Characterization of Weissella Koreensis SK Isolated From

microorganisms

Article Characterization of Weissella koreensis SK Isolated from Kimchi Fermented at Low Temperature ◦ (around 0 C) Based on Complete Genome Sequence and Corresponding Phenotype

So Yeong Mun and Hae Choon Chang * Department of Food and Nutrition, Kimchi Research Center, Chosun University, 309 Pilmun-daero, Dong-gu, Gwangju 61452, Korea; [email protected] * Correspondence: [email protected]

Received: 17 June 2020; Accepted: 28 July 2020; Published: 29 July 2020

Abstract: This study identiﬁed lactic acid bacteria (LAB) that play a major role in kimchi fermented at low temperature, and investigated the safety and functionality of the LAB via biologic and genomic analyses for its potential use as a starter culture or probiotic. Fifty LAB were isolated from 45 kimchi samples fermented at 1.5~0 C for 2~3 months. Weissella koreensis strains were determined as the − ◦ dominant LAB in all kimchi samples. One strain, W. koreensis SK, was selected and its phenotypic and genomic features characterized. The complete genome of W. koreensis SK contains one circular chromosome and plasmid. W. koreensis SK grew well under mesophilic and psychrophilic conditions. W. koreensis SK was found to ferment several carbohydrates and utilize an alternative carbon source, the amino acid arginine, to obtain energy. Supplementation with arginine improved cell growth and resulted in high production of ornithine. The arginine deiminase pathway of W. koreensis SK was encoded in a cluster of four genes (arcA-arcB-arcD-arcC). No virulence traits were identiﬁed in the genomic and phenotypic analyses. The results indicate that W. koreensis SK may be a promising starter culture for fermented vegetables or fruits at low temperature as well as a probiotic candidate.

Keywords: Weissella koreensis; low-temperature; fermentation; kimchi; ADI pathway

1. Introduction Members of the Weissella genus belonging to the Leuconostocacea family are gram-positive, non-spore forming, heterofermentative, and short-rod bacteria [1]. Weissella can be isolated from a variety of sources, including fermented foods, milk, vegetable, soil, and the gastrointestinal tracts of humans and animals [2]. To date, the Weissella genus comprises 19 validly described species [2]. Weissella koreensis isolated from kimchi, a traditional Korean fermented food, was first proposed as a novel species belonging to the Weissella genus in 2002 by Lee et al. [1]. Several health benefits or functions related with Weissella species, including anti-tumor, anti-obesity, and immuno-modulatory effects, have been reported [3–5]. Although human infections caused by Weissella species are rarely reported, some Weissella species were reported to be involved in disease outbreaks, including endocarditis, sepsis, and fish mortality [6,7]. Thus, any strain intended to be used as a starter culture or probiotic should be properly evaluated in terms of safety. Members of the Weissella genus have been reported as one of the major lactic acid bacteria (LAB) responsible for kimchi fermentation [8]. Among Weissella species, Weissella confusa and Weissella cibaria species are being extensively studied for their use as probiotics based on functional and genomic characteristics [2,9], whereas only few studies have been conducted on the functional, genomic, and metabolic features of W. koreensis [4,10].

Microorganisms 2020, 8, 1147; doi:10.3390/microorganisms8081147 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 1147 2 of 17

The aim of this study was to identify a dominant LAB species in kimchi fermented at 1ow temperature as well as investigate its safety and functionality via biologic and genomic analyses for its potential use as a starter culture or probiotic. For this, we isolated a total of 50 LAB strains as dominant LAB from various kimchi samples, which were fermented at 1.5~0 C for 2~3 months. One strain − ◦ was selected among the isolates, after which its whole genome was sequenced, and genomic features analyzed. Furthermore, its phenotypic characteristics such as carbohydrate assimilation, growth at diﬀerent temperatures, acid-alkali tolerance, amino acid (arginine) catabolism, and virulence traits were investigated and further compared with its genome analysis data.

2. Materials and Methods

2.1. Isolation and Identiﬁcation of LAB Freshly made kimchi was collected from different locations in South Korea and fermented at 1.5~0 C (Kimchi refrigerator, LG, Changwon, Korea) for 2~3 months. The resulting kimchi sample was − ◦ macerated, and the kimchi slurry was filtered through a sterile thin cloth [11]. The kimchi filtrate was then serially diluted with sterile-distilled water and spread onto de Man, Rogosa, and Sharpe (MRS; Difco, Sparks, MD, USA) +2% CaCO3 agar. The plates were incubated at 30 ◦C for 2~3 days, after which colonies forming a clear zone on the MRS+2% CaCO3 agar were tentatively selected as a LAB strain. The isolates were identiﬁed by gram-staining, catalase test, morphological observation under a microscope (Eclipse 55i, Nikon, Tokyo, Japan), and determination of 16S rRNA gene sequences [12,13]. The determined 16S rRNA gene sequences were compared with sequences available in the GenBank database (https: //www.ncbi.nlm.nih.gov/) using CLUSTAL W (https://www.genome.jp/tools-bin/clustalw).

2.2. Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DGGE) The prepared kimchi filtrate was centrifuged (13,475 g, 5 min, 4 C), the pellet washed, and DNA × ◦ extracted using a genomic DNA preparation kit (Qiagen, Hilden, Germany). The extracted DNA was then used as a template for PCR amplification of the 16S rRNA gene using 27F and 1492R 16S universal primers [14]. The purified amplicon was used as a template for nested PCR targeting the V3 region of the 16S rRNA gene using the DGGE primers of Lac1 and GC Lac2 [15]. DGGE was conducted using 8% (w/v) acrylamide with the Dcode system (BioRad, Hercules, CA, USA). Bands of interest were extracted from gels, reamplified using the DGGE primers of Lac1 and GC Lac 2, and sequenced using an automated DNA sequencer (Applied Biosystem, Forster City, CA, USA). The determined partial ribosomal DNA sequences were queried by Blast searches of the GenBank database to identify the closest known relatives.

2.3. Whole Genome Sequencing, Assembly, and Annotation Whole genomic DNA from the LAB isolate was extracted using DNeasy Blood & Tissue Kits (Qiagen). The extracted DNA was completely sequenced by Macrogen (Seoul, Korea) using PacBio RS single-molecule real-time (SMRT) sequencing and Illumina HiseqXten sequencing. The PacBio SMRT reads were assembled using the hierarchical genome assembly process, and errors in PacBio sequencing were corrected by paired-end reads obtained by Illumina sequencing. The complete genome of the LAB isolate consisting of a chromosome and plasmid were deposited in GenBank under the accession numbers CP043431-2. Sequences were further annotated by RAST server using the classic scheme (https://rast.theseed.org/FIG/rast.cgi). Protein coding sequences (CDSs) encoded on the plasmid were annotated by using NCBI BlastX (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.4. Plasmid Preparation and Digestion Plasmid in the LAB isolate was extracted by using a QIAprep spin miniprep kit (Qiagen). The isolated plasmid DNA was digested with restriction enzymes, Bgl II and Blp I, at 37 ◦C for 1 h. Microorganisms 2020, 8, 1147 3 of 17

The plasmid digestion mixtures were separated by electrophoresis on 1% (w/v) agarose gel stained with ethidium bromide.

2.5. Phenotypic Properties

2.5.1. Carbohydrate Assimilation Carbohydrate assimilation of the LAB isolate was investigated using an API 50 CHL (BioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. W. koreensis KACC 11853T, obtained from the Korean Agricultural Culture Collection (Wanju, Korea), was used as a control.

2.5.2. Growth at Different Temperatures Growth of the LAB isolate at different temperatures was observed in MRS broth at 1.5, 5, 10, 15, − 25, and 30 ◦C for 24~2016 h. Growth was determined by monitoring the absorbance at 600 nm (A600; Ultrospec 2100 pro, Biochrom, Cambridge, UK). Growth of viable cells after cultivation at different temperatures was also measured; briefly, the culture was serially diluted, spread on MRS (Difco) agar, and incubated at 30 ◦C, after which the colonies on the plate were counted.

2.5.3. Cell Viability According to pH Level To determine the acid and alkali tolerances of the LAB isolate, the isolate was adjusted to pHs ranging from 3.0~10.0 for 24~48 h. Speciﬁcally, the isolate was cultivated in 5 mL of MRS broth for 24 h, centrifuged (9950 g, 5 min, 4 C), and then resuspended in 5 mL of MRS broth adjusted to pHs × ◦ of 3.0, 4.0, 5.0, 8.0, 9.0, and 10.0. MRS broth without pH adjustment (pH 6.5) was used as a control. Subsequently, the suspensions were incubated at 30 ◦C for 24 or 48 h, after which cell viabilities of suspensions were determined. Homofermentative LAB, Lactobacillus plantarum AF1 (GenBank accession No. FJ386491), which shows strong acid tolerance [16], was used as a control LAB.

