cell division and/or the cell enlargement and subsequently growth (Taiz and Zeiger 1998). It is well known that salicylic acid induces flowering, increases flower life, retard senescence and increases cell metabolic rate. In addition, salicylic acid may be a prerequisite for synthesis

Treatments (ppm) Benzoic acid Tarns-cinnamic acid 5 10 20 5 10 20 1.1 3.9 2.2 0.4 0.9 1.2 3.7 1.9 0.4 1.6 14.8 24.3 9.9 11.4 24.4 22.6 0.8 4.5 2.1 0.3 1.1 35.0 25.9 21.1 27.4 36.5 34.6 11.9 16.7 9.6 13.8 19.3 21.1 1.0 4.8 7.0 0.2 3.2 0.8 4.3 5.3 0.5 2.3 2.1 15.2 7.0 8.0 0.8 1.7 11.5 5.2 3.8 11.2 2.7 1.4 3.7 2.7 0.7 1.1 1.0 5.3 1.6 1.2 10.4 15.0 5.9 7.2 2.8 3.3 3.2 2.9 5.7 2.7 2.4 0.9 3.8 5.4 2.9 0.1 1.7 100 100 100 100 100 100 96.2 100 94.6 97.1 99.9 98.3 3.8 5.4 2.9 0.1 1.7

Salicylic acid 5 10 20 1.5 1.2 3.9 2.1 1.4 4.9 6.4 16.5 15.6 1.1 2.5 2.6 59.0 34.4 38.2 6.4 11.3 13.8 3.7 4.7 3.5 0.9 0.8 2.0 6.7 3.7 5.7 0.9 6.9 4.5 2.2 0.9 3.1 2.4 6.0 8.7 5.3 2.2 2.4 100 100 100 100 97.6 100 2.4 -

of auxin and /or cytokinin. (Metwally et al. 2003, Gharib 2006). Furthermore, these increments in growth regulating substances might be a prerequisite for acceleration of growth resumption of sunflower plant. In addition,salicylic acid effects on abscisic acid (Senaranta et al. 2000), gibberellins (Traw and Bergelson 2003) regulate many physiological process and plant growth. Moreover, Dawood et al. (2012) reported that SA caused marked increments in IAA, GA3, zeatin and zeatin riboside, in the meantime decrease in ABA content comparing with untreated controls.

TALAAT et al. – Growth, hormones levels and essential oil content of Ammi visnaga

Figure 10 indicated that the fruit yield, oil yield percentage and oil yield (ml plant-1) increased in plants treated with phenolic compounds and amino acids. The maximum levels of oil yield percentage (ml plant-1) were recorded in seeds exposed to 20 mg L-1 benzoic acid. The increment in oil% and protein% might be due to the increase in vegetative growth and nutrients uptake. Similar results were reported by Gharib (2006) and Çag et al. (2009). In addition, Noreen and Ashraf (2010) mentioned that high doses of salicylic acid caused marked increases in sunflower achene oil content as well as some key fatty acids and significant decrease in stearic acid. Table 2 represents the compounds of essential oil obtained from A. visnaga as detected by GC-MS. the relative levels of various constituents of oil yield were increased, decreased or disappeared in A. visnaga fruits of plants treated with amino acids and phenolic compounds compared with untreated control plants. 2,2dimethylbutanoic acid, isobutyl isobutyrate, linalool, thymol and croweacin are the major constituents of A. visnaga fruits. These results are similar to those obtained by Khalfallah et al. (2011) who found that the major component of essential oil in A. visnaga are 2, 2dimethylbutanoic acid, isobutyl isobutyrate, croweacin, linalool and thymol. The effect of different treatments on essential oil and its constituents may be due to its effect on enzyme activity and metabolism of essential oil production (Burbott and Loomis 1969). SA has a role in controlling gene expression (He et al. 2005) reported that most of the genes regulated by SA are defense related genes and many of them participate in plant responses to biotic and abiotic stresses. Therefore SA may change secondary metabolites and its pathway by effects on plastid, chlorophyll level and represent stress conditions. The SA like stress manipulated quality and quantity of essential oil of Salvia macrosiphon. The yield of essential oil was increased. The useful component such as Linalool was increased. Seventeen components were identified in SA-treated plants (Rowshan et al. 2010).

CONCLUSION Finally, it is apparently clear that phenolics treatments were more effective in enhancing growth and productivity of A. visnaga. Moreover, the greatest increase in the growth parameters and chemical constituents obtained at 20 mg L-1 of benzoic acid. On the other hand, the major component of essential oil gave the best percentage (59%) was assayed in seeds exposed to salicylic acid.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 5, No. 2, pp. 65-69 November 2013

Effect of water regime on the growth, flower yield, essential oil and proline contents of Calendula officinalis SAMI ALI METWALLY1, KHALID ALI KHALID2,♥, BEDOUR H. ABOU-LEILA3 1

Department of Ornamental Plants and Woody Trees, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. 2 Department of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. Tel. +202-3366-9948, +202-33669955, Fax: +202-3337-0931, email: ahmed490@gmail.com 3 Department of Water Relation and Field Irrigation, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. Manuscript received: 10 May 2013. Revision accepted: 16 July 2013.

Abstract. Metwally SA,Khalid KA, Abou-Leila BH. 2013. Effect of water regime on the growth, flower yield, essential oil and proline contents of Calendula officinalis. Nusantara Bioscience 5: 65-69. The effects of water regime on the growth, content of essential oil and proline of Calendula officinalis L. plants were investigated. Water regimes of 75% of field water capacity increased certain growth characters [i.e. plant height (cm), leaf area (cm2), flower diameter (cm) and spike stem diameter] and vase life (day). Water regime promoted the accumulation of essential oil content and its main components as well as proline contents. Key words: Calendula officinalis, essential oil, flower yield, growth, proline, vase life, water regime

Abstrak. Metwally SA,Khalid KA, Abou-Leila BH. 2013. Pengaruh tata air terhadap pertumbuhan, hasil panen bunga, kandungan minyak atsiri dan prolin pada Calendula officinalis. Nusantara Bioscience 5: 65-69. Pengaruh tata air terhadap pertumbuhan, kandungan minyak atsiri dan prolin dari tanaman Calendula officinalis L. diteliti. Pengaturan tata air sebanyak 75% dari kapasitas air lapangan meningkatkan karakter-karakter pertumbuhan tertentu [yaitu: tinggi tanaman (cm), luas daun (cm2), diameter bunga (cm) dan diameter tangkai bulir] dan masa hidup bunga (hari). Pengaturan tata air menyebabkan akumulasi kadar minyak atsiri dan komponen utamanya serta kandungan prolin. Kata kunci: Calendula officinalis, minyak atsiri, hasil panen bunga, pertumbuhan, proline, tata air

INTRODUCTION Calendula officinalis L. (English marigold, pot marigold; Figure 1) belongs to the Asteraceae (Compositae) family; it is an annual with bright or yellow orange daisy-like flowers which are used for ornamental and medicinal purposes (Bcerentrup and Robbelen 1987; Cromack and Smith 1988). Calendula officinalis can be broadly applied as an antiseptic, antiinflammatory and cicatrizing (Correa Júnior 1994) aswell as a light antibacterial (Chiej 1988) and antiviral (Bogdanova and Farmakol 1970) agent. Many Calendula species have a characteristic scent or taste caused by mono and sesquiterpenes within the essential oil, which in many cases are the reason for their application in folk medicine (Yoshikawa et al. 2001). Recently, many attempts have been made to better characterize their therapeutic properties and to enhance the production of these useful compounds within their essential oils. Selected Calendula chemo-types growing in soil or in vitro, for example, flowers of the cadinol chemo-type, are very important in European and western Asian folk medicines and are used to treat inflammatory conditions (Yoshikawa et al. 2001). Distinct subspecies of C. officinalis have been reported from various countries (Chalchat et al. 1991; Nicoletta et al. 2003), i.e. Herbaria, Mecsek, Melius,

Golden Dragon and Adamo (Bakó et al. 2002). Calendula officinalis can be used as a colorant because it primarily contains two classes of pigments, the flavonoids and carotenoids, which can be used as yellow and orange natural colors, respectively. Natural colors are gaining considerable attention since several synthetic colorants have given rise to allergic, toxic and carcinogenic effects (Lea 1988). Flavonoids have antioxidant activities which play an important role in food preservation and human health by combating damage caused by oxidizing agents (Meda et al. 2005). Carotenoids are important to humans and other animals as precursors of vitamin A and retinoids. In addition, they act as antioxidants, immune-enhancers, inhibitors of mutagenesis and transformation, inhibitors of premalignant lesions, screening pigments in primate fovea, and non photochemical fluorescence quenchers (Castenmiller and West 1998). In aromatic plants, growth and essential oil production are influenced by various environmental factors, such as water deficit (Burbott and Loomis 1969; Sabih et al. 1999). Solinas and Deiana (1996) reported that secondary products of plants can be altered by environmental factors and that water deficit is a major factor affecting the synthesis of natural products. Water deficit resulted in a significant reduction of fresh and dry matter, and essential

5 (2): 65-69, November 2013

foreseeable future. Reclamation of desert lands has been a top priority and challenge for the Egyptian government over the last few decades. In this study, we investigate the possible effect of water defect on the flower yield, essential oil composition and proline content of C. officinalis flowers, an economically important medicinal and ornamental plant in Egypt.

MATERIALS AND METHODS Experiments were carried out in a greenhouse at the National Research Centre, Egypt, during 2010/2011 and 2011/2012. Calendula officinalis seedlings were obtained from the Medicinal and Aromatic department, Agriculture Research Centre, Egypt. Uniform seedlings were transplanted into plastic pots (30 cm diameter and 50 cm height). In the first week of November during both seasons, the pots were Figure 1. Calendula officinalis L. (English marigold, pot marigold) transferred to a greenhouse adjusted to the natural conditions. Each pot was filled with 10 kg of air-dried Typic Torrifluvents soil oil yield of mint (Mentha sp.) plants (Mirsa and Strivastava (USDA 1999), with a field water capacity (FWC) of 62.5% 2000). Fresh and dry weights of Ocimum basilicum L. based on the weight of the soil. Physical and chemical decreased as plant water deficit increased while the linalool properties of the soil used in this study were determined and methyl chavicol contents increased (Simon et al. 1992). according to Jackson (1973) and Cottenie et al. (1982) and The essential oil yield and proline contents of basil are presented in Table 1. Three weeks after transplanting, (Ocimum sp.) increased by subjecting plants to water the seedlings were thinned to three plants per pot. deficit just before harvesting (Baeck et al. 2001). Khalid Calendula officinalis plants were divided into four main (2006) reported that fresh and dry weights of Ocimum sp. groups were subjected to different levels of water regime: were significantly decreased by water deficit. Meanwhile, 25, 50, 75, or 100% (the control) corresponding to the essential oil percentage, as well as the main constituents of FWC determined in the soil by weight. All agricultural the essential oil, proline content increased. Baher et al. practices, other than the experimental treatments were done (2002) showed that water deficit reduced the fresh and dry according to the recommendation of the Ministry of weights of Satureja hortensis L. plants, while severe water Agriculture, Egypt. deficit increased essential oil content more than moderate water deficit. The main constituents, such as carvacrol, Table 1. Physical and chemical properties of the soil used increased under moderate water deficit, while ď Ą-terpinene content decreased under moderate and severe water deficit. Soil Total Total Total Org-C OM Hendawy and Khalid (2005) showed that essential oil, and texture pH EC* N P K proline contents showed a pronounced increased by % Sandy increasing the water stress levels of Salvia officinalis L. 7.2 0.6 0.9 1.9 0.3 0.1 0.1 loam plants. On the other hand, Petropoulos et al. (2007) noted that water deficit had relatively little effect on the essential Note: EC* = electronic conductivity (salinity), Org-C = organic C, OM = organic matter oil composition of parsley (Petroselinum crispum). The Egyptian climate is mostly arid and semi-arid, where water availability is a major problem for crop Harvesting Fresh flowers were collected from each treatment at production (Abou El-Fadl et al. 1990). In such conditions three flowering stages, start flowering or flower bud cultivation of resistant plants is one way to utilize these initiation (25 days after bud formation), full flowering (86 lands and therefore the selection of suitable crops, which days after bud formation) and end of flowering (119 days could cope with these conditions, is a necessity. In arid and semi-arid regions, where water availability is a major after bud formation) in both seasons, all of which were air limitation in crop production, using alternative water dried.â&#x2C6;&#x2019;1 Yield (dry weights of flower) was recorded (g growth characters resources. The major challenge facing water management plant ). On the other hand the vegetative 2 [Plant height (cm), leaf area (cm ), flower diameter (cm) is the availability of water. Its amount is fixed, but its and spike stem diameter] and vase life (day) were recorded demand will continue to increase steadily into the during the start flowering stage.

METWALLY et al. – Effect of water regime on Calendula officinalis

Essential oil isolation Fresh flowers were collected from each treatment and from the three flowering stages in both seasons, air dried and weighed to extract the essential oil. Dry flowers (500 g) from each of these treatments were hydro-distilled for 3 h using a Clevenger-type apparatus (Clevenger 1928). The essential oil content was calculated as a percentage. Also g essential oil plant −1 was calculated according to the dry weight of flowers per plant. Gas Chromatography-Mass Spectrophotometric (GC-MS) analysis The ADELSIGLC MS system, equipped with a BPX5 capillary column (0.22 mm id × 25 m, film thickness 0.25 µm) was used. Analysis was carried out using He as the carrier gas, with a flow rate of 1.0 mL/min. The column temperature was programmed from 60 to 240°C at 3°C/min. The sample size was 2 µl, the split ratio 1: 20; injector temperature was 250°C; ionization voltage applied was 70 eV, mass range m/z 41-400 amu. Kovat’s indices were determined by co-injection of the sample with a solution containing a homologous series of nhydrocarbons in a temperature run identical to that described above. Identification of essential oil components The separated components of the essential oil were identified by matching with the National Institute of Standards and Technology (NIST) mass spectral library data, and by comparison with Kovat’s indices of authentic components and with published data (Adams 2001). Quantitative determination was carried out based on peak area integration. Proline determination Proline content was determined in fresh leaves during three flowering stages using the method of Bates et al. (1973). Statistical analysis In this experiment, 2 factors were considered: water deficit (100, 75, 50 and 25 % FWC) and flowering stages. For each treatment there were 4 replicates, each of which had 8 pots; in each pot 3 individual plants were planted. The experimental design followed a complete random block design. According to Snedecor and Cochran (1990) the averages of data for two seasons were statistically analyzed using 2-way analysis of variance (ANOVA-2) for flower yield (g plant-1), Proline (µm mg-1) and

Table 2. Effect of water regime on the vegetative growth characters and vase life Vegetative growth characters Water stress Plant Flower Spike stem Leaf area treatment height diameter diameter 2 (cm ) (cm) (cm) (cm) 100 36.3 79.3 5.0 0.3 75 39.3 95.0 5.7 0.5 50 38.3 85.3 5.5 0.4 25 35.7 52.7 4.5 0.2 F. values 0.3 0.1 *** 0.1 0.1 ** LSD at 0.05 NS 6.4 NS 0.04 Note: *P≤0.05, **P<0.01, ***P<0.001 according to F-values of analysis of variance (ANOVA-1).

Vase life (day) 8.3 10.0 9.3 6.7 0.1 ** 1.4 the 2-way

Table 3. Effect of water regime, flowering stages and their interactions on flower yield, proline and essential oil content Treatments Water regime 100 75 50 25 Over all

Flower yield (g plant -1) 9.3 113.1 50.4 44.1 54.2

Full

100 75 50 25 Over all

End

100 75 50 25 Over all

Stages Start

Proline (µm mg-1)

Essential oil %

mL plant -1

2.1 2.5 3.8 8.6 4.3

0.1 0.2 0.2 0.3 0.2

0.009 0.226 0.101 0.132 0.117

135.7 233.6 222.9 100.3 173.1

3.4 3.9 5.8 10.9 6.0

0.2 0.2 0.3 0.4 0.3

0.271 0.467 0.669 0.401 0.452

69.1 152.6 103.1 58.8 95.9

2.4 2.9 4.8 9.8 5.0

0.2 0.3 0.4 0.5 0.4

0.138 0.458 0.412 0.294 0.326

Over all water regime 100 71.4 2.6 0.2 0.139 75 166.4 3.1 0.2 0.384 50 125.5 4.8 0.3 0.394 25 67.7 9.8 0.4 0.276 F values Water regime 2466.2*** 2555.9*** 9.727*** 31606.9*** Stages 15248.6*** 247.4*** 7.364*** 140934.9*** Water regime Ҳ stages 3796.1 *** 5.7 *** 0.455 5553.3*** LSD at 0.05 Water stress 38.8 0.6 0.04 0.04 Stages 22.8 0.3 0.03 0.03 Water stress Ҳ stages 21.7 0.2 NS 0.02 Note: *P≤0.05, **P<0.01, ***P<0.001 according to F-values of the 2-way analysis of variance (ANOVA-2). Table 4. Effect of water regime on the chemical constitution of essential oil Water regime treatments F. values LSD 100 75 50 25 γ-Cadinene 1514 2.5 2.6 2.7 2.8 1.3 NS ∆ -Cadinene 1523 18.8 18.9 19.3 19.8 8.9** 0.2 β-Calacorene 1564 4.4 4.5 4.7 4.9 1.3 NS Nerolidol (E) 1565 6.9 7.1 7.8 7.9 11.9*** 0.2 β-Acorenol 1635 4.9 5.2 5.3 5.5 1.4 NS α-Eudesmol 1653 10.8 10.9 11.8 12.9 36.9*** 0.1 α-Cadinol 1663 33.9 34.8 34.9 35.2 1.6 NS Pentacosane 2501 3.8 4.2 4.4 4.9 6.3* 0.2 Total identified 86.0 88.2 90.9 93.9 Note: *P≤0.05, **P<0.01, ***P<0.001 according to F-values of the 2-way analysis of variance (ANOVA-2). Constituents (%)

5 (2): 65-69, November 2013

essential oil (% and mL plant-1); while using 1-way analysis of variance (ANOVA-1) for plant height (cm), leaf area (cm2),flower diameter (cm),Spike stem diameter, Vase life (day) and essential oil constituents. The applications of that technique were according to the STAT-ITCF program (Foucart 1982).

RESULTS AND DISCUSSION Effect of water regime on the vegetative growth characters and vase life Vegetative growth characters [Plant height (cm), leaf area (cm2), flower diameter (cm) and spike stem diameter] and vase life (day) of Calendula plants were affected by changes of soil moisture. The highest values of these measurements were recorded when plants subjected to 75% of FWC with the values of 39.3, 95.0, 5.7, 0.5 and 10.0 respectively. On the other hand the lowest values were recorded when the plants were subjected to 25% of FWC. ANOVA indicated that the changes in plant height and flower diameter were insignificant but highly significant for leaf area while more significant for pike stem diameter and Vase life (Table 2). The inhibition of plant growth characters and flower yield under water deficit treatment (25% of FWC) may be due to exposure to injurious levels of drought causing a decrease of turgor which would result in a decrease of growth and development of cells, especially in stems and leaves (Merrill and Eckard 1971). Cell growth is the most important process and is affected by water stress. Plant size is indicated by a decrease in height or smaller size of leaves when there is a decrease in the growth of cells (Hsiao 1973). When leaf size is smaller, the capacity to trap light decreases too and the capacity of total photosynthesis decreases, i.e. photosynthesis is restricted in water shortage conditions, with a subsequent reduction in plant growth and performance (Hsiao 1973). Water stress resulted in significant reductions in CO2 exchange rate, total assimilatory area, fresh and dry matter and chlorophyll in Japanese mint (Mentha arvensis L. cv. MS 77) (Misra and Srivastava 2000). The loss of photosynthesis in drought stress conditions results in a loss of dry matter production at the leaf level of mungbean, bean, topiary bean, Sesuvium portulacastrum (ambiguously) and Pesquisa Agropeularia (embrapa) plants (Cox and Jolliff 1987; Abdul-Hamid et al. 1990; Castonguay and Markhart 1991; Nunez-Barrios 1991; Viera et al. 1991; Slama et al. 2007). However the decrease in flowers vase life under stress condition may be due to loss of turgidity (Hirt and Shinozaki 2003). Effect of water regime, flowering stages and their interactions on the flower yield Water regime and/or flowering stage affected the total flowers (g plant-1) (Table 3). Thus, various characteristics of the flowers decreased under the various water regime levels, especially at 25 % of FWC at the end of flowering stages. Greatest yields were obtained at 75 % of FWC, especially at full flowering stage (Table 3). The decrease in flower heads was highly significant for water regime

treatments and for flowering stages. In addition, the changes in this variable were highly significant for the water regime × flowering stage interaction (Table 3). Our results showed that growth and flower yield of C. officinalis plants was clearly affected by the different water regime, where the growth parameters recorded the highest values when plants irrigated after adaptation of 75 % of FWC. These superiority may be due to treatment provide the plant all time of growth with adequate supply of water which accelerate physiological processes and plant growth. In this respect Tayel and Sabreen (2011) indicated that soil water potential through the growing season is necessary to maintain crop growth. Moreover, addition of adequate water decreased abssic acid and increased cytokinins, gibrellin and indole acetic acid hormones, which reflecting good plant growth and finally yield (Hayat 2007). Effect of water regime, flowering stages and their interactions on proline content The accumulation of proline in C. officinalis leaves was promoted by applying various levels of soil moisture, flowering stages and their interaction (Table 3). The highest proline content resulted from 25 % FWC treatment at full flowering stage (Table 3). The increase in proline content was highly significant for water regime flowering stages and their interaction treatments (Table 3). The results of proline content agree with those of Salama et al. (2001) and Blum and Ebercon (1993) who indicated that proline is regarded as a source of energy, carbon, and nitrogen for recovering tissues under stress conditions. Effect of water regime, flowering stages and their interactions on essential oil content and its chemical composition Data in table indicates that the highest percentage of C. officinalis essential oil was obtained from flower heads at the full flowering stage, i.e. full flowering stage, therefore, this stage was investigated to identify the essential oil components. Treatment with 25% (FWC) caused the most pronounced increase in the essential oil percentage at all flowering stages; however, this percentage decreased at control treatments. The essential oil yield (g plant-1) increased in the most water regime treatments compared with the control treatment in. The essential oil% and yield of flower heads was greatest at full flowering. Water regime treatments and flowering stage, when assessed separately, had a greater effect on essential % and yield of flowers than their interaction, i.e. water regime treatments × flowering stages. The changes in essential oil yield were highly significant for water regime treatments and flowering stages (Table 3). A qualitative and quantitative comparison of the main constituents present with the water regime treatments in hydro-distilled C. officinalis essential oil was studied (Table 4). A total of 8 compounds, accounting for 86.094.3% of the oil, were identified. The effects of soil moisture levels on the chemical composition of essential oil extracted are shown in Table 4. The main components were γ-cadinene, ∆-cadinene, β-calacorene, nerolidol, β-acoreno, α-eudesmol, α-cadinol and pentacosane. Moreover, the

METWALLY et al. – Effect of water regime on Calendula officinalis

highest percentages of the main components resulted from the treatment of 25% FWC. These percentages decreased as soil moisture levels increased. The changes in nerolidol and α-eudesmol constituents were highly significant for water regime treatments while the changes in ∆-cadinene constituent were more significant. The changes in pentacosane were significant. On the other hand, changes in γ-cadinenem, β-calacorene, β-acorenol and α-cadinol were insignificant for water regime treatments (Table 4). The effect of different treatments on essential oil and its constituents may be due to its effect on enzyme activity and metabolism of essential oil production (Burbott and Loomis 1969).

CONCLUSION It may be concluded that water stress affects on growth, flower yield, vase life. essential oil composition and proline contents of Calendula officinalis L. plants.

REFERENCES Abou El-Fadl, IA, Abd-Ella MK, Hussein EH.1990. Effect of irrigation by saline water on the growth and some principal compounds of peppermint and spearmint in two types of soil. J Agric Res Tanta Univ 16: 276-295. Abdul-Hamid AF, Kubota FA, Morokuma M. 1990. Photosynthesis, transpiration, dry matter accumulation and yield performance of mungbean plant in response to water stress. J Fac Agric Kyushu Univ 1 (2): 81-92. Adams RP. 1995. Identification of Essential Oil Components by Gas Chromatogra-phy/Mass Spectroscopy. Allured, Carol Stream, IL, USA. Bakó E, Delia J, Tóth G. 2002. HPLC study on the carotenoid composition of Calendula products. J Biochem Biophys Meth 53: 241-250. Bcerentrup H, Robbelen G.1987. Calendula and Coriandrum new potential oil crops for industrial use. Fat Sci Technol 89: 227-230. Bogdanova X, Farmakol T. 1970. Biological effects of some aromatic plants. Toksikol 33: 349-355. Burbott AJ, Loomis D. 1969. Evidence for metabolic turnover monoterpene in peppermint. Plant Physiol 44: 173-179. Baeck H, Kuenwoo P, Baeck HW, Park KW. 2001. Effect of watering on growth and oil content of sweet basil (Ocimum americanum L.). Kor J Hort Sci Technol 19 (1): 81-86. Baher ZF, Mirza M, Ghorbanil M, Rezaii MZ. 2002. The influence of water stress on plant height, herbal and essential oil yield and composition in Satueja hortensis L., Flav Frag J 17: 275-277. Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline of water stress studies. Plant and Soil 39: 205-207. Cromack H, Smith J. 1988. Calendula officinalis production potential and crop agronomy in southern England. Ind Crops Prod 7: 223 229. Correa Júnior C. 1994. Cultivo de plantas medcinais, condimentares e aromáticas, 2nd ed. FUNEP, Japoticabal. Chiej R. 1988. The Macdonald Encycopedia of Medicinal Plants. Macdonald & Co., London. Cottenie A, Verloo M, Kiekens L, Velghe G, Camerlynck R. 1982. Chemical Analysis of Plant and Soil. Laboratory of Analytical and Agrochemistry, State University, Ghent, Belgium. Chalchat JC, Ph R, Mechet AG. 1991. Chemical composition of essential oil of Calendula officinalis L. (pot marigold). Flav Frag J 6: 189-192. Castenmiller JJM, West CE. 1998. Bioavailability and bioconversion of carotenoids. Ann Rev Nutr 18: 19-38. Clevenger JF, 1928. Apparatus for determination of essential oil. J Amer Pharm17: 346-349.

