The addition of jogi, Micropogonias undulates, affects amino acid content in kimchi fermentation.

The effects of jogi (the fish Atlantic croaker, Micropogonias undulatus) on the production of physicochemical components, such as color, organic acids, and amino acids, in kimchi, a traditional fermented vegetable food of Korea, were determined. As fermentation progressed, the color change of jogi-added kimchi increased, but in comparison with that of the control group without jogi-added kimchi, was difficult to distinguish with the naked eye. Reducing sugar decreased in all experimental groups, and as fermentation progressed, kimchi with jogi showed a lower value. Acetic acid, citric acid, lactic acid, and ethanol, were highly produced in both types of kimchi, and above all, the jogi-baechu-kimchi group showed higher acetic acid and lactic acid contents than the control group. The increase and decrease of amino acids were similar in both types of kimchi. However, significantly, immediately after manufacture, the savory components aspartic acid and glutamic acid were detected higher than the control group. Subsequently, the fermentation tended to decrease as it progressed, but the content was higher than that of the control group. The above results show that jogi addition has a greater effect on the contents of amino acid, especially the savory component, than on the physicochemical components.

Postbiotic potential of Bacillus velezensis KMU01 cell-free supernatant for the alleviation of obesity in mice.

Attention toward the preventive effects of postbiotics on metabolic diseases has increased because of greater stability and safety over probiotics. However, studies regarding the bioactive effects of postbiotics, especially from probiotic Bacillus strains, are relatively limited. The anti-obesity effects of the cell-free culture supernatant of Bacillus velezensis KMU01 (CFS-B.vele) were evaluated using high-fat-diet (HFD)-induced mice. HFD-induced mice (n = 8 per group) received equal volumes of (1) CFS-B.vele (114 mg/kg) in PBS, (2) Xenical in PBS, or (3) PBS alone by oral gavage daily for 13 weeks. The results demonstrated that CFS-B.vele changed the gut microbiota and showed anti-obesity effects in HFD-induced obese mice. The elevated Firmicutes/Bacteroidota ratio induced by HFD was decreased in the CFS-B.vele group compared to the other groups (p < 0.05). The CFS-B.vele intervention led to the enrichment of SCFA-producers, such as Roseburia and Eubacterium, in the cecum, suggesting their potential involvement in the amelioration of obesity. Due to these changes, the various obesity-related biomarkers (body weight, fat in tissue, white adipose tissue weight and size, serum LDL-cholesterol level, hepatic lipid accumulation, and adipogenesis/lipogenesis-related gene/protein expression) were improved. Our findings suggest that CFS-B.vele has potential as a novel anti-obesity agent through modulation of the gut microbiota.

Anti-Inflammatory Effect and Signaling Mechanism of Glycine max Hydrolyzed with Enzymes from Bacillus velezensis KMU01 in a Dextran-Sulfate-Sodium-Induced Colitis Mouse Model.

The purpose of this study was to investigate the effect that Glycine max hydrolyzed with enzymes from Bacillus velezensis KMU01 has on dextran-sulfate-sodium (DSS)-induced colitis in mice. Hydrolysis improves functional health through the bioconversion of raw materials and increase in intestinal absorption rate and antioxidants. Therefore, G. max was hydrolyzed in this study using a food-derived microorganism, and its anti-inflammatory effect was observed. Enzymatically hydrolyzed G. max (EHG) was orally administered once daily for four weeks before DSS treatment. Colitis was induced in mice through the consumption of 5% (w/v) DSS in drinking water for eight days. The results showed that EHG treatment significantly alleviated DSS-induced body weight loss and decreased the disease activity index and colon length. In addition, EHG markedly reduced tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 production, and increased that of IL-10. EHG improved DSS-induced histological changes and intestinal epithelial barrier integrity in mice. Moreover, we found that the abundance of 15 microorganisms changed significantly; that of Proteobacteria and Escherichia coli, which are upregulated in patients with Crohn's disease and ulcerative colitis, decreased after EHG treatment. These results suggest that EHG has a protective effect against DSS-induced colitis and is a potential candidate for colitis treatment.

Prevalence of Indigenous Antibiotic-Resistant Salmonella Isolates and Their Application to Explore a Lytic Phage vB_SalS_KFSSM with an Intra-Broad Specificity.

