In vivo functional effects of Weissella confusa VP30 exopolysaccharides on loperamide-induced constipation in rats.

In this work, the in vivo functionalities of milk fermented with Weissella confusa VP30 (VP30-EPS) and purified exopolysaccharide (pEPS) from the milk fermented with Weissella confusa VP30 were evaluated for their effect on constipation using an experimental constipated rat model. Rats were randomly divided into four groups: (i) control group (PBS administered normal group), (ii) loperamide treated group (constipation group), (iii) constipation with loperamide plus VP30-EPS (1 g/kg), and (iv) constipation with loperamide plus pEPS (0.6 g/kg) groups. Loperamide treatment induced animal constipation and significantly reduced the frequency of defecation, intestinal transit ratio, and water content of feces. However, all four fecal parameters were improved in both the loperamide plus VP30-EPS and pEPS administered groups as compared to the loperamide group. These results suggest that the addition of VP30-EPS potentially improves the functional laxative effects of commercial products. This study suggests the possibility that VP30-EPS can be applied to fermented and/or functional foods to relieve constipation.

A recombinant Bifidobacterium bifidum BGN4 strain expressing the streptococcal superoxide dismutase gene ameliorates inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a gastrointestinal disease characterized by diarrhea, rectal bleeding, abdominal pain, and weight loss. Recombinant probiotics producing specific proteins with IBD therapeutic potential are currently considered novel drug substitutes. In this study, a Bifidobacterium bifidum BGN4-SK strain was designed to produce the antioxidant enzymes streptococcal superoxide dismutase (SOD) and lactobacillus catalase (CAT), and a B. bifidum BGN4-pBESIL10 strain was proposed to generate an anti-inflammatory cytokine, human interleukin (IL)-10. In vitro and in vivo efficacy of these genetically modified Bifidobacterium strains were evaluated for colitis amelioration. RESULTS: In a lipopolysaccharide (LPS)-stimulated HT-29 cell model, tumor necrosis factor (TNF)-α and IL-8 production was significantly suppressed in the B. bifidum BGN4-SK treatment, followed by B. bifidum BGN4-pBESIL10 treatment, when compared to the LPS-treated control. Synergistic effects on TNF-α suppression were also observed. In a dextran sodium sulphate (DSS)-induced colitis mouse model, B. bifidum BGN4-SK treatment significantly enhanced levels of antioxidant enzymes SOD, glutathione peroxidase (GSH-Px) and CAT, compared to the DSS-only group. B. bifidum BGN4-SK significantly ameliorated the symptoms of DSS-induced colitis, increased the expression of tight junction genes (claudin and ZO-1), and decreased pro-inflammatory cytokines IL-6, IL-1ß and TNF-α. CONCLUSIONS: These findings suggest that B. bifidum BGN4-SK ameliorated DSS-induced colitis by generating antioxidant enzymes, maintaining the epithelial barrier, and decreasing the production of pro-inflammatory cytokines. Although B. bifidum BGN4-pBESIL10 exerted anti-inflammatory effects in vitro, the enhancement of IL-10 production and alleviation of colitis were very limited.

Butyl-fructooligosaccharides modulate gut microbiota in healthy mice and ameliorate ulcerative colitis in a DSS-induced model.

Butyl-fructooligosaccharides (B-FOSs) are newly synthesized prebiotics composed of short-chain FOS (GF2, 1-kestose; GF3, nystose; GF4, fructofuranosyl-nystose; GF5, 1-F-(1-b-D-fructofuranosyl)-2-nystose) bound with one or two butyric groups by ester bonds. Previous in vitro studies have shown that B-FOS treatment increases butyrate production and protects the growth of butyrate-producing bacteria during fermentation. The aim of this study was to further test B-FOS as a novel prebiotic compound by evaluating the effect of B-FOS on gut microbiota via 16S rRNA metagenomic analysis in an Institute of Cancer Research (ICR) mouse model and examining its anti-inflammatory efficacy in a mouse model of colitis induced by dextran sodium sulphate (DSS). In the healthy ICR mouse study, linear discriminant analysis effect size results revealed that Bifidobacterium was the representative phylotype in the B-FOS treatment compared to the control group. Furthermore, the cecal butyrate concentration of the B-FOS group was significantly higher than that of the control (P < 0.05). The high concentration of butyrate in the B-FOS treatment was probably associated with the high relative abundance of clusters of orthologous group (COG) 4770 (acetyl/propionyl-CoA carboxylase). In the DSS-induced infection study, B-FOS significantly ameliorated the symptoms of DSS-induced colitis, increased the mRNA expression of occludin, decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin (IL-8) in the colon tissues, and significantly increased cecal butyrate concentrations. These findings suggest that B-FOS ameliorated DSS-induced colitis by maintaining the epithelial barrier and reducing the secretion of inflammation related cytokines.

