Original Research

Biodiversity of Lactobacillus from traditional Indian Cow milk Churpi cheese

Dhiraj Kumar Nanda1, 2 , Reeti Chaudhary1*, Karan Veer Singh2, Dinesh Kumar 2, 3

1Department of Biotechnology, D C R University of Science & Technology, Murthal - 131039, Haryana
2National Bureau of Animal Genetic Resources, Karnal – 132001, Haryana
3Centers for Agricultural Bioinformatics, Indian Agricultural Statistics Research Institute, New Delhi -110012

J Innov Biol (2014) Volume 1, Issue 3: Pages: 168-174

Abstract: Churpi cheese is one of the traditional varieties of fermented dairy product prepared from cow milk, and consumed by ethnic groups of people residing in alpine regions of India, Sikkim and Darjeeling. The aim of current study was to investigate the biodiversity of Lactobacilli in indigenous Churpi cheese prepared from cow milk. Twenty strains of Lactobacilli were isolated from eight different samples of Churpi cheese procured from different regions of Sikkim (India), and were analysed by phenotypic and genotypic methods. For molecular characterization of these isolates 16 S r DNA sequencing was carried out which confirmed species of these isolates. Nine isolates were confirmed as Lactobacillus casei, followed by four as L. plantarum, three as L. delbrueckii, two as L. paracasei, and two as L. brevis. The results of this study shows a high level of biodiversity among Lactobacilli isolated from Churpi cheese and offered a remarkable reservoir of ‘natural’ microbes. Isolates obtained in this study can be potentially used for the development of defined strain starter for Churpi cheese and other dairy products.

Received: 03 September 2014
Accepted: 27 September 2014
Published: 30 September 2014


Corresponding Author:
Chaudhary R.,
National Bureau of Animal Genetic Resources, Karnal – 132001, Haryana
email: reeti.malik@gmail.com

Keywords: Cow milk; Churpi cheese; Lactobacillus; 16S rDNA

IntroductionMaterial & MethodsResults & DiscussionFigures & TablesReferences
Churpi is a traditional variety of cheese like dairy product which is mainly prepared and consumed in the Himalayan regions of Darjeeling and Sikkim, India (Dewan and Tamang, 2007; Tamang et al. 2000). Churpi can be prepared from milk of cow and yak, churpi prepared out of cow milk is softer than yak Churpi and has a mild to strong flavoured taste and is consumed as curry mix, pickles and condiments. It has been reported that per capita average consumption of soft-variety churpi in the Darjeeling hills is 6.9 g/day, and in Sikkim is 9.9 g/day (Yonzan and Tamang, 1998). Traditional process of Churpi preparation involves fermentation of boiled or unboiled cow milk, such traditional dairy products have their natural microflora is from non-pasteurized milk and raw materials which can be the sources of new strains of microbes (Weerkamp et al. 1996; Wouters et al. 2002). The fermentation in such food products is carried out by a class of microbes known as lactic acid bact-eria (LAB), comprising a group of numerous bacterial genera including Lactobacillus, Lactococcus, Strepto-coccus, and Leuconostoc (Holzapfel et al. 2001). LAB are commonly found in foods and fermented products (O'sullivan et al. 2002) including dairy products (Beresford et al. 2001). Pure microbial community isolated from traditionally fermented foods exhibits div-erse metabolic activities which can be compared with commercial strains used as industrial starters (Klijn et al. 1995; Randazzo et al. 2002). Thus, efforts are needed to isolate, characterize and pre-serve the microbial diversity of raw milk and traditional dairy products.
Lactobacillus have been characterized by phenol-typic methods as morphology, Sugar fermen-tation analysis (Kandler and Weiss, 1986; Coeuret et al. 2003) fatty acid methyl ester (FAME) analysis (Klein et al. 1998; Giraffa and Neviani, 2000) and whole-cell protein profiling for certain lactobacilli at both species and sub-species level (Giraffa and Neviani, 2000). These phenotypic methods include poor reproducibility, ambiguity of some techniques, extensive logistics for large-scale investigations and poor discriminatory power (McCartney, 2002). Thus these phenotypic protocols have been supplemented by using various DNA fingerprinting techniques like RAPD-PCR, RFLP, ribotyping, and rep-PCR (Nguyen et al. 2013; Massi et al. 2004; Gevers et al. 2001). Sequencing of 16S rRNA gene have been found more reliable for molecular ecology and diversity analysis study because it is difficult to identify minor components of microbiota by other methods (Kim and Chun, 2005; Lane, 1991; Collins and Wallbanks, 1992; Babalola, 2004).
In the context of Churpi cheese studies on diversity of Lactobacilli in Yak Churpi has been carried out (Prashant et al. 2009) but report concerning the geno-typic and phenotypic characterization of the micro-flora from Churpi prepared from cow milk is lacking behind. Hence, the current study was taken up with the objective to isolates, characterize, and study the diversity within the Lactobacillus from cow milk Churpi cheese. Natural biodiversity of Lactobacilli from Churpi can be of immense importance and can be used to formulate defined starter strains for promoting and commercialization of Churpi cheese.

