Lactobacillus brevis OPK-3 isolated from kimchi inhibits adipogenesis and exerts anti-inflammation in 3T3-L1 adipocyte.

BACKGROUND: Kimchi is a traditional fermented food in Korea that contains various unique microorganisms. Diverse bacteria are involved in the process of Kimchi fermentation and the healthful advantages; one of the major species is Lactobacillus. We investigated whether lactic acid bacteria isolated from Kimchi (KLAB) are capable of reducing intracellular lipid accumulation by downregulating the expression of adipogenesis and lipogenesis promoting genes in differentiating 3T3-L1 cells. RESULTS: KLAB (Lactobacillus brevis OPK-3) mediated dose-dependent inhibition of adipocyte differentiation, intracellular triglyceride accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity. The expression of transcription factors such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α involved in adipogenesis was markedly decreased by the KLAB treatment. Terminal adipogenic marker, e.g. adipocyte fatty acid binding protein (aP2), lipoprotein lipase, liver X receptor α, leptin and GPDH were significantly downregulated by KLAB treatment compared to untreated control. Moreover, cytokine genes, such as tumor necrosis factor-α and interleukin-6 mRNA expressions level were also decreased, whereas adiponectin mRNA level was upregulated by KLAB. CONCLUSION: These results suggest that the KLAB inhibits lipid accumulation in the differentiating adipocyte through downregulating the expression of adipogenic transcription factors and other specific genes involved in lipid metabolism.

Antioxidant and Longevity-Related Properties of the Ethyl Acetate Fraction of Cnidium officinale Makino in Caenorhabditis elegans.

Reactive oxygen species (ROS) are produced from energy metabolism and may cause diseases or cell death. Antioxidation refers to the suppression of ROS production and is considered beneficial in preventing diseases. This study aimed to examine the antioxidative effects of Cnidium officinale Makino (COM) extracts and fractions using Caenorhabditis elegans as an experimental model. The COM ethanol extract was fractionated according to polarity. The results showed that the ethyl acetate fraction of COM showed powerful radical scavenging activities and increased the activities of superoxide dismutase (SOD) and catalase in C. elegans in a concentration-dependent manner. Moreover, the ethyl acetate fraction reduced the ROS production rate in C. elegans and increased the cell survival rate, suggesting oxidative and thermal stress resistance. In addition, the SOD-3::green fluorescent protein (GFP) expression level in the transformed cells of C. elegans (CF1553) increased, suggesting oxidative stress resistance. Similarly, the HSP-16.2::GFP expression level increased, suggesting thermal stress resistance. In conclusion, the ethyl acetate fraction of COM demonstrated the strongest antioxidative effects, indicating that it may help extend longevity.

Kimchi and Leuconostoc mesenteroides DRC 1506 Alleviate Dextran Sulfate Sodium (DSS)-Induced Colitis via Attenuating Inflammatory Responses.

Ulcerative colitis (UC) is caused by inflammation only in the mucosa of the colon, and its incidence is increasing worldwide. The intake of probiotics is known to have a beneficial effect on the development of UC. In this study, we investigated the alleviating effects of kimchi (KC), a fermented food rich in probiotics, and Leuconostoc mesenteroides DRC 1506 (DRC) isolated from kimchi on UC. A freeze-dried kimchi suspension and DRC were orally given to mice at a dose of 1 × 109 CFU/day for 3 weeks. Furthermore, 3% dextran sulfate sodium (DSS) in drinking water was given to induce UC. The KC and DRC groups reduced symptoms of colitis, such as disease activity index, decrease in colon length, colon weight-to-length ratio, and pathological damage to the colon caused by DSS treatment. The KC and DRC groups decreased the levels of pro-inflammatory cytokine (TNF-α) and increased anti-inflammatory cytokine (IL-10) in the colon tissues. At the mRNA and protein expression levels in the colon tissue, KC and DRC groups downregulated inflammatory factors and upregulated tight junction-related factors. Therefore, DRC, as well as KC supplementation, are potent in alleviating UC by improving the inflammatory response and mucosal barrier function in the colon.

Two-color, ultra-sensitive fluorescent strategy for Ochratoxin A detection based on hybridization chain reaction and DNA tweezers.

