Exploring the Potential Probiotic Properties of Bifidobacterium breve DSM 32583-A Novel Strain Isolated from Human Milk.

Human milk is the best nutrition for infants, providing optimal support for the developing immune system and gut microbiota. Hence, it has been used as source for probiotic strain isolation, including members of the genus Bifidobacterium, in an effort to provide beneficial effects to infants who cannot be exclusively breastfed. However, not all supplemented bifidobacteria can effectively colonise the infant gut, nor confer health benefits to the individual infant host; therefore, new isolates are needed to develop a range of dietary products for this specific age group. Here, we investigated the beneficial potential of Bifidobacterium breve DSM 32583 isolated from human milk. We show that in vitro B. breve DSM 32583 exhibited several characteristics considered fundamental for beneficial bacteria, including survival in conditions simulating those present in the digestive tract, adherence to human epithelial cell lines, and inhibition of growth of potentially pathogenic microorganisms. Its antibiotic resistance patterns were comparable to those of known beneficial bifidobacterial strains, and its genome did not contain plasmids nor virulence-associated genes. These results suggest that B. breve DSM 32583 is a potential probiotic candidate.

Bifidobacterium breve DSM 32583 and Limosilactobacillus fermentum CECT5716 postbiotics attenuate S. aureus and IL-33-induced Th2 responses.

Over the past decades, the prevalence of allergic diseases noticeably increased in industrialized countries. The Th2 immune response plays a central role in these pathologies and its modulation using pro-/postbiotics constitutes a promising approach to prevent or alleviate disease symptoms. The aim of this in vitro study, was to investigate the ability of human milk-derived Bifidobacterium breve DSM 32583 (Bb) and Limosilactobacillus fermentum CECT5716 (Lf), to modulate the Th2 induced responses. To this end, Th2 cells were generated by co-culturing of human naïve Th cells with monocyte-derived dendritic cells (moDCs) either stimulated with Staphylococcus aureus or IL-33. The immunomodulatory effects of pro-/postbiotic preparations of Bb and Lf on moDCs and Th2 cells were evaluated in terms of maturation markers expression and cytokines production. Remarkably, the tested strains induced the anti-inflammatory cytokine IL-10 in moDCs, in a strain-, dose- and viability-dependent manner with no significant upregulation of IL-12p70 nor CD83, CD86 or HLA-DR. Interestingly, Bb and Lf postbiotics were able to dampen the Th2/Th1 response induced upon S. aureus- or IL-33 stimulation. They were also able to synergistically induce IL-10 in moDCs and T cells, upon co-stimulation with LPS. Finally, we observed that live probiotics triggered a mild Th1 response that was attenuated in the presence of galacto-oligosaccharides. Altogether, Bb and Lf pro-/postbiotics exhibited remarkable immune regulatory effects on both moDCs and Th2 cells. Therefore, further in vivo studies should be considered to validate these findings and assess their ability to prevent allergy or alleviate its symptoms in affected patients.

Oral Administration of Probiotic Bifidobacterium breve Ameliorates Tonic-Clonic Seizure in a Pentylenetetrazole-Induced Kindling Mouse Model via Integrin-Linked Kinase Signaling.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures that affects over 70 million people worldwide. Although many antiepileptic drugs that block seizures are available, they have little effect on preventing and curing epilepsy, and their side effects sometimes lead to serious morbidity. Therefore, prophylactic agents with anticonvulsant properties and no adverse effects need to be identified. Recent studies on probiotic administration have reported a variety of beneficial effects on the central nervous system via the microbiota-gut-brain axis. In this study, we investigated the effects of the oral administration of Bifidobacterium breve strain A1 [MCC1274] (B. breve A1) on tonic-clonic seizure in a pentylenetetrazole (PTZ)-induced kindling mouse (KD mouse) model. We found that the oral administration of B. breve A1 every other day for 15 days significantly reduced the seizure score, which gradually increased with repetitive injections of PTZ in KD mice. The administration of B. breve A1, but not saline, to KD mice significantly increased the level of Akt Ser473 phosphorylation (p-Akt) in the hippocampus; this increase was maintained for a minimum of 24 h after PTZ administration. Treatment of B. breve A1-administered KD mice with the selective inhibitor of integrin-linked kinase (ILK) Cpd22 significantly increased the seizure score and blocked the antiepileptic effect of B. breve A1. Moreover, Cpd22 blocked the B. breve A1-induced increase in hippocampal p-Akt levels. These results suggest that the ILK-induced phosphorylation of Akt Ser473 in the hippocampus might be involved in the antiepileptic effect of B. breve A1.

