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In order to analyze the differences in functional genes of Bifidobacterium pseudocatenulatum. Comparative genomics analysis was performed on 104 strains of Bifidobacterium pseudocatenulatum isolated from China, Japan and America. The results showed that the number of coding sequences of Japanese isolates were significantly higher than Chinese and American isolates in genome level (P<0.01).Phylogenetic tree based on core gene and average nucleotide identity clustering heat map showed that strains isolated from the same country were more similar. The results of functional gene annotation further showed that the isolation could affect the functional genes distribution of strains. The annotation of carbohydrate active enzymes revealed that 15 carbohydrate active enzymes had regional differences. The number of antibiotic resistance genes and adhesion virulence factors carried by Chinese isolates were more abundant than Japanese isolates by the annotation of antibiotic resistance genes database and virulence factors database. The number of Replication\recombination\repair and phage associated mobile genetic elements carried by the Chinese and Japanese isolates were different from American isolates. In this study, it was found that the location of strains will affect the functional genes of Bifidobacterium pseudocatenulatum, and Bifidobacterium pseudocatenulatum in different locations have different functional genes. This study provides theoretical basis for the analysis of the functional differences of Bifidobacterium pseudocatenulatum. [ABSTRACT FROM AUTHOR]

BackgroundBifidobacteria represent an important gut commensal in humans, particularly during initial microbiome assembly in the first year of life. Enrichment of Bifidobacterium is mediated though the utilization of human milk oligosaccharides (HMOs), as several human-adapted species have dedicated genomic loci for transport and metabolism of these glycans. This results in the release of fermentation products into the gut lumen which may offer physiological benefits to the host. Synbiotic pairing of probiotic species with a cognate prebiotic delivers a competitive advantage, as the prebiotic provides a nutrient niche.MethodsTo determine the fitness advantage and metabolic characteristics of an HMO-catabolizing Bifidobacterium strain in the presence or absence of 2'-fucosyllactose (2'-FL), conventionally colonized mice were gavaged with either Bifidobacterium pseudocatenulatum MP80 (B.p. MP80) (as the probiotic) or saline during the first 3 days of the experiment and received water or water containing 2'-FL (as the prebiotic) throughout the study.Results16S rRNA gene sequencing revealed that mice provided only B.p. MP80 were observed to have a similar microbiota composition as control mice throughout the experiment with a consistently low proportion of Bifidobacteriaceae present. Using 1H NMR spectroscopy, similar metabolic profiles of gut luminal contents and serum were observed between the control and B.p. MP80 group. Conversely, synbiotic supplemented mice exhibited dramatic shifts in their community structure across time with an overall increased, yet variable, proportion of Bifidobacteriaceae following oral inoculation. Parsing the synbiotic group into high and moderate bifidobacterial persistence based on the median proportion of Bifidobacteriaceae, significant differences in gut microbial diversity and metabolite profiles were observed. Notably, metabolites associated with the fermentation of 2'-FL by bifidobacteria were significantly greater in mice with a high proportion of Bifidobacteriaceae in the gut suggesting metabolite production scales with population density. Moreover, 1,2-propanediol, a fucose fermentation product, was only observed in the liver and brain of mice harboring high proportions of Bifidobacteriaceae.ConclusionsThis study reinforces that the colonization of the gut with a commensal microorganism does not guarantee a specific functional output. Video Abstract.

IntroductionSeveral studies indicated that depression is associated with liver injury. The role of probiotics in alleviating depression is focused on improving the abnormalities of the central nervous system through the gut-brain axis, while the effect on liver injury is still unclear. The aim of this study was to elucidate the potential link between the antidepressant effect of a potential probiotic strain Bifidobacterium pseudocatenulatum W112 and its effect on alleviating liver injury.MethodsThe 4-week-old Kunming mice were exposed to chronic stress for 4 weeks to establish a depression model.ResultsThe depression-like behavior and related biomakers in chronic unpredictable mild stress (CUMS) mice were altered by supplemented with W112 for 2 weeks. Meanwhile, the modulation effect of W112 the gut microbiota in CUMS mice also result in an increase in the abundance of beneficial bacteria and a decrease in the abundance of harmful bacteria. Significantly, liver injury was observed in CUMS model mice. W112 improved liver injury by reducing AST/ALT in serum. Quantitative PCR results indicated that the mechanism of action of W112 in ameliorating liver injury was that the altered gut microbiota affected hepatic phospholipid metabolism and bile acid metabolism.DiscussionIn short, W112 could significantly improve the depressive and liver injury symptoms caused by CUMS. The gut-liver-brain axis is a potential connecting pathway between the antidepressant effects of W112 and its alleviation of liver injury.

