Molecular discrimination of <i>Lactobacillus brevis</i> strains isolated from food products in South Korea using multilocus sequence typing

Sharma, Anshul,Kaur, Jasmine,Lee, Sulhee,Park, Young-Seo Elsevier 2017 FOOD SCIENCE AND TECHNOLOGY -ZURICH- Vol.86 No.-

<P><B>Abstract</B></P> <P> <I>Lactobacillus brevis</I> plays an important role in the manufacture of fermented foods. The aim of the present study was to know the genetic relationships among <I>L. brevis</I> isolates using multi-locus sequence typing of seven different housekeeping genes. 13 isolates were collected from different food products of South Korea. The results of the sequence analysis indicated 13 different sequence types (STs), each represented by a single strain. Among the seven loci, a total of 83 polymorphic sites were detected. The number of polymorphic sites varied from 4 (<I>rpoB</I>) to 24 (<I>recA</I>), whereas nucleotide diversity per site among the seven genes varied from 0.00161 (<I>rpoB</I>) to 0.01585 (<I>recA</I>). The number of alleles varied from 2 (<I>dnaK</I>) to 13 (<I>recA</I>). SplitsTree recombination was also performed for all seven loci and concatenated sequences. Phylogenetic analysis divided the 13 strains into three major groups based on their relationships. The minimum spanning tree revealed no clear relationship between the and isolation source or location and <I>L. brevis</I> isolates. This is the first genetic diversity study of <I>L. brevis</I> isolates from South Korea.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An MLST technique was developed for characterization of 13 <I>L. brevis</I> strains from South Korea. </LI> <LI> Seven housekeeping genes were selected and analyzed for MLST. </LI> <LI> A total of 83 polymorphic sites varying from 4 to 24 in each species were identified. </LI> <LI> Thirteen sequence types were generated with allele numbers varying from 2 to 13. </LI> </UL> </P>

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing

Sharma, Anshul,Kaur, Jasmine,Lee, Sulhee,Park, Young-Seo Springer-Verlag 2018 Applied Microbiology and Biotechnology Vol.102 No.11

RAPD typing of Lactobacillus brevis isolated from various food products from Korea

Sharma, Anshul,Kaur, Jasmine,Lee, Sulhee,Park, Young-Seo Korean Society of Food Science and Technology 2016 Food Science and Biotechnology Vol.25 No.6

In the present study, the fingerprinting technique, random amplified polymorphic DNA-PCR was evaluated to characterize 13 strains of Lactobacillus brevis, isolated from different vegetable products of South Korea. Two primers i.e. 239 and KAY3 were used. The primer 239 produced bands ranged from 500-4,000 bp and KAY3 primer produced bands with sizes from 600-4,000 bp. Both primers produced thirteen different RAPD profiles. Phylogenetic dendrogram showed that all the isolates could be divided into six major clusters both the primers. However, a few strains of L. brevis had similar profiles and were not well differentiated by RAPD.

DNA profiling of Leuconostoc mesenteroides strains isolated from fermented foods and farm produce in Korea by repetitiveelement PCR

Jasmine Kaur,이설희,Anshul Sharma,박영서 한국식품과학회 2017 Food Science and Biotechnology Vol.26 No.6

Lactic acid bacteria are known for their preservative effects on food products like meat and sausage. Since they are related to humans, these bacteria require proper characterization and identification among various other bacteria in the surroundings. For their identification, several typing methods have already been applied of which the genotyping methods provide reproducible and unambiguous results. In this study, PCR-based method called repetitive element PCR was used for typing 37 Leuconostoc mesenteroides with three primers, REP, ERIC, and (GTG)5, annealing to repetitive sequences present in the bacterial genome. Different fingerprints were obtained for the isolates showing distinguishing profiles. Further phylogenetic analysis was performed using UPGMA method of clustering which provided proper identification with genetic relatedness of all the isolates. It was finally observed that, out of the three primers used, (GTG)5 discriminated the strains precisely than the other two.

Characterization of Lactobacillus brevis isolates using PCR-based molecular typing techniques

Jasmine Kaur,Anshul Sharma,Sulhee Lee,Young-Seo Park 한국산업식품공학회 2017 학술대회 및 심포지엄 Vol.2017 No.04

Fermented food consists of a variety of lactic acid bacteria (LAB) that are also used in the industry as starter cultures. This genus comprises of different species that find importance as a preservatives in food like meat and sausages. Likewise, Lactobacillus brevis has been recognized as GRAS and is a potential probiotic strain extensively being used on an industrial scale. Since such bacteria are directly related to human health, there has been a need to identify and characterize them on the molecular level. In this study, LAB was identified and characterized from various fermented food samples available in South Korea. Two types to PCR-based molecular typing methods were used to analyze the 13 Lactobacillus brevis isolates, of which one was based on difference in the banding patterns originated on agarose gel and the other was related to the sequence analyses of various housekeeping genes in the particular strain. The former rep-PCR technique used three primers namely, REP, ERIC and (GTG)5 that amplified repetitive sequences in the genome and provided characteristic fingerprint profile for each isolate. This clustered the strains in 3 groups with the help of UPGMA method of clustering distinguishing between closely related strains. However, the latter multilocus sequencing typing (MLST) technique provided definite identification of the strains. A set of 7 housekeeping genes were determined as groEL, gyrB, rpoA, rpoB, pheS, recA and dnaK. These genes were amplified and sequenced and were subjected to comparative analysis. Discrete allelic profiles and 13 sequence types (STs) were resolved and minimum spanning tree (MST) was constructed, revealing the genetic relatedness among the isolates. On comparing the results from both the techniques, MLST proved to generate accurate and precise fingerprints owing to the sequence analysis of conserved genes thus providing a scope for research in the monitoring related species.

