Device-Physics Realization of ZnO-MWCNT Nanostructure-Based Field-Effect Biosensor for Ultrasensitive Simultaneous Genomic Detection of Foodborne Pathogens.

The complex and versatile interactions among the wide variety of the nanostructures and the target analytes have primarily limited the detailed investigation of the transduction mechanism of nanomaterial-assisted electrical signal-based biosensors despite their high sensitivity, low-cost, portability, and ease of deployment. Hence, no common ground is formed detailing the principle of operation, demanding a strong need for systematic examination instead of hit and trial. Therefore, a maiden mechanistic investigation has been carried out in this paper for a field-effect-based biosensor device relying on the energy band diagram and the surface potential profile. To demonstrate the experimental evidence and appreciate the importance of food safety, three hazardous foodborne pathogens (Proteus mirabilis, Escherichia coli, and Clostridium botulinum) have been detected herein. The biosensor device, built on a hydrothermally synthesized zinc oxide and MWCNT (ZnO-MWCNT) composite nanostructure, simultaneously incorporates three fairly specific ss-DNA probes. Furthermore, the unmet challenge of biosensor device variability is addressed through the optimum selection of operating voltage of the device via a unique "voltage-selection-algorithm". We believe that the rigorous experimentation and the insightful device-physics realization demonstrated in this work will pave the way for a future decisive biosensor platform.

Selective extracellular secretion of small double-stranded RNA by Tetragenococcus halophilus.

Small double-stranded RNAs (dsRNAs) abundantly produced by lactic acid bacteria demonstrate immunomodulatory activity and antiviral protective immunity. However, the extracellular secretion of dsRNA from lactic acid bacteria and their compositional and functional differences compared to the intracellular dsRNA is unknown. In this study, we compared the intracellular and secreted extracellular dsRNA of the lactic acid bacteria, Tetragenococcus halophilus, commonly present in fermented foods, by growing in RNA-free and RNase-free media. We used RNA deep sequencing and in-silico analysis to annotate potential regulatory functions for the comparison. A time series sampling of T. halophilus culture demonstrated growth phase-dependent dynamics in extracellular dsRNA secretion with no major change in the intracellular dsRNA profile. The RNA deep sequencing resulted in thousands of diverse dsRNA fragments with 14-21 nucleotides in size from T. halophilus culture. Over 70% of the secreted extracellular dsRNAs were unique in their sequences compared to the intracellular dsRNAs. Furthermore, the extracellular dsRNA abundantly contains sequences that are not T. halophilus genome encoded, not detected intracellularly and showed higher hits on human transcriptome during in-silico analysis, which suggests the presence of extrachromosomal mobile regulatory elements. Further analysis showed significant enrichment of dsRNA target genes of human transcriptome on cancer pathways and transcription process, indicating the extracellular dsRNA of T. halophilus is different not only at the sequence level but also in function. Studying the bacterial extracellular dsRNA is a promising area of future research, particularly for developing postbiotic fermented functional foods and understanding the impact of commensal gut bacteria on human health.

Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension.

Fermented milk is a potential source of various biologically active peptides with specific health benefits. Angiotensin converting enzyme inhibitory (ACE-I) peptides are one of the most studied bioactive peptides produced during milk fermentation. The presence of these peptides is reported in various fermented milk products such as, yoghurt, cheese, sour milk, etc., which are also available as commercial products. Many of the ACE-I peptides formed during milk fermentation are resistant to gastrointestinal digestion and inhibit angiotensin converting enzyme (ACE) in the rennin angiotension system (RAS). There are various factors, which affect the formation ACE-I peptides and their ability to reach the target tissue in active form, which includes type of starters (lactic acid bacteria (LAB), yeast, etc.), substrate composition (casein type, whey protein, etc.), composition of ACE-I peptide, pre and post-fermentation treatments, and its stability during gastrointestinal digestion. The antihypertensive effect of fermented milk products has also been proved by various in vitro and in vivo (animal and human trials) experiments. This paper reviews the literature on fermented milk products as a source of ACE-I peptides and various factors affecting the production and activity of ACE-I peptides.

Three-phase succession of autochthonous lactic acid bacteria to reach a stable ecosystem within 7 days of natural bamboo shoot fermentation as revealed by different molecular approaches.

