Complete Genome Sequence of Lactobacillus plantarum EM, A Putative Probiotic Strain with the Cholesterol-Lowering Effect and Antimicrobial Activity.

Lactobacillus plantarum EM is a probiotic strain with antimicrobial activity, cholesterol-lowering effects, and tolerance to acid and bile. To understand the genetic basis of the probiotic characteristics of this strain, genome sequencing and probiotic-related genetic analysis were performed. The genomic characteristics of L. plantarum EM were confirmed by comparative genomic analysis with 41 probiotic lactic acid bacteria, including 10 L. plantarum strains. L. plantarum EM was shown to contain a circular chromosome of 3,184,808 bp and eight plasmids with various lengths from 5,027 to 76,369 bp. The L. plantarum EM genome had a total of 3560 protein-coding genes, including probiotic-related genes, such as tolerance to acid and bile, temperature stress, and oxidative stress. Comparative genomic analysis showed that L. plantarum EM contained plantaricin and bovicin gene clusters, which are related to antimicrobial activity, and five bile salt hydrolase genes related to serum cholesterol-lowering effects. The genomic analysis confirmed the probiotic properties of L. plantarum EM, and our results indicated that this strain has potential application for use as an industrially important probiotic.

Characterization of starter kimchi fermented with Leuconostoc kimchii GJ2 and its cholesterol-lowering effects in rats fed a high-fat and high-cholesterol diet.

BACKGROUND: The hypocholesterolemic effects of lactic acid bacteria and kimchi have been demonstrated previously. However, the kimchi fermentation process still relies on naturally present microorganisms. To obtain functional kimchi with consistent quality, we validated the capacity of Leuconostoc kimchii GJ2 as a starter culture to control kimchi fermentation. Moreover, cholesterol-lowering effects of starter kimchi as a health-promoting product were explored. RESULTS: Bacteriocin production by Lc. kimchii GJ2 was highly enhanced in the presence of 5% Lactobacillus sakei NJ1 cell fractions. When kimchi was fermented with bacteriocin-enhanced Lc. kimchii GJ2, Lc. kimchii GJ2 became overwhelmingly predominant (98.3%) at the end of fermentation and maintained its dominance (up to 82%) for 84 days. Growing as well as dead cells of Lc. kimchii GJ2 showed high cholesterol assimilation (in vitro). Rats were fed a high-fat and high-cholesterol diet supplemented with starter kimchi. The results showed that feeding of starter kimchi significantly reduced serum total cholesterol, triglyceride and low-density lipoprotein cholesterol levels. Additionally, atherogenic index, cardiac risk factor and triglyceride and total cholesterol levels in liver and epididymal adipose tissue decreased significantly in rats fed starter kimchi. CONCLUSION: Kimchi fermented with Lc. kimchii GJ2 as a starter culture has efficient cholesterol-lowering effects.

Purification and characterization of antifungal compounds from Lactobacillus plantarum HD1 isolated from kimchi.

Strain HD1 with antifungal activity was isolated from kimchi and identified as Lactobacillus plantarum. Antifungal compounds from Lb. plantarum HD1 were active against food- and feed-borne filamentous fungi and yeasts in a spot-on-the-lawn assay. Antifungal activity of Lb. plantarum HD1 was stronger against filamentous fungi than yeast. Antifungal compounds were purified using solid phase extraction (SPE) and recycling preparative-HPLC. Structures of the antifungal compounds were elucidated by electrospray ionization-mass spectrometry and nuclear magnetic resonance. Active compounds from Lb. plantarum HD1 were identified as 5-oxododecanoic acid (MW 214), 3-hydroxy decanoic acid (MW 188), and 3-hydroxy-5-dodecenoic acid (MW 214). To investigate the potential application of these antifungal compounds for reduction of fungal spoilage in foods, Korean draft rice wine was used as a food model. White film-forming yeasts were observed in control draft rice wine after 11 days of incubation. However, film-forming yeasts were not observed in draft rice wine treated with SPE-prepared culture supernatant of Lb. plantarum HD1 (equivalent to 2.5% addition of culture supernatant) until 27 days of incubation. The addition of antifungal compounds to Korean draft rice wine extended shelf-life up to 27 days at 10 °C without any sterilization process. Therefore, the antifungal activity of Lb. plantarum HD1 may lead to the development of powerful biopreservative systems capable of preventing food- and feed-borne fungal spoilage.

