Original article Microbial diversity and stability during primary cultivation and subcultivation processes of Tibetan kefir Wei Gao, 1 Lanwei Zhang, 1 * Zhen Feng, 2 Hui Liu, 1 Nditange Shigwedha, 1 Xue Han, 1 Huaxi Yi, 1 Wenli Liu 1 & Shuang Zhang 1 1 School of Food Science and Engineering, Harbin Institute of Technology, 73 Huanghe Road, Harbin 150090, Heilongjiang, China 2 College of Food Science, Northeast Agricultural University, 59 Mucai Road, Harbin 150030, Heilongjiang, China (Received 25 November 2014; Accepted in revised form 7 March 2015) Summary In this study, we have investigated the microbial diversity and stability in the Tibetan kefir during the primary cultivation and subcultivation processes via a combination of culture-independent and culture- dependent methods. According to the culture-independent methods, the profiles of PCR-DGGE (polymer- ase chain reaction–denaturing gradient gel electrophoresis) indicated that nine microbial species were pre- dominant at different cultivation stages; seventy four isolates of seven predominant species were obtained and identified via the culture-dependent methods. PCR-DGGE further confirmed that Lactobacillus kefir- anofaciens, Lactobacillus kefiri, Lactobacillus paracasei, Streptococcus thermophilus, Kazachstania unispora and Saccharomyces cerevisiae were the most dominant species in the Tibetan kefir during the process of primary cultivation, among which Lb. kefiri, Lb. paracasei and K. unispora showed relative strong stability during both the processes of primary cultivation and subcultivation. These findings suggested that some isolates of the three species possessed the potentiality of being used in the development of direct vat set (DVS) starters for the production of Tibetan kefir and the related products. Keywords Microflora, polymerase chain reaction–denaturing gradient gel electrophoresis, primary cultivation, subcultivation, Tibetan kefir. Introduction Tibetan kefir grains are unique and valuable natural starter cultures for milk fermentation in Tibetan pla- teau, which contain a complex symbiotic diversity of microorganisms, such as lactic acid bacteria (LAB) and yeasts (Diniz et al., 2003; Zhou et al., 2009). Tibe- tan kefir is a fermented milk beverage made with Tibe- tan kefir grains (Gao et al., 2013). The microorganisms that exist in the Tibetan kefir or Tibe- tan kefir grains have been proved to possess some health benefits including anti-inflammatory activity, cholesterol-lowering activity and adhesion ability (Diniz et al., 2003; Wang et al., 2009; Zheng et al., 2013). Various kefir beverages can be made by adding kefir grains directly into the cow and goat’s milk (Hsieh et al., 2012), cocoa pulp (Puerari et al., 2012), sugar solution (Magalh~ aes et al., 2010a), cheese whey and other substrates (Magalh~ aes et al., 2010b). Earlier on, research made on the microbial ecology of complexity systems typically relied on the culture-dependent methods. With the update of molecular technologies, a culture-independent method, polymerase chain reac- tion–denaturing gradient gel electrophoresis (PCR- DGGE) has been developed and proved to be a useful tool for rapid fingerprint analysis of microbial commu- nity composition, diversity and dynamic. In recent years, this technology has been applied to the food detection area (Masoud et al., 2011; Cocolin et al., 2013). Researchers from different countries have inves- tigated the microflora structure of kefir grains or related products via PCR-DGGE (Garbers et al., 2004; Wang et al., 2006; Leite et al., 2012; Londero et al., 2012). Zhou et al. (2009) investigated the micro- flora structures in three samples of Tibetan kefir grains by PCR-DGGE. Their results indicated that the predominant microorganisms were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lacto- bacillus kefiri, Lactobacillus casei, Kazachstania unis- pora, Kluyveromyces marxianus, Saccharomyces cerevisiae and Kazachstania exigua. *Correspondent: Fax: +86-451-86282906; e-mail: zhanglw@hit.edu.cn International Journal of Food Science and Technology 2015 doi:10.1111/ijfs.12801 © 2015 Institute of Food Science and Technology 1