Inflammatory bowel disease (IBD) is a chronic disease associated with overactive inflammation and gut dysbiosis. Owing to the beneficial effects of bifidobacteria on IBD treatment, this study aimed to investigate the anti-inflammation effects of an exopolysaccharide (EPS)-producing strain Bifidobacterium pseudocatenulatum Bi-OTA128 through a dextran sulfate sodium (DSS)-induced colitis mice model. B. pseudocatenulatum treatment improved DSS-induced colitis symptoms and maintained intestinal barrier integrity by up-regulating MUC2 and tight junctions' expression. The oxidative stress was reduced after B. pseudocatenulatum treatment by increasing the antioxidant enzymes of SOD, CAT, and GSH-Px in colon tissues. Moreover, the overactive inflammatory responses were also inhibited by decreasing the pro-inflammatory cytokines of TNF-α, IL-1β, and IL-6, but increasing the anti-inflammatory cytokine of IL-10. The EPS-producing strain Bi-OTA128 showed better effects than that of a non-EPS-producing stain BLYR01-7 in modulating DSS-induced gut dysbiosis. The Bi-OTA128 treatment increased the relative abundance of beneficial bacteria Bifidobacterium and decreased the maleficent bacteria Escherichia-Shigella, Enterorhabuds, Enterobacter, and Osillibacter associated with intestinal inflammation. Notably, the genera Clostridium sensu stricto were only enriched in Bi-OTA128-treated mice, which could degrade polysaccharides to produce acetic acid and butyrate in the gut. This finding demonstrated a cross-feeding effect induced by the EPS-producing strain in gut microbiota. Collectively, these results highlighted the anti-inflammatory effects of the EPS-producing strain B. pseudocatenulatum Bi-OTA128 on DSS-induced colitis, which could be used as a candidate probiotic supporting recovery from ongoing colitis. [ABSTRACT FROM AUTHOR]

Human milk enriches members of the genus Bifidobacterium in the infant gut. One species, Bifidobacterium pseudocatenulatum, is found in the gastrointestinal tracts of adults and breastfed infants. In this study, B. pseudocatenulatum strains were isolated and characterized to identify genetic adaptations to the breastfed infant gut. During growth on pooled human milk oligosaccharides (HMOs), we observed two distinct groups of B. pseudocatenulatum, isolates that readily consumed HMOs and those that did not, a difference driven by variable catabolism of fucosylated HMOs. A conserved gene cluster for fucosylated HMO utilization was identified in several sequenced B. pseudocatenulatum strains. One isolate, B. pseudocatenulatum MP80, which uniquely possessed GH95 and GH29 α-fucosidases, consumed the majority of fucosylated HMOs tested. Furthermore, B. pseudocatenulatum SC585, which possesses only a single GH95 α-fucosidase, lacked the ability to consume the complete repertoire of linkages within the fucosylated HMO pool. Analysis of the purified GH29 and GH95 fucosidase activities directly on HMOs revealed complementing enzyme specificities with the GH95 enzyme preferring 1-2 fucosyl linkages and the GH29 enzyme favoring 1-3 and 1-4 linkages. The HMO-binding specificities of the family 1 solute-binding protein component linked to the fucosylated HMO gene cluster in both SC585 and MP80 are similar, suggesting differential transport of fucosylated HMO is not a driving factor in each strain's distinct HMO consumption pattern. Taken together, these data indicate the presence or absence of specific α-fucosidases directs the strain-specific fucosylated HMO utilization pattern among bifidobacteria and likely influences competitive behavior for HMO foraging in situ. IMPORTANCE Often isolated from the human gut, microbes from the bacterial family Bifidobacteriaceae commonly possess genes enabling carbohydrate utilization. Isolates from breastfed infants often grow on and possess genes for the catabolism of human milk oligosaccharides (HMOs), glycans found in human breast milk. However, catabolism of structurally diverse HMOs differs between bifidobacterial strains. This study identifies key gene differences between Bifidobacterium pseudocatenulatum isolates that may impact whether a microbe successfully colonizes an infant gut. In this case, the presence of complementary α-fucosidases may provide an advantage to microbes seeking residence in the infant gut. Such knowledge furthers our understanding of how diet drives bacterial colonization of the infant gut.