Identification of lactic acid bacteria in salted Chinese cabbage by SDS-PAGE and PCR-DGGE.

BACKGROUND: Lactic acid bacteria (LAB) in salted Chinese cabbage, the main ingredient of kimchi, were analyzed by culture-dependent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by sequencing of the 16S rRNA gene and by culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), followed by sequencing of the V3 region of the 16S rRNA gene. The results were compared to those of LAB that had previously been found in kimchi. RESULTS: The two identification methods produced distinct overall LAB profiles. The PCR-DGGE method detected a more diverse microflora, including non-LAB strains. The culture-dependent method uniquely detected Weissella sp. and was able to provide the quantitative distribution of LAB in samples. However, Leuconostoc mesenteroides, Lactobacillus curvatus and Leuconostoc carnosum, which had also been reported as the dominant LAB in kimchi in previous studies, were identified by both methods. CONCLUSION: The two identification methods gave different bacterial profiles, while both methods were sufficient to identify the most prevalent LAB in salted Chinese cabbage samples. The quantitative feature of the culture-dependent identification method would make it preferable for studying and monitoring LAB viability in kimchi at each fermentation stage. The availability of the culture-independent identification method to identify a broader bacterial profile, including non-LAB, would make it a more effective tool for controlling contamination of undesirable bacteria during kimchi fermentation.

Construction and evaluation of shuttle vector, pGYC4α, based on pYC2 from Lactobacillus sakei.

The shuttle vector pGYC4α (6,157 bp) was constructed based on the sigma-replicon plasmid pYC2 from Lactobacillus sakei BM5 isolated from kimchi. The vector contained inserts of the ColE1 replicon, α-amylase gene from Bacillus licheniformis containing its own signal peptide, and lactococcal promoter P32. Transformation and expression of a selection marker gene (α-amylase) with pGYC4α were demonstrated in Escherichia coli and several lactic acid bacteria (LAB). The highest α-amylase activity in LAB transformants was obtained in M17/0.25% glucose media with 0.5% CaCO(3). The segregational stability of the shuttle vector in LAB was 100% for more than 100 generations in the absence of antibiotic pressure. The developed vector might be useful as a genetic tool for food industries.

Rice Bran Fermented with Kimchi-Derived Lactic Acid Bacteria Prevents Metabolic Complications in Mice on a High-Fat and -Cholesterol Diet.

This aim of this study was to investigate the potential beneficial effects of rice bran powder, fermented by Weissella koreensis DB1 isolated from kimchi, to protect against obesity and dyslipidemia induced by a high-fat and high-cholesterol diet, in a mouse model. Male mice were fed a modified AIN-93M diet containing high fat/high-cholesterol (HFCD), or same diet supplemented with non-fermented rice bran powder (HFCD-RB) or fermented rice bran powder (HFCD-FRB) for 10 weeks. In the HFCD-FRB group, body weight, liver and white fat pads weights, triglyceride (TG), total cholesterol (TC), non-high-density lipopreotein cholesterol (non-HDL-C), insulin, glucose and leptine levels in serum, TG levels and the ratio of fat droplets in the liver, TG levels and fat cell size in adipose tissue were decreased, and (high-density lipopreotein cholesterol) HDL-C and adiponectin levels in serum were increased, compared with the HFCD group. The HFCD-FRB group had significantly lower CCAAT-enhancer-binding potein α (C/EBPα), sterol regulatory element-binding transcription protein-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl CoA carboxylase (ACC) gene expression when compared to the HFCD group. The anti-obesity and hypolipidemic effects were marginally greater in the HFCD-FRB group than in the HFCD-RB group. These results suggest that fermented rice bran powder by Weissella koreensis DB1 may have potential beneficial effects on the obesity-related abnormalities and the dysfunction of lipid metabolism.

