Effect of Germination and Roasting on Nutritive Composition and Anti-Nutrients in Fenugreek Seeds

IJSTE - International Journal of Science Technology & Engineering | Volume 3 | Issue 06 | December 2016 ISSN (online): 2349-784X

Effect of Germination and Roasting on Nutritive Composition and Anti-Nutrients in Fenugreek Seeds Manju GJU Hisar Haryana

Dr. B. S. Khatkhar GJU Hisar Haryana Mrs. Parveen GJU Hisar Haryana

Abstract Fenugreek seeds were processed by germination and roasting. Raw and processed fenugreek seeds were analyzed for total polyphenolic content, antioxidant activity and tannins in 80% methanolic extract and absolute methanolic extract. Total polyphenolic content, antioxidant activity and tannins were significantly (P ≤ 0.05) different in 80% methanolic extract and absolute methanolic extract. Germination process significantly (P ≤ 0.05) increased total polyphenolic content and antioxidant activity. The maximum level of phenolic content (93.27 mg/g) was found in fenugreek seeds germinated for 48 hrs. Total polyphenolic content, antioxidant activity and tannins were also significantly (P ≤ 0.05) different in roasted fenugreek seeds.

Keywords: Fenugreek seeds, Germination process, Estimation of phytic acid ________________________________________________________________________________________________________ I.

INTRODUCTION

Herb and spices have been used in food as a food additives due to their natural antioxidant property. Fenugreek (Trigonella foenum-gracum) is an annual leguminous bean member of Fabaceae family. Its seeds and green leaves are having many medicinal applications (Thomas et al., 2011; Paridar et al., 2011; Vaidya etal., 2013). In India, it is extensively used as Ayurvedic medicine and in China as traditional medicine (Prasad et al., 2014). Interestingly, in herbal medicine, it is used in the treatment of diabetes (Leela and Shafeekh, 2008).Fenugreek leaves provide a good amount of various minerals and vitamins.

They are rich in choline.

Medicinally seeds are the most important and useful part of fenugreek plant .Seeds are aromatic, bitter, carminative, galactogogue and have antibacterial properties.These seeds are golden-yellow in colour, small in size, hard, having four-faced stone like structure. Fenugreek seeds have high protein (25 %), lysine (5.7 g/16 g N), soluble (20 %) and insoluble (28 %) dietary fiber besides being rich in calcium, iron and betacarotene. The biological and pharmacological actions of fenugreek seeds are mostly attributed to the variety of its bioactive chemical constituents that serve as raw materials for the manufacturing of various hormonal and therapeutic drugs (Mehrafarin etal., 2010; Priya et al., 2011). Their application are limited due to bitter taste (Sharma 1986). However these days various processing techniques are applied for debittering of fenugreek seeds.

Secondary metabolites flavonoids and phenolic compounds are widely distributed in plant and it exert multiple biological effects including free radical scavenging, anti-inflammatory, and anticarcinogenic (Miller A. L.,1996).T. foenum-graecum contain high phenolic contents having high antioxidant activity(Kaur.C.et al.,2002).Among the plethora of bioactive compounds found in fenugreek seeds the major chemical constituents are polyphenolic compounds, galactomannan (fiber), diosgenin (saponin) , quercetin (flavonoid), trigonelline (alkaloid) and 4-hydroxyisoleucine (unusual amino acid). Germinated fenugreek seed are rich in polyphenolic compounds. Being a rich source of mucilaginous fiber and other dietary essential, fenugreek seeds used as a functional and nutritional food.

Therefore, the present study was planned to evaluate raw, germinated and roasted fenugreek seed extracts as a source of natural antioxidant. In this study, the effect of germination and roasting on their chemical composition was also investigated.

II. MATERIALS AND METHODS Fenugreek seeds were purchased from local market of Hissar, Haryana. Seeds were cleaned to remove any extraneous material.

Raw seeds were dried at 40±5 °C in a hot air oven to increase its keeping quality and stored in an airtight containers at ambient temp.