Bacterial biofilm is an emerging form of life that involves cell populations living embedded in a self-produced matrix of extracellular polymeric substances (EPS). Currently, little is known about the molecular mechanisms of Bifidobacterium biofilm formation. We used the Bifidobacterium biofilm fermentation system to preparation of biofilms on wheat fibers, and multi-omics analysis of both B. pseudocatenulatum biofilms and planktonic cells were performed to identify genes and metabolites involved in biofilm formation. The average diameter of wheat fibers was around 50 μm, while the diameter of particle in wheat fibers culture of B. pseudocatenulatum was over 260 μm at 22 h with 78.96% biofilm formation rate (BR), and the field emission scanning electron microscopy (FESEM) results showed that biofilm cells on the surface of wheat fibers secreted EPS. Transcriptomic analysis indicated that genes associated with stress response (groS, mntH, nth, pdtaR, pstA, pstC, radA, rbpA, whiB, ybjG), quorum sensing (dppC, livM, luxS, sapF), polysaccharide metabolic process (rfbX, galE, zwf, opcA, glgC, glgP, gtfA) may be involved in biofilm formation. In addition, 17 weighted gene coexpression network analysis (WGCNA) modules were identified and two of them positively correlated to BR. Metabolomic analysis indicated that amino acids and amides; organic acids, alcohols and esters; and sugar (trehalose-6-phosphate, uridine diphosphategalactose, uridine diphosphate-N-acetylglucosamine) were main metabolites during biofilm formation. These results indicate that stress response, quorum sensing (QS), and EPS production are essential during B. pseudocatenulatum biofilm formation. [ABSTRACT FROM AUTHOR]

The developing infant gut microbiome is highly sensitive to environmental exposures, enabling its evolution into an organ that supports the immune system, confers protection from infection, and facilitates optimal gut and central nervous system function. In this study, we focus on the impact of maternal psychosocial stress on the infant gut microbiome. Forty-seven mother–infant dyads were recruited at the HEAL Africa Hospital in Goma, Democratic Republic of Congo. Extensive medical, demographic, and psychosocial stress data were collected at birth, and infant stool samples were collected at six weeks, three months, and six months. A composite maternal psychosocial stress score was created, based on eight questionnaires to capture a diverse range of stress exposures. Full-length 16S rRNA gene sequences were generated. Infants of mothers with high composite stress scores showed lower levels of gut microbiome beta diversity at six weeks and three months, as well as higher levels of alpha diversity at six months compared to infants of low stress mothers. Longitudinal analyses showed that infants of high stress mothers had lower levels of health-promoting Lactobacillus gasseri and Bifidobacterium pseudocatenulatum at six weeks compared to infants of low stress mothers, but the differences largely disappeared by three to six months. Previous research has shown that L. gasseri can be used as a probiotic to reduce inflammation, stress, and fatigue, as well as to improve mental state, while B. pseudocatenulatum is important in modulating the gut–brain axis in early development and in preventing mood disorders. Our finding of reduced levels of these health-promoting bacteria in infants of high stress mothers suggests that the infant gut microbiome may help mediate the effect of maternal stress on infant health and development. [ABSTRACT FROM AUTHOR]

Early intervention in rheumatoid arthritis (RA) is critical for optimal treatment, but initiation of pharmacotherapy to prevent damage remains unsatisfactory currently. Manipulation of the gut microbiome and microbial metabolites can be effective in protecting against RA. Thus, probiotics can be utilized to explore new strategies for preventing joint damage. The aim of this study was to explore the metabolites and mechanisms by which Bifidobacterium pseudocatenulatum affects RA. Based on 16S rRNA sequencing and UPLC-MS/MS assays, we focused on bile acid (BA) metabolism. In a collagen-induced arthritis (CIA) mouse model, B. pseudocatenulatum prevented joint damage by protecting the intestinal barrier and reshaped gut microbial composition, thereby elevating bile salt hydrolase (BSH) enzyme activity and increasing the levels of unconjugated secondary BAs to suppress aberrant T-helper 1/17-type immune responses; however, these benefits were eliminated by the Takeda G protein-coupled receptor 5 (TGR5) antagonist SBI-115. The results suggested that a single bacterium, B. pseudocatenulatum, can prevent RA, indicating that prophylactic administration of probiotics may be an effective therapy. [ABSTRACT FROM AUTHOR]

