• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.25-33
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• 2019

Lactobacillus rhamnosus BFE5264, isolated from a Maasai fermented milk product ("kule naoto"), was previously shown to exhibit bile acid resistance, cholesterol assimilation, and adhesion to HT29-MTX cells in vitro. In this study, we re-annotated and analyzed the previously reported complete genome sequence of strain BFE5264. The genome consists of a circular chromosome of 3,086,152 bp and a putative plasmid, which is the largest one identified among L. rhamnosus strains. Among the 2,883 predicted protein-coding genes, those with carbohydrate-related functions were the most abundant. Genome analysis of strain BFE5264 revealed two consecutive CRISPR regions and no known virulence factors or antimicrobial resistance genes. In addition, previously known highly variable regions in the genomes of L. rhamnosus strains were also evident in strain BFE5264. Pairwise comparison with the most studied probiotic strain L. rhamnosus GG revealed strain BFE5264-specific deletions, probably due to insertion sequence-mediated recombination. The latter was associated with loss of the spaCBA pilin gene cluster and exopolysaccharide biosynthetic genes. Comparative genomic analysis of the sequences from all available L. rhamnosus strains revealed that they were clustered into two groups, being within the same species boundary based on the average nucleotide identities. Strain BFE5264 had a sister group relationship with the group that contained strain GG, but neither ANI-based hierarchical clustering nor core-gene-based phylogenetic tree construction showed a clear distinctive pattern associated with the isolation source, implying that the genotype alone cannot account for their ecological niches. These results provide insights into the probiotic mechanisms of strain BFE5264 at the genomic level.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.721-730
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• 2019

The probiotic Lactobacillus acidophilus KBL409 was encapsulated with alginate (Al) and alginate-chitosan (Al/Chi) through extrusion method. The sizes and zeta potentials of microspheres were measured to confirm encapsulation. To evaluate the protective effect of microspheres against gastrointestinal fluids, all the samples were exposed to simulated gastric fluids (SGFs, pH 1.5) at $37^{\circ}C$ for 1 or 2 h, followed by incubation with simulated intestinal fluids (SIFs, pH 6.5) for 2 h. The mucoadhesive ability of microspheres was evaluated using the intestinal epithelial cell line HT29-MTX. To extend the shelf-life of probiotics, lyoprotectants such as disaccharide and polysaccharide were mixed with free or encapsulated cells during the freeze-drying process. The size of the microspheres demonstrated a narrow distribution, while the zeta potentials of Al and Al/Chi-microspheres were $-17.9{\pm}2.3$ and $20.4{\pm}2.6mV$, respectively. Among all the samples, Al/Chi-encapsulated cells showed the highest survival rate even after exposure to SGF and SIF. The mucoadhesive abilities of Al and Al/Chi-microspheres were higher than 94%, whereas the free L. acidophilus showed 88.1% mucoadhesion. Ten percent of sucrose showed over 80% survival rate in free or encapsulated cells. Therefore, L. acidophilus encapsulated with Al and Al/Chi-microspheres showed higher survival rates after exposure to the gastrointestinal tract and better mucoadhesive abilities than the free cells. Also, sucrose showed the highest protective effect of L. acidophilus during the freeze-drying process.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.432-439
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• 2021

Microbial community plays important roles in the culture environment of mud crab Scylla serrata. One of the environmental management efforts for the cultivation of S.serrata is by stabilizing microorganisms involved in nitrogen cycle process. The availability of dissolved inorganic nitrogen in its culture environment under a recirculating system closely relates to the nitrogen cycle, which involves both anaerobic and aerobic bacterial activities. Anaerobically, there are two major nitrogen compound degradation processes, i.e., denitrification and dissimilatory nitrate reduction to ammonium (DNRA). This study aimed to identify denitrifying and DNRA bacteria isolated from the recirculating cultivation of S. serrata. The water samples were collected from anaerobic filters called close filter system, which is anaerobically conditioned with the addition of varying physical filter materials in the recirculating mud crab cultures. The results showed that three denitrifying bacterial isolates and seven DNRA bacterial isolates were successfully identified. The phylogenetic analysis based on 16S rRNA gene of the denitrifying bacteria revealed that HIB_7a had the closest similarity to Stenotrophomonas daejeonensis strain MJ03. Meanwhile, DNRA bacterial isolate of HIB_92 showed a 100% similarity to Bacillus sonorensis strain N3, Bacillus vallismortis strain VITS-17, Bacillus tequlensis strain TY5, Geobacillus sp. strain DB24, Bacillus subtilis strain A1, and Bacillus mojavensis strain SSRAI21. This study provides basic information denitrifying and DNRA bacterial isolates identity which might have the potential to be applied as probiotics in aquaculture systems in order to maintain optimal environmental conditions.