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Effect of Germination and Roasting on Nutritive Composition and Anti-Nutrients in Fenugreek Seeds (IJSTE/ Volume 3 / Issue 06 / 007)

Germination Fenugreek seeds (20 g) were soaked overnight in water at the ratio of 1:5 (w/v). The excess water was drained and seeds were germinated (tied in a muslin cloth) at room temperature for 24 h, 36 h and 48 h. The germinated seeds were dried in an oven at 50 °C til the constant weight.

Roasting Fenugreek seeds (20 g) were roasted in an open pan at 130±5 °C ,150±5 °C and 200±5°Cfor 7 min,5 min and 2 min respectively.

Continuous stirring was done with laddle for proper and uniform roasting.

Preparation of Fenugreek Seeds Extract Raw and processed (germination and roasting) fenugreek seeds were ground in Wall Mill. Ground sample were collected in air tight container separately for further analysis at room temperature. The prepared sample were extracted in absolute methanol and 80% methanol using orbital shaker for 4 hrs at 45°C. In extraction process, 3 g of prepared sample were weighed in universal bottle and 75 ml solvent was added .After extraction process supernatant were collected for further analysis.

Determination of Total Phenolic Content The total phenols in extracts was measured by UV spectrophotometry based on a colorimetric oxidation/reduction reaction. The oxidizing agent used was Folin-Ciocalteu reagent (AOCS,1990). To 0.2 ml of extract,7.5 ml water was added in a test tube and after that 0.5 ml of 1 N Folin-Ciocalteu reagent was added and, then, 1 ml of Na 2CO3 were added. The sample was incubated for 30 min at room temperature . For a control sample, 0.2 ml of distilled water was used. The absorbance of the resulting bluecolored solutions was measured at 760 nm.Quantitative measurements were performed, based on a standard calibration curve of gallic acid in water. The results were expressed as gallic acid equivalents (GAE) in milligrams per gram of sample.

Determination of Total Flavonoid Content Total flavonoid content was determined using a method by (Liu et al., 2008).Briefly, 2 ml of the extracts was taken in a test tube and 0.2 ml of 5% sodium nitrite was mixed.After 5 min 0.2 ml of 10%aluminium chloride was added and after 6 min 1 ml of 1M sodium hydroxide was added. The absorbance was measured at 510 nm.The resultswere expressed as Quarcetin equivalents (QE) in milligrams per gram of sample.

Total Dietary Fiber The dietary fiber content in processed fenugreek seeds was estimated by rapid enzymatic assay method. Briefly, 1 g processed fenugreek seeds sample was subjected to sequential enzymatic digestion by heat-stable α-amylase, protease and amyloglucosidase. Insoluble dietary fiber (IDF) filtered, and then residue were washed with warm distilled water. Combined solution of filtrate and water washings was precipitated with 4 volumes of 95% ethanol (EtOH) for soluble dietary fiber (SDF) determination. Precipitates was filtered and dried. Both SDF and IDF residues were estimated for protein, ash and blank, for the final calculation of SDF and IDF values. SDF was precipitated with EtOH,and residue was filtered, dried and weighed. Total dietary fiber (TDF) value was calculated for protein and ash content (AOAC, 1991).

Total Antioxident Activity Antioxidant activity was estimated by DPPH free radical scavenging method (Maizura et al.,2011). To 0.2 ml of extract taken, 2 ml DPPH in methanol was added and volume was made 3 ml. After that sample was incubated for 30 min in dark. Absorbance was measured at 517nm.

Total antioxidant activity was expressed as the % of DPPH decrease using the equation: % Antioxidant activity= (Control absorbance−sample absorbance) × 100 Control absorbance Estimation of phytic acid Phytic acid was estimated by the method of Davies and Reid (1979). One g material was ground and extracted with 0.2 N HCl. To 0.2 ml of the extract, distilled water to make volume 1.4 ml was added. After that 1 ml of ferric ammonium sulphate solution was added, mixed and kept in a boiling water bath for 20 min. The contents were cooled and 5 ml of isoamyl alcohol was added and mixed. To this, 0.1 ml ammonia solution was added, shaken thoroughly and centrifuged at 3000 rpm for 10 min. The alcoholic layer was separated and the colour intensity was read at 465 nm against amyl alcohol blank after 15 min. Sodium phytate standards were run along with the sample. The results were expressed as mg phytic acid/100 g sample.