2.5.4. Arginine Catabolic Ability The LAB isolate was cultivated in MRS, MRS+1% arginine, and MRS+1% citrulline broths (pH 6.5) at 30 ◦C for 12, 18, 24, 48, and 72 h. Thereafter, viable cells, total cells (A600), pH level, and acidity of the culture were measured. Simultaneously, organic acids, ethyl alcohol, amino acids (arginine, citrulline, and ornithine), and ammonia contents of the culture supernatant were measured. Organic acids and amino acids were analyzed by HPLC (Ultimate 3000, Thermo Scientific Dionex, Sunnyvale, CA, USA). For organic acid and ethyl alcohol analysis, an Aminex 87H column (300 10 mm; Bio-Rad) was used, and the elution solvent was 0.01 N H SO at a flow rate of × 2 4 0.5 mL/min. Organic acids and ethyl alcohol were detected using an RI detector (RefractoMAX520, Tokyo, Japan) [17]. For amino acid analysis, the filter-sterilized culture broth was derivatized using O-phthalaldehyde (OPA)-fluorenylmethyl chloroformate (FMOC) [18] and then analyzed using the HPLC system (Ultimate 3000) equipped with a UV detector and FL detector (Agilent 1260, Agilent, Santa Clara, CA, USA). An Inno C column (5 µm, 4.6 150 mm; Youngjin, Seongnam, Korea) was 18 × used. The mobile phase was prepared with A solution (40 mM sodium phosphate (pH 7.0)) and B solution (water:acetonitrile:methanol = 10:45:45). The program was initiated using solutions A:B = 95:5 (v/v) for 0~24 min, 45:55 for 24~25 min, 10:90 for 25~34.5 min, and 95:5 after 34.5 min at a flow rate of 1.5 mL/min [17]. Ammonia was analyzed by cation-chromatography (Dionex ICS 5000, Thermo Scientific Dionex) equipped with a suppressed conductivity detector and cation self-regenerating suppressor (CSRS-Ultra, 4 mm; Thermo Scientific Dionex) in recycle mode. An Ionpac CS16A column (4 250 mm, Thermo Scientific Dionex) was used. Mobile phase was 40 mM methane sulfonic acid at × a flow rate of 1 mL/min. Microorganisms 2020, 8, 1147 4 of 17

2.5.5. Biogenic Amine Production LAB were incubated in MRS broth supplemented with a precursor amino acid (1% arginine, ornithine, or histidine) at 30 C for 48 h, after which the culture was centrifuged (9950 g, 15 min, ◦ × 4 ◦C). The supernatant was further ﬁlter-sterilized (0.4 µm pore size, Advantec, Dublin, CA, USA), freeze-dried, and then concentrated 5-fold in distilled water. The concentrated sample (2 mL) was added to 10 mL of 5% trichloroacetic acid-0.4 M perchloric acid, after which 0.2 mg of 1,7-diaminoheptane was added as an internal standard. The mixture was homogenized for 5 min, derivatized using benzoyl chloride, dissolved in 0.5 mL of 50% methanol, and then injected (20 µL) for HPLC analysis [19]. The HPLC system (Waters 2695, Waters, Boston, MA, USA) consisted of Waters 2996 photodiode array detector (254 nm) along with Waters Empower software. A SunFire C column (3.5 µm, 4.6 150 mm; 18 × Waters) was used at a ﬂow rate of 0.4 mL/min. The program was initiated with 50% methanol, maintained at 50% methanol for 15 min, increased to 90% methanol for 10 min, decreased to 50% methanol for 5 min, and then maintained at 50% methanol for an additional 5 min [20].

2.6. Statistical Analysis Data are presented as the means and standard deviations (means SD) of two independent ± experiments performed in duplicate. Statistical analyses on the data were performed using the Duncan’s multiple range test of SPSS version 26.0 for Windows (SPSS, Chicago, IL, USA) with the signiﬁcance determined at p < 0.05.

3. Results and Discussion

3.1. Kimchi Samples and Isolation of LAB Mesophilic LAB play a key role in lactic acid fermentation of dairy foods, which occurs at 37~42 C [21]. On the other hand, kimchi is generally fermented at low temperature ( 1~10 C), ◦ − ◦ although sometimes it is fermented at room temperature (15~20 ◦C) to accelerate the fermentation process [8,11]. In Korea, to ensure and maintain quality, a special kimchi refrigerator equipped with a fermentation mode at 6~7 C and storage mode at 2~ 1 C is used for the long-term storage ◦ − − ◦ of kimchi [22]. Analyses have shown that LAB communities belonging to the genera Leuconostoc, Lactobacillus, and Weissella are mainly responsible for kimchi fermentation [23]. However, the dominant LAB strain in fermented kimchi can vary depending on the fermentation temperature, as LAB present in kimchi materials have different growth properties according to temperature [8,22]. In this study, we tried to identify and characterize a key LAB in kimchi fermented at low temperature (around 0 ◦C) for long-term storage. Thus, a total of 45 freshly made kimchi samples (pH 5.81~6.39) were collected from 38 different regions in Korea and then immediately fermented at 1.5~0 C for 2~3 months − ◦ (Supplementary Table S1). After fermentation, pHs of the kimchi samples ranged between 4.18~4.39 (data not shown), which indicates ripened kimchi. Isolation of LAB from the kimchi samples was performed next. Relatively tiny-shaped colonies with a faint clear zone on MRS+2% CaCO3 plate were detected as dominant LAB (accounting for 38~96% of the plates) from all 45 kimchi samples, whereas the others showed definitely larger colonies with a distinct clear zone (data not shown). A total of 50 LAB strains were isolated from 45 kimchi samples (Supplementary Table S1). All isolates were gram-positive, catalase-negative, and short rod-shaped cells. When 16S rRNA gene sequences (1,268~1,523 bp) of the 50 isolates were compared with those of LAB type strains in GenBank, all 50 sequences showed 100% homology with those of W. koreensis JCM11263T (Supplementary Table S1). This result is very unique, considering the 16S rRNA gene sequences of other LAB species such as Leuconostoc and Lactobacillus species show high similarities (greater than 99.54% but not 100%) with those of type strains in GenBank [24,25]. The reason that all 50 16S rRNA gene sequences of the isolates showed 100% homology with each other along with type strain in GenBank should be investigated via further experimentation. Microorganisms 2020, 8, x FOR PEER REVIEW 5 of 16

16S rRNA gene sequences of other LAB species such as Leuconostoc and Lactobacillus species show high similarities (greater than 99.54% but not 100%) with those of type strains in GenBank [24,25]. The reason that all 50 16S rRNA gene sequences of the isolates showed 100% homology with each other along with type strain in GenBank should be investigated via further experimentation. MicroorganismsAmong2020 the, 8 50, 1147 isolates, we selected 15 isolates from 45 kimchi samples according to sampling5 of 17 location; LAB 1, 3, and 6 from Seoul/Gyeonggi, LAB 11, 14, and 18 from Gangwon, LAB 23, 26, and 31 from Chungcheong, LAB 34 and 37 from Busan/Gyeongsang, and LAB 41, 43, 45, and 48 from Gwangju/JeollaAmong the (Supplementary 50 isolates, we selected Table S1). 15 Subsequently, isolates from 45 to kimchiconfirm samples that the accordingselected 15 to isolates sampling are location;indeed dominant LAB 1, 3, LAB and 6in from each Seoulkimchi/Gyeonggi, sample, LABanalysis 11, 14,of bacterial and 18 from communities Gangwon, inLAB the 23,kimchi 26, andsamples 31 from was Chungcheong, performed by LABPCR-DGGE 34 and 37(Figure from Busan1). Microbial/Gyeongsang, population and LABprofiles 41, 43,of 45,the andkimchi 48 fromsamples Gwangju were /differentJeolla (Supplementary from each other. Table However, S1). Subsequently, the arrow-indicated to confirm bands that the identified selected (with 15 isolates 100% areidentity) indeed as dominant W. koreensis LAB were in each detected kimchi as sample,a ticker band analysis from of all bacterial 15 kimchi communities samples (Figure in the kimchi1). The samplesresults demonstrate was performed that by W. PCR-DGGE koreensis was (Figure a dominant1). Microbial LAB population in kimchi fermented profiles of theat low kimchi temperature samples were different from each other. However, the arrow-indicated bands identified (with 100% identity) (−1.5~0 °C for 2~3 months). Kim also reported that W. koreensis is easily detected in kimchi stored at as W. koreensis were detected as a ticker band from all 15 kimchi samples (Figure1). The results demonstrate−1 °C for 1~2 that monthsW. koreensis [26]. was a dominant LAB in kimchi fermented at low temperature ( 1.5~0 C − ◦ for 2~3Finally, months). one Kimisolate also (LAB reported 6) designated that W. koreensis as W. koreensisis easily SK detected among in the kimchi 15 isolates stored was at 1selectedC for − ◦ 1~2based months on carbohydrate [26]. assimilation (Supplementary Table S2) for further experimentation.

FigureFigure 1.1. PCR-DGGEPCR-DGGE patterns of 16S V3 rRNA gene gene sequences sequences in in the the kimchi kimchi samples. samples. 1: 1: Kimchi Kimchi 1, 1,2: 2:Kimchi Kimchi 3, 3,3: Kimchi 3: Kimchi 5, 4: 5, Kimchi 4: Kimchi 10, 5: 10, Kimchi 5: Kimchi 13, 6: 13, Kimchi 6: Kimchi 17, 7: 17,Kimchi 7: Kimchi 21, 8: Kimchi 21, 8:Kimchi 23, 9: Kimchi 23, 9: Kimchi27, 10: Kimchi 27, 10: Kimchi30, 11: Kimc 30, 11:hi 33, Kimchi 12: Kimchi 33, 12: 37, Kimchi 13: Kimchi 37, 13: 39, Kimchi 14: Kimchi 39, 14: 41, Kimchi 15: Kimchi 41, 15:43. KimchiThe closest 43. Therelative closest of relativethe fragments of the fragments (arrow-indicated) (arrow-indicated) was determined was determined and compared and compared using usingsequences sequences from fromGenBank GenBank as described as described in the in Materials the Materials and Methods. and Methods.

