Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan

Dahi and datshi are common naturally fermented milk (NFM) products of Bhutan. Population of lactic acid bacteria (LAB) in dahi (pH 3.7) and datshi (pH 5.2) was 1.4 × 107 and 3.9 × 108 cfu/ml, respectively. Based on 16S rRNA gene sequencing isolates of LAB from dahi and datshi were identified as Enterococcus faecalis, E. faecium, Lactococcus lactis subsp. lactis. LAB strains were tested for some technological properties. All LAB strains except E. faecalis CH2:17 caused coagulation of milk at both 30°C for 48 h. Only E. faecium DH4:05 strain was resistant to pH 3. No significant difference (P > 0.05) of viable counts was observed in MRS broth with and without lysozyme. All LAB strains grew well in 0.3% bile showing their ability to tolerate bile salt. None of the LAB strains showed >70% hydrophobicity. This study, being the first of its microbiological analysis of the NFM of Bhutan, has opened up to an extent of research work that gives a new insight to the products.


INTRODUCTION
Naturally fermented milk (NFM) products are prepared by the practice of one of the oldest techniques of milk fermentation known as the 'back-sloping' method in which a previous batch of a fermented product is used to inoculate the new batch (Josephsen and Jespersen, 2004;Tamang et al., 2016b). NFM products are prepared and consumed daily in Bhutan. Some NFM products of Bhutan are dahi, datshi, mohi, gheu, hard-chhurpi (chugo/churkam) and hitpa. Dahi ( Figure 1A) is a yogurt-like NFM product of Bhutan, which is traditionally prepared by allowing the boiled milk to undergo spontaneous fermentation at room temperature for 2-3 days with the inoculation of the previous dahi sample. Dahi is drunk as a refreshing non-alcoholic beverage in Bhutan. Datshi ( Figure 1B) is a cottage cheese like product, which is prepared by churning dahi for 10-15 min until a clumping product; butter (locally called gheu) is extracted. The butter is collected in another vessel and the buttermilk, locally called mohi is then heated for 15-20 min for the curdling of the product, called datshi, which is made into round small balls. It is consumed as curry in main meals in Bhutan. Most of these NFM products are occasionally used for religious ceremonies in Bhutan. Some people are economically dependent upon these NFM products where they sell at local markets. Some NFM products of other countries were well studied such as dahi, misti dahi, shrikhand, chhu, chhurpi, philu and somar of India, Nepal, Pakistan, and Bangladesh (Tamang et al., 2000;Tamang, 2006, 2007;Harun-ur-Rashid et al., 2007;Sarkar, 2008;Patil et al., 2010;Tamang, 2010), kurut of China (Sun et al., 2010), aaruul, airag, byasulag, chigee, tarag, and khoormog of Mongolia (Watanabe et al., 2008;Takeda et al., 2011;Oki et al., 2014), ergo of Ethiopia, lben, rayeb, zabady, and zeer of Morocco and Northern African and Middle East countries, rob (from camel milk), biruni, mish (cow/camel milk) of Sudan, amasi (hodzeko, mukaka wakakora) of Zimbabwe, nunu of Ghana (Akabanda et al., 2013), filmjölk and långfil of Sweden (Mayo et al., 2010), and koumiss or kumis or kumys or kymys of the Caucasian area (Wu et al., 2009). Among species of lactic acid bacteria (LAB), Lactococcus lactis subsp. cremoris, and Lc. lactis subsp. lactis are the dominant microbiota along with other mesophilic lactobacilli (Lactobacillus casei/Lb. paracasei, Lb. fermentum, Lb. helveticus, Lb. plantarum, and/or Lb. acidophilus), Enterococcus faecium, species of Leuconostoc and Pediococcus in NFMs (Tamang et al., 2000(Tamang et al., , 2016bMathara et al., 2004;Tamang, 2006, 2007;Patrignani et al., 2006;Watanabe et al., 2008;Wu et al., 2009;Hao et al., 2010;Yu et al., 2011;Akabanda et al., 2013;Oki et al., 2014). Technological properties including probiotics characters have been extensively studied in some NFM products of the world (Patrignani et al., 2006;Dewan and Tamang, 2007;Harun-ur-Rashid et al., 2007;Wu et al., 2009;Tamang et al., 2016a). Till date, there has been no report on the microbiological analysis and technological properties of the NFM from Bhutan, making this research the first of this kind. This paper is aimed to determine some technological properties of the LAB isolates from two popular NFM products of Bhutan-dahi and datshi such as acidification and coagulation, resistance to low pH, tolerance against bile, lysozyme tolerance and hydrophobicity assay, and also to isolate and identify LAB species by 16S rRNA sequencing.