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Effect of Germination and Roasting on Nutritive Composition and Anti-Nutrients in Fenugreek Seeds (IJSTE/ Volume 3 / Issue 06 / 007)

Estimation of tannins Tannins were estimated by Vanillin-HCl method (Price et al.,1978). One ml of suitably diluted extract was taken in a test tube and 5 ml of freshly prepared vanillin-HCl reagent was added slowly with mixing and colour developed was read at 525 nm.

Catechin standards were run simultaneously along with sample. The results were expressed as mg/100g.

Statistical analysis Each experiment was conducted in triplicates. The results were expressed as mean±SD. Analysis of variance was applied to analyze data and significance was accepted at p≤0.05 level.

III. RESULT AND DISCUSSION Total Phenolic Content Data pertaining to total phenolic contents presented in Table 1.1.Total phenolic content of raw and processed fenugreek seeds was significantly (P ≤ 0.05) different in absolute methanolic extract and 80% methanolic extract. This difference might be due difference in polarity of extracting solvent (Rostagno.M.A.et.al., 2003). Total phenolic content of raw fenugreek seeds was 67.32 mg/g and 59.72 mg/g in 80% methanolic extract and aqueous methanolic extract respectively. Total phenolic contents significantly (P ≤ 0.05) increase by germination process. In 80% methanolic extract phenolics content increased from 67.32 mg/g to 75.17 mg/g, 80.25 mg/g and 93.27 mg/g in samples germinated for 24 hr, 36hr and 48 hr respectively. In absolute methanolic extract phenolic content increased from 59.72 mg/g to 89.54 mg/g in germinated fenugreek seeds.The highest level of phenolic content (93.27 mg/g) was observed in fenugreek seeds germinated for 48 hr in 80% methanolic extract. Increases might be due to the biosynthesis of phenolic compounds during germination process (Randhir et al., 2004).Total phenolic content in roasted fenugreek seeds increased significantly (P ≤ 0.05) from 73.12mg/g to 86.43mg/g in 80% methanolic extract as presented in table 1.1. In roasting process TPC increased with roasting temp. and was oberved maximum (86.43 mg/g) at 200°C temp (Jeong et al.,2004).

Solvent 80%Methanol Methanol

Raw 67.32±0.22b 59.72±0.01a

Table – 1 Effect of processing on TPC in processed fenugreek seeds.

TPC(mg/g) Germination Roasting 24 hr 36 hr 48 hr 130°C 150°C 75.17±0.06b 80.25±0.13b 93.27±0.33b 73.12±0.06b 81.30±0.44b 66.64±0.14a 72.60±0.10a 89.54±0.05a 61.34±0.36a 64.12±0.09a

200°C 86.43±0.16b 76.64±0.22a

Means in the same column with different superscripts differ significantly at (P < 0.05).

Antioxident activity Antioxidant activities of raw and germinated fenugreek seeds as shown in Table 1.2. DPPH radical-scavenging activity expressed in % inhibition of raw and germinated fenugreek seeds ranged from 39.11% to 73.65% in 80% methanolic extract.

Antioxident activity is significantly (P ≤ 0.05) different in both extracting solvent of raw and processed fenugreek seeds. In absoulte methanolic extract antioxidant activity ranged from 16.47% to 63.72%. Germinated fenugreek seeds had significantly (P ≤ 0.05) higher DPPH radical-scavenging activity compared to raw seeds. Polyphenols have high free radical scavenging activity.This increase might be dueto the synthesis of compounds and tocopherols which are responsible for antioxidant activity (Sharma and Gujral, 2010).