Oral probiotic activities and biosafety of Lactobacillus gasseri HHuMIN D.

BACKGROUND: Lactobacillus spp. have been researched worldwide and are used in probiotics, but due to difficulties with laboratory cultivation of and experimentation on oral microorganisms, there are few reports of Lactobacillus spp. being isolated from the oral cavity and tested against oral pathogens. This research sought to isolate and determine the safety and inhibitory capabilities of a Lactobacillus culture taken from the human body. RESULTS: One organism was isolated, named "L. gasseri HHuMIN D", and evaluated for safety. A 5% dilution of L. gasseri HHuMIN D culture supernatant exhibited 88.8% inhibition against halitosis-producing anaerobic microorganisms and the organism itself exhibited powerful inhibitory effects on the growth of 11 oral bacteria. Hydrogen peroxide production reached 802 µmol/L after 12 h and gradually diminished until 24 h, it efficiently aggregated with P. catoniae and S. sanguinis, and it completely suppressed S. mutans-manufactured artificial dental plaque. L. gasseri HHuMIN D's KB cell adhesion capacity was 4.41 cells per cell, and the cell adhesion of F. nucleatum and S. mutans diminished strongly in protection and displacement assays. CONCLUSION: These results suggest that L. gasseri HHuMIN D is a safe, bioactive, lactobacterial food ingredient, starter culture, and/or probiotic microorganism for human oral health.

Isolation, Characterization and Biosafety Evaluation of Lactobacillus Fermentum OK with Potential Oral Probiotic Properties.

It has been reported that certain probiotic bacteria have inhibitory effects against oral pathogens. Lactobacillus spp. have been studied and used as probiotics globally, but due to difficulties with laboratory cultivation and experimentation with oral microorganisms, there are few studies on Lactobacillus spp. isolated from the oral cavity being used against oral pathogens. The purpose of this study was to evaluate the biosafety and inhibitory effects of Lactobacillus fermentum OK as a potential oral biotherapeutic probiotic against oral pathogens. L. fermentum OK was evaluated based on microbial and genetic characteristics. A 5% dilution of L. fermentum OK culture supernatant showed that 60% inhibition against the growth of S. mutans and L. fermentum OK displayed significant inhibitory effects against the growth of Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus gordonii, and Streptococcus sanguinis. However, proliferation of L. fermentum OK, when co-cultured with harmful oral bacteria, was retarded. L. fermentum OK was shown to produce 1130 µmol/L hydrogen peroxide, aggregate efficiently with Streptococcus sobrinus, S. gordonii, S. mutans, S. sanguinis, and P. gingivalis, and reduce S. mutans that produced artificial dental plaque by 97.9%. The in vitro cell adhesion capacity of L. fermentum OK to an oral epithelial cell line was 3.1 cells per cell and the cell adhesion of F. nucleatum and S. mutans decreased strongly in protection and displacement assays. L. fermentum OK was evaluated for safety using ammonia production, biogenic amine production, hemolytic property, mucin degradation testing, antibiotic susceptibility, and whole genome sequencing (WGS). Based on this study, L. fermentum OK appears to be a safe and bioactive lactobacterial food ingredient, starter culture, and/or probiotic microorganism for human oral health.

Production of biologically active human interleukin-10 by Bifidobacterium bifidum BGN4.

BACKGROUND: Bifidobacterium spp. are representative probiotics that play an important role in the health of their hosts. Among various Bifidobacterium spp., B. bifidum BGN4 exhibits relatively high cell adhesion to colonic cells and has been reported to have various in vivo and in vitro bio functionalities (e.g., anti-allergic effect, anti-cancer effect, and modulatory effects on immune cells). Interleukin-10 (IL-10) has emerged as a major suppressor of immune response in macrophages and other antigen presenting cells and plays an essential role in the regulation and resolution of inflammation. In this study, recombinant B. bifidum BGN4 [pBESIL10] was developed to deliver human IL-10 effectively to the intestines. RESULTS: The vector pBESIL10 was constructed by cloning the human IL-10 gene under a gap promoter and signal peptide from Bifidobacterium spp. into the E. coli-Bifidobacterium shuttle vector pBES2. The secreted human IL-10 from B. bifidum BGN4 [pBESIL10] was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western Blotting, and enzyme-linked immunosorbent assay (ELISA). More than 1,473 ± 300 ng/mL (n = 4) of human IL-10 was obtained in the cell free culture supernatant of B. bifidum BGN4 [pBESIL10]. This productivity is significantly higher than other previously reported human IL-10 level from food grade bacteria. In vitro functional evaluation of the cell free culture supernatant of B. bifidum BGN4 [pBESIL10] revealed significantly inhibited interleukin-6 (IL-6) production in lipopolysaccharide (LPS)-induced Raw 264.7 cells (n = 6, p < 0.0001) and interleukin-8 (IL-8) production in LPS-induced HT-29 cells (n = 6, p < 0.01) or TNFα-induced HT-29 cells (n = 6, p < 0.001). CONCLUSION: B. bifidum BGN4 [pBESIL10] efficiently produces and secretes significant amounts of biologically active human IL-10. The human IL-10 production level in this study is the highest of all human IL-10 production reported to date. Further research should be pursued to evaluate B. bifidum BGN4 [pBESIL10] producing IL-10 as a treatment for various inflammation-related diseases, including inflammatory bowel disease, rheumatoid arthritis, allergic asthma, and cancer immunotherapy.

