Isolation of Latilactobacillus curvatus with Enhanced Nitric Oxide Synthesis from Korean Traditional Fermented Food and Investigation of Its Probiotic Properties.

Nitric oxide (NO) is a free radical associated with physiological functions such as blood pressure regulation, cardiovascular health, mitochondrial production, calcium transport, oxidative stress, and skeletal muscle repair. This study aimed to isolate Latilactobacillus curvatus strains with enhanced NO production from the traditional Korean fermented food, jangajji, and evaluate their probiotic properties for industrial purposes. When cells were co-cultured with various bacterial stimulants, NO production generally increased, and NO synthesis was observed in the range of 20-40 mg/mL. The selected strains of Lat. curvatus were resistant to acid and bile conditions and with variable effectiveness (1-14%) in adhering to Caco-2 cells. Most bacterial strains can inhibit the growth of various pathogens. In addition, they are capable of reducing cholesterol levels via assimilation of cholesterol at 10-50%. Among the selected NO synthases from Lat. curvatus strains, the strain JBCC38 showed the highest capacity to scavenge ABTS (30.1%) and DPPH radicals (39.4%). Moreover, these strains exhibited immunomodulatory properties. The production of TNF-α and IL-6 in the macrophages treated with various bacterial stimulants was induced in all the selected strains.

Probiotic Properties of Pediococcus pentosaceus JBCC 106 and Its Lactic Acid Fermentation on Broccoli Juice.

To understand the biological roles of Pediococcus pentosaceus strains as probiotics isolated from the traditional Korean fermented food, Jangajji, Pediococcus pentosaceus was selected based on its high cinnamoyl esterase (CE) and antioxidant activities. The acid and bile stability, intestinal adhesion, antagonistic activity against human pathogens, cholesterol-lowering effects, and immune system stimulation without inflammatory effects were evaluated. Nitric oxide (NO) levels were measured in co-culture with various bacterial stimulants. Fermentation ability was measured by using a broccoli matrix and the sulforaphane levels were measured. Resistance to acidic and bilious conditions and 8% adherence to Caco-2 cells were observed. Cholesterol levels were lowered by 51% by assimilation. Moreover, these strains exhibited immunomodulatory properties with induction of macrophage TNF-α and IL-6 and had microstatic effects on various pathogens. Co-culture with various bacterial stimulants resulted in increased NO production. Fermentation activity was increased with the strains, and higher sulforaphane levels were observed. Therefore, in the future, the applicability of the selected strain to broccoli matrix-based fermented functional foods should be confirmed.

Microbial Diversity and Volatile Flavor Changes during Gayangju Fermentation, a Traditional Korean House Rice Wine.

Physicochemical changes in fermented alcoholic beverages are significantly related to microbial community development during fermentation. Due to its unusually long fermentation, Gayangju, a traditional Korean house rice wine fermented with nuruk as the traditional starter, gives rise to a strong yeast community and, therefore, a high ethanol concentration and different flavors. However, no detailed analysis has been examined. Changes in microbial community structure during Gayangju fermentation were examined using both culture-dependent and culture-independent methods. During fermentation, Saccharomyces cerevisiae and Saccharomycopsis fibuligera were dominant during all stages of the fermentation. In contrast, Candida parapsilosis, Hanseniaspora guilliermondii, Pichia anomala, Malassezia cuniculi and P. fermentans were identified as minor. P. anomala appeared after the second brewing and then remained constant. Among the 19 compounds identified in this study as order-active compounds, 2-methyl-1-butanol (isoamyl alcohol) was the major compound that increased during the long fermentation stage. Most of the odor-active compounds such as 2,3-butanediol, 3-methyl-1-butanol, ethyl tetradecanoate, ethyl decanoate, ethyl dodecanoate, butanoic acid, 3-methylbutanoic acid (isovaleric acid), 2-methylbutanoic acid, 2-methyl-1-propanol, ethyl acetate, ethyl caprylate, 2-phenylethanol, and 3-methylbutyl acetate increased as the fermentation progressed during 68 days of fermentation, which showed significant differences in the concentrations of odor-active compounds of commercially fermented makgeolli.

