Central Journal of Human Nutrition & Food Science

Cite this article: Keishing S, Banu T, Umadevi M (2015) Effect of Fermentation on the Nutrient Content, Antioxidant and Antidiabetic Activities of Hawaijar, an Indigeneous Fermented Soya of Manipur, India. J Hum Nutr Food Sci 3(3): 1066.

*Corresponding authorSomishon Keishing, Department of Home Science, Faculty of Rural Oriented Sciences, Gandhigram Rural Institute- Deemed University, Gandhigram-624302, Tamil Nadu, India, Tel: 91 9600206738; E-mail:

Submitted: 06 September 2014

Accepted: 02 June 2015

Published: 04 June 2015

ISSN: 2333-6706

Copyright© 2015 Keishing et al.

OPEN ACCESS

Research Article

Effect of Fermentation on the Nutrient Content, Antioxidant and Antidiabetic Activities of Hawaijar, an Indigeneous Fermented Soya of Manipur, IndiaSomishon Keishing*, Thahira Banu and Umadevi MDepartment of Home Science, Gandhigram Rural University, India

Abstract

Hawaijar, an indigenous fermented soya product is produced and consumed in Manipur, India. In this present study, the proximate composition, and phytochemicals were analyzed and the antioxidant and antidiabetic activity was determined for both raw soya and hawaijar. Soyabean were collected from local market and fermented traditionally at household level and used for the analysis. Results indicate an increase in the nutrient composition of hawaijar than the unfermented counterpart. The antioxidant and antidiabetic activity of hawaijar also showed promising result than the unfermented soya. Thus hawaijar can be used as a diet supplement to overcome malnutrition and other related diseases.

Keywords•Hawaijar•Fermented Soya•Nutrient Composition•Antioxidant activity•Antidiabetic activity

INTRODUCTIONManipur is one of the eight states of North east India, bounded

by Nagaland in the north, Mizoram in the south, Assam in the west and by the borders of the country Myanmar in the east as well as the part of south. The state lies at latitude of 230 83’ N -250 68’N and longitude of 93003’E 94078’E [1]. The major ethnic groups living in Manipur are Meitei, Naga, Kuki, Meitei Pangal and Gorkha [2]. Ethnic, fermented foods are foods produced by ethnic people using their native knowledge from locally available raw materials of plant or animal sources either naturally or by adding starter culture(s) containing functional microorganisms that modify the substrates biochemically and organoleptically into edible products that are culturally and socially acceptable to the consumers [3]. Hawaijar is an indigeneous traditional fermented soya bean with characteristic flavour and stickiness. It is consumed commonly in the local diet as a low cost source of high protein food and plays an economical, social and cultural role in Manipur [4]. Hawaijar is similar to natto of Japan and thaunao of Thailand: kinema in Sikkim, aakhuni in Nagaland, tungrymbai in Meghalaya and bekangthu in Mizoram [5]. Fermentation is a very important process that allows the utilization of microorganisms

to break down complex compounds to yield a unique taste and aroma. Not only the process of fermentation preserve foods, it also improves digestibility by breaking down proteins within foods and have been known to enrich nutrients such as vitamins, amino acids and fatty acids [6]. The present study was conducted to evaluate the nutrient composition, antioxidant and anti-diabetic activity of raw soya bean and hawaijar (fermented soy bean).

MATERIALS AND METHODS

Collection and preparation of the sample

Small sized soybean grown locally was purchased from local market of Imphal District, Manipur, India. The purchased seeds were cleaned, shade dried and the procured seeds were fermented traditionally at household level. The seeds were soaked overnight and cooked till tender and excess water was drained off and washed with normal tap water. The cooked and washed soya were packed in fig (Ficus hispida) leaves and fermented in bamboo basket lined with straw for 3 day during the month of December. The maturity of the Hawaijar was identified with the formation of sticky white slimy substances on the beans

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with a pungent ammonia cal smell. The raw and fermented soyabean were analyzed for proximate nutrients, antioxidant and antidiabetic activity.