Anti-Menopausal Effect of Heat-Killed Bifidobacterium breve HDB7040 via Estrogen Receptor-Selective Modulation in MCF-7 Cells and Ovariectomized Rats.

Menopause is induced by spontaneous ovarian failure and leads to life quality deterioration with various irritating symptoms. Hormonal treatment can alleviate these symptoms, but long-term treatment is closely associated with breast and uterine cancer, and stroke. Therefore, developing alternative therapies with novel anti-menopausal substances and improved safety is needed. In our study, heat-killed Bifidobacterium breve HDB7040 significantly promoted MCF-7 cell proliferation in a dose-dependent manner under estrogen-free conditions, similar to 17ß-estradiol. This strain also triggered ESR2 expression, but not ESR1, in MCF-7 cells. Moreover, administrating HDB7040 to ovariectomized (OVX) Sprague-Dawley (SD) female rats reduced estrogen deficiency-induced weight gain, fat mass, blood triglyceride, and total cholesterol levels. It also recovered collapsed trabecular microstructure by improving trabecular morphometric parameters (bone mineral density, bone volume per tissue volume, trabecular number, and trabecular separation) and decreasing blood alkaline phosphatase levels with no significant changes in uterine size and blood estradiol. HDB7040 also significantly regulated the expression of Tff1, Pgr, and Esr2, but not Esr1 in uteri of OVX rats. Heat-killed B. breve HDB7040 exerts an anti-menopausal effect via the specific regulation of ERß in vitro and in vivo, suggesting its potential as a novel substance for improving and treating menopausal syndrome.

Effectiveness of Psychobiotic Bifidobacterium breve BB05 in Managing Psychosomatic Diarrhea in College Students by Regulating Gut Microbiota: A Randomized, Double-Blind, Placebo-Controlled Trial.

Diarrhea of college students (DCS) is a prevalent issue among college students, affecting their daily lives and academic performance. This study aims to explore the potential effect of Bifidobacterium breve BB05 supplements on the DCS. Initially, fifty healthy and fifty diarrheal students were recruited in the observational experiment and allocated into control and diarrhea groups, respectively. Subsequently, one hundred diarrheal students were newly recruited in the intervention experiment and randomly allocated into placebo and probiotic groups, both treated for 2 weeks. Questionnaires (BSS, HAMA-14, and HDRS-17) were performed to assess the students' diarrheal states and mental health at baseline and post-treatment. Fecal samples underwent 16S rRNA sequencing and Enzyme-Linked Immunosorbent Assay to evaluate gut microbiota and fecal metabolite alternations. Results indicated that B. breve BB05 supplementation significantly enriched (p < 0.05) the reduced gut microbial diversity caused by diarrhea. Diarrhea resulted in notable alterations in gut microbiota composition, as exhibited by elevated Collinsella and Streptococcus, alongside substantially decreased Bifidobacterium, Bacteroides, and Prevotella, while B. breve BB05 supplementation partially restored the compromised gut microbiota at both the phylum and genus levels, particularly by increasing Bifidobacterium and Roseburia (p < 0.05). Importantly, questionnaire results suggested that B. breve BB05 administration achieved superior efficacy in relieving diarrhea symptoms and the associated anxiety and depression in college students. An increased fecal concentration of 5-hydroxytryptamine (5-HT) was also observed in the probiotic group, while Acetylcholine (ACH), Epinephrine (EPI), and Noradrenaline/Norepinephrine (NANE) reduced, revealing the potential of B. breve BB05 in alleviating anxiety and depression via modulating the microbiota-gut-brain axis. Furthermore, correlation analysis suggested that the altered microbiota and fecal neurotransmitters were closely associated with the mental symptoms. These results endorse B. breve BB05 intervention as a promising and innovative approach to alleviate both diarrhea and mental health conditions among college students.