[Display omitted] • High genetic diversity and regional clustering in Bifidobacterium pseudocatenulatum. • Significant differences in genome characteristics and phylogeny. • Regional variation in plant-derived carbohydrate metabolism genes (GH13, GH43, GH5). • Antibiotic resistance genes (tet O, tet W) transmitted through genomic islands. • Gene specialization drives adaptive evolution, likely shaped by diet and lifestyle. Bifidobacterium pseudocatenulatum is a prevalent gut microbe in humans of all ages and plays a crucial role in host health. However, its adaptive evolutionary characteristics remain poorly understood. This study analyzed the genome of 247 B. pseudocatenulatum isolates from Chinese, Vietnamese, Japanese and other region populations using population genomics and functional genomics. Our findings revealed high genetic heterogeneity and regional clustering within B. pseudocatenulatum isolates. Significant differences were observed in genome characteristics, phylogeny, and functional genes. Specifically, Chinese and Vietnamese isolates exhibited a higher abundance of genes involved in the metabolism of plant-derived carbohydrates (GH13, GH43, and GH5 enzyme families), aligning with the predominantly vegetable-, wheat- and fruit-based diets of these populations. Additionally, we found widespread transmission of antibiotic resistance genes (tet O and tet W) through mobile genetic elements, such as genomic islands (GIs), resulting in substantial intra-regional differences. Our findings highlight distinct adaptive evolution in B. pseudocatenulatum driven by gene specialization, possibly in response to regional variations in diet and lifestyle. This study sheds light on bifidobacteria colonization mechanisms in the host gut. Gut microbiota, as a key link in the gut-brain axis, helps to maintain the health of the organism, among which, Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) is an important constituent member of the gut microbiota, which plays an important role in maintaining the balance of gut microbiota. The probiotic properties of B. pseudocatenulatum have been widely elaborated, and in order to excavate its evolutionary features at the genomic level, here we focused on the genetic background and evolutionary mechanism of the B. pseudocatenulatum genomes isolated from the intestinal tracts of different populations. Ultimately, based on the phylogenetic tree, we found that B. pseudocatenulatum has high genetic diversity and regional clustering phenomenon, in which plant-derived carbohydrate metabolism genes (GH13, GH43, GH5) showed significant regional differences, and this genetic differentiation drove the adaptive evolution, which likely shaped by diet and lifestyle. [ABSTRACT FROM AUTHOR]

Human longevity has a strong familial and genetic component. Dynamic characteristics of the gut microbiome during aging associated with longevity, neural, and immune function remained unknown. Here, we aim to reveal the synergistic changes in gut microbiome associated with decline in neural and immune system with aging and further obtain insights into the establishment of microbiome homeostasis that can benefit human longevity. Based on 16S rRNA and metagenomics sequencing data for 32 longevity families including three generations, centenarians, elderly, and young groups, we found centenarians showed increased diversity of gut microbiota, severely damaged connection among bacteria, depleted in microbial-associated essential amino acid function, and increased abundance of anti-inflammatory bacteria in comparison to young and elderly groups. Some potential probiotic species, such as Desulfovibrio piger, Gordonibacter pamelaeae, Odoribacter splanchnicus, and Ruminococcaceae bacterium D5 were enriched with aging, which might possibly support health maintenance. The level of Amyloid-β (Aβ) and brain-derived neurotrophic factor (BDNF) related to neural function showed increased and decreased with aging, respectively. The elevated level of inflammatory factors was observed in centenarians compared with young and elderly groups. The enriched Bacteroides fragilis in centenarians might promote longevity through up-regulating anti-inflammatory factor IL-10 expression to mediate the critical balance between health and disease. Impressively, the associated analysis for gut microbiota with the level of Aβ, BDNF, and inflammatory factors suggests Bifidobacterium pseudocatenulatum could be a particularly beneficial bacteria in the improvement of impaired neural and immune function. Our results provide a rationale for targeting the gut microbiome in future clinical applications of aging-related diseases and extending life span.Abbreviations: 16S rRNA: 16S ribosomal RNA; MAGs: Metagenome-assembled genomes; ASVs: Amplicon sequence variants; DNA: Deoxyribonucleic acid; FDR: False discovery rate: KEGG: Kyoto Encyclopedia of Genes and Genomes; PCoA: Principal coordinates analysis; PCR: Polymerase chain reaction; PICRUSt: Phylogenetic Investigation of Communities by Reconstruction of Unobserved States; Aβ: Amyloid-β (Aβ); BDNF: Brain-derived neurotrophic factor