A two-color fluorescent DNA tweezers was developed for ultrasensitive detection of Ochratoxin A (OTA) based on hairpin-locked aptamer and hybridization chain reaction (HCR) amplification strategy. OTA can bind with hairpin-locked aptamer and then trigger the HCR reaction to produce a long double-strand DNA. The side-chains of the long duplex can separately hybridize with the two locker sequences of DNA tweezer, causing the opening of DNA tweezer and the recovery of two-color fluorescent signals. It shows a good linear range from 0.02 to 0.8 ppb with limit of detection of 0.006 ppb for FAM and 0.014 ppb for Cy5, which is beyond the requirement of actual application. In addition, the two-color fluorescent strategy can greatly reduce the false positive rate. It shows excellent performance for detection of OTA in practical food sample.

Lactobacillus Brevis OPK-3 from Kimchi Prevents Obesity and Modulates the Expression of Adipogenic and Pro-Inflammatory Genes in Adipose Tissue of Diet-Induced Obese Mice.

Our previous study reported that lactic acid bacteria (L. brevis OPK-3) isolated from kimchi ameliorated intracellular lipid accumulation in 3T3-L1 adipocyte. The current study explored potential roles of L. brevis OPK-3 (KLAB) on preventing body weight gain and its effect on the inflammatory response of adipose tissue. Male C57BL/6 mice (n = 10) were divided into four groups: normal diet with distilled water (NDC), high-fat diet with distilled water (HDC), high-fat diet with L-ornithine (OTC) or high-fat diet with KLAB. The KLAB supplement resulted in significantly lower body weight, lower epididymal fat tissue mass, and lower serum and hepatic TG levels than the HDC. KLAB supplementation improved serum cytokines, and real-time polymerase chain reaction (PCR) analysis showed significantly lower inflammatory cytokine mRNA levels in epididymal adipose tissue. These results suggest that the administration of KLAB inhibits the induction of inflammation in adipose tissue along with the inhibition of weight gain. Therefore, this study demonstrates the therapeutic and beneficial value of this strain produced during the fermentation of kimchi.

Lactobacillus Aggravate Bile Duct Ligation-Induced Liver Inflammation and Fibrosis in Mice.

Lactobacillus (LAB) have been reported to exert both harmful and beneficial effects on human and animal health. Recently, it has been reported that dysbiosis and bacterial translocation contribute to liver fibrosis. However, the role of Gram-positive LAB in the situation of chronic liver diseases has not been yet elucidated. Liver injury was induced by bile duct ligation (BDL) in LAB or control-administered mice. Liver fibrosis was enhanced in LAB-administered mice compared with control-treated mice as demonstrated by quantification of Sirius-red positive area, hydroxyproline contents and fibrosis-related genes (Col1α1, Acta2, Timp1, Tgfb1). Moreover, LAB-administered mice were more susceptible to BDL-induced liver injury as shown by increased ALT and AST level of LAB group compared with control group at 5 days post BDL. Consistent with serum level, inflammatory cytokines (TNF-α, IL-6 and IL-1ß) were also significantly increased in LAB-treated mice. Of note, LAB-treated liver showed increased lipoteichoic acid (LTA) expression compared with control-treated liver, indicating that LAB-derived LTA may translocate from intestine to liver via portal vein. Indeed, responsible receptor or inflammatory factor (PAFR and iNOS) for LTA were upregulated in LAB-administered group. The present findings demonstrate that administration of LAB increases LTA translocation to liver and induces profibrogenic inflammatory milieu, leading to aggravation of liver fibrosis. The current study provides new cautious information of LAB for liver fibrosis patients to prevent the detrimental effect of LAB supplements.

Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3.

Lactobacillus brevis OPK-3, having 84.292 mg/L/h of gamma-aminobutyric acid (GABA) productivity, was isolated from Kimchi, a traditional fermented food in Korea. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from the L. brevis OPK-3, using primers based on two highly conserved regions of GAD. A full-length GAD (LbGAD) clone was subsequently isolated through rapid amplification of cDNA ends (RACE) PCR. Nucleotide sequence analysis revealed that the open reading frame (ORF) consisted of 1401 bases and encoded a protein of 467 amino acid residues with a calculated molecular weight of 53.4 kDa and a pI of 5.65. The amino acid sequence deduced from LbGAD ORF showed 83%, 71%, and 60% identity to the Lactobacillus plantarum GAD, Lactococcus lactis GAD, and Listeria monocytogenes GAD sequences, respectively. The LbGAD gene was expressed in Escherichia coli strain UT481, and the extract of transformed E. coli UT481 contained an induced 53.4 kDa protein and had significantly enhanced GAD activity.