Antioxident activity increased from 39.11% to 49.03%,54.19% and 61.29% in germinated and roasted at 130°C,150°C and 200°C respectively in 80% methanolic extract. In absoult methanolic extract, antioxidant activity ranged from 29.11% to 44.31%.

Solvent 80%Methanol Methanol

Table – 2 Effect of processing on AOA in processed fenugreek seeds.

Antioxident Activity(%) Germination Roasting Raw 24 hr 36 hr 48 hr 130°C 150°C 39.11±0.08a 50.74±0.14a 53.87±0.12a 73.65±0.29a 49.03 ±0.14a 54.19 ±0.09a 16.47±0.19b 35.37±0.13b 53.41±0.21b 63.72±0.31b 29.11 ±0.11b 41.06±0.07b

200°C 61.29 ±0.04a 44.31±0.23b

Means in the same column with different superscripts differ significantly at (P < 0.05).

Total Flavonoid Content Data pertaining total flavonoids content is presented in Table 2. Total flavonoids content of raw fenugreek seeds was 3.48 mg/ g in 80% methanolic extract. Total flavonoids content in 80% methanolic extract was increased to 5.29 mg/g and in absoulte methanolic extract it increased to 5.10 mg/g in processed fenugreek seeds.

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Effect of Germination and Roasting on Nutritive Composition and Anti-Nutrients in Fenugreek Seeds (IJSTE/ Volume 3 / Issue 06 / 007)

Solvent 80%Methanol Methanol

Table – 3 Effect of processing on total flavonoids content of processed fenugreek seeds.

TFC(mg/g) Germination Roasting Raw 24 hr 36 hr 48 hr 130°C 150°C 3.48±0.08b 4.98±0.04b 5.17 ±0.06b 5.29±0.10b 3.61±0.08b 3.72 ±0.23b 3.15±0.09a 4.20 ±0.14a 4.40 ±0.09a 5.10±0.04a 3.19±0.17a 3.38 ±0.02a

200°C 3.92 ±0.01b 3.54±0.19a

Means in the same column with different superscripts differ significantly at (P < 0.05).

Total flavonoids content of raw and processed fenugreek seeds significantly (P ≤ 0.05) different in two different extracting solvent. Flavonoids content in raw fenugreek seeds was significantly (P ≤ 0.05) lower than germinated seeds. Significantly (P ≤ 0.05) increase in the total flavonoids content of fenugreek seeds was observed as a result of germination process. A gradual increase was observed in flavanoid content with increase in germination time from3.48 mg/g to 5.29 mg/g . The highest level (5.29 mg/g ) was recorded for fenugreek seeds germinated for 48 hr. Roasting process also increased the flavonoids content from 3.48 mg/g to 3.92 mg/g in 80%methanolicextract.In absoulte methanolic extract, flavonoids content also increased from 3.15 mg/g to 3.54 mg/g.

Antinutritional Factors Significant (P ≤ 0.05) difference was observed in tannin content extractced in two solvents in as shown in Table 3. Tannins content was higher in absolute methanolic extract (41.76mg/100g) and reduced to 27.23 mg/100g during germination process. In germination process, tannins content was decreased from 12.52 mg/100g to 9.40mg/100g in fenugreek seed in 80% methanolic extract. As the germination period increased tannin content gradually decreased. The reduction might be due to leaching of tannins into water (Shimelis and Rakshit, 2007) and bonding of polyphenols with carbohydrate or protein (Saharan et al., 2002).

In roasting process, tannins were decreased from 12.52mg/100g to 8.42mg/100g in 80% methanolic extract and from 41.76 mg/100g to 20.37 mg/100g in absolute metabolic extract. During roasting breakdown of the bond between phytate and might takes place which results in destruction of phytates, tannins and oxalates (Reddy et al., 1978)

Solvent 80%Methanol Methanol

Table – 4 Effect of processing on tannins content in processed fenugreek seeds.