Probiotic Properties of Lactic Acid Bacteria with High Conjugated Linoleic Acid Converting Activity Isolated from Jeot-Gal, High-Salt Fermented Seafood.

Conjugated linoleic acid (CLA) isomers are potent health-promoting fatty acids. This study evaluated the probiotic properties of 10 strains of high CLA-producing lactic acid bacteria (LAB) isolated from Jeot-gal, a high-salt, fermented seafood. Two isolates, Lactiplantibacillus plantarum JBCC105683 and Lactiplantibacillus pentosus JBCC105676, produced the largest amounts of CLA (748.8 and 726.9 µg/mL, respectively). Five isolates, L. plantarum JBCC105675, L. pentosus JBCC105676, L. pentosus JBCC105674, L. plantarum JBCC105683, and Lactiplantibacillus paraplantarum JBCC105655 synthesized more cis-9, trans-11-CLA than trans-10, cis-12-CLA (approximately 80:20 ratio). All the strains survived severe artificial acidic environments and showed antimicrobial activity and strong adhesion capability to Caco-2 cells as compared to the commercial strain Lactocaseibacillus rhamnosus GG. Among them, Pediococcus acidilactici JBCC105117, L. paraplantarum JBCC105655, and L. plantarum JBCC105683 strongly stimulated the immunological regulatory gene PMK-1 and the host defense antimicrobial peptide gene clec-60 in Caenorhabditis elegans. Moreover, three strains showed a significant induction of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-12, and IL-10 production in RAW 264.7 macrophages, indicating that they were promising candidates for probiotics with high CLA-converting activity. Our results indicate that the newly isolated CLA-producing LAB might be useful as a functional probiotic with beneficial health effects that modulate the immune system.

Evaluation of Probiotic Properties of Pediococcus acidilactici M76 Producing Functional Exopolysaccharides and Its Lactic Acid Fermentation of Black Raspberry Extract.

This study aimed to determine the probiotic potential of Pediococcus acidilactici M76 (PA-M76) for lactic acid fermentation of black raspberry extract (BRE). PA-M76 showed outstanding probiotic properties with high tolerance in acidic GIT environments, broad antimicrobial activity, and high adhesion capability in the intestinal tract of Caenorhabditis elegans. PA-M76 treatment resulted in significant increases of pro-inflammatory cytokine mRNA expression in macrophages, indicating that PA-M76 elicits an effective immune response. When PA-M76 was used for lactic acid fermentation of BRE, an EPS yield of 1.62 g/L was obtained under optimal conditions. Lactic acid fermentation of BRE by PA-M76 did not significantly affect the total anthocyanin and flavonoid content, except for a significant increase in total polyphenol content compared to non-fermented BRE (NfBRE). However, fBRE exhibited increased DPPH radical scavenging activity, linoleic acid peroxidation inhibition rate, and ABTS scavenging activity of fBRE compared to NfBRE. Among the 28 compounds identified in the GC-MS analysis, esters were present as the major groups. The total concentration of volatile compounds was higher in fBRE than that in NfBRE. However, the undesirable flavor of terpenes decreased. PA-M76 might be useful for preparing functionally enhanced fermented beverages with a higher antioxidant activity of EPS and enhanced flavors.

Application of indigenous Saccharomyces cerevisiae to improve the black raspberry (Rubus coreanus Miquel) vinegar fermentation process and its microbiological and physicochemical analysis.

In order to improve the slow ethanol fermentation during acetic acid fermentation process of black raspberry vinegar (BRV), the microbiological and physicochemical aspects of the effects of indigenous Saccharomyces cerevisiae JBCC-21A were examined. The selected S. cerevisiae JBCC-21A showed better growth and ethanol production rates than the commercial yeast strains. The ethanol production rate was 3-times faster than the traditional method. Acetic acid fermentation by S. cerevisiae JBCC-21A began 10 days earlier than the traditional method and reached up to 60 g/L acetic acid. Bacterial counts revealed Acetobacter pasteurianus was the only dominant species throughout the inoculated acetic acid fermentation. The physicochemical and functional properties of the fermented vinegar using indigenous S. cerevisiae JBCC-21A maintained a high quality similar to the traditional method, while being the faster fermentation process. Thus, it is suggested that inoculation of the indigenous S. cerevisiae strain in order to shorten the fermentation time without affecting the quality of traditional BRV.