The consumption of fresh produce has led to increase in antibiotic-resistant (AR) Salmonella outbreaks. In this study, indigenous Salmonella was isolated from a total of two hundred-two samples including fresh produce and agricultural environmental samples in Korea. After biochemical confirmation using the Indole, Methyl Red, Voges-Proskauer, Citrate tests, presumable Salmonella isolates were identified by 16S rRNA sequencing. Identified Salmonella isolates were evaluated for antibiotic susceptibility against twenty-two antibiotics. The specificity and the efficiency of plating (EOP) of vB_SalS_KFSSM were evaluated against fifty-three bacterial strains. Twenty-five suspected Salmonella were isolated and confirmed by the positive result for methyl red and citrate, of which ten were identified as Salmonella spp. through 16S rRNA gene sequencing. Eight Salmonella isolates (4.0%, n = 8/202) were resistant to at least one antibiotic, among which five were multi-drug resistant. As a lytic phage against Salmonella spp. CMGS-1, vB_SalS_KFSSM was isolated from cow manure. The phage was observed as a tailed phage belonging to the class Caudoviricetes. It exhibited an intra-broad specificity against four indigenous AR Salmonella isolates, two indigenous Salmonella isolates, and five other Salmonella serotypes with great efficiencies (EOP ≥ 0.75). Thus, this study suggested the potential of vB_SalS_KFSSM to combat indigenous AR Salmonella.

Postbiotics Enhance NK Cell Activation in Stress-Induced Mice through Gut Microbiome Regulation.

Recent studies have revealed that probiotics and their metabolites are present under various conditions; however, the role of probiotic metabolites (i.e., postbiotics in pathological states) is controversial. Natural killer (NK) cells play a key role in innate and adaptive immunity. In this study, we examined NK cell activation influenced by a postbiotics mixture in response to gut microbiome modulation in stress-induced mice. In vivo activation of NK cells increased in the postbiotics mixture treatment group in accordance with Th1/Th2 expression level. Meanwhile, the Red Ginseng treatment group, a reference group, showed very little expression of NK cell activation. Moreover, the postbiotics mixture treatment group in particular changed the gut microbiome composition. Although the exact role of the postbiotics mixture in regulating the immune system of stress-induced mice remains unclear, the postbiotics mixture-induced NK cell activation might have affected gut microbiome modulation.

Bacterial Community of Galchi-Baechu Kimchi Based on Culture-Dependent and - Independent Investigation and Selection of Starter Candidates.

In this study, the bacterial community of galchi-baechu kimchi was determined using culture-based and culture-independent techniques (next generation sequencing:NGS), and showed discrepancies between results. Weissella koreensis and Pediococcus inopinatus were the dominant species according to the NGS results, while Bacillus species and P. inopinatus were dominant in the culture-dependent analysis. To identify safe starter candidates, sixty-five Bacillus strains isolated from galchi-baechu kimchi using culture-dependent methods were evaluated for their antibiotic resistance, presence of toxin genes, and hemolytic activity. Strains were then assessed for salt tolerance and protease and lipase activity. As a result, four strains-B. safensis GN5_10, B. subtilis GN5_19, B. velezensis GN5_25, and B. velezensis GT8-were selected as safe starter candidates for use in fermented foods.

Functional Genomic Insights into Probiotic Bacillus siamensis Strain B28 from Traditional Korean Fermented Kimchi.

Bacillus siamensis strain B28 was previously isolated from traditional Korean fermented kimchi and inhibited expression of the microphthalmia-associated transcription factor and ß-catenin in human embryonic kidney 293 cells. Here, we determined the complete genome sequence of strain B28 and compared it with other strains to elucidate its potential probiotic properties. Strain B28 does not contain antibiotic resistance-, hemolysin- or enterotoxin-encoding genes. The genome includes genes related to survival in extreme conditions, adhesion in the gut, and synthesis of the bacteriocin. Considering the potential for enhancement of human health, the strain B28 genome encodes genes related to production of eight essential amino acids, γ-aminobutyric acid, branched-chain fatty acids, γ-glutamyltransferase, and subtilisin. There are genes for the synthesis of uracil, lipoteichoic acid, glutathione, and several reactive oxygen species-scavenging enzymes. Experimentally, strain B28 exhibited sensitivity to eight antibiotics and antibacterial activity against seven foodborne pathogens. B. siamensis B28 is a safe strain with potential for development as a probiotic.