Rice Bran Fermentation Using Lactiplantibacillus plantarum EM as a Starter and the Potential of the Fermented Rice Bran as a Functional Food.

Rice bran was fermented using a functional starter culture of Lactiplantibacillus plantarum EM, which exhibited high cholesterol removal and strong antimicrobial activity. Highest viable cell counts (9.78 log CFU/mL) and strong antimicrobial activity were obtained by fermenting 20% rice bran supplemented with 1% glucose and 3% corn steep liquor (pH 6.0) at 30 °C for 48 h. The fermented rice bran slurry was hot air-dried (55 °C, 16 h) and ground (HFRB). HFRB obtained showed effective cholesterol removal (45-68%) and antimicrobial activities (100-400 AU/mL) against foodborne pathogenic bacteria and food spoilage fungi. Phytate levels were significantly reduced during fermentation by 53% due to the phytase activity of L. plantarum EM, indicating HFRB does not present nutrient deficiency issues. In addition, fermentation significantly improved overall organoleptic quality. Our results indicate that HFRB is a promising functional food candidate. Furthermore, HFRB appears to satisfy consumer demands for a health-promoting food and environmental and legal requirements concerning the re-utilization of biological byproducts.

Characterization of the Psychrotrophic Lactic Acid Bacterium Leuconostoc gelidum subsp. aenigmaticum LS4 Isolated from Kimchi Based on Comparative Analyses of Its Genomic and Phenotypic Properties.

With the aim of developing a new food starter culture, twenty-three psychrotrophic lactic acid bacteria (LAB) were isolated from 16 kimchi samples. One strain, Leuconostoc gelidum subsp. aenigmaticum LS4, which had typical psychrotrophic characteristics, was selected, and its phenotypic and genomic properties as a starter culture were investigated. The complete genome of L. aenigmaticum LS4 consisted of one circular chromosome (1,988,425 bp) and two plasmids (19,308 bp and 11,283 bp), with a guanine-cytosine content of 36.8%. L. aenigmaticum LS4 could grow at 5 °C but not at 37 °C, and maximum cell growth was obtained at 15~25 °C. L. aenigmaticum LS4 did not show any harmful characteristics such as hemolysis, undesirable enzyme activities, biogenic amine production, or antibiotic resistance. L. aenigmaticum LS4 was investigated for its suitability for technological processes (pH, temperature and NaCl treatment). L. aenigmaticum LS4 exhibited strong antimicrobial activity caused by the production of organic acids and bacteriocin, and it produced an exopolysaccharide composed of glucose with a molecular weight of 3.7 × 106 Da. Furthermore, L. aenigmaticum LS4 improved the organoleptic qualities of kimchi juice. Our results indicate that L. aenigmaticum LS4 could be used as a functional starter culture for food (vegetable or fruit) fermentation at low temperatures.

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

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.

Effects of Cabbage-Apple Juice Fermented by Lactobacillus plantarum EM on Lipid Profile Improvement and Obesity Amelioration in Rats.

