Elsevier

Food Bioscience

Volume 35, June 2020, 100570
Food Bioscience

Physiological and genomic characterization of an exopolysaccharide-producing Weissella cibaria CH2 from cheese of the western Himalayas

https://doi.org/10.1016/j.fbio.2020.100570Get rights and content

Highlights

  • The exopolysaccharide producing strain CH2 was isolated from a fermented milk product.

  • The complete genome of Weissella cibaria CH2 comprised a chromosome (2.6 Mb) and 6 plasmids.

  • The strain had attributes that would qualify it as a potential probiotics in food formulations.

  • Genomics provided a genetic basis for the various functional proteins responsible for probiotic attributes.

Abstract

Weissella cibaria CH2 is an exopolysaccharide (EPS) producing bacterium, isolated from cheese. Maximum EPS production (81.3%) was observed with sucrose at 20 °C. The CH2 strain was tolerant of simulated gastric juice (pH 3) with a 5.3% decrease in the viable count after 180 min, but not of pH 2. Similarly, no significant decrease in the viable count was observed in the presence of simulated intestinal juices (pH 8) and bile salts (0.3%) after 240 min. The CH2 strain showed good aggregation ability and antimicrobial activity against representative pathogenic bacteria that may promote the colonization of the CH2 strain in the gastrointestinal tract. Complete genome sequencing supplemented the experimental evidence as genes encoding for EPS production, gastrointestinal tolerance, and adhesion were observed in the CH2 genome. Genomic analysis also showed the presence of functional traits such as cholesterol reduction, β-galactosidase production, vitamin, and essential amino acid biosynthesis. The absence of transferable antibiotic resistance genes and virulence factors in the CH2 strain was further confirmed using genome sequencing. The present study highlights the potential of the CH2 strain for use as a probiotic.

Introduction

Probiotic fermented foods are considered an important component of the human diet due to their nutritive value and purported health benefits. Probiotics not only influence gut microbiota and maintain intestinal integrity, but also strengthens the intestinal immune responses (Hemarajata & Versalovic, 2013). However, the beneficial health effects of probiotics are specific and variations are observed even within different strains of a particular species (Campana, van Hemert, & Baffone, 2017). Most of the commercial probiotic strains available are of either Western or European origin (Kaushik et al., 2009). Therefore, there is a growing scope for identification of indigenous probiotic strains from different geographical areas with specific health benefits. The western Himalayas represent relatively unexplored environments particularly with respect to their probiotic diversity. People living in these regions are generally healthier even under the extreme stress of a high-altitude environment, with a diet mainly based on traditional fermented foods (Kanwar, Gupta, Katoch, Kumar, & Kanwar, 2007; Tamang, Okumiya, & Kosaka, 2010). This suggested that the traditional fermented foods of the western Himalayan region of India might be studied for isolation of potential probiotic strains. Indigenous microbiota belonging to the genera Lactobacillus, Leuconostoc, Pediococcus, Enterococcus, Bacillus, Saccharomyces, Kluveromyces, and Candida sp. have been found in the traditional fermented food of H.P., India (Sharma, Gupta, & Handa, 2014).

The WHO has formulated specific guidelines for screening of a potential probiotic strain (FAO and WHO, 2002). However, the physiological characterization of probiotic bacteria alone may not provide sufficient information about the unrecognized metabolic capacities and functionalities of a particular probiotic. Therefore, the recent emphasis on genomic characterization of probiotics strains may allow better understanding of additional functions and properties of the microbes. Further, genomics allowed the identification of genes responsible for virulence and antibiotic resistance (Oniciuc et al., 2018), thus, it has been used as an important tool for assessing the safety of lactic acid bacteria (LAB). In the present study, an exopolysaccharide (EPS) producing LAB strain was isolated from cheese; a traditional cow's milk-based product from the Lahaul valley of H.P. LAB with EPS production have been preferred as a probiotic in functional foods (Patel & Prajapat, 2013). EPS contributes to the probiotic characteristics of bacteria by enhancing their colonization and health benefits in the gastrointestinal tract (GIT) (Lebeer, Claes, Verhoeven, Vanderleyden, & De Keersmaecker, 2011; Nwodo, Green, & Okoh, 2012; Stack, Kearney, Stanton, Fitzgerald, & Ross, 2010). EPS from LAB have also been used to improve the texture and stability of fermented foods, as well as being used as therapeutics and prebiotics.

An EPS-producing LAB strain, W. cibaria CH2 was obtained from cheese and checked for its probiotic attributes, i.e., gastrointestinal tolerance, antimicrobial activity, cell surface hydrophobicity, autoaggregation and antibiotic sensitivity. To understand the genetic basis of probiotic attributes, whole genome sequence analysis was done. Genomic data was explored to get insight into other health beneficial attributes of the CH2 strain.

Section snippets

Sample collection and isolation of bacteria

The cheese was prepared locally and collected from the village of Tandi (elev. 3002 m), Lahaul and Spiti district of H.P, India. The sample was hygienically prepared from fresh cow's milk using a mixed strain of Lactobacillus microbial coagulant and collected in a sterilized container, brought to the laboratory with an icebox and kept at 4 °C for a maximum of two days. For isolation of LAB, samples were processed using serial dilution and spread plate technique on MRS agar (de Man, Rogosa, and

Identification of CH2 strain

The colonies of the CH2 strain were opaque, slimy and circular with entire margins. The bacteria were seen to be Gram-positive small rods and catalase-negative. On the basis of partial 16S rRNA gene sequencing, the CH2 strain (1477 bp length) showed maximum similarity with sp. W. cibaria KACC 11862(T) (100%) in the EzBioCloud database. Using phylogenetic clustering based on 16S rRNA gene sequences, the CH2 forms a monophyletic cluster with the type strain W. cibaria KACC 11862(T) with bootstrap

Conclusion

The EPS-producing CH2 strain qualified as a potential probiotic bacterium while testing for the requisite attributes. The complete genome analysis provided information about its acid/bile tolerance, adhesion, antimicrobial activity, and other health-promoting properties. This supported the strains efficiency to be used as a potential probiotic bacterium. An important advantage of W. cibaria CH2 strain is the absence of transferable antibiotic resistance genes and virulence factors. However, in

Nucleotide sequence accession number

The complete genome sequence of the CH2 strain has been deposited with the DDBJ/EMBL/GenBank (www.ncbi.nlm.nih.gov/genbank/) with the accession no. CP012873-CP012878 and CP012880.

CRediT authorship contribution statement

Madhu Kumari: Formal analysis, Data curation, Software. Rakshak Kumar: Methodology. Dharam Singh: Resources. Shriya Bhatt: Investigation. Mahesh Gupta: Conceptualization, Writing - review & editing, Supervision.

Declaration of competing interest

The authors confirm that they have no conflicts of interest with respect to the study described in this manuscript.

Acknowledgments

MK is thankful to CSIR-HRDG for the senior research fellowship, CSIR-India. The authors acknowledge financial support from the CSIR network project BSC0125. Authors also acknowledge the technical support by Mohit Kumar Swarnkar to carry out the whole genome sequencing. This manuscript represents CSIR-IHBT communication number 4138.

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