Analysis of proximate composition

The nutrients namely carbohydrate [7], protein, fat and moisture [8] and the total ash and crude fiber [9] were analysed. pH was measured using pH meter (Elico L.I 120).

EVALUATION OF PHYTOCHEMICALS AND ANTIOXIDANT ACTIVITYEthanol extracts

Extracts of the samples, raw Soyabean and hawaijar were taken using ethanol. Two gram of the each sample was ground and blended with 25ml of ethanol water (4:1) at room temperature (25-300C) using a shaker for 3-4hours at 150 rpm. The mixture was filtered through Whatmann filter paper No. 4 and 5. The filtrate was kept in freezing condition. The extract was used for analysis of phytonutrients, antioxidant and anti diabetic activity.

Aqueous extract

Five gram of the samples were ground and blended with 20ml of aqueous water (4:1) at room temperature (400C) kept in a shaker for 2 hours at 150rpm. The mixer was filtered through the Whattman filter paper No. 4 and 5. The filtrate was kept in freezing condition. Aqueous extract was used for analysis of phytonutrients and evaluation of antioxidant and antidiabetic activity because the sample used are foods consumed by the people and not a pharmaceutical agent hence to understand the activity in aqueous extract the study was undertaken.

Flavonoid content

The flavonoid content was determined as described by Boham and Abyazan, (1974) [10]. 0.5g of powdered sample was added to 75 ml of distilled water and the mixture was heated gently for 30 minutes. The solution was centrifuged at 2000 rpm for 20 minutes and the supernatant was collected. 1ml of extracted sample was transferred to 100ml volumetric flask containing 75 ml water. 5 ml of Folin-Denis reagent and 10ml of sodium carbonate solution was mixed and diluted to 100ml with water. After mixing the solution was shaken well and absorbance was read at 700nm after 30 minutes.

Total poly phenolic content (TPC)

The total poly phenolic content was determined using the Folin- Ciocalteu reagent [11]. A 0.3ml of extract made up to 1ml with distilled water was mixed with 2.25ml of Folin-Ciocalteu reagent diluted (1:10) in distilled water and allowed to stand at room temperature for 25 minutes; 2.25ml of sodium carbonate (60g/1) solution was added to the mixture. After 90 minutes at 280C, the absorbance was measured at 725nm using spectrophotometer (UV Spectrophotometer, Evolution 201). The total phenolic content was calculated and expressed as Gallic acid equivalents based on the Gallic acid (100µg/ml) standard curve.

DPPH radical scavenging activity

The radical scavenging activity of the extract was determined using DPPH [12]. Ethanolic extract (0.1ml) was added to 3ml of

a 0.001M DPPH in methanol. The absorbance of the solution was determined using spectrophotometer (UV Spectrophotometer, Evolution 201) at 517 nm after 30 minutes, and the percent inhibition of activity was calculated as:

Radical scavenging activity = [(Ao-Ae) / Ao] × 100

(Ao = absorbance without extract; Ae = absorbance with extract)

Antidiabetic activity

α- Amylase activity was determined by the method of MuCue and Shetty (2004) [13], using starch as a substrate in a colorimetric reaction using 3, 5-dinitro salicylic acid.

The bio assay method was adopted and modified from Sigma-Aldrich (www.sigmaaldrich.com). A starch solution (0.1%w/v) was obtained by stirring 0.1 g of potato starch in 100ml of 16mM of sodium acetate buffer. The enzyme solution was prepared by mixing 27.5mg of α-amylase in 100ml of distilled water. The colorimetric reagent was prepared by mixing sodium potassium tartarate solution and 96mM 3, 5-dinitro salicylic acid solution. Both control and ethanol extracts (0.5mg/ml & 1.0mg/ml) were added with starch solution and left to react with α-amylase solution under alkaline condition at 25ºC. The reaction was measured over 3 minutes. The generation of maltose was quantified by the reduction of 3, 5-dinitro salicylic acid to 3- amino-5-nitro salicylic acid. The reaction is detectable at 540 nm. The anti-diabetic activity was investigated through the inhibition of α-amylase, an enzyme that made the digestion of starch and so reduced the glucose absorption.