Cox WJ, Jolhff GD. 1987.Water relations of sunflower and soybean under irrigated and dry land conditions. Crop Sci 27: 553-557. Castonguay Y, Markhart AH. 1991. Leaf gas exchange in water stress common bean and topiary bean. Crop Sci 32 (4): 980. Blum A, Ebercon A. 1976. Genotype responses in sorghum to drought stress. III. Free proline accumulation and drought resistance. Crop Sci 16: 379-386. Foucart T. 1982. Analyse factorielle, programmatiol sur micro-ordinateur. Masson, ITCF, Paris. Hayat AEH. 2007. Physiological studies on Hibiscus sabdariffa L., production I new recleaned Soils. M Sc Thesis Fac Agric Zagazig Univ., Egypt. Hirt, H. and k. Shinozaki.2003.Plant responses to a biotic stress. Asian J Pl Sci 6: 1072-1079. Hsiao T C. 1973. Plant responses to water stress. Annual Review. Plant Physiol 24: 519- 570. Hendawy SF, Khalid KA. 2005. Response of sage (Salvia Officinalis L.) plants to zinc application under different salinity levels. J App Sci Res 1 (2): 147-155. Jackson M. 1973. Soil Chemical Analysis. Prentice Hall of India Pvt., Ltd., New Delhi. Khalid KA. 2006. Influence of water stress on growth, essential oil and chemical composition of herbs (Ocimum sp.). Int Agrophys 20 (4): 289-296. Lea AGH. 1988. HPLC in Food Analysis. Academic Press, London. Merrill RK, Eckard AN. 1971. Evaluation of water stress control with polyethylene glycols by analysis of guttation. Plant Physiol 47: 453-456. Misra A, Srivastava NK. 2000. Influence of water stress on Japanese mint. J Herbs Spices Med Plants 7 (1): 51-58. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma GO. 2005. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem 91: 571-577. Nunez-Barrious A. 1991. Effect of soil water deficits on the growth and development of dry bean (Phaseoulus Vulgaris L.) at different stages of growth. [Dissertation]. Department of Crop and Soil Sciences, Michigan State University, Michigan. Nicoletta CB, Marongiu PA, Pivetta T, Procedda S. 2003. Extraction, separation, and isolation of volatiles and dyes from Calendula officinalis L. And Aloysia tryphylla(L’Her) britton by supercritical CO2. J Ess Oil Res 15: 272-277. Petropoulos SA, Dimitra D, Polissiou MG, Passam HC. 2007. The effect of water deficit stress on the growth, yield and composition of essential oils of parsley. Scient Hort 115: 393-397. Sabih F, Abad Farooki AH, Ansari SR, Sharama S. 1999. Water stress on growth and essential oil metabolism in Cymbopogon martinii (Palmarosa) cultivars. J Ess Oil Res 1: 151-157. Simon JE, Reiss BD, Joly RJ, Charles DJ. 1992. Water stress induced alternations in essential oil content of sweet basil. J Ess Oil Res 1: 71-75. Solinas V, Deiana S. 1996. Effect of water and nutritional conditions on the Rosmarinus offfcinalis L., phenolic fraction and essential oil yields. Rivista Italiana EPPOS 19: 189-198. Slama I, Ghnaya T, Hessini K, Messedi D, Savoure A, Abdelly C. 2007. Comparative study of the effects of mannitol and PEG osmotic stress on growth and solute accumulation in Sesuvium portulacastrum. Environ Exp Bot 61: 10-17. Snedecor GW, Cochran WG. 1990. Statistical Methods, 11th ed. Iowa State Univ. Press, Ames, Iowa, USA. Tayel MY, Sabreen KHP. 2011. Effect of irrigation regemes, phosphoous level and two Vicia faba varieties on yield, water and phosphorus use efficiency. J app Sci Res 7 (11): 1518-1526. USDA. 1999. Soil Taxonomy. Natural resources conservation service. Washington D.C., USA. Viera HJ, Bergamaschi H, Angelocci LR, Libardi PL. 1991. Performance of two bean cultivars under two water availability regimes. II. Stomatal resistance to vapour diffusion, transpiration flux density and water potential in the plant. Pesquisa Agropeularia Brasileria 9: 10351045. [Portugal] Yoshikawa M. Murakami T, Kishi A, Kageura T, Matsuda H. 2001. Medicinal flowers. III. Marigold (1): hypoglycemic, gastric emptying inhibitory and gastroprotective principles and new oleanane type triterpene oligoglycoside, calenda saponins A, B, C, and D, from Egyptian Calendula officinalis. Chem Pharm Bull 49: 863-870.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 5, No. 2, pp. 70-74 November 2013

Phenotypic recurrent selection on herb growth yield of citronella grass (Cymbopogon nardus) grown in Egypt MOHAMED M. IBRAHIM1,â&#x2122;Ľ, KHALID A. KHALID2 1

Department of Genetics and Cytology, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. Tel. +202-3371615. ď&#x201A;Š email: mohamed_mostafa480@yahoo.com 2 Department of Medicinal and Aromatic Plants, National Research Centre, El Buhouth St., Dokki 12311, Giza, Cairo, Egypt. Manuscript received: 26 June 2013. Revision accepted: 16 July 2013.

Abstract. Ibrahim MM,Khalid KA. 2013. Phenotypic recurrent selection on herb growth yield of citronella grass (Cymbopogon nardus) grown in Egypt. Nusantara Bioscience 5: 70-74. This investigation was conducted in four generations: base population (G0, G1, G2) and G3 (clone selection generation) to evaluate the genetic variability of citronella clones. Thirteen clones were selected from base population to study the herb growth yield characters and oil production as well as genetic parameters, correlation and regression. Results were recorded for herb growth characters (i.e. plant high (PH), no. of tillers (NOT), dry yield (DY), viability percentage (VP) and oil production. Significant variation was observed among citronella clones in base population for most studied traits. Wide range of mean values was observed among the characters for generations and cuts in most of traits. High heritability values (0.95, 0.93, 0.89 and 0.72) were estimated in NOT, LG, HY and VP., respectively. Clone code no. 39/3, 17/4 and 8/1 gave highest values of dry weight, oil yield and viability percentage. Selected clones showed significant positive regression and correlation between dry weight and each of number of tillers and linear growth. On contrary, viability percentage had significant negative correlation and regression with other characters. These results raveled high yielding selected citronella clones will be utilized in medicinal plant breeding program. Key words: citronella, essential oil, heritability, selection

Abstrak. Ibrahim MM,Khalid KA. 2013. Seleksi fenotipik berulang pada pertumbuhan herba rumput serai (Cymbopogon nardus) yang ditanam di Mesir. Nusantara Bioscience 5: 70-74. Penelitian ini dilakukan dalam empat generasi: populasi dasar (G0, G1, G2) dan G3 (generasi pilihan klon) untuk mengevaluasi keragaman genetik klon-klon serai (sereh wangi). Tiga belas klon dipilih dari populasi dasar untuk mempelajari karakter hasil pertumbuhan herba dan produksi minyak serta parameter genetik, dengan korelasi dan regresi. Hasil yang dicatat berupa karakter hasil pertumbuhan herba (yaitu: tinggi tanaman (PH), jumlah anakan (NOT), hasil berat kering (DY), persentase viabilitas (VP) dan produksi minyak. Variasi yang signifikan teramati diantara klon-klon serai dalam populasi dasar untuk sebagian besar sifat-sifat yang dipelajari. Nilai rata-rata yang berjangkauan luas teramati diantara sifat-sifat untuk generasi dan pemotongan di sebagian besar sifat. Nilai heritabilitas tinggi (0,95, 0,93, 0,89 dan 0,72) diperkirakan secara berturut-turut pada NOT, LG, HY dan VP. Kode klon no. 39/3, 17/4 dan 8/1 memiliki nilai tertinggi dalam berat kering, hasil minyak dan persentase viabilitas. Klon terpilih menunjukkan regresi dan korelasi positif yang signifikan antara berat kering dan jumlah anakan serta pertumbuhan linear. Sebaliknya, persentase viabilitas memiliki korelasi dan regresi negatif yang signifikan dengan karakter lain. Hasil ini memberikan klonklon serai terseleksi dengan hasil panen yang tinggi yang akan digunakan pada program pemuliaan tanaman obat. Kata kunci: serai, minyak esensial, heritabilitas, seleksi

INTRODUCTION Citronella, Cymbopogon nardus L. is a tufted perennial grass with long narrow leaves and numerous stems arising from short rhizome roots (Figure 1) which are indigenous to India (Weiss 1997). The importance of citronella grass is related to widely use in perfumes, soaps, insect repellent. It is also used in Chinese medicine and traditional medicine for the treatment of rheumatism, digestive problems, fever, and intestinal problems and in aromatherapy to treat colds, flu, and headaches (Akhila 2010). Few efforts had been carried out on the crop improvement through the clone selection of herb and essential oil yield. Plant breeders primarily estimate variability in initial population of its importance in

choosing the most efficient breeding procedures. Kulkarni (1994) studied the herb growth characters, oil yield and variability in lemon grass though phenotypic recurrent selection, found that realized gains from selection were slightly smaller or smaller to predicted gains. Kole and Sen (1986) studied the selection strategy of yield and yield component in lemon grass clones in base population and selected clones. Selection is more efficient to improvement essential oil and yield component. The importance of clone selection in genus Cymbopogon and genetic variability were studied by many researchers (Patra et al. 1991; Rao and sobti 1991; Siugh and pathak 1994). They found recently a few clonal varieties have been developed to overcome wide fluctuation in quantity production of genus Cymbopogon.

IBRAHIM & KHALID – Phenotypic recurrent selection on Cymbopogon nardus

Statistical analysis Statistical analysis of data was computed according to Steel and Torrie (1965). Heritability estimates were according to Robinson et al. (1951).Correlation and regression analysis were estimates with according to Mode and Robinson (1959). Extraction of essential oils The essential oils were extracted by harvesting the plants of selected clone's basis on dry weight. 30 grams of dried leaves from each of three replications for each selected clone were hydro distilled using Clevenger apparatus (Gunter 1962) for three hours. The obtained oil was measured and then computed as percentage value.

RESULTS AND DISCUSSION

Figure 1. Cymbopogon nardus L. (citronella grass)

Information on genetic variability, heritability estimates and interclass correlations for important characters are essential for plant breeding program. The present work aims to study genetic variability in population of citronella grass and then attempt to select some characterized and improved clones in herb growth yield and essential oil production.

MATERIALS AND METHODS Cultivation method This work was carried out in the Experimental farm of Nation Research Centre (NRC) in El Nobaria, Egypt, during four successive seasons 2008 - 2011 seasons or generations. Clones of citronella grass (Cymbopogom nards L.) were obtained from the groups of genetics and breeding of medicinal plants, Nation Research Centre (NRC), Egypt. All tented clones were cultivated using tillers witch separated from mother plants. On the 1st of May in base population and selected clones over two trials. A randomized – complete block design with 3 replications was used. Each replicate had one line of 3m long and 60 cm in between. Each line had six ridges with 50 cm space. All cultural practices were followed. The first cut was taken after six months after planting, while the second cut was taken three months later. The plants of base population and selection experiment were carried out though four generation. The plant records included linear growth, number of tillers, herb dry yield and viability percentages of 13 clones were cultivated.

Rang, mean values, coefficient of variation. (%) analysis of variance, heritability and L.S.D. of four herb growth characters (no. of tillers.linear growth, herb dry yield and viability percentage) at three generations (G0, G1 and G2) of citronella clones population are presented in Table 1. Significant differences were showed in all studied characters. Wide rang observed in all studied traits among three generations. Mean values ranged from(8.3±0.6) to (87.3±0.6), (43.4±0.9) to (108.5±5.1), (32.3±2.7) to (165.9±2.48), (77.0±1.9) to (98±0.07) % of numbers of tillers, linear growth, herb dry yield and viability percentage respectively. Coefficient of variation (C.V. %) varied in all generations and all studied characters. Heritability estimate revealed highest values (0.9528, 0.9303, 0.8935 and 0.725) in no. of tillers,linear growth, herb dry yield and viability percentage., respectively,while the values 0.4253, 0.1589, 0.372 and 0.1986 were the lowest in the same characters., respectively. Moreover, the first cut had higher heritability values comparing with the second cut values for all studied characters. Evaluation of selected citronella clones Thirteen clones of citronella were selected from earlier population to study clonal variation parameters and evaluate herb growth characters and yield components Analysis of variance Highly significant differences were shown among generations in all studied characters and only related to linear growth among clones Covariance analysis Average values, coefficient of variation and parentprogeny regression of 13 selected clones over four different generations presented in Table (3). clonal variations were found for herb growth yield characters in the four

5 (2): 70-74, November 2013

generations. Average values of clones cod no. 39/3, 15/4, 17/4, 1/2and 14/3 had the highest values of herb dry character.Linear growth had the highest spatial regression (bij) in case of clone 25/1 followed by clone 39/3 in dry weight and no. of tillers Interclass correlation and regression Interclass correlation and regression among four growth herb yield in the mean of 13 citronella clones was presented in Table 4. Viability percentage showed high negative correlation with each of linear growth, no. of tillers and herb dry yield. One the other hand herb dry yield was high positive correlation with linear growth and no. of tillers. Viability percentage with each of no. of tillers, linear growth and herb dry yield revealed negative regression values (-0.52, 1.01, -13.25) respectively. Herb dry yield with each of linear growth and no. of tillers had high (25.23, 18.66) and positive values of regression respectively. Variation in oil production in selected citronella clones Data presented in Table 5 showed variation in the oil content variation and oil yield of the thirteen selected clones basis on dry weight..Oil percentage ranged from 2.00 to 2.66 for clones (24/4, 14/3) and 36/3 respectively. Clones no. 15/4, 17/4 and 1/2 had highest values of oil yield. Oil yield ranged from 2.96 in clone 34/3 to 11.99 in case of clone 15/4 respectively.

DISCUSSION Cymbopogon species display wide variation in morphological attributes and essential oil composition at inter- and intra specific levels. Germplasm diversity is important for plant conservation and improvement, therefore there is interest in determining the genetic diversity in Cymbopogon germplasm. Although, morphological traits can be used to assess genetic diversity they are strongly influenced by environment conditions and show

Table 1. Biometrical genetic parameters of four major herb yield component in citronella grass under clone selection Mean

C.V. ANOVA % ơ2g

ơ2e

Number of tillers 1st gen (G0) 5.5-11.3 2nd gen (G1) 11.4-17.7 1st Cut 5.1-13.0 2nd Cut 16.0-28.5 3rd gen(G2) 42.5-119.3

8.3±0.6 14.9±0.6 8.3±0.6 21.1±0.8 75.7±1.2

25 14 25 14 24

24.43** 226.38** 77.15** 143.48** 1289.07**

Linear growth (cm) 1st gen (G0) 39.6-49.0 2nd gen (G1) 51.0-63.1 1st Cut 37.5-52.5 2nd Cut 62.3-74.5 3rd gen (G2) 77.5-127.5

43.4±0.9 57.0±0.6 46.4±1.7 63.6±1.3 108.5±5.1

7 8 12 6 15

Herb dry yield (g/plant) 1st gen (G0) 18.4-95.0 2nd gen (G1) 30.0-78.1 1st Cut 18.9-52.7 2nd Cut 32.5-121.7 3rd gen (G2) 420.344

35.3±1.2 56.0±3.6 32.3±2.7 80.0±1.3 1659±248

Parentage viability 1st gen (G0) 75-100 2ndgen (G1) 42-100 3rd gen (G2) 68-89

98±0.7 79±4.2 77±1.9

Items

Range

h2b

LSD 0.05 0.01

33.64 11.33 10.15 39.48 105.44

0.4253 0.9528 0.8828 0.7837 0.5508

4.36 1.71 2.29 4.6 44.94

5.74 2.25 3.01 6.06 59.16

41 37 37 37 16

29.78* 216.92** 147.22** 46.04* 435.26**

157.69 52.6 105.35 121.92 32.63

0.1589 0.7574 0.5829 0.2741 0.9303

8.99 3.7 7.6 7.9 7.9

0.0 4.9 10 10.4 10.4

41 37 37 37 16

56 23 30 28 54

1328.92** 2021.91* 1083.82** 1048.57** 8.79**

678.41 1197.83 596.35 1799.3 0.26

0.662 0.372 0.6451 0.3682 0.8935

20 17.9 17.8 30.9 70.7

26.4 23.5 23.4 40.7 930

41 37 37 37 16

7 19 9

429.59** 75.23**

164.99 315.57

0.725 20.6 0.1986 22

27.1 29

41 41

Table 2. Analysis of variance of 13 citronella clones grown in four generations under clone selection S.O.V.A

DF No. of tillers DF Linear growth DF Herb dry yield Viability %

Clones 12 117.93 10 140.57** Generations 3 13435.49*** 3 9822.34 *** Error 36 101.13 36 51.07

12 203566.21 3 8423104.3** 36 199424.21

82.57 1187.16*** 123.467

Table 3. Average values, coefficient of variation and regression on the median index of 13 citronella clones grown in four generations Viability % C.V. bij Average % 1.02 16 87 1.22 20 83 0.98 16 85 1.72 40 74 1.16 19 86 1.16 25 73 0.91 14 88 1.01 19 88 0.69 11 92 0.74 11 90 1.30 12 88 1.20 17 84 0.04 12 77 84.23±1.66 7.42

Herb dry yield (gm.) C.V. bij Average % 2.10 18.7 905 0.98 18.7 424 1.84 18.7 794 1.54 18.4 673 0.63 16.4 309 0.94 17.0 443 0.75 17.8 341 1.01 17.9 455 0.24 13.9 137 0.58 15.4 293 0.42 15.1 245 1.29 18.6 560 0.63 17.7 287 451±62.53 49.97

Linear growth (cm.) C.V. bij Average % 1.26 52 75 1.28 57 68 00 00 000 1.09 46 72 1.27 49 74 0.75 35 64 5.85 32 56 1.26 51 74 0.83 43 59 0.73 33 66 00 00 00 1.03 52 64 1.01 49 62 66.59±1494 44.57

Number of tillers C.V. bij Average % 1.62 13.1 40 0.79 5.6 27 0.92 12.1 25 1.04 10.4 32 1.09 12.2 29 0.92 12.1 25 1.04 12.6 27 0.61 8,9 23 0.47 7.4 21 0.05 11.5 32 0.87 10.1 24 1.36 12.7 35 1.05 12.4 28 28.0±15.94 11.3.85

Code no. 39/3 54/4 15/4 17/4 24/1 22/1 25/1 14/3 34/3 8/1 36/3 1/2 18/2 Mean CV%

IBRAHIM & KHALID – Phenotypic recurrent selection on Cymbopogon nardus

little variation at the intra-specific level (Dhar et al. 1981; Kulkarni and Rajagopol 1986; Kulkarni 1997). The fluctuation of viability percentage of citronella clones population from generation to other due to compensation among yield component, fitness characters and genotype – environmental interaction Adams and Gutfuls (1971). The analysis of variance and covariance in the actual and predicted selection in base population and selected top 30% of citronella clones were studied in herb growth characters and oil yield production. Highly significant differences were shown among generations in three characters only (number of tillers, linear growth and viability percentage). This finding revealed that clones carried genes with different additive effects. Meanwhile, in citronella clones reveled highly significant differences between clones in case of linear growth character, these results were reflected the role of gene type–environment interaction and seasonal variations (Western and Lawrence 1970). The result of clonal variation for herb growth yield characters in the three generations are in agreement with the investigation of Omkhafe and Alika (2004) and Jezowski (2008). Table 4. Estimates of interclass correlation (above) and regression (down) coefficients among four herb growth yield components. Characters

No. of tillers (X1) Linear growth ( X2) Herb dry yield ( X3) Viability% ( X4)

1.000 0.360 25.23 -0.52

0.3368* 1.000 18.66 -1.01

0.5911** 0.4699** 1.0000 -13.25

-0.5202** -0.85414** -03706** 1.0000

Table 5. Average values, coefficient of variation in oil production for selected citronella clones at two generations. Code no. 39/3 24/4 15/4 17/4 24/1 22/1 25/1 14/3 34/3 8/1 36/3 1/2 18/2 Mean C.V.%

Oil percent % Average C.V.% 2.05 17.02 2.00 13.20 2.05 4.80 2.10 7.20 2.46 14.30 2.03 28.70 2.08 16.23 2.00 16.06 2.16 11.03 2.14 10.25 2.66 16.22 2.12 12.80 2.16 10.30 2.16 ± 0.053 13.70

Oil yield ml./plant Average C.V.% 6.33 24.22 8.02 67.21 11.99 61.12 10.43 50.93 7.43 39.04 8.43 56.50 7.09 40.30 9.12 60.12 2.96 53.61 6.22 24.30 7.33 40.15 10.40 61.30 6.22 25.26 7.85 ± 0.645 46.47

In citronella studied clones the correlation analysis confirms the role of clones x environment interaction Viability percentage showed high negative correlation with each of linear growth, no. of tillers and herb dry yield. One the other hand herb dry yield was high positive correlation with linear growth and no. of tillers. These results reflected compensation in yield components and variation in excretion. That is confirmed clear homeostasis of genetic

background and differential expression in these characters expression (El- Balal et al. 1983). Comparison of oil content and yield of the selected clones in the four seasons revealed apparent interaction (Harridy et al. 2001). Herb dry yield express of growth activity and essential oil production in grasses (Barrister and GutBurg 1965). Variations in oil production and accumulation (oil/yield ml/plant) due to many factors, such as growth stage, elongation of tillers and dry matter production were influenced. These finding is in agreement with Misra and Srivastava (1991) and Behura et al. (1991).

CONCLUSION

The results from this study leads to the conclusion that there are significant genetic variability among citronella clones has been made through phenotypic recurrent selection for studied traits. Wide rang were showed in all studied characters among three generations. Heritability estimates revealed highest values in no. of tillers, linear growth, and herb dry yield and viability percentage. Coefficient of variation (C.V. %) varied in all generations and all studied characters. One the other hand herb dry yield was high positive correlation with linear growth and no. of tillers. Highly significant differences were shown among generations in three characters only (number of tillers, linear growth and viability percentage). This finding revealed that clones carried genes with different additive effects. Meanwhile, in citronella clones reveled highly significant differences between clones in case of linear growth character, these results were reflected the role of gene type–environment interaction and seasonal variations

REFERENCES Adams MW, Gafuis JR. 1971.Yield compensation –alternative inter pertains. Crop Sci 11: 33-39. Akhila A. 2010. Essential oil bearing grasses. The genus Cymbopogon CRC Press. New York. Behura BS, Sahoo NK, Dutta PK. 1991. Cymbopogon natural hybrid Jamarose the aromatic grass suitable for chromite overburden plantation. Indian Perfumer 35 (2): 90-92. Burmeister VA, Gutenberg A. 1960 On the formation of essential oils in plants. Planta Medica 8: 1-30. Dhar AK, Thappa RK, Tel CKA. 1981. Variabililty in yield and composition of essential oil in Cymbopogon jawarancusa. Planta Medica 41: 366-388 El-ballal AS. 1983. Compensation pattern of essential oil yield component in the selected caraway genotypes. Acta Hort 36 (1,4): 132 - 171. El-balal AA, Mandour MS, Nofel M, Tawfik MSH. 1983. Physiological homestasis of essential oil production in lemon grass (Cymbopogon citratus L). The IX Inter. Cong. Ess. Oils Singapore 147-151. Guenther E. 1962. Essential Oils vol.1.4D Van Nostrand Co., New Jersey. Harridy IM, Gabr AS, Shalan MN. 2001. Comparative study on some species of the genus Cymbopogon grown in Egypt. Egypt J Res 79 (4) 48-1469. Jezoaski S. 2008. yield traits of six clones of Misconstrues in the first 3 year following planting in Poland. Industr Crops Prod 87: 65-68. Kole CR. 1986. Path- coefficient analysis in citronella. Indian Journal of Agric Sci 56 (4) 241-244. Kole CR, Sen S. 1986. Selection strategy for improvement of oil yield of citronella Cymbopogon winterianus. Environ Ecol 4 (4) 613-618.

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Kulkarni RN. 1994. Phenotypic recurrent selection for oil content in East Indian Lemongrass. Euphytica 78 (1-2): 103-107. Kulkarni RN. 1997. Developmental and performance of a high yielding synthetic Variety of lemon grass. J Herb Spice Med Plants 3 (3) 2331. Kukarni RN, Rajagopol K. 1986. Broad and narrow sense heritability estimates of leaf yield, leaf width, tiller number and oil contact East Indian lemon grass. Z Phar 96: 135-139. Mode CJ, Robinson HF. 1959. Pleitropism and genetic variance and covariance. Biometrics 15: 518-537. Omokokhafe KO, Alika JE. 2004. Clonal variation and correlation of seed characters in Heave brasiliensis Muel.Arg. Industr Crops Prod 19: 173- 184. Patra NK, Sharma S, Ram RS. 1991. correlation and several plant traits to citral content in lemongrass (Cymbopogon spp.). Exp Genet 7: 102106 Rao BL, Sobti SN. 1991. CkP 25 a hybrid lemon grass. Indian Perfumer 35: 48-149.

Robinson HF, Comstock RE, Harvy PH. 1951. Genotypic and phenotypic correlation in corn and their implication to selection. Argon J 43: 282287. Samuel CJK, Hill J, Breese EI, Davies A. 1970. Assessing and predicting environmental response in Lolium perenum. J Agric Sci Comb 75: 19. Sing RS, Pathak MC. 1994. Variability in herb yield and volatile constituents of Cymbpobogon jawarancusa (Jones) Schull cultivars. Ind Crops Prod 2 (3) 197-199. Steel A, Torrie F. 1965. Principles and procedures of statistical,with special references to the biological science. 2nd ed. McGraw-Hill Co. New York. Weiss EA. 1997. Essential oil Crops. CAB International, New York U.S.A Westerman JM., Lawrance G. 1970. Genotype- environmental interaction developmental regulation in Aabidapsis thaliana L. Heredity 25: 1457-1465

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 5, No. 2, pp. 75-85 November 2013

Experimental effect of temperature and sedimentation on bleaching of the two Red Sea corals Stylophora pistillata and Acropora humilis MOHAMMED S.A. AMMAR1,♥, AHMED H. OBUID-ALLAH2, MONTASER A.M. AL-HAMMADY3 1

Department of Hydrobiology, National Institute of Oceanography and Fisheries, P.O. Box 182, Suez, Egypt. Tel. +20 111 1072982, Fax. +20 623360016, E-mail: shokry_1@yahoo.com 2 Department of Zoology, Faculty of Science, Assiut University, Assiut, Egypt 3 National Institute of Oceanography and Fisheries, Hurghada, Egypt Manuscript received: 30 July 2013. Revision accepted: 15 August 2013.