• Journal of Microbiology and Biotechnology
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• v.31 no.8
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• pp.1144-1153
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• 2021

A released exopolysaccharide (rEPS)-producing strain (LM187) with good acid resistance, bile salt resistance, and cholesterol-lowering properties was isolated from Sichuan paocai and identified as Leuconostoc mesenteroides subsp. mesenteroides. The purified rEPS, designated as rEPS414, had a uniform molecular weight of 7.757 × 10⁵ Da. Analysis of the monosaccharide composition revealed that the molecule was mainly composed of glucose. The Fourier transform-infrared spectrum showed that rEPS414 contained both α-type and β-type glycosidic bonds. ¹H and ¹³C nuclear magnetic resonance spectra analysis showed that the purified rEPS contained arabinose, galactose, and rhamnose, but less uronic acid. Scanning electron microscopy demonstrated that the exopolysaccharide displayed a large number of scattered, fluffy, porous cellular network flake structures. In addition, rEPS414 exhibited strong in vitro antioxidant activity. These results showed that strain LM187 and its rEPS are promising probiotics with broad prospects in industry.

• Journal of Microbiology and Biotechnology
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• v.31 no.7
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• pp.949-955
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• 2021

Previously, our research group isolated Bifidobacterium breve IDCC4401 from infant feces as a potential probiotic. For this study, we evaluated the safety of B. breve IDCC4401 using genomic and phenotypic analyses. Whole genome sequencing was performed to identify genomic characteristics and investigate the potential presence of genes encoding virulence, antibiotic resistance, and mobile genetic elements. Phenotypic analyses including antibiotic susceptibility, enzyme activity, production of biogenic amines (BAs), and proportion of D-/L-lactate were evaluated using E-test, API ZYM test, high-performance liquid chromatography (HPLC), and D-/L-lactic acid assay respectively. The genome of B. breve IDCC4401 consists of 2,426,499 bp with a GC content of 58.70% and 2,016 coding regions. Confirmation of the genome as B. breve was provided by its 98.93% similarity with B. breve DSM20213. Furthermore, B. breve IDCC4401 genes encoding virulence and antibiotic resistance were not identified. Although B. breve IDCC4401 showed antibiotic resistance against vancomycin, we confirmed that this was an intrinsic feature since the antibiotic resistance gene was not present. B. breve IDCC4401 showed leucine arylamidase, cystine arylamidase, α-galactosidase, β-galactosidase, and α-glucosidase activities, whereas it did not show production of harmful enzymes such as β-glucosidase and β-glucuronidase. In addition, B. breve IDCC4401 did not produce any tyramine, histamine, putrescine, cadaverine, or 2-phenethylamine, which are frequently detected BAs during fermentation. B. breve IDCC4401 produced 95.08% of L-lactate and 4.92% of D-lactate. Therefore, our findings demonstrate the safety of B. breve IDCC 4401 as a potential probiotic for use in the food industry.

• Korean Journal of Community Nutrition
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• v.26 no.4
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• pp.259-269
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• 2021

Objectives: This study aimed to investigate the changes in the eating habits and lifestyle of middle-aged women in Gwangju during the COVID-19 pandemic. Methods: A total of 428 middle-aged women aged between 40 and 60 participated in a survey relating to general information, food and lifestyle, health functional food, and menopausal symptoms. The correlation between the variables was analyzed. Results: In the positive habits, the intake of nutritional supplements for immunity enhancement increased the most, followed by the use of media to learn healthy eating tips, and diets including healthy food. Negative habits increased in the order of frequency of taking delivery orders, levels of stress or anxiety, and time spent sitting or watching movies. In the case of recommended foods, the intake increased the most in the order of eggs, fruits, vegetables, milk/dairy products, and seaweed. Non-recommended foods increased in the order of meat, bread, rice, and noodles. The awareness of health functional foods was in the increasing order of interest, knowledge, consumption experience, and purchase amount. The type of health functional food intake was in the increasing order of probiotics, multivitamin and mineral supplements, vitamin C, collagen, and omega-3. Menopausal symptoms were in the increasing order of bone and joint pain, poor sleep quality, emotional ups and downs, loneliness, and feeling of emptiness. In the correlation of major variables, positive habits showed a significant positive correlation with recommended food intake and the recognition of health functional foods. Negative habits showed a significant positive correlation with non-recommended food intake and a significant positive correlation with menopausal symptoms. Recommended food intake showed a significant positive correlation with health functional food recognition and intake and menopausal symptoms. Conclusions: This study suggests that it is necessary to establish social measures to reduce the negative effects of the COVID-19 pandemic on middle-aged women and to ensure effective self-management through a healthy lifestyle since the pandemic has a long-term impact.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.717-725
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• 2021