Collection of cheese samples
Eight samples of four different varieties of Churpi cheese samples A: Dry powder Churpi cheese, B: Wet Churpi cheese farmer made, C: Wet Churpi cheese machine made, D: Dry Churpi cheese garland were collected from Local market of Gangtok, Sikkim, India. During transportation the cheese samples were kept in refrigerated condition and brought to lab, and kept in same condition till analysis.

Isolation of Lactobacillus
In order to recover a majority of Lactobacillus diversity for subsequent characterization purpose, cheese samples were subjected to microbiological analysis for total bacterial counts. Ten gram of cheese sample was homogenized with 90 mL of 2% sodium citrate solution, which was further serially diluted (10−1 – 10−8) in normal saline solution (0.85% NaCl) and one mL of decimal dilutions of the samples were pour plated with culture media De Man Rogosa Sharpe (De Man et al. 1960) (MRS) agar (Himedia, Bombay, India) and incubated for 48 h at 37 °C. Colonies grown on the plates were counted, the suitable plates with well isolated discrete colonies were then selected for isolation and 10–15 individual colonies were picked randomly from each sample plate with help of sterile toothpick and transferred into MRS broth tubes and incubated at 37 °C. After incubation for 24-48 hours, the MRS broth cultures were examined microscopically for purity. The tubes showing negative for catalase test and gram positive rods were selected and further purified by successive streaking on MRS agar plate. All colonies thus isolated were presumptively screened on the basis of gram reaction, morphology, and catalase test.
All these isolates were preserved as glycerol stocks (MRS broth with 15% glycerol) and stored at −20 °C, for long term preservation freeze dried ampoules were prepared by lyophilization using freeze dryer (Edwards High Vacuum International, Sussex, England) and were stored for further characterization by biochemical and molecular methods.

Phenotypic characterization
Negative staining and gram staining were used to check the morphology of Isolates. For Biochemical characterization of these putative Lactobacillus isolates a battery of few biochemical tests was carried out as per the scheme suggested in Bergey’s Manual (Kandler and Weiss, 1986). The ability of cultures to ferment and produce acid from various sugars was tested by using HiCarbohydrate™ Kit KB009 (Hi Media Laboratories Ltd., Mumbai) comprising of 35 different carbohydrate sources, Cultures were inoculated to the kit as per instruction provided by manufacturer. This fermentation profile obtained was used to prepare a dendrogram of isolates by using NTSYSpc 2.02 software package (Applied Biostatistics Inc., NY, USA).

Molecular characterization

DNA extraction
Genomic DNA of all isolates was extracted from 2 mL samples of overnight cultures grown in MRS broth at 37 °C as previously described (Pospiech and Neumann, 1995) with slight modification.