Samples
A total number of eight fresh samples of dahi (4) and datshi (4) were collected from Tabthangbu village, Bhutan in pre-sterilized sampling bags and were transported to the laboratory in an icebox carrier, stored at 4 • C and analyzed within a week.

Microbiological Analysis
Samples (10 ml) were homogenized with sterile physiological saline (90 ml) in a stomacher lab-blender (400, Seward, London, UK) for 1 min, and were serially diluted in the same diluent. LAB were enumerated on MRS agar (M641, HiMedia, Mumbai, India) plates under anaerobic conditions in an anaerobic gaspack system (LE002, HiMedia, Mumbai, India) and incubated at 30 • C for 48-72 h (Dewan and Tamang, 2007). Colonies were selected randomly from the plates which contained less than 10 colonies, according to Leisner et al. (1997). Purity of the isolates was checked by streaking again and sub-culturing on fresh agar plates of the isolation media, followed by microscopic examinations. LAB isolates were preserved at −20 • C in MRS broth (M369, HiMedia, Mumbai, India) mixed with 20% (v/v) glycerol.

Determination of pH
The pH of samples was determined using a pH meter (Crison basic 20, Barcelona, Spain) calibrated with standard buffers.

Phenotypic Characterization
Cell morphology of all isolates and their motility was determined using a phase contrast microscope (Olympus CH3-BH-PC, Japan). Isolates were Gram-stained and tested for catalase production, and were preliminarily identified based on the phenotypic properties including sugar fermentations, following the methods of Schillinger and Lücke (1987) and Dykes et al. (1994).

DNA Extraction
Based on similar sugar fermentation and other phenotypic characteristics criteria, six representative strains of LAB were randomly selected from 44 strains of LAB. Total genomic DNA of six representative strains of LAB was extracted from 2-ml samples of overnight cultures grown in MRS broth at 30 • C according to the methods of Martín-Platero et al. (2007). DNA was quantified using fluorometer (Qubitol R 3.0, Fisher Scientific, USA).

16S rRNA Gene Sequencing
The 16S rRNA gene was amplified by PCR mixtures (25 µL) contained approximately 30-50 ng template DNA, 1 µM forward primer 27F and 1 µM reverse primer 1492R (Lane, 1991)   using a PCR Master Mix (Promega, Canada) performed under the standard PCR amplification procedure in a SimpliAmp TM Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). The PCR amplicons were checked for their purity on 1% agarose gel electrophoresis in the presence of ethidium bromide (10 mg/mL), which was later analyzed by the Gel Doc System (Ultra-Violet Products Ltd, UK). Sequencing service was outsourced.

Phylogenetic Analysis
The BLAST (Basic Phylogenetic Local Alignment Search Tool) program was used for comparing DNA databases for sequence similarities available in the NCBI database. Five different strains/species from each BLAST results were chosen for phylogenetic analysis using Molecular Evolutionary genetics Analysis software (MEGA version 6).

Acidification and Coagulation
Acidification and coagulation ability of LAB strains were assayed by inoculating 10% skim milk (RM1254, HiMedia, Mumbai, India) at 1% level and incubated at 30 • C for 72 h. Observation was made for commencement of clotting, followed by pH measurement (Olasupo et al., 2001).

Tolerance against Bile
MRS broth containing 0.3% bile was inoculated with active cultures for 4 h (Prasad et al., 1998) and viable cells were enumerated in MRS agar plates after 24 h incubation and growth was recorded.
Lysozyme Tolerance 10 mL of MRS broth with lysozyme (MB098-1G, HiMedia, India) and without lysozyme, respectively, was inoculated with 1 mL of both culture suspensions of 10 8 cfu/ml cell concentration and incubated at 30 • C for 24 h and viable cells were enumerated in MRS agar plates after 24 h incubation (Brennan et al., 1986).

