Nano Techniques & Applications Enhance Activity in Food Science

IJIRST –International Journal for Innovative Research in Science & Technology| Volume 3 | Issue 10 | March 2017 ISSN (online): 2349-6010

Nano Techniques & Applications Enhance Activity in Food Science Dr. A. Jaganathan Principal / Secretary Department of Hotel Management Food Craft Institute, Hoshiarpur, Punjab, India

S. Muguntha Kumar Assistant Professor Department of Hotel Management & Catering Science Muthayammal College of Arts & Science, Namakkal, Tamilnadu, India

Abstract This paper aims at investigating the effect of the nanoemulsion and nanoencapsulation delivery systems on the antioxidant and antimicrobial activity of different bioactive components. Thymol, carvacrol, linalool and eugenol hydrophobic compounds were encapsulated in dextran of oil-in-water (o/w) nanoemulsions prepared by ultrasonic homogenization method and stabilized by different types of emulsifier Tween 20, 40 or 80 and distilled water. The results showed that the reducing power, antioxidant capacity, H2O2 scavenging and antioxidant activity of un-encapsulated eugenol was the highest percent compared others essential oils. The highest essential oils nanoencapsulated yield (EY %) and efficiency (EE %) were observed for eugenol 91.74 and 97.81 %, respectively by using Dex/T20 treatment. Nanoencapsulation of eugenol can enhance the aqueous solubility of eugenol at 25, 35 and 45 °C. Thermal stability of eugenol increased from 63.5 to 143 °C after nanoenc capsulation by using Differential Scanning Coliremetry (DSC). Antioxidant activity of nanoencapsulated eugenol 0.5 mg/ml in linolic acid system compared with BHT activity at different periods storage were 98.97, 98.42, 98.12 and 97.89 % at 0, 60, 120 and 180 days, respectively. Sensory evaluation of jelly prepared with different levels of nano-encapsulated eugenol compared to 1% its un-encapsulated showed the (0.3 %) formation was the most accepted by testers. The antimicrobial activity of nano-encapsulated eugenol was measured against three different microorganisms, such as Molds & yeasts, coliforms and salmonella. Significance and Impact of the Study: nanoencapsulation affects positively essential oils main compounds, improves antioxidant activity and retains antimicrobial activity, enhancing the quality of the oils.

Keywords: Nano-Emulsion, Thymol, Carvacrol, Diet, Food Science _______________________________________________________________________________________________________ I.

INTRODUCTION

In plant phenolic and polyphenolic compounds are widespread components of the human diet found in vegetables, fruits, soybean, cereals, tea, coffee, red wine and natural herb extracts. The extracts of medicinal plant and culinary herb sources possess very strong antioxidant activity in vitro. This antioxidant activity is due mainly to phenolic compounds present in plants (Sahaa et al, 2008). The strong antioxidant activity of the volatile extract of thyme is mostly due to its main volatile components, thymol and carvacrol. In the case of the volatile extract of basil, eugenol, reported as a major volatile component, probably contributes significantly to the strong antioxidant activity of the extract. Eugenol showed high antioxidant activity in the aldehyde/carboxylic acid assay (Teissedre & Waterhouse 2000; and Lee, & Shibamoto 2001).

Conventional emulsions, that is, emulsions with microdroplets, are thermodynamically unstable liquid–l iquid dispersions.

Emulsions with particle sizes higher than 100 nm are usually prepared by high-energy methods. As in the case of nanoemulsions, the O/W systems consist of lipid droplets dispersed in an aqueous medium, with each lipid droplet being surrounded by a thin emulsifier layer. The initial droplet concentration and size distribution of this type of delivery system can be controlled, as can the nature of the emulsifier used to stabilize the droplets. A careful selection of emulsifier type enables one to control interfacial properties such as charge, thickness (dimensions), rheology, and response to environmental stresses (such as pH, ionic strength, temperature, and enzyme activity) (McClements and Li 2010). To form conventional emulsions, usually a preemulsion with coarse particles, obtained by a mechanical device such as an Ultra-Turrax, is prepared. This system is further homogenized to obtain a micro droplet emulsion. In general, a variety of homogenizers are available to prepare emulsions, including high-shear mixers, high-pressure homogenizers, colloid mills, ultrasonic homogenizers, and membrane homogenizers.

Encapsulation of bioactive compounds represents a feasible and efficient approach to modulate drug release, increase the physical stability of the active substances, protect them from the interactions with the environment, decrease their volatility, enhance their bioactivity, reduce toxicity, and improve patient compliance and convenience (Ravi 2000).

Encapsulation of Essential Oils deals with micrometric size capsules, which are used for the protection of the active compounds against environmental factors (e.g, oxygen, light, moisture, and pH), to decrease oil volatility and to transform the oil into a powder.