Impact of Combined Prebiotic Galacto-Oligosaccharides and Bifidobacterium breve-Derived Postbiotic on Gut Microbiota and HbA1c in Prediabetic Adults: A Double-Blind, Randomized, Placebo-Controlled Study.

The complex interactions between intestinal microbiota and metabolic disorders are well-documented, with implications for glucose metabolism, energy expenditure, and intestinal permeability. Prebiotics induce beneficial changes in gut microbiota composition in prediabetes, while postbiotics can enhance gut barrier function, complementing each other to improve glucose metabolism and insulin sensitivity. This study investigated the effects of a 12-week dietary fibre (DF) supplement on gut health, metabolic function, and diet. The supplement contained konjac glucomannan (KGM), galacto-oligosaccharides (GOSs), and exopolysaccharides (EPSs) from Bifidobacterium breve. In a randomised, double-blind, placebo-controlled, parallel-group clinical trial, 53 prediabetic volunteers were randomly assigned to either a daily DF supplement (YMETA) or a placebo (cellulose microcrystalline) for 12 weeks, followed by a 4-week follow-up. Measurements included gut microbiota composition, glycated haemoglobin (HbA1c), fasting plasma glucose (FPG), plasma lipids, anthropometry, body composition, blood pressure, and dietary intake. The intervention group showed a significant increase in alpha diversity and butyrate-producing bacteria, with reductions in HbA1c and FPG levels below prediabetes thresholds. No significant changes were observed in the placebo group. This study suggests that manipulating the human gut microbiome through dietary interventions could be a promising therapeutic approach to managing prediabetes and preventing or delaying diabetes.

Early-Life Supplementation Enhances Gastrointestinal Immunity and Microbiota in Young Rats.

Immunonutrition, which focuses on specific nutrients in breast milk and post-weaning diets, plays a crucial role in supporting infants' immune system development. This study explored the impact of maternal supplementation with Bifidobacterium breve M-16V and a combination of short-chain galacto-oligosaccharide (scGOS) and long-chain fructo-oligosaccharide (lcFOS) from pregnancy through lactation, extending into the early childhood of the offspring. The synbiotic supplementation's effects were examined at both mucosal and systemic levels. While the supplementation did not influence their overall growth, water intake, or food consumption, a trophic effect was observed in the small intestine, enhancing its weight, length, width, and microscopic structures. A gene expression analysis indicated a reduction in FcRn and Blimp1 and an increase in Zo1 and Tlr9, suggesting enhanced maturation and barrier function. Intestinal immunoglobulin (Ig) A levels remained unaffected, while cecal IgA levels decreased. The synbiotic supplementation led to an increased abundance of total bacteria and Ig-coated bacteria in the cecum. The abundance of Bifidobacterium increased in both the intestine and cecum. Short-chain fatty acid production decreased in the intestine but increased in the cecum due to the synbiotic supplementation. Systemically, the Ig profiles remained unaffected. In conclusion, maternal synbiotic supplementation during gestation, lactation, and early life is established as a new strategy to improve the maturation and functionality of the gastrointestinal barrier. Additionally, it participates in the microbiota colonization of the gut, leading to a healthier composition.

Impact of maternal Bifidobacterium breve M-16V and scGOS/lcFOS supplementation during pregnancy and lactation on the maternal immune system and milk composition.

Introduction: Maternal synbiotic supplementation during pregnancy and lactation can significantly influence the immune system. Prebiotics and probiotics have a positive impact on the immune system by preventing or ameliorating among others intestinal disorders. This study focused on the immunomodulatory effects of B. breve M-16V and short chain galacto-oligosaccharides (scGOS)/long chain fructo-oligosachairdes (lcFOS), including systemic and mucosal compartments and milk composition. Methods: Lewis rats were orally administered with the synbiotic or vehicle during pregnancy (21 days) and lactation (21 days). At the weaning day, small intestine (SI), mammary gland (MG), adipose tissue, milk, mesenteric lymph nodes (MLN), salivary gland (SG), feces and cecal content were collected from the mothers. Results: The immunoglobulinome profile showed increased IgG2c in plasma and milk, as well as elevated sIgA in feces at weaning. The supplementation improved lipid metabolism through enhanced brown adipose tissue activity and reinforced the intestinal barrier by increasing the expression of Muc3, Cldn4, and Ocln. The higher production of short chain fatty acids in the cecum and increased Bifidobacterium counts suggest a potential positive impact on the gastrointestinal tract. Discussion: These findings indicate that maternal synbiotic supplementation during gestation and lactation improves their immunological status and improved milk composition.