This study aimed to investigate the potential of cabbage-apple juice, fermented by Lactobacillus plantarum EM isolated from kimchi, to protect against obesity and dyslipidemia that are induced by a high-fat diet in a rat model. Male rats were fed a modified AIN-93M high-fat diet (HFD), the same diet supplemented with non-fermented cabbage-apple juice, or the same diet supplemented with fermented cabbage-apple juice for eight weeks. In the HFD-fermented cabbage- apple juice administered groups the following parameters decreased: body weight, liver and white fat pad weights, serum triglyceride (TG), total cholesterol (TC), LDL-cholesterol, insulin, glucose and leptin levels, TG levels, while HDL-C and adiponectin levels in serum increased as compared with the HFD group. The HFD-fed rats that were supplemented with fermented cabbage-apple juice exhibited significantly lower fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and malic enzyme gene expression levels when compared to the exclusively HFD-fed rats. The anti-obesity and hypolipidemic effects were marginally greater in the fermented juice administered group than in the non-fermented juice administered group. These results suggest that cabbage-apple juice-especially fermented cabbage-apple juice-might have beneficial effects on lipid metabolism dysfunction and obesity-related abnormalities. However, further studies are necessary for analyzing the biochemical regulatory mechanisms of fermented juice for obesity amelioration and lipid metabolic homeostasis.

Identification of a replicon from pCC3, a cryptic plasmid from Leuconostoc citreum C4 derived from kimchi, and development of a new host-vector system.

Analysis of the structural properties of pCC3, a cryptic plasmid from Leuconostoc citreum C4 isolated from kimchi, determined its length as 3,338 bp and revealed three open reading frames (ORFs): ORF1-ORF3. ORF3 showed high homology with a replication initiation protein of the theta-type plasmid pTXL1. The fragment encompassing ORF3 and its upstream sequences (nt 1,299-1,634) was found to contain a functional plasmid replicon. A new shuttle vector, pUCC3E1, was constructed based on pCC3. Using Southern hybridization analysis, no single-stranded DNA intermediate was detected from Leu. citreum harboring pUCC3E1, which indicates that pCC3 replicated via the theta mechanism. The pUCC3E1 could be replicated in E. coli TG1 (5.8 x 10(4) CFU/microg DNA) and the developed cloning hosts, Leu. citreum C16 (2.1 x 10(2) CFU/microg DNA) and Leu. citreum GJ7 (8.0 x 10(1) CFU/microg DNA). pUCC3E1 was stably maintained in Leu. citreum C16 (for 100 generations, ca. 94.2%) in the absence of erythromycin (5 microg/ml).

Analysis of pYC2, a cryptic plasmid in Lactobacillus sakei BM5 isolated from kimchi.

Analysis of the structural properties of pYC2, a cryptic plasmid from Lactobacillus sakei BM5 isolated from kimchi, determined its length as 1,970 bp with a G + C content of 34%. The double-strand origin (dso) and single-strand origin (sso) of rolling-circle replicating plasmids were found in the nucleotide sequence of the pYC2 plasmid. Sequence analysis of pYC2 revealed that ORF 1 and ORF 2 showed high homology with the Cop and Rep proteins encoded by the pMV158 family of plasmids replicating via the rolling-circle mechanism. pYC2 also replicates by this mechanism, as confirmed by Southern hybridization analysis.

Characterization of juice fermented with Lactobacillus plantarum EM and its cholesterol-lowering effects on rats fed a high-fat and high-cholesterol diet.

The aim of this study was to investigate the ability of Lactobacillus plantarum EM as a starter culture to control cabbage-apple juice fermentation and to explore the cholesterol-lowering effects of the fermented juice (EM juice) in rats. L. plantarum EM produced strong antimicrobial activities against bacteria and fungi, suppressing other microorganisms in the fermented juice, and was the dominant organism during fermentation and storage. The EM juice also showed strong and broad-spectrum antimicrobial activity. Rats fed a high-fat and high-cholesterol diet and administered EM juice showed significantly reduced total cholesterol (TC), triglyceride, and LDL-cholesterol levels, as well as a reduced atherogenic index, lower cardiac factors in serum, and lower TC levels in the liver, while total lipid and TC levels in the rat feces increased. Reverse transcription-polymerase chain reaction analysis revealed that the hepatic mRNA expression of HMG-CoA reductase decreased and the expressions of cholesterol 7α-hydroxylase and low-density lipoprotein receptor increased in rats administered EM juice. The effects of EM juice on rats included inhibition of cholesterol synthesis as well as enhancement of cholesterol uptake and cholesterol excretion. The results of this study indicate that the use of L. plantarum EM as a functional starter culture for juice fermentation exerts microbial control, enhances sanitary safety, and provides beneficial food effects against hypercholesterolemia.