Functional Annotation Genome Unravels Potential Probiotic Bacillus velezensis Strain KMU01 from Traditional Korean Fermented Kimchi.

Bacillus velezensis strain KMU01 showing γ-glutamyltransferase activity as a probiotic candidate was isolated from kimchi. However, the genetic information on strain KMU01 was not clear. Therefore, the current investigation was undertaken to prove the probiotic traits of B. velezensis strain KMU01 through genomic analysis. Genomic analysis revealed that strain KMU01 did not encode enterotoxin genes and acquired antibiotic resistance genes. Strain KMU01 genome possessed survivability traits under extreme conditions such as in the presence of gastric acid, as well as several probiotic traits such as intestinal epithelium adhesion and the production of thiamine and essential amino acids. Potential genes for human health enhancement such as those for γ-glutamyltransferase, nattokinase, and bacteriocin production were also identified in the genome. As a starter candidate for food fermentation, the genome of KMU01 encoded for protease, amylase, and lipase genes. The complete genomic sequence of KMU01 will contribute to our understanding of the genetic basis of probiotic properties and allow for the assessment of the effectiveness of this strain as a starter or probiotic for use in the food industry.

Comparison of the Stability of Poly-γ-Glutamate Hydrogels Prepared by UV and γ-Ray Irradiation.

Poly-γ-glutamate (γ-PGA) has various applications due to its desirable characteristics in terms of safety and biodegradability. Previous studies have been conducted on γ-PGA hydrogels produced by γ-ray irradiation, but these hydrogels have proved unstable in solutions. This study was conducted to enable the γ-PGA hydrogel to maintain a stable form in solutions. The γ-PGA mixture for UV-irradiation was prepared with a cross-linker (N,N,N-trimethyl-3-[(2- methylacryloyl)amino]propan-1-aminium). Both γ-PGA hydrogels' characteristics, including stability in solutions, were examined. The UV-irradiated γ-PGA hydrogel maintained a stable form during the nine weeks of the study, but the γ-ray irradiated hydrogel dissolved after one week.

Protection of Radiation-Induced DNA Damage by Functional Cosmeceutical Poly-Gamma-Glutamate.

This study compared the radioprotective effects of high-molecular-weight poly-gamma-glutamate (γ-PGA, average molecular mass 3,000 kDa) and a reduced form of glutathione (GSH, a known radioprotector) on calf thymus DNA damage. The radiation-induced DNA damage was measured on the basis of the decreased fluorescence intensity after binding the DNA with ethidium bromide. All the experiments used 6°Co gamma radiation at 1,252 Gy, representing 50% DNA damage. When increasing the concentration of γ-PGA from 0.33 to 1.65 µM, the DNA protection from radiation-induced damage also increased, with a maximum of 87% protection. Meanwhile, the maximal DNA protection when increasing the concentration of GSH was only 70%. Therefore, γ-PGA exhibited significant radioprotective effects against gamma irradiation.

Microbial ß-Galactosidase of Pediococcus pentosaceus ID-7: Isolation, Cloning, and Molecular Characterization.

Pediococcus pentosaceus ID-7 was isolated from kimchi, a Korean fermented food, and it showed high activity for lactose hydrolysis. The ß-galactosidase of P. pentosaceus ID-7 belongs to the GH2 group, which is composed of two distinct proteins. The heterodimeric LacLM type of ß-galactosidase found in P. pentosaceus ID-7 consists of two genes partially overlapped, lacL and lacM encoding LacL (72.2 kDa) and LacM (35.4 kDa). In this study, Escherichia coli MM294 was used for the production of LacL, LacM, and LacLM. These three types of recombinant proteins were expressed, purified, and characterized. The specific activities of LacLM and LacL were 339 and 31 U/mg, respectively. However, activity was not detected with LacM alone. The optimal pH of LacLM and LacL was pH 7.5 and pH 7.0, and the optimal temperature of LacLM and LacL was 40°C and 50°C, respectively. The optimal temperature changes indicate that LacLM is able to achieve higher activity at a relatively lower temperature. LacLM was strongly activated by Mg2+, Mn2+, and Zn2+, which was not true for LacL. Consistent with this, EDTA strongly inactivated LacLM and LacL, but the presence of reducing agents did not dramatically alter the activity. Taken together, multiple alignment of amino acid sequences and phylogenetic analysis results of LacL and LacM of P. pentosaceus ID-7 suggest the evolution of LacL into LacLM and that the use of divalent metal ions results in higher activity.