Bifidobacterium breve-derived indole-3-lactic acid ameliorates colitis-associated tumorigenesis by directing the differentiation of immature colonic macrophages.

Aim: To elucidate dynamics and functions in colonic macrophage subsets, and their regulation by Bifidobacterium breve (B. breve) and its associated metabolites in the initiation of colitis-associated colorectal cancer (CAC). Methods: Azoxymethane (AOM) and dextran sodium sulfate (DSS) were used to create a CAC model. The tumor-suppressive effect of B. breve and variations of macrophage subsets were evaluated. Intestinal macrophages were ablated to determine their role in the protective effects of B. breve. Efficacious molecules produced by B. breve were identified by non-targeted and targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The molecular mechanism was further verified in murine bone marrow-derived macrophages (BMDMs), macrophages derived from human peripheral blood mononuclear cells (hPBMCs), and demonstrated in CAC mice. Results: B. breve alleviated colitis symptoms, delayed colonic tumorigenesis, and promoted phenotypic differentiation of immature inflammatory macrophages into mature homeostatic macrophages. On the contrary, the ablation of intestinal macrophages largely annulled the protective effects of B. breve. Microbial analysis of colonic contents revealed the enrichment of probiotics and the depletion of potential pathogens following B. breve supplementation. Moreover, indole-3-lactic acid (ILA) was positively correlated with B. breve in CAC mice and highly enriched in the culture supernatant of B. breve. Also, the addition of ILA directly promoted AKT phosphorylation and restricted the pro-inflammatory response of murine BMDMs and macrophages derived from hPBMCs in vitro. The effects of ILA in murine BMDMs and macrophages derived from hPBMCs were abolished by the aryl hydrocarbon receptor (AhR) antagonist CH-223191 or the AKT inhibitor MK-2206. Furthermore, ILA could protect against tumorigenesis by regulating macrophage differentiation in CAC mice; the AhR antagonist largely abrogated the effects of B. breve and ILA in relieving colitis and tumorigenesis. Conclusion: B. breve-mediated tryptophan metabolism ameliorates the precancerous inflammatory intestinal milieu to inhibit tumorigenesis by directing the differentiation of immature colonic macrophages.

Investigating the therapeutic potential of Bifidobacterium breve and Lactobacillus rhamnosus postbiotics through apoptosis induction in colorectal HT-29 cancer cells.

Background and Objectives: Colorectal cancer (CRC) is a prevalent form of cancer worldwide. Recent studies suggest that postbiotics derived from probiotic bacteria have the potential as an adjunct therapy for CRC. This study investigates the anti-cancer effects of Bifidobacterium breve (B. breve) and Lactobacillus rhamnosus (L. rhamnosus) postbiotics on the HT-29 cell line. Materials and Methods: Through MTT and scratch assay, we investigated the anti-proliferation and anti-migration effects of B. breve and L. rhamnosus postbiotics on HT-29 cells. Furthermore, postbiotic-mediated apoptosis was assessed by analyzing the expression of Bax, Bcl-2, and caspase-3. We also investigated the effects of B. breve postbiotics on the expression of three important genes involved in metastasis, including RSPO2, NGF, and MMP7. Consequently, we validated the expression of selected genes in twelve adenocarcinoma tissues. Results: The results demonstrated the significant impact of postbiotics on HT-29 cells, highlighting their ability to induce anti-proliferation, anti-migration, and apoptosis-related effects. Notably, these effects were more pronounced using B. breve postbiotics than L. rhamnosus. Additionally, B. breve postbiotics could inhibit metastasis through upregulation of RSPO2 while downregulating NGF and MMP7 expression in HT-29 cells. Conclusion: Our research suggests that postbiotic metabolites may be effective biological products for the prevention and treatment of cancer.