Isolation and molecular identification for autochthonous starter Saccharomyces cerevisiae with low biogenic amine synthesis for black raspberry (Rubus coreanus Miquel) wine fermentation.

Biogenic amines (BAs) are widely present in nearly all fermented foods and beverages, and excess consumption can cause adverse health effects. To prepare BA-free Korean black raspberry wine (BRW), four autochthonous starter yeast strains without hazardous BA synthesis activity were selected and their physiological and biochemical properties were examined. The selected strains were identified as Saccharomyces cerevisiae based on 26S rDNA sequencing and microsatellite analysis. Molecular fingerprinting revealed that isolates were quite different from commercial wine yeast S. cerevisiae (52.4% similarity), but genetically relevant to commercial beer yeasts. The four S. cerevisiae strains produced over 10% ethanol during BRW fermentation. In addition, the fermented BRW with these strains showed higher levels of total flavonoids and similar antioxidant activity compared to the control sample. Potentially hazardous BAs that commonly occur in black raspberry extract (BRE) such as cadaverine, histamine, and spermidine were also not detected in the fermented BRW. Thus, we suggest that our strains are promising fermentation tools to ensure high quality and enhanced functionality in the production of BA-free BRW.

Oral administration of lactobacilli isolated from Jeotgal, a salted fermented seafood, inhibits the development of 2,4-dinitrofluorobenzene-induced atopic dermatitis in mice.

Certain strains of lactobacilli have been reported to exert favorable effects on atopic dermatitis (AD). Jeotgal, a traditional Korean food, is a salted fermented seafood known to harbor many lactic acid bacteria. In the present study, two novel lactobacillus strains were isolated from Jeotgal, and their anti-AD effects were investigated. Lactobacilli isolated from Jeotgal were identified, according to conjugated linoleic acid-producing activity, as Lactobacillus plantarum (JBCC105645 and JBCC105683). AD-like skin lesions were induced in BALB/c mice using dinitrofluorobenzene (DNFB). Ear swelling, histological analysis and serum immunoglobulin E (IgE) levels in mice were evaluated to investigate the anti-AD effects of lactobacilli. Cytokine production of ex vivo cluster of differentiation (CD)4+ T cells, and interleukin (IL)-12 production of in vitro macrophages were also evaluated to establish a putative mechanism of the action of lactobacilli. Administration of JBCC105645 or JBCC105683 suppressed ear swelling and serum IgE levels in DNFB-treated mice (P<0.05). Notably, JBCC105645 was more effective than JBCC105683 (P<0.05). Treatment with the lactobacilli also induced a significant decrease in IL-4 production with concomitant increase in interferon (IFN)-γ production in DNFB-exposed CD4+ T cells, and an increase in IL-12 production in macrophages (P<0.05). Taken together, the lactobacilli isolated from Jeotgal may suppress the development of AD-like skin inflammation in mice by modulating IL-4 and IFN-γ production in CD4+ T cells, presumably via enhancing IL-12 production by macrophages.

Molecular cloning, purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223.

A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4 kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0-10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4 min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4 min at 60, 62, and 65°C, respectively. The Km and Vmax values for ChA were 0.153 mM and 559.6 µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca2+, Cu2+, and Fe2+ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.