Genus-specific PCR
Lactobacillus genus-specific primer (Dubernet et al. 2002) as in Table 1, targeting 16S rRNA gene was used for the confirmation of Lactobacillus genus. The Polymerase chain reaction (PCR) was performed in 25 μL of reaction volume, containing 50–100 ng of genomic DNA, 1X Taq buffer, 1.5 mmol/L MgCl2, 10 mmol/L of each deoxyribonucleotide tri phosphate (dNTP), 50 ng of each primer, and 1 unit of Taq DNA polymerase (Fermentas, Germany). Amplification was performed on Eppendorf Mastercycler (Hamburg, Germany) according to the program of earlier published literature (Dubernet et al. 2002). Amplification was verified by electrophoresis on 1.5% (w/v) agarose gel in 1X TAE buffer using GeneRuler 100 bp DNA ladder (Fermentas, Germany) as a molecular weight marker, and stained with ethidium bromide (1 mg/meL).

16S rRNA gene (partial) Sequencing and phylogenetic analysis
For sequencing of 16S rRNA gene (partially) PCR was carried out for all isolates by using primer 7F (Lane, 1991) and S-G-Lab-0677-R (Heilig et al. 2002) as in Table 1. The PCR products of 750 bp size (25 μL) amplified were custom sequenced by using primer 7F (Lane, 1991) by Automated DNA Sequencing Services provided by Vimta Labs Pvt. Ltd, Hyderabad, India. The chromatogram of sequences obtained from the service provider were analysed, trimmed and converted to FASTA format by using BioEdit Sequence alignment editor Version 7.0.9.0. Basic Local Alignment Search Tool (BLAST) analysis was performed to check the identity of DNA sequence in the database and for species determination. The sequences thus generate after analysis with species identity were submitted to the GENBANK database NCBI (Bethesda, MD. USA) by using Sequin Application Version 12.10 (NCBI, NIH). The phylogenetic analysis of these sequences was done with help of MEGA 5.0 (Tamura et al. 2011), and a phylogenetic tree was constructed with reference sequences from GenBank database NCBI, by using unweighted pair-group method with arithmetic averages (UPGMA).

In all of the collected samples LAB as well as aerobic mesophilic counts were found to be in the range of 1.6 x 10¬¬5 – 4.0 x 105 CFU/ gm of cheese. Yeast and mold were detected only in samples D. A total of 103 colonies were picked up initially from MRS plates, these isolates were presumptively screened for genus Lactobacillus. Finally only 20 isolates (2 from Churpi cheese-A, 7 from Churpi cheese-B and 11 from Churpi cheese-C) were confirmed to be Lactobacillus on basis of preliminary screening strategies, all of the 20 isolates conformed to the general phenotypic characteristics of genus Lactobacillus i.e. Gram positive, rod shaped, nonmotile, non-sporulating, and catalase negative. None of isolate from Churpi cheese-D sample could be confirmed to be Lactobacillus. The generic status of isolates was further confirmed by PCR, all these 20 isolates gave a specific band of 250 bp with Lactobacillus genus specific PCR (Fig 1) as described by (Dubernet et al. 2002), confirming for Lactobacillus at genus level.
Phenotypic characters especially sugar fermentation pattern of all the isolates was used to prepare a dendrogram (Fig 2) with help of NTSys software using SM coefficient and clustered by UPGMA method, based on sugar fermentation profile these isolates were clustered into 2 major groups at 0.75 coefficient level the cluster-I was further divided into 3 subgroups mainly comprising of isolates from Churpi cheese-C and B, cluster-II was divided in 2 subgroups and comprised isolates from sample B and A as in figure 2. These isolates were found ability to ferment Lactose, Maltose, Fructose, dextrose, Trehalose, Mannose, Inulin, Salicin and Cellobiose.
For species confirmation, partial sequencing of 16S rDNA (V1–V3 region) was performed. For sequencing all the 20 isolates gave a positive PCR product of 750 bp (Fig 3). BLAST analysis of the sequences obtained was carried out to find similarity of our sequence with the sequence deposited in GENBANK, and species of all isolates was confirmed. The sequence data generated have been submitted to GENBANK database under accession numbers as in Table 2. Among the isolates Lactobacillus casei (9) was found to be most prevalent in Churpi cheese samples followed by L. plantarum (4), L. delbrueckii (3), L. paracasei (2) and L. brevis (2).