Resistance to Low pH
Active cultures were harvested by centrifugation and pellets were washed once in phosphate-saline buffer (PBS, pH 7.2), resuspended in PBS (pH 3) and incubated in MRS agar plates at 30 • C for 24 h, and growth was recorded (Prasad et al., 1998).

Hydrophobicity Assay
Bacterial affinity to hydrocarbons was determined and results were expressed according to Perez et al. (1998), modified by Tamang et al. (2009) as follows. Fresh cultures were grown in MRS broth at 30 • C for 24 h and centrifuged at 8,000 g for   Data represent an average of three sets of experiments. +, indicates growth (>10 6 cfu/ml) of LAB strains; a no significant difference (P > 0.05) of viable LAB counts in MRS broth with and without lysozyme after incubation (30 • C/24 h) was considered as a strain resistant to lysozyme.; b MRS broth with 0.3% bile. 5 min. The pellet was washed with 9 ml of Ringer solution (Merck, Germany) and thoroughly mixed. Suspension (1 ml) was taken and the absorbance at 580 nm was measured. Then, 1.5 ml of suspension was mixed with equal volume of n-hexadecane (RM 2238, HiMedia, Mumbai, India) in duplicates and mixed thoroughly. Phases were allowed to separate for 30 min at room temperature, after which aqueous phase was carefully transferred to a new tube and absorbance at 580 nm was measured. The percentage hydrophobicity was expressed as follows: where A 0 and A are the absorbance values of the aqueous phase before and after contact with n-hexadecane.

RESULTS AND DISCUSSION
Dahi and datshi are acidic fermented milk products showing an average pH of 3.7 ± 0.17 and 5.2 ± 0.12, respectively. Isolation of LAB was performed on the classical media i.e., Lactobacillus MRS Agar media under anaerobic conditions at 30 • C incubation for 48 h. The microbial load of LAB in dahi was 1.4 × 10 7 cfu/ml and in datshi was 3.9 × 10 8 cfu/mL, respectively. A total of 44 LAB isolates were isolated from dahi and datshi and phenotypically characterized and were randomly grouped into six representative strains based on similar sugar fermentation and other phenotypic characteristics ( Table 1).
These isolates were tentatively identified as Enterococcus and Lactococcus (Table 1).
Lactic acid bacteria strains were tested for some technological properties ( Table 3). All LAB strains except E. faecalis CH2:17 caused coagulation of milk at both 30 • C for 48 h with a significant drop in pH ( Table 3). Coagulation of milk by LAB strains reveals their potential as starters or adjunct cultures in the production of NFM of Bhutan. Only E. faecium DH4:05 strain showed positive result indicating its resistance to pH 3 in applied method ( Table 3). Resistance to pH 3 is often used in vitro assays to determine the resistance to stomach pH (Prasad et al., 1998). Resistances to the lysozyme by all six strains of LAB were evaluated in MRS broth with and without lysosome at 30 • C for 24 h ( Table 3). Lysozyme is capable of lysing bacteria, but it doesn't impair activities of LAB (Saran et al., 2012). Tolerance against bile was also tested and found that all LAB strains grew well in 0.3% bile showing their ability to tolerate bile salt. The mean intestinal bile concentration is 0.3% (w/v) and the staying time of food in small intestine is suggested to be 4 h (Prasad et al., 1998). The probiotic bacteria survival in the gastrointestinal transit is primordial, and implies in the ability of microorganisms to survive at the stomach acidity and bile, so that they can exert their beneficial effects on the host (Pozza et al., 2011).
Bacterial affinity to hydrocarbons, such as hexadecane, proved to be a simple method to determine cell surface hydrophobicity (van Loosdrecht et al., 1987). None of the LAB strains showed >70% hydrophobicity (Table 3). A percent hydrophobic index greater than 70% was classified as hydrophobic (Nostro et al., 2004). Hence, LAB strains from dahi and datshi do not show hydrophobic character in the applied method. However, these limited technological properties are not enough to validate the potential probiotic uses of these isolates.

CONCLUSION
Based on 16S rRNA gene sequencing isolates of LAB, isolated from dahi and datshi of Bhutan, were identified as E. faecalis, E. faecium, Lactococcus lactis subsp. lactis and some strains showed promising technological properties. This is the first report on NFM of Bhutan, which may be used as baseline data for further research on NFM products.