Encapsulation in nanometric particles is an alternative for overcoming these problems but additionally, due to the sub cellular size, may increase the cellular absorption mechanisms and increasing bioefficacy (Anna et al, 2014).

Although the antimicrobial effects of essential oils are well established and despite the several studies on their antimicrobial activity (Bakkali et al, 2008; Ben Arfa et al, 2006; Ceylan and Fung, 2004; Helander et al, 1998; Lambert et al, 2001; Ultee et al,

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Nano Techniques & Applications Enhance Activity in Food Science (IJIRST/ Volume 3 / Issue 10/ 012)

2000, 2002; Sikkema et al, 1995), it must be highlighted that their mechanisms of action are poorly understood, even though different theories were proposed. In particular, it appears that the different essential oil components are characterized by a peculiar mechanism of action, which depends on the nature of the specific bioactive molecule (Di Pasqua et al, 2007). The objective of this work was to try increase the efficiency and stability of some essential oils using nanotechnology (nano-emulsion and nanoencapsulation) techniques on the antioxidant and antimicrobial activity for application in the food industry.

II. MATERIALS AND METHODS Reducing Power The reducing power of thymol, carvacrol, linalool, and eugenol was determined according to the method of Oyaizu, (1986). A volume of 0.4 ml of ethanolic solutions with different concentration (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml) form each tested compound were added to 1 ml potassium ferricyanide 1% and 1 ml phosphate buffer 0.2 M, pH = 6.6. Mixture was incubated at 50 oC for 20 min. Then, trichloroacetic acid 10% was added to mixture, and centrifuged at 650 g for 10 min. Two ml of the supernatant was mixed with 2 ml distilled water and 0.4 ml ferric chloride 0.1% and the absorbance was read spectrophotometrically (Shimadzu UV-VIS 160 A) at 700 nm. Higher absorbance of the reaction mixture indicated greater reducing power.

Total Antioxidant Capacity as Equivalents of α-Tocopheroel The total antioxidant capacity of thymol, carvacrol, linalool and eugenol was determined according to the method of Prieto et al.

(1999). Ethanolic solution with different concentration (10, 20, 30, 40, 50, 70, 100 and 500 ppm) was added to 3.5 ml reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4mM Ammonium molybdate). Then, the tubes were capped and incubated in a water bath at 95 oC for 90 min. After cooling to room temperature, the absorbance was measured at 695 nm against a blank. The antioxidant capacities expressed as equivalents of α-tocopheroel.

The Antioxidant Capacity of Thymol, Carvacrol, Eugenol and Linalool by DPPH Method The ability of tested compounds to scavenge DPPH radical was determined according to the method of (Hatano, et al, 1988). The reaction mixture had contained 0.1 ml of DPPH radical solution (5 mM) and different concentration of tested compounds (from 0.0026 mM to 83 mM). Total volume of the reaction mixture was 3ml. Absorption of DPPH radical at 515 nm was determined after 10 min against a blank solution that contained only methanol. % DPPH radical scavenging activity = ((Ac-As)/Ac) × 100 Scavenging of Hydrogen Peroxide The ability of tested compounds to scavenge hydrogen peroxide was determined according to the method of Ruch et al, (1989). A solution (20 mM) of hydrogen peroxide was prepared in ethanol 95%. Hydrogen peroxide concentration was determined spectrophotometrically from absorption at 230 nm by using the molar absorptive of 81M -1 cm-1. Ethanolic solution of each sample with different concentration 10, 20, 40, 60, 80, 100, 120,140 ppm was added to a hydrogen peroxide solution. Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank. % scavenging of hydrogen peroxide effect = (As/Ac -1) ×100 Antioxidant Activity in a Linoleic Acid System An antioxidant activity assay was carried out by using the linoleic acid system Osawa and Namiki (1981). Tested compounds in ethanolic solution with different concentration (125, 250, 375,500 ppm) were added to 2 ml ethanolic solution of linoleic acid 0.2 M. Then, the total volume was adjusted to 4 ml with ethanol. And the reaction mixture incubated at 40 oC. the degree of oxidation was measured according to the thiocyanate method Mitsuda et al, (1996) by adding 0.1 ml reaction mixture , 0.1 ml ammonium thiocyanate 30% , 3 ml solvent (n butanol :ethanol 1:1) and 0.1 ml ferrous chloride (20 mM in 3.5% HCl). After mixing was stirred for 3 min, the absorbance measured at 500 nm, and the percentage of antioxidant activity was calculated from the following equation: Antioxidant activity %= 100 - [(absorbance of sample / absorbance of control) ×100] All tests and analyses were run in triplicate and averaged Microbiological Analyses For microbiological evaluation, the following three analyses were performed: yeast and mold counting, coliforms at 45°C and Salmonella sp. research using the techniques outlined in the current legislation, RDC # 12 (BRASIL, 2001).

