Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR

Abstract

Kefir grains are complex microbial systems of several groups of microorganisms. The identification and quantification of the microbial composition of milk kefirs was described in several studies, which provided an insight into the microbial consortia in this complex ecosystem. Nevertheless, the current methods for identification and quantification are not appropriate for deeper studies on kefir consortia, e.g., population dynamics and microbial interactions in kefir grains. This requires another sensitive and reliable quantitative method. Therefore, this study aims to develop multiplexed qPCR assays to specifically detect and quantify, as an example, several microorganisms of the milk kefir microbial community. Primer-probe sets, which target species-specific genes in six bacteria and five yeasts, were designed, and their sensitivity and specificity to the target species was analyzed in simplex as well as four multiplex qPCR assays. The self-designed multiplex assays were applied for the detection of target bacteria and yeast species in milk kefirs, in both, grain and beverage fractions. Detection of all target microorganisms in simplex and multiplex qPCR was achieved by good linearity, efficiency, repeatability and reproducibility in all assays. When the designed assays were applied on six kefirs, all target microorganisms were detected in different samples, but not all in one kefir sample. The two ubiquitous lactobacilli Lactobacillus kefiranofaciens and Lb. kefiri were present in all six kefirs studied, but were associated with different other yeasts and bacteria. Especially on the yeast community a significant diversity was observed. In general, multiplex TaqMan qPCR as developed here was proven to have high potential for specific identification of target microorganisms in kefir samples and for the first time, eleven target bacteria and yeasts of kefir microbiota were rapidly detected and quantified. This study, thus, provides a fast and reliable protocol for future studies on kefir and other similar microbial ecosystems.

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR

“…in various foods (Hong et al 2007 ; Tebbs et al 2011 ; Forghani et al 2016 ; Liu et al 2017 ; Heymans et al 2018 ; Parichehr et al 2019 ). In addition, several applications deal with probiotic or beneficial organisms, such as yeasts, Acetobacter spp., or different lactic acid bacteria (LAB) in kefir or starter cultures in cheese production (Bottari et al 2013 ; Nejati et al 2020 ). Moreover, the detection of food-spoiling bacteria, e.g., Clostridium spp.…”

Novel Insights for Metabiotics Production by Using Artisanal Probiotic Cultures

“…As a result, their growth can be stimulated or altered. A mutualism relationship was identified between a nitrogen formation yeast strain and a Lactococcus lactis, another important observation being related to the lactose that contributes to an easiest interaction between L. lactis and Saccharomyces cerevisiae [84]. Stadie et al contrasted the growth of the co-cultivation vs. a pure culture of key representative water kefir isolates.…”

Lactobacillus kefiranofaciens: From Isolation and Taxonomy to Probiotic Properties and Applications

“…Wang et al [28] reported the use of fluorescence in situ hybridization (FISH) and real-time quantitative PCR (RT-qPCR), demonstrating that L. kefiranofaciens was the only dominant bacterial species in Tibetan kefir grains (Table 1). Recently, multiplexed qPCR assays, using dual-labeled oligoprobes of TaqMan assay, which anneal specifically to a target region, were developed in order to specifically detect and quantify several microorganisms in kefir grains and beverages' microbial communities and consequently evaluate their population dynamics and microbial interactions [38]. Primer-probe sets targeting species-specific genes were designed for six bacteria and five yeasts, among them L. kefiranofaciens (Table 1).…”

“…Grains, China CD Ropy colonies [21] Grains, China CI - [45] Grains and beverages, Brazil CI - [47] Grains and beverages, Ireland CI - [31] Grains and beverages, Turkey CD, CI Not detected [29] Grains, Turkey CI - [55] Grains, Brazil CI - [46] Grains, Slovenia CD, CI 40 [48] Grains, Argentina CD, CI 11 [27] Grains, Turkey CI - [51] Grains, China CD, CI 11 [44] Grains, Italy CD, CI - [23] Grains, Korea CI - [37] Grains, Belgium CI - [50] Grains, Brazil CD, CI 5 [22] Grains, USA CI - [56] Grains, Russia CD, CI Not detected [30] Grains and beverages, Ireland, France, United Kingdom CI - [34] Grains, Malaysia CI - [57] Grains, Turkey CI - [35] Grains, China CD, CI 18 [28] Kefir and beverages, China CI - [58] Beverages, Korea CI - [59] Grains, China CI - [60] Grains, Turkey CD 3 [61] Grains, Greece CI - [62] Beverage, Korea CI - [63] Grains and beverages, Germany, Italy CI - [38] Grains and beverages, United Kingdom, Caucuses region, Ireland, Lithuania, South Korea, USA CI - [64] Grains, China, Germany, USA CI - [65] Grains, China CD, CI 1 [66] CD: culture-dependent method; CI: culture-independent method; -: isolation not performed.…”

“…L. kefiranofaciens has also been detected in the commercial kefir grains of a biotechnology company (Bionova snc, Villanova sull'Arda, Piacenza, Italy) [23]. Nejati et al [38] reported the detection of L. kefiranofaciens in two commercial kefir grains and their respective beverages (Primal Life UG, Berlin, Germany). Moreover, Wang et al [65] studied commercial grains purchased from Huacheng Biological Corporation (Changchun, Jilin, China), Mr.…”