Acute and sub-chronic (28-day) oral toxicity profiles of newly synthesized prebiotic butyl-fructooligosaccharide in ICR mouse and Wistar rat models.

B-FOS (butyl-fructooligosaccharide) is a newly synthesized prebiotic molecule, formed by the combination of FOS and butyrate by ester bonds. B-FOS has been reported to have the potential prebiotic effect of promoting intestinal flora diversity and enhancing butyrate production. The aim of this study was to investigate the potential acute and sub-chronic toxicity of B-FOS in Institute of Cancer Research (ICR) mice and Wistar rats to verify its biosafety. ICR mice were administered a single oral gavage of B-FOS at doses of 0, 500, 1000, and 2000 mg/kg body weight and observed for signs of acute toxicity for 14 days. Sub-chronic toxicity was evaluated by repeated oral administration of B-FOS at 2000 mg/kg for 28 days, in accordance with Organization for Economic Co-operation and Development (OECD) protocol test numbers 420 and 407. No mortality or abnormal clinical signs were observed during the experimental periods after B-FOS administration. Furthermore, no significant changes in body weight, organ weight, serum biochemical parameters, or tissue histology were observed after animal sacrifice. These in vivo results indicate that B-FOS does not exert any acute or sub-chronic toxicity at a dose of 2000 mg/kg, and this novel molecule can be regarded as a safe prebiotic substance for use in the food and nutraceutical industries.

Production and Characterization of Anti-Inflammatory Monascus Pigment Derivatives.

The prevention and treatment of chronic inflammation using food-derived compounds are desirable from the perspectives of marketing and safety. Monascus pigments, widely used as food additives, can be used as a chronic inflammation treatment. Orange Monascus pigments were produced by submerged fermentation in a 5 L bioreactor, and multiple orange Monascus pigment derivatives with anti-inflammatory activities were synthesized using aminophilic reaction. A total of 41 types of pigment derivatives were produced by incorporating amines and amino acids into the orange pigments. One derivative candidate that inhibited nitric oxide (NO) production in Raw 264.7 cells and exhibited low cell cytotoxicity was identified via in vitro assay. The 2-amino-4 picoline derivative inhibited NO production of 48.4%, and exhibited cell viability of 90.6%. Expression of inducible NO synthase, an important enzyme in the NO synthesis pathway, was suppressed by such a derivative in a dose-dependent manner. Therefore, this derivative has potential as a functional food colorant with anti-inflammatory effects.

Production, Structural Characterization, and In Vitro Assessment of the Prebiotic Potential of Butyl-Fructooligosaccharides.