3.2. GenomeFinally, oneProperties isolate of (LAB W. koreensis 6) designated SK as W. koreensis SK among the 15 isolates was selected based on carbohydrate assimilation (Supplementary Table S2) for further experimentation. A total of four complete genome sequences of W. koreensis (SK in this study, KACC 15510, 3.2.WiKim0080 Genome Properties and CBA3615) of W. koreensis can be SKobtained from GenBank as of now. The genomes of the four strains were compared and their general features are summarized (Table 1). The complete A total of four complete genome sequences of W. koreensis (SK in this study, KACC 15510, genome of W. koreensis SK was found to consist of a circular single chromosome (1,451,607 bp) WiKim0080 and CBA3615) can be obtained from GenBank as of now. The genomes of the four and one circular plasmid (10,481 bp) with 35.5% and 36.9% G+C contents, respectively (Figure strains were compared and their general features are summarized (Table1). The complete genome of 2A, Table 1). A total of 1318 protein coding genes were identified among the 1389 predicted total W. koreensis SK was found to consist of a circular single chromosome (1,451,607 bp) and one circular genes, along with 71 RNA genes on the chromosome and 11 coding genes on the plasmid. The plasmid (10,481 bp) with 35.5% and 36.9% G+C contents, respectively (Figure2A, Table1). A total of K 1318general protein features coding of genes the four were strains identiﬁed are among slightly the different 1389 predicted from totaleach genes,other; alongWi im0080 with 71 strain RNA genesharbors on the chromosomebiggest genome and (1,537,013 11 coding genesbp) along on the with plasmid. two plasmids, The general while features KACC of the 15510 four harbors strains aretheslightly smallest di genomeﬀerent from (1,441,470 each other; bp). WiKim0080 strain harbors the biggest genome (1,537,013 bp) along with two plasmids, while KACC 15510 harbors the smallest genome (1,441,470 bp).

Table 1. Comparison of general genome features of W. koreensis SK and other W. koreensis strains.

SK KACC 15510 WiKim0080 CBA3615 Feature Chromo- Chromo- Chromo- Chromo- Plasmid Plasmid Plasmid Plasmid Some Some Some Some 31,842 Genome size (bp) 1,451,607 10,481 1,422,478 18,992 1,495,237 1,491,842 19,971 9934 Microorganisms 2020, 8, 1147 40.0 6 of 17 G+C content (%) 35.5 36.9 35.5 36.7 35.5 35.4 38.7 27.5 32 Total genes (no.) 1389 11 1379Table 1. Cont. 24 1454 1443 27 11 Protein coding 32 1318 SK11 1308 KACC 1551024 1382 WiKim0080 1371 CBA3615 27 Feature sequences (no.) Chromo- Chromo- Chromo- 11 Chromo- Plasmid Plasmid Plasmid Plasmid rRNAs (no.) Some15 - Some15 - Some15 - Some15 - tRNAs (no.) 56 - 56 - 57 - 57 - Protein coding 32 GenBank 1318 11 1308 24 1382 CP026848 1371 27 sequences (no.) CP043431 CP043432 CP002899 CP002900 CP026847 11 CP046070 CP046071 AccessionrRNAs (no.) No. 15 - 15 - 15CP026849 - 15 - AssemblytRNAs (no.) level 1 56Complete - 56 Complete - 57 Complete - Chromosome57 - GenBank CP026848 Source CP043431Kimchi CP043432 CP002899 Kimc CP002900hi CP026847Kimchi CP046070Kimchi CP046071 Accession No. CP026849 Reference This study 2 [27] [10] [28] Assembly level 1 Complete Complete Complete Chromosome 1 Complete;Source all chromosomes Kimchi are gapless and Kimchihave no runs of 10 or more Kimchi ambiguous bases, Kimchi there are noReference unplaced or unlocalizedThis study scaffo2 lds, and all the[27 ][expected chromosomes10][ are present. Chromosome;28] there1 Complete; is sequence all chromosomes for one or are more gapless chromosomes. and have no runs This of 10could or more be a ambiguous completely bases, sequenced there are nochromosome unplaced or withoutunlocalized gaps sca fforolds, a chromosome and all the expected containing chromosomes scaffolds are or present. contigs Chromosome; with gaps between there is sequencethem. There for one may or more chromosomes. This could be a completely sequenced chromosome without gaps or a chromosome containing 2 alsoscaffolds be unplaced or contigs withor unlocalized gaps between scaffolds. them. There W. may koreensis also be unplaced was deposited or unlocalized in The sca Koreanffolds. 2 CollectionW. koreensis wasfor Typedeposited Cultures in The (Jeongeup, Korean Collection Korea) for under Type Culturesthe accession (Jeongeup, No. Korea)KCTC14235BP. under the accession No. KCTC14235BP.

Figure 2. Genomic features features of of W. koreensis SK. (A) Circular representation of the chromosome and W. koreensis plasmid genomegenome ofof W. koreensisSK. SK. Marked Marked characteristics characteristics are are shown shown from from outside outside to theto the center; center; CDS CDS on forward strand, CDS on reverse strand, tRNA, rRNA, GC content and GC skew. (B) Subsystem feature on forward strand, CDS on reverse strand, tRNA, rRNA, GC content and GC skew. (B) Subsystem of the genomic sequence of W. koreensis SK analyzed with RAST server. Out of 1334 coding sequences feature of the genomic sequence of W. koreensis SK analyzed with RAST server. Out of 1334 coding predicted by RAST server, the subsystem coverage is 51% (the green bar), which contributes to a total sequences predicted by RAST server, the subsystem coverage is 51% (the green bar), which of 237 subsystems. The blue bar refers to the % of proteins that are not included in the subsystems. contributes to a total of 237 subsystems. The blue bar refers to the % of proteins that are not included (C) Agarose gel electrophoresis of pSK. M, size marker; 1, ccc pSK; 2, linear pSK digested with Bgl II, 3; in the subsystems. (C) Agarose gel electrophoresis of pSK. M, size marker; 1, ccc pSK; 2, linear pSK linear pSK digested with Blp I. (D) Physical and genetic map of pSK from W. koreensis SK. Orientation digested with Bgl Ⅱ, 3; linear pSK digested with Blp Ⅰ. (D) Physical and genetic map of pSK from W. of deduced CDSs are marked by black arrows. koreensis SK. Orientation of deduced CDSs are marked by black arrows. All protein coding sequences (CDSs) of W. koreensis SK were functionally annotated by subsystems based on RAST server (Figure2B).The function annotated results indicate that genes in the W. koreensis SK chromosome were related with protein metabolism (15.57%), carbohydrates (10.97%), DNA metabolism (9.07%), cell wall and capsule (8.82%), RNA metabolism (7.28%), nucleosides and nucleotides (6.83%), amino acids and derivatives (6.61%), etc. Genes for protein metabolism, which account for the highest percentage of the SK strain genome, were shown to be mainly related with protein biosynthesis to maintain cell metabolism. Regarding RAST functional annotation for carbohydrates, W. koreensis SK was shown to harbor genes for glucose metabolism as a central carbohydrate, followed by chitin/N-acetylglucosamine, lactose/galactose, glycerate, lactate, glycerol/glycerol-3-phosphate, Microorganisms 2020, 8, 1147 7 of 17 mannose, ribose, xylose, gluconate/ketogluconate, deoxyribose/deoxynucleoside, and arabinose. Regarding functional annotation for amino acids and derivatives, W. koreensis SK was found to harbor genes related with arginine degradation (37.29%), polyamine metabolism (22.03%), methionine biosynthesis and degradation (22.03%), as well as glutamine, glutamate, and asparagine biosynthesis (8.47%). For the safety assessment of W. koreensis SK, genes related with biogenic amine production, hemolysis, and disease and defense such as antibiotic resistance were investigated. RAST analysis identified a gene encoding a hemolysin III in W. koreensis SK. There are multiple reports that most Weissella species are vancomycin-resistant, as they harbor a D-alanine-D-alanine ligase gene [9]. The D-alanine-D-alanine ligase gene contributing to vancomycin resistance was also identified in W. koreensis SK. However, the analysis did not detect any other genes related with biogenic amine production, toxins, production of superantigens, antibiotic resistance except for vancomycin, or disease and defense in the genome of W. koreensis SK (Figure2B). On the other hand, restriction enzyme sites on plasmid pSK were investigated using NEB Cutter V2.0 tools (http://nc2.neb.com/Nebcutter2/), and 31 single-cutter restriction sites were identified. pSK was extracted from W. koreensis SK by digestion with Bgl II and Blp I at the predicted single-cutter restriction site on pSK. One circular plasmid comprising 10,481 bp in W. koreensis SK was confirmed by this plasmid digestion (Figure2C). Among the 11 coding genes on pSK, seven putative proteins (designated ORF 1 to ORF 7) showing greater than 98% homologies with the CDSs in BlastX with 100% query cover were functionally annotated as mobilization protein, RepB for replication of plasmid, copper transport repressor, heavy-metal associated domain-containing protein, cadmium-translocating P-type ATPase, glyoxalase/extradiol dioxygenase family protein, and fructose permease (Table2).