Cell-Free Supernatant of Lactobacillus rhamnosus and Bifidobacterium breve Ameliorates Ischemic Stroke-Generated Neurological Deficits in Rats.

The beneficial effects of probiotics, postbiotics, and paraprobiotics have already been registered in managing ischemic stroke-generated neuroinflammation and gut dysbiosis. Herein, we examined the impact of cell-free supernatant (CFS) obtained from probiotics (Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01) in a rat transient middle cerebral artery occlusion (MCAO) model of focal cerebral injury. Pre-MCAO supplementation of probiotics (2 × 109 CFU/mL) for 21 days or CFS (1 mL/rat) for 7 days protect the MCAO-induced somatosensory and motor impairments recorded at 24 h and 72 h after reperfusion in foot-fault, rotarod, adhesive removal, and vibrissae-evoked forelimb placing tests. We also noted the reduced infarct area and neuronal degradation in the right hemisphere of probiotics- and CFS-recipient MCAO-operated animals. Moreover, MCAO-induced altered concentrations of glial-fibrillary acidic protein, NeuN, zonula occludens-1 (ZO-1), TLR4, IL-1ß, IL-6, and TNF-α, as well as matrix metalloproteinase-9 (MMP9) were reversed in the treatment groups. Probiotics and CFS treatment ameliorated the elevated levels of IL-6, IL-1ß, and MMP9 in the blood plasma of rats. The disrupted microbial phyla, Firmicutes-to-Bacteroides ratio, villi/crypt ratio, and decreased mucin-producing goblet cells, ZO-1, and occludin in the colon of MCAO-operated rats were recovered following probiotics and CFS treatment. NMR characterization of CFS and rat blood plasma revealed the presence of several important bacterial metabolites. These findings suggest that the CFS obtained from Lactobacillus rhamnosus UBLR-58 and Bifidobacterium breve UBBr-01 has the propensity to improve MCAO-generated neurological dysfunctions in rats by dampening neuroinflammation and modulating the gut-brain axis modulators.

Evaluation of Safety and Beneficial Health Effects of the Human-Milk Strain Bifidobacterium breve DSM32583: An Infant Pilot Trial.

Human milk promotes the growth of bifidobacteria in the infant gut. Adding bifidobacterial species to infant formula may contribute to increasing their presence in the gut of formula-fed infants. Therefore, the safety and anti-infectious effects of Bifidobacterium breve DSM32583, a breast milk isolate, were assessed in a pilot trial involving 3-month-old infants. The infants were randomly assigned to either the probiotic (PG) or the control (CG) groups. All the infants consumed the same formula, although it was supplemented with the strain (1 × 107 cfu/g of formula) in the PG. Overall, 160 infants (80 per group) finished the intervention. Infants in CG gained more weight compared to PG (p < 0.05), but the weights for age Z-scores at 6 months were within the normal distribution for this age group. The rates of infections affecting the gastrointestinal and respiratory tracts and antibiotic therapy were significantly lower in the PG. The bifidobacterial population and the level of short-chain fatty acids were higher (p < 0.05) in the fecal samples of PG infants. No adverse events related to formula consumption were observed. In conclusion, the administration of an infant formula with B. breve DSM32583 was safe and exerted potential beneficial effects on gut health.

Genomic Characterization and Safety Assessment of Bifidobacterium breve BS2-PB3 as Functional Food.

