Storage study of Pre-cooked and Post-cooked Tungrymbai (Fermented Soy Food of Khasi Hills, Meghalaya)

Tungrymbai is a traditional fermented food of Khasi and Jaintia tribes of Meghalaya. In this study Tungrymbai was analyzed for physico-chemical and microbial quality of pre-cooked and post-cooked samples prepared by using Lactobacillus fermentum and Lactobacillus plantarum culture combination in 1:1 ratio at 1, 2 and 3% culture combination. Shelf-life of the product was carried out by storing at 6 ̊C and 33 ̊C for 4 days. pH was found to decrease while titratable acidity increases as the number of storage days increases. Protein content was highest in Tungrymbai with 3% cell biomass. pH and titratable acidity was more in pre-cooked samples, whereas protein content was more in post-cooked samples. Lactobacillus count was found in all the samples, yeast and mold was observed only in the 4th day of storage and coliforms was absent in precooked samples. However, microbes were absent in post-cooked sample I and only Lactobacillus strain were detected in post-cooked samples II which indicates that the cooking procedure and heat intensity while cooking affect the Lactobacillus strains. Therefore, awareness can be created for the preparation method of Tungrymbai without cooking the product, to get the beneficial properties of Tungrymbai prepared with Lactobacillus strains.

Soybean is a major leguminous crop in the world, and its application as food nutrients are generally confined to Asia (Shurtleff et al. 2010). Tungrymbai is a naturally fermented soybean food prepared by Khasi and Jaintia tribes of Meghalaya in India (Tamang et al. 2009). Tungrymbai is prepared by fermenting the soybean seeds for 3-4 days. It is primarily consumed as a side dish throughout winter season. The North-Eastern region of India is well known for the preparation and consumption of many diversities of native fermented food. These fermented foods provide essential components beneficial for the health with nutritious values, flavour, palatability and texture (Sohliya et al. 2009;Tamang et al. 2009. Traditional fermentation is a method of food processing done by using microorganisms, especially lactic acid bacteria (LAB), and yeast (Guarner et al. 2008). Unlike their unfermented counterparts, fermented foods are preferred by consumers because of their typical taste, texture, and colour (Nout et al. 1997). The function of LAB in increasing the shelf life and nutritional quality of fermented foods and beverages, controlling diarrhea, in addition to their antimicrobial properties have been established (Thokchom et al. 2012). These microorganisms are harmless and have enzymes such as proteases, amylases and lipases that hydrolyze food complexes into simple non-toxic products (Alokail et al. 2013;Steinkraus 1997). LAB, involved in the fermentation, is associated with substrate utilization, flavour promotion, food preservation and probiotic properties (Leroy andDe Vuyst, 2004, Chao et al. 2009;Tu et al. 2010;Liu et al. 2011). The present paper aim to analyse the physico-chemical and microbial quality of pre-cooked and post-cooked Print ISSN:  Online ISSN: 2321-712X Tungrymbai, to study the shelf-life and the effect of cooking processes in the preparation of post-cooked Tungrymbai.

Collection of soybean sample and other materials
Local variety of soybean (Glycine max (L.) Merill), ginger, garlic, black sesame seeds, mustard oil, salt and chillies were obtained from the local market of Meghalaya.

Microorganisms and Media
The strains used in this study were obtained from fermented foods of Meghalaya by the department of RDAP, NEHU Tura Campus. Pure bacterial strain Lactobacillus fermentum and Lactobacillus plantarum was transfer in de Mann, Rogosa and Sharpe (MRS broth) (M255, HiMedia, India) and incubate at 37˚C for 24 hours. The activated culture was inoculated in MRS broth and incubated at 37˚C for 16 hours. These working cultures were then transferred into skim milk medium to check their activity in this medium thereby evaluating the growth of these cultures in skim milk (Gajbhiye et al. 2012).