DNA Profiling of Leuconostoc citreum Strains in Fermented Foods by Repetitive Element Polymerase Chain Reaction

( Jasmine Kaur ),( Anshul Sharma ),( Sulhee Lee ),( Young-seo Park ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.10

To identify and discriminate the bacterial species at the subspecific level, rep-PCR is a reliable genomic fingerprinting tool. Fourteen strains of bacteria were isolated from different food sources, identified as Leuconostoc citreum using 16S rRNA gene sequencing, and amplified using rep-primers (REP, ERIC, and (GTG)₅). Fingerprinting patterns generated bands in the range of 300-6,000 bp with REP, 150-6,000 bp with ERIC, and 200-1,700 bp with (GTG)₅ primers. In UPGMA dendrogram analysis, 14 strains were clustered into three clades (I, II, and III) with all the primers, thus differentiating them at the molecular level. The present study revealed the differentiation of L. citreum strains using rep-PCR.

Molecular Typing of Leuconostoc citreum Strains Isolated from Korean Fermented Foods Using a Random Amplified Polymorphic DNA Marker

( Jasmine Kaur ),( Sulhee Lee ),( Anshul Sharma ),( Young-seo Park ) 한국산업식품공학회 2017 산업 식품공학 Vol.21 No.2

For preliminary molecular typing, PCR-based fingerprinting using random amplified polymorphic DNA (RAPD) is the method of choice. In this study, 14 bacterial strains were isolated from different Korean food sources, identified using 16S rRNA gene sequencing, and characterized through RAPD-PCR. Two PCR primers (239 and KAY3) gen-erated a total of 130 RAPD bands, 14 distinct PCR profiles, 10 polymorphic bands, one monomorphic band, and four unique bands. Dendrogram-based analysis with primer 239 showed that all 14 strains could be divided into seven clades out of which clade VII had the maximum of seven. In contrast, dendrogram analysis with the primer KAY3 divided the 14 L. citreum strains into four clades out of which clade IV consisted of a maximum of 10 strains out of 14. This research identified and characterized bacterial populations associated with different Korean foods. The proposed RAPD-PCR method, based on sequence amplification, could easily identify and discriminate the lactic acid bacteria species at the strain-specific level and could be used as a highly reliable genomic fingerprinting tool.

Analysis of Leuconostoc citreum strains using multilocus sequence typing

Anshul Sharma,Jasmine Kaur,이설희,박영서 한국식품과학회 2018 Food Science and Biotechnology Vol.27 No.6

The objective of this study was to perform genetic diversity analysis of 13 strains isolated from South Korean foods by multilocus sequence typing (MLST). For typing, seven housekeeping loci (atpA, dnaA, dnaK, gyrB, pheS, pyrG, and rpoA) were selected, amplified and analyzed. Fifty-one polymorphic sites varying from 1 to 22 in each species were identified. Thirteen sequence types were generated with allele numbers ranged from 2 to 10. The overall relationship between strains was assessed by unweighted pair group method with arithmetic mean dendrogram and minimum spanning tree. In addition, combined spits tree analysis revealed intragenic recombination. No clear relationship was observed between the isolation sources and strains. The developed MLST scheme enhanced our knowledge of the population diversity of Leu. citreum strains and will be used further for the selection of industrially important strain.

RAPD typing of Lactobacillus brevis isolated from various food products from Korea

Anshul Sharma,Jasmine Kaur,이설희,박영서 한국식품과학회 2016 Food Science and Biotechnology Vol.25 No.6

In the present study, the fingerprinting technique, random amplified polymorphic DNA-PCR was evaluated to characterize 13 strains of Lactobacillus brevis, isolated from different vegetable products of South Korea. Two primers i.e. 239 and KAY3 were used. The primer 239 produced bands ranged from 500-4,000 bp and KAY3 primer produced bands with sizes from 600-4,000 bp. Both primers produced thirteen different RAPD profiles. Phylogenetic dendrogram showed that all the isolates could be divided into six major clusters both the primers. However, a few strains of L. brevis had similar profiles and were not well differentiated by RAPD.

RAPD analysis of <i>Leuconostoc mesenteroides</i> strains associated with vegetables and food products from Korea

Kaur, Jasmine,Lee, Sulhee,Park, Young-Seo,Sharma, Anshul Elsevier 2017 LWT- Food science and technology Vol.77 No.-

<P>Lactic acid bacteria play a very important role in food fermentation. For strain identification, characterization and protection, it is important to use highly discriminatory identification methods. In the present study, 37 strains isolated from vegetables and food products of Korea were identified using 16S rDNA gene sequencing and were found to be Leuconostoc mesenteroides. Further, molecular characterization of all the strains was performed using two RAPD primers, i.e. 239 and KAY3. All strains have shown RAPD profiles and the amplified products of the profiles ranged from 300 to 4000 bp with both the primers and only small differences in banding pattern were observed. Out of 37, maximum bands were observed in strain 11436 with 239 and strain 11260 with KAY3. With both the primers, the phylogenetic analysis revealed seven clades which could be further subdivided into groups. The dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA). The results showed that 16S rDNA sequencing and RAPD-PCR are suitable preliminary molecular tools for identification and characterization of bacteria. (C) 2016 Elsevier Ltd. All rights reserved.</P>