Abstract. Ammar MSA, Obuid-Allah AH, Al-Hammady MAM. 2013. Experimental effect of temperature and sedimentation on bleaching of the two Red Sea corals Stylophora pistillata and Acropora humilis. Nusantara Bioscience 5: 75-85. At 26°C (the control sample), the loss of zooxanthellae by each of the two studied corals Stylophora pistillata and Acropora humilis was very low. Cell viability of the two studied corals was similar at 26 and 29°C, but depicted a sharp decline of zooxanthellae lost at 31°C through time. As the temperature increased to 35°C, the loss of zooxanthellae from each host increased both with time and temperature elevation. The coral A. humilis had a higher decrease in its zooxanthellae densities than S. pistillata at the same treatment. Bleaching temperature threshold was 33°C or less for the two species S. pistillata and A. humilis where 51% of their zooxanthellae were lost after 24 h of exposure. In samples exposed to sediment concentration of 0.1 mg/cm2/L, zooxanthellae densities of A. humilis and S. pistillata did not show any decrease after 1 day. However, after 1 days of exposure to 0.5 mg/cm2/L, zooxanthellae densities were significantly different from those of the controls. Increases in sediment concentration to 1 mg/cm2/L caused a decrease in zooxanthellae densities that vary greatly over time. Measurements of zooxanthellae densities of A. humilis and S. pistillata at this stage revealed a highly significant difference between exposed and control sample. At 1 g/cm2/L, the number of zooxanthellae lost from A. humilis was higher than those lost from S. pistillata at same time. It is suggested that, the normal sedimentation rate for A. humilis and S. pistillata to be in an order of 1 mg/cm2/L or less. Key words: Acropora humilis, bleaching, Red Sea corals, sedimentation, Stylophora pistillata, temperature Abstrak. Ammar MSA, Obuid-Allah AH, Al-Hammady MAM. 2013. Pengaruh perlakuan suhu dan sedimentasi terhadap pemutihan dua jenis karang dari Laut Merah Stylophora pistillata dan Acropora humilis. Nusantara Bioscience 5: 75-85. Pada suhu 26°C (kontrol), hilangnya zooxanthellae dari dua jenis karang yang diteliti Stylophora pistillata dan Acropora humilis sangat rendah. Viabilitas sel kedua jenis karang tersebut serupa pada suhu 26 dan 29°C, tetapi pada suhu 31°C terjadi penurunan tajam zooxanthellae sejalan dengan bertambahnya waktu. Pada saat suhu meningkat menjadi 35°C, hilangnya zooxanthellae dari masing-masing inang meningkat sejalan dengan bertambahnya waktu maupun suhu. Karang A. humilis mengalami penurunan kepadatan zooxanthellae yang lebih tinggi daripada S. pistillata pada perlakuan yang sama. Batas suhu pemutihan karang adalah 33°C atau kurang untuk kedua jenis karang, S. pistillata dan A. Humilis, dimana 51% dari zooxanthellae-nya hilang setelah 24 jam paparan. Pada sampel yang terpapar sedimen dengan konsentrasi 0,1 mg/cm2/L, kepadatan zooxanthellae A. humilis dan S. pistillata tidak menunjukkan penurunan apapun setelah 1 hari. Namun, setelah 1 hari paparan sedimen 0,5 mg/cm2/L, kepadatan zooxanthellae secara signifikan berbeda dari kontrol. Peningkatan konsentrasi sedimen 1 mg/cm2/L menyebabkan penurunan kepadatan zooxanthellae yang sangat bervariasi dari waktu ke waktu. Pada tahap ini, pengukuran kepadatan zooxanthellae A. humilis dan S. pistillata menunjukkan perbedaan yang sangat signifikan antara yang terpapar dan kontrol. Pada konsentrasi sedimen 1 g/cm2/L, jumlah zooxanthellae yang hilang dari A. humilis lebih tinggi daripada yang hilang dari S. pistillata pada waktu yang sama. Hal ini menunjukkan bahwa, tingkat sedimentasi normal untuk A. humilis dan S. pistillata berada pada kisaran 1 mg/cm2/L atau kurang. Kata kunci: Acropora humilis, pemutihan, karang Laut Merah, sedimentasi, Stylophora pistillata, suhu

INTRODUCTION Bleaching (loss of pigmentation by corals) is a widespread phenomenon in coral reef ecosystems. Despite this, the underlying of some forms of bleaching are poorly understood. This study explores the conditions that induced bleaching in two zooxanthellate, reef coral species Acropora humilis and Stylophora pistillata collected from the middle-reef in front of National institute of oceanography and fisheries (NIOF), Hurghada Branch.

Environmental extremes, such as high temperature or irradiance, damage the symbionts’ photosynthetic machinery, resulting in the over production of oxygen radicals. This leads to eventual cellular damage in the symbionts and/or their hosts, and can lead to the expulsion of symbionts and the eventual break down of the symbiosis (Lesser 2006). The loss of zooxanthellae (and/or a reduction in their pigment concentrations) as a result of this process is referred to as‘‘bleaching’’ (Brown et al. 1999; Fitt et al. 2000).

5 (2): 75-85, November 2013

An important factor affecting coral communities is the widespread, periodic bleaching of coral colonies (Celliers and Schleyer 2002). Coral bleaching results in the breakdown of a mutualistic symbiosis that is essential for the survival of corals, since the polyp receives a substantial part of its energy from the zooxanthellae (Muller-Parker and D’Elia 1997), and any disruption of this relationship will affect photosynthetic potential, coral growth, reproductive output and may eventually kill the coral (Richmond 1997). Because of the intensity of recent bleaching events and associated mortalities, bleaching is considered by most reef scientists to be a serious and relatively new challenge to the health of world's coral reef (Celliers and Schleyer 2002; Nesa and Hidaka 2009; Obura 2009; Miller et al. 2011). The widespread bleaching of corals on individual reefs has largely been correlated with elevated sea temperature (Celliers and Schleyer 2002; Mc Clanahan et al. 2005). Seasonal cycles in the quantum yields of chlorophyll fluorescence of corals have also been observed (Warner et al. 2002), revealing seasonal acclimatization in solar irradiance and seawater temperature. Moreover, Nesa and Hidaka (2009) detected a negative correlation between survival time and the zooxanthella density of tissue balls at 31°C in both Fungia sp. and P. divaricata. Elevated temperature was found to significantly reduce the amount of zooxanthellae in primary polyps (Anlauf et al. 2010). Exposure to elevated temperatures reduces the photosynthetic rate of zooxanthellae and predisposes their photosynthetic apparatus to further damage (Bhagooli and Hidaka 2004). Reef building corals and their zooxanthellae had shown two broad ways that may be able to cope with elevated temperature (Clark 1983). Firstly, by micro-adaptive combinations of symbiotic algae (Rowan 1997), secondly by biochemical defense mechanisms, such as the induction of heat shock proteins (Sharp et al. 1997; Ammar and Mueller 2001). Despite their ability to acclimatize to their thermal limits, reef building corals do not appear to have acclimatized to the rapid increase in sea temperature over the past 20 years. Sedimentation is among the factors that leads to escape of symbiotic zooxanthellae from the host coral (Dubinsky and Stambler 1996). Fabricius (2005) regarded sedimentation as an increasing threat to coral reefs. The impacts associated with sedimentation and sediment burial include reduced photosynthesis and increased respiration (Philipp and Fabricius 2003; Weber et al. 2006), tissue mortality (Lirman and Manzello 2009), reduced growth (Lirman and Manzello 2009), and reduced fertilization, larval survivorship, and recruitment (Babcock and Smith 2000). The degree of coral mortality and bleaching depends on the amount of sediment in the coral communities following a tropical storm in the tropical Atlantic (Nowlis et al. 1997). Burial of corals by sediments for 20 hours resulted in increased discoloration of coral tissue, after 68 hours of burial, up to 98% of the tissue bleached in the first days, about 50% of this tissue disappeared subsequently and bare coral skeleton became exposed or were covered with algae (Wesseling et al. 1999). Cruz-Pinio et al. (2003) found that, high sedimentation rates, low light availability and

anthropogenic influence lead to cellular damage and deteriorated coral skeletal density. Turbidity reduces light levels, photosynthetic potential and possibly coral growth rates (Yentsch et al. 2002; Anthony and Hoegh-Guldberg 2003) while elevated net sedimentation rates increase abrasion and smothering (Fabricius 2005), inhibiting coral growth and reef accretion. In contrast, Palmer et al. (2010) found that, near shore environments directly influenced by fluvial sediments and dominated by terrigenoclastic sedimentation are generally considered marginal for coral reef growth. The energetic costs of sediment clearing can be considerable (Riegl and Branch 1995), and the inability to clear sediments exposes corals to further stress as anoxic conditions under sediments can cause tissue bleaching and subsequent mortality (Weber et al. 2006). For the purposes of this study, the release of algal symbionts at various temperatures and sediment concentrations (“bleaching response”) was studied on the two reef corals S. pistillata and A. humilis using the protocols of Hoegh-Guldberg and Smith (1989) and Anlauf et al. (2010). The focus here on increasing seawater temperature reflects the choice of an environmental factor that is an integral component of global climate change effects and is tractable to experimental manipulation. Moreover both temperature and sedimentation have a wellestablished effect on coral bleaching.

MATERIALS AND METHODS Two experiments were carried out in the NIOF laboratories using the hermatypic coral S. pistillata and A. humilis in which corals were exposed to elevated water temperatures and different concentrations of sediments. Corals were collected from the middle reef in front of the National institute of Oceanography and Fisheries (NIOF), Hurghada Branch, Egypt. Experiments were conducted in open topped glass aquaria, under controlled conditions. Seawater was aerated using air pumps and heated and recirculated using submersible pumps and aquarium heaters. Refrigerated coolers and refrigerated water-baths were used to control the temperatures to be within ±0.3°C of the desired level. Temperature readings were made every 5-15 min between 8.00 a.m. and 6.00 p.m. During the night, the readings were made only every 2 hours since the temperature was more stable. The temperatures were increased and decreased following the diurnal variation in water temperature in the field by approximately 6°C. Temperature in the field was between 29°C and 30°C at midday (at 3 m depth). Small colonies of the two studied species were collected from individual colonies located at 3 m depth from the Middle reef, which is located 200 m offshore between the northern reef and the crescent reef, directly in front of NIOF, Hurghada, Red Sea. Samples were then transferred directly into glass aquaria supplied with air pumps for aeration. Two corals for studying the effect of temperature were incubated at each temperature test (24, 29, 31, 33 and

AMMAR et al. – Effect of temperature and sedimentation on corals bleaching

35˚C). Control samples were placed at room temperature (26˚C). An air stone was placed in each aquarium for water circulation and the sample was put in a good light condition during the day for photosynthesis. Three colonies from each species were then taken after 6, 12 and 24 hours and their tissues were removed using sea water jet (Water Pik Teledyne) for counting the zooxanthellae and measuring the chlorophyll concentration. Zooxanthellae were counted using count Rafter cell and the chlorophyll concentration was measured by spectrophotometer method of Jeffrey and Humphrey (1975). Branches for studying the effect of sediments were put in glass aquaria, exposed to 0.1, 0.5, 1 mg/cm2/L and 1g/cm2/L different concentrations of sediments, an air stone was placed in each aquarium for aeration in a good lighting conditions, then three branches of each colony were taken after 1, 5 and 10 days for counting zooxanthellae and measuring chlorophyll concentration. Biomass measurements For zooxanthellae density, tissues were striped from the skeletons (Plate 15) with a jet of recirculated 0.45 µm membrane filtered sea water using a water pikTM (Johannes and Wiebe 1970). The slurry produced from the tissuestripping process was homogenated in a blender for 30s and the volume of homogenate was recorded. The number of zooxanthellae in 10 ml aliquotes of homogenate was measured in triplicate by light microscope (X 400) using Count Rafter Cell. The total number of zooxanthellae per coral was measured after correcting the volume of homogenate. Zooxanthellae density was calculated as a number per unit surface area. Zooxanthellae number / cm2 = counted cells / cell surface area x cell depth x dilution For chlorophyll analysis, 10-20 ml sub samples of the homogenate were filtered through Whatman GF/C 0.45 filters, which were then homogenized for 30s using a tissue homogenizer. Chlorophyll was extracted twice with 10 ml of acetone 90% for 24 h in darkness at-4 C°. Extracts were centrifuged at 6000 g rev/min for 20 minutes to remove filter fiber from suspension and the supernatants read on a spectrophotometer at 630, 645, 665 and 750 wave lengths. Chlorophyll concentrations were calculated according to the equation of Jeffery and Humphrey (1975), as follow: Ch.a (mg m3) = 11.85D663-665-1.54 D647-0.08D630 ) v I-1 V-1 D = absorbance at wave length incubated by subscript, after correction by the cell to cell bank and subtraction of the cell-to-cell blank corrected absorbance at 750 nm V = volume of acetone (ml) I = cell (cuvette) length (cm) V = volume of filtered water (L) Spectrophotometric reading When possible, the absorption spectra in the range 630750 nm were collected. Our spectrophotometer SPECORD M40 was connected with pc computer. Some of the earlier

data were registered on the paper. The length of cuvettes was chosen according to chlorophyll range (usually L 2cm). Surface area of the bare skeletons remaining after removal of tissue was measured independently using the paraffin wax technique (Stambler et al. 1991), by immersing the skeleton bar in hot wax; the mass of wax added to the skeleton bare was determined by weighing the skeleton bare before and after immersion. A relationship between change in mass and surface area was obtained by immersing a known surface area cubes in the wax.

RESULTS AND DISCUSSION Experimental effect of elevated temperature on the population density of zooxanthellae of A. humilis The changes observed in zooxanthellae densities lost from A. humilis indicate clearly increasing susceptibility to both elevated temperature and prolonged exposure. The control samples maintained at 26°C exhibited no variation in symbiont density at both the beginning and the end of the experiment. In samples exposed to 24°C, zooxanthellae densities were highly significantly different from those at the control sample (P < 0.01, HSD = 0.324), where zooxanthellae densities were 0.78±0.015x106 cells/cm2 after 6 hours, 0.77±0.014x106 cells/cm2 after 12 hours and 0.64±0.015x106 cells/cm2 after 24 hours. A. humilis at 29°C had lower counts of symbiotic algae (0.71±0.023 x106 cells/cm2 after 6 hours, 0.6±0.015x106 cells/cm2 after 12 hours and 0.55±0.015x106 cells/cm2 after 24 hours) within host tissue compared to control sample. Moreover, zooxanthellae densities of A. humilis were significantly different from those at the control sample (P < 0.01, HSD = 0.18). Increases in temperature to 31°C caused a decrease in zooxanthellae densities and vary greatly over time. Measurements of zooxanthellae densities at this stage revealed a highly significant difference between exposed and control sample (ANOVA, P < 0.01, HSD = 0.296). Where, zooxanthellae densities were 0.62±0.015 x106 cells/cm2, 0.5±0.015x106 cells/cm2, 0.43±0.015 x106 cells/cm2 after 6 hours, 12 hours and 24 hours respectively. At 33°C, the number of zooxanthellae lost from A. humilis was decreased to 38% (content after loss= 0.5±0.015x106 cells/cm2) after 6 hours, 48% (content after loss= 0.42±0.015x106 cells/cm2) after 12 hours and to 51% (content after loss= 0.4±0.015x106 cells/cm2) after 24 hours compared to control sample. This indicates that, the physiological state of zooxanthellae is clearly influenced by elevated temperatures and the duration of heat exposure. In samples exposed to 35°C, the density of zooxanthellae was (0.41±0.015x106 cells/cm2). This represents a 50% decrease compared to controls after 6 hours. Samples exposed to 35°C and analyzed after 12 hours, showed a zooxanthellae density of (0.3±0.015x106 cells/cm2) (63% decrease) compared to (0.81±0.0125x106 cells/cm2) for controls. While after 24 hours the loss of zooxanthellae was about 75% (content after loss= 0.2±0.015x106 cells/cm2) compared to the control sample (ANOVA, P < 0.01, HSD= 0.503).

5 (2): 75-85, November 2013

Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of zooxanthellae at the different time of exposure (Table 1). It revealed that, the mean value of zooxanthellae densities after 6 hours of exposure was significantly different with those after 12 hours . However, the difference was highly significant between zooxanthellae densities after 12 hours of exposure and 24 hours. This was driven from the data that zooxanthellae densities after 6 hours exposure (0.638x106 cells/cm2) were higher than those after 12 hours (0.616 106 cell/cm2), which in turn were higher than those after 24 hours (0.503x106 cells/cm2). Table 1. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in temperature (°C) on zooxthancellae density (10 6 cells/cm2) of A. humilis by using the sampling frequency as dependent variables. After 6 h. (0.638)

After 12 h. (0.766)

After 24 h. (0.503)

After 6 h. (0.638) After 12 h. 0.128 (Sig) (0.766) After 24 h. 0.135 (Sig) 0.263 (H. Sig) (0.503) Note: Number in parentheses = Zooxthancellae density (10 6 cells/cm2). Minimum significant difference 0.0102. H. = Highly significant differences. Sig. = Significant difference

Experimental effect of elevated temperature on chlorophyll contents of A. humilis The amount of chlorophyll per zooxanthellae was inversely related to increased temperature. All chlorophyll concentrations showed a significant decrease with increased temperature (P < 0.01 for all cases). Control samples (26°C) had higher content of chlorophyll (2.1±0.11, 2.1±0.07 and 1.99±0.04 µg/cm2 after 6, 12 and 24 hours, respectively) within host tissue compared to test samples. In samples exposed to 24°C, the content of chlorophyll was (2±0.09, 1.97±0.04 and 1.91±0.06 µg/cm2 after 6, 12 and 24 hours respectively). Although the difference was statistically significant (ANOVA, P < 0.01, HSD = 0.108). Colonies immediately sampled after 6 hours exposure to 29°C, showed a slight decrease in chlorophyll contents (1.98±0.0106 µg/cm2). While, chlorophyll contents in colonies collected after 12 hours were 1.9±0.04 µg/cm2, and after 24 hours were 1.87±0.106 µg/cm2. Colonies exposed to 29°C showed a significant difference relative to the control samples (ANOVA, P < 0.01, HSD = 0.15). Measurements of chlorophyll contents at 31°C of exposure revealed a significant difference between exposed and control sample (ANOVA, P < 0.01, HSD = 0.42). However, chlorophyll contents were 1.82±0.0104 µg/cm2, 1.6±0.08 µg/cm2, 1.5±0.02 µg/cm2 after 6 hours, 12 hours and 24 hours respectively. An increase in temperature to 33°C caused an increase in the amount of chlorophyll lost from A. humilis. Chlorophyll content decreased from 2.1±0.11 µg/cm2 in controls to 1.43±0.039 µg/cm2 in exposed samples after 6 hours (P < 0.01, HSD = 0.73). An analyzed A. humilis, after 12 hours of exposure to 33°C, showed a chlorophyll content of 1.37±0.03 µg/cm2

compared to 2.1±0.07 µg/cm2 in controls. While after 24 hours it was 1.21±0.08 µg/cm2. Exposure of A. humilis to 35°C reduced the content of chlorophyll sharply compared to the control sample. However, the difference was statistically highly significant (P < 0.0001, HSD = 1.166). About 48% of chlorophyll content was lost from A. humilis after 6 hours when incubated at 35°C (1.1±0.13 µg/cm2), after 12 hours about 57% of chlorophyll content (0.9±0.03 µg/cm2) was lost. While after 24 hours the loss of chlorophyll content was about 65% (0.7±0.019 µg/cm2) relative to the control sample. Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of chlorophyll contents at different times of exposure (Table 2). It was revealed that, the mean value of chlorophyll contents after 6 hours of exposure was significantly different with those after 12 hours and 24 hours Also the difference was significant between chlorophyll contents after 12 hours of exposure and 24 hours where the contents of chlorophyll after 6 hours exposure (1.74 µg/cm2) were higher than those after 12 hours (1.65 µg/cm2), which in turn were higher than those after 24 hours (1.53 µg/cm2). In general, the physiological state of Symbiodinium is clearly influenced by elevated temperatures and duration of heat exposure. Table 2. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in temperature (°C) on chlorophyll concentration (µg/cm2) of A. humilis by using the sampling frequency as dependent variables. After 6 h. (1.74)

After 12 h. (1.65)

After 24 h. (1.53)

After 6 h. (1.74) After 12 h. 0.087 (Sig) (1.65) After 24 h. 0.206 ( H. Sig) 0.11 (Sig) (1.53) Note: Number in parentheses = Chlorophyll concentration (µg/cm2). Minimum significant difference 0.043 .H. = Highly significant differences. Sig. = Significant difference

Experimental effect of elevated temperature on the population density of zooxanthellae of S. pistillata The number of zooxanthellae showed a significant decrease with increasing temperature (ANOVA, P < 0.01) and exposure time (P < 0.01), additionally there was interaction between temperatures and exposure time (P < 0.01). To detect the different effect of changes in temperatures on zooxanthellae densities after different exposure times, Turkey’s Studentized Rang Statistical Analysis (HSD) was applied. It was shown that the mean value of zooxanthellae densities exposed to 24°C was significantly different from those in controls (HSD = 0.083). However, the control samples (26°C) had higher densities of zooxanthellae (0.83±0.015 after 6 and 12 hours and 0.82±0.018x106 cells/cm2 after 24 hours) within host tissue compared to test samples. In samples exposed to 24°C, the densities of zooxanthellae were (0.81±0.012,

AMMAR et al. – Effect of temperature and sedimentation on corals bleaching

0.73±0.015 and 0.69±0.018x106 cells/cm2 after 6, 12 and 24 hours, respectively). Zooxanthellae densities of S. pistillata at 29°C were also significantly different from those of the control sample (P < 0.01, HSD = 0.118). Stylophora pistillata had lower counts of symbiotic algae cells (0.76±0.015x106 cells/cm2 after 6 hours, 0.75±0.015 x106 cells/cm2 after 12 hours and 0.62±0.018x106 cells/cm2 after 24 hours) within host tissue compared to control sample. While, the difference between zooxanthellae densities at 31°C and those in control samples were highly significant (P < 0.01, HSD = 0.273), zooxanthellae densities at this stage were 0.66±0.015x106 cells/cm2, 0.53±0.015x106 cells/cm2, 0.47±0.015x106 cells/cm2 after 6 hours, 12 hours and 24 hours respectively. Measurements of zooxanthellae densities at 33°C revealed a highly significant difference between the exposed and the control sample (ANOVA, P < 0.01, HSD = 0.373). Increases in temperature to 33°C caused a decrease in zooxanthellae densities and vary greatly over time. Zooxanthellae densities at this stage (33°C) were 0.51±0.015x106 cells/cm2, 0.44±0.015x106 cells/cm2, 0.41±0.015x106 cells/cm2 after 6 hours, 12 hours and 24 hours respectively. In samples exposed to 35°C, the density of zooxanthellae was (0.43±0.015x106 cells/cm2). This represents a 48% decrease compared to the control sample after 6 hours. Exposure to 35°C for 12 hours, showed a zooxanthellae density of (0.35±0.015x106 cells/cm2) compared to (0.83±0.015x106 cells/cm2) in controls recording a decrease of 58%. While after 24 hours the loss of zooxanthellae was about 67% (content after loss= 0.27±0.015x106 cells/cm2) relative to the control sample (ANOVA, P < 0.01, HSD= 0.476). Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of zooxanthellae at the time of exposure (Table 3). It was detected that, the mean value of zooxanthellae densities after 6 hours of exposure was significantly different from those after 12 hours. However, the difference was highly significant between zooxanthellae densities after 12 hours and 24 hours of exposure. This revealed that zooxanthellae densities after 6 hour exposure (0.67x106 cells/cm2) were higher than those after 12 hours (0.605x106 cells/cm2) while zooxanthellae densities after 12 hours were higher than those after 24 hours (0.546x106 cells/cm2).

Experimental effect of elevated temperature on chlorophyll contents of S. pistillata The amount of chlorophyll concentration showed a significant decrease with increasing temperature (ANOVA, P < 0.01) and prolonged exposure (P < 0.01). Additionally, there was interaction between the treatments. Control samples (26°C) had chlorophyll contents of (2.2±0.11, 2.1±0.088 and 2±0.015 µg/cm2 after 6, 12 and 24 hours, respectively). The contents of chlorophyll in samples exposed to 24°C was (1.98±0.082, 1.94±0.051 and 1.9±0.018 µg/cm2 after 6, 12 and 24 hours, respectively). Also, the difference between theses samples was statistically significant (ANOVA, P < 0.01, HSD = 0.163). Six hours exposure to 29°C showed a slight decrease in chlorophyll contents (1.84±0.056 µg/cm2). While, chlorophyll content in colonies exposed for 12 hours was 1.83±0.046 µg/cm2, while it was 1.8±0.015 µg/cm2 after 24 hours. Colonies exposed to 29°C showed a significant difference from the control samples (ANOVA, P < 0.01, HSD = 0.28). Measurements of chlorophyll content at 31°C of exposure revealed also a significant difference between exposed and control sample (ANOVA, P < 0.0001 and HSD = 0.43). Also, chlorophyll contents were 1.7±0.016 µg/cm2, 1.67±0.045 µg/cm2, 1.62±0.018 µg/cm2 after 6 hours, 12 hours and 24 hours respectively. An increase in temperature to 33°C caused an increase in chlorophyll content loss from S. pistillata. Chlorophyll content decreased from 2.2±0.11 µg/cm2 in controls to 1.5±0.028 in samples exposed for 6 hours (P < 0.01, HSD = 0.73). An analyzed S. pistillata after 12 hours of exposure to 33°C showed a chlorophyll content of 1.1±0.035 µg/cm2 compared to 2.1±0.088 µg/cm2 in controls. While after 24 hours it was 0.9±0.015 µg/cm2 compared to 2±0.015 for the controls. Exposure of S. pistillata to 35°C sharply reduced the content of chlorophyll relative to the control sample. In addition, the difference was statistically highly significant between both samples (P < 0.0001, HSD = 1.36). About 63% of chlorophyll content was lost from S. pistillata after 6 hours when incubated at 35°C (content after loss= 0.81±0.023 µg/cm2), while it was 65% after 12 hours (content after loss= 0.74±0.007 µg/cm2). While after 24 hours, the loss of chlorophyll content was about 66% (content after loss= 0.68±0.015 µg/cm2) relative to the control sample.

Table 3. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in temperature (°C) on zooxthancellae density (10 6 cells/cm2) of S. pistillata by using the sampling frequency as dependent variables.

Table 4. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in temperature (°C) on chlorophyll concentration (µg/cm2) of S. pistillata by using the sampling frequency as dependent variables.

After 6 h. (0.666)

After 12 h. (0.605)

After 24 h. (0.546)

After 6 h. (0.666) After 12 h. 0.061 (Sig) (0.605) After 24 h. 0.12 (Sig) 0.342 (H. Sig) (0.546) Note: Number in parentheses = Zooxthancellae density (10 6 cells/cm2). Minimum significant difference 0.01; H. = Highly significant differences . Sig. = Significant difference.

After 6 h. (1.67)

After 12 h. (1.56)

After 24 h. (1.48)

After 6 h. (1.67) After 12 h. 0.11 (Sig) (1.56) After 24 h. 0.19 (Sig) 0.08 (Sig) (1.48) Note: Number in parentheses = Chlorophyll concentration (µg/cm2). Minimum significant difference 0.0304. Sig. = Significant difference

5 (2): 75-85, November 2013

Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of chlorophyll contents at different times of exposure (Table 4). It was revealed that, the mean value of chlorophyll contents after 6 hours of exposure was significantly different from those after 12 hours (ANOVA, P < 0.0001, HSD = 0.11) and 24 hours. Also the difference was significant between chlorophyll contents after 12 hours of exposure and 24 hours, this revealed the recorded data that the contents of chlorophyll after 6 hours of exposure (1.67 µg/cm2) were higher than those after 12 hours (1.56 µg/cm2), and chlorophyll contents after 12 hours were higher than those after 24 hours (1.48 µg/cm2).

Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of zooxanthellae densities after different times of exposure (Table 5). It was revealed that, the mean value of zooxanthellae densities after 1 day of exposure was significantly different from those after 5 days. However, zooxanthellae densities after 10 days of exposure were highly significantly different from the sample of 1 day of exposure and 5 days. This revealed the result that zooxanthellae densities after 1 day exposure (0.755x106 cells/cm2) were higher than those after 5 days (0.706x106 cells/cm2), also those after 5 days were higher than those after 10 days (0.58x106 cells/cm2).