Bacteria isolated from Korean black raspberry vinegar with low biogenic amine production in wine

Abstract A high concentration of histamine, one of the biogenic amines (BAs) usually found in fermented foods, can cause undesirable physiological side effects in sensitive humans. The objective of this study is to isolate indigenous Acetobacter strains from naturally fermented Bokbunja vinegar in Korea with reduced histamine production during starter fermentation. Further, we examined its physiological and biochemical properties, including BA synthesis. The obtained strain MBA-77, identified as Acetobacter aceti by 16S rDNA homology and biochemical analysis and named A. aceti MBA-77. A. aceti MBA-77 showed optimal acidity % production at pH 5; the optimal temperature was 25 °C. When we prepared and examined the BAs synthesis spectrum during the fermentation process, Bokbunja wine fermented with Saccharomyces cerevisiae showed that the histamine concentration increased from 2.72 of Bokbunja extract to 5.29 mg/L and cadaverine and dopamine was decreased to 2.6 and 10.12 mg/L, respectively. Bokbunja vinegar prepared by A. aceti MBA-77 as the starter, the histamine concentration of the vinegar preparation step was decreased up to 3.66 mg/L from 5.29 mg/L in the wine preparation step. To our knowledge, this is the first report to demonstrate acetic acid bacteria isolated from Bokbunja seed vinegar with low spectrum BA and would be useful for wellbeing vinegar preparation.

Bacteria isolated from Korean black raspberry vinegar with low biogenic amine production in wine.

A high concentration of histamine, one of the biogenic amines (BAs) usually found in fermented foods, can cause undesirable physiological side effects in sensitive humans. The objective of this study is to isolate indigenous Acetobacter strains from naturally fermented Bokbunja vinegar in Korea with reduced histamine production during starter fermentation. Further, we examined its physiological and biochemical properties, including BA synthesis. The obtained strain MBA-77, identified as Acetobacter aceti by 16S rDNA homology and biochemical analysis and named A. aceti MBA-77. A. aceti MBA-77 showed optimal acidity % production at pH 5; the optimal temperature was 25°C. When we prepared and examined the BAs synthesis spectrum during the fermentation process, Bokbunja wine fermented with Saccharomyces cerevisiae showed that the histamine concentration increased from 2.72 of Bokbunja extract to 5.29mg/L and cadaverine and dopamine was decreased to 2.6 and 10.12mg/L, respectively. Bokbunja vinegar prepared by A. aceti MBA-77 as the starter, the histamine concentration of the vinegar preparation step was decreased up to 3.66mg/L from 5.29mg/L in the wine preparation step. To our knowledge, this is the first report to demonstrate acetic acid bacteria isolated from Bokbunja seed vinegar with low spectrum BA and would be useful for wellbeing vinegar preparation.

Microbial community, and biochemical and physiological properties of Korean traditional black raspberry (Robus coreanus Miquel) vinegar.

BACKGROUND: The aim of this study was to elucidate the changes in microbial community and biochemical and physiological properties of traditional Muju black raspberry (Robus coreanus Miquel) vinegar (TMBV) during fermentation by culture-independent methods. RESULTS: During vinegar fermentation, ethanol produced up to 120 g L(-1) until day 35, with continuously increasing yeast concentration to a total of log 7.6 CFU mL(-1) . After day 35, acetic acid bacteria (AAB) concentrations rose to log 5.8 CFU mL(-1) until day 144. Denaturing gradient gel electrophoresis analysis showed that Saccharomyces cerevisiae was detected until day 87 of the fermentation, at which point Acetobacter pasteurianus gradually took over as the dominant species. Total sugar was reduced to 6.6 °Brix and total acidity produced up to 44 g L(-1) . CONCLUSION: In this study, we established the physicochemical analysis and growth dynamics of yeast and AAB during alcoholic and acetic acid fermentation of black raspberry by a traditional method. Overall, S. cerevisiae and A. pasteurianus species appeared to dominate the TMBV fermentation. In conclusion, this study demonstrated a suitable fermentation system for TMBV by the static surface method. © 2015 Society of Chemical Industry.

Monitoring of Yeast Communities and Volatile Flavor Changes During Traditional Korean Soy Sauce Fermentation.