Microbial community structure and population dynamics during spontaneous bamboo shoot fermentation for production of 'soidon' (indigenous fermented food) in North-east India were studied using cultivation-dependent and cultivation-independent molecular approaches. Cultivation-dependent analyses (PCR-amplified ribosomal DNA restriction analysis and rRNA gene sequencing) and cultivation-independent analyses (PCR-DGGE, qPCR and Illumina amplicon sequencing) were conducted on the time series samples collected from three independent indigenous soidon fermentation batches. The current findings revealed three-phase succession of autochthonous lactic acid bacteria to attain a stable ecosystem within 7 days natural fermentation of bamboo shoots. Weissella spp. (Weissella cibaria, uncultured Weissella ghanensis) and Lactococcus lactis subsp. cremoris predominated the early phase (1-2 days) which was joined by Leuconostoc citreum during the mid-phase (3 days), while Lactobacillus brevis and Lactobacillus plantarum emerged and became dominant in the late phase (5-7 days) with concurrent disappearance of W. cibaria and L. lactis subsp. cremoris. Lactococcus lactis subsp. lactis and uncultured Lactobacillus acetotolerans were predominantly present throughout the fermentation with no visible dynamics. The above identified dominant bacterial species along with their dynamics can be effectively utilized for designing a starter culture for industrialization of soidon production. Our results showed that a more realistic view on the microbial ecology of soidon fermentation could be obtained by cultivation-dependent studies complemented with cultivation-independent molecular approaches. Moreover, the critical issues to be considered for reducing methodological biases while studying the microbial ecology of traditional food fermentation were also highlighted with this soidon fermentation model.

Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting.

BACKGROUND: Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes. RESULTS: We targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping. CONCLUSIONS: We herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used as a reliable tool for rapid and accurate identification of closely related species of M. guilliermondii complex and for differentiating emerging infectious yeasts of the Saccharomycotina CTG clade.

Endophytic fungi of Panax sokpayensis produce bioactive ginsenoside Compound K in flask fermentation.

Endophytes of Panax have the potential to produce their host plant secondary metabolites, ginsenosides. Panax sokpayensis, an endemic traditional medicinal plant of the Sikkim Himalayas was explored for the isolation of endophytic fungi. In the present study, we have isolated 35 endophytic fungal cultures from the rhizome of P. sokpayensis and screened for ginsenosides production by HPLC by comparing the peak retention time with that of standard ginsenosides. The HPLC analysis revealed that out of 35 isolates, the mycelial extracts of four fungal endophytes (PSRF52, PSRF53, PSRF49 and PSRF58) exhibited peaks with a similar retention time of the standard ginsenoside, Compound K (CK). LC-ESI-MS/MS analysis led to the confirmation of ginsenoside CK production by the four fungal endophytes which showed a compound with m/z 639.6278, similar to that of standard ginsenoside CK with yield in potato dextrose broth flask fermentation ranging from 0.0019 to 0.0386 mg/g of mycelial mass in dry weight basis. The four prospective fungal endophyte isolates were identified as Thermothielavioides terrestris PSRF52, Aspergillus sp. PSRF49, Rutstroemiaceae sp. strain PSRF53, and Phaeosphaeriaceae sp. strain PSRF58 based on ITS sequencing. The present finding highlights the need for further study on growth optimization and other culture parameters to exploit the endophytes as an alternative source for ginsenoside CK production.

Comparative analyses of the bacterial communities present in the spontaneously fermented milk products of Northeast India and West Africa.

Introduction: Spontaneous fermentation of raw cow milk without backslopping is in practice worldwide as part of the traditional food culture, including "Doi" preparation in earthen pots in Northeast India, "Kindouri" of Niger and "Fanire" of Benin prepared in calabash vessels in West Africa. Very few reports are available about the differences in bacterial communities that evolved during the spontaneous mesophilic fermentation of cow milk in diverse geographical regions. Methods: In this study, we used high throughput amplicon sequencing of bacterial 16S rRNA gene to investigate 44 samples of naturally fermented homemade milk products and compared the bacterial community structure of these foods, which are widely consumed in Northeast India and Western Africa. Results and discussion: The spontaneous milk fermentation shared the lactic acid bacteria, mainly belonging to Lactobacillaceae (Lactobacillus) and Streptococcaceae (Lactococcus) in these two geographically isolated regions. Indian samples showed a high bacterial diversity with the predominance of Acetobacteraceae (Gluconobacter and Acetobacter) and Leuconostoc, whereas Staphylococcaceae (Macrococcus) was abundant in the West African samples. However, the Wagashi cheese of Benin, prepared by curdling the milk with proteolytic leaf extract of Calotrophis procera followed by natural fermentation, contained Streptococcaceae (Streptococcus spp.) as the dominant bacteria. Our analysis also detected several potential pathogens, like Streptococcus infantarius an emerging infectious foodborne pathogen in Wagashi samples, an uncultured bacterium of Enterobacteriaceae in Kindouri and Fanire samples, and Clostridium spp. in the Doi samples of Northeast India. These findings will allow us to develop strategies to address the safety issues related to spontaneous milk fermentation and implement technological interventions for controlled milk fermentation by designing starter culture consortiums for the sustainable production of uniform quality products with desirable functional and organoleptic properties.