Starter culture(s) preparation
A loopful culture of Lactobacillus fermentum and Lactobacillus plantarum was inoculated in 10 ml MRS broth (M255, HiMedia) and incubated overnight at 37˚C. One ml of each culture was centrifuge at 10,000 RPM for 15 minutes, the supernatant was discarded and one ml of sterile saline was added to the pellet, cells were resuspended and again centrifuged at 10,000 RPM for 10 minutes, the supernatant was discarded and one ml of sterile distilled water was added. Through this procedure the desired inoculum was achieved (Hati et al. 2014).

Preparation of Tungrymbai in traditional way (control)
Local variety of soybean was used and about 50g of soybean was cleaned, washed and soaked in 100ml Reverse Osmosis (RO) water and kept overnight at room temperature. Soaked soybeans was cleaned and without dehulling it was boiled in pressure cooker for 15 minutes at 100˚C till it softens. The cooked soybeans were transferred into a pre-sterile bamboo basket aligned with fresh leaves of Clinogyne dichotoma locally known as "slamet", and covered on top by the same leaves. The bamboo basket was wrapped with sterile muslin cloth and kept for fermentation for 3-4 days at 37˚C (Thokchom and Joshi 2012).

Laboratory scale preparation of Tungrymbai using different culture combinations
About 50g of soybean was cleaned, washed and soaked in 100ml Reverse Osmosis (RO) water and kept overnight at room temperature. Soaked soybeans was cleaned and without dehulling it was boiled in pressure cooker for 15 minutes at 100˚C till it softens. The cooked soybean is allowed to cool till it reaches 30˚C. It is then transferred into a sterile bamboo basket aligned with fresh leaves of Clinogyne dichotoma; inoculate with the cell biomass of Lactobacillus fermentum and Lactobacillus plantarum in 1: 1 ratio in different percentages of 1, 2, and 3%. Slamet leaves are then covered on top of the soybean. The whole basket was wrapped with sterile muslin cloth and kept for fermentation in an incubator at 37˚ C for 3-4 days (Thokchom and Joshi 2012).

Preparation of post-cooked Tungrymbai:
The sample was divided into two parts Sample I and Sample II.
Sample I: Firstly, Mustard oil was heated in a pan at 100˚C; garlic paste was added and fried until golden brown. Next, pre-cooked Tungrymbai sample was added and fried until brownish in colour followed by grounded chillies, black sesames seeds and salt, 50ml of RO water was poured for mixing the ingredients properly. The product was cooked for 5-10 minutes and ginger was added for garnishing.
Sample II: Mustard oil was heated in a pan at 100˚C; garlic paste was added and fried until golden brown, followed by spices like grounded chillies, black sesames seeds and salt were added. 50ml of RO water was poured for mixing the ingredients properly. The mix was cooked for 5-10 minutes. The spices was allowed to cool down till 25-30˚C and pre-cooked Tungrymbai sample was mixed with it, ginger was added for garnishing.
Physico-chemical analysis: The samples were analyzed for their pH, total titratable acidity and total protein content according to AOAC, 1990.

Determination of pH
This experiment was carried out with the help of digital pH meter (Hanna instruments, Model HI96107) after fermenting soybean for 3-4 days of incubation at 37 o C. The readings for all the samples were taken accordingly for 1, 2, 3 and 4 days.

Determination of Total titratable acidity
Total titratable acidity was determined by using 0.1N Sodium Hydroxide solution. 5ml of liquid soybean sample was taken in a beaker (previously obtained by homogenizing 10gm of soybean sample with 90ml of sterile distilled water) and mixed with 10ml distilled water and two drops of phenolphthalein indicator was added. Each sample was titrated with 0.1N NaOH solution until the sample turn light pink in colour. The reading was taken accordingly and recorded for each sample. The percent acidity was expressed in terms of % lactic acid. The titratable acidity was calculated as percent lactic acid as follows: % of acidity = [mls of NaOH used] × [0.1 N NaOH] × [0.09g (milliequivalent factor)] × [100]/ grams of sample used.