Production of yogurt with enhanced levels of gamma-aminobutyric acid and valuable nutrients using lactic acid bacteria and germinated soybean extract.

Yogurt with high levels of gamma-aminobutyric acid (GABA), free amino acids and isoflavones was developed using lactic acid bacteria (LAB) and germinated soybean extract. Fermented soya milk (GABA soya yogurt) produced with starter and substrate had the GABA concentration of 424.67 microg/gDW, whereas fermented milk produced by a conventional method had GABA less than 1.5 microg/gDW. The GABA soya yogurt also contained significantly high levels of free amino acids and isoflavones compared with other conventional yogurts. The results suggested that the Lactobacillus brevis OPY-1 and germinated soybean possessed a prospect to be applied in dairy and other health products with high nutritive values and functional properties.

G-Aminobutyric acid promotes methionine-choline deficient diet-induced nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is one of the most common liver diseases and a major cause of liver fibrosis worldwide. G-Aminobutyric acid (GABA) is one of the most abundant inhibitory neurotransmitters in the central nervous system. Recently, it has been reported that GABAergic signaling pathways are found in various non-neuronal tissues including the immune system and play a functional role. In the present study, we investigated whether administration of GABA has effects on NASH through its immunomodulatory effects. To test this hypothesis, C57BL/6 mice were fed a methionine-choline-deficient (MCD) diet for 8 weeks. After four weeks into MCD feeding, mice were provided with plain water (control) or water containing 2 mg/mL of GABA for the subsequent 4 weeks. Using this MCD diet-induced NASH model, we found that mice receiving GABA showed more severe steatohepatitis and liver fibrosis than control mice. This increased liver damage was confirmed by higher levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) compared to the control group. In accordance with increased liver steatohepatitis, NASH-related and inflammatory gene expression (collagen α1, tissue inhibitor of metalloproteinase-1, TNF-α) in the liver was markedly increased in GABA-treated mice. Furthermore, GABA directly enhanced production of inflammatory cytokines including IL-6 and TNF-α in LPS activated RAW macrophage cells and increased TIB-73 hepatocyte death. Such effects were abolished when GABA was treated with bicuculline, a competitive antagonist of GABA receptors. These results suggest that oral administration of GABA may be involved in changes of the liver immune milieu and conferred detrimental effects on NASH progression.

Cloning and characterization of a rice cDNA encoding glutamate decarboxylase.

In this study, we have isolated a rice (Oryza sativa L.) glutamate decarboxylase (RicGAD) clone from a root cDNA library, using a partial Arabidopsis thaliana GAD gene as a probe. The rice root cDNA library was constructed with mRNA, which had been derived from the roots of rice seedlings subjected to phosphorus deprivation. Nucleotide sequence analysis indicated that the RicGAD clone was 1,712 bp long, and harbors a complete open reading frame of 505 amino acids. The 505 amino acid sequence deduced from this RicGAD clone exhibited 67.7 % and 61.9 % identity with OsGAD1 (AB056060) and OsGAD2 (AB056061) in the database, respectively. The 505 amino acid sequence also exhibited 62.9, 64.1, and 64.2 % identity to Arabidopsis GAD (U9937), Nicotiana tabacum GAD (AF020425), and Petunia hybrida GAD (L16797), respectively. The RicGAD was found to possess a highly conserved tryptophan residue, but lacks the lysine cluster at the C-proximal position, as well as other stretches of positively charged residues. The GAD sequence was expressed heterologously using the high copy number plasmid, pVUCH. Our activation analysis revealed that the maximal activation of the RicGAD occurred in the presence of both Ca(2+) and calmodulin. The GAD-encoded 56 approximately 58 kDa protein was identified via Western blot analysis, using an anti-GAD monoclonal antibody. The results of our RT-PCR analyses revealed that RicGAD is expressed predominantly in rice roots obtained from rice seedlings grown under phosphorus deprivation conditions, and in non-germinated brown rice, which is known to have a limited phosphorus bioavailability. These results indicate that RicGAD is a Ca(2+)/ calmodulin-dependent enzyme, and that RicGAD is expressed primarily under phosphate deprivation conditions.

Stimulation of gamma-aminobutyric acid synthesis activity in brown rice by a chitosan/glutamic acid germination solution and calcium/calmodulin.