Flavor development in soy sauce is significantly related to the diversity of yeast species. Due to its unique fermentation with meju, the process of making Korean soy sauce gives rise to a specific yeast community and, therefore, flavor profile; however, no detailed analysis of the identifying these structure has been performed. Changes in yeast community structure during Korean soy sauce fermentation were examined using both culture-dependent and culture-independent methods with simultaneous analysis of the changes in volatile compounds by GC-MS analysis. During fermentation, Candida, Pichia, and Rhodotorula sp. were the dominant species, whereas Debaryomyces, Torulaspora, and Zygosaccharomyces sp. were detected only at the early stage. In addition, Cryptococcus, Microbotryum, Tetrapisispora, and Wickerhamomyces were detected as minor strains. Among the 62 compounds identified in this study, alcohols, ketones, and pyrazines were present as the major groups during the initial stages, whereas the abundance of acids with aldehydes increased as the fermentation progressed. Finally, the impacts of 10 different yeast strains found to participate in fermentation on the formation of volatile compounds were evaluated under soy-based conditions. It was revealed that specific species produced different profiles of volatile compounds, some of which were significant flavor contributors, especially volatile alcohols, aldehydes, esters, and ketones.

Effects of indigenous yeasts on physicochemical and microbial properties of Korean soy sauce prepared by low-salt fermentation.

This study deals with understanding the effects of salt reduction on both the physicochemical and microbiological properties of soy sauce fermentation and also the application of indigenous yeast starters to compensate for undesirable changes occurring in salt-reduced processes. Fermentation was tested in situ at a Korean commercial soy sauce processing unit. Salt reduction resulted in higher acidity as well as lower pH and contents of residual sugar and ethanol. Moreover, undesired flavor characteristics, due to a lack of distinctive compounds, was observed. In addition, putrefactive Staphylococcus and Enterococcus spp. were present only during salt-reduced fermentation. To control these adverse effects, a single or mixed culture of two indigenous yeasts, Torulaspora delbrueckii and Pichia guilliermondii, producing high ethanol and 3-methyl-1-butanol, respectively, were tested. Overall, all types of yeast applications inhibited undesirable bacterial growth despite salt reduction. Of the starter cultures tested, the mixed culture resulted in a balance of more complex and richer flavors with an identical flavor profile pattern to that obtained from high salt soy sauce. Hence, this strategy using functional yeast cultures offers a technological option to manufacture salt-reduced soy sauce while preserving its typical sensory characteristics without affecting safety.

Lipid-lowering effects of Pediococcus acidilactici M76 isolated from Korean traditional makgeolli in high fat diet-induced obese mice.

The effect of Pediococcus acidilactici M76 (lactic acid bacteria) isolated from makgeolli on mice fed a high fat diet was investigated to clarify the lipid lowering function. C57BL/6J male mice were randomly divided into a normal diet (ND) group, high fat diet (HD) group, HD plus Pediococcus acidilactici DSM 20284 reference strain (PR) group, and HD plus Pediococcus acidilactici M76 strain (PA) groups. The lyophilized PA and PR strain were dissolved in distilled water at a final concentration of 1.25 × 109 cfu/mL and was given orally to animals at a dose of 4 mL/kg body weight for 12 weeks. The PA group had a lower final body weight, adipose tissue weight, and lipid profile than those in the HD group. Additionally, level of ACC, FAS and PPAR-γ, a key lipid synthesis enzyme, was markedly suppressed in the PA compared to those in the HD group. These data suggest that P. acidilactici M76 may exert a lipid-lowering effect in high fat diet-induced obese mice.

Identification of novel non-metal haloperoxidases from the marine metagenome.