Estimation of protein
This experiment was performed by using Biuret method. Freshly prepared solution of standard bovine serum albumin (0.1g of BSA is diluted in 5 ml sterile distilled water) is used and freshly prepared biuret reagent is taken. A standard solution of BSA with a concentrations range of 0, 2, 4, 6, 8 and 10 mg/ ml was taken in test-tubes by keeping the volume of BSA solution 1ml in each test-tube), 1ml of unknown sample is taken in each test tubes and 5ml of Biuret reagent is added into all the test tubes and was properly mixed. The samples were kept in the dark at room temperature for 20 minutes and absorbance was recorded using spectrophotometer at 550nm within 10-15 minutes. A graph is plotted, with protein concentration on X axes and absorbance at Y axes and a standard graph was prepared. The values of protein in unknown samples were calculated by using slope value of the graph.

Microbial analysis
10 grams of soybean sample was mixed well in a sterile mortar and pestle and homogenised with 90 mL of sterile distilled water. From this solution, 1ml of liquid sample was taken and was mixed thoroughly with 4ml of sterile 0.1% peptone water and serial dilutions was performed.  Bristone et al. 2016 in the study of yoghurt fermentation, the decrease in pH and increase in titratable acidity was found to be a desired quality and characteristics of good yoghurt. The decrease in pH may be due to the production of organic acids. Similar results were observed by Maftei et al. 2012 in which soymilk was combined with different percentages of sea buckthorn syrup and fermented with a culture of Bifidobacterium bifidus.    Tungrymbai (control) with 98.52-80.20 mg/ml at 6˚C and 106.983 -90.137 mg/ml at 33˚C. The protein content of laboratory prepared Tungrymbai was found to be more than the traditional product. It can be assumed that fermentation with Lactobacillus cell biomass slightly increases the protein content of Tungrymbai as reported by Mohiedeen et al. 2010 there was a slight gain of protein content during microbial growth in maize fermentation. From the result, it was observed that there was a decrease of protein content as the number of storage days increases, this could be due to protein degradation by proteolytic activities of microorganisms. Similar reports were found by Khetarpaul and Chauhan et al. 1989 in the study of effect of fermentation on protein of pearl millet, and in the study of soy proteins during fermentation of thuanao inoculated with Bacillus subtilis (Visessanguan et al. 2005).

Microbial analysis of pre-cooked Tungrymbai samples
The microbial analysis was determined through Lactobacillus, Coliforms, Yeast and mold viable cell count. Analysis was done for the study of shelf-life and to examine the presence of pathogenic organisms in the product. Samples were stored for 1, 2, 3 and 4 th days at 6˚C and 33˚C. Lactobacillus counts were found to be highest in Tungrymbai prepared with 3% cell biomass ranging from 8.199 ± 0.109 to 8.158 ± 0.150 log cfu/ml at 6˚C and 9.057 ± 0.200 to 8.882 ± 0.208 log cfu/ml at 33˚C. Yeast and mold count was absent in 1, 2 and 3 rd day of storage, but found only on 4 th day. Coliforms were not observed in all four days of storage. From the results it can be concluded that the product was free from any contamination, while only on the 4 th day of storage contamination was observed which may arise from the surrounding environment. Thus, we can assume that all the samples were safe and have the characteristics of good quality product. Similar results was observed by Thokchom and Joshi., 2013 in which Enterobacteriaceae and LAB count was highest in Tungrymbai sample, while coliforms, yeast and mold count was comparatively less than the bacterial counts. Other reports were observed that LAB population was more compared to microbial load of yeasts in log CFU/ml in the Tungrymbai sample (Chettri and Tamang., 2014).
Physico-chemical analysis of post-cooked Tungrymbai samples:

pH and Titratable acidity of post-cooked Tungrymbai samples
It was observed that the pH and titratable acidity was more in sample II than in sample I of Tungrymbai. In Sample I, the pH was found to be 7.0 ± 0.078 and 7.05 ± 0.141 for traditional sample and 1% cell biomass samples. The titratable acidity was observed to be 0.061 ± 0.141 and 0.060 ± 0.012 for traditional and 1% cell biomass sample. In Sample II, the pH was found to be 7.10 ± 0.124 and 7.12 ± 0.070 for traditional and 1% cell biomass sample. The titratable acidity was observed to be 0.054 ± 0.056 and 0.063 ± 0.070 for traditional and 1% cell biomass sample. The pH and titratable acidity was found to be lesser in post cooked sample than in pre-cooked sample. This could be due to the preparation method and ingredients used when cooking, which was similar with other studies done by Thokchom and Joshi., 2013 in which the pH and titratable acidity was lower in the post-cooked Tungrymbai sample.

Total protein content of post-cooked Tungrymbai samples
The total protein content was found to be more in sample II than in sample I. In sample I, the protein content was 116.094 and 121.309 mg/ml for traditional and 1% cell biomass. Whereas, in sample II, the protein content was 136.679 and 142.242 mg/ ml for traditional and 1% cell biomass samples. During protein analysis it was found that Tungrymbai samples inoculated with Lactobacillus cell biomass has slightly more protein content than traditional Tungrymbai samples this may be due to the microbial growth during fermentation which was in agreement with Mohiedeen et al. 2010 in the study of maize fermentation there was a slight gain of protein content during microbial growth. Sample II Tungrymbai has more protein content than sample I, this could be due to the preparation method and heat intensity while cooking the samples. Similar reports were found by Sohliya et al. 2009 in the study of chemical changes during preparation of Tungrymbai.

Microbial Analysis of Post cooked Tungrymbai samples
The microbial analysis of post-cooked sample showed that Lactobacillus count was present only in Sample II and not in Sample I of Tungrymbai. In sample II the microbial load was found to be 6.65 ± 0.150 and 7.31 ± 0.112 (log CFU/ml) for traditional and Tungrymbai sample prepared with 1% cell biomass. This can be due to the heat generated from cooking temperature in which the Lactobacillus species cannot survive. Coliforms, Yeast and mold viable cell count were found to be absent in all the samples. Other findings have been reported by Thokchom and Joshi., 2012 were Lactobacillus species are absent in the postcooked samples of Tungrymbai. Absence of coliform implies that the samples were contamination free, and has good quality standard. Other findings have been reported by Oyeniyi et al. 2014 in the study of soy-yoghurt sample. Hence, sample II shows better result compared to sample I.

CONCLUSION
Lactic acid bacteria have been widely used in food industries due to their probiotic attributes. In the present investigation, physico-chemical and microbial compositions of Tungrymbai were analyzed for both pre-and post-cooked samples. It was found that the pH and titratable acidity decrease in postcooked Tungrymbai samples, whereas, the protein content was more in post-cooked samples which may be due to the preparation method and spices used while cooking the samples. Through microbial analysis, it was found that Tungrymbai prepared with Lactobacillus fermentum and Lactobacillus plantarum was more in Lactobacillus count as compared to the traditional sample. However, it was observed that the microbial load was absent in sample I post-cooked Tungrymbai and present only in sample II, this could be due to the heat generated while cooking, in which all the beneficial microbes did not survived. Shelflife study revealed that the product is free from contamination and pre-cooked Tungrymbai could be kept for 4 days without getting spoiled. Further, Tungrymbai prepared with Lactobacillus fermentum and Lactobacillus plantarum was found to be more beneficial with probiotic attributes than the traditional Tungrymbai and could be used as an alternative for the preparation of traditional product. Moreover, awareness can be created for the preparation method of Tungrymbai, so that consumers can get the beneficial properties of Tungrymbai prepared with Lactobacillus fermentum and Lactobacillus plantarum.

ACKNOWLEDGMENTS
The authors are grateful to North-Eastern Hill University for the financial support for the research fellowship.