The α-amylase inhibition was expressed as a percentage of inhibition and calculated by the following equation.

(Maltose) Test

% Reaction = -------------------- X 100

(Maltose) Control

% Inhibition = 100 - % reaction ±SD

Statistical analysis

Each measurement was carried out in triplicates. All the data are expressed as mean ± standard deviation.

RESULTS AND DISCUSSION

Proximate analysis

The proximate nutrient composition of soybean and hawaijar was analysed and the results are presented in Table 1.

The moisture content of soybean and hawaijar was analysed and it was found that moisture content in soya was 8.4%±0.1 whereas in hawaijar it was 10.3 % ± 0.8. As hawaijar are prepared by soaking soya and cooked prior to fermentation, hawaijar had high moisture content [14].

The carbohydrate content of soya (19.6 ± 0.4g/100g) was found to decrease to 15.0 ± 1g/100g after fermentation. The result is in line with the similar studies that states that the carbohydrate content decreases during fermentation due to the utilization by the growing microorganism [15].

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The most significant change in hawaijar is noticed by increase in protein content during fermentation. The protein content increased from to 39.8 ± 0.5g /100g to 42.2 ± 2.9 g/100g after fermentation. The protein content of the fermented foods was marginally higher than their unfermented counterparts [16]. Similar observations were noted while working with fermentation of Hawaijar [14,15], soya [12], tungrynbai [16] and kinema [17,18] all had reported that increase in protein may be attributed due to the high protease activity that has led to protein hydrolysis during fermentation.

The fat content of hawaijar was higher than the unfermented soya. The result is in agreement with work done earlier [14] that reports that the fat content increases from 20g/100g in raw soya to 23g/100g on the third day of fermentation. Studies on similar oil beans suggested that fermentation probably enhances oil extraction [19].

Ash content of hawaijar decreased to 2.3 ± 0.5g from 4.88 ± 0.2g in soya. The result is in line with a study on tempeh and raw soyabean where the ash content of raw soya bean was significantly higher than tempeh [20]. The crude fiber content was low in soya (4.54 ± 0.6g/100g) whereas in hawaijar it increased to 11.23 ± 0.4g/100g. Similarly, the fibre content of tungrymbai was found to be 12.8g/100g [16]. The pH of soya (4.07±0.07) increased with fermentation and was recorded to be 8.36±0.5 in hawaijar. The pH of fermented soya bean increased with fermentation time [17]. Increasing pH during fermentation has been attributed to proteolytic activities and release of ammonia following the utilization of amino acids by microorganisms involved in fermentation [21].

ANTIOXIDANT ACTIVITIES

Flavanoid content and total polyphenolic content (TPC)

The result of the quantitative phytonutrients analysis of soya and hawaijar is presented in Table 2. The quantitative phytonutrient analysis of the samples reveals that the flavonoid content decreased in hawaijar (3.88±0.13 mg) when compared to its unfermented counterpart.

As shown in Table-2 the total polyphenol content was higher in ethanol extract (0.930±0.01mg) after fermentation. The increased in total phenol content of Hawaijar in the present study, is in agreement with the findings reported by other investigators [22,23] who suggested that the release of aglycones from the bean

substrate during fermentation increase their phenolic content.

Phenolics have biological properties such as anti-oxidant, anti- apoptosis, anti- aging, anti-carcinogen, anti-inflamation, anti-atherosclerosis, cardiovascular protection, improvement of the endothelial function, as well as inhibition of angiogenesis and cell proliferation activity [24].

Antioxidant activity of Hawaijar and unfermented soya

DPPH assay was used as a rapid method to detect from the samples for their antioxidant activity of ethanol and aqueous extract. The results indicate a positive DPPH radical scavenging activity for soya bean and hawaijar as depicted in Table -3. The radical scavenging activity of ethanol extract was 90.58±0.44 % for soya bean and 75.65±0.04 % for hawaijar. In aqueous extract the antioxidant activity was 96.54 ± 0.02 % for soybean and 66.77±0.08 % for hawaijar. The increase in scavenging effect was observed in the present study, is consistent with the findings, of the study on kinema a fermented soya inhibited DPPH absorption and was superior to cooked non fermented soya [25].