Short-chain fatty acids (SCFAs), especially butyrate, produced in mammalian intestinal tracts via fermentation of dietary fiber, are known biofunctional compounds in humans. However, the variability of fermentable fiber consumed on a daily basis and the diversity of gut microbiota within individuals often limits the production of short-chain fatty acids in the human gut. In this study, we attempted to enhance the butyrate levels in human fecal samples by utilizing butyl-fructooligosaccharides (B-FOS) as a novel prebiotic substance. Two major types of B-FOS (GF3-1B and GF3-2B), composed of short-chain fructooligosaccharides (FOS) bound to one or two butyric groups by ester bonds, were synthesized. Qualitative analysis of these B-FOS using Fourier transform infrared (FT-IR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), nuclear magnetic resonance (NMR) and low-resolution fast-atom bombardment mass spectra (LR-FAB-MS), showed that the chemical structure of GF3-1B and GF3-2B were [O-(1-buty-ß-D-fru-(2→1)-O-ß-D-fru-(2→1)-O-ß-D-fru-O-α-D-glu] and [O-(1-buty)-ß-D-fru-(2→1)-O-ß-D-fru-(2→1)-O-(4-buty)-ß-D-fru-O-α-D-glu], respectively. The ratio of these two compounds was approximately 5:3. To verify their biofunctionality as prebiotic oligosaccharides, proliferation and survival patterns of human fecal microbiota were examined in vitro via 16S rRNA metagenomics analysis compared to a positive FOS control and a negative control without a carbon source. B-FOS treatment showed different enrichment patterns on the fecal microbiota community during fermentation, and especially stimulated the growth of major butyrate producing bacterial consortia and modulated specific butyrate producing pathways with significantly enhanced butyrate levels. Furthermore, the relative abundance of Fusobacterium and ammonia production with related metabolic genes were greatly reduced with B-FOS and FOS treatment compared to the control group. These findings indicate that B-FOS differentially promotes butyrate production through the enhancement of butyrate-producing bacteria and their metabolic genes, and can be applied as a novel prebiotic compound in vivo.

Isolation and characterization of high exopolysaccharide-producing Weissella confusa VP30 from young children's feces.

BACKGROUND: Lactic acid bacteria (LAB) are known to have a significant ability to colonize the human intestinal tract and adhere to the surface of intestinal epithelial cells. Among the various lactic acid bacteria, exopolysaccharide (EPS) producing strains are known to provide a variety of health benefits for their hosts (e.g. anti-inflammatory, antioxidant, antitumor and stress tolerant effects). Recently, EPSs and EPS-producing lactic acid cultures have gained interest within the food industry and are playing important roles as biothickeners and texturizing agents due to their hydrocolloidal nature. In this study, 156 lactic acid bacterial strains isolated from fecal samples of healthy young children were screened and evaluated for active EPS-production capability. RESULTS: Among the various human origin lactic acid flora isolated, Weissella confusa VP30 showed the highest EPS productivity and its EPS producing properties were characterized under various cultural conditions in this research. To document the safety of W. confusa VP30, antibiotic resistance, hemolytic, and ammonia production properties were evaluated in addition. No significant negative results were observed. The maximum EPS production by W. confusa VP30 was 59.99 ± 0.91 g/l after 48 h of cultivation in media containing 10% sucrose, far exceeding EPS production by other bacterial strains reported elsewhere. Based on gel permeation chromatography (GPC), the molecular weight of EPS produced by W. confusa VP30 was 3.8 × 106 Da. Structural analysis of the released EPS fraction by 13C and 1H nuclear magnetic resonance (NMR) spectroscopy revealed that W. confusa VP30 can produce dextran with glucose units linked with 96.5% α (1 → 6) glycosidic bonds and 3.5% α (1 → 3) branches. CONCLUSION: The high EPS production capability and safety of W. confusa VP30 justify food industry consideration of this cell strain for further evaluation and potential industrial use.

Biocatalysis of Fucodian in Undaria pinnatifida Sporophyll Using Bifidobacterium longum RD47 for Production of Prebiotic Fucosylated Oligosaccharide.

Fucosylated oligosaccharide (FO) is known to selectively promote the growth of probiotic bacteria and is currently marketed as a functional health food and prebiotic in infant formula. Despite widespread interest in FO among functional food customers, high production costs due to high raw material costs, especially those related to fucose, are a significant production issue. Therefore, several actions are required before efficient large-scale operations can occur, including (i) identification of inexpensive raw materials from which fucosylated oligosaccharides may be produced and (ii) development of production methods to which functional food consumers will not object (e.g., no genetically modified organisms (GMOs)). Undaria pinnatifida, commonly called Miyeok in Korea, is a common edible brown seaweed plentiful on the shores of the Korean peninsula. In particular, the sporophyll of Undaria pinnatifida contains significant levels of l-fucose in the form of fucoidan (a marine sulfated polysaccharide). If the l-fucose present in Undaria pinnatifida sporophyll was capable of being separated and recovered, l-fucose molecules could be covalently joined to other monosaccharides via glycosidic linkages, making this FO manufacturing technology of value in the functional food market. In our previous work, ß-galactosidase (EC 3.2.2.23) from Bifidobacterium longum RD47 (B. longum RD47) was found to have transglycosylation activity and produce FO using purified l-fucose and lactose as substrates (reference). In this research, crude fucodian hydrolysates were separated and recovered from edible seaweed (i.e., U. pinnatifida sporophyll). The extracted l-fucose was purified via gel permeation and ion exchange chromatographies and the recovered l-fucose was used to synthesize FO. B. longum RD47 successfully transglycosilated and produced FO using l-fucose derived from Undaria pinnatifida and lactose as substrates. To the best of our knowledge, this is the first report of synthesized FO using Bifidobacterium spp.