Changes in the concentrations of gamma-aminobutyric acid (GABA), soluble calcium ions, glutamic acid, and the activity of glutamate decarboxylase (GAD) were investigated in non-germinated vs. germinated brown rice. Brown rice was germinated for 72 h by applying each of the following solutions: (1) distilled water, (2) 5 mM lactic acid, (3) 50 ppm chitosan in 5 mM lactic acid, (4) 5 mM glutamic acid, and (5) 50 ppm chitosan in 5 mM glutamic acid. GABA concentrations were enhanced in all of the germinated brown rice when compared to the non-germinated brown rice. The GABA concentration was highest in the chitosan/glutamic acid that germinated brown rice at 2,011 nmol/g fresh weight, which was 13 times higher than the GABA concentration in the non-germinated brown rice at 154 nmol/g fresh weight. The concentrations of glutamic acid were significantly decreased in all of the germinated rice, regardless of the germination solution. Soluble calcium and GAD were higher in the germinated brown rice with the chitosan/glutamic acid solution when compared to the rice that was germinated in the other solutions. GAD that was partially purified from germinated brown rice was stimulated about 3.6-fold by the addition of calmodulin in the presence of calcium. These data show that the germination of brown rice in a chitosan/glutamic acid solution can significantly increase GABA synthesis activity and the concentration of GABA.

Effects of germinated brown rice extracts with enhanced levels of GABA on cancer cell proliferation and apoptosis.

In the present work we investigated the effects of brown rice extracts on proliferation and apoptosis of cancer cells. Brown rice extracts were prepared using nongerminated brown rice versus germinated brown rices. Mouse leukemia L1210 cells, human acute lymphoblastic leukemia Molt4 cells, and human cervical cancer HeLa cells were treated with either nongerminated brown rice extract (N ex), water-germinated extract (W ex), chitosan-germinated extract (C ex), glutamic acid-germinated brown rice extract (G ex), or chitosan/glutamic acid-germinated brown rice extract (CG ex). The concentrations of gamma-aminobutyric acid (GABA) in the G ex and CG ex were three and 3.3 times higher than the GABA concentration in the N ex, respectively. The G ex and CG ex retarded significantly the proliferation rates of L1210 and Molt4 cells, and the highest retardation rate was with CG ex. In addition, the G ex and CG ex enhanced significantly apoptosis of the cultured L1210 cells, but no significant apoptosis was seen with the other extracts, which have lower concentrations of GABA than G ex and CG ex. These results show that brown rice extracts with enhanced levels of GABA have an inhibitory action on leukemia cell proliferation and have a stimulatory action on the cancer cell apoptosis.

Carnitine profiles during differentiation and effects of carnitine on differentiation of 3T3-L1 cells.

These experiments were conducted to investigate carnitine profiles during differentiation and the effects of carnitine on the differentiation of 3T3-L1 cells. To induce cell differentiation, undifferentiated 3T3-L1 cells were treated with dexamethasone, 1-methyl-3-isobutylxanthine, and d-biotin. Carnitine was also exogenously added to the cells to test its effect on cell differentiation. Triglyceride, total lipid, total protein, nonesterified carnitine, acid-soluble acylcarnitine, and acid-insoluble acylcarnitine were analyzed during the differentiation of 3T3-L1 cells. Total lipid, triglyceride, and total protein increased during the 3T3-L1 cell differentiation. However, nonesterified carnitine, acid-soluble acylcarnitine, and acid-insoluble acylcarnitine concentrations were lower in the differentiated 3T3-L1 cells. In addition, the exogenously added carnitine inhibited the increases in triglyceride and total lipid levels. These results suggest that carnitine may have an inhibitory role on the early stage of 3T3-L1 cell differentiation.

Germinated brown rice extract shows a nutraceutical effect in the recovery of chronic alcohol-related symptoms.

Chronic ethanol abuse can cause liver damage and unfavorable lipid profiles in humans and rodents. Phytonutrients have the potential to partially reverse some of the adverse effects of alcoholism. In this study, a germinated brown rice grown under conditions that favor high concentrations of gamma-aminobutyric acid (GABA) was evaluated for protective effects against the toxic consequences of chronic ethanol use. Serum and hepatic lipid concentrations and enzymes indicative of liver damage were determined in mice chronically administered ethanol. Balb/c mice were fed with either AIN-76 diet (control), control diet plus ethanol, or control diet plus ethanol and supplemental brown rice extract for 30 days. The extract naturally contained 841 nmol GABA per milliliter and was prepared from germinated brown rice. Serum low-density lipoprotein cholesterol (LDL-C), liver aspartate aminotransferase, and liver alanine aminotransferase levels were increased in mice administered ethanol, but not in mice given ethanol and brown rice extract. The brown rice extract significantly increased serum and liver high-density lipoprotein cholesterol (HDL-C) concentrations. Furthermore, administration of the extract prevented ethanol-induced increases in liver triglyceride and total cholesterol concentrations. These findings raise the possibility that brown rice extracts containing a high level of GABA may have a nutraceutical role in the recovery from and prevention of chronic alcohol-related diseases.