Tannins(mg/100g) Germination Roasting Raw 24 hr 36 hr 48 hr 130°C 150°C 12.52±0.21a 11.22±0.22a 10.31±0.38a 9.40±0.57a 9.22±0.23a 8.45±0.49a 41.76±0.30b 33.37±0.28b 31.75±0.34b 27.23±0.15b 38.61±0.07b 32.17 ±0.14b

200°C 8.42 ±0.15a 20.37±0.24b

Means in the same column with different superscripts differ significantly at (P < 0.05).

Germination of fenugreek seeds reduced the phytic acid content from 190.30-117.74 mg/100 g. It was reported that phytase activity originates after germination and the phosphatase activity was increased in the germinated seeds which results in the reduction of phytic acid content in fenugreek seeds after germination and roasting ( El Mahdy and El-Sebaiy .,1982).During germination enzymatic hydrolysis of phytate phosphorus takes place which decreases phytic acid content (Gupta et al., 2001).During roasting phytic acid reduced from298.77-177.18 mg/100g.Decrease in phytic acid during roasting may be due to thermoability of phytic acid.Germination effects phytic acid more than roasting as shown in Table.4 Total Dietary Fibre Data regarding TDF, IDF and SDF presented in Table 4. Significant(P ≤ 0.05) reduction was noted in total dietary fibre (TDF),soluble dietary fibre (SDF) and insoluble dietary fibre (IDF) upon germination as shown in Table 4.Total dietary fibre reduced from 47.35% to 40.20% during germination process.

Table – 5 Effect of processing on Dietary Fibre and phytic acid in processed fenugreek seeds.

Sample Raw Germination 24 hr 36 hr 48 hr Roasting 130°C 150°C 200°C

TDF (%) 50.20±0.14ͣa

IDF (%) 31.70±0.14a

SDF (%) 18.40±0.14b

PA (mg/100g) 317.18±0.14a

47.35±0.21b 43.40±0.28d 40.20±0.14f

30.30±0.14b 28.20±0.14c 28.45±0.35c

17.60±0.14c 15.65±0.21d 11.50±0.28e

190.30±0.25c 157.44±0.14f 117.74±0.24g

45.45±0.2c 41.40±0.28e 32.60±0.28g

24.45±0.35c 23.45±0.35e 20.65±0.21f

20.75±0.21a 17.45±0.35c 11.55±0.35e

298.77±0.31b 182.25±0.07d 177.18±0.21e

Means in the same column with different superscripts differ significantly at (P < 0.05).

There was no significant (P ≤ 0.05) change in insoluble dietary fibre germinated for 36 hr and 48 hr. Soluble dietary fibre also significantly (P ≤ 0.05) reduced from 17.60% to 11.50% upon germination process. Reduction in dietary fiber content during germination may be due to enzymatic breakdown of the galactomannan units. Shakuntala et al.(2011) concluded that decrease in soluble dietary fiber content on germination of fenugreek seeds. An enzyme α-galactosidase during germination of fenugreek seeds partially attacks galactomannan to produce galactose. Decrement in total dietary fiber, insoluble dietary fiber and soluble

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Effect of Germination and Roasting on Nutritive Composition and Anti-Nutrients in Fenugreek Seeds (IJSTE/ Volume 3 / Issue 06 / 007)

dietary fiber also noticed during roasting process of fenugreek seed as shown in Table 4. Total dietary fibre (TDF) reduced from 45.45% to 32.60% during roasting process. Significant (P ≤ 0.05) reduction in soluble dietary fibre (SDF) and insoluble dietary fibre (IDF) also determined as listed in table 4.Reduction in IDF content during roasting might bedue to retrogradation of starch (Mathur and Chaudhary.,2009).

IV. CONCLUSION It may be concluded from the present study that nutritional and functional quality of fenugreek seeds can be improved by various processing methods e.g germination and roasting. Antioxidant activity also increases significantly after processing which are found to be responsible for functional properties of processed fenugreek seeds. Therefore, the use of processed fenugreek seeds can be exploited in functional foods as well as a therapeutic agent.

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