Production of Selenomethionine-Enriched Bifidobacterium bifidum BGN4 via Sodium Selenite Biocatalysis.

Selenium is a trace element essential for human health that has received considerable attention due to its nutritional value. Selenium's bioactivity and toxicity are closely related to its chemical form, and several studies have suggested that the organic form of selenium (i.e., selenomethionine) is more bioavailable and less toxic than its inorganic form (i.e., sodium selenite). Probiotics, especially Bifidobacteriium and Lactobacillus spp., have received increasing attention in recent years, due to their intestinal microbial balancing effects and nutraceutical benefits. Recently, the bioconversion (a.k.a biotransformation) of various bioactive molecules (e.g., minerals, primary and secondary metabolites) using probiotics has been investigated to improve substrate biofunctional properties. However, there have been few reports of inorganic selenium conversion into its organic form using Bifidobacterium and Lactobacillus spp. Here we report that the biosynthesis of organic selenium was accomplished using the whole cell bioconversion of sodium selenite under controlled Bifidobacterium bifidum BGN4 culture conditions. The total amount of organic and inorganic selenium was quantified using an inductively coupled plasma-atomic emission spectrometer (ICP-AES). The selenium species were separated via anion-exchange chromatography and analyzed with inductively coupled plasma-mass spectrometry (ICP-MS). Our findings indicated that the maximum level of organic selenium was 207.5 µg/g in selenium-enriched B. bifidum BGN4. Selenomethionine was the main organic selenium in selenium-enriched B. bifidum BGN4 (169.6 µg/g). Considering that B. bifidum BGN4 is a commercial probiotic strain used in the functional food industry with clinically proven beneficial effects, selenium-enriched B. bifidum BGN4 has the potential to provide dual healthy functions as a daily supplement of selenium and regulator of intestinal bacteria. This is the first report on the production of organic selenium using B. bifidum spp.

Biocatalysis of Platycoside E and Platycodin D3 Using Fungal Extracellular ß-Glucosidase Responsible for Rapid Platycodin D Production.

Platycodi radix (i.e., Platycodon grandiflorum root) products (e.g., tea, cosmetics, and herbal supplements) are popular in East Asian nutraceutical markets due to their reported health benefits and positive consumer perceptions. Platycosides are the key drivers of Platycodi radixes' biofunctional effects; their nutraceutical and pharmaceutical activities are primarily related to the number and varieties of sugar side-chains. Among the various platycosides, platycodin D is a major saponin that demonstrates various nutraceutical activities. Therefore, the development of a novel technology to increase the total platycodin D content in Platycodi radix extract is important, not only for consumers' health benefits but also producers' commercial applications and manufacturing cost reduction. It has been reported that hydrolysis of platycoside sugar moieties significantly modifies the compound's biofunctionality. Platycodi radix extract naturally contains two major platycodin D precursors (platycoside E and platycodin D3) which can be enzymatically converted to platycodin D via ß-d-glucosidase hydrolysis. Despite evidence that platycodin D precursors can be changed to platycodin D in the Platycodi radix plant, there is little research on increasing platycodin D concentrations during processing. In this work, platycodin D levels in Platycodi radix extracts were significantly increased via extracellular Aspergillus usamii ß-d-glucosidase (n = 3, p < 0.001). To increase the extracellular ß-d-glucosidase activity, A. usamii was cultivated in a culture media containing cellobiose as its major carbon source. The optimal pH and temperature of the fungal ß-d-glucosidase were 6.0 and 40.0 °C, respectively. Extracellular A. usamii ß-d-glucosidase successfully converted more than 99.9% (w/v, n = 3, p < 0.001) of platycoside E and platycodin D3 into platycodin D within 2 h under optimal conditions. The maximum level of platycodin D was 0.4 mM. Following the biotransformation process, the platycodin D was recovered using preparatory High Performance Liquid Chromatography (HPLC) and applied to in vitro assays to evaluate its quality. Platycodin D separated from the Platycodi radix immediately following the bioconversion process showed significant anti-inflammatory effects from the Lipopolysaccharide (LPS)-induced macrophage inflammatory responses with decreased nitrite and IL-6 production (n = 3, p < 0.001). Taken together, these results provide evidence that biocatalysis of Platycodi radix extracts with A. usamii may be used as an efficient method of platycodin D-enriched extract production and novel Platycodi radix products may thereby be created.

Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI.

Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.