Experimental effect of increased sediment concentrations on the population density of zooxanthellae density of A. humilis The changes observed in zooxanthellae densities lost from A. humilis indicated an increasing susceptibility to both increased sediment concentrations and prolonged exposure. The control samples exhibited slight variations in algal density at the beginning and the end of the experiment. In samples exposed to 0.1 mg/cm2/L, zooxanthellae densities did not show any decrease after 1 day (0.8±0.025x106 cells/cm2 in controls and exposed samples). However, after 5 days of exposure to 0.1 mg/cm2/L, zooxanthellae densities decreased from 0.79±0.015x106 cells/cm2 (controls) to 0.76±0.007 x106 cells/cm2, while after 10 days zooxanthellae densities were 0.73±0.014x106 cells/cm2 compared to 0.77±0.01 x106 cells/cm2 for the controls. The mean of zooxanthellae densities in samples exposed to 0.1 mg2/cm/L were significantly different from those of the controls (P < 0.01, HSD = 0.023). Acropora humilis at 0.5 mg/cm2/L had lower counts of symbiotic alga cells (0.75±0.015x106 cells/cm2 after 1 day, 0.7±0.015x106 cells/cm2 after 5 days and 0.65±0.01x106 cells/cm2 after 24 hours) within host tissue compared to the control sample. Moreover, zooxanthellae densities were significantly different from those of the control sample (P < 0.01, HSD = 0.086). Increases in sediment concentration to 1 mg2/cm/L caused a decrease in zooxanthellae densities and vary greatly over time. Measurements of zooxanthellae densities at this stage revealed a highly significant difference between exposed and control sample (ANOVA, P < 0.0001 and HSD = 0.173). Where, zooxanthellae densities were 0.72±0.015 x106 cells/cm2, 0.68±0.015x106 cells/cm2, 0.44±0.01 x106 cells/cm2 after 1 day, 5 days and 10 days, respectively. At 1g/cm2/L, the number of zooxanthellae lost from A. humilis was decreased to 11% (content after loss= 0.71±0.012x106 cells/cm2) compared to control sample after 1 day, 24% (content after loss= 0.6±0.015 x106 cells/cm2) after 5 days and to 60% (content after loss= 0.31±0.012x106 cells/cm2) after 10 days, indicating that the physiological state of zooxanthellae is clearly influenced by elevated sedimentation rate and the duration of exposure. In addition, the differences in zooxanthellae densities between samples exposed to 1 g/cm2/L and controls were highly significant (ANOVA, P < 0.01, HSD= 0.246).

Table 5. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in sedimentation on zooxthancellae density (106 cells/cm2) of A. humilis by using the sampling frequency as dependent variables. After 1 day (0.755)

After 5 days (0.706)

After 10 days (0.58)

After 1 day (0.755) After 5 days 0.049 (Sig) (0.706) After 10 days 0.175 ( H. Sig) 0.126 (H. Sig) (0.58) Note: Number in parentheses = Zooxthancellae density (10 6 cells/cm2). Minimum significant difference 0.01. H. = highly significant differences. Sig. = Significant difference

Experimental effect of increased sediment concentrations on chlorophyll contents of A. humilis Chlorophyll contents within the host tissue showed a significant decrease with increased sedimentation rate (P < 0.01 for all cases). Control samples had higher content of chlorophyll (1.99±0.15, 1.73±0.015 and 1.31±0.018 µg/cm2 after 1, 5 and 10 days, respectively) compared to the test samples. In samples exposed to 0.1 mg/cm2/L the content of chlorophyll was (1.99±0.02, 1.71±0.015 and 1.27±0.01 µg/cm2 after 1, 5 and 10 days, respectively). In addition, the difference between 1, 5 and 10 days samples was statistically significant (ANOVA, P < 0.01, HSD = 0.02). Colonies immediately sampled after 1 days exposure to 0.5 mg/cm2/L showed a slight decrease in chlorophyll contents (1.91±0.01 µg/cm2). While chlorophyll contents were 1.66±0.015 µg/cm2 in colonies collected after 5 days and 1.1±0.05 µg/cm2 after 10 days. Colonies exposed to 0.5 mg/cm2/L showed a significant difference in chlorophyll contents compared to the control samples (ANOVA, P < 0.001, HSD = 0.121). Chlorophyll contents of samples exposed to 1 mg/cm2/L was highly significantly different from the control sample (ANOVA, P < 0.01, HSD = 0.242). Chlorophyll contents were 1.84±0.015 µg/cm2, 1.58±0.015 µg/cm2 and 0.89±0.01 µg/cm2 after 1 day, 5 days and 10 days respectively. Exposure of A. humilis to 1 g/cm2/L sharply reduced the content of chlorophyll in relative to the control sample, giving a high significance of difference between both treated and control sample (P < 0.01, HSD = 0.374). About 10% of chlorophyll content was lost from A. humilis after 1 day when exposed to 1 g/cm2/L (content after loss =

AMMAR et al. – Effect of temperature and sedimentation on corals bleaching

1.79±0.02 µg/cm2), after 5 days for about 18% of chlorophyll content (content after loss = 1.42±0.012 µg/cm2) was lost. While after 10 days the loss of chlorophyll content was about 47% (content after loss = 0.7±0.019 µg/cm2) relative to the control sample. Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of chlorophyll contents at the different times of exposure (Table 6). It was revealed that, the mean value of chlorophyll contents after 1 day of exposure to different concentrations of sediments was significantly different from those after 5 days. However, the difference was highly significant between chlorophyll contents of 10 days samples and controls. In addition, the difference was highly significant between chlorophyll contents of 10 days and 5 days of exposure. Contents of chlorophyll after 1 day of exposure (1.904 µg/cm2) were higher than those after 5 days (1.62 µg/cm2), which in turn were higher than those after 10 days (1.056 µg/cm2). Table 6. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in sedimentation rates (mg/cm2/L) on chlorophyll concentration (µg/cm2) of A. humilis by using the sampling frequency as dependent variables. After 1 day (1.904)

After 5 days (1.62)

After 10 days (1.056)

After 1 day (1.904) After 5 days 0.284 (Sig) (1.62) After 10 days 0.848 ( H. Sig) 0.564 (H. Sig) (1.056) Note: Number in parentheses = Chlorophyll concentration (µg/cm2). Minimum significant difference 0.015. H. = highly significant differences. Sig. = Significant difference

Experimental effect of increased sediment concentrations on the population densities of zooxanthellae of S. pistillata The number of zooxanthellae showed a significant decrease with increasing sediment concentrations (ANOVA, P < 0.01) and exposure time (P < 0.01). Additionally, there was an interaction between temperature and exposure time (P < 0.01). To detect the distinct different effects of changes in sediment concentrations on zooxanthellae densities after different exposure times, Turkey’s Studentized Rang Statistical Analysis (HSD) was applied. It was shown that the mean value of zooxanthellae densities exposed to 0.1 mg/cm2/L was significantly different from those in the controls (HSD = 0.02). However, control samples had higher densities of zooxanthellae (0.81±0.015, 0.8±0.015 and 0.78±0.015x106 cells/cm2 after 1, 5 and 10 days respectively) compared to test samples. While, in samples exposed to 0.1 mg/cm2/L, the densities of zooxanthellae were (0.8±0.015 after 1 day, 0.78±0.007 after 5 days and 0.75±0.01x106 cells/cm2 after 10 days, respectively). Zooxanthellae densities of S. pistillata exposed to 0.5 mg/cm2/L had also a significant difference from those of the control sample (P < 0.01, HSD = 0.073). Recorded data revealed that, S. pistillata had lower counts of symbiotic algae cells (0.77±0.015x106 cells/cm2, 0.72±0.015x106 cells/cm2 and 0.68±0.01x106

cells/cm2 after 1, 5 and 10 days respectively) within host tissue compared to control sample. The difference was highly significant between zooxanthellae densities at 1 mg/cm2/L and those in control samples (P < 0.01, HSD = 0.156). At this stage zooxanthellae densities were 0.74±0.015x106 cells/cm2, 0.69±0.018x106 cells/cm2, and 0.49±0.01x106 cells/cm2 after 1 day, 5 days and 10 days respectively. An increases in sediment loading to 1 g/cm2/L caused a decrease in zooxanthellae densities which vary greatly over time. Measurements of zooxanthellae densities after exposure to 1 g/cm2/L revealed a high significant difference between exposed and control samples (ANOVA, P < 0.01, HSD = 0.226). Zooxanthellae densities at this stage were 0.71±0.014x106 cells/cm2 after 1 day, 0.63±0.015 x106 cells/cm2 after 5 days and 0.37±0.015x106 cells/cm2 after 5 10 days. This represents a 13.4% decrease in zooxanthellae density after 1 day and 31% after 5 days compared to controls. While after 24 hours the loss of zooxanthellae was about 53.6% relative to the control sample. Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of zooxanthellae at the time of exposure (Table 7). It was revealed that, the mean value of zooxanthellae densities after 5 days of exposure was significantly different from those after 1 day. However, the difference was highly significant between zooxanthellae densities after 10 days of exposure and those after 1 and 5 days. However, zooxanthellae densities after 1 day of exposure were 0.766 x106 cells/cm2, being higher than those after 5 days (0.724 x106 cells/cm2). However, which in turn were higher than those after 10 days (0.614x106 cells/cm2). Table 7. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in sedimentation rates (mg/cm2/L) on zooxthancellae density (10 6 cells/cm2) of S. pistillata by using the sampling frequency as dependent variables. After 1 day (0.766)

After 5 days (0.724)

After 10 days (0.614)

After 1 day (0.766) After 5 days 0.042 (Sig) (0.724) After 10 days 0.152 ( H. Sig) 0.11 (H. Sig) (0.614) Note: Number in parentheses = Zooxthancellae density (10 6 cells/cm2). Minimum significant difference 0.0096. H. = highly significant differences. Sig. = Significant difference.

Experimental effect of increased sediment concentrations on chlorophyll contents of S. pistillata The amount of chlorophyll showed a significant decrease with increasing sediment concentration (ANOVA, P < 0.01) and prolonged exposure (P < 0.01). Additionally, there was interaction between the treatments. Control samples had chlorophyll contents 2±0.015, 1.84±0.018 and 1.34±0.015 µg/cm2 after 1, 5 and 10 days respectively. The contents of chlorophyll in samples exposed to 0.1 mg/cm2/L was (1.98±0.015 µg/cm2 after 1 day, 1.74±0.013 µg/cm2 after 5 days and 1.32±0.013 µg/cm2 after 10 days). The difference between exposed and controls was

5 (2): 75-85, November 2013

statistically significant (ANOVA, P < 0.01, HSD = 0.044). One day exposure to 0.5 mg/cm2/L showed a slight decrease in chlorophyll contents (1.93±0.01 µg/cm2). However, chlorophyll contents in colonies analyzed after 5 and 10 days were 1.69±0.013 µg/cm2 and 1.21±0.013 µg/cm2 respectively. Colonies exposed to 0.5 mg/cm2/L showed a significant difference relative to the control samples (ANOVA, P < 0.0001, HSD = 0.114). While, measurements of chlorophyll content in samples exposed to 1 mg/cm2/L expressed a high significant difference between exposed and control sample (ANOVA, P < 0.01, HSD = 0.242). Where is chlorophyll contents were 1.87±0.013 µg/cm2, 1.61±0.013 µg/cm2, 0.97±0.013 µg/cm2 after 1 day, 5 days and 10 days respectively. An increase in sediment concentration to 1 g/cm2/L caused a sharp loss in chlorophyll content from S. pistillata. Chlorophyll content decreased from 2±0.15 µg/cm2 in controls to 1.83±0.016 µg/cm2 in exposed samples after 1 day (8.5% decrease). An analyzed A. humilis after 5 days of exposure to 1 g/cm2/L, showed a chlorophyll content of 1.47±0.007 µg/cm2 compared to 1.84±0.088 µg/cm2 in controls (20% decreased). After 10 days it was 0.81±0.016 µg/cm2 compared to 1.34±0.013 µg/cm2 in controls (40% decrease). The difference between 1 g/cm2/L exposure and controls was statistically highly significant (P < 0.01, HSD = 0.354). Turkey’s Studentized Rang Statistical Analysis (HSD) was applied to detect the distinct variance between means of chlorophyll contents at different times of exposure. It was revealed that, the mean value of chlorophyll contents after1 day of exposure was significantly different from those after 5 days but it was highly significantly different from those after 10 days In addition, the difference between chlorophyll contents after 5 and 10 days exposure was highly significant. However, the measured chlorophyll content after 1 day of exposure (1.92 µg/cm2) was higher than those after 5 days (1.66 µg/cm2) which in turn was higher than those after 10 days (1.129 µg/cm2). Table 8. Turkey’s studentized rang statistical analysis (HSD) for the experimental effect of changes in sedimentation rates (mg/cm2/L) on chlorophyll concentration (µg/cm2) of S. pistillata by using the sampling frequency as dependent variables. After 1 day (1.921)

After 5 days (1.669)

After 10 days (1.129)

After 1 day (1.921) After 5 days 0.252 (Sig) (1.669) After 10 days 0.792 ( H. Sig) 0.54 (H. Sig) (1.129) Note: Number in parentheses = Chlorophyll concentration (µg/cm2). Minimum significant difference 0.01. H. = highly significant differences. Sig. = Significant difference.

Experimental effect of changes in temperature on bleaching The changes observed in zooxanthellae lost from the two corals A. humilis and S. pistillata clearly indicate increasing susceptibility to both elevated temperature and prolonged exposure. At 26°C (the control sample), the loss

of zooxanthellae by each of these corals was very low. Cell viability of these corals was similar at 26 and 29°C, but depicted a sharp decline of zooxanthellae lost from these corals at 31°C through time. This result confirms the result of Strychar et al. (2004) that zooxanthellae lost from Acropora hyacinthus, Favites complanata, and Porites solida at 32°C was greater than that at 28°C. However, Berkelmans and Willis (1999) found a temperature increase of 2-4°C is to have caused coral bleaching within days while a temperature increase of 1-2°C caused bleaching within weeks. Moreover, Nesa and Hidaka (2009) detected a negative correlation between survival time and the zooxanthella density of tissue balls at 31°C in both Fungia sp. and Porites divaricata. This relationship was clearly observed in the Caribbean basin during the 1980s and 1990s, when annual coral bleaching increased logarithmically with sea surface temperature anomalies (McWilliams et al. 2005). A 0.1°C rise in regional sea surface temperature resulted in a 35% increase in the number of areas that reported bleaching, and mass bleaching events occurred at regional sea surface temperature anomalies of 0.2°C and above (Baker et al. 2008). As the temperature increased to 35°C in the present experiment, the loss of zooxanthellae from each host increased both with time and temperature elevation. This result agrees with the finding of Riegl (2002) that bleaching mortalities were reported in Abu Dhabi at 1996 when temperatures remained above 35°C for 3 weeks. Elevated temperature was found to significantly reduce the amount of zooxanthellae in primary polyps (Anlauf et al. 2010). The result reported in the present experiment indicated that, A. humilis had a higher decrease in its zooxanthellae densities than S. pistillata at the same treatment; where 50% of zooxanthellae in the host tissue of A. humilis were lost after 6 hours while S. pistillata lost 48% of zooxanthellae after equal time of exposure. Differences in the response of these species of coral to thermal stress may result from difference in hospite irradiances driven by the combination of skeletal architecture and light scattering properties (Enríquez et al. 2005). An additional source of variation in the response to thermal stress in these corals may originate from differences in tissue thickness that is associated with difference in the initial protein content (Warner et al. 2002). Fitt et al. (2009) found physiological and biochemical differences of both symbiont and host origin in the response to high-temperature stress of Porites cylindrica and S. pistillata. Ferrier-Pagès et al. (2010) studied the changes in feeding rates of three scleractinian coral species between normal and short-term stress conditions, and assessed the effect of feeding on the photosynthetic capacity of corals exposed to a thermal stress. He found that, S. pistillata significantly decreased its feeding rates at 31°C, while rates of Turbinaria reniformis and Galaxea fascicularis were increased between 26 and 31°C. Exposure to elevated temperatures reduces the photosynthetic rate of zooxanthellae and predisposes their photosynthetic apparatus to further damage (Jones et al. 1998; Bhagooli and Hidaka 2004). In the present experiment, the bleaching temperature threshold was 33°C or less for the two species S. pistillata

AMMAR et al. – Effect of temperature and sedimentation on corals bleaching

and A. humilis where 51% of their zooxanthellae were lost after 24 h of exposure. This result confirms that of Leletkin (2002) that water temperature of 32°C and above inevitably caused coral bleaching. However, the thermal bleaching threshold for primary polyps might be below that reported (30-31°C) from most adult coral species in the eastern Paciﬁc (D'Croz et al. 2001; Hueerkamp et al. 2001). Bleaching temperature thresholds vary locally, and conditions that result in coral mortality in some regions can have no effect on corals in others. For example, while 30.5°C and 30.8°C represent bleaching thresholds for at least some regions of the Caribbean and Great Barrier Reef, respectively (Berkelmans et al. 2004; Manzello et al. 2007), temperatures as high as 35.5°C do not affect corals in the Arabian Gulf or in the Samoan Manu’a Islands (Craig et al. 2001; Riegl 2002; Birkeland et al. 2008). Indeed, individual corals have been reported surviving in Abu Dhabi at temperatures up to 40°C (Kinsman 1964), although mortality did occur in this region in 1996 when temperatures remained above 35°C for 3 weeks (Riegl 2002). Experimental effect of changes in sedimentation rate on bleaching In samples exposed to 0.1 mg/cm2/L, zooxanthellae densities of A. humilis and S. pistillata did not show any decrease after 1 day. However, after 1 days of exposure to 0.5 mg/cm2/L, zooxanthellae densities were significantly different from those of the controls. This result agrees with the laboratory finding of Peters and Pilson (1985) who examined the effect of heavy sedimentation rate on corals using both symbiotic and asymbiotic colonies of Astrangia danae at a rate of 200 mg. cm.−2 day-1 for 4 weeks. He found no difference from controls while slight adverse effects relative to controls were noted after increasing the sand applications to three times per day. The effects of varying rates of sedimentation (0.5 to 325 mg cm-2d-1) on settlement rates of Acropora millepora larvae were examined experimentally, in aquaria. Higher sedimentation rates reduced the number of larvae settling on upper surfaces, but total numbers of settled larvae were not significantly affected by sedimentary regime (Babcock and Davies 1990). Increases in sediment concentration to 1 mg/cm2/L caused a decrease in zooxanthellae densities and vary greatly over time. Measurements of zooxanthellae densities of A. humilis and S. pistillata at this stage revealed a highly significant difference between exposed and control sample. However, Sofonia and Anthony (2008) found no effect of sediment loads greater than 110 mg cm−2 on any of the physiological variables of Turbinaria mesenterina, that tolerant to sediment loads an order of magnitude higher than most severe sediment conditions in situ. Likely mechanisms for such tolerance are that: (1) colonies covered in sediment in low-flow were able to clear themselves rapidly (within 4-5 h) and (2) sediment provides a source of food. These results suggest that intensified sediment regimes on coastal reefs may shift coral communities towards dominance by a few welladapted species (Weber et al. 2006; Palmer et al. 2010). Cruz-Pinion et al. (2003) found that, high sedimentation rates, low light availability and anthropogenic influence

lead to cellular damage and deteriorated coral skeletal density. At 1 g/cm2/L, the number of zooxanthellae lost from A. humilis was higher than that was lost from S. pistillata at same time. This result agrees with the finding of Fabricius et al. (2007). Who reported a contrast between species susceptibility at 39.6 mg cm-2 day-1 sedimentation rate in Ngardmau, Palau, Micronesia, that small polyp corals such as Porites rus suffered greatest mortality while damage in Galaxea fascicularis was less severe as sediments were shifted and removed by the large polyps. However, Acropora spp. Appeared partially bleached although little time sediment remained on branches. With reference to the present experiment, we can suggest that the normal sedimentation rate for A. humilis and S. pistillata to be in an order of 1 mg/cm2/L or less. Chronic rates and concentrations above these values are high. This result is conflicted with the finding of Rogers (1990), who concluded that the mean sediment concentration was < 10 mg/L at reefs subject to stresses from human activities. Sedimentation is regarded as an increasing threat to coral reefs (reviewed by Fabricius 2005). The impacts associated with sedimentation and sediment burial include reduced photosynthesis and increased respiration (Philipp and Fabricius 2003; Weber et al. 2006), tissue mortality, reduced growth (Rice and Hunter 1992; Lirman and Manzello 2009), and reduced fertilization, larval survivorship, and recruitment (Babcock and Smith 2000). Turbidity reduces light levels, photosynthetic potential and possibly coral growth rates (Yentsch et al. 2002; Anthony and HoeghGuldberg 2003); however, elevated net sedimentation rates increases abrasion and smothering (Rogers 1990; Fabricius 2005). On the other hand, Palmer et al. (2010) found that, near shore environments directly influenced by fluvial sediments and dominated by terrigenoclastic sedimentation are generally considered marginal for coral reef growth. The same author did not mention if there is some way of washing or cleaning of these sediments. The energetic costs of sediment clearing can be considerable (Riegl and Branch 1995), and the inability to clear sediments will expose corals to further stress because anoxic conditions due to sediments can cause tissue bleaching and subsequent mortality (Weber et al. 2006). Increased temperature and sedimentation rate are both known to cause physiological stress in corals (Weber et al. 2006; Lirman and Manzello 2009; Obura 2009). Corals are not homoiothermic (Hoegh-Guldberg and Smith 1989) and short-term temperature and sediment stress can cause changes in basal metabolism, such as respiration and zooxanthellae photosynthesis (Nyström et al. 2001; Philipp and Fabricius 2003; Weber et al. 2006). Some key questions on the present work and their answers Q: How does the fact that “the temperature treatments were just 6, 12 or 24 hours long” relate to the aims and how does this design advance our understanding of temperature change as a result of climate change effects? A: These experimental choices should not be construed to mean that the response of reef corals to global climate change can be fully understood without addressing other facets of global climate change and through a comprehensive analysis of

5 (2): 75-85, November 2013

the coral holobiont. This study addresses mainly the early effects of heat stress on symbiotic dinoflagellates (zooxanthellae) within the tissues of a common reef-coral. Q: Why were the experiments conducted under constant temperature conditions and how does this design advance our understanding of temperature change as a result of climate change effects? A: The experiments were conducted under constant temperature conditions as they were conducted in an aquarium, and to maintain a stable reef tank ecosystem, a constant temperature is required. Also, as the reef tank is a closed system and most types of coral can thrive a few degrees above or below the ideal level in such a closed system, the study examined the early effects of heat stress on symbiotic dinoflagellates (zooxanthellae) within the tissues of a common reef-coral. Q: How are results from a constant temperature experiment reconciled with the normal cyclic conditions experienced by the corals in their natural habitat? A: The temperatures in the present experiment were increased and decreased following the diurnal variation in water temperature in the field by approximately 6 ˚C. Temperature in the field was between 29 ˚C and 30 ˚C at midday (at 3 m depth). Therefore, corals for studying the effect of temperature were incubated at each temperature test (24, 29, 31, 33 and 35 ˚C). Control samples were placed at room temperature (26˚C). Since coral bleaching occurs when the thermal tolerance of corals and their photosynthetic symbionts (zooxanthellae) is exceeded, corals in the present experiment, beside being subjected to the normal cyclic temperature, they were subjected to some few dgrees below and above the normal cyclic temperature. Q: How quickly was ambient temperature changed during experimentation and how does this affect interpretation of the outcome? A: Bleaching can be induced by short-term exposure (i.e. 1 days) at temperature elevations of 3°C to 4°C above normal summer ambient or by long-term exposure (i.e. several weeks) at elevations of 1°C to 2°C. Temperature elevations above summer ambient, but still below the bleaching threshold (as in the present work), could impair growth and reproduction by the effect of increased zooxanthellae expulsion. In 1998 Red Sea corals were perilously close to their bleaching threshold during the summer months, and localized bleachings did occur. In some cases, local warming of surface water on shallow reef flats exceeded this threshold temperature and caused localized coral bleaching. Q: What is briefly the design of the sedimentation experiments and how do they allow the conclusions that are postulated. A: The present experiment examined the effect of different sedimentation rates on corals. For detection of the short-term sediment, branches for studying the effect of sediments were put in glass aquaria, exposed to 0.1, 0.5, 1 mg/cm2/L and 1g/cm2/L different concentrations of sediments. The present experiment postulated that, the normal, safe sedimentation rate for A. humilis and S. pistillata to be in an order of 1mg/cm2/L or less. Q: Exactly how do the outcomes of this work advance our understanding? A: Threshold temperatures as well as normal safe sedimentation rate determined in the present work can be applied directly to reef aquaria where they are

closed systems exactly like those used in our experiments. These experimental choices should not be construed to mean that the response of reef corals to global climate change or to natural or human-made sedimentation in the sea can be fully understood without a comprehensive analysis of the coral holobiont and the surrounding environmental conditions.

CONCLUSIONS The result reported in the present experiment indicated that, Acropora humilis had a higher decrease in its zooxanthellae densities than Stylophora pistillata at the same treatment after equal time of exposure to temperature. In the present experiment, the bleaching temperature threshold was 33°C or less for the two species S. pistillata and A. humilis where 51% of their zooxanthellae were lost after 24 h of exposure. On exposure to sedimentation, number of zooxanthellae lost from A. humilis was higher than those lost from S. pistillata at same time. The normal sedimentation rate for A. humilis and S. pistillata was found to to be in an order of 1 mg/cm2/L or less.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 5, No. 2, pp. 86-103 November 2013

Review: Physical, physical chemistries, chemical and sensorial characteristics of the several fruits and vegetables chips produced by low-temperature of vacuum frying machine AHMAD DWI SETYAWANâ&#x2122;Ľ, SUGIYARTO, SOLICHATUN, ARI SUSILOWATI Department of Biology, Faculty of Mathematic and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36a Surakarta 57126, Central Java, Indonesia. Tel./Fax.: +92-271-663375. â&#x2122;Ľemail: volatileoils@gmail.com Manuscript received: 12 November 2012. Revision accepted: 4 May 2013.