Our group had isolated Bifidobacterium breve strain BS2-PB3 from human breast milk. In this study, we sequenced the whole genome of B. breve BS2-PB3, and with a focus on its safety profile, various probiotic characteristics (presence of antibiotic resistance genes, virulence factors, and mobile elements) were then determined through bioinformatic analyses. The antibiotic resistance profile of B. breve BS2-PB3 was also evaluated. The whole genome of B. breve BS2-PB3 consisted of 2,268,931 base pairs with a G-C content of 58.89% and 2,108 coding regions. The average nucleotide identity and whole-genome phylogenetic analyses supported the classification of B. breve BS2-PB3. According to our in silico assessment, B. breve BS2-PB3 possesses antioxidant and immunomodulation properties in addition to various genes related to the probiotic properties of heat, cold, and acid stress, bile tolerance, and adhesion. Antibiotic susceptibility was evaluated using the Kirby-Bauer disk-diffusion test, in which the minimum inhibitory concentrations for selected antibiotics were subsequently tested using the Epsilometer test. B. breve BS2-PB3 only exhibited selected resistance phenotypes, i.e., to mupirocin (minimum inhibitory concentration/MIC >1,024 µg/ml), sulfamethoxazole (MIC >1,024 µg/ml), and oxacillin (MIC >3 µg/ml). The resistance genes against those antibiotics, i.e., ileS, mupB, sul4, mecC and ramA, were detected within its genome as well. While no virulence factor was detected, four insertion sequences were identified within the genome but were located away from the identified antibiotic resistance genes. In conclusion, B. breve BS2-PB3 demonstrated a sufficient safety profile, making it a promising candidate for further development as a potential functional food.

Potential Therapeutic Effects of Bifidobacterium breve MCC1274 on Alzheimer's Disease Pathologies in AppNL-G-F Mice.

We previously demonstrated that orally supplemented Bifidobacterium breve MCC1274 (B. breve MCC1274) mitigated Alzheimer's disease (AD) pathologies in both 7-month-old AppNL-G-F mice and wild-type mice; thus, B. breve MCC1274 supplementation might potentially prevent the progression of AD. However, the possibility of using this probiotic as a treatment for AD remains unclear. Thus, we investigated the potential therapeutic effects of this probiotic on AD using 17-month-old AppNL-G-F mice with memory deficits and amyloid beta saturation in the brain. B. breve MCC1274 supplementation ameliorated memory impairment via an amyloid-cascade-independent pathway. It reduced hippocampal and cortical levels of phosphorylated extracellular signal-regulated kinase and c-Jun N-terminal kinase as well as heat shock protein 90, which might have suppressed tau hyperphosphorylation and chronic stress. Moreover, B. breve MCC1274 supplementation increased hippocampal synaptic protein levels and upregulated neuronal activity. Thus, B. breve MCC1274 supplementation may alleviate cognitive dysfunction by reducing chronic stress and tau hyperphosphorylation, thereby enhancing both synaptic density and neuronal activity in 17-month-old AppNL-G-F mice. Overall, this study suggests that B. breve MCC1274 has anti-AD effects and can be used as a potential treatment for AD.

Bifidobacterium breve Protects the Intestinal Epithelium and Mitigates Inflammation in Colitis via Regulating the Gut Microbiota-Cholic Acid Pathway.

In this study, we aimed to explore the protective effects of Bifidobacterium in colitis mice and the potential mechanisms. Results showed that Bifidobacterium breve (B. breve) effectively colonized the intestinal tract and alleviated colitis symptoms by reducing the disease activity index. Moreover, B. breve mitigated intestinal epithelial cell damage, inhibited the pro-inflammatory factors, and upregulated tight junction (TJ)-proteins. Gut microbiota and metabolome analysis found that B. breve boosted bile acid-regulating genera (such as Bifidobacterium and Clostridium sensu stricto 1), which promoted bile acid deconjugation in the intestine. Notably, cholic acid (CA) was closely associated with the expression levels of inflammatory factors and TJ-proteins (p < 0.05). Our in vitro cell experiments further confirmed that CA (20.24 ± 4.53 pg/mL) contributed to the inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression (49.32 ± 5.27 pg/mL) and enhanced the expression of TJ-proteins (Occludin and Claudin-1) and MUC2. This study suggested that B. breve could be a probiotic candidate for use in infant foods.

Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.

Psychobiotics Regulate Purine Metabolism to Influence Host Emotional Behavior.