Table 2. ORFs and their deduced proteins on plasmid pSK from W. koreensis SK.

ORF Related Protein Accession No. ORF Identity (%) Length (a.a) (Length of a.a) of Related Protein mobilization protein 1 411 99.76 WP_104914736.1 [Weissella koreensis] (411) MULTISPECIES: RepB family plasmid replication 2 384 98.18 WP_102753750.1 initiator protein [Leuconostocaceae] (384) CopY/TcrY family copper 3 151 transport repressor 100.00 WP_135469337.1 [Weissella confusa] (151) heavy-metal-associated 4 58 domain-containing protein 100.00 WP_147153977.1 [Weissella soli] (58) MULTISPECIES: cadmium-translocating 5 692 99.86 WP_004909401.1 P-type ATPase [Leuconostocaceae] (692) MULTISPECIES: glyoxalase/bleomycin 6 127 resistance/extradiol 100.00 WP_104914740.1 dioxygenase family protein [Weissella] (127) MULTISPECIES: fructose 7 242 98.76 WP_071952482.1 permease [Leuconostoc] (242) Functional annotation of coding sequences (CDSs) on plasmid pSK was performed using basic local alignment search tool (BlastX). Microorganisms 2020, 8, 1147 8 of 17

3.3. Phenotypic Characteristics of W. koreensis SK

3.3.1. Carbohydrate Assimilation Regarding carbohydrate assimilation, W. koreensis strains were positive for 6~8 carbohydrates, which is a substantially lower number than other LAB species. For instance, Lactobacillus or Leuconostoc species can utilize numerous (15~23) carbohydrates [25,29,30]. The 15 isolates and W. koreensis KACC 11853T as a control (Supplementary Table S2) were positive for ribose, D-glucose, D-mannose, and N-acetyl glucosamine as well as weakly positive for gluconate. Differences in carbohydrate assimilation among LAB were observed for L-arabinose, D-xylose, and D-fructose; 12 isolates showed the same carbohydrate assimilation profile as W. koreensis KACC 11853T, while LAB 3 was only negative for L-arabinose, LAB 23 was only negative for D-xylose, and strains LAB 3, 6, and 23 were negative for D-fructose. From the results of carbohydrate assimilation, we selected LAB 6, identified and designated as W. koreensis SK in Section 3.1, which showed the same carbohydrate assimilation profile as W. koreensis KACC 11853T except for fructose assimilation, for further experiments. When the results of carbohydrate assimilation were compared with gene annotation for carbohydrates, W. koreensis SK was shown to ferment L-arabinose, ribose, D-xylose, D-glucose, D-mannose, N-acetylglucosamine, and gluconate along with harboring genes for utilization of those carbohydrates. W. koreensis SK was also found to harbor genes related with glycerol, galactose, and lactose utilization, but these genes were not phenotypically expressed. Specifically, in the case of lactose utilization, it is well known that LAB can utilize lactose, but it is more common in dairy originated LAB. As kimchi does not contain lactose, LAB in kimchi might not require lactose as a carbon source. We previously reported the lactose-negative metabolism of kimchi-originated LAB; most LAB such as Leuconostoc and Weissella species isolated from kimchi are lactose-negative [16,30]. We also previously reported that the more evolutionally developed strains might have deleted or turned off expression of lactose metabolic genes [16]. However, the current study indicates that kimchi-originated LAB, W. koreensis SK, still retain lactose utilization genes (eight copies) without any phenotypic gene expression. On the other hand, W. koreensis SK was determined to be fructose-negative, whereas the other 13 W. koreensis strains, including KACC 11853T, were fructose-positive (Supplementary Table S2). For LAB to metabolize fructose as a carbon source, fructose should be transported inside of the cell by the fructose transport system, after which fructose can be converted into fructose-6-phosphate by fructose kinase (fk) or mannitol by mannitol dehydrogenase (mdh)[23]. However, neither of these fructose-metabolizing genes (fk and mdh) were detected in the genome of W. koreensis SK, whereas one copy of fructose-permease gene (ORF 7) was identified in plasmid pSK (Table2). In addition, most Lactobacillus and Leuconostoc species reported to be fructose-positive [23] were found to usually harbor several copies of fructose-permease genes encoded on their chromosomes (https://rast.theseed.org/ FIG/rast.cgi). The results suggest that some W. koreensis strains may have deleted fructose-metabolizing genes during their evolutionary process and now utilize other carbon sources instead of fructose to obtain energy. Thus, a fructose-permease gene encoded on plasmid pSK may be considered as an evolutionary trace of the past fructose-metabolizing ability of W. koreensis SK.

3.3.2. Growth at Diﬀerent Temperatures Growth of W. koreensis SK at different temperatures was investigated (Figure3). Optimum growth temperatureofW.koreensisSKwas25~30 ◦C,butW.koreensisSKgrewwellat5 ◦C(A600:1.90, and 8.58 CFU/mL for 264 h of cultivation) and even at 1.5 C (A :0.96 and 8.45 log CFU/mL for 672 h). As shown − ◦ 600 in Figure3, W. koreensis SK, reported as a psychrophilic bacterium [8], was able to grow well under mesophilic as well as psychrophilic conditions. Microorganisms 2020, 8, x FOR PEER REVIEW 9 of 16

Microorganisms 2020, 8, 1147 9 of 17 Microorganisms 2020, 8, x FOR PEER REVIEW 9 of 16

MicroorganismsMicroorganisms 2020Microorganisms 2020, 8,, x8 ,FOR x FOR PEER 2020 PEER ,REVIEW 8, REVIEWx FOR PEER REVIEW 9 of9 17of 17 9 of 17 Microorganisms 2020, 8, x FOR PEER REVIEW 9 of 17

Figure 3. Growth of W. koreensis SK at different temperature. W. koreensis SK was incubated in MRS broth at –1 °C (●); 5 °C (▲); 10 °C (■); 15 °C (○); 25 °C (△); 30 °C (□) for 24~2016 h.

