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Monitoring of Lactobacillus sanfranciscensis strains during wheat and rye sourdough fermentations by CRISPR locus length polymorphism PCR.
International Journal of Food Microbiology ( IF 5.4 ) Pub Date : 2019-12-16 , DOI: 10.1016/j.ijfoodmicro.2019.108475
Esther Rogalski 1 , Rudi F Vogel 1 , Matthias A Ehrmann 1
Affiliation  

Lactobacillus (L.) sanfranciscensis is a competitive key species in sourdough fermentations. However, the principles involved in establishing the commonly observed phenomenon of strain dominance are unresolved. This has been studied little because the methods for fast and reliable differentiation of strains and their monitoring during fermentation are tedious and cannot be done with large numbers of isolates. In this contribution, we present a strain-specific, PCR-based typing method that uses length heterogeneities of the clustered regularly interspaced short palindromic repeats (CRISPR) loci as they occur in the genomes of different strains. In silico analysis of 21 genomes revealed 14 different CRISPR genotypes. We then designed a primer set to simultaneously detect different strains in a multiplex PCR assay designated CRISPR locus length polymorphism PCR (CLLP-PCR). The usefulness of this method was evaluated in lab-scale sourdough fermentations conducted with rye and wheat flours. First, the flour was mixed with water to a dough yield of 200. Then each dough was inoculated with four different L. sanfranciscensis strains (TMW 1.1150, TMW 1.392, TMW 1.2142, and TMW 1.2138) at levels of 109 cfu/g each. Sourdoughs were propagated at 28 °C for 5 days by back slopping 5% to the flour mass every 24 h. Samples were collected each day; DNA was isolated, and the presence of strains was detected qualitatively in the sourdoughs with PCR. L. sanfranciscensis TMW 1.392 became dominant as early as 2 days into the fermentation and remained the only detectable strain for the rest of the sampling period. CLLP-PCR proved to be useful in investigating the assertiveness of different strains of L. sanfranciscensis in sourdoughs. Therefore, CLLP-PCR may be used as a tool to investigate assertiveness of microorganisms in food fermentations at the strain level.

中文翻译:

通过CRISPR基因座长度多态性PCR监测小麦和黑麦酸面团发酵过程中的旧金山乳杆菌菌株。

Sanfranciscensis乳酸杆菌(L.tobaccobacillus(L.)Sanfranciscensis)是酵母发酵中的关键竞争品种。但是,建立普遍观察到的应变优势现象所涉及的原理尚未解决。由于对菌株进行快速可靠的分化及其在发酵过程中进行监测的方法很繁琐,而且不能用大量的分离株完成,因此很少进行研究。在这项贡献中,我们提出了一种基于菌株的,基于PCR的分型方法,该方法使用了簇状规则间隔的短回文重复序列(CRISPR)位点的长度异质性,因为它们出现在不同菌株的基因组中。在对21个基因组的计算机分析中发现了14种不同的CRISPR基因型。然后,我们设计了引物组,以在称为CRISPR基因座长度多态性PCR(CLLP-PCR)的多重PCR分析中同时检测不同的菌株。在用黑麦和小麦粉进行的实验室规模的酵母发酵中评估了该方法的有用性。首先,将面粉与水混合,使面团的产量达到200。然后,将每个面团分别以109 cfu / g的水平接种4种不同的桑弗朗西斯菌L. Sanfranciscensis菌株(TMW 1.1150,TMW 1.392,TMW 1.2142和TMW 1.2138)。面团在28°C下繁殖5天,方法是每24小时向面粉团块中倒入5%的面粉。每天收集样品;分离DNA,并通过PCR在酵母中定性检测菌株的存在。L.sanfranciscensis TMW 1。392早在发酵开始的2天就成为主要菌种,并且在其余采样期间仍是唯一可检测到的菌株。事实证明,CLLP-PCR可用于研究酵母中不同菌株L. sanfranciscensis的确证性。因此,CLLP-PCR可以用作在菌株水平上研究食品发酵中微生物的权威性的工具。
更新日期:2019-12-17
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