Purine metabolism plays a pivotal role in numerous biological processes with potential implications for brain function and emotional regulation. This study utilizes gene-edited probiotics and pseudo-germ-free mice to unravel this intricate interplay. Transcriptomic analysis identified a ribonucleoside-diphosphate reductase ß chain (nrdB) as a pivotal gene in purine metabolism within Bifidobacterium breve CCFM1025. Comparative evaluation between the wild-type and nrdB mutant strains revealed CCFM1025's effective reduction of xanthine and xanthosine levels in the serum and brain of stressed mice. Concomitantly, it downregulated the expression of the adenosine receptor gene (Adora2b) and inhibited the overactivation of microglia. These findings emphasize the potential of psychobiotics in modulating emotional responses by regulating purine metabolites and adenosine receptors. This study sheds light on novel pathways that influence emotional well-being through gut microbiota interactions and purine metabolic processes.

Bifidobacterium breve Probiotic Compared to Lactobacillus casei Causes a Better Reduction in Demyelination and Oxidative Stress in Cuprizone-Induced Demyelination Model of Rat.

Despite the anatomical separation, strong evidence suggested a bidirectional association between gut microbiota and central nervous system. Cross-talk between gut microbiota and brain has an important role in the pathophysiology of neurodegenerative disorders and regenerative processes. However, choosing the appropriate probiotics and combination therapy of probiotics to provide a synergistic effect is very crucial. In the present study, we investigated the effect of Lactobacillus casei (L. casei) and Bifidobacterium breve (B. breve) on alternation performance, oxidant/antioxidant biomarkers, the extent of demyelination, and the expression level of HO-1, Nrf-2, Olig2, MBP, PDGFRα, and BDNF in cuprizone (CPZ)-induced demyelination model of rat corpus callosum. In order to induce this model, rats received oral administration of CPZ 0.6% w/w in corn oil for 28 days. Then, L. casei, B. breve, or their combinations were orally administrated for 28 days. Y maze test was performed to investigate the alternation performance. Oxidant/antioxidant biomarkers were determined by colorimetric methods. Extent of demyelination was investigated using FluoroMyelin staining. The genes' expression levels of antioxidant and myelin lineage cells were assessed by quantitative real time PCR. The results showed the probiotics supplementation significantly improve the alternation performance and antioxidant capacity in demyelinated corpus callosum. Interestingly, B. breve supplementation alleviated demyelination and oxidative stress levels more than the administration of L. casei alone or the combination of two probiotics. These observations suggest that B. breve could provide a supplementary strategy for the treatment of multiple sclerosis by increasing antioxidant capacity and remyelination.

Structural Characterization and Immunomodulatory Activity of a Mannose-Rich Polysaccharide Isolated from Bifidobacterium breve H4-2.

In this study, a novel homogeneous mannose-rich polysaccharide named EPS-1 from the fermentation broth of Bifidobacterium breve H4-2 was isolated and purified by anion exchange column chromatography and gel column chromatography. The primary structure of EPS-1 was analyzed by high-performance liquid chromatography, Fourier-transform infrared spectroscopy, gas chromatography-mass spectrometry, and nuclear magnetic resonance. The results indicated that EPS-1 had typical functional groups of polysaccharides. EPS-1 with an average molecular weight of 3.99 × 104 Da was mainly composed of mannose (89.65%) and glucose (5.84%). The backbone of EPS-1 was →2,6)-α-d-Manp-(1→2)-α-d-Manp-(1→2,6)-α-d-Manp-(1→2)-α-d-Manp-(1→2,6)-α-d-Manp-(1→6)-α-d-Glcp-(1→ simultaneously containing two kinds of branched chains (α-d-Manp-(1→3)-α-d-Manp-(1→ and α-d-Manp-(1→). Besides, EPS-1 had a triple-helical conformation and exhibited excellent thermal stability. Moreover, the immunomodulatory activity of EPS-1 was evaluated by RAW 264.7 cells. Results indicated that EPS-1 significantly enhanced the viability of RAW 264.7 cells. EPS-1 could also be recognized by toll-like receptor 4, thereby activating the nuclear factors-κB (NF-κB) signaling pathway, promoting phosphorylation of related nuclear transcription factors, improving cell phagocytic activity, and promoting the secretion of NO, IL-6, IL-1ß, and TNF-α. Thus, EPS-1 could activate the TLR4-NF-κB signaling pathway to emerge immunomodulatory activity on macrophages. The above results indicate that EPS-1 can serve as a potential immune-stimulating polysaccharide.