Lactobacillus plantarum LG42 isolated from gajami sik-hae inhibits adipogenesis in 3T3-L1 adipocyte.

We investigated whether lactic acid bacteria isolated from gajami sik-hae (GLAB) are capable of reducing the intracellular lipid accumulation by downregulating the expression of adipogenesis-related genes in differentiated 3T3-L1 cells. The GLAB, Lactobacillus plantarum LG42, significantly decreased the intracellular triglyceride storage and the glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner. mRNA expression of transcription factors like peroxisome proliferator-activated receptor (PPAR) γ and CCAAT/enhancer-binding protein (C/EBP) α involved in adipogenesis was markedly decreased by the GLAB treatment. Moreover, the GLAB also decreased the expression level of adipogenic markers like adipocyte fatty acid binding protein (aP2), leptin, GPDH, and fatty acid translocase (CD36) significantly. These results suggest that the GLAB inhibits lipid accumulation in the differentiated adipocyte through downregulating the expression of adipogenic transcription factors and other specific genes involved in lipid metabolism.

Expression, purification, and biochemical properties of arginase from Bacillus subtilis 168.

The arginine-degrading and ornithine-producing enzymes arginase has been used to treat arginine-dependent cancers. This study was carried out to obtain the microbial arginase from Bacillus subtilis, one of major microorganisms found in fermented foods such as Cheonggukjang. The gene encoding arginase was isolated from B. subtilis 168 and cloned into E. coli expression plasmid pET32a. The enzyme activity was detected in the supernatant of the transformed and IPTG induced cell-extract. Arginase was purified for homogeneity from the supernatant by affinity chromatography. The specific activity of the purified arginase was 150 U/mg protein. SDS-PAGE analysis revealed the molecular size to be 49 kDa (Trix·Tag, 6×His·Tag added size). The optimum pH and temperature of the purified enzyme with arginine as the substrate were pH 8.4 and 45°C, respectively. The Km and Vmax values of arginine for the enzyme were 4.6 mM and 133.0 mM/min/mg protein respectively. These findings can contribute in the development of functional fermented foods such as Cheonggukjang with an enhanced level of ornithine and pharmaceutical products by providing the key enzyme in arginine-degradation and ornithine-production.

Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt.

Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO(2) production. The isolates grew well on MRS broth at 30-37 degrees C and a pH of 6.5-8.0. The optimum temperature and pH for growth are 37 degrees C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0-6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.

Enhancement of gamma-aminobutyric acid production in Chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis.

For a foreign glutamate decarboxylase (GAD) to be expressed in Bacillus host system, a recombinant DNA (pLip/LbGAD) was constructed by ligating an LbGAD gene from Lactobacillus brevis OPK-3 into Escherichia coli-Bacillus shuttle vector, pLip. The pLip/LbGAD construct was then transformed into Bacillus subtilis. The culture of the transformed Bacillus strain with the pLip/LbGAD construct had higher GAD activity and gamma-aminobutyric acid (GABA) concentration than those of untransformed Bacillus counterpart. In addition, Chungkukjang, a traditional Korean fermented soybean product prepared by the transformed Bacillus subtilis, contained a significantly higher level of GABA than conventional ones. Thus, by introducing a foreign GAD gene, Bacillus strains have been genetically engineered to produce high levels of GAD and GABA.

Expression of rice glutamate decarboxylase in Bifidobacterium longum enhances gamma-aminobutyric acid production.

Bifidobacteria are important for the production of fermented dairy products and probiotic formulas but have a low capacity for gamma-aminobutyric acid (GABA) production. To develop a Bifidobacterium strain with an enhanced GABA production, we transformed Bifidobacterium longum with a rice glutamate decarboxylase (OsGADC(-)) gene by electroporation. When the transformed strain was cultured in medium containing monosodium glutamate, the amount of GABA increased significantly compared with those of untransformed Bifidobacterium. Thus, by introducing a plant derived GAD gene, a Bifidobacterium strain has been genetically engineered to produce high levels of GABA from glutamate.