DISCUSSION

Fermentation of any traditional food products is carried out by the natural, wild-type LAB which can come from the raw materials or from the environment. During studying the natural microbial diversity of Churpi cheese in the present study, Lactobacilli were found to be dominating other LAB with predominance of L. casei in sample C followed by L. plantarum in sample B, L. delbrueckii in samples A and B, L. paracasei in sample C and L. brevis in sample B. These species L. casei, L. plantarum, and L. brevis have been reported to be predominating Lactobacilli of nonstarter lactic acid bacteria (NSLAB) in Cheddar cheese (Peterson and Marshall, 1990) our result supports the similar predominance in Churpi cheese. Similarly earlier studies on Churpi cheese of yak milk clearly supports the presence of L. paracasei and L. plantarum as the dominating LAB species (Tamang et al. 2000; Prashant et al. 2009). L. delbrueckii has been found dominating Lactobacilli in various types of artisanal cheese as Ragusano cheese (Randazzo et al. 2002) Italian hard and semi-hard cheeses (Giraffa et al. 2004) Serbian cheese (Begovic et al. 2011) and camel cheese (Nanda et al. 2011). Thus the species of Lactobacilli obtained from Churpi cheese are in agreement with the findings of other workers in different varieties of cheese. The Lactobacilli grow as secondary microflora particularly during maturation process and influence the organoleptic properties of any cheese (Veljovic et al. 2007), thus the peculiar flavour of Churpi can be correlated with these isolates. Lactobacillus species, viz. L. brevis, L. fermentum, L. rhamnosus, and L. coryn-iformis, are less commonly found in cheese where NSLAB densities are initially lower and build up with time during maturation (Crow et al. 2001). The presence of L. brevis in sample B (farmer made Churpi cheese) may be due to maturation of cheese.
In the present study it was tried to isolate and identify the isolates genotypically by comparing their partial DNA sequence of 16S r RNA gene with the sequences with the existing sequences in the public database GENBANK (NCBI). The sequence results were able to successfully characterize the species of isolates. The dendrogram derived from the sequences of isolates along with reference sequences retrieved from database clearly grouped the isolates with reference sequence representing the species (Fig 4). But the two isolates of L. paracasei and reference sequence could not be grouped separately and were grouped along with L. casei, this result was obtained because L. casei and L. paracasei are phylogenetically closely related to each other (Felis et al. 2001; Diancourt et al. 2007) and they have similarities in 16 S r DNA sequences.
No discrepancy was detected in phenotypic traits and molecular data. By comparing the dendrogram generated by phenotypic and genotypic data Fig 2 and Fig 4 it was found that the isolates were clustered in similar pattern except one isolates CCC-6. A polyphasic approach involving the combination of biochemical and molecular techniques is a method of choice for characterization of Lactobacilli from dairy and non-dairy sources (Vandamme et al. 1996; Gancheva et al. 1999; Lombardi et al. 2002; Nanda et al. 2011) in the current study also polyphasic approach allowed the identification of Lactobacilli in Churpi cheese and establish the biodiversity within these isolates. These strains of Lactobacilli can be further explored for the development of primary and adjunct starter cultures for Churpi and other varieties of cheeses and dairy products.

Conclusion
In the current study, 20 isolates of Lactobacillus were identified and characterized by a combination of conventional and molecular techniques. The study provides data on microflora of Churpi cheese. The technological properties of these isolates can further be explored with good starter activity and flavour production for industrial use as a novel starter culture for the preparation of cheese and other fermented dairy products. These isolates could also be studied for their possible probiotic applications.

Acknowledgements
The help provided by Chairman, Department of Biotechnology DCRUST is sincerely acknowledged. Authors also acknowledge the help provided by Directors of NBAIM, NDRI and NBAGR. Fellowship to DKN and financial support by Indian Council of Agriculture Research (ICAR), Government of India, New Delhi in the form of Network Project Application of Microbes in Agriculture and Allied Sectors (AMAAS) is thankfully acknowledged.

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