3.3.3. Tolerance to Acid and Alkali Conditions AsFigureFigure shown 3.3. Growth in Figure of W.W. 4, koreensiskoreensisthe acid SK SKtolerance atat didifferentﬀerent of W. temperature.temperature. koreensis SK W.W. was koreensiskoreensis significantly SKSK waswas incubatedincubated weaker than inin MRSMRS that of broth at 1 ◦C( ); 5 ◦C(N▲); 10 ◦C(■); 15 ◦C( ○); 25 ◦C( △); 30 ◦C() for 24~2016 h. L. plantarumbroth at AF1,−–1 °C which (•●); 5 ° Cshowed ( ); 10 strong°C ( ); 15acid °C tolerance ( ); 25 °C (9(44.3~99.6%); 30 °C (□) survival for 24~2016 rate) h. after treatment with phosphate-buffered FigureFigure saline 3. Growth3. Growth orFigure simulated of ofW. 3. W. Growthkoreensis koreensis gastricof SK W.SK at koreensis at#different differentjuice SK temperature.(pH temperature.at different 2.5) for W.temperature. W. koreensis1 koreensish [16]. SK W. SK Cellwas koreensiswas incubated viability incubated SK was in inMRSof incubated MRS W. koreensis in MRS 3.3.3. Tolerance tobroth Acidbroth at at–1 and –1°C ° (C● Alkali );(● 5); ° 5C ° (C▲ Conditions ();▲ 10); 10°C ° (C■ );(■ 15▲); 15°C ° (C ○ );(○ 25);■ 25°C ° (C △ );(△ 30);○ 30°C ° (C□ )( □for)△ for 24~2016 24~2016 h. h. Figure 3. Growth of W. koreensis SK at different temperature. W. koreensis SK was incubatedSK3.3.3. was inTolerance MRSdramatically to Acid reduced andbroth Alkali between at –1 °ConditionsC (● );pH 5 °C 3.0~4.0; ( ); 10 °C no ( ); surviving15 °C ( ); 25 ° CW. ( );koreensis 30 °C (□) for SK 24~2016 cells h. were detected broth at –1 °C (●); 5 °C (▲); 10 °C (■); 15 °C (○); 25 °C (△); 30 °C (□) for 24~2016 h. As shown in Figure4, the acid tolerance of W. koreensis SK was significantly weaker than that of uponAs treatment shown3.3.3.3.3.3. inat Tolerance FigurepH Tolerance 3.03.3.3. 4,forto thetoToleranceAcid 24Acid acid andh, and and Alkalitoleranceto Alkali Acid reduced Conditions andConditions ofAlkali (approximatelyW. Conditionskoreensis SK was90-folds) significantly cell viability weaker was than observed that of L. plantarum AF1, which showed strong acid tolerance (94.3~99.6% survival rate) after treatment with 3.3.3. Tolerance to Acid and Alkali Conditions uponL. plantarum treatment AF1, atAs which AspH shown shown 4.0 showed in for inAsFigure Figure shown48 h4,strong 4,thecompared in the Figureacid acid acid tolerance tolerance4, tolerance theto acidthe of ofW. tolerancecontrol W. koreensis (9 koreensis4.3~99.6% of(pH SK W. SK was koreensis6.5) was survival significantly atsignificantly SK48 wash. rate) On significantlyweaker weaker afterthe thanother treatmentthan weakerthat that hand, of of than with the that of phosphate-buffered saline or simulated gastric juice (pH 2.5) for 1 h [16]. Cell viability of W. koreensis As shown in Figure 4, the acid tolerance of W. koreensis SK was significantly weakerstrongphosphate-buffered than acid-tolerant that L.of L.plantarum plantarum LABsaline AF1,L. AF1,plantarumstrain orwhich which simulated L. showed AF1, plantarumshowed which strong gastric strong showed AF1 acid acid juice tolerancewas strong tolerance (pH stable acid (9 2.5) 4.3~99.6%(9 tolerance 4.3~99.6%within for 1 survival h (9the survival4.3~99.6%[16]. pH Cellrate) rangerate) survival afterviability after oftreatment treatmentrate)3.0~6.5 of after W. with for withtreatment koreensis 24~48 with SK was dramatically reduced between pH 3.0~4.0; no surviving W. koreensis SK cells were detected L. plantarum AF1, which showed strong acid tolerance (94.3~99.6% survival rate) afterh.SK Ittreatment was has dramaticallybeen withphosphate-buffered reported phosphate-buffered reduced phosphate-bufferedthat members saline betweensaline or orsimulated simulatedof salinepH Lactobacillus 3.0~4.0; orgastric gastricsimulated juice no juice and (pHsurvivinggastric (pH 2.5)Weissella 2.5) juice for for 1 (pH W. h1 ,[16].h including 2.5)koreensis[16]. Cell for Cell 1viability h viability [16].SK L. cellssakei Cellof ofW. viability W. , were koreensisL. koreensis plantarum detectedof W. koreensis, phosphate-buffered saline or simulated gastric juice (pH 2.5) for 1 h [16]. Cell viabilityupon of W. treatment koreensisSK SK was at was pH dramatically dramatically 3.0SK forwas 24 dramaticallyreduced reduced h, and between between reduced reduced pH pH between3.0~4.0; (approximately 3.0~4.0; no pH no surviving 3.0~4.0;surviving 90-folds)no W. W.surviving koreensis koreensis cell SKW. SK viability cellskoreensis cells were were SK detected was cellsdetected observed were detected andupon W. treatment koreensis at, showpH 3.0 strong for 24 h,acid and tolerance, reduced (approximatelyand they become 90-folds) more celldominant viability as was the observed kimchi SK was dramatically reduced between pH 3.0~4.0; no surviving W. koreensis SK cellsupon were treatment detectedupon upon at treatment treatment pHupon 4.0 at for atpHtreatment pH 483.0 3.0 for h for atcompared24 pH24 h, h, and3.0 and forreduced reduced 24 to h, the and(approximately (approximately controlreduced (approximately (pH 90-folds) 90-folds) 6.5) atcell cell 48 90-folds)viability viability h. On cellwas was theviability observed observed other was hand, observed fermentationupon treatment uponenvironmentupon at treatment pHtreatment 4.0upon at forbecomes attreatmentpH pH48 4.0 4.0h for compared formoreat 48 pH48 h compared h 4.0 acidiccompared for to 48 [23].the toh comparedtothe control theHowever, control control to(pH (pH the (pH according 6.5)6.5)control 6.5) at at at48 (pH 48 h. h. Ontoh.6.5) On Onthethe at the 48othertheresults otherh. On otherhand, hand, thein thehand,otherFigure the hand, the 4, the upon treatment at pH 3.0 for 24 h, and reduced (approximately 90-folds) cell viabilitythe was strong observed acid-tolerant LAB strain L. plantarum AF1 was stable within the pH range of 3.0~6.5 for upon treatment at pH 4.0 for 48 h compared to the control (pH 6.5) at 48 h. On theL.strong plantarumother hand,acid-tolerant theAF1strong strong was acid-tolerant acid-tolerantLAB stronglystrong strain acid-tolerantLAB acid-tolerantLAB L. strain plantarum strain L. LAB L.plantarum plantarum strainwhile AF1 AF1L. was W.AF1plantarum was koreensis stablewas stable stable AF1 within within SK waswithin was stable the the the pHnot. pHwithinpH range rangerange the of of 3.0~6.5pH of3.0~6.5 range 3.0~6.5 for for of 24~48 3.0~6.524~48 for 24~48 for 24~48 24~48 h. It has been reported that members of Lactobacillus and Weissella, including L. sakei, L. plantarum, strong acid-tolerant LAB strain L. plantarum AF1 was stable within the pH range ofh. 3.0~6.5 It L.has plantarumfor been 24~48h. h.reportedIt IthasAF1 has been showedbeenh. thatreported It reported has members significantlybeen that that reported members members of Lactobacillus that higher of ofLactobacillusmembers Lactobacillus stability ofand andLactobacillus and inWeissella Weissella MRSWeissella andfor, ,including ,including including 48Weissella h than L., L.includingsakei L. W.sakei ,sakei L.koreensis, L.plantarum ,L.plantarum L. sakei plantarum SK., , L., plantarum Cell, , h. It has been reported that members of Lactobacillus and Weissella, including L. sakeiand, W.L. plantarum koreensisand, and, showW. W. koreensis koreensis strongand , W.show, acid showkoreensis tolerance,strong strong, show acid acid andstrongtolerance, tolerance, they acid becomeand tolerance,and they they more become becomeand dominant they more more become dominant dominant as themorekimchi as dominant asthe the kimchi fermentation kimchi as the kimchi viabilityand W. ofkoreensis L. plantarum, show AF1 strong slightly acid was tolerance, reduced and depending they become on the increasemore dominant in pH from as the8.0 tokimchi 10.0. and W. koreensis, show strong acid tolerance, and they become more dominantenvironment as the kimchifermentation becomes fermentation morefermentation environment environment acidic [environment 23becomes becomes]. However, more more becomes acidic acidic according [23].more [23]. However, acidic However, to the[23]. according results accordingHowever, into toaccording Figurethe the results results4, to L.in the inFigure plantarum Figure results 4, 4, in AF1Figure 4, However,fermentation theL. environmentalkali L.plantarum plantarum tolerance AF1L. AF1plantarum wasbecomes was ofstrongly strongly W.AF1 koreensis morewas acid-tolerant acid-tolerant strongly acidic SK acid-tolerant while was[23]. while W. determinedHowever, W. koreensis koreensis while SK W. SKaccording wastokoreensis was be not. not.quite SK to was high,the not. results and reduction in Figure of 4, fermentation environment becomes more acidic [23]. However, according to the resultswas strongly in Figure acid-tolerant4, while W. koreensis SK was not. L. plantarum AF1 was strongly acid-tolerant while W. koreensis SK was not. SKL. plantarumviable cells AF1 at L. pHwas L.plantarum plantarum 9.0~10.0 strongly AF1L. AF1 plantarumwas showedacid-tolerant showed significantly AF1significantly significantly showed while less significantlyhigher higher W. compared stabilitykoreensis stability higher in toin MRSSK stability MRSthat was for for below48 in not.48 h MRS thanh than pH forW. W. 8.0.48koreensis koreensis h than SK. W. SK. Cellkoreensis Cell SK. Cell L. plantarum AF1 showed significantly higher stability in MRS for 48 h than W. koreensisL. plantarum SK. Cellviability viability AF1 of ofL.showedviability L.plantarum plantarum ofsignificantly AF1L. AF1plantarum slightly slightly AF1was higher was reducedslightly reduced stability wasdepending depending reduced in MRS on depending on the thefor increase increase 48 on h inthethan inpH increase pH fromW. from koreensis 8.0 in 8.0 topH to10.0. from 10.0. SK. 8.0 Cell to 10.0. viability of L. plantarum AF1 slightly was reduced depending on the increase in pH from 8.0 to 10.0.However, However, the theHowever, alkali alkali tolerance tolerancethe alkali of of W.tolerance W. koreensis koreensis of SKW. SK waskoreensis was determined determined SK was todetermined tobe be quite quite high, to high, be and quite and reduction reductionhigh, and of ofreduction of viability of L. plantarum AF1 slightly was reduced depending on the increase in pH from 8.0 to 10.0. However, the alkali tolerance of W. koreensis SK was determined to be quite high, and reduction SKof SK viable viable cells cellsSK at viable atpH pH 9.0~10.0 cells9.0~10.0 at waspH was 9.0~10.0significantly significantly was significantlyless less compared compared less to comparedtothat that below below to pH thatpH 8.0. 8.0.below pH 8.0. SK viable cells at pH 9.0~10.0 was significantly less compared to that below pH 8.0.However, the alkali tolerance of W. koreensis SK was determined to be quite high, and reduction of SK viable cells at pH 9.0~10.0 was significantly less compared to that below pH 8.0.

