Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain\'s Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30-70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5-9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens\' plots showed the protease had Vmax of 1.001 U/ml with corresponding Km value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them.

Kinema is a traditional food prepared by the natural fermentation of cooked soybeans. The fermented Kinema is known to have several bioactive constituents, however, only limited reports on the effect of fermentation time on the bioactivity of Kinema are available. Therefore, in this work, changes in phenolics content and radical scavenging activity of Kinema at different fermentation times were explored. Furthermore, the optimum fermentation time for maximum bioactivities (total phenolic content, total flavonoid content, and DPPH radical scavenging activity) was determined using one-factor response surface methodology. The numerical optimization suggested an optimum fermentation time of 29.6 h with significantly higher total phenolics and flavonoid contents of 62.84 ± 0.89 mg GAEs/g dry extract, 45.41 ± 0.57 mg QEs/g dry extract, respectively (p < 0.05) compared to traditionally fermented Kinema. Similarly, the IC50 concentration for DPPH radical scavenging activity of 1.78 ± 0.01 mg dry extract/mL, was significantly lower than those for traditionally prepared Kinema (p < 0.05). Moreover, optimized Kinema had significantly higher overall sensory scores compared to the traditional sample. The results suggested that fermentation time affects the amount of bioactive constituents of Kinema. Further studies are needed to explore the changes in the type of phenolic and flavonoid compounds.

COVID-19, which is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), is the deadliest outbreak of this millennium. Despite adopting several precautionary strategies and guidelines, COVID-19 has spread rapidly, and the number of cases is still in escalation across the world. The various immune-boosting drugs with severe side effects and the vaccines approved after negotiated clinical trials have been struggling to cope with the emergence of new variants of the virus. Nevertheless, given a large number of asymptomatic cases, a high magnitude of recovery rate, and a relatively higher prevalence of morbidity and mortality among immunologically compromised individuals, those affected by an illness, and the elderly, it appears that a healthy microbiome and the associated immune responses are the key factors for survival. Incidentally, the consumption of traditionally popular and nutritious fermented foods, which are composed of biologically functional ingredients and several health-promoting probiotics, offers promising health benefits through the improvement of the immune system in general. Given the progress in functional food research, it has become crucial to understand the impact of a healthy microbiome and the immunomodulatory roles of fermented foods on the battles to combat infectious diseases. Based on the evidence of the impact of probiotics-based fermented foods, the beneficial roles of a few frequently consumed fermented foods in the management of various infections have been resolutely discussed in the present study, with a focus on their antagonistic and immune-modulating effects in the context of the current COVID-19 pandemic.

Bacterial and fungal communities in kinema, a naturally fermented soybean food of the Eastern Himalayan regions of India, Nepal and Bhutan were profiled by high-throughout sequence analysis. Firmicutes (78.4%) was the most abundant phylum in kinema, followed by Proteobacteria (14.76%) and other phyla. Twenty seven species of Bacillus were detected, among which Bacillus subtilis (28.70%) was the most abundant bacterium, followed by B. licheniformis, B. thermoamylovorans, B. cereus, Ignatzschineria larvae, Corynebacterium casei, B. sonorensis, Proteus vulgaris, Brevibacillus borstelensis, Thermoactinomyces vulgaris, Lactobacillus fermentum and Ignatzschineria indica. Ascomycota was the most abundant fungal phylum in kinema. Wallemia canadensis, Penicillium spp., Aspergillus spp., Exobasidium spp., Arthrocladium spp., Aspergillus penicillioides, Mortierella spp., Rhizopus arrhizus and Mucor circinelloides, were major moulds, and Pichia sporocuriosa, Trichosporon spp., Saccharomycopsis malanga and Rhodotorula cycloclastica were abundant yeasts in kinema. We detected 277 species of bacteria among which, 99.09% were culturable and 0.91% were unculturable; and 80 fungal species among which, 33.72% were culturable and 66.28% were unculturable. Several unique bacterial genera to each country were observed, whereas no unique fungal genus was observed in kinema. Maximum coverage of sequencing depth was observed in all samples. Based on PCA plot, close relation was observed between samples of India and Nepal, whereas samples of Bhutan was clearly distinctive. Predictive functional features of bacterial and fungi related to metabolisms were inferred by the KEGG Orthology and MetaCyc databases, respectively.

In this study, the lipopeptide biosurfactant was extracted, purified and characterized from the Bacillus isolate LK5.4 obtained from kinema samples of Sikkim. Plant growth-promoting property of the biosurfactant producing bacterium was also evaluated. Out of fifty-seven isolates, only ten were biosurfactant producer as determined by the oil displacement test. Bacillus isolate LK5.4 showed the maximum emulsification index (52.3 ± 0.02), reduced surface tension up to 40% and produced 754 mgL-1 biosurfactant in the nutrient broth. Based on 16S  rRNA gene sequencing, the isolate LK5.4 was identified as B. tequilensis. Biosurfactant was purified by Thin Layer Chromatography (TLC). Evaluation of the chemical characteristics by TLC, Liquid Chromatography-Mass Spectrometry, Fourier Transform Infrared Spectroscopy and Nuclear Magnetic Resonance Spectroscopy identified the biosurfactant as surfactin. The effect of different concentration of biosurfactant in maize seed germination was evaluated under in vitro condition. It showed the fastest growth of seedlings at 300 µg/ml biosurfactant solution. Similar results were shown by the potted plant experiment, where the soil was directly treated with biosurfactant producing bacterium LK5.4. The LK5.4 treated plants showed a mean height of 29.17 ± 0.47 cm and mean leaf length of 18.42 ± 0.17 cm while the mean height and mean length of the leaf were 15.48 ± 0.98 cm and 11.12 ± 0.40 cm respectively in the control plants. The treated plants had higher moisture content (68.48 ± 2.79%) than the control plants (50.53 ± 1.63%), which is because of higher bioadsorption in the treated plants. These results provided indirect evidence of plant growth-promoting property of the biosurfactant.