Cloning of four genes involved in limonene hydroxylation from Enterobacter cowanii 6L.

Genes encoding proteins responsible for limonene catabolism were cloned from a limonene-degrading microorganism, Enterobacter cowanii 6L, which was isolated from citron (Citrus junos) peel. The 8.6, 4.7, and 7.7 kb fragments (CD3, CD4, and CD6) of E. cowanii 6L chromosomal DNA that confer to E. coli the ability to grow on limonene have been cloned and their corresponding DNA sequences were determined. Nine open reading frames (ORFs) were identified, and the four ORFs (921 bp of CD3-2; 1515 bp of CD4-1; 1776 bp of CD6-1; and 1356 bp of CD6-2) that encode limonene hydroxylase were confirmed by independently expressing these genes in E. coli. FAD and NADH were found to stimulate the hydroxylation reaction if added to cell extracts from E. coli recombinants, and multiple compounds (linalool, dihydrolinalool, perillyl alcohol, alpha-terpineol, and gamma-terpineol) were the principal products observed. Our results suggest that the isolate E. cowanii 6L has a broad metabolic capability including utilization of limonene. This broad metabolic ability was confirmed by identifying four novel limonene hydroxylase functional ORFs in E. cowanii 6L.

Production of gamma-aminobutyric acid (GABA) by Lactobacillus buchneri isolated from kimchi and its neuroprotective effect on neuronal cells.

Lactic acid bacteria that accumulated gamma-aminobutyric acid (GABA) in culture medium were screened to identify strains with high GABA-producing ability. One strain, MS, which was isolated from kimchi, showed the highest GABA-producing ability among the screened strains. MS was identified as Lactobacillus buchneri based on Gram-staining, metabolic characteristics, and 16S rDNA sequence determination. Optimum culture conditions for GABA production were determined: MRS broth containing 5% MSG, 1% NaCl, and 1% glucose, at an initial pH of 5.0, the incubation temperature at 30 degrees C for 36 h. Under these conditions, MS produced GABA at a concentration of 251 mM with a 94% GABA conversion rate. Moreover, culture extracts of Lb. buchneri MS partially or completely protected neuronal cells against neurotoxicant-induced cell death.

Characterization of paraplantaricin C7, a novel bacteriocin produced by Lactobacillus paraplantarum C7 isolated from kimchi.

A Lactobacillus paraplantarum strain producing a bacteriocin was isolated from kimchi using the spot-on-the lawn method and named L. paraplantarum C7. The bacteriocin, paraplantaricin C7, was found to inhibit certain Lactobacillus strains, including L. plantarum, L. pentosus, and L. delbrueckii subsp. lactis. It also inhibited Enterococcus faecalis, yet did not inhibit most of the other LAB (lactic acid bacteria) tested. The maximum level of paraplantaricin C7 activity was observed under the culture conditions of 25 degrees C and a constant pH of 4.5. Paraplantaricin C7 retained 90% of its activity after 10 min of treatment at 100 degrees C and remained stable within a pH range of 2-8. Based on a culture supernatant, paraplantaricin C7 was purified by DEAE-Sephacel column chromatography and C18 reverse-phase HPLC. SDS-PAGE and activity staining were then conducted using the purified paraplantaricin C7, and its molecular mass determined to be about 3,800 Da. The 28 N-terminal amino acids from the purified paraplantaricin C7 were determined, and the structural gene encoding paraplantaricin C7, ppnC7, was cloned by PCR using degenerate primers based on the N-terminal amino acid sequence. The nucleotide sequences for ppnC7 and other neighboring orfs exhibited a limited homology to the previously reported plantaricin operon genes. Paraplantaricin C7 is a novel type II bacteriocin containing a double glycine leader sequence.