Dynamics of microbial populations and metabolites of fermenting saps throughout tapping process of ron and oil palm trees in Côte d'Ivoire.

Palm wine fermentation is a complex microbial process that evolves with tapping times. The dynamics in microbiota and metabolites throughout palm wine tapping days is still not established, which are critical for the distinctive characteristics of palm wine taste and quality, and thus the mastery of the daily quality fluctuation during tapping. We analyzed the changes in microbial community structure by amplicon sequencing of bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region, and metabolite profile changes using mass spectrometry in palm wine collected over 25-30 days tapping of ron (Borassus aethiopum) and oil palms (Elaeis guineensis) from Côte d'Ivoire. The stage-wise collected palm wine samples showed distinct changes in microbial diversity and pH, supporting microbial community dynamics during palm wine tapping. Results highlighted the dominance of Saccharomyces cerevisiae in early stages and the emergence of non-Saccharomyces yeasts, particularly Hanseniaspora spp. in the later stages of oil palm wine tapping, vice versa in the case of ron palm wine tapping, with a unique presence of Saccharomycodes in the later stages (15-30 days). Fructophilic lactic acid bacteria (FLAB), mainly Fructobacillus and Leuconostoc, encountered in both types of palm wine tapping showed a decline at later stages of oil palm wine tapping. In this type of palm wine, acetic acid bacteria with genera Acetobacter and Glucanoacetobacter, by surpassing Lactobacillus in the last stage become dominant, whereas Lactobacillus remained dominant in ron palm wine throughout tapping days. The decline in the relative abundance of gevotroline and essential amino acids during the later stages of palm wine tapping (15-25 days) supports the difference in the health benefits of the palm wine obtained from different days of tapping, indicating that early stages of tapping is more nutritional and healthy than the later stages. The microbial dynamics may be a potential indicator of metabolite changes during palm sap fermentation, thus contributing to establish particular features of palm wines in different stages of tapping. This understanding of microbial ecology and chemical composition changes during palm wine tapping can be used as biomarkers to assess palm wine's quality and help to design an optimum starter culture.

Geographical markers for Saccharomyces cerevisiae strains with similar technological origins domesticated for rice-based ethnic fermented beverages production in North East India.

Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.

Diversity of beneficial microorganisms and their functionalities in community-specific ethnic fermented foods of the Eastern Himalayas.

The Eastern Himalayan regions of India, Nepal and Bhutan have more than 200 varieties of unsurpassed ethnic fermented foods and alcoholic beverages, which are lesser known outside the world. However, these ethnic foods are region- and community-specific, unique and some are exotic and rare, which include fermented vegetables, bamboo shoots, soybeans, cereals, milk (cow and yak), meats, fishes, and cereal-based alcoholic beverages and drinks. Ethnic communities living in the Eastern Himalayas have invented the indigenous knowledge of utilization of unseen microorganisms present in and around the environment for preservation and fermentation of perishable plant or animal substrates to obtain organoleptically desirable and culturally acceptable ethnic fermented food and alcoholic beverages. Some ethnic fermented products and traditionally prepared dry starters for production of alcoholic beverages of North Eastern states of India and Nepal were scientifically studied and reported till date, and however, limited publications are available on microbiological and nutritional aspects of ethnic fermented foods of Bhutan except on few products. Most of the beneficial microorganisms isolated from some ethnic fermented foods of the EH are listed in microbial food cultures (MFC) safe inventory. This study is aimed to review the updates on the beneficial importance of abundant microbiota and health-promoting benefits and functionalities of some ethnic fermented foods of the Eastern Himalayan regions of North East India, Nepal and Bhutan.

Small double-stranded RNA with anti-HIV activity abundantly produced by Bacillus subtilis MTCC5480 isolated from fermented soybean.