Bifidobacteria have been investigated due to their mutualistic microbe–host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of Bifidobacterium pseudocatenulatum JCLA3. By multilocus analysis, the species of B. pseudocatenulatum JCLA3 was established as pseudocatenulatum. It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The ack gene was found as part of an operon formed by xfp and pta genes. Two genes of ldh were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were demonstrated by B. pseudocatenulatum JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5–5. The cbh gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. B. pseudocatenulatum JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of B. pseudocatenulatum JCLA3. [ABSTRACT FROM AUTHOR]

Blautia is a genus of anaerobic bacteria that is widely distributed in the mammalian gut. Recently, an increasing body of research has demonstrated a link between this genus and human health, suggesting applications as a novel probiotic strain. Moreover, we have previously shown that 2′-fucosyllactose (2′-FL), a major component of human milk oligosaccharides, increases the relative abundance of Blautia sp., particularly Blautia wexlerae , in the cultured fecal microbiota of healthy adults using a pH-controlled single-batch fermenter. However, the effects of 2′-FL on Blautia proliferation vary among individuals. In this study, we assessed the impact of the intrinsic gut microbiota on the prebiotic effects of 2′-FL. Metagenomic analysis of feces collected from all donors showed that the homolog of the intracellular GH95 α-l-fucosidase gene was considerably enriched in two non-responders (individuals who showed no increase in Blautia proliferation), whereas the homologous genes encoding extracellular α-l-fucosidase were more abundant in responders, suggesting that lactose and fucose released into the environment could be substrates mediating the growth of Blautia. In vitro assays confirmed the ability of B. wexlerae to utilize the two carbohydrates but not 2′-FL. We also observed that B. wexlerae utilized fucose released from 2′-FL by Bifidobacterium bifidum , which possessed extracellular GH95 α-l-fucosidase, in co-cultures of these two organisms. Finally, increasing the proportion of extracellular GH95 by the addition of a B. bifidum strain led to Blautia proliferation by 2′-FL in fecal cultures of the two non-responders. These findings provided valuable perspectives on individualized nutritional approaches to properly control the gut microbiota. Future clinical trials are needed to obtain further insights into the characteristics of responders vs. non-responders. [ABSTRACT FROM AUTHOR]

Bifidobacterium pseudocatenulatum grows well in the early stages of cultivation in medium containing sucrose (Suc), whereas its growth in medium containing the analogue disaccharide N -acetylsucrosamine (SucNAc) tends to exhibit a considerable delay. To elucidate the cause of this phenomenon, we investigated the proliferation pattern of B. pseudocatenulatum in medium containing D-glucose (Glc) and SucNAc and identified the enzyme that degrades this disaccharide. We found that B. pseudocatenulatum initially proliferates by assimilating Glc, with subsequent growth based on SucNAc assimilation depending on production of the β-fructofuranosidase, which can hydrolyze SucNAc, after Glc is completely consumed. Thus, B. pseudocatenulatum exhibited a diauxic growth pattern in medium containing Glc and SucNAc. In contrast, when cultured in medium containing Glc and Suc, B. pseudocatenulatum initially grew by degrading Suc via the phosphorolysis activity of Suc phosphorylase, which did not react to SucNAc. These observations indicate that B. pseudocatenulatum proliferates by assimilating Suc and SucNAc via different pathways. The β-fructofuranosidase of B. pseudocatenulatum exhibited higher hydrolytic activity against several naturally occurring Suc-based tri- or tetrasaccharides than against Suc, suggesting that this enzyme actively catabolizes oligosaccharides other than Suc. [ABSTRACT FROM AUTHOR]

Eighty-eight Bifidobacterium pseudocatenulatum strains, which were isolated from human, chicken and cow fecal samples from different niches of China, were compared genomically in this study to evaluate their diversity. It was found that B. pseudocatenulatum displayed a closed pan-genome, including abundant glycoside hydrolase families of the carbohydrate active enzyme (CAZy). A total of 30 kinds of glycoside hydrolases (GHs), 14 kinds of glycosyl transferases (GTs), 13 kinds of carbohydrate-binding modules (CBMs), 6 kinds of carbohydrate-esterases (CEs), and 2 kinds of auxiliary activities (AAs) gene families were identified across the genomes of the 88 B. pseudocatenulatum strains. Specifically, this showed that significant differences were also present in the number of 10 carbohydrate-active enzyme gene families (GT51, GH13_32, GH26, GH42, GH121, GH3, AA3, CBM46, CE2, and CE6) among the strains derived from the hosts of different age groups, particularly between strains from infants and those from other human age groups. Twelve different individuals of B. pseudocatenulatum from four main clusters were selected for further study to reveal the genetic diversity of carbohydrate metabolism-related genes within the same phylogenetics. The animal experiment showed that 3 weeks of oral administration and 1 week after cessation of administration of these strains did not markedly alter the serum routine inflammatory indicators in mice. Furthermore, the administration of these strains did not significantly cause adverse changes in the gut microbiota, as indicated by the α- and β-diversity indexes, relative to the control group (normal diet). Beyond that, FAHBZ9L5 significantly increased the abundance of B. pseudocatenulatum after 3 weeks and significantly increased the abundance of acetic acid and butyric acid in the host's intestinal tract 3 and 4 weeks after the first administration, respectively, compared with the control group. Corresponding to this, comparative genomic analyses of 12 B. pseudocatenulatum suggest that FAHBZ9L5-specific genes were rich in ABC transporters and carbohydrate esterase. Combining the results of comparative genomics analyses and animal experiment, it is suggested that the strains containing certain gene clusters contribute to another competitive growth advantage of B. pseudocatenulatum, which facilitates its intestinal carbohydrate metabolism in a host. [ABSTRACT FROM AUTHOR]

Microbial exopolysaccharides (EPSs) have attracted extensive attention for their biological functions in antioxidant activities. In this study, we characterized a novel EPS produced by Bifidobacterium pseudocatenulatum Bi-OTA128 which exhibited the highest antioxidant capacity compared to nine other ropy bacterial strains, achieving 76.50 % and 93.84 % in DPPH· and ABTS·+ scavenging activity, and ferric reducing power of 134.34 μM Fe2+. Complete genomic analysis identified an eps gene cluster involved in the EPS biosynthesis of Bi-OTA128 strain, which might be responsible for its ropy phenotype. The EPS was then isolated and purified by a DEAE-Sepharose Fast Flow column. A single elution part EPS 128 was obtained with a recovery rate of 43.5 ± 1.78 % and a total carbohydrate content of 93.6 ± 0.76 %. Structural characterization showed that EPS 128 comprised glucose, galactose, and rhamnose (molar ratio 4.0:1.2:1.1), featuring a putative complex backbone structure with four branched chains and an unusual acetyl group at O -2 of terminal rhamnose. Antioxidant assay in vitro indicated that EPS 128 exhibited antioxidant potential with 50.52 % DPPH· and 65.40 % ABTS·+ scavenging activities, reaching 54.3 % and 70.44 % of the efficacy of standard Vitamin C at 2.0 mg/L. Furthermore, EPS 128 showed protective effects against H 2 O 2 -induced oxidative stress in HepG2 cells by reducing cellular reactive oxygen species (ROS) and increasing cell viability. These findings present the first comprehensive report of an antioxidant EPS from B. pseudocatenulatum , highlighting its potential as a natural antioxidant for applications in the food industry and clinical settings. [Display omitted] • Bifidobacterium pseudocatenulatum Bi-OTA128 exhibited high antioxidant activity in vitro. • A novel eps gene cluster involved in EPS biosynthesis was found in Bi-OTA128. • A single EPS component, namely EPS 128 , was purified from Bi-OTA128 strain. • EPS 128 showed a novel structure as a high-molecular-mass bifidobacterial EPS. • EPS 128 showed hepatoprotective effects on H 2 O 2 -induced oxidative stress in HepG2 cells. [ABSTRACT FROM AUTHOR]

Blautia is a genus of anaerobic bacteria that is widely distributed in the mammalian gut. Recently, an increasing body of research has demonstrated a link between this genus and human health, suggesting applications as a novel probiotic strain. Moreover, we have previously shown that 2′-fucosyllactose (2′-FL), a major component of human milk oligosaccharides, increases the relative abundance of Blautia sp., particularly Blautia wexlerae, in the cultured fecal microbiota of healthy adults using a pH-controlled single-batch fermenter. However, the effects of 2′-FL on Blautia proliferation vary among individuals. In this study, we assessed the impