Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains

Chon

Journal of Food Protection

et al. 2015

Lactobacillus kefiranofaciens is an indicator microorganism for kefir and a key factor in kefir grain formation and kefiran production. We designed a novel real-time PCR primer and probe set, LKF_KU504, for the rapid detection of L. kefiranofaciens. In inclusivity and exclusivity tests, only 14 L. kefiranofaciens strains were positive among 61 microorganisms, indicating 100 % sensitivity and specificity. The LKF_KU504 set also differentiated kefir milk from 30 commercial nonkefir yogurts. The levels of L. kefiranofaciens in kefir grain and kefir milk were significantly different, indicating L. kefiranofaciens was more concentrated in kefir grain than in kefir milk.

Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Rapid Detection of Lactobacillus kefiranofaciens in Kefir Grain and Kefir Milk Using Newly Developed Real-Time PCR

Chon

Journal of Food Protection

et al. 2015

“…Lb. kefiranofaciens , a heterofermentative bacterium, has also been reported as a kefiran (exopolysaccharide) producer [35] and is used as the starter for kefir beverage [36] and Caucasian cultured milk [37]. According to Cheirsilp et al [37], S. cerevisiae could assimilate kefiran production rates of Lb.…”

Section: Discussionmentioning

confidence: 99%

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

Mei

Guo

et al. 2014

PLoS ONE

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.

Journal of Dairy Science

“…The kefir beverage is produced by mixing milk, water, or fruit juice with kefir grains, which have the appearance of small cauliflowers (Lopitz-Otsoa et al, 2006). These grains are a symbiotic combination of bacteria (mainly lactobacilli) and yeast that are protected by an exopolysaccharide structure called kefiran, which is mainly produced by Lactobacillus kefiranofaciens in conjunction with yeasts (Lopitz-Otsoa et al, 2006;Hamet et al, 2013). More precisely, the Codex Alimentarius commission states that kefir is a combination of bacteria belonging to several genera (Lactobacilli, Leuconostoc, Lactococcus, and Acetobacter) and yeasts.…”

Section: Short Communicationmentioning

confidence: 99%

Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments

Korsak

Taminiau

Leclercq

et al. 2015

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques. Key words: kefir, microbiota, metagenetics, 16S rRNA sequencing, 26S rRNA sequencing Short CommunicationOriginally from the Caucasus Mountains (Otles and Cagindi, 2003), kefir is widely consumed in Eastern Europe but now encountered all over the world. The word "kefir" means "good feeling" in Turkish, due to the cooling nature of this beverage. The kefir beverage is produced by mixing milk, water, or fruit juice with kefir grains, which have the appearance of small cauliflowers (Lopitz-Otsoa et al., 2006). These grains are a symbiotic combination of bacteria (mainly lactob...