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; Dewan and 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, 2016b; Mathara et al., 2004; Dewan and 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.

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 × 10⁷ cfu/ml and in datshi was 3.9 × 10⁸ 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).

Total genomic DNA of 6 representative strains of LAB was extracted and amplified and were identified by partial 16S rRNA gene sequencing which were compared to the NCBI database for their phylogenetic relationship by using the software MEGA 6 (Figure 2). On the basis of molecular identification, the following species of LAB were identified from dahi and datshi of Bhutan with percentage similarity of LAB: E. faecalis CH1:14 (99%), E. faecalis CH2:02 (99%), E. faecalis CH2:17 (99%), E. durans CH3:03 (99%), Lactococcus lactis subsp. cremoris CH4:01 (99%), and E. faecium DH4:05 (99%; Table 2).

Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris, E. faecium, E. faecalis, Leuconostoc mesenteroides and Pediococcus and lactobacilli (Lactobacillus casei, Lb. fermentum, Lb. helveticus, Lb. plantarum, and/or Lb. acidophilus), were reported from many NFM products of different countries (Tamang et al., 2000; Mathara et al., 2004; Dewan and 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).

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.

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.

HS: Molecular analysis of LAB isolates. SS: Isolation and phenotypic characterization. RR: Determination of technological properties of isolates. JT: Compilation of data and preparation of manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.