Figure 4. Acid and alkali tolerances of W. koreensis. Overnight-cultured W. koreensis SK (A) and Figure 4. AcidFigure Figureand 4.alkali 4.Acid AcidFigure andtolerances and alkali 4. alkali Acid tolerances tolerancesofand W. alkali ofkoreensis of W.tolerances W. koreensis koreensis. Overnight-cultured of. Overnight-cultured .W. Overnight-cultured koreensis. Overnight-cultured W. W. W. koreensis koreensis SK W.SK ( ASKkoreensis ()A and )( andA )L. SKandL. (A L.) and L. L. plantarum AF1 (B) in MRS broth (pH 6.5) were harvested, and each cell pellet was resuspended Figure 4. Acid and alkali tolerances of W. koreensis. Overnight-cultured W. koreensis SK (Aplantarum) and L. AF1plantarum (plantarumB) in MRSAF1 AF1plantarum (B broth)( Bin) inMRS MRSAF1(pH broth (broth B6.5)) in(pH (pHMRSwere 6.5) 6.5) broth wereharvested, were (pHharvested, harvested, 6.5) wereand and and harvested,each each each cellcell cell pelletand pellet pellet each was was wascell resuspended resuspendedpellet resuspended was inresuspended in in in plantarum AF1 (B) in MRS broth (pH 6.5) were harvested, and each cell pellet was resuspendedMRSin MRS inbroth broth adjustedMRS adjustedMRS broth broth to adjustedpH toadjustedMRS 3.0 pH broth to(□ 3.0topH), pH4.0adjusted 3.0 ( 3.0 (),▨□ ),( 4.0),□ 4.0 ),to 5.0 4.0 (pH(▨ ((), ▩▨3.0 5.0), 5.0), 5.0( □6.5(▩), (( ),4.0▩ (6.5),■ (6.5,▨ 6.5(as■), (, 5.0■ asa (, ascontrol),a(▩ ,control), a), as control), 6.5 a (■ control), ,8.08.0 as 8.0 (a ■( control),■(),■ 9.0),), 9.0 8.0 (▧ ( ), ▧(8.0 (▧or), ), (),or10.0■ or ), 9.010.0 9.0 (10.0▦ ( ().▦▧ The).), ( The▦or or ).10.0 10.0The (▦ ). The MRS broth adjusted to pH 3.0 (□), 4.0 (▨), 5.0 (▩), 6.5 (■, as a control), 8.0 (■), 9.0 (▧), or 10.0 ((▦). The The suspensionsuspensionsuspension was was suspensionwas incubated incubated incubated was at at at 30incubated 30 30°C ° CCfor for 24 at 24 24or30 or 48 or° C 48h. 48for h.Thereafter, h.24Thereafter, Thereafter,or 48 h.viable viableThereafter, cells viable cells of viableofthe cells the suspension cellssuspension of theof the suspensionwere suspensionwere were suspension was incubated at 30 °C for 24◦ or 48 h. Thereafter, viable cells of the suspension were suspension was incubated at 30 °C for 24 or 48 h. Thereafter, viable cells of the suspension were determined.determined. determined. determined.were determined. determined. Figure 4. Acid and alkali tolerances of W. koreensis. Overnight-cultured W. koreensis SK (A) and L. 3.3.4.3.3.4. Biogenic Biogenic3.3.4. Amine Amine Biogenic (BA) (BA) ProductionAmine Production (BA) and Production and Other Other Virulence andVirulence Other Traits TraitsVirulence Traits L.plantarum plantarum AF1AF1 (B) in showed MRS broth signiﬁcantly (pH 6.5) were higher harvested, stability and in each MRS cell for pellet 48 hwas than resuspendedW. koreensis in SK. 3.3.4. Biogenic Amine (BA) Production and Other Virulence Traits 3.3.4. Biogenic AmineAlthoughAlthough (BA) BAs BAs AlthoughProduction are are required required BAs forareand for manyrequired manyOther critical critical for Virulence many bi ologicalbiological critical Traitsfunctions, functions,biological exce excefunctions,ssivessive human human exce ssiveconsumption consumption human consumption Cell viabilityMRS broth of adjustedL. plantarum to pH AF13.0 (□ slightly), 4.0 (▨), was 5.0 (▩ reduced), 6.5 (■, dependingas a control), on 8.0the (■), increase 9.0 (▧), or in 10.0 pH (▦ from). The 8.0 to Although BAs are required for many critical biological functions, excessive human consumptionof ofBAs BAs can can haveof have BAs toxicological toxicological can have toxicologicaleffe effectscts [31]. [31]. BAseffe BAs ctssuch such [31]. as asBAsagmatine, agmatine, such asputrescine, putrescine,agmatine, and putrescine, and histamine histamine and can canhistamine be be can be 10.0.Althoughsuspension However, BAs thewas alkaliare incubated required tolerance at for30 of°manyCW. for koreensis critical24 or 48 biSK h.ological Thereafter, was determined functions, viable exce tocells be ssiveof quite the human suspension high, andconsumption reductionwere of BAs can have toxicological effects [31]. BAs such as agmatine, putrescine, and histamine can be of SKBAsdetermined. viable can have cells toxicological at pH 9.0~10.0 effe wascts signiﬁcantly[31]. BAs such less as compared agmatine, to putrescine, that below and pH 8.0.histamine can be

Microorganisms 2020, 8, 1147 10 of 17

3.3.4. Biogenic Amine (BA) Production and Other Virulence Traits Although BAs are required for many critical biological functions, excessive human consumption of BAs can have toxicological effects [31]. BAs such as agmatine, putrescine, and histamine can be produced by microbial decarboxylation of amino acids, arginine, ornithine, and histidine, respectively [31]. In this study, we examined whether or not W. koreensis SK produces BAs via amino acid decarboxylation or in combination with decarboxylation and the ADI pathway. As shown in Supplementary Figure S1, no BA was detected among the W. koreensis SK cultures, which supports the gene analysis in which W. koreensis SK was demonstrated as lacking biogenic amine-forming genes. However, some Weissella strains have been reported to produce BAs such as histamine and putrescine [32]. The selection of LAB starters lacking pathways for BA accumulation is essential to develop high-quality foods with reduced contents of these toxic compounds. Thus, it was proposed that the inability of a strain to synthesize BAs is an important selection criterion for starter cultures [32]. In our previous study, the potential virulence of W. koreensis SK was investigated; W. koreensis SK did not show α- or β-hemolysis activity [33]. However, in this study, a gene encoding hemolysin III was identified in W. koreensis SK. Most LAB species have been recognized as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration [34] or have attained Qualified Presumption of Safety (QPS) status by the European Commission-European Food Safety Authority (EFSA) [35]. However, Weissella species have not yet attained QPS status [36], and thus breakpoints for antibiotics for Weissella species have not been made available by the EFSA. The minimal inhibitory concentrations (MICs) (0.5~8 µg/mL) [33] of antibiotics (ampicillin, gentamycin, kanamycin, streptomycin, erythromycin, tetracycline, and chloramphenicol) for W. koreensis SK have been reported to be significantly lower than those for other Weissella species, including W. koreensis, as determined by previous reports [37,38]. Breakpoints (MICs) of vancomycin for LAB such as Lactobacillus and Leuconostoc species are not required according to the guidelines of the EFSA [39], as most LAB are intrinsically vancomycin-resistant with MICs greater than 512 µg/mL [16,33]. Regarding vancomycin, W. koreensis SK showed a significantly lower MIC (128 µg/mL) [33], although our results show that it harbors a vancomycin-resistant gene. These results indicate that that W. koreensis SK is susceptible to all antibiotics tested except vancomycin. Based on these results, it can be concluded that W. koreensis SK is safe for human consumption as a starter culture or a probiotic.

3.3.5. Growth of W. koreensis SK in MRS Supplemented with Arginine and Citrulline To investigate the arginine catabolic features of W. koreensis SK, strain SK was cultivated in MRS, MRS+1% arginine, and MRS+1% citrulline. Viable cells (CFU/mL), total cells (A600), pH level, and acidity of the culture were measured during 72 h (Figure5). Cell viabilities of the three cultures were almost identical during 12~24 h, with a maximum cell viability of 8.99 log CFU/mL. However, cell viability of MRS dramatically decreased after 24 h, and 1.96 log CFU/mL was detected at 72 h. Viable cell counts among the others gradually decreased after 24 h; 6.60 log CFU/mL in MRS+1% arginine and 7.35 log CFU/mL in MRS+1% citrulline were detected at 72 h (Figure5A). Total cell growth levels of the three different cultures were significantly different (Figure5B). Total cell growth of MRS+1% arginine (A600:3.70) was substantially higher than those of the other two cultures (A600:2.57 for MRS and A600:2.53 for MRS+1% citrulline). All three W. koreensis SK cultures reached stationary phase from 24 h, and the highest A600 values were obtained between 18~24 h. The pHs (acidity %) of the cultures were pH 4.36 (1.05%) for MRS culture, pH 5.10 (0.65%) for MRS+1% citrulline culture, and pH 6.40 (0.31%) for MRS+1% arginine culture at 72 h (Figure5C,D). Organic acid production due to cell growth in MRS resulted in a pH decrease. However, the pH of MRS+1% arginine culture slightly decreased until 12 h, after which it increased and reached its peak at 48 h, even though its total cell growth was significantly higher than those of the other two cultures (Figure5). Microorganisms 2020, 8, x FOR PEER REVIEW 11 of 16 carbamate kinase [40]. Genes for the ADI pathway were identified in W. koreensis SK by RAST functional annotation (Figure 2B). Ammonia production by the ADI pathway of W. koreensis SK may have caused the increase in pH in MRS supplemented with arginine or citrulline. However, the pH of MRS culture decreased due to lack of ammonia production, which resulted in rapid reduction of cell viability at 72 h due to the weak acid tolerance of W. koreensis SK, as supported by Figure 4. The pH of W. koreensis SK culture in MRS supplemented with arginine (pH 6.40) at 72 h was clearly higher than those of the other two cultures (pH 4.36 for MRS, pH 5.10 for MRS+1% citrulline), and SK culture showed higher total cell growth (Figure 5). These results definitively show that arginine facilitates theMicroorganisms growth 2020of W., 8, 1147koreensis SK, whereas citrulline has no such effect even though citrulline11 of 17 supplementation caused a rise in pH along with ammonia production.