High-molecular-weight poly-gamma-glutamate protects against hypertriglyceridemic effects of a high-fructose diet in rat.

We studied the effects of 2 different dosages of highmolecular- weight poly-γ-glutamic acid (hm γ-PGA) derived from Bacillus subtilis chungkookjang on lipid metabolism in a high-fructose diet-induced hypertriglyceridemic animal model. For 4 weeks, rats were fed either AIN-93 diet (normal control, NC; n = 10) or modified AIN-93 diet in which cornstarch was substituted with 63% fructose (n = 30) to induce hypertriglyceridemia. After 4 weeks, the hypertriglyceridemic rats were treated with daily oral doses of 0 mg (hypertriglyceridemic control, HC), 2.5 mg (hypertriglyceridemic, low hm γ-PGA, HL), or 5 mg·kg·bw(-1)·d(-1) (hypertriglyceridemic, high hm γ-PGA, HH) hm γ-PGA for 4 weeks. The HL and HH groups exhibited significantly lower levels of serum triglyceride, total cholesterol, LDL cholesterol, and free fatty acids than the HC group. The administration of hm γ-PGA reduced serum ALT and AST levels. The activities of lipogenic enzymes such as hepatic malic enzyme and glucose-6-phosphate dehydrogenase as well as glucose-6-phosphate dehydrogenase mRNA expression were significantly decreased by hm γ-PGA administration (p < 0.05). These results indicate that hm γ- PGA has an anti-hypertriglyceridemic effect in highfructose diet-induced hypertriglyceridemic rats.

New biological functions and applications of high-molecular-mass poly-gamma-glutamic acid.

Ultra-high-molecular-weight poly-gamma-glutamic acid (gamma-PGA) is a most promising biodegradable polymer that is produced by Bacillus subtilis (chungkookjang). Attractive properties of gamma-PGA are that it is water soluble, anionic, biodegradable, and edible. Development of gamma-PGA has pursued in terms of cosmetics/skin care, bone care, nanoparticle for drug delivery system, hydrogel, and so on. Very recently, our research has shown that gamma-PGA can be used as an immune-stimulating agent, especially at high molecular weight. This review presents the synthesis and production of high-molecular-weight gamma-PGA and its various applications in industrial fields.

Functional and structural characterization of thermostable D-amino acid aminotransferases from Geobacillus spp.

D-amino acid aminotransferases (D-AATs) from Geobacillus toebii SK1 and Geobacillus sp. strain KLS1 were cloned and characterized from a genetic, catalytic, and structural aspect. Although the enzymes were highly thermostable, their catalytic capability was approximately one-third of that of highly active Bacilli enzymes, with respective turnover rates of 47 and 55 s(-1) at 50 degrees C. The Geobacillus enzymes were unique and shared limited sequence identities of below 45% with D-AATs from mesophilic and thermophilic Bacillus spp., except for a hypothetical protein with a 72% identity from the G. kaustophilus genome. Structural alignments showed that most key residues were conserved in the Geobacillus enzymes, although the conservative residues just before the catalytic lysine were distinctively changed: the 140-LRcD-143 sequence in Bacillus D-AATs was 144-EYcY-147 in the Geobacillus D-AATs. When the EYcY sequence from the SK1 enzyme was mutated into LRcD, a 68% increase in catalytic activity was observed, while the binding affinity toward alpha-ketoglutarate decreased by half. The mutant was very close to the wild-type in thermal stability, indicating that the mutations did not disturb the overall structure of the enzyme. Homology modeling also suggested that the two tyrosine residues in the EYcY sequence from the Geobacillus D-AATs had a pi/pi interaction that was replaceable with the salt bridge interaction between the arginine and aspartate residues in the LRcD sequence.

Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1.

A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+). The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).