Molecular Analysis Of Microbial Community Structure And Identification Of The Predominant Species In Shubat From Xinjiang

Shubat is a special fermented product, prepared from unheated two-humped camel milk through indigenous fermentation process. Although it is more or less similar to yoghurt in appearance, there are important differences between these two products since shubat is liquid rather than creamy, sparking due to its CO2production and has a high degree of sourness. In the Xinjiang in China, shubat have been widely used both as beverage and folk medicine. The indigenous populations have believed that fermented camel milk (shubat) is safe and even has medicinal properties such as antidiabetic, anti-cancer and anti-tuberculosis activities.Traditionally, shubat is homemade by using a semi-continuous or fed-batch fermentation process that was handed down throughout generations. Whenever part of the product is withdrawn for consumption, a portion of raw camel milk is added to make up volume and this process of retrieval and replacement of milk continues for months. The spontaneous fermentation of unheated milk takes advantage of natural microflora inherent in milk and environmental contaminants. However, many reports about traditional dairy products have shown that they have unique and different microflora dependent on the production technology as well as on the ecological localities where they have been produced. It is well known that specific characteristics of dairy products depend on the present microflora. Therefore, this knowledge can contribute to improving the process of the shubat manufacture, as well as to the fermentation condition for obtaining the product of better quality.The present study aimed to isolate and identify predominant microflora in homemade shubat by using phenotypic and molecular methods, which can further developed starter cultures for camel milk fermentation. To the best of our knowledge, no similar investigation into microflora of shubat from two-humped camel milk in China has been carried out so far.The main results are given as follows: 1. Microbiological and chemical characters of shubat prepared by natural fermention procotol were characteried in this study, including LAB, yeast and mould analysis as well as protein and fat ash analysis. The results showed that number of LAB and yeast were4.3×107and2.8×106CFU/mL, respectively. The coliform was more than102cfu/ml at fresh camel milk, but disappeared at Shubat. The moulds were not detected in Shubat. The protein, fat and ash contents of Shubat were4.21%,2.81%and0.98%, respectively. According to the pH, viscosity and acidity, Shubat from defferent regions were divided into two types:Type I have a low viscosity and mild acidity; while Type II have a high viscosity and strong acidity.2. The microbial communities structures in traditional fermented camel milk (shubat) were analysed by using the PCR-DGGE fingerprint technique. The results showed that bacteria and yeast community similarity were78%-84%and80-92%among shubat samples from different regions, respectively. By sequencing V6-V8region of16s rDNA, bacteria community were composed of Macrococcus caseolyticus, Lactobacillus helveticus, Lactobacillus brevis, Weissella hellenica, Lactobacillus sakei, Enterococcus durans, Enterococcus faecium and Leuconostoc lactis et al. Yeast community were found to be Kluyveromyces marxianus, Kazachstania unispora, Candida ethanolica and Geotrichum sp. By compared with sequence similarity of D1region of26s rDNAgene. This results provided a theoretical foundation for developing the culture starter of shubat.3. A total of48LAB and15yeast isolates were isolated from fermented camel milk(shubat),which collected from Kanas areas and Borjin county, Xinjiang China. According to their observed features such as microscopy appearance, CO2production from glucose, NH3production from arginine, the ability to grow under different temperature and salt conditions, these isolates were grouped into six groups,named group Ⅰ,Ⅱ,Ⅲ,Ⅳ Ⅴ and VI. By use of API50CH system and16s rDNA sequence analysis, the six groups were clearly identified as Lactobacillus sakei, Lb. helvelticus, Enterococcus feacium, Ec. feacalis, Lb. brevis and Leuconstoc lactis, respectively. Among the LAB microfora, Lb. sakei were the most frequently isolated species, then were Enterococcus faecium (14%) and Lactobacillus helveticus (13%). The yeast microfora in shubat were identified as Kluyveromyces marxianus and Kazachstania unispora as well as Candida ethanolica, with Kl. marxianus being dominant yeast.4. Based on special colony character combined with detecting katA gene sequence of the pure culture strains, a new detection procotol specific for Lb. sakei have been developed in this study. Using this new method, we have confirmed that Lb. sakei played important role in Shubat fermentation. It is the growth of Lb. sakei that Camel milk have a mild acidity and lower viscosity, which forms the flavor of Shubat. It can be reduced if the existence LB.sakei in Shubat.