Figure 5. Growth, pH, and acidity changes of W. koreensis SK. W. koreensis SK was cultivated in MRS Figure 5. Growth, pH, and acidity changes of W. koreensis SK. W. koreensis SK was cultivated in MRS ( ); MRS+1% arginine (N); MRS+1% citrulline () at 30 C for 72 h. Viable cell (A), total cell (B), pH (C), (●•); MRS+1% arginine (▲); MRS+1% citrulline (■) at 30◦ °C for 72 h. Viable cell (A), total cell (B), pH and acidity (D) were determined. (C), and acidity (D) were determined. Supplementation with arginine or citrulline has been shown to enhance bacterial growth, as bacteria 3.4. Arginine and Glucose Metabolism by W. koreensis SK use arginine and citrulline as energy, carbon, and nitrogen sources [40]. In this study, growth enhancementWe determined of W. koreensis the arginineSK was catabolic observed products upon supplementation of W. koreensis SK with in arginineMRS, MRS+1% but not arginine, citrulline and(Figure MRS+1%5B). Arginine citrulline deiminase cultures (ADI)(Table converts 3). Added L-arginine arginine into and L-citrulline citrulline andwere ammonia, converted after into which their metabolitesL-citrulline and is transferred reached their to ornithine highest amounts transcarbamoylase, at 72 h. Most which of the yields added ornithine arginine and (MRS-A) carbamoyl was completelyphosphate. converted Carbamoyl into phosphate citrulline (676.13 is then mg/L), converted ornithine into ammonia, (7174.73 mg/L), carbon and dioxide, ammonia and (2321.34 ATP by mg/L)carbamate at 72 kinase h. Added [40]. citrulline Genes for in theMRS-C ADI (6687. pathway78 mg/L) were identifiedgradually indecreasedW. koreensis in concentrationSK by RAST (2155.59functional mg/L) annotation and was (Figure converted2B).Ammonia into ornithine production (4254.92 by mg/L) the ADI at 72 pathway h. Glutam of icW. acid koreensis levelsSK of maythe threehave cultures caused the gradually increase increased in pH in MRSduring supplemented 72 h of cultivation. with arginine or citrulline. However, the pH of MRS culture decreased due to lack of ammonia production, which resulted in rapid reduction of cell viability at 72 h due to the weak acid tolerance of W. koreensis SK, as supported by Figure4. The pH of W. koreensis SK culture in MRS supplemented with arginine (pH 6.40) at 72 h was clearly higher than those of the other two cultures (pH 4.36 for MRS, pH 5.10 for MRS+1% citrulline), and SK culture showed higher total cell growth (Figure5). These results definitively show that arginine facilitates the growth of W. koreensis SK, whereas citrulline has no such effect even though citrulline supplementation caused a rise in pH along with ammonia production.

3.4. Arginine and Glucose Metabolism by W. koreensis SK We determined the arginine catabolic products of W. koreensis SK in MRS, MRS+1% arginine, and MRS+1% citrulline cultures (Table3). Added arginine and citrulline were converted into their metabolites and reached their highest amounts at 72 h. Most of the added arginine (MRS-A) was completely converted into citrulline (676.13 mg/L), ornithine (7174.73 mg/L), and ammonia (2321.34 mg/L) at 72 h. Added citrulline in MRS-C (6687.78 mg/L) gradually decreased in concentration (2155.59 mg/L) and was converted into ornithine (4254.92 mg/L) at 72 h. Glutamic acid levels of the three cultures gradually increased during 72 h of cultivation. Microorganisms 2020, 8, 1147 12 of 17

Table 3. ADI and glucose metabolites produced by W. koreensis SK. Unit: mg/L

Incubation Time Substrate Culture Content 0 h 24 h 72 h Glutamic acid 614.69 17.96 a,C 666.74 9.16 a,B 719.00 16.48 a,A ± ± ± Glutamine N.D 1 N.D N.D Citrulline 11.38 3.85 c,A 3.07 0.85 c,B 3.91 0.20 c,B MRS ± ± ± Arginine 301.38 62.79 b,A 7.48 2.65 b,B 5.09 1.74 b,B ± ± ± Ornithine 38.78 10.09 b,B 93.25 13.10 c,A 99.16 8.37 c,A ± ± ± NH 453.06 49.30 a,A 462.52 46.27 b,A 443.97 10.23 c,A 3 ± ± ± Glutamic acid 611.92 17.25 a,C 677.89 5.39 a,B 739.63 11.00 a,A ± ± ± Arginine/ Glutamine N.D N.D N.D Citrulline 47.90 11.75 b,C 841.70 75.64 b,A 676.13 12.60 b,B Citrulline MRS-A ± ± ± Arginine 7713.34 34.57 a,A 62.25 9.10 a,B 41.68 1.07 a,B ± ± ± Ornithine 55.81 21.42 b,B 6569.52 327.28 a,A 7174.73 148.56 a,A ± ± ± NH 465.11 8.64 a,B 2334.97 182.86 a,A 2321.34 143.57 a,A 3 ± ± ± Glutamic acid 617.07 16.83 a,C 676.51 12.01 a,B 725.21 10.52 a,A ± ± ± Glutamine N.D N.D N.D Citrulline 6687.78 5.11 a,A 4504.69 199.02 a,B 2155.59 69.07 a,C MRS-C ± ± ± Arginine 229.10 0.08 b,A 5.01 1.60 b,B 4.95 0.24 b,B ± ± ± Ornithine 1010.71 63.32 a,A 3048.81 76.68 b,B 4254.92 193.10 b,C ± ± ± NH 479.51 32.06 a,B 697.32 112.75 b,B 938.11 29.22 b,A 3 ± ± ± Lactic acid 209.00 9.85 a,C 5917.63 201.77 b,B 7556.81 31.06 a,A ± ± ± Formic acid 144.36 1.12 a,B 180.37 1.05 b,A 198.10 17.70 a,A ± ± ± MRS Acetic acid 4396.08 49.64 a,C 4538.84 4.50 a,B 4730.90 28.87 a,A ± ± ± EtOH N.D 354.56 10.03 b,B 431.56 1.16 b,A ± ± Citric acid 1947.97 38.88 a,A 1870.40 22.09 a,A 1895.47 38.77 a,A ± ± ± Lactic acid 210.14 2.50 a,B 6965.00 255.56 a,A 7078.32 9.72 b,A ± ± ± Formic acid 145.69 19.66 a,B 233.16 4.01 a,A 228.98 15.42 a,A Glucose ± ± ± MRS-A Acetic acid 4351.16 54.43 a,B 4539.66 25.54 a,A 4534.56 34.99 bA ± ± ± EtOH N.D 458.74 23.58 a,A 464.78 4.60 a,A ± ± Citric acid 1932.38 57.61 a,B 1817.38 7.87 a,A,B 1832.65 13.49 a,b,A ± ± ± Lactic acid 194.64 3.72 a,C 5339.90 192.18 b,B 7166.88 223.21 a,b,A ± ± ± Formic acid 148.02 30.62 a,A 207.75 14.47 a,b,A 197.35 3.49 a,A ± ± ± MRS-C Acetic acid 4220.48 238.05 a,A 4475.87 2.93 b,A 4481.72 69.85 b,A ± ± ± EtOH N.D 323.44 15.38 b,B 444.94 5.91 b,A ± ± Citric acid 1825.29 108.46 a,A 1823.65 35.44 a,A 1767.00 41.11 a,A ± ± ± W. koreensis SK was cultivated in MRS, MRS+1% arginine (MRS-A), and MRS+1% citrulline (MRS-C) at 30 ◦C for 72 h. Each content from the culture supernatant was measured at 0, 24, and 72 h as described in Materials and Methods. Values are mean SD from duplicate determination. Means with different letters are significantly different (p < 0.05) by one-way ANOVA,± followed by Duncan’s multiple range test; a~c: means for culture media (row); A–C: means for incubation time (column). 1 N.D: Not detected.

