Denaturing Gradient Gel Electrophoresis Analysis of Lactic Acid Bacteria and Yeasts in Traditional Mongolian Fermented Milk

Abstract

We analyzed lactic acid bacteria (LAB) and yeast populations in samples of airag, undaa, and hoormog, which are varieties of traditional fermented milk produced in Mongolia. To effectively detect lactobacilli, lactococci/streptococci/enterococci, and leuconostocs for complete analysis of the complex LAB population in airag, we developed two new primer sets and examined these primer sets in three different combinations using denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed the diversity of the LAB and yeast populations in these traditional fermented milk samples; six to 11 LAB and yeast species were detected. The species common to all samples include Lactobacillus helveticus, Leuconostoc mesenteroides, Lactococcus lactis, and Kluyveromyces marxianus. Lc. raffinolactis, Enterococcus faecium/ hirae/durans/villorum/ratti, Streptococcus thermophilus, L. kefiranofaciens, L. kefiri/buchneri, Leu. citreum, and Leu. lactis were detected in multiple samples, while lactose non-fermenting Saccharomyces cerevisiae was only detected in one sample of hoormog.Keywords: traditional fermented milk, denaturing gradient gel electrophoresis, lactic acid bacteria, yeast *To whom correspondence should be addressed. E-mail: mari-miya@snowbrand.co.jp IntroductionAirag is the most popular traditional fermented milk in Mongolia. It is made using horse milk and has a low alcohol content (normally less than 3%). Similar types of traditional fermented milk are also consumed in Inner Mongolia (chigee and tsege) and Russia (koumiss). Hoormog and undaa, respectively prepared using the milk of camel and cow, are less popular types of Mongolian fermented milk.Airag is generally fermented by adding fresh raw milk to a small quantity of previously prepared airag, and the mixture is then stirred several thousand times. At the end of the horse lactation period, Mongolian people prepare hurunge, naturally dried airag which is stored for use as a starter culture in the following summer.

Sequencing-Based Analysis of the Bacterial and Fungal Composition of Kefir Grains and Milks from Multiple Sources

“…With respect to other species, it was also notable that Torulaspora delbreuckii was not detected in this study despite the fact that both it [10], [11] and its anamorph form, Candida colliculosa , have previously been detected in kefir [8]. There were also many instances whereby we identified species not previously detected in kefir milks, for instance while Dekkera anomala (anamorph: Brettanomyces anomalus ) has been isolated from kefir [8], Dekkera bruxellensis (anamorph: Brettanomyces bruxellensis ) has not been isolated from kefir before now (but has been found in traditional fermented Mongolean and Zimbabwean milks [64], [65]). Other species which had not previously been detected, but were present in lower abundance and few (often just one) milk sample(s) included Cryptococcus sp.…”

Interaction between lactic acid bacteria and yeasts in airag, an alcoholic fermented milk

“…Bacterial genomic DNA of the two strains was extracted and purified according to the lysozyme‐SDS (sodium dodecylsulfate) method as described in Miyamoto et al . (2010).…”

Bacterial Community of Koumiss from Mongolia Investigated by Culture and Culture-Independent Methods

“…On the other hand, several other studies have reported high proportion of Lb. helveticus in koumiss (Uchida et al, 2007;Watanabe et al, 2008;Wu et al, 2009;Miyamoto et al, 2010;Sun et al, 2010 a;Yu et al, 2011). The reason for this difference is unclear and merits further investigations.…”

“…Based on this fact, various independent cultivation methods have been developed during the last two decades (Theron and Cloete, 2000;Jany and Barbier, 2008;Pogacic et al, 2010). During the last decade, numerous studies have evaluated the bacterial communities of fermented milk products using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) but less information is available on fermented milk products from Inner Mongolia and Mongolia (Hao et al, 2010;Miyamoto et al, 2010;Liu et al, 2012). Furthermore, minor knowledge is accessible on the construction of 16S rRNA clone libraries to evaluate the microbial communities of untreated and treated milk products (Callon et al, 2007;Rasolofo et al, 2010;Raats et al, 2011;Osvik et al, 2013).…”