Anti-viral RNA therapy is on high demand nowadays due to the emergence of several new viral infections. The small non-coding regulatory RNAs (dsRNA) from the microbial sources are not yet explored for anti-viral activity. In this study, we assessed the anti-HIV activity of the small dsRNA produced by 12 different microbial species isolated from naturally fermented foods of North-East India. For this, we selectively extracted the dsRNA from the microbial culture, confirmed its double-stranded nature by immunoblotting, and deep sequenced the cDNA library using Illumina platform. Further, we used conventional algorithms to predict the potential targets of the dsRNA sequences within the 3'-UTR region of HIV-1. A small dsRNA fragment with 34 bases in size with a sequence of 3'-UUGGUACACGAGAUGGUUCGACUCGAUGAAGGGC-5' produced abundantly (9.17% of the total dsRNA fraction) by Bacillus subtilis MTCC5480 showed a much higher base complementarity values than previously reported miRNAs analysed against HIV-1. We separated the dsRNA fraction and validated the anti-HIV activity against human peripheral blood mononuclear cells (PBMC) infected with JRCSF strain of HIV-1 virus and the EC50 value ranges from 0.2-0.3 µM. This small dsRNA abundantly produced by B. subtilis could be studied further for its application as an anti-viral therapeutic agent.

Microbial Diversity and Metabolite Profiles of Palm Wine Produced From Three Different Palm Tree Species in Côte d'Ivoire.

Palm wine, the most commonly consumed traditional alcoholic beverage in Western Africa, harbours a complex microbiota and metabolites, which plays a crucial role in the overall quality and value of the product. In the present study, a combined metagenomic and metabolomic approach was applied to describe the microbial community structure and metabolites profile of fermented saps from three palm species (Elaeis guineensis, Raphia hookeri, Borassus aethiopum) in Côte d'Ivoire. Lactobacillaceae (47%), Leuconostocaceae (16%) and Acetobacteriaceae (28%) were the most abundant bacteria and Saccharomyces cerevisiae (87%) the predominant yeasts in these beverages. The microbial community structure of Raphia wine was distinctly different from the others. Multivariate analysis based on the metabolites profile clearly separated the three palm wine types. The main differentiating metabolites were putatively identified as gevotroline hydrochloride, sesartemin and methylisocitrate in Elaeis wine; derivative of homoserine, mitoxantrone in Raphia wine; pyrimidine nucleotide sugars (UDP-D-galacturonate) and myo-Inositol derivatives in Borassus wine. The enriched presence of gevotroline (an antipsychotic agent) and mitoxantrone (an anticancer drug) in palm wine supports its therapeutic potential. This work provides a valuable insight into the microbiology and biochemistry of palm wines and a rationale for selecting functional microorganisms for potential biotechnology applications.

Toxigenic and pathogenic potential of enteric bacterial pathogens prevalent in the traditional fermented foods marketed in the Northeast region of India.

The microbial risk involved with natural food fermentation is largely unknown. Here, we report the prevalence of enteric bacterial pathogens in the traditional fermented foods marketed in Northeast region of India. A total of 682 samples of 39 food types (broadly categorized into fermented soybean, bamboo shoot, fish, milk and pork products) collected over four different seasons from seven states of India were analyzed in this study. Cultivation-independent analysis by MiSeq amplicon sequencing of V4-V5 region of the 16S rRNA gene showed the bacterial community structure in the foods. Among the WHO prioritized foodborne bacterial pathogens, we detected the prevalence of phylotypes related to Clostridium botulinum, Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, Listeria monocytogenes, and Escherichia coli in these ethnic foods. We also observed the occurrence of other well known human enteric pathogens like Proteus mirabilis, Clostridium difficile, and Yersinia enterocolitica. Further pathogen-specific qPCR assays confirmed a higher population (>107 cells/g) of B. cereus, P. mirabilis, and a C. botulinum related phylotype in the fermented soybean, fish, and pork products. We noticed a general trend of higher pathogen occurrence during the colder months without any seasonal variation of total bacterial load in the fermented foods. Further qPCR analysis on toxigenic and pathogenic potential, and toxins production by immunoassays showed that all the soybean samples and the isolated B. cereus cultures were positive for diarrheal toxins (Nhe and Hb1), and nearly half of the samples were positive for emetic toxin (cereulide). Similarly, the food samples and associated swarming P. mirabilis cultures were positive with the pathogenic factors like hemolysin (hpm), urease (ure) and multidrug resistance. However, we could not confirm the presence of botulinum neurotoxin (toxins A, B, E, and F) in the C. botulinum positive food samples. This is the first baseline data of the enteric bacterial pathogens prevalent in the traditional fermented foods of India, which will support the sustained effort of WHO to estimate the global foodborne disease burden. The unusual presence of P. mirabilis in the fermented foods marketed in the Indian region with high incidence of urolithiasis cases is a concern. Our study emphasizes the need of the hour to have a coordinated action to control and prevent the spread of enteric bacterial pathogens through fermented foods marketed in India. Moreover, replacing the indigenous process with a defined starter culture based controlled fermentation will enhance the safety of Indian fermented foods.