The RAST annotation results confirm arginine degradation via the ADI pathway, which is composed of ADI encoded by arcA, ornithine transcarbamoylase (encoded by arcB), and carbamate kinase (encoded by arcC), in W. koreensis SK. The ADI pathway of W koreensis SK was shown to be encoded by a cluster of four genes (arcA-arcB-arcD-arcC) and one arginine repressor gene (argR) located downstream of the cluster transcribed in the opposite direction (Figure6A). It has been reported that clusters containing the structural genes encoding the ADI pathway are quite diverse, and the order of the genes differs among different species [41]. Analysis of the arc gene cluster found in the genomes of 1281 different bacterial species revealed the presence of 124 arc gene clusters and two types of L-arginine/L-ornithine exchangers, which belong to two different gene families (arcD and arcE) in bacteria. The majority of the arc gene clusters analyzed contain either arcD or arcE, but few (3/124) contain both [40]. Analysis of gene order in the 124 different bacterial clusters showed that the order of transcription was arcA followed by arcB and then arc with few exceptions. The arcD or arcE gene Microorganisms 2020, 8, 1147 13 of 17 Microorganisms 2020, 8, x FOR PEER REVIEW 13 of 16

(andgene other(and arcothergenes) arc genes) is added is added to this to sequence this sequence or inserted or inserted in between. in between. For example, For example, the ADI the gene ADI clustergene cluster in Lactobacillus in Lactobacillus brevis containsbrevis containsarcE1- arcAarcE1-arcB-arcA-arcD-arcB-arcT-arcD-arcC-arcT-argR-arcCand-argR2CS and-arcE2 2CS--arcDarcE2in-arcD the oppositein the opposite direction direction [42], whereas [42], whereas the ADI the pathway ADI pathway of Lactobcoccus of Lactobcoccus lactis is encodedlactis is encoded by a cluster by a ofcluster nine genes,of ninearcD2-arcT-arcC2-arcC1-arcD1-arcB-arcA-argS genes, arcD2-arcT-arcC2-arcC1-arcD1-arcB-arcA-argSwith -argR encodedwith -argR in theencoded opposite in directionthe opposite [40]. Thedirection order [40]. and directionThe order of andarcA direction, arcB, arcC of arcA,, and arcDarcB, inarcC,W. koreensisand arcDSK in W. were koreensis shown SK to bewere the shown same asto thosebe the in same other as bacteria, those in but other the bacteria, numbers but of these the numbers genes encoding of these the genes ADI encoding pathway the were ADI found pathway to be diwereﬀerent. found In particular,to be different. duplicated In particular, genes and duplicated other associated genes and genes other mostly associated of unknown genes function mostly inof otherunknown bacteria function [40] were in other not foundbacteria in [40]W. koreensiswere notSK. found in W. koreensis SK.

FigureFigure 6. Schematic 6. Schematic representation representation of of ADI ADI pathway-related pathway-related gene gene cluster cluster (A ()A and) and ADI ADI pathway pathway (B) (B) in inW.W. koreensis koreensis SKSK.. Red Red character: character: identified identiﬁed compound oror gene,gene, green green character: character: cannot cannot identify, identify, andand black character:character: did did not not try try to identify.to identify. Numbers Numb iners A indicatein A indicate start and start stop and bases stop in thebases genome in the genomeof W. koreensis of W. koreensisSK. Solid SK. arrow: Solid proven arrow: pathway proven and pathway dotted arrow: and dotted unproven arrow: pathway. unproven pathway.

Furthermore,Furthermore, carbamoyl phosphate phosphate synthetase synthetase (carA (carA and and carB carB), glutamine), glutamine synthetase synthetase (glnA (glnA), and), andglutamine-fructose-6-phosphate glutamine-fructose-6-phosphate transaminase transaminase (glmS (glmS) were) were detected detected in in W.W. koreensis koreensis SKSK (Figure (Figure 66A).A). GlutamineGlutamine cancan bebe synthesizedsynthesized fromfrom glutamateglutamate andand ammoniaammonia byby glutamateglutamate synthetase.synthetase. However, glutamineglutamine waswas notnot detecteddetected inin anyany ofof thethe threethree culturescultures atat anyany timetime duringduring cultivationcultivation (24(24 hh andand 7272 hh inin TableTable3 3as as well well as as in in MRS MRS+1%+1% glutamateglutamate culture;culture; datadata notnot shown)shown) inin thethe presencepresence ofof glutamate,glutamate, ammonia,ammonia, andand ATP.ATP. BasedBased onon thethe results,results, thethe argininearginine cataboliccatabolic pathwaypathway ofof W.W. koreensiskoreensis SKSK waswas constructedconstructed (with(with solidsolid line)line) inin FigureFigure6 6BB based based on on genome genome analysis analysis and and determination determination of of arginine arginine cataboliccatabolic products.products. GlucoseGlucose metabolitesmetabolites byby W.W. koreensiskoreensis SKSK werewere alsoalso determineddetermined (Table(Table3 ).3). Five Five compounds compounds were were detected;detected; lacticlactic acidacid andand ethanolethanol werewere signiﬁcantlysignificantly elevatedelevated duringduring incubation,incubation, whereaswhereas thethe otherother compounds showed no significant changes. This result indicates that W. koreensis SK showed typical heterofermentative fermentation, in which glucose is converted into lactic acid, ethanol, and carbon

Microorganisms 2020, 8, 1147 14 of 17 compounds showed no significant changes. This result indicates that W. koreensis SK showed typical heterofermentative fermentation, in which glucose is converted into lactic acid, ethanol, and carbon dioxide. The highest amount of lactic acid was detected in MRS+1% arginine at 24 h and MRS at 72 h. While the highest amount of ethanol was detected in MRS+1% arginine, followed by MRS+1% citrulline, the lowest amount was detected in MRS culture at 24~72 h. The results of ethanol production by W. koreensis SK among the three cultures are consistent with those of total cell growth in Figure5B. The improved cell growth caused high production of lactic acid and ethanol, followed by strong expression of the ADI pathway. This resulted in high production of ammonia, which was further neutralized by the lactic acid produced. As a consequence of neutralization with lactic acid and ammonia produced after 24 h, the lowest amount of lactic acid (7078.32 mg/L) was detected in MRS+1% arginine at 72 h. During arginine catabolism (Figure6B), the concomitant production of ammonia by W. koreensis SK caused an increase in pH, which may promote survival of SK strain in an acidic environment. Based on the results, we can conclude that the detection of W. koreensis strains in the middle or late stage of kimchi fermentation (acidic condition) [23] could be attributed to their ammonia production ability, which neutralizes the produced acids according to cell growth, not to their acid tolerance ability. L-Ornithine is an amino acid with a variety of significant functions, including stress reduction, improvement of sleep quality, physical fatigue reduction, wrinkle improvement, anti-obesity effect, and others [43]. Moreover, ornithine is a metabolic intermediate that plays a role in the urea cycle by detoxifying ammonia in order to reduce the level or ammonia in the bloodstream [43]. L-Citrulline has been reported to retard high glucose-induced endothelial senescence as well as provide nutritional support in malnourished patients, especially those who are aging and have sarcopenia [44]. In this study, ornithine production (7174.73 mg/L) by W. koreensis SK supplemented with 1% arginine was significantly higher than those (45~46 mg/L) of other W. koreensis strains [45]. Production of ornithine and citrulline by W. koreensis SK can be enhanced by optimization of this process through further experimentation. This unique property of W. koreensis SK implies that SK strain has potential as a functional starter culture or probiotic. The ADI pathway is widely utilized in bacteria, most abundantly in Lactobacillales [40]. We believe that this study is the first report to verify the ADI pathway of W. koreensis based on genome analysis and determination of arginine catabolic products.

4. Conclusions W. koreensis strains were identiﬁed as dominant microorganisms in kimchi fermentation at low temperature (around 0 ◦C). The result indicates that W. koreensis strains play a major role in kimchi fermentation at low temperature. Regarding carbohydrate assimilation, W. koreensis strains showed a lesser degree (positive for 6~8 carbohydrates) of carbohydrate assimilation compared to Lactobacillus or Leucnostoc species. W. koreensis strains were found to utilize an alternative carbon source, the amino acid arginine, to obtain energy. In particular, W. koreensis SK selected among the 50 isolates in this study showed advanced arginine utilization. Accordingly, arginine supplementation improved bacterial growth and resulted in high production of ornithine. W. koreensis SK was able to grow well under mesophilic as well as psychrophilic conditions. Furthermore, W. koreensis SK was found to be safe for human consumption and beneﬁcial due to its functional material production (ornithine) ability. The results of this study indicate that W. koreensis SK may be a promising starter culture for fermented vegetables or fruits at low temperature as well as a probiotic candidate.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/8/8/1147/s1, Figure S1: HPLC analysis of biogenic amine from W. koreensis, Table S1: Isolated LAB strains and their 16s rRNA gene sequences similarities with sequences in the GenBank database, Table S2: Carbohydrate assimilation of LAB. Author Contributions: Conceptualization, H.C.C.; Funding acquisition, H.C.C.; Investigation, S.Y.M.; Project administration, H.C.C.; Supervision, H.C.C.; Validation, S.Y.M.; Visualization, S.Y.M.; Writing—original draft, H.C.C.; Writing—review & editing, H.C.C. All authors have read and agreed to the published version of the manuscript. Microorganisms 2020, 8, 1147 15 of 17

Funding: This research was funded by the National Research Foundation of Korea (NRF), grant number 2019R1A2C1002643. Acknowledgments: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (No. 2019R1A2C1002643). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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