Isolation of antifungal activity of Leuconostoc mesenteroides TA from kimchi and characterization of its antifungal compounds.

Strain TA harboring antifungal activity was isolated from kimchi and identified based on Gram-staining, biochemical properties using an API 50 CHL, determination of rRNA gene sequences, and RAPD analysis. The determined gene sequences and RAPD pattern showed 100% homology with those of Leuconostoc mesenteroides ATCC 8293T. However, their properties were slightly different from each other. Especially, TA could not utilize lactose while strain ATCC 8293 could use lactose. Leu. mesenteroides ATCC 8293 showed higher antibacterial activity against Bacillus cereus than Leu. mesenteroides TA, whereas the antifungal activities of these 2 LAB against Aspergillus fumigatus were the same. Leu. mesenteroides TA showed broad antimicrobial activities against Gram-positive and - negative bacteria as well as molds. We determined that the responsible antifungal compounds from Leu. mesenteroides TA are lactic acid, acetic acid and unidentified hydrophobic compound(s). Additionally, synergistic interactions involving acetic acid, lactic acid, phenyllactic acid, and unidentified hydrophobic compound(s) contribute to the overall inhibitory activity of Leu. mesenteroides TA.

Acute and Subacute Oral Toxicity Evaluation of Crude Antifungal Compounds Produced by Lactobacillus plantarum HD1 in Rats.

The aim of this study was to investigate the acute and subacute oral toxicity of crude antifungal compounds produced by Lactobacillus plantarum HD1 in Sprague-Dawley rats. In the acute toxicity study, the crude antifungal compounds (0.625, 1.25, 2.5, and 5.0 g/kg) did not produce mortality, significant changes in general behavior, or changes in the gross appearance of the organs. In the subacute toxicity study, the crude antifungal compounds were administered orally to rats at doses of 0, 0.5, 1.0, and 2.0 g/kg daily for 28 days. There were no test article-related deaths, abnormal clinical signs, or body weight changes. The study also showed no significant differences between the control and treated groups in hematological and serum biochemical parameters, histopathological examination, or any other findings. These results suggest that acute or subacute oral administration of crude antifungal compounds from L. plantarum HD1 is not toxic in rats.

New integration vector using a cellulase gene as a screening marker for Lactobacillus.

The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker. pJC4 was transformed into four Lactobacillus species, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus, and Lb. plantarum. In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants. Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs. The celA gene could be a useful screening marker for other lactic acid bacteria.

Comparison of bacterial community changes in fermenting kimchi at two different temperatures using a denaturing gradient gel electrophoresis analysis.

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique followed by sequencing of the 16S rDNA fragments eluted from the bands of interest on denaturing gradient gels was used to monitor changes in the bacterial microflora of two commercial kimchi, salted cabbage, and ingredient mix samples during 30 days of fermentation at 4°C and 10°C. Leuconostoc (Lc.) was the dominant lactic acid bacteria (LAB) over Lactobacillus (Lb.) species at 4°C. Weissella confusa was detected in the ingredient mix and also in kimchi samples throughout fermentation in both samples at 4°C and 10°C. Lc. gelidum was detected as the dominant LAB at 4°C in both samples. The temperature affected the LAB profile of kimchi by varing the pH, which was primarily caused by the temperature-dependent competition among different LAB species in kimchi. At 4°C, the sample variations in pH and titratable acidity were more conspicuous owing to the delayed growth of LAB. Temperature affected only initial decreases in pH and initial increases in viable cell counts, but affected both the initial increases and final values of titratable acidity. The initial microflora in the kimchi sample was probably determined by the microflora of the ingredient mix, not by that of the salted cabbage. The microbial distributions in the samples used in this study resembled across the different kimchi samples and the different fermentation temperatures as the numbers of LAB increased and titratable acidity decreased.