High-Throughput Illumina MiSeq Amplicon Sequencing of Yeast Communities Associated With Indigenous Dairy Products From Republics of Benin and Niger.

Traditional Wagashi cheese and fermented cow milk are among the most popular dairy products appreciated by people from Benin, Niger, and the neighboring region. These products are the main source of protein in the diet of the low-income population in the region. The fermented milk is prepared by spontaneous fermentation without back-slopping. Whereas, the leaf extract of Calotropis procera is used for curdling the milk to prepare the soft Wagashi cheese. The present study aims to provide in-depth analysis of yeast communities associated with these traditional milk products by high-throughput Illumina MiSeq amplicon sequencing of internal transcribed spacer (ITS) region of fungal rRNA genes. A total of 60 samples, 20 samples of fermented milk each from Benin and Niger, and 20 samples of Wagashi cheese from Benin were used for analysis. The metagenomic investigation revealed that Kluyveromyces marxianus, Saccharomyces cerevisiae, Candida parapsilosis, and Sagenomella keratitidis were the predominant yeast species present in the traditional milk products. Furthermore, we noticed a high presence of K. marxianus (61.1% relative abundance) in the Wagashi cheese and S. cerevisiae (28.4% relative abundance) in the fermented milk of Niger. The presence of potential pathogenic yeast C. parapsilosis and S. keratitidis in these African milk products calls for further investigation to assess their safety. The predominant yeast K. marxianus and S. cerevisiae, recognized with generally regarded as safe (GRAS) status, could be further selected as starter culture along with lactic acid bacteria for developing controlled fermentation processes with enhanced product quality and safety.

Comparative analysis of the gut microbiota in centenarians and young adults shows a common signature across genotypically non-related populations.

Gut microbiota is among the factors that may be involved in healthy aging. Broader and geographically spread studies on gut microbiota of centenarians can help in identifying a common signature of longevity. We identified an endogamous Indian population with high centenarian prevalence. Here, we compared the gut microbiota composition and fecal metabolites of a centenarians group (˜100 years) with young people (25-45 years) of the region with the high centenarian prevalence and the nearby region of low centenarian prevalence to decipher microbial-related longevity signatures. Also, we compared our results with publicly available datasets of similar groups including 125 centenarians from three countries (Italy, Japan, China). Our comparative analysis resulted in higher biodiversity within Ruminococcaceae in centenarians, with respect to younger adults, irrespective of their nationality. We observed bacterial signatures that are common among extremely old people of different nationality. Comparative metabolites profiling identified the fecal metabolic signature of extreme aging in the Indian study population. Our analysis of the co-occurrence network and bimodal distribution of several taxa suggested the establishment of a pervasive change in the gut ecology during extreme aging. Our study might pave the way to develop gut microbiota based biomarkers for healthy aging.

Production of exopolysaccharide by strains of Lactobacillus plantarum YO175 and OF101 isolated from traditional fermented cereal beverage.

Lactobacillus plantarum YO175 and OF101 isolates from Nigerian traditional fermented cereal gruel 'ogi', were investigated on the basis of their capability to produce exopolysaccharide (EPS) on sucrose modified deMan Rogosa Sharpe medium (mMRS). Functional groups analysis of the EPSs produced (EPS-YO175 and EPS-OF101) by Fourier-transform infrared (FT-IR) spectroscopy revealed the presence of -OH, C=O and C-H groups. The chemical composition of EPS-YO175 and EPS-OF101 showed the presence of 87.1% and 80.62% carbohydrates and 1.21% and 1.47% protein. For maximum EPS yield, three significant factors were optimized using central composite design and response surface methodology, the predicted maximum EPS produced was 1.38 g/L and 2.19 g/L, while the experimental values were 1.36 g/L and 2.18 g/L for EPS-YO175 and EPS-OF101. The EPS samples showed strong antioxidant activities in-vitro. The scale-up of the production process of the EPS will find its potential application in food industries.