Sensory Evaluation An acceptance test of the jelly samples was carried out at the Sensory Analysis Laboratory of the Food Science and Technology Department, National Research Center (NRC) with the participation of 41 volunteer members of the NRC community from both sexes and of all ages. Volunteers were selected based on their interest and availability to take part in the tests. Samples were

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Nano Techniques & Applications Enhance Activity in Food Science (IJIRST/ Volume 3 / Issue 10/ 012)

evaluated through tests of preference and intention to purchase in accordance with NBR 12806/1993 (ABNT, 1993). The testers assessed the samples using a 9-point structured hedonic scale, as follows: 1 - disliked very much; 2 - disliked quite a lot; 3 - fairly disliked; 4 - slightly disliked; 5 - neither liked nor disliked; 6 - slightly liked; 7 - fairly liked; 8 - liked quite a lot; 9 - liked very much. The samples were marked with random three digit numbers and served to testers in individual booths under white light.

Plain mineral water and biscuit means cookie were provided for rinsing of the palate between sample evaluations.

Statistical Analyses Experimental results were mean ± S.D. of three parallel measurements and analyzed by SPSS 10 (SPSS Inc. Chicago, IL).

Differences between means were determined using Tukey multiple comparisons and least significant difference (LSD).

Correlations were obtained by Pearson correlation coefficient in bivariate correlations. P values <0.05 were regarded significant.

III. RESULTS AND DISCUSSIONS Antioxidant Activity of the Basil and Essential Oils Major Components Reducing Power The reducing power of thymol (T), carvacrol (C), linalool (L), and eugenol (E) was measured by the ferricyamide method. Different concentrations of thymol, carvacrol, linalool and eugenol 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml were investigated. Higher absorbance at 700 nm of the reaction mixture indicated greater reducing power. Table 1 represents the reducing power of T, C, L and E measured as absorbance at 800 nm. Table 1 reveals that T, C, E exhibited a reducing power, but to varying degrees while L has no significant reducing power. A positive correlation has been observed between concentrations and reducing power for T, C, E especially E. There is a significant difference among the tested compounds in their reducing power.

Table - 1 Reducing Power of Thymol, Carvacrol, Linalool and Euganol as Absorbance at 800 nm Concentration Absorbance at 800 nm (mg/ml) Thymol Carvacrol Linalool Eugenol Ascorbic 0.6 0.36±0.01 0.23±0.02 0.04±0.01 0.96±0.03 0.83±0 .01 0.2 0.66±0.02 0.53±0.02 0.09±0.01 1.64±0.02 1.36±0. 11 0.3 1.05±0.01 0.87±0.01 0.14±0.01 2.15±0.05 1.68±0. 06 0.5 1.48±0.20 1.26±0.02 0.18±0.01 2.73±0.03 1.87±0. 03 0.6 1.64±0.06 1.41±0.04 0.20±0.01 2.91±0.07 2.03±0. 05 0.7 1.73±0.04 1.56±0.06 0.21±0.01 3.37±0.13 2.05±0. 12 1.0 1.78±0.05 1.68±0.03 0.22±0.01 3.92±0.11 2.07±0. 07

The reducing power of E was higher than T than C in the same order, we have notice that the reducing power of E was about 7fold the reducing power of T and C. The Antioxidant Activity of Thymol, Carvacrol, Linalool and Eugenol in LInoleic Acid System The antioxidant activity of T, C, L and E were determined in linoleic acid system and compared with BHT activity. Different concentrations used in determination 125, 250, 375 and 500 ppm for the tested compounds and 200 ppm for BHT. The linoleic acid and tested compounds samples mixtures were incubated at 40ºC for 3 days. Every day, samples were taken for determination of the antioxidant activity.

IV. CONCLUSIONS Nano-encapsulation offers great advantage for an improved stability of bioactive compounds in certain food formulations. Jelly as a staple food is considered to be a useful model to develop new generation of functional products, and to test the stability of nanoencapsulated bioactive compounds. In this study, bioactive compounds were encapsulated with dextran and added to jelly formulation to test their stability during the process. The results revealed that nano-encapsulation significantly improved the thermal stability of eugenol at elevated temperatures applied during process. Addition of the particles of encapsulated bioactive compounds at amounts up to 0.02 % in jelly formulation seemed reasonable in terms of general quality parameters and sensory properties. Presence of bioactive compounds in a formulation may bring important changes in the chemical reactions occurring in food during processing. The most bioactive compounds bear functional groups that may react with other compounds in food. These reactions may lead to the formation of processing contaminants especially in products in which processing conditions are severe.

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