Kinema is an ethnic, naturally fermented soybean product consumed in the Sikkim Himalayan region of India. In the present study, the whole metagenome sequencing approach was adopted to examine the microbial diversity and related functional potential of Kinema, consumed in different seasons. Firmicutes was the abundant phylum in Kinema, ranging from 82.31 to 93.99% in different seasons, followed by Actinobacteria and Proteobacteria. At the species level, the prevalent microorganisms were Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Corynebacterium glutamicum, Bacillus pumilus, and Lactococcus lactis. The abundance of microbial species varied significantly in different seasons. Further, the genomic presence of some undesirable microbes like Bacillus cereus, Proteus mirabilis, Staphylococcus aureus, Proteus penneri, Enterococcus faecalis, and Staphylococcus saprophyticus, were also detected in the specific season. The metagenomic analysis also revealed the existence of bacteriophages belonging to the family Siphoviridae, Myoviridae, and Podoviridae. Examination of the metabolic potential of the Kinema metagenome depicted information about the biocatalysts, presumably involved in the transformation of protein and carbohydrate polymers into bioactive molecules of health-beneficial effects. The genomic resource of several desirable enzymes was identified, such as β-galactosidase, β-glucosidase, β-xylosidase, and glutamate decarboxylase, etc. The catalytic function of a novel glutamate decarboxylase gene was validated for the biosynthesis of γ-aminobutyric acid (GABA). The results of the present study highlight the microbial and genomic resources associated with Kinema, and its importance in functional food industry.

The objective of this work was to optimise different parameters (solvent concentration, time and temperature) for antioxidant extraction from kinema, demonstrated by total phenol content (TPC) and DPPH-scavenging activity (DSA), using response surface methodology. A central composite design was performed to determine the effect of solvent concentration (methanol, 30-100%), temperature (30-60°C) and time (30-150min) on the TPC and DSA of the extract. The solvent concentration and temperature had the most significant (p<0.05) effect. The optimum conditions for the TPC extraction and DSA were 100% methanol, 50°C and 30min, in which 140mg of gallic acid equivalent (GAE) g-1 lyophilised extract and 52% DSA were predicted, while 135mg GAE g-1 lyophilised extract and 56% DSA were experimentally obtained. No significant difference (p<0.05) was found between the experimental and predicted values of the response variables.

Kinema, an ethnic fermented, non-salted and sticky soybean food is consumed in the eastern part of India. The stickiness is one of the best qualities of good kinema preferred by consumers, which is due to the production of poly-γ-glutamic acid (PGA). Average load of Bacillus in kinema was 10(7) cfu/g and of lactic acid bacteria was 10(3) cfu/g. Bacillus spp. were screened for PGA-production and isolates of lactic acid bacteria were also tested for degradation of PGA. Only Bacillus produced PGA, none of lactic acid bacteria produced PGA. PGA-producing Bacillus spp. were identified by phenotypic characterization and also by 16S rRNA gene sequencing as Bacillus subtilis, B. licheniformis and B. sonorensis.

OBJECTIVE: Optimization of traditional processing of soybeans using response surface methodology (RSM) to achieve a minimum level of antinutritional factors (ANFs) in kinema.
RESULTS: Central composite rotatable designs were used to optimize the processing stages of kinema preparation. In each stage, the linear or quadratic effects of independent variables were significant in minimizing ANF levels. The predicted optimum condition for soaking was when the raw beans-water ratio was 1 : 10, and the soaking temperature, time and pH were 10°C, 20 h and 8·0 respectively. Here, tannins content (TC), phytic acid content (PAC) and trypsin inhibitor activity (TIA) decreased (P < 0·05). While haemagglutinating activity (HA) level remained unchanged (P < 0·05), total biogenic amines content (TBAC) increased. The optimum condition for cooking was optimally soaked beans-water ratio of 1 : 5, and cooking pressure and time were 1·10 kg cm(-2) and 20 min respectively. Here, TC, PAC, TIA and HA decreased (P < 0·05), but TBAC remained unchanged compared to optimally soaked beans. TC and HA went below the level of detection. The optimum condition for fermentation was obtained when inoculum load was 10(3) total cells g(-1) grits, and fermentation temperature and time were 37°C and 48 h respectively. Fermentation of optimally cooked beans caused a reduction (P < 0·05) of PAC. While TIA remained unchanged (P < 0·05), TBAC increased. In kinema, TC, PAC, TIA and HA decreased (P < 0·05) over raw beans by 100, 61, 71 and 100% respectively. Good agreement was observed between predicted values and experimental values.
CONCLUSIONS: The processing treatments significantly minimized the level of ANFs in soybeans.
CONCLUSIONS: RSM was successfully deployed to obtain the optimum condition for kinema-making with a minimum level of ANFs without impairing sensory attributes of the product. The results are useful for commercial production of kinema.