Antidiabetic activity

Antidiabetic activity of hawaijar extract showed alpha-amylase activity more pronounced than in the soybean aqueous extract as shown in Table –4.

Fermented soya products may be better for preventing or delaying the progression of type II diabetes compared with non fermented soyabean [26]. Hawaijar showed 65% antidiabetic activity in aqueous extract and the unfermented soyabean showed 40% of antidiabetic activity in aqueous extract.

Phenolic rich extracts of soybean may have the potential to act as a source of anti-diabetic agent to control post-prandial hyperglycemia and anti-ACE agents to control hypertension, a known complication of long-term diabetes and / or hyperglycemia [27].

Proximate Nutrient/ 100g Soya Hawaijar

Moisture % 8.4 ± 0.1 10.3 ± 0.8

Carbohydrate (g) 19.6 ± 0.4 15.0 ± 1

Protein(g) 39.8 ± 0.5 42.2 ± 2.9

Fat (g) 18.55 ± 1.2 19.3 ± 0.7

Total ash (g) 4.88 ± 0.2 2.3 ± 0.5

Crude fibre (g) 4.54 ± 0.6 11.23 ± 0.4

pH 4.07 ± 0.07 8.36 ± 0.5

Table 1: Proximate nutrient composition of soybean and hawaijar.

Data represent mean of three sample analysis (n=3) ± s.d

Phytonutrients Ethanol extract Aqueous extract

Samples Soyabean Hawaijar Soyabean Hawaijar

Flavonoids (mg) 4.28 ± 0.03 3.88 ± 0.13 - -Total polyphenol (mg)

0.826 ±0.001 0.930±0.01 0.43±0.03 0.253±

Table 2: Phytonutrient analysis of processed soyabean.

Data represent mean of three sample analysis (n=3) ± s.d

SamplesAntioxidant capacity (%)

Soya bean Hawaijar

Ethanol extract 90.58 ± 0.44 75.65 ± 0.04

Aqueous extract 96.54 ± 0.02 66.77 ± 0.08

Table 3: Antioxidant activity of the processed soyabean.

Data represent mean of three sample analysis (n=3) ± s.d

Samples Soyabean HawaijarAqueous extract % of

inhibition 40% 65%

Table 4: Antidiabetic activity of processed soyabean.

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Study has reported that α-Amylase is responsible for cleaving starch during digestive process, which is important to manage postprandial blood glucose levels [28].

Natural α-amylase inhibition from food-grade plant sources offer an attractive therapeutic approach to the treatment of post-prandial hyperglycemia by decreasing glucose release from starch and delaying carbohydrates absorption in the small intestine and may have potential for use in the treatment of diabetes mellitus and obesity [29].

Chungkookjang, a fermented unsalted soybean predominantly with Bacillus subtilis generated isoflavonoid aglycones and small peptides, which improved insulinotropic action in islets of type 2 diabetic rats which exhibited better anti-diabetic action [30]. A study suggested that meju fermented in the standardized and meju fermented in the traditional way improved glycemic control by potentiating insulinotropic actions and alleviating hepatic insulin resistance in diabetic rats [31].

CONCLUSIONThe present study on soya bean and hawaijar reveals

that the nutrient composition of hawaijar enhanced after the fermentation process. The antioxidant and antidiabetic activity of hawaijar were significantly higher than the unfermented soya bean, suggesting the role of fermentation in improving these properties. Thus, hawaijar can act as a potential diet supplement to overcome malnutrition and degenerative diseases.

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Keishing S, Banu T, Umadevi M (2015) Effect of Fermentation on the Nutrient Content, Antioxidant and Antidiabetic Activities of Hawaijar, an Indigeneous Fer-mented Soya of Manipur, India. J Hum Nutr Food Sci 3(3): 1066.

Cite this article

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