Haloperoxidase (HPO, E.C.1.11.1.7) is a metal-containing enzyme oxidizing halonium species, which can be used in the synthesis of halogenated organic compounds, for instance in the production of antimicrobial agents, cosmetics, etc., in the presence of halides and H2O2. To isolate and evaluate a novel non-metal HPO using a culture-independent method, a cassette PCR library was constructed from marine seawater in Japan. We first isolated a novel HPO gene from Pseudomonas putida ATCC11172 by PCR for constructing the chimeric HPO library (HPO11172). HPO11172 showed each single open-reading frame of 828 base pairs coding for 276 amino acids, respectively, and showed 87% similarity with P. putida IF-3 sequences. Approximately 600 transformants screened for chimeric genes between P. putida ATCC11173 and HPO central fragments were able to identify 113 active clones. Among them, we finally isolated 20 novel HPO genes. Sequence analyses of the obtained 20 clones showed higher homology genes with P. putida or Sinorhizobium or Streptomyces strains. Although the HPO A9 clone showed the lowest homology with HPO11172, clones in group B, including CS19, showed a relatively higher homology of 80%, with 70% identy. E. coli cells expressing these HPO chimeric genes were able to successfully bioconvert chlorodimedone with KBr or KCl as substrate.

Exopolysaccharide produced by Pediococcus acidilactici M76 isolated from the Korean traditional rice wine, makgeolli.

This work is aimed to increase knowledge of the functional exopolysaccharide (EPS) from lactic acid bacteria (LAB) in makgeolli, a Korean fermented rice wine. Among LAB strains isolated from makgeolli, strain M76 was selected as a functional strain producing a bioactive EPS, based on its antioxidative activity on the DPPH radical. The 16S rRNA gene sequencing analysis showed a high sequence similarity (99.0%) with P. acidilactici, but had different biochemical properties with the already known P. acidilactici type strains in the aspect of carbohydrates utilization. The obtained P. acidilactici M76 produced a soluble EPS above 2 g/l. One-step chromatography using gel filtration after ethanol precipitation from the supernatant of P. acidilactici M76 was enough to obtain purified EPS with a single peak, showing a molecular mass of approximately 67 kDa. Componential and structural analyses of EPS by TLC, HPLC, and FT-IR indicated that the EPS is a glucan, consisting of glucose units. The purified EPS had antioxidant activity on the DPPH radical of 45.8% at a concentration of 1 mg/ml. The purified EPS also showed proliferative effect on the pancreatic RIN-m5F cell line and remarkable protection activity on alloxan-induced cytotoxicity. This potent antioxidant and antidiabetic EPS by LAB in makgeolli may contribute to understanding the functionality of makgeolli.

Optimal production of L-threo-2,3-dihydroxyphenylserine (L-threo-DOPS) on a large scale by diastereoselectivity-enhanced variant of L-threonine aldolase expressed in Escherichia coli.

This study examined the efficient production and optimal separation procedures for pure L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) from a mixture of diastereomers synthesized by whole-cell aldol condensation reaction, harboring diastereoselectivity-enhanced L-threonine aldolase in Escherichia coli JM109. The addition of the reducing agent sodium sulfite was found to stimulate the production of L-threo-DOPS without affecting the diastereoselectivity ratio, especially at the 50 mM concentration. The optimal pH for diastereoselective synthesis was 6.5. The addition of Triton X-100 also strongly affected the synthesis yield, showing the highest conversion yield at a 0.75% concentration; however, the diastereoselectivity of the L-threonine aldolase was not affected. Lowering the temperature to 10°C did not significantly affect the diastereoselectiviy without affecting the synthesis rate. At the optimized conditions, a mixture of L-threo-DOPS and L-erythro-DOPS was synthesized by diastereoselectivity-enhanced L-threonine aldolase from E. coli in a continuous process for 100 hr, yielding an average of 4.0 mg/mL of L-threo-DOPS and 60% diastereoselectivity (de), and was subjected to two steps of ion exchange chromatography. The optimum separation conditions for the resin and solvent were evaluated in which it was found that a two-step process with the ion-exchange resin Dowex 50 W × 8 and activated carbon by washing with 0.5 N acetic acid was sufficient to separate the L-threo-DOPS. By using two-step ion-exchange chromatography, synthesized high-purity L-threo-DOPS of up to 100% was purified with a yield of 71%. The remaining substrates, glycine and 3,4-dihydroxybenzaldehyde, were recovered successfully with a yield of 71.2%. Our results indicate this potential procedure as an economical purification process for the synthesis and purification of important L-threo-DOPS at the pharmaceutical level.