Bacterial community in naturally fermented milk products of Arunachal Pradesh and Sikkim of India analysed by high-throughput amplicon sequencing.

Naturally fermented milk (NFM) products are popular ethnic fermented foods in Arunachal Pradesh and Sikkim states of India. The present study is the first to have documented the bacterial community in 54 samples of NFM products viz. chhurpi, churkam, dahi and gheu/mar by high-throughput Illumina amplicon sequencing. Metagenomic investigation showed that Firmicutes (Streptococcaceae, Lactobacillaceae) and Proteobacteria (Acetobacteraceae) were the two predominant members of the bacterial communities in these products. Lactococcus lactis and Lactobacillus helveticus were the predominant lactic acid bacteria while Acetobacter spp. and Gluconobacter spp. were the predominant acetic acid bacteria present in these products.

Quantifying the biases in metagenome mining for realistic assessment of microbial ecology of naturally fermented foods.

Cultivation-independent investigation of microbial ecology is biased by the DNA extraction methods used. We aimed to quantify those biases by comparative analysis of the metagenome mined from four diverse naturally fermented foods (bamboo shoot, milk, fish, soybean) using eight different DNA extraction methods with different cell lysis principles. Our findings revealed that the enzymatic lysis yielded higher eubacterial and yeast metagenomic DNA from the food matrices compared to the widely used chemical and mechanical lysis principles. Further analysis of the bacterial community structure by Illumina MiSeq amplicon sequencing revealed a high recovery of lactic acid bacteria by the enzymatic lysis in all food types. However, Bacillaceae, Acetobacteraceae, Clostridiaceae and Proteobacteria were more abundantly recovered when mechanical and chemical lysis principles were applied. The biases generated due to the differential recovery of operational taxonomic units (OTUs) by different DNA extraction methods including DNA and PCR amplicons mix from different methods have been quantitatively demonstrated here. The different methods shared only 29.9-52.0% of the total OTUs recovered. Although similar comparative research has been performed on other ecological niches, this is the first in-depth investigation of quantifying the biases in metagenome mining from naturally fermented foods.

Bacterial dynamics during yearlong spontaneous fermentation for production of ngari, a dry fermented fish product of Northeast India.

Ngari is the most popular traditionally processed non-salted fish product, prepared from sun-dried small cyprinid fish Puntius sophore (Ham.) in Manipur state of Northeast India. The microbial involvement in ngari production remained uncertain due to its low moisture content and yearlong incubation in anaerobically sealed earthen pots without any significant change in total microbial count. The culture-independent PCR-DGGE analysis used during this study confirmed a drastic bacterial community structural change in comparison to its raw material. To understand the bacterial dynamics during this dry fermentation, time series samples collected over a period of nine months through destructive sampling from two indigenous ngari production centres were analysed by using both culture-dependent and culture-independent molecular methods. A total of 210 bacteria isolated from the samples were identified by amplified ribosomal DNA restriction analysis (ARDRA) based grouping and 16S rRNA gene sequence similarity analysis. The dominant bacteria were Staphylococcus cohnii subsp. cohnii (38.0%), Tetragenococcus halophilus subsp. flandriensis (16.8%), a novel phylotype related to Lactobacillus pobuzihii (7.2%), Enterococcus faecium (7.2%), Bacillus indicus (6.3%) and Staphylococcus carnosus (3.8%). Distinct bacterial dynamics with the emergence of T. halophilus at third month (10(6)CFU/g), L. pobuzihii at sixth month (10(6)CFU/g), S. carnosus at three to six months (10(4)CFU/g) and B. indicus at six to nine months (10(5)CFU/g) in both the production centres was observed during ngari fermentation. However, the other two dominant bacteria S. cohnii and E. faecium were isolated throughout the fermentation with the population of 10(6)CFU/g and 10(4)CFU/g respectively. Culture-independent PCR-DGGE analysis further showed the presence of additional species, in which Kocuria halotolerans and Macrococcus caseolyticus disappeared during fermentation while Clostridium irregulare and Azorhizobium caulinodans were detected throughout the fermentation. Principal component analysis showed a drastic bacterial community structural change at the sixth month of fermentation. These identified dominant bacterial cultures of T. halophilus, L. pobuzihii, S. carnosus and B. indicus could be effectively utilised for designing starter culture and optimizing fermentation technology for industrialisation of ngari production.

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.