This study aimed to optimize medium composition and culture conditions for enhancing the biomass of Lactobacillus plantarum 200655 using statistical methods. The one-factor-at-a-time (OFAT) method was used to screen the six carbon sources (glucose, sucrose, maltose, fructose, lactose, and galactose) and six nitrogen sources (peptone, tryptone, soytone, yeast extract, beef extract, and malt extract). Based on the OFAT results, six factors were selected for the Plackett-Burman design (PBD) to evaluate whether the variables had significant effects on the biomass. Maltose, yeast extract, and soytone were assessed as critical factors and therefore applied to response surface methodology (RSM). The optimal medium composition by RSM was composed of 31.29 g/l maltose, 30.27 g/l yeast extract, 39.43 g/l soytone, 5 g/l sodium acetate, 2 g/l K₂HPO₄, 1 g/l Tween 80, 0.1 g/l MgSO₄·7H₂O, and 0.05 g/l MnSO₄·H₂O, and the maximum biomass was predicted to be 3.951 g/l. Under the optimized medium, the biomass of L. plantarum 200655 was 3.845 g/l, which was similar to the predicted value and 1.58-fold higher than that of the unoptimized medium (2.429 g/l). Furthermore, the biomass increased to 4.505 g/l under optimized cultivation conditions. For lab-scale bioreactor validation, batch fermentation was conducted with a 5-L bioreactor containing 3.5 L of optimized medium. As a result, the highest yield of biomass (5.866 g/l) was obtained after 18 h of incubation at 30℃, pH 6.5, and 200 rpm. In conclusion, mass production by L. plantarum 200655 could be enhanced to obtain higher yields than that in MRS medium

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.280-289
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• 2021

Genetic markers currently used for the discrimination of Lactobacillus delbrueckii subspecies have low efficiency for identification at subspecies level. Therefore, our objective in this study was to select novel genetic markers for accurate identification and discrimination of six L. delbrueckii subspecies based on pangenome analysis. We evaluated L. delbrueckii genomes to avoid making incorrect conclusions in the process of selecting genetic markers due to mislabeled genomes. Genome analysis showed that two genomes of L. delbrueckii subspecies deposited at NCBI were misidentified. Based on these results, subspecies-specific genetic markers were selected by comparing the core and pangenomes. Genetic markers were confirmed to be specific for 59,196,562 genome sequences via in silico analysis. They were found in all strains of the same subspecies, but not in other subspecies or bacterial strains. These genetic markers also could be used to accurately identify genomes at the subspecies level for genomes known at the species level. A real-time PCR method for detecting three main subspecies (L. delbrueckii subsp. delbrueckii, lactis, and bulgaricus) was developed to cost-effectively identify them using genetic markers. Results showed 100% specificity for each subspecies. These genetic markers could differentiate each subspecies from 44 other lactic acid bacteria. This real-time PCR method was then applied to monitor 26 probiotics and dairy products. It was also used to identify 64 unknown strains isolated from raw milk samples and dairy products. Results confirmed that unknown isolates and subspecies contained in the product could be accurately identified using this real-time PCR method.

• Journal of Periodontal and Implant Science
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• v.51 no.3
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• pp.199-212
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• 2021

Purpose: This randomized clinical placebo-controlled trial was conducted to evaluate the effectiveness of Lactobacillus reuteri as a probiotic in guided pocket recolonization (GPR) for the treatment of chronic periodontitis (CP) adjunctive to scaling and root planing (SRP). Methods: Forty-eight CP patients were randomly assigned to 3 treatment groups: group 1 (SRP+placebo), group 2 (SRP+single application of probiotic), and group 3 (SRP+incremental application of probiotic). Clinical parameters were evaluated at baseline and at 8, 12, and 24 weeks, whereas biochemical parameters were measured at baseline and 12 weeks. Results: At 24 weeks, the probing pocket depth and clinical attachment level improved in all 3 groups from baseline with no significant intergroup differences; however, a statistically significant difference was observed in localized plaque and gingival scores between groups 1 and 3 (P<0.05). At 12 weeks, matrix metalloproteinase-8 (MMP-8), nitric oxide (NO), and gingipains-R (Rgps) levels improved in all 3 groups, with statistically significant differences between groups 1 and 3 for MMP-8 and NO (P<0.05), but no difference for Rgps levels. Conclusions: Within its limitations, the results of this study show that incremental 3-time application of L. reuteri as a probiotic led to improvements in clinical and biochemical parameters. This protocol can be a useful adjunct to SRP in the non-surgical management of CP.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.92-103
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• 2021

Lactobacillus casei, one of the most widely used probiotics, has been reported to alleviate multiple diseases. However, the effects of this species on intestinal diseases are strain-specific. Here, we aimed to screen L. casei strains with inflammatory bowel disease (IBD)-alleviating effects based on in vitro physiological characteristics. Therefore, the physiological characteristics of 29 L. casei strains were determined, including gastrointestinal transit tolerance, oligosaccharide fermentation, HT-29 cell adhesion, generation time, exopolysaccharide production, acetic acid production, and conjugated linoleic acid synthesis. The effects of five candidate strains on mice with induced colitis were also evaluated. The results showed that among all tested L. casei strains, only Lactobacillus casei M2S01 effectively relieved colitis. This strain recovered body weight, restored disease activity index score, and promoted anti-inflammatory cytokine expression. Gut microbiota sequencing showed that L. casei M2S01 restored a healthy gut microbiome composition. The western blotting showed that the alleviating effects of L. casei M2S01 on IBD were related to the inhibition of the NF-κB pathway. A good gastrointestinal tolerance ability may be one of the prerequisites for the IBD-alleviating effects of L. casei. Our results verified the efficacy of L. casei in alleviating IBD and lay the foundation for the rapid screening of L. casei strain with IBD-alleviating effects.