Abstract. Setyawan AD, Sugiyarto, Solichatun, Susilowati A. 2013. Review: Physical, physical chemistries, chemical and sensorial characteristics of the several fruits and vegetables chips by produced low-temperature of vacuum frying machine. Nusantara Bioscience 5: 86-103. Frying process is one of the oldest cooking methods and most widely practiced in the world. Frying process is considered as a dry cooking method because it does not involve water. In the frying process, oil conduction occured at high temperature presses water out of food in the form of bubbles. Fried foods last longer due to reduced water contents leading to less decomposition by microbes, even fried foods can enhance nutritional value and beautify appearance. Food frying technology can extend the shelf life of fruits and vegetables, while the frying oil are used to enhance the flavor of the products, but the use of improper frying oil can have harmful effects on human health. Vacuum frying is a promising technology and may become an option for the production of snacks such as fruit and vegetable crisps that present the desired quality and respond to the new health trends. This technique of frying food at a low temperature and pressure makes the nutritional quality of the food is maintained and the quality of the used oil is not quickly declined to become saturated oils that are harmful for human health. This technique produces chips that have physical, physico-chemical, chemical, and sensorial properties generally better than chips produced by conventional deep-fat frying methods. Key words: chips, food, frying, preservation, vacuum frying

Abstrak. Setyawan AD, Sugiyarto, Solichatun, Susilowati A. 2013. Review: Karakteristik fisik, kimia fisik, kimia dan sensoris beberapa keripik buah-buahan dan sayuran yang dihasilkan dengan mesin vacuum frying bersuhu rendah. Nusantara Bioscience 5: 86-103. Proses penggorengan merupakan salah satu metode memasak yang paling tua dan paling banyak dilakukan di dunia. Proses penggorengan dianggap sebagai metode memasak kering karena proses ini tidak memerlukan air. Dalam proses penggorengan, terjadi konduksi minyak bersuhu tinggi yang mendesak air keluar dari bahan makanan dalam bentuk gelembung-gelembung. Makanan yang digoreng tahan lebih lama karena berkurangnya kadar air yang menyebabkan tidak terjadinya pembusukan oleh mikroba, bahkan makanan yang digoreng dapat ditingkatkan nilai gizi dan kualitas penampakannya. Teknologi penggorengan makanan dapat memperpanjang umur simpan buah-buahan dan sayuran, sementara itu minyak goreng yang digunakan meningkatkan cita rasa produk, namun penggunaan minyak goreng yang tidak tepat dapat merugikan kesehatan. Penggorengan hampa udara (vacuum frying) adalah teknologi penggorengan yang menjanjikan dan dapat menjadi pilihan untuk produksi makanan ringan seperti keripik buah dan sayuran dengan kualitas yang diinginkan dan memenuhi kecenderungan kesehatan saat ini. Teknik ini menggoreng makanan pada suhu dan tekanan rendah sehingga kualitas gizi makanan terjaga dan minyak yang digunakan tidak cepat rusak dan menjadi minyak jenuh yang berbahaya bagi kesehatan manusia. Teknik ini menghasilkan keripik yang memiliki sifat-sifat fisik, fisika-kimia, kimia, dan sensoris yang umumnya lebih baik daripada keripik yang dihasilkan dengan metode penggorengan konvensional. Kata kunci: keripik, makanan, menggoreng, pengawetan, penggorengan hampa udara, vacuum frying

INTRODUCTION All foods require preservation for several reasons, such as to prevent spoilage, to maintain the availability throughout the year, to retain the nutritional value and to make value-added products (higher prices). Food spoilage or damage may occur during handling process due to the influence of physical, physiological, chemical or microbial damage. Chemical and microbial factors are the main causes of food spoilage. Several chemical and enzymatic reactions can occur during processing and storage of food (Mujumdar and Jangam 2012). Food preservation is usually

done by preventing the growth of bacteria, fungi (e.g. yeast), and other microbes (although in some method, benign bacteria or fungi has been used to make certain foods, such as tempeh, oncom and tape), as well as retarding the oxidation of fats which cause rancidity. Preservation of food can also include inhibition of visual deterioration during food preparation, such as the enzymatic browning reaction in salaks, apples and potatoes after peeling. Maintaining or creating nutritional value, texture and flavor are important aspects of food preservation, although, historically, some methods drastically change the character of the food which is

SETYAWAN et al. â&#x20AC;&#x201C; Chips characteristic by vacuum frying process

preserved. In many cases, these changes have come to be seen as a desirable quality, including cheese, yoghurt and pickled onions. To preserve food, some methods are sometimes used together. Preserving fruit by turning into jam, for example, involves boiling (to reduce the water content of fruit and to kill microbes), the provision of sugar (to prevent their re-growth) and sealing in an airtight jar (to prevent recontamination) (Vivante 2009). There are various ways to preserve food, including canning, freezing, pickling, salting, sugaring (providing sugar syrup), irradiation, vacuum packaging, etc. The pre and/or postprocessing steps are critical to reduce the drying load as well as to make better quality product. The commonly methods used for pre-treatment are osmotic dehydration, blanching, salting and soaking. While post-processing such as coating, blending, packaging, etc. are also important after drying of food (Mujumdar and Jangam 2012). Water content is a major cause of food spoilage, therefore the drying process is often done to reduce levels of water and extend the shelf life of food (Potter 1973). Drying or dehydration process by thermal is one of the most ancient food preservation and the most frequently used, which reduces water activity sufficiently to prevent bacterial growth; although some loss of quality occurs during dehydration. Drying has been applied to grains, seafood, and meat products as well as fruits, tubers and vegetables. Food products can have wide ranges of water content; as low as 35% in grains and as high as 90% or more in some fruits (e.g. 93% in water melon) which needs to be reduced to an acceptable value so as to avoid microbial growth. There is reported that microbes have different water activity (which means free water available for microbial growth in solids) (Mujumdar and Devahastin 2008). In addition, each food product must be dried using various types of suitable dryers and also using appropriate pre- and post-processing to obtain a satisfactory value addition to the dried product (Chen and Mujumdar 2008; Mujumdar and Devahastin 2008). Traditionally, food products were commonly dried by open sun drying method. Recently various advanced drying methods have been practised for food application as a result of the increased demand for high quality products and to reduce energy consumption which is one of the highest costs in the food processing industries (Kudra and Mujumdar 2009). The use of some techniques, such as solar cabinet dryers, tray dryers, fluid bed dryers, vacuum dryers, freeze dryers, etc has resulted in a better product quality (Potter 1973; Chen and Mujumdar 2008; Jangam et al. 2010). These processes can also be made efficient costs in terms of energy consumption (Mujumdar and Jangam 2012). Dehydration is one of the main processes in food preservation. Frying is the most widely practiced cooking method and the most cost-effective techniques for food preservation as well as for the production of traditional and innovative products such as processed snacks with desired quality (Mujumdar and Devahastin 2008). Food frying technology can extend the shelf life of fruits and vegetables and frying oil can increase the flavors of the products, however, improper frying oil can have harmful effects on the

consumer health (Inprasit 2011). Fruit into chips processing requires technological support so that the qualities of the resulting chips are acceptable for consumers. One way to produce healthy food without changing its original form is by using the vacuum frying technology (Siregar et al. 2004). This article begins by reviewing some of the conventional drying techniques used in food preservation. Later, it will focus on recent advances in vacuum frying technique for food production; as well as physical, physical chemistries, chemical and sensory characteristics of some chips of fruits and vegetables (incl. tubers) processed by low-temperature vacuum frying machine.

FOOD FRYING PRESERVATION Fresh fruits and vegetables are highly perishable, shelf life is so short. If not handled properly, fruits and vegetables that have been harvested will undergo physiological, physical, chemical, and microbiological changes that become damaged or rotten. Meanwhile, the tubers usually have a longer shelf life, although some will soon rot in storage, such as cassava. One effort to maintain quality and shelf life of fruit and have a pretty good market is processed into chips. Chips are more durable than the stored fresh fruits or vegetables (incl. tubers) because of the low water contents and no longer occurring physiological processes such as fresh crops (Antarlina and Rina 2005; Shidqiana 2012). Deep-fat (ordinary, atmospheric) frying is one of the oldest food preparation processes and is widely used in the food industry. Frying is a complex operation process which is basically the immersion of food pieces in hot vegetable oil, at a temperature of above the boiling point of water (Amany et al. 2009). This condition causes high rates of heat transfer, so that water evaporates and an oil layer covering the product surface (Hubbard and Farkas 1999; Bouchon et al. 2003). Several models have been developed to describe the moisture evaporation and oil absorption in deep-fat frying (Moreira and Bakker-Arkema 1989; Rice and Gamble 1989; Kozempel et al. 1991). Oil temperature and frying time are the main frying operation variables controlling mass transfer in deep-fat frying (Mittelman et al. 1984). The deep-fat frying seals the food by immersing in hot oil so that all the flavors and juices are retained by a crisp crust (Moreira et al. 1995; Troncoso et al. 2009). Fried is generally processed under atmospheric pressure at high temperatures. During the process, food is rapidly cooked, browned, and the texture and flavor is developed (Farkas et al. 1996a). Therefore, deep-fat frying is often selected as a method for creating unique flavors, colors, and textures in processed foods. Due to the higher heat treatment, surface darkening and many other adverse reactions may occur before the food is fully cooked (Blumenthal and Stier 1991). Frying temperature can range from 130-190Â°C, but the most common temperatures are 170-190Â°C. The high temperature of the frying fat typically leads to the appreciated surface color and mechanical characteristics of

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fried foods; and besides that, heating the reducing sugar affects a complex group of reactions, called caramelization, which leading to browning development that defines the color of the final product (Arabhosseini et al. 2009). Some fat and oil decomposition products have also been involved in producing adverse health effects when frying oils degraded with continued use (Taylor et al. 1983; Hageman et al. 1989). In addition, heat toxic compound acrylamide can be formed during this process (Gokmen and PalazoÄ&#x;lu 2008; Pedreschi et al. 2004). Frying is considered a dry cooking method because it does not require water. In the frying process, the food is immersed in a container of oil at a temperature of above the boiling point of water. High temperature and high heat conduction of oil, causing the cooked food preserved and even enhanced nutritional value and reduced degradation. The high temperature causes partial evaporation of water, which is moving away from foods and through the surrounding oil; and a certain amount of oil absorbed by the food replaces water lost (Inprasit 2011). Frying is often chosen as a method to create a unique flavor and texture in processed foods that can improve their overall tasty and palatability (Moreira et al. 1999). High heat transfer rates lead to the development of desirable sensory properties in fried foods (Farkas et al. 1996). During the frying process, physicochemical, physical, chemical and sensory properties of food will be modified. Texture, color and oil content are the main quality parameters of fried foods (Aguilera 1997; Moreira et al. 1999). Texture is important for prominent sensory attributes of food preferences (Thygesen et al. 2001), and is a critical parameter for the quality of fried chips (Ross and Scanlon 2004). The texture of fried chips is known to be directly associated with a specific gravity, total solids, starch content, cell size, surface area and pectin (Moyano et al. 2007). Textural changes during frying are the result of many physical, chemical and structural changes resulting in a complex process operation unit, which includes heat and mass transfer together with chemical reactions. Good quality chips should have a crispy crust about 1-2 mm, whereas most of the oil is located and awed, a soft center, like a cooked potato. For potato chips, a very crunchy texture is expected all the way through since crispness is an indicator of freshness and high quality (Troncoso and Pedresch 2007). It is a well known fact that texture of this product depends on the quality of raw potato and technological parameters used in the production process (Kita 2002). Crisp texture is associated with the dry matter of raw potato tubers (Thygesen et al. 2001). Crisps obtained from potatoes which is rich in dry matter (above 25%) can exhibit hard texture, whereas crisps of too low a specific gravity (low in dry matter), containing too much oil, are characterized by greasy and sticky texture. The dry matter of potato tubers is composed of various substances, i.e.: starch (15%), sugar, nitrogen compounds, lipids, organic acids, phenolic compound, mineral substances and non-starch polysaccharides (Amany et al. 2009). Color development begins when a sufficient amount of drying has occurred in the chips and depends also on the drying rate and heat transfer coefficients during the various

stages of frying. Color is visually regarded as one of the most important parameters in determining the quality of fried chips (Scanlon et al. 1994) and is the result of the Maillard reaction that depends on the content of reducing sugars and amino acids or proteins on the surface, as well as the temperature and frying time (Marquez and Anon 1986). The reduction in weight and size of dehydrated product and the increase in shelf stability can reduce product storage and distribution costs (Toledo et al. 1991). Da Silva and Moreira (2008) shows that the vacuum fried snacks (blue potato, green bean, mango and sweet potato chips) retain more of their natural colors and flavors due to the less oxidation and lower frying temperature. Oil absorption is one of the most important quality parameters of fried foods, but this is incompatible with consumer trend recently towards healthier foods and lowfat products (Bouchon and Pyle 2004). However, the oil consumption derived from salty snack products is very high (Kuchler et al. 2004). Consumption of oils and saturated fats are specifically related to significant health problems, including coronary heart disease, cancer, diabetes, and hypertension (Saguy and Dana 2003). Other undesirable effects due to high temperatures in frying process and exposure to oxygen are the degradation of essential nutrient compounds and the formation of toxic molecules in the foodstuff or the frying oil itself (Fillion and Henry 1998). This information has raised a red flag on the human consumption of fried foods and has a significant impact on the snack food industry (Dueik et al. 2010). As a result, healthy low-fat snack products have acquired a new level of importance in the snack food industry (Moreira et al. 1999). However, even the health-conscious consumers are not willing to sacrifice organoleptic properties, an intense full-flavor snacks continue to play an important role in the salty snacks market (Mariscal and Bouchon 2008). Traditional deep-fat frying and vacuum frying are two common types of applied frying processes (Garayo and Moreira 2002). Vacuum frying is an alternative technique to improve the quality of dehydrated food (Song et al. 2007). It operates at relatively lower temperatures (e.g. 130oC), thus the texture, color, flavor, and nutritional value is more preserved and naturally. Apart from high quality retention in the final product obtained by vacuum frying, the main difference of these two techniques is the investment cost and operational cost. For vacuum frying, specially designed machinery and equipment are required. Both frying techniques have different benefits and disadvantages, therefore, it should carefully consider the required properties of raw materials and desired characteristics of the final product to avoid wasteful investment (Inprasit 2011).

DISADVANTAGES OF FRIED FOODS Snack foods and especially fried chips, are popular forms of refreshment among consumers, because frying in oil helps to create a great flavor and texture. Frying is one of the oldest and most popular cooking methods in the world. Deep-fat frying is a method to produce dried food

SETYAWAN et al. – Chips characteristic by vacuum frying process

where an edible fat heated above the boiling water serves as a heat transfer medium, fats also migrate into food, providing nutrients and flavor (Fan et al. 2005; Tarmizi and Niranjan 2013). Unfortunately, deep-frying foods have some shortage. This process causes the foods contain a lot of oils and saturated fats that are related to coronary heart disease, cancer, diabetes, and hypertension, as well as leads to formation of acrylamide on foods containing amino acid and reducing sugars (starch) that is carcinogenic and neurotoxic for human health. Oil content Oil absorption is one of the most important quality parameters of fried food, which is incompatible with recent consumer trends towards healthier food and low-fat products (Bouchon and Pyle 2004). Repeated use of oil at high temperature causes quality degradation through chemical reactions, such as oxidation, hydrolysis and polymerization. The resulting decomposition products affect the flavor and color of the frying oil and fried products. These reactions impair the oil quality by increasing the amount of free fatty acids and polar compounds that affect consumers’ health by causing a higher risk of developing cancer, hypertension and coronary heart disease. In addition, food labors are also in danger, as they may breathe the oil vapor that can cause lung cancer (Hein et al. 1998; Goburdhun et al. 2000). Consumer’s preference for low-fat products have become the driving force of the food industry to produce lower oil content fried potatoes that still retain the desired texture and flavor. In order to obtain a low-fat product, it is critical to understand when, how and where the oil absorption occurs, so that the oil migration into the structure can be minimized. Several studies have revealed that most of the oil is confined to the surface area of fried product and is restricted to a depth of a few cells (Keller et al. 1986; Lamberg et al. 1990). Potato chips have become popular salty snacks for 150 years and retail sales in many countries are around 6 billion/year, representing 33% of total sales in the market fried foods (Garayo and Moreira 2002). However, potato chips have an oil content ranging from 35 to 45g/100g (wet basis), which is a major factor affecting consumer acceptance for oil-fried products today (Dueik and Bouchon 2011). Rimac-Brncis et al. (2004) reported that the osmotic dehydration pretreatment can be an effective operation to produce low-fat chips. Pre-drying of potatoes is a common way to reduce fat uptake in the final fried product (Moreira et al. 1999, Krokida et al. 2001, Moyano et al. 2002). Drying step following the blanching step reduces the absorbed oil on potato chips (Pedreschi and Mayano 2005). The application of a proper coating is promising to reduce the oil content (Mellema 2003). The major oil fraction is suctioned by the microstructure of the potato piece when this is removed from the fryer during the cooling period, indicating that the tight oil absorption associated with the loss of moisture (Erdogdu and Dejemek 2010). Oil absorption is mainly a surface phenomena and most of the oil is absorbed by the fried product in the post-frying period (Doran 2007; Dueik et al. 2011). Oil absorption will

result from the competition between drainage and suction into the porous crust once the food is removed from the oil (Bouchon et al. 2003). However, oil absorption during vacuum frying follows transport mechanisms are more complexly than those elucidated in conventional frying and is currently the subject of intensive study (Amany et al. 2009). Oil absorption is a surface phenomenon that happens as the product is removed from the fryer due to temperature difference between the product and ambient temperature. Changes in temperature cause an increase in capillary pressure in the product pores, which causes the oil to flow into the opened pore spaces. The de-oiling process becomes more important during vacuum frying because of the pressurization process. Chip has increased the oil content following vacuum frying and depressurization due to rapid change in pressure (vacuum to atmospheric) (Moreira et al. 1997). De-oiling is one of the most important unit operation steps in vacuum deep-fat frying to ensure best quality products (Da Silva et al. 2009). Vacuum frying at 120˚C under a pressure of 5.37kpa produce potato chips with acceptable quality and improved the quality of frying oil. De-oiling mechanisms are generally centrifuges, installed in a special vacuum dome attached to the vacuum frying (Amany et al. 2012a,b,c,d). However, it is sometimes manufactured separately. Vacuum fried products of apple and potato chips significantly absorb lower oil than atmospheric fried (56.7% and 18% less oil in vacuum fried potatoes and apples, respectively). There are large differences in the drainage capacity of the two products. Apples significantly drained more oil from their surface than potato chips; and had smoother surfaces with a higher drainage. Surface roughness and drainage capacity are inversely related (r2= 0.949). For potato products, vacuum fried chips have a rougher surface than the atmospheric fried ones (29% rougher), which may explain the lower drainage of the first ones, along with the higher oil viscosity at a lower temperatures. The higher roughness of vacuum fried potatoes can be a result of pressure changes that suffer the tissue during depressurization and pressurization steps. Certainly, the surface roughness is a key factor determining its capacity to drain oil during the post frying period, but there might be other factors such as crust micro structural parameters that affect the final oil content of fried products (Dueik et al. 2011). Moreno et al. (2010) determined that products with higher surface roughness absorbed more oil. However, this relationship is restricted to products of similar nature (either gluten or potato-flake based products categories) and cannot be extended when comparing different product categories. Vacuum frying is a promising technology that is potential to produce low-fat chips (Dueik et al. 2010; Dueik and Bouchon 2011; Mariscal and Bouchon 2008). It is an efficient method to reduce the oil content in fried chips, maintain nutritional quality of products, and reduce damage to the oil deterioration. It is a technology that can be used to produce fruits and vegetables chips with the necessary degree of dehydration without excessive darkening or scorching of the product. Vacuum frying is a deep-frying

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process, which is carried out in a closed system, under pressures well below atmospheric levels (preferably lower than 7-8 KPa), making possible to substantially reduce the boiling-point of water, and therefore, the frying temperature. The low temperatures employed and minimal exposure to oxygen account for most of its benefits, which include nutrient preservation (Da Silva and Moreira 2008), oil quality protection (Shyu et al. 1998) and a reduction in the generation of toxic compounds (Granda et al. 2004). Compared with other dehydration methods for fruits and vegetables, vacuum frying is a viable option to obtain high quality dried foods in a much shorter process. Vacuum fried products can absorb between 25 and 55% less oil than atmospheric fried products (Garayo and Moreira 2002; Dueik and Bouchon, 2010). Vacuum frying (driving from 60°C) can reduce the oil content of carrot crisps by nearly 50% (d.b.) compared with atmospheric fried crisps produced using the same driving force (Passos and Ribeiro 2009; Da Silva and Moreira 2008; Dueik et al. (2009). Acrylamide formation Deep-fat frying is one of the oldest and most common unit operations used in the preparation of foods. However, consumer fears have started to arise as acrylamide, a possible carcinogen, has been detected in foods exposed to high temperatures, including fried and baked foods (Tareke et al. 2002). Acrylamide is classified as possibly carcinogenic and neurotoxic to humans. It has been found in starch-rich foods cooked at high temperatures (Granda and Moreira 2005). Acrylamide was accidentally discovered in foods in April 2002 by scientists in Sweden when they found the chemical in starchy foods, such as potato chips, french fries, and bread that had been heated. Boiled foods and raw or unheated foods did not exhibit any formation of acrylamide (Mottram et al. 2002; Stadler et al. 2002; Tareke et al. 2002). Although the researchers are still unsure of the precise mechanisms by which acrylamide formed in foods, many believe it is a by-product of the Maillard reaction. Development of acrylamide as a by-product of the Maillard browning is currently the most accepted theory (Stadler et al. 2002; Yaylayan et al. 2003). In fried foods, acrylamide can be produced by the reaction between asparagine and reducing sugars (fructose, glucose, etc.) or reactive carbonyls at temperatures above 120°C (Mottram et al. (2002). Acrylamide formation in fried foods found to depend on the composition of raw materials as well as frying time and temperature. In potato chips, acrylamide is rapidly formed at more than 160°C, with the amount proportional to the heating duration and temperature. Free amino acids are used to reduce acrylamide, with lysine, glycine, and cysteine having the greatest effects in aqueous system. Lysine and glycine are effective at inhibiting the formation of acrylamide in wheat-flour snacks. In potato chips, the addition of 0.5% glycine to pallets reduced acrylamide by more than 70%. Soaking potato slices in a 3% solution of either lysine or glycine reduces acrylamide formation more than 80% in potato chips fried for 1.5 minutes at 185°C. These results indicate that the addition of certain amino

acids by soaking the raw products in appropriate solutions is an effective way to reduce acrylamide in processed foods (Kim et al. 2005). Granda and Moreira (2005) shows that during traditional frying potato, higher temperatures are used (150 to 180°C) and acrylamide produced after some time but started to degrade, resulting in a constant rate on the acrylamide content at longer times. In addition, during vacuum frying (10 Torr), acrylamide increases exponentially (but at lower levels) for all frying times. Decreasing in pH is a way to reduce the Maillard reaction when it is undesirable (Schwartzberg and Hartel 1992). Jung et al. (2003) proposed a theory of acrylamide reduction by lowering the pH of the raw product prior to cooking. Stadler et al. (2002) observed that when pyrolyzed at 180°C in the presence of glucose, asparagine formed significant amounts of acrylamide (368 ppm). Appropriate reactants enhanced interaction when water is added to the reaction mixture, and there was an increase of the product (acrylamide) in reaction (960 ± 210 ppm). Schwartzberg and Hartel (1992) suggested that one way to inhibit the Maillard reaction in cases where it is undesirable is the maintenance of lowest possible temperatures. Tareke et al. (2002) showed that acrylamide formation depends on temperature; it increases as the increasing of temperature. Mottram et al. (2002) indicated that acrylamide formation increases with temperature from about 120-170oC and then decreases. Surdyk et al. (2004) found that not only the temperature (above 200oC) but also heating time increased acrylamide content in yeastleavened wheat bread crust. When the bread is baked at 270oC for 18 and 32 minutes, the acrylamide content increased from about 300 ppb to 1200 ppb, respectively.

VACUUM FRYING TECHNOLOGY Due to the increasing health concern and the trends toward healthier food snacks, vacuum fried foods have become a common product that can be found in the local markets. Various kinds of fried chips are now offered on the local market shelves, such as bananas, jackfruits, pineapples, salaks, mangoes, cassavas, potatoes, sweet potatoes, etc. Nonetheless, the vacuum frying process, similar to the atmospheric frying process, is quite complicated, involving coupled heat and mass transfer through a porous media, crust formation, product shrinkage and expansion, and so forth. These mechanisms all contribute to the difficulties in predicting the physical and structural appearance of the final product. Therefore, a better understanding of the frying mechanism and the heat and mass transport phenomena would be useful for food processors to produce and develop new fried and vacuum fried snack foods for growing allegiance of healthy consumers (Yamsaengsung et al. 2008). Vacuum frying is a promising technology that could be an option for the production of novel snacks such as fruit and vegetable crisps that present the desired quality attributes and respond to new health trends (Dueik et al. 2010). Fruits and vegetables are important sources of vitamins and antioxidants. However, average consumption

SETYAWAN et al. â&#x20AC;&#x201C; Chips characteristic by vacuum frying process

of fruits and vegetables in modern societies is low due to early decay and rather high price (Da Silva and Moreira 2008). Fruits and vegetables are high in sugar content and heat sensitive, thus they are usually burned in temperature of usual frying process and lose their natural colors and flavors, unless the frying process takes place at low temperature (Shyu and Hwang 2001). One of the modern methods for fruits and vegetables processing in the world is the vacuum frying that can be performed at low temperatures and minimal exposure to oxygen (Maadyrad et al. 2011). This allows us to create products with the desired crispy texture and high nutritional value (Escaladapla et al. 2007). In vacuum-frying, food is heated under a reduce pressure that lowered the boiling points of frying oil and water in food (Troncoso et al. 2009). Water can be removed from the fried food rapidly once the oil temperature reaches the boiling point of water. Colors and flavors can be better preserved in vacuum-fried food, because the food is heated at a lower temperature and oxygen content (Hidaka et al. 1991; Shyu et al. 1998). The absence of air during frying can inhibit oxidation including lipid oxidation and enzymatic browning; therefore, the color and nutrients of food can be largely preserved (Xu 1996; Gao and Liang 1999; Tarzi et al. 2011). Dehydrated food produced by vacuum frying can have crunchy texture, good color and flavor and good retention of nutrients. Vacuum frying also has less adverse effects on the quality of oil (Kato and Sato 1991). During frying, the heat from the oil is confected to the product surface and then conducted to the productâ&#x20AC;&#x2122;s center, thus increasing its temperature. Water evaporates as the product reaches the boiling-point temperature. This process is generally regarded a Stephan type of heat transfer problem, which is characterized by the presence of a moving interface that divides two areas of physical and thermal properties (Farkas et al. 1996a). Farkas et al. (1996a, b) gives separate equations for the two regions: the crust and the core, with a moving boundary. The study of these transport mechanism have led to the investigation on the effect of vacuum frying on the transport processes. For instance, several studies have shown that less oil is absorbed during the vacuum frying process (Garayo and Moreira 2002; Krupanyamat and Bhumiratana 1994; Choodum and Rojwatcharapibarn 2002; Yamsaengsung and Rungsee 2003). It has been suggested that the pressure difference between the internal pressure of the product and the vacuum pressure of the fryer help to reduce the amount of surface oil present at the end of the frying process, which in turn limits the total amount of oil absorbed. Another important advantage of the vacuum frying is the reduced temperature which helps to maintain the natural color of the product while minimizing the loss of vitamins and minerals. In atmospheric frying, the products are generally fried at 160-190 oC, and the water inside the product evaporates at approximately 100oC depending on the presence of dissolved components. On the other hand, under vacuum frying, the boiling point of water can be reduced to as low as 35-40oC, thus the frying temperature can be as low as 90-100oC. Shyu and Hwang (2001) found

that the optimum conditions for frying apple chips are at a pressure of 3.115 kPa, a frying temperature of 100-110oC, a frying time of 20-25 minutes, and a concentration of immersing fructose solution of 30-40%. Garayo and Moreira (2002) found that potato chips fried under vacuum conditions (3.115 kPa and 144oC) have more shrinkage volume and slightly softer, and lighter in color than potato chips fried under atmospheric conditions (165oC). Vacuum frying can reduce levels of fat fries to 26.63% (which is normally 35.3-44.5% by deep-fat frying). Yamsaengsung and Rungsee (2003) also found that compared to atmospheric frying, vacuum fried potato chips retained in a more intense flavor and color. Fruits and vegetables are generally dehydrated by freeze drying, a process that can maintain their original flavor and color (Luh et al. 1975), but it is energy and time consuming (Flink et al. 1977). Many fruits and vegetables with high nutritional value, such as cauliflower, carrots, mangoes, and pineapples, cannot be processed by ordinary frying methods. However, they can be processed by a vacuum frying due to low temperature (Mariscal and Bouchon 2008). This technology is expected to improve the nutrition and health by producing products that taste good, keep most of their nutrition values, have lower fat contents than conventional fried chips, they also safer with little or no acrylamide formation, and they can be kept longer (Kemp et al. 2009). Compared with other dehydration technologies for fruits and vegetables, vacuum frying is a viable option to obtain high quality dried products within a much shorter process (Laura and Claudio 2009). It has been shown that vacuum fried chips (blue potato, green bean, mango and sweet potato chips) retain more of their natural colors and flavors because of less oxidation and lower frying temperature (Da Silva and Moreira 2008). Vacuum fried potato chips and sliced guava have lower oil content and more natural colorations than the conventional frying process (Yamsaengsung and Rungsee 2003). Instead, Diamante et al. (2010) reported that hot air drying of green and gold kiwifruits at increasing temperatures (60 to 100oC) leads to increased browning and ascorbic acid loss. Vacuum frying may be a good alternative for the production of fruit and vegetable dehydrated slices (Shyu et al. 1998, 2005).

DESCRIPTION OF VACUUM FRYING The main factors influencing the fried products are combinations of time and temperature of the cooking process; the correct combination is required in producing food product with acceptable physical attributes (such as color, appearance, texture and flavor) as well as preserving nutritional, but not stable, compounds such as vitamin C (Inprasit 2011). Several processes have been developed to manufacture low-fat products that possess the desired quality attributes of deep-fat fried food whilst preserving their nutritional and better sensory properties; such as extrusion, drying, and baking, which can be applied to raw food or formulated products. Unfortunately, none of them have been as successful as expected because they are still

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unable to impart the desired quality attributes of deep-fat fried food, such as amour, texture, appearance and mouth feel (Dueik et al. 2010). Vacuum frying is a promising technology that could be an option for the production of novel snacks such as fruit and vegetable crisps that present the desired quality attributes and respond to new health trends. This process is carried out in a closed system under pressures well below atmospheric levels, which makes it possible to substantially reduce the boiling point of water and thus the frying temperature (Garayo and Moreira 2002). In fact, most of the beneďŹ ts of this technology are the result of the low temperatures employed and minimal exposure to oxygen. Said the beneďŹ ts include: (i) reduction of adverse effects on oil quality (Shyu et al. 1998), (ii) preservation of natural color and amours (Shyu and Hwang 2001), (iii) reduced levels of acrylamide (Granda et al. 2004), and (iv) preservation of nutritional compounds (Da Silva and Moreira 2008). Vacuum frying is the technique of deep-fat frying foods under pressures well below atmospheric levels, preferably below 6.65 kPa, which serves to reduce oil content, discoloration and loss of vitamins and other nutrients that are usually associated with oxidation and high temperature processing (Garayo and Moreira 2002). One of its first applications was to reduce the formation of acrylamide in potato crisps, as this tends to occur during hightemperature processing of high carbohydrate foods (Granda et al. 2004). It has also shown some success in producing vacuum fried products with other foods, including pineapple, apples, carrots, blue potato, sweet potato, beans, mangoes and jackfruit (Da Silva and Moreira 2008; Diamante 2009; Mariscal and Bouchon 2008; Perez-Tinoco et al. 2008; Fan et al. 2005). Figure 1 shows the schematic diagram of vacuum frying system.

Figure 1. Schematic of the vacuum frying system (Garayo and Moreira 2002)

When the frying is carried out under atmospheric pressure, boiling point of water reduces; hence, higher

temperatures are not required to remove moisture from the food. Deteriorating effect on the food due to heat will be less. The following is a brief explanation of governing theories: (i) Water evaporation under vacuum. Boiling point of water is 100oC at atmospheric pressure. Evaporation of water at this temperature occurred together with the loss of some food nutrients. Under vacuum water is boiled and evaporated at lower temperature even at 0 oC so that nutrients loss is reduced especially for heat sensitive nutrients. (ii) Heat transfer. For hot air dryers, heat is transferred by convection using hot air as the medium. Air has relatively lower heat transfer coefficient. Contrast to the air, in a vacuum frying, vegetable oil has higher heat transfer coefficient. Therefore shorter time is required to reduce water content. (iii) Frying temperature. Automatic temperature control system, when used, provides a mechanism by which moisture is reduced while food temperature is controlled by the system. Constant temperature of the food results in uniformity of product quality (Inprasit 2011). Stages using vacuum frying machine is as follows: The material to be fried is prepared (peeled and sliced with a thickness of 0.50-1 cm). If the water content is high, the spinner machine can be used to reduce the water content. Fryer tube is filled with frying oil. To 4 kg of fresh fruit, it is required 40 liters of frying oil. The raw materials are put into the frying basket; the basket position is appointed (not submerged in oil). The frying machine and gas stove is turned on and the temperature is set. Furthermore, fryer tube is closed to get the vacuum condition. After the pressure needle pointing at-680 mm Hg, the basket is lowered down into the submerged position. Raw material is fried until drying. After completed frying, the position of the basket is moved up (not submerged in oil), and the electric instalation and stove is turned off. Tap of the fryer tube is opened until the pressure needle pointing at 0 mm Hg. Than, the fryer tube is opened; and chips is removed and dried by using spinner machine. Chips is cooled and packaged in plastic (PP 0.80 mm) or alumunium foil bags and then sealed (Kamsiati 2010). The oil uptake mechanism of vacuum frying is still not fully understood. During normal operation, the product is placed inside the frying basket once the oil reaches the target temperature. The lid is then closed and the chamber is depressurized. Subsequently, the basket is immersed in the oil bath, where it remains for the required amount of time. It is then lifted out and the vessel is pressurized using a pressure release valve. This results in a sudden increase in the surrounding pressure, which may force the vapor inside of the pores to condense, which means that oil absorption may precede cooling. The low pressure may allow air to diffuse faster into the porous structure, obstructing oil passage and leading to lower oil absorption than is observed in atmospheric frying. Vacuum fried potato crisps absorb less than half the oil of crisps fried under atmospheric conditions (Garayo and Moreira 2002). Mariscal and Bouchon (2008) states vacuum fried apple slices absorbed slightly less oil, and presented better results for color preservation than atmospheric fried samples.

SETYAWAN et al. – Chips characteristic by vacuum frying process

BENEFIT OF VACUUM FRYING Vacuum frying may be an option for the production of fruits and vegetables with low oil content and the desired texture and flavor characteristics. It is defined as the frying process that is carried out under pressures below atmospheric levels, preferably below 50 Torr (6.65 kPa). Due to the lowering pressure, boiling points both of oil and moisture in the foods lowered (Garayo and Moreira 2002). Vacuum frying technology has the advantage of low oxygen existed in the system resulted in the low rates of frying oil oxidation and lower boiling point of below 100ºC. Vacuum fried products have better aroma and flavor similar to that of fresh fruit and tubers (vegetables). In fact most of the benefits of this technology are the results of low temperatures used and minimal exposure to oxygen. The benefits include: (i) reduction of adverse effects on the oil quality (Shyu and Hwang 1998; Shyu et al. 1998), (ii) the preservation of natural colors and flavors (Shyu and Hwang 2001), (iii) decreased of the acrylamide content (Granda et al. 2004), and (iv) the preservation of nutritional compounds (Da Silva and Moreira 2008). Vacuum frying was tested as an alternative technique to develop low oil content of potato chips. During vacuum frying, oil temperature and vacuum pressure had a significant effect on the drying rate and oil absorption rate of potato chips. Fried potato chips at low vacuum pressure and higher temperature had less volume shrinkage. Color was not significantly affected by oil temperature and vacuum pressure. Hardness values increased with increasing oil temperature and decreasing vacuum levels. Potato chips fried under vacuum (3.115 kPa, 144ºC) had more volume shrinkage, slightly softer, and lighter in color than the potato chips fried under atmospheric conditions (165ºC). Vacuum frying is a process that could be a feasible alternative to produce potato chips with lower oil content and the desirable color and texture (Garayo and Moreira 2002). Vacuum fried products have low water content (<6%) and low water activity (aw<0.3) (Tawong 2000; Piamkhla 2004; Wongsuwan and Laosuksuwan 2006), so it has a long shelf life. Under vacuum condition, frying temperature is constant and not higher than 100oC and frying time is not longer than 2 hours. Obviously, vacuum frying is an energy efficient process. The products are crispy and retain its original color, taste and odor as of the natural foods (Granda et al. 2004). Vacuum frying can process heat-sensitive commodities such as fruits being processed in the form of crisps (chips), such as jackfruit chips, apple chips, banana chips, pineapple chips, melon chips, and papaya chips, etc. Compared with conventional fryers, vacuum systems produce a much better product in terms of color appearance, aroma, and taste like a fruit because it is relatively original (Siregar et al. 2004). In vacuum frying, vegetable oil is used as a heat transfer medium. Oil may be absorbed by the foods, therefore, there will be oil remaining in fried products making them undesirable and may raise health concern among consumers. There are many research works which

has been described techniques to reduce oil absorption during vacuum frying. Pre-coating with guar gum is one of the recommended techniques. Banana coated with guar gum before frying under vacuum has lower oil content of 8% compared to vacuum fried banana without guar gum pre-coating, which has 12% oil content. Oil absorption, however, varies with the chemical properties of raw materials. Different pre-treatment techniques and processes will be applied to produce a good quality of vacuum fried products (Inprasit 2011). In financial terms, the investment cost of vacuum frying process is much higher than that of deep frying. This is because the vacuum frying technique is basically designed for large-scale industry. There is a lack of vacuum fryer design for small scale production. Small scale producers such as farmers' group, small community enterprise, and cooperatives are difficult to buy vacuum frying machinery and equipment without financial support from the government. High investment costs are substantial disadvantage in applying vacuum frying in small-scale production (Inprasit 2011). Although, it is the costinvestment, among several deep-fat frying technologies, vacuum frying has significant strategic importance for the future fried food manufacturing. This technology offers significant benefits such as improved product safety and quality cooking oil and oxidation is reduced due to low temperature processing (Granda et al. 2004).

RAW MATERIALS FOR VACUUM FRYING Raw materials that are not eligible for fresh consumption and are not eligible to be sold as fresh fruits and vegetables such as too big or too small, not smooth on the surface or have defects, are selected for vacuum frying. Unqualified fruits and vegetables are cheap. They should be washed, peeled, and trimmed to remove defects and uneatable parts before vacuum frying. Defects are not detected in fried fruit products. Therefore vacuum frying should be applied to reduce waste because they can use unqualified raw material for processing. Ripe fruits, which have high sugar content, are popularly fried under vacuum because it is not possibly fried at atmospheric pressure. In the fruit and vegetable industry, much kind of fruits and vegetables are wasted from pruning. These parts are in good quality but their sizes and shapes do not meet the processing standards. Vacuum frying help improve the commercial value of the waste (Inprasit 2011). Figure 2 shows the flow diagram for the processing of vacuum fried pineapple. The selection of fruits suitable for vacuum frying is based on the followings (Inprasit 2011): Variety. Chemical and physical properties of fruits and vegetables vary depending on the variety. These properties affect the quality of fried products. Thin flesh jackfruits can be fried as a lump of pulp whereas thick flesh jackfruits should be chopped into small pieces before frying. The thickness of raw materials affects the vacuum frying process. Crispy and soft texture of vacuum fried jackfruits is obtained together with uniformity of the water content

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when raw material thickness is efficiently controlled. Freezing is a recommended pre-treatment to obtain crispy vacuum fried products.

Pineapple Washing Peeling and core removing Trimming

PROCEDURE FOR HANDLING

Washing

Slicing

Water content. It is usually difficult to fry high moisture fruits. Plenty of water must be removed during frying. Fruits will be burned before they dry and shrinkage will occur during water evaporation. Freezing of high moisture raw materials before vacuum frying such as longan and lychee is recommended to keep the water evaporation at a lower temperature. Seasonal fruits. Some fruit are seasonally grown and harvested. Domestic and export markets cannot always absorb the entire production. Excess fruit during the season reduce the price. This is an important issue in the developing agricultural countries. Frying can be applied to extend the shelf life of seasonal fruits.

Tidbiting

Freezing (if any) Vacuum frying Cooling Nitrogen packing in gas barrier plastic bags Storing in cold room Figure 2. Flow diagram for processing of vacuum-fried pineapple (Inprasit 2011)

Ripeness. Vacuum frying is effectively used for frying high sugar fruits that has sweet taste fried products. Fruit subjected to vacuum frying should not have any astringent taste. Fruits should be ripe but not be too ripe because the high sugar content induces high oil absorption during frying. Likewise, ripe fruits should not have too soft texture due to breaking of fruits to small pieces occurs from rapid evaporation of water during vacuum frying. Vacuum frying of too ripe durian produce small pieces of fried products. Taste. Vacuum frying is a process of evaporating of water at low temperature to retain natural flavor and minimize nutrient losses. Original fruit flavors should be considered for the selection of raw material. Stronger taste of fried products is observed when compared to the taste of raw materials. This is due to very little excreting saliva during eating fried products, therefore, a strong taste of the high concentration of flavor components is observed. Vacuum fried pineapple has very sour taste when made from sour pineapple. Also too sweet ripe fruits should not be selected as raw materials for vacuum frying due to its high content of sugar cause caramelisation.

Vacuum fried product is much susceptible to spoilage during storage especially in tropical and humid conditions. It can easily absorb moisture from storage environments. Therefore, manufacturers should separate package of fried products that have different water content because, in the same packaging, moisture can move out from high moisture product and be absorbed by low moisture product. Then product crispiness decreases and consumer will reject the products. Fried product quality changes when storing at high temperature such as shop standing in open space or in cars and containers parking in the sun. This also causes loss of product crispiness (Inprasit 2011). Among the important properties of deep and vacuum fried products are nutrition values, consumer acceptability and safety of frying oil. These qualities can be controlled by proper selection of raw materials. One of properties that affect consumer acceptance is crispiness of fried products. Crispiness of deep fried product changes rapidly after frying because it mainly consists of sugar and easy to absorb moisture. Whereas crispiness of vacuum fried product does not change much because it consists of starch, which absorbs less moisture after frying. In addition, the type of raw material thickness affects product crispiness. Thin slices required to obtain soft crispy and dried texture after frying. Manufacturers have to change frying oil when oxidized and unsafe for consumers. During the frying process, quality of frying oil should continue to be monitored. Frying oil should be changed frequently due to changes in the physicochemical properties of the oil that will affect the quality of the product and oil uptake during frying. In addition, toxic compounds can be generated in deteriorated frying oil, which is harmful to human health (Inprasit 2011). Evaluation of frying oils can perform the following methods: (i) Sensory evaluation: Used frying oils are generally considered as deteriorated when they clearly indicate an objection smell or taste, for example strong mildew, strongly gritty, rancid, vanish, or bitter; and showed intense smoke and foam formation during frying. These sensory impressions are objectified through further analytical criteria such as polar compounds and polymer triglycerides. Intensify the dark, however, is not a measure of deterioration. This color change is caused by the reaction

SETYAWAN et al. – Chips characteristic by vacuum frying process

of proteins with fat or sugars components. (ii) Quick tests: Colorimetric procedures to determine the amount of degradation products of fatty acids (carbonyl compounds). Color reaction aims to determine portion of the polar compounds or acid value. Redox reaction determines the amount of oxidized fatty acids. It also needs to measure the height of the foam, viscosity and dielectric properties. (iii) Analytical methods: Physical methods include determining the smoking point, viscosity, conductivity, constant dielectricity and the Lovibond color index. Chemical methods include the determination of free fatty acids (acid value) by acid-base titration, polar compounds by chromatographic procedures, and also triglyceride polymers and oxidized fatty acids (Inprasit 2011). Manufacturers should follow the following guidelines to control the quality of fried chips products (Inprasit 2011): (i) Manufacturers should use control sheet presented to control frying operation and also responsible operators. (ii) After frying, manufacturers should remove high moisture fried product by pressing and observing the soft texture and then bring that product to fry once more to get the low water content and desire crispiness. (iii) For storing fried products prior to packaging, manufacturers should use containers that can be sealed to prevent moisture transfer and store products in the shade at low temperatures. The best storing method is packaged in a double polypropylene (PP) plastic bag and then placed in a sealed plastic bucket. (iv) For retail packaging, manufacturers should use containers that can be sealed to prevent moisture transfer such as aluminum foil bag, aluminum foil bag in a paper box, paper can coated inside with aluminum foil and metal can. Fried product shelf life is not less than six months when the manufacturer follows the above guidelines. Long shelf life can be obtained when using nitrogen gas packaging. According to Piamkhla (2004), the shelf life of vacuum fried ripe banana is six months when packaged in a plastic bag that can be flushed with nitrogen or putting moisture absorbent.

LESSON LEARNED FROM SEVERAL CHIPS Many vacuum fried products are introduced in the markets. Fruits, tubers and other vegetables are the most widely processed food with vacuum frying method, but some types of meat and fish are also treated with this method, such as shrimp, squid, green shell mussels (Taryana 2012), sepat-siam (Suwanchongsatit et al. 2004), lemuru (Manurung 2011), tongkol (Nufzatussalimah 2012), beefs (Shofiyatun 2012) and others. Some types of fruits and vegetables that have been processed into chips with vacuum frying method are: banana (Garcia and Barette 2002), banana peel (Dewantara 2012), jackfruit (Alamsyah et al. 2002), durian, mango, pineapple, taro, yam, baby corn (Inprasit 2011), carrot (Fan et al. 2005), okra (Arlai 2009), garlic, sapodilla (Paramita 1999), gembili (Dioscorea aculeata) (Wibowo 2012), chickpea (Widaningrum et al. 2008), salaks (Maulana 2012), apple (Shidqiana 2012), tapioca (Binti Zahroni2012), cassava (Aprillia 2007), eggplant (Nur-Aeny 2012), potato (Granda et al. 2004),

kiwi fruits (Diamante et al. 2011), pumpkin (Mehrjardi et al. 2012), melon (Arum 2012), sweet potato (Abdullatif 2012), tempeh (Kato and Sato 1991), etc. Potato (Solanun tuberosum) Potato (Solanum tuberosum L.) is one of the world’s major agricultural crops and it is consumed daily by millions of people from diverse cultural backgrounds (NPC1988). The potato is best known for its carbohydrate content (approximately 26 grams in a medium potato). The potato contains vitamins and minerals, as well as an assortment of phytochemicals, such as carotenoids and natural phenols. Large variation in suitability of potato for processing of crisp and French fries have special quality demands compared to ware potatoes. Unfortunately, potato chips fried conventionally produce acrylamide that harmful to human health. Potatoes and other foods that have a high content of the amino acid asparagine and a high accumulation of reducing sugars are subject to the formation of acrylamide during frying (Granda et al. 2004). Acrylamide has been classified as probably carcinogenic in humans (Rosen and Hellenas 2002; Tareke et al. 2002). Reducing acrylamide in food industry can only help the public perception about safety, which has suffered in recent years. Acrylamide formation can be diminished by adding amino acids such as lysine, glycine and cysteine (Kim et al. 2005). Lowering the pH with citric acid before frying was effective in diminishing acrylamide formation (by about 73%) in French fries when fried for 6 minutes at 190 °C in an atmospheric fryer (Jung et al. 2003). However, according to Pedreschi et al. (2004), the effect of citric acid immersion on acrylamide reduction was not obvious in their experiment with potato chips fried at 170°C and 190°C. On the other hand, the blanching process led to a significant reduction in acrylamide content of their chips. Haase et al. (2003) reported that by lowering the frying temperature of potato chips from 185°C to 165°C, it was possible to reduce the acrylamide formation by half. Among several deep-fat frying technologies, vacuum frying has a significant strategic importance for future fried manufacturing and in reducing acrylamide formation (Garayo and Moreira 2002; Granda et al. 2004). Vacuum frying reduced acrylamide formation in fried potatoes by 94%. As the frying temperature decreased from 180°C to 165°C, acrylamide content in potato chips reduced by 51% during traditional frying and by 63% as the temperature decreased from 140°C to 125°C in vacuum frying. Increased frying time increased acrylamide formation during frying for all temperatures and frying methods analyzed. However, the effect on acrylamide concentration was greater for the traditional frying than the vacuum frying (Granda et al. 2004). Acrylamide formation decreased dramatically as the frying temperature decreased from 190 to 150°C for all the pre-treatments tested. Color represented by the parameters L* and a* showed high correlations (r2 of 0.79 and 0.83, respectively) with French fry acrylamide content (Pedreschi et al. 2006).

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Carrot (Daucus carota) Carrot (Daucus carota L. var. sativa D.C.) has the highest carotene content of any human foods (Desobry et al. 1998). Carotene, a source of pro-vitamin A, may play a role in protecting the body from numerous diseases that are associated with oxidative stress and damage (Handelman 2001), and it also has many non-antioxidant properties that affect cellular signaling pathways, modify the expression of some genes and can act as inhibitors of regulatory enzymes (Stahl and Ale-Agha 2002). To maximize the use of carrot as a source of pro-vitamin A, it is important to find an appropriate processing method to manufacture products that are not only highly preferred by consumers but also are good nutritional sources of pro-vitamin A. Vacuum-fried carrots may be a promising snack category due to the fact that this technology makes it possible to overcome major carotenoids degradation pathways due to isomerization and oxidation and thus preserve biological activity. Vacuum fried crisps (driving force of 60°C) may reduce the oil content of carrot crisps by nearly 50% (d.b.) compared to atmospheric fried crisps produced using the same driving force. Furthermore, they preserve around 90% of trans a-carotene and 86% trans bcarotene, which leads to the preservation of the color of raw carrots. This is reflected by L*, a*, b* color coordinate analyses, excellent linear correlations between a* and trans b-carotene content (r2 = 0.95), b* and trans a-carotene content (r2 = 0.78), and hue and total carotenoids content (r2 = 0.91), when comparing values of fried crisps at bubble-end point. As a result, vacuum frying may be a useful process in the production of novel snacks that present desired quality attributes and respond to new health trends (Dueik et al. 2010). Bananas (Musa paradisiaca) Bananas (Musa x paradisiaca L.) are one of the world’s most traded fruit in both fresh and processed forms. Bananas are an excellent source of vitamin B6, soluble fiber, and contain moderate amounts of vitamin C, manganese and potassium (USDA NND 2012). Along with other fruits and vegetables, consumption of bananas may be associated with a reduced risk of colorectal cancer (Deneo-Pellegrini et al. 1996) and in women, breast cancer (Zhang et al. 2009) and renal cell carcinoma (Rashidkhani et al. 2005). The market quality and consumer acceptability of fresh banana and processed banana are significantly influenced by the fruit color. For vacuum-fried, banana slice products are prepared by peeling and slicing before vacuum-frying. In this preparation step, as a result in slicing and waiting for processing, there is accumulation of cell fluids, especially the phenolic compounds, on the cut surface and their exposion to oxygen, leading to browning (Garcia and Barette 2002). Phenolic compounds undergo oxidation to brown compounds that discolor fruits, reducing their quality (Rocha and Morais 2001). Discoloration is known as enzymatic browning which results from the action of a group of enzymes called polyphenol oxidase (PPO). PPO has been reported to occur in all plants and exists in particularly high amounts in mushroom, banana, apple,

pear, potato, avocado and peach (Garcia and Barette 2002). PPO catalyzes, in the presence of oxygen, the oxidation of mono-and di-phenols to o-quinones; these products are highly reactive and can either polymerize spontaneously to form high-molecular-weight compounds or brown pigments, or react with amino acids and proteins to enhance the brownish color produced (Vamos-Vigyazo 1981; McEvily et al. 1992). Inhibition of enzymatic browning can be achieved by a number of strategies that can be divided into three classes, depending on whether they affect the enzymes, substrates or reaction products, although in some cases, two or three targets can be affected at the same time. In addition, enzymatic inhibition can be reversible or irreversible; the latter case often achieved by physical treatment (heat), while chemicals may act in one or another way. The control of enzymatic browning has always been a challenge to the food industry. For using chemical treatments, several types of chemicals are used in the control of browning; some act directly as inhibitors of PPO, others by rendering the medium inadequate for the development of the browning reaction, still others act by reacting with the products of the PPO reaction before these can lead to the formation of dark pigments (Nicolas et al. 1994). Banana chips coated with an edible coating and produced using the higher speed during the oil centrifuge step in the vacuumfrying process maintained a good quality with low oil content, representing a healthier snack for consumers (Sothornvit 2011). Banana peel is a byproduct of the use of bananas that can be used as snack foods like banana peel chips. The banana peel contains a lot of water (68.90%) and carbohydrate (18.50%). To produce chips with good quality in terms of color, aroma, and taste, the temperature setting should not exceed 85◦C and vacuum pressure between 65-76 cm Hg (Dewantara 2012). Kiwi fruit (Actinidia deliciosa) Kiwi fruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson) is native to southern China (Scott et al. 1986). Kiwi fruit is a highly nutritional fruit due to its high level of vitamin C and it has a strong antioxidant activity due to carotenoids, lutein, flavonoids and chlorophyll contents (Cassano et al. 2006). Furthermore, kiwi fruits have a very short shelf-life due to their highly perishable nature, and they are not only consumed as fresh fruits but also as processed foods in the form of jams, juices, canned fruits, frozen and dehydrated products (Abedini 2004; Emamjome and Alaedini 2005). The color and the shrinkage of kiwi fruit chips were significantly (p<0.05) correlated with the frying temperature and time, while the crispiness was affected only by the frying temperature. There was no significant relation between the vacuum pressure and the responses except the shrinkage. Sensory evaluation indicated that there were no significant differences (p<0.05) between the vacuum fried kiwi slices and the dried kiwi chips except flavor. The optimum conditions for the vacuum frying of kiwi slices were found to be: 105ºC, 62 mbar, and 8

SETYAWAN et al. – Chips characteristic by vacuum frying process

minutes, for the frying temperature, the vacuum pressure and the frying time, respectively (Maadyrad et al. 2011). Shallot (Allium cepa var. aggregatum) Shallot (Allium cepa L. var. aggregatum G. Don) is an elementary spice of Southeast Asia as well as the world. Shallots appear to contain more flavonoids and phenols than other members of the onion genus (Yang et al. 2004). It was proven to increase high density lipoprotein (HDL) cholesterol, reduce low density lipoprotein (LDL), reduce cholesterol in the blood and control blood sugar. Thus it would be beneficial to develop a snack from shallot. Deepfat fried snack is one of the most tasteful products. However, high fat content could reduce consumption due to a health concern issue; and uses high temperature in an opened system speeds up oxidation and thereby rancidity development. Vacuum frying could be used to reduce fat content, frying temperature and slow down rancidity of oil. To minimize fat content of fried snack, shallot should be fried under vacuum 551 mm Hg and 108°C for 13 minutes. The optimal vacuum frying condition was conducted to compare with the deep-fat frying. Vacuum fried shallot showed the improvement of product color as well as a decrease in fat content in the finished product. After 7 continuous vacuum frying processes, a slight change in acid value for the oil was found. Therefore, the optimal vacuum frying condition could be applied to produce fried snack from shallot (Therdthai et al. 2007). Salaks (Salacca edulis) Salaks or snake fruit (Salacca edulis Reinw.) is one of the horticultural commodities that have high potential to be explored and developed in Indonesia. Salaks fruit contain nutrients such as protein, carbohydrates, dietary fiber, calcium, phosphorus, iron, carotene, and thiamine that are good for body health. The mass production of salaks makes an excess amount of salaks distributed in the market; salaks become wasted and priceless. To prevent the decreasing value of salaks, it can be proceed in to fruit chips. The process of making salaks chips start from the frying stage. In order to pretend the composition and the taste of salaks chips, vacuum frying is used. After fried, salaks must be packaged with the suitable packaging to provide a longer shelf life of salaks chips. Aluminum foil is the best packaging for salaks chips comparing to polypropylene and laminated plastic because alumunium foil has the lowest transmission rate of water and oxygen (Maulana 2012). Melon (Cucumis melo) Melon (Cucumis melo L.) is an annual plant that is pervasive or a year or vines. Melon fruit is an excellent source of vitamin A and vitamin C, and a good source of potassium. Optimum temperature for the manufacture of melon chips is 75°C with a time of 55 minutes. Chips with this variable has a sweet, brownish orange color, crisp, and has aromas of melon and entrained water content is equal to 92.406% (Arum 2012).

Papaya (Carica papaya) Papaya (Carica papaya L.) is pretty much cultivated in Indonesia. Papaya fruit is a source of nutrients such as provitamin A carotenoids, vitamin C, folate and dietary fiber. Papaya skin, pulp and seeds also contain a variety of phytochemicals, including lycopene and polyphenols (Echeverri et al. 1997). Generally, processed papaya products on an industrial scale are still a household nata and candy. In fact, papaya has a huge production to be processed into other products, such as fruit chips. In vacuum frying process, administration of CaCl2 can improve the texture of papaya chips. Calcium chloride is widely used to improve texture of the processed fruit and vegetable products; it can also be used for texture chips, because it can reduce the decomposition of the cells that cause tissue softening (Indera-Sari 2012). Sweet potato (Ipomoea batatas) Sweet potatoes (Ipomoea batatas (L.) Lam.) have been an important part of the diet in the world and are a staple of human consumption, led by New Guinea at about 500 kg per person per year. Considering fiber content, complex carbohydrates, protein, vitamins A and C, iron, and calcium, the sweet potato ranked highest in nutritional value to other vegetables (CSPI 1992). For vacuum frying, sweet potato was sliced into 2 mm thickness and pretreated with 1% (w/w) NaCl solution and 1% (w/w) CaCl2 solution for 1 hour prior to frying process. Pre-treated slices were fried at atmospheric condition (180°C) and vacuum condition (120°C, 130°C, 140°C) for 5 minutes. In general, pre-treatments gave a significant effect to the texture, color and oil contents of atmospheric fried crisps. NaCl pretreated crisps showed the best crispness and color quality compared to the control. For vacuum fried crisps, breaking force (N) increased with increasing oil temperature. Oil absorption of control slices showed no significant difference (p<0.05) at all frying temperature, while NaCl pre-treated slices showed an increase with temperature increased. In contrast, CaCl2 pre-treatment increased oil absorption with increasing frying temperature. As frying temperature increased, the lightness of crisps was decreased, while a* and b* value were increased in all pretreatment. The best texture of crisps was obtained at 130°C vacuum frying temperature. Comparing between atmospheric and vacuum fried crisps, there is no significant difference (p<0.05) in terms of fracturability of crisps. However, oil absorption of vacuum fried crisps is 7.12% less than atmospheric fried. The color of vacuum fried crisps was also lighter than atmospheric fried. Sensory evaluation revealed that, consumer can accept the quality attributes of vacuum fried crisps. There is no significant difference between vacuum fried and atmospheric fried crisps in terms of color, crispness and overall acceptability (binti Ismail 2011). In Cilembu sweet potato chips, the optimum quality of the vacuum frying is 35 minutes treatment; and is obtained flavor, color, and crispness to the optimum water content of 17.4% (Abdullatif 2012).

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Okra (Abelmoschus esculentus) Okra (Abelmoschus esculentus L. Moench) is an important economic vegetable of the world. Okra is a popular health food due to its high fiber, vitamin C, and folate content. Okra is also known for being high in antioxidants. Okra is also a good source of calcium and potassium (Duvauchelle 2011). Processed okra is an important agricultural product. During processing, many of important quality compounds in okra maybe lost. The vacuum frying treatment reduced the physical and chemical quality of okra, but increased beta-carotene content. The moisture heating and vacuum fry processing affected the quality of okra chips. The processing appeared to affect the chemical quality of organically grown okra less than conventionally grown okra, especially the vitamin C and beta-carotene contents. The rate of chemical decline was lower with the blanching process, especially the vitamin C content, whilst vacuum frying resulted in the highest levels of beta-carotene. The growing area, environmental conditions and climate where the different okras grew may partially affect to those of physical and chemical qualities (Arlai 2009). Gembili (Dioscorea aculeata) Gembili (Dioscorea aculeata L.) is one of the types of tubers that have not been cultivated and not many people know. The nutritional value is not known yet. This plant is widely grown in the rural areas which are usually used as a substitute food for rice, snack, even just left alone to grow. In this time, the processing gembili as food only to the process of boiling or steaming, so the need for the utilization gembili processed into new products that have high sales value through the manufacture of chips such as food diversity efforts. From the experiments conducted with gembili weight 300 g, frying temperature 75◦C and changing variables such as frying time of 20, 25, 30, 35 and 40 minutes in the manufacture of vacuum frying chips of gembili showed that the longer the frying pan, the water content is more and more vaporized. Water content contained in the chips greatly affect the quality of the chips which the less water content of chips have a longer shelf life and more crisp (Wibowo 2012). Beans (Phaseolus vulgaris) Bean (of the Dutch, boontjes, Phaseolus vulgaris L.) is a kind of beans that can be eaten. Bean is high in starch, protein and dietary fiber and is an excellent source of iron, potassium, selenium, molybdenum, thiamine, vitamin B6, and folate. The fruits, seeds, and beans are rich in protein and vitamin that helps lower blood pressure and escort blood sugar metabolism and very suitable food by those who suffer from diabetes or hypertension. The optimum temperature for the manufacture of chips beans by using vacuum fryer is 90oC for 30 minutes. Chips with this variable has a low bitter taste, greenish brown, very crisp, and has a very strong smell of beans and water content are 8.62% (Septiyani 2012).

Manggo (Mangifera indica) Mango (Mangifera indica L.) is a horticultural commodity in Indonesia. Mango fruit is rich in vitamin C and carbohydrate. Much-loved mango consumers because it can be consumed fresh or in processed form. Mango is a seasonal fruit which the product will be abundant in the harvest season and rare outside of the harvest season. Mango is a perishable commodity (have a relatively short shelf life), hence the need for an alternative treatment that mango production in large quantities can be consumed to the all year round. Vacuum frying mango chips with frying temperature 80◦C, for 45 minutes is the best yield that produces low water content and good organoleptic chips (Sulistyaningrum 2012). Chickpea (Cicer arietinum) Chickpeas (Cicer arietinum L.) are a source of zinc, folate and protein. Chickpeas are low in fat and most of this is polyunsaturated. Chickpeas also provide dietary phosphorus (168 mg/100 g), which is higher than the amount found in a 100 grams serving of whole milk (NDLUSDA 2008). They can assist in lowering cholesterol in the bloodstream (Pittaway et al. 2008). In processing technology of young chickpea, it is soaked in CaCl2 solution (1000 ppm, t=30’). For wet flavoring method, young chickpea was boiled with flavor, meanwhile for dry flavoring method, young chickpea was steam blanched. Then, young chickpea vacuum fried at 65, 75, and 85°C with vacuum pressure 72 cm Hg, and packaged in alumunium foil. Yield of young chickpea chips were 13.58-14.17% with vacuum frying time range from 1.08-1.41 hours. For both flavoring methods on young chickpea chips, moisture was 6.33-7.39%; ash 4.456.10%; fat 33.95-42.93%; protein 10.86-12.24%; crude fiber 11.94-14.10%; free fatty acid (FFA) 0.62-0.70%; vitamin C 0.27-0.46 mg/100g; and vitamin A 135.54265.39 ppm. Sensory evaluation showed that different treatment of flavor and temperature did not have significant effect (P>0.05) to all parameter (color, odor, texture, taste, crispiness and acceptability of chickpea chips). But, flavor had significant effect (P<0.05) to chickpea chips taste, and temperature had significant effect (P<0.05) to crispiness of chickpea chips (Widaningrum et al. 2008) Apple (Malus domestica) Apple (Malus domestica Borkh.) is one of the most widely cultivated tree fruits, and the most widely known of the many members of genus Malus that are consumed by humans. Apple peels are a source of various phytochemicals with unknown nutritional value (Boyer and Liu 2004) and possible antioxidant activity in vitro (Lee et al. 2004). The predominant phenolic phytochemicals in apples are quercetin, epicatechin, and procyanidin B2 (Lee et al. 2003. Shidqiana (2012) had been conducted a study to look for water content and organoleptic on apple chips processed by vacuum fryer. The study was conducted in 5 variables change, the longer of frying time were 35, 40, 45, 50 and 55 minutes, while the fixed variable was 750 g heavy material and frying temperature was 80°C. The

SETYAWAN et al. – Chips characteristic by vacuum frying process

results indicates that water content of the chips had declined 9.45%, 7.46%, 6.44%, 5.47%, and 4.97 respectively, and of the organoleptic test the most preferred apple chips were processed with a temperature of 80°C and time of 50 minutes; at this variable the color was well, the flavor was delicious and the crispness was crisp. Shyu and Hwang (2001) studied the eﬀect of processing conditions on the quality of vacuum fried apple chips. They used a single vacuum pressure condition, 3.115 kPa, and three levels of temperature, 90, 100, and 110 ºC to fry the chips. After frying, the chips were centrifuged for 30 minutes at 350 rpm to remove the surface frying oil and then packed in polyethylene bags and stored at 30ºC. Using texture (hardness) as an indicator of product quality, the optimum conditions were vacuum frying temperature of 100-110ºC, vacuum time of 20-25 minutes, and a concentration of immersing fructose solution of 30-40%.

process, soaking the product in CaCl2 is also needed in order to maintain the texture of the product during heat process (Virgiawan 2011; Nur-Aeny 2012). Meyer (1987) states that CaCl2 including material hardening or firming agent for fruits and vegetables. CaCl2 significantly affect the fracture but does not affect the color, flavor and yield. Immersion on CaCl2 significantly affects the water content and crispy chips and all organoleptic parameters (taste, color, appearance, crispness) (Nur-Aeny 2012). The soaking of the prepared eggplant in CaCl2 has significant effects on the breaking strength but has no significant effects on water content and yield. The CaCl2 soaking factor combined with freezing time have significant effects on all organoleptic parameters. Best treatment was chosen using effective index method and marks the soaking of product in 1.5% CaCl2 and the freezing time of 12 hours as the best treatment (Virgiawan 2011).

Sapodilla or sawo (Manilkara zapota) Sapodilla (Manilkara zapota (L.) P.Royen) is a meso American tropical fruit crop which is widespread in Indonesia, and can harvest throughout the year. Sapodilla fruit is generally consumed as a table fruit, rarely further processing. The fruit has an exceptionally sweet, malty flavor. The unripe fruit is hard to the touch and contains high amounts of saponin, which has astringent properties similar to tannin, drying out the mouth. After ripening, sapodilla cannot survive long, easily damaged and rot. For vacuum fried chips, acquired conditions which make good chips, starting with slicing fruit with a stainless steel blade in a uniform thickness, immersing the slices in a brown solution of sodium bisulfite (1000 ppm) to prevent enzymatic browning reactions. Frying temperatures cause decreased significantly (p <0.05) on water content, hardness and brightness (L value). Water content ranged from 3.45 to 5.15% (dry basis). Hardness ranged between 2.73-4.50 kg/7mm. While L values ranged between 42.58 and 50.92. Factors of frying temperature, frying time and the interaction between these two factors did not affect significantly (p> 0.05). On the other observations that yield, fat content, and color parameters (a and b values), yield provided the range between 24.05-26.01%. 27.35; fat content ranged from 31.05% (dry basis); value of a (redness) was 3.79-8.46 while the value of b (yellowness) was 14.78-20.00 (Paramita 1999).

Jackfruit (Artocarpus heterophyllus) Jackfruit (Artocarpus heterophyllus Lam.) is commonly used in Southeast Asian cuisines. It can be eaten raw when ripe, but as the raw unripe fruit is considered inedible, it is best cooked. The ripe jackfruit is naturally sweet with subtle flavoring, and contains a lot of energy (95 calories/100 g) and the antioxidant vitamin C (13.7 mg/100g) (NDL 1998). For vacuum frying of jackfruit, the frying condition is vacuum pressure of-70 cm Hg and temperature level of 75◦C and 80◦C. Such condition was done to minimize the heat used and therefore reduce changes in composition, color, taste and flavor of the jackfruit. The fried product was 22% and the product has low water content of 3.58% (wet basis) with the taste, flavor, color and volume similar to the fresh jackfruit. Financial analysis of the jackfruit production capacity of 30 kg per day showed that NPV (Net Present Value) was IDR 52.391.000 which was bigger than investment cost, IRR (Internal Rate Return) was 51% and PBP (Pay Back Period) was 1.95 years, thus jackfruit fried chips was viable to be established (Alamsyah et al. 2002).

Eggplant (Solanum melongena) Eggplant (Solanum melongena L.) is one of the favorite fruit among the people that it tastes good. Nutritionally, eggplant is low in fat, protein, and carbohydrates. It also contains relatively low amounts of most important vitamins and minerals. Eggplant juice can significantly reduce weight, cholesterol levels, and aortic cholesterol (Jorge et al. 1998). In general, eggplant is consumed in the fresh form or cooked vegetables. One great way to reduce the water content is to process them into fruit crisps. To improve the crispness of the product, the freezing process is conducted. Freezing process increase the level of crispness and reduce the shrinkage. Besides the freezing

Cassava (Manihot esculenta) Cassava (Manihot esculenta Crantz) root is essentially a carbohydrate source. Its composition shows 60-65% moisture, 20-31% carbohydrate, 1-2% crude protein and a comparatively low content of vitamins and minerals. However, the roots are rich in calcium and vitamin C and contain a nutritionally significant quantity of thiamine, riboflavin and nicotinic acid. Cassava starch contains 70% amylopectin and 20% amylose. Cooked cassava starch has a digestibility of over 75% (Tewe 2004). After harvest, cassava conditions quickly change, that needs processing to longer shelf life, such as for making vacuum fried chips. Cassava varieties very significant effect on the water content, HCN levels, fat content, yield, broken power and color of chips. Blanching and freezing treatments significantly affect on water contents, levels of HCN, starch content, fat content, the fracture, taste, color, appearance and crispness of cassava sticks. The best treatment was obtained from treatment of butter, with freezing and blanching with HCN levels of 3.54 ppm, and

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the chips have starch content of 32.43%, the fracture 2284.19 N/m, flavor score 4.2 (good), appearance score 3.22 (rather dense) and crispness score 4.04 (crispy) (Aprillia 2007). Another product of cassava is tapioca chips from cassava starch. In atmospheric frying, NaCl pre-treatment had greatly reduced the oil absorption of tapioca crisps but did not provide improvement on color and texture. Under vacuum frying, the oil absorption for control and pretreated sample shows a significant different (p<0.05) at all frying time range. For color values, L*, a* and b* was not affected by the NaCl pre-treatment, but was affected by the frying time. While for texture, NaCl pre-treatment increases hardness and breaking force as the frying time increases. The most suitable time of vacuum frying for tapioca crisps is at 2 minutes as it gives the best quality of crisps in terms of oil absorption, color and texture. There was a significant different in all physical and sensory properties between atmospheric and vacuum fried tapioca crisps. The vacuum fried tapioca crisps had absorbed 53.36% less oil compared to atmospheric fried crisps. Vacuum fried tapioca crisps also had lighter color and better texture compared to atmospheric frying. However for sensory evaluation, consumer prefers the atmospheric fried crisps rather than vacuum fried crisps (Binti Zahroni 2012).

CONCLUSION Vacuum fried chips are potential to increase the added value of fruits and vegetables, both nutritionally and economically. Fruits and vegetables are processed with vacuum frying have better nutritional value than traditional deep-fat frying, as well as the texture, color, and other sensory character is also better. This process prevent or reduce the formation of harmful substances in the traditional deep-fat frying, such as acrylamide and excessive saturated oil, thus meet the demands of modern public health. This process also adds value to fruits and vegetables are not eligible to be sold because of defects and prevent the waste of fruits and vegetables during the harvest because of the large supply.

ACKNOWLEDGEMENTS This paper was generously supported by the Sebelas Maret University through IbM BOPTN for financial year 2012 and IbM Dikti for financial year 2013. Without the kindness, this paper would not have been possible. The authors thanks to the administrative village head (lurah) and residents of Kampung Kejiwan in Wonosobo, Central Java, Indonesia for supporting the application of vacuum frying program.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 5, No. 2, pp. 104-107 November 2013

Short Communication: Global warming – Problem with environmental and economical impacts SHIVANI M. RAI Kesharbai Lahoti College of Commerce, Amravati-444 602, Maharashtra, India. Tel. +91-9737057775, ♥email: pmkrai@hotmail.com, shelldreams@rocketmail.com Manuscript received: 16 April 2013. Revision accepted: 10 May 2013.

Abstract. Rai SM. 2013. Short Communication: Global warming – Problem with environmental and economical impacts. Nusantara Bioscience 5: 102-105. The present article is focused on global warming, which is an important global problem being faced by the humankind. The article discusses about the causes of the global warming, such as green house gases. The earth receives energy from the Sun in the form of solar radiations with small amount of infra red and ultraviolet rays. A part of these radiations is absorbed by green house gases which results into warming of the earth. These radiations increase temperature on the universe and are one of the most important global problems. The efforts from all the countries of the world are required for reduction of emissions of green house gases. Key words: economy, environment, global warming, green house gases

Abstrak. Rai SM. 2013. Komunikasi singkat: Pemanasan global – Permasalahan dengan dampak lingkungan dan ekonomi. Nusantara Bioscience 5: 102-105. Artikel ini difokuskan pada pemanasan global, yang merupakan masalah global penting yang dihadapi oleh umat manusia. Artikel ini membahas tentang penyebab pemanasan global, seperti gas rumah kaca. Bumi menerima energi dari matahari dalam bentuk radiasi surya dengan sejumlah kecil sinar infra merah dan ultraviolet. Sebagian dari radiasi ini diserap oleh gas-gas rumah kaca yang mengakibatkan pemanasan bumi. Radiasi-radiasi ini meningkatkan suhu alam semesta dan merupakan salah satu masalah global yang paling penting. Upaya dari semua negara di dunia diperlukan untuk mengurangi emisi gas rumah kaca. Kata kunci: ekonomi, lingkungan hidup, pemanasan global, gas rumah kaca

The temperature of the earth is rising with fast pace. Since 1975, the global surface temperature has been increased by 0.5oC (Hansen et al. 1999; Jones et al. 1999; Mann et al. 1999; Jansen et al. 2000), The main reasons for rise in temperature include green house gases, deforestation, etc. According to the U.S. Environmental Protection Agency (2009): “Global warming is an average increase in the temperature of the atmosphere near the Earth’s surface and in the troposphere, which can contribute to changes in global climate patterns”. Global warming can occur from a variety of causes, both natural and human induced. In common usage, “global warming” often refers to the warming that can occur as a result of increased emissions of greenhouse gases from human activities.” In fact, due to global warming sea-level is rising and has become a great puzzle (Woodsworth 1990; Douglas and Peltier 2002; Woodworth and Player 2003; Holgate and Woodworth 2004). The basic cause of global warming is increase in temperature due to the greenhouse gases. A certain amount of these greenhouse gases maintain the earth's climate congenial to live but it has been observed that over the years these gases have increased and do not allow the solar heat to escape into space keeping the earth's temperature warmer than needed and allowing the polar caps to melt a little more each year causing a rise in the oceans.

Agriculture is the main occupation across the globe with 1.2-1.5 billion hectare as a crop land and 3.5 billion as a grass land (Howden et al. 2007; Thangarajan et al. 2013). Agriculture contributes up to 10-12% of the total emissions of green house gas emissions (IPCC 2007).The higher input of the modern chemical fertilizers have created problems like degradation of quality of soil, loss of biodiversity and contamination of ground water. Excessive irrigation is also the cause of climate change and global warming (Puma and Cook 2010). According to a survey, in the past three decades global warming is 0.6°C and 0.8°C in the past century. It is not appropriate to claim that ‘‘most global warming took place before 1940”. Up to 1975, there was slow global warming, with large fluctuations, over the century up to 1975, followed by rapid warming at a rate 0.2°C per decade. Global warming was 0.7°C between the late 19th century (the earliest time at which global mean temperature can be accurately defined) and 2000, and continued warming in the first half decade of the 21st century is consistent with the recent rate of 0.2°C per decade (Hansen et al. 2006). Around the areas of ocean, quite away from the anthropogenic activities warming occurs. The gradual rise in earth temperature is a matter of great concern. General public, politicians and environmentalists are interested to solve this issue at global

RAI â&#x20AC;&#x201C; Environmental and economical impacts of global warming

level. There are various reports of efforts made to solve this problem all over the world. There have been public awareness and a great concern towards the problem of global warming and it is realized that it should be tackled meticulously in order to save the mankind. This article is aimed to discuss the global warming problem, its causes and pragmatic approach to solve this problem. Causes of global warming There are many greenhouse gases (GHGs) responsible for warming. Due to the anthropogenic activities the gases are emitted in different ways. Most of these gases are produced by modern agricultural practices, from the combustion of fossil fuels in industries, cars, and by generation of electricity. The most important among these gases is carbon dioxide. The carbon dioxide (about 20 %) produced due to the anthropogenic activities remains in the environment for thousands of years. Other gas which contributes a major part includes methane released from land and agriculture, nitrous oxide from fertilizers, gases used in refrigerators and freezers. There has been much deforestation which is really a cause of worry because these forests are responsible for binding carbon-dioxide. There are ten primary green house gases including water vapor, carbon dioxide, methane, and nitrous oxide which are occurs naturally. Perfluorocarbon, hydrofluorocarbons, and sulfur hexafluoride are found in the atmosphere due to emissions from different kind of industries. Among these, water vapor is the most abundant kind of green house gases present in the atmosphere. Carbon-dioxide is the primary anthropogenic greenhouse gas, accounting for 77% of the human contribution to the greenhouse effect. It is estimated that from 10,000 years ago until 150 years ago, atmospheric concentrations of carbon-dioxide, methane and nitrogen-dioxide were relatively stable. Unfortunately, during the last 150 years, concentrations of methane and nitrogen dioxide increased 148% and 18%, respectively. There are various sources of Greenhouse Gas Emissions. Due to human activities (anthropogenic source) carbon-dioxide is emitted from burning fossil fuels, cement industries and due to rapid deforestation. Methane and nitrogen dioxides emissions are both man-made and natural. Agriculture accounts for major contribution of methane and nitrous dioxide gases. Many hydrofluorocarbons used in refrigeration, cooling, and as solvents in place of ozone depleting chlorofluorocarbons. There are different heat-trapping abilities of the green house gases. It is worthy of note that a molecule of methane gas produces more than 20 times the warming of a molecule of carbon dioxide. Another example is nitrous oxide, which is 300 times more powerful than carbon dioxide. There are other gases also which include chlorofluorocarbons. These have been banned in most of the countries of the world because they are responsible for degradation of the ozone layer. This ozone layer has heattrapping capacity -thousand of times greater than carbon dioxide. There are various reports, which provide evidence that carbon dioxide is a major contributor of global warming.

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There was no concrete decision in Copenhagen in December 2009 to reach to final conclusion to extend and broaden the Kyoto Protocol raises the prospect that attempts to limit atmospheric concentrations of carbon dioxide (CO2) and other greenhouse gases (GHGs), as a consequence of which global temperature increases. It is really difficult politically. Nordhaus (2010) reported improved estimates of the likely trajectories of global output, GHG emissions, climate change, and damages in the coming decades. Deforestation is a major problem Forests play a major role in balancing the carbondioxide in the atmosphere in several ways. The plants of the forests remove carbondioxide from the atmosphere and absorb carbon into different parts of the plants, such as wood, leaves, where it can be stored for a large period. However, due to deforestation, stored carbon may be released into the atmosphere, depending in part on how much of the wood is destroyed. For example, forest fires destroy many plants. In addition human also fell trees for timber and other uses. Deforestation is sometimes manmade because for construction purpose cleaning of forests is required. A huge amount of carbon stored in forests worldwide indicates the significant role of forests in climate change and global warming. According to an estimate, the forest trees are estimated to store the equivalent of roughly 760 billion metric tons of carbondioxide worldwide over one hundred times the United Statesâ&#x20AC;&#x2122; emissions of Carbon-dioxide and other greenhouse gases in 2009. Ecological effects of global warming Droughts and floods Between the ninth and fourteenth centuries (Medieval Warm Period) the global temperatures rose up to 2oC (Acemoglu et al. 2012). Fagan (2008) stated that this brought bounty to some areas, but others suffered from droughts. There will be drastic changes of tropical rainfall on a regional basis (Allen and Ingram 2002) Due to the rise in temperature water evaporates rapidly. This evaporated water will quickly condense to form clouds and fall on the earth as rains.. Unfortunately, this rainfall is not evenly distributed. The rapid evaporation of water may generate several problems particularly in developing countries where availability of water is a great problem. Plant life depends on water from rivers and lakes if water evaporates with faster rate; the life is threatened due to drought. The drought will affect indirectly the crops and if there will not be proper crop yield, there will be food problem. On the other hand in wet area the evaporation would be much higher and this would cause untimely rainfall and flood. Drought is a main natural cause of agricultural, economic, and environmental damage (Burton et al. 1978; Wilhite and Glantz 1985; Wilhite 1993). Rising sea level Constant increase in temperature would be responsible for melting ice in North and South poles (Steffensen et al. 2008). There are various reports concerning sea level rise (Douglas 1991, 1992; Maul and Martin, 1993; Church et al.

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5 (2): 104-107, November 2013

2001; Chambers et al. 2002; Douglas and Peltier 2002; Brohan et al 2006). The report of melting ice in Antarctica is a burning example. The surface melting was recorded in ice sheet (Velicogna 2010; Buis and Cole 2012; Vinas 2012) The glaciers melts and causes land-slide as a consequence of which the sea level is rising. It is estimated that in future 1-4 m water level will rise. If the ice sheets of Greenland and Antarctica fully melts, the sea level will rise by 64 m. During the period of 1993-2003 there has been loss of 50-100 tons of ice. The low-lying areas in general and coastal areas in particular will be flooded and may be submerged. It is really a matter of great concern that 29% of the worldâ&#x20AC;&#x2122;s population which lives in coastal areas will be affected. According to Church and White (2006) between 1870 and 2004, global average sea levels rose 195 mm. The results of the sea-level rise would affect badly particularly coastal flooding and storm damage, eroding shorelines, salt water contamination of fresh water supplies, flooding of coastal wetlands and barrier islands, and an increase in the salinity of estuaries are all realities of even a small amount of sea level rise (Lambeck and Chappell 2001). There are about 30 countries which would be affected by rising sea level. There is an alarming repot by Bennartz et al. (2013) in July 2012, an historically rare melting was recorded across the entire Greenland ice sheet, raising questions about the frequency and spatial extent of such events. Extreme weather Climate is the average of many weather events over of a span of years (Huber et al. 2011). In fact, climate change can be described in terms of average changes in temperature or precipitation (Karl et al. 2008). After 1880, globally 2005 and 2010 were the warmest year (NCDCNOAA 2010), both years are known for exceptionally damaging weather events, for example, Hurricane Katrina in 2005 and the deadly Russian heat wave in 2010. The year 2005 have been considered as the warmest year globally, 19 countries set new national high-temperature records. In 2010, global precipitation was also far above normal and it was the wettest year since 1900 (Huber et al. 2011). It was Rio de Janeiro which received the heaviest rainfall in 30 years causing nearly 300 mudslides and killing at least 900 people (Cabral 2010). There would be a remarkable change in weather owing to temperature rise. The high temperature can increase winds, rains and storms and finally there would change in overall climate of Earth. The climate of the future will be entirely different from the one we are having now. We are already experiencing the change of weather all over the world and it is a matter of discussion among scientists, politicians and common people. Economic impact As far as the global warming is concerned, itâ&#x20AC;&#x2122;s essential economic elements can be explained in a simple economic model which include four elements: (i) the consumption of the present generation, (ii) the consumption of future generations, (iii) the conventional capital stock resulting

from the investment of the current generation, and (iv) the climatological capital stock representing the reduction in the stock of greenhouse gases in the atmosphere due to investments of the current generation in the mitigation of global warming (Foley 2007). There are severe economic impacts of global warming and climate change. The loss of crops, forests, and animals are most important. Due to the sea-level rise there will be huge migration of the inhabitants of as low-lying countries which would be affected with flood. Moreover, there will be disruptions to global trade, transport, energy supplies and labor markets, banking and finance, investment and insurance, would all create havoc on the stability of both developed and developing nations. Consequently, there will be adverse effect on markets by increased volatility. Due to rise in temperature and change in climatic conditions, several diseases, such as, Malaria, dengue and viral diseases will spread, which will be responsible for huge economic loss in treatment of the diseases. There will be excessive economic loss due to hurricane, floods and diseases. The problem will not only be faced by developing countries, it will be the problem of the developed world also. The main problem in coastal areas particularly would be of potable water, energy and transportation. These all problems would indirectly affect the economy of the people. Public perception It is really very important for public to understand that global warming is manmade According to a recent survey in the U.S. by Rabe and Borick (2012) provides evidence that public opinion for the global warming depends mainly on their perceptions of local climate variations. In 1988, Hansen and his coworkers suggested that by the early 21st century the informed public should be able to recognize that the frequency of unusually warm seasons had increased. In 2011, heat waves in Texas and Oklahoma in the summer raise the question of whether these extreme events are related to the on-going global warming trend, which has been attributed with a high degree of confidence to human-made greenhouse gases. The change of global temperature may have the greatest practical impact via effects on the water cycle. Indeed climate changes occurring with global warming involve intimate interactions of the energy and water cycles. The extreme rise in temperature causing heat waves and frequent floods has received public attention. However, the common public has no perception of why the global warming is taking place. It is the need of the hour to generate awareness about the environmental problems in general and rise in temperature. Solutions Investing in clean energy industries, such as wind and solar, as well as energy efficiency programs, can lead us out of crisis and into a new clean energy economy. We should focus on vehicles which can be run on solar light. It puts about 10,000 miles a year on the car, running it purely on sunlight. The solar panels that provide all the electricity homes also charge the car battery. By using solar energy-

RAI – Environmental and economical impacts of global warming

based cars, the use of oil can be reduced. Government should focus on use of solar energy and it should be imperative that new buildings should meet energyefficiency standards that maximize energy savings. It should be mandatory for new buildings existing and commercial spaces to save energy by installing energy efficient heating, cooling and lighting systems. The garbage should be recycled in order to avoid methane gas production. Garbage should not be burnt because it releases carbon dioxide and hydrocarbons into the atmosphere. There should be plantation program at large scale. There is a greater need to develop non-fossil fuel energy sources. Solar, wind and hydroelectric power, which are the free gift of almighty can reduce greenhouse gases. It can be concluded that the greenhouse effect is one of the most important global problems. The efforts from all the countries of the world are required for reduction of emissions of green house gases. However, it has been experienced from the past that there are meetings, conventions, and discussions by the scientists and politicians regarding the global warming and climate change but the efforts are not focused. The most important is that if reductions are not controlled, we should try to go for mass plantation programs by public participations. References Acemoglu D, Aghion P, Bursztyn L, Hemous D. 2012. The environment and directed technical change. Amer Econ Rev102: 131-166. Allen MR, Ingram WJ. 2002. Constraints on future changes in climate and the hydrologic cycle. Nature 419: 224-232 Bennartz R, Shupe MD, Turner DD, Walden VP, Steffen K, Cox CJ, Kulie MS, Miller NB, Pettersen C. 2013. July 2012 Greenland melt extent enhanced by low-level liquid clouds. Nature 496: 83-86. Brohan P, Kennedy JJ, Harris I, Tett SFB, Jones PD. 2006. Uncertainty estimates in regional and global observed temperature changes: A new data set from 1850. J Geophys Res Atmos 111: D12106, 10.1029/2005JD006548. Buis A, Cole S 2012. Satellites see unprecedented Greenland Ice Sheet melt. Jet Propulsion Laboratory, Pasadena, Calif. and NASA Headquarters, Washington, DC. www.jpl.nasa.gov/news/news.cfm? release=2012-217&cid=release_2012-217 Burton I, Kates RW, White GF. 1978. The Environment as Hazard. Oxford University Press, London. Cabral P. 2010. Flooding in Rio de Janeiro state kills scores. BBC News, Rio deJaineiro, 7 April 2010. Chambers DP, Melhaff CA, Urban TJ, Fuji D, Nerem RS. 2002. Lowfrequency variations in global mean sea level: 1950-2000. J Geophys Res 107 (C4), 3026, doi: 10.1029/2001JC001089. Church JA, White NJ. 2006. A 20th century acceleration in global sealevel rise. Geophys Res Lett 33, doi: 10.1029/2005GL024826. Church JA, Gregory JM, Huybrechts P, Kuhn M, Lambeck K, Nhuan MT, Qin D, Woodworth PL. 2001. Changes in sea level. In: Houghton JT, Ding Y, Griggsis DJ, Noguer M, van der Linden PJ, Dai X, Maskell K, Johnson CA (eds). Climate Change 2001: The Scientific Basis. Cambridge Univ. Press, Cambridge, UK. Douglas BC, Peltier WR. 2002. The puzzle of global sea-level rise. Phys Today 55: 35-40. Douglas BC. 1991. Global sea level rise. J Geophys Res 96: 6981-6992. Douglas BC. 1992. Global sea level acceleration. J Geophys Res 97: 699706. EPA [US Environmental Protection Agency]. 2009. Frequently Asked Questions about Global Warming and Climate Change: Back to Basics. Office of Office of Air and Radiation US EPA, Washington, DC. Fagan B. 2008. The Great Warming: Climate Change and the Rise and Fall of Civilizations. Bloomsbury Press, New York. Foley DK. 2007. The economic fundamentals of global warming. SFI Working Paper: 2007-12-044

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Hansen J, Ruedy, R, Glascoe J, Sato M. 1999. GISS analysis of surface temperature change. J Geophys Res 104: 30997-31022. Hansen J, Sato M, Ruedy R, Lacis A, Oinas V. 2000. Global warming in the twenty-first century: An alternative scenario. Proc Natl Acad Sci USA 97 (18): 9875-9880 Holgate SJ, Woodworth PL. 2004. Evidence for enhanced coastal sea level rise during the 1990s. Geophys Res Lett 31: L07305, doi: 10.1029/2004GL019626. Howden SM, Soussana JF, Tubiello FN, Chhetri N, Dunlop M, Meinke H. 2007. Adapting agriculture to climate change. Proc Natl Acad Sci USA 104: 19691–19696. Huber DG, Gulledge J. 2011. Extreme weather and climate change: Understanding the link and managing the risk. Science and Impacts Program. Center for Climate and Energy Solutions, Arlington, VA. IPCC [Intergovernmental Panel on Climate Change]. 2007. The physical science basis: contribution of working group I to the IV assessment report of the intergovernmental panel on climate change. Cambridge University Press, Cambridge, UK. Jones PD, New M, Parker DE, Martin S, Rigor IG. 1999. Surface air temperature and its changes over the past 150 years. Rev Geophys 37: 173-199. Karl TR, Meehl GA, Miller CD, Hassol SJ, Waple AM, Murray WL. 2008. Weather and Climate Extremes in a Changing Climate; Regions of Focus: North America, Hawaii, Caribbean, and U.S. Pacific Islands. A Report by the U.S. Climate Change Science Program and the Subcommittee on Global Change Research. NOAA’s National Climatic Data Center, Washington, D.C., USA. Lambeck K, Chappell J. 2001. Sea level change through the last glacial cycle. Science 292: 679-686. Mann ME, Bradley RS, Hughes MK. 1999. Northern hemisphere temperatures during the past millennium: Inferences, uncertainties and limitations. Geophys Res Lett 26: 759-762. Masters J. 2010. Asia records its hottest temperature in history; Category 4 Phet threatens Oman. Weather Underground. Jeff Masters' WunderBlog. Retrieved 21 July 2010. http://bit.ly/bDAvx2 Maul GA, Martin DM. 1993. Sea level rise at Key West, Florida, 18461992: America’s longest instrument record. Geophys Res Lett 20: 1955-1958. NCDC-NOAA [National Climatic Data Center-National Oceanic and Atmospheric Administration]. 2010. State of the Climate Global Analysis Annual 2010. NOAA’s National Climatic Data Center, Washington, D.C., USA. Nordhaus WD. 2010. Economic aspects of global warming in a postCopenhagen environment. Proc Natl Acad Sci USA 107 (26): 1172111726 Puma MJ, Cook BI. 2010. Effects of irrigation on global climate during the 20th century. J Geophys Res 115: D16120. Rabe BG, Borick CP. 2012. Fall 2011 national survey of American public opinion on climate change. Issues in Governance Studies. Brookings Institution, Washington, DC. Steffensen JP, Andersen KK, Bigler M, Clausen HB, Dahl-Jensen D, et al. 2008. High-resolution Greenland ice core data show abrupt climate change happens in few years. Science 321: 680-684. Thangarajan R, Bolan NS, Tian G, Ravi Naidu, Kunhikrishnan A. 2013. Role of organic amendment application on greenhouse gas emission from soil. Sci Total Environ. dx.doi.org/10.1016/ j.scitotenv.2013.01.031 Velicogna I. 2009. Increasing rates of ice mass loss from the Greenland and Antarctic ice sheets revealed by GRACE. Geophys Res Lett 36: L19503. Vinas MJ. 2012. Satellites see unprecedented Greenland Ice Sheet surface melt (07.24.12). NASA's Earth Science News Team, Goddard Space Flight Center, Greenbelt, Md. www.nasa.gov/topics/earth/features/ greenland-melt.html Wilhite DA, Glantz MH. 1985. Understanding the drought phenomenon: The role of dentitions. Water Intl 10: 111-120. Wilhite DA. 1993. Drought Assessment, Management, and Planning: Theory and Case Studies. Natural Resource Management and Policy Series, Vol. 2. Kluwer, Amsterdam. Woodworth PL, Player R. 2003. The Permanent Service for Mean Sea Level: An update to the 21st century. J Coastal Res 19: 287-295. Woodworth PL. 1990. A search for accelerations in records of European mean sea level. Intl J Climatol 10: 129-143.

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Authors Index Abdelazim H Abou-Leila BH Ahmed AM Al-Hammady MAM Ameh JB Ammar MSA Ashour F Atawodi SE Azadfar D Borah RK El-Mergawi RA Hendawy SF Hussein MS Ibrahim MM Khalid KA Khattab HI Khazaeian A Kumar R Malik N Metwally SA

35 65 57 75 51 35, 75 35 51 30 1 22 22 22 70 15, 65, 70 57 30 1 8 65

Negedu A Obuid-Allah AH Pandey S Pyasi A Rai MK Rai SM Setyawan AD Singh RK Singh S Solichatun Soni KK Sugiyarto Susilowati A Talaat IM Tapwal A Umoh VJ Verma RK Youssef AA Zoghi Z

51 75 1 44 51 104 86 8 8 86 44 86 86 57 1 51 44 22 30

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Subject Index Acropora humilis amino acids Ammi visnaga Anise autoclaving B. cepacia bio fertilization biometry properties bleaching Calendula officinalis carbohydrate carpophores castor seeds chips citronella coral disease coriander distribution economy ectomycorrhiza environment

essential oil

ethnomycology Fagus orientalis fiber fixed oil flower yield food

free fatty acids frying global warming green house gases growth

growth criteria

35, 37, 39, 41, 75, 76, 77, 78, 80, 81, 82, 83, 84 57, 58, 59, 60, 62, 63, 64 57, 58, 59, 60, 62, 63, 64 15, 16, 17, 18, 19, 20 51, 52, 53, 54, 55, 56 8, 9, 10, 11, 12, 13 22, 23 30, 31 36, 38, 39, 41, 75, 76, 82, 83, 84 25, 65, 66, 68, 69, 1, 3, 6, 7, 15, 17, 19, 20, 25, 45, 53, 54, 55, 92, 96, 97, 99 1 51, 52, 53, 54, 55 86, 87, 88, 89, 90, 91, 93, 95, 96, 97, 98, 99 70, 71, 72, 73 35, 36, 37, 38, 40, 41, 42 15, 16, 17, 18, 19, 20 6, 35, 42, 88 104, 106 44, 46, 47, 48 7, 15, 30, 31, 35, 36, 40, 41, 42, 65, 73, 75, 76, 83, 84, 94, 97, 104, 105, 106 15, 16, 17, 18, 19, 20, 27, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 1, 2 22, 30, 31, 32, 2, 3, 30, 31, 32, 33, 34, 54, 97, 98 15, 16, 18, 19, 20 65, 66, 67, 68, 69 1, 2, 6, 7, 16, 65, 83, 86, 87, 88, 89, 90, 91, 92, 93, 95, 96, 98, 99, 104 51, 54, 55, 56, 89, 95, 98, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 7, 9, 104, 105, 106, 107 104, 105, 107 1, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 36, 38, 39, 40, 41, 42, 44, 45, 46, 48, 65, 66, 67, 68, 69, 70, 71, 72, 73, 76, 83, 84, 86, 87 57

Gulf of Aqaba heritability hormones inoculum irrigation intervals mycorrhiza nitrate nutrient uptake peroxide value phenolic compounds plant growth plus trees preservation proline protein

proximate analysis proximate composition R. undicola Ras Mohammed Red Sea Red Sea corals Rhizobium leguminosarum bv. phaseoli sal forest sedimentation selection silymarin Stylophora pistillata sweet fennel Sylibium marianum synthesis temperature

vacuum frying vase life water regime Zea mays

35, 36, 38, 40 70, 71 57, 59, 62, 64 44, 46 22, 23, 24, 25, 26, 27, 28, 1, 23, 24, 25, 26, 27, 28, 44, 45, 46, 48 8, 9, 11, 12, 13, 24 12, 44 51, 54, 55 57, 58, 59, 60, 62, 63, 64 8, 12, 18, 20, 23, 25, 27, 28, 68 30, 32, 33, 34 52, 53, 65, 86, 87, 90, 92, 93, 96 2, 65, 66, 67, 68, 69 1, 2, 3, 6, 7, 11, 15, 16, 18, 19, 20, 51, 52, 53, 54, 56, 76, 82, 88, 95, 97, 98, 99 1, 2 6, 7, 51, 52 8, 9, 10, 11, 12, 13 35, 36, 37, 38, 40 35, 36, 38, 40, 41, 76 75, 84 8, 9, 11, 13

44, 46, 48, 22, 40, 41, 42, 75, 76, 80, 81, 82, 83, 84 30, 31, 32, 33, 34, 66, 70, 71, 72, 73, 93, 94 22, 24, 26, 27, 28 38, 39, 42, 75, 76, 78, 79, 81, 82, 83, 84 15, 16, 17, 18, 19, 20 22, 23, 25, 26, 27, 8, 15, 18, 20, 23, 25, 40, 43, 48, 65, 68, 76, 77, 83, 2, 6, 9, 17, 18, 41, 51, 52, 67, 75, 76, 77, 78, 79, 81, 82, 83, 84, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 103, 104, 105 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 100 65, 66, 67, 68, 69 65, 66, 67, 68, 69 8

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List of Peer Reviewer Abdala G. Diédhiou

Laboratoire Commun de Microbiologie, IRD/ISRA/UCAD, Centre de Recherche de BelAir, BP 1386 CP 18524 Dakar-Sénégal

Ahmad Dwi Setyawan

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Andrea Patriarca

Departamento de Quimica Organica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab II, Piso 3, 1429-Buenos Aires, Argentina

Ashok Kumar

Department of Botany, Dr. Bhim Rao Ambedkar Government Degree College, Maharajganj, Maharajganj-273303, Uttar Pradesh, India

Claudia L. Prins

Plant Production Laboratory, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro (CCTA/UENF), 28013-602, Campos dos Goytacazes, RJ, Brazil

Daniel Isidoro

Unidad de Suelos y Riegos (unidad asociada EEAD-CSIC), CITA, Avda. Montañana 930, 50059-Zaragoza, Spain

Federica Aureli

Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy

Goodarz Hajizadeh

Department of Forestry, Faculty of Natural Resources, Sari University of Agricultural Sciences and Natural Resources, Sari, Mazandaran, Iran

Jay P. Verma

Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India

Kateryna Kon

Department of Microbiology, Virology, and Immunology, Kharkiv National Medical University, 61022 Pr. Lenina, 4, Kharkiv, Ukraine

Mahdi Reyahi Khoram

Department of Environment, Hamadan Branch, Islamic Azad University, P.O. Box 65155-184, Hamadan, Iran

Mahendra K. Rai

Department of Biotechnology, Sant Gadge Baba (SGB) Amravati University, Amravati 444602, Maharashtra, India

Sugiyarto

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia

Wiryono

Department of Forestry, Faculty of Agriculture, University of Bengkulu. Bengkulu 38371A, Bengkulu, Indonesia.

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Table of Contents Vol. 5, No. 1, Pp. 1-49, May 2013 Macro-fungal diversity and nutrient content of some edible mushrooms of Nagaland, India RAJESH KUMAR, ASHWANI TAPWAL, SHAILESH PANDEY, RAJIB KUMAR BORAH Impact of rhizobial inoculation and nitrogen utilization in plant growth promotion of maize (Zea mays L.) RAMESH K. SINGH, NAMRATA MALIK, SURENDRA SINGH

1-7 8-14

Effect of nitrogen fertilization on morphological and biochemical traits of some Apiaceae crops under arid regions in Egypt KHALID ALI KHALID

15-21

Response of Silybum marianum plant to irrigation intervals combined with fertilization SABER F. HENDAWY, MOHAMED S. HUSSEIN, ABD-ELGHANI A.YOUSSEF, REYAD A. EL-MERGAWI

22-29

Study of altitude and selection on fiber biometry properties of Fagus orientalis Lipsky ZOHREH ZOGHI, DAVOUD AZADFAR, ALI KHAZAEIAN

30-34

Coral disease distribution at Ras Mohammed and the Gulf of Aqaba, Red Sea, Egypt MOHAMMED SHOKRY AHMED AMMAR, FEKRY ASHOUR, HODA ABDELAZIM

35-43

Effect of ectomycorrhizae on growth and establishment of sal (Shorea robusta) seedlings in central India ABHISHEK PYASI, KRISHNA KANT SONI, RAM KEERTI VERMA

44-49

Vol. 4, No. 2, Pp. 51-107, November 2013 Effects of autoclaving on the proximate composition of stored castor (Ricinus communis) seeds ANTHONY NEGEDU, JOSEPH B. AMEH, VERONICA J. UMOH, SUNDY E. ATAWODI, MAHENDRA K. RAI

51-56

Changes in growth, hormones levels and essential oil content of Ammi visnaga plants treated with some bioregulators IMAN M. TALAAT, HEMMAT I. KHATTAB, AISHA M. AHMED

57-64

Effect of water regime on the growth, flower yield, essential oil and proline contents of Calendula officinalis SAMI ALI METWALLY, KHALID ALI KHALID, BEDOUR H. ABOU-LEILA

65-69

Phenotypic recurrent selection on herb growth yield of citronella grass (Cymbopogon nardus) grown in Egypt MOHAMED M. IBRAHIM, KHALID A. KHALID

70-74

Experimental effect of temperature and sedimentation on bleaching of the two Red Sea corals Stylophora pistillata and Acropora humilis MOHAMMED S.A. AMMAR, AHMED H. OBUID-ALLAH, MONTASER A. M. AL-HAMMADY

75-85

Review: Physical, physicochemical, chemical and sensorial characteristics of the several fruits and vegetables chips by low-temperature vacuum frying machine AHMAD DWI SETYAWAN, SUGIYARTO, SOLICHATUN, ARI SUSILOWATI Short Communication: Global warming â&#x20AC;&#x201C; Problem with environmental and economical impacts SHIVANI M. RAI

86-103

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Guidance for Authors Aims and Scope Nusantara Bioscience (Nus Biosci) is an official publication of the Society for Indonesian Biodiversity (SIB). The journal encourages submission of manuscripts dealing with all aspects of biological sciences that emphasize issues germane to biological and nature conservation, including agriculture, animal science, biochemistry and pharmacology, biomedical science, ecology and environmental science, ethnobiology, genetics and evolutionary biology, hydrobiology, microbiology, molecular biology, physiology, and plant science. Manuscripts with relevance to conservation that transcend the particular ecosystem, species, genetic, or situation described will be prioritized for publication. Article The journal seeks original full-length research papers, short research papers (short communication), reviews, monograph and letters to the editor about material previously published; especially for the research conducted in the Islands of the Southeast Asian reign or Nusantara, but also from around the world. Acceptance The acceptance of a paper implies that it has been reviewed and recommended by at least two reviewers, one of whom is from the Editorial Advisory Board. Authors will generally be notified of acceptance, rejection, or need for revision within 2 to 3 months of receipt. Manuscript is rejected, if the content is not in line with the journal scope, dishonest, does not meet the required quality, written in inappropriate format, has incorrect grammar, or ignores correspondence in three months. The primary criteria for publication are scientific quality and biological or natural conservation significance. The accepted papers will be published in a chronological order. This journal is published in May and November. Copyright Submission of a manuscript implies that the submitted work has not been published before (except as part of a thesis or report, or abstract); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors. If and when the manuscript is accepted for publication, the author(s) agree to transfer copyright of the accepted manuscript to Nusantara Bioscience. Authors shall no longer be allowed to publish manuscript without permission. Authors or others are allowed to multiply article as long as not for commercial purposes. For the new invention, authors are suggested to manage its patent before published. Submission The journal only accepts online submission, through e-mail to the managing editor at unsjournals@gmail.com. The manuscript must be accompanied with a cover letter containing the article title, the first name and last name of all the authors, a paragraph describing the claimed novelty of the findings versus current knowledge, and a list of five suggested international reviewers (title, name, postal address, email address). Reviewers must not be subject to a conflict of interest involving the author(s) or manuscript(s). The editor is not obligated to use any reviewer suggested by the author(s). Preparing the Manuscript Please make sure before submitting that: The manuscript is proofread several times by the author (s); and is criticized by some colleagues. The language is revised by a professional science editor or a native English speaker. The structure of the manuscript follows the guidelines (sections, references, quality of the figures, etc). Abstract provides a clear view of the content of the paper and attracts potential citers. The number of cited references complies with the limits set by Nus Biosci (around 20 for research papers). Microsoft Word files are required for all manuscripts. The manuscript should be as short as possible, and no longer than 7000 words (except for review), with the abstract < 300 words. For research paper, the manuscript should be arranged in the following sections and appear in order: Title, Abstract, Key words (arranged from A to Z), Running title (heading), Introduction, Materials and Methods, Results and Discussion, Conclusion, Acknowledgements, and References. All manuscripts must be written in clear and grammatically correct English (U.S.). Scientific language, nomenclature and standard international units should be used. The title page should include: title of the article, full name(s), institution(s) and address(es) of author(s); the corresponding authors detailed postal and email addresses, and phone and fax numbers. References Author-year citations are required. In the text give the authors name followed by the year of publication and arrange from oldest to newest and from A to Z. In citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the first author is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or (Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and

Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation as shown with word “cit” should be avoided. Reference to unpublished data and personal communication should not appear in the list but should be cited in the text only (e.g., Rifai MA 2007, personal communication; Setyawan AD 2007, unpublished data). In the reference list, the references should be listed in an alphabetical order. Names of journals should be abbreviated. Always use the standard abbreviation of a journal’s name according to the ISSN List of Title Word Abbreviations (www.issn.org/222661-LTWA-online.php). The following examples are for guidance. Journal: Saharjo BH, Nurhayati AD. 2006. Domination and composition structure change at hemic peat natural regeneration following burning; a case study in Pelalawan, Riau Province. Biodiversitas 7: 154-158. The usage of “et al.” in long author lists will also be accepted: Smith J, Jones M Jr, Houghton L et al. 1999. Future of health insurance. N Engl J Med 965: 325–329 Article by DOI: Slifka MK, Whitton JL. 2000. Clinical implications of dysregulated cytokine production. J Mol Med. Doi:10.1007/s001090000086 Book: Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds. Elsevier, Amsterdam. Book Chapter: Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. In: Carson W, Schnitzer S (eds) Tropical forest community ecology. Wiley-Blackwell, New York. Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th annual symposium of the International Association for Vegetation Science, Swansea, UK, 23-27 July 2007. Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in Java island. Sebelas Maret University, Surakarta, 17-20 July 2000. [Indonesia] Thesis, Dissertation: Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon. [Dissertation]. Brawijaya University, Malang. [Indonesia] Online document: Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Q u a k e S R , Y o u L . 2 0 0 8 . A s yn t h e t i c E s c h e r i c h i a c o l i p r e d a t o r - p r e y e c o s y s t e m . M o l S ys t B i o l 4 : 1 8 7 . www.molecularsystemsbiology.com Tables should be numbered consecutively and accompanied by a title at the top. Illustrations Do not use figures that duplicate matter in tables. Figures can be supplied in digital format, or photographs and drawings, which can be ready for reproduction. Label each figure with figure number consecutively. Uncorrected proofs will be sent to the corresponding author by e-mail as .doc or .docx files for checking and correcting of typographical errors. To avoid delay in publication, proofs should be returned in 7 days. A charge The cost of each manuscript is IDR 250,000,- plus postal cost or IDR 150,000,- for SIB members. There is free of charge for non Indonesian author(s), but need to pay postal cost for hardcopy. Reprints Two copies of journal will be supplied to authors; reprint is only available with special request. Additional copies may be purchased by order when sending back the uncorrected proofs by e-mail. Disclaimer No responsibility is assumed by publisher and co-publishers, nor by the editors for any injury and/or damage to persons or property as a result of any actual or alleged libelous statements, infringement of intellectual property or privacy rights, or products liability, whether resulting from negligence or otherwise, or from any use or operation of any ideas, instructions, procedures, products or methods contained in the material therein.

NOTIFICATION: All communications are strongly recommended to be undertaken through email.

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Effects of autoclaving on the proximate composition of stored castor (Ricinus communis) seeds ANTHONY NEGEDU, JOSEPH B. AMEH, VERONICA J. UMOH, SUNDY E. ATAWODI, MAHENDRA K. RAI

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Changes in growth, hormones levels and essential oil content of Ammi visnaga plants treated with some bioregulators IMAN M. TALAAT, HEMMAT I. KHATTAB, AISHA M. AHMED

57-64

Effect of water regime on the growth, flower yield, essential oil and proline contents of Calendula officinalis SAMI ALI METWALLY, KHALID ALI KHALID, BEDOUR H. ABOU-LEILA

65-69

Phenotypic recurrent selection on herb growth yield of citronella grass (Cymbopogon nardus) grown in Egypt MOHAMED M. IBRAHIM, KHALID A. KHALID

70-74

75-85

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Short Communication: Global warming â&#x20AC;&#x201C; Problem with environmental and economical impacts SHIVANI M. RAI

104-107

Society for Indonesian Biodiversity

Sebelas Maret University Surakarta

Published semiannually PRINTED IN INDONESIA

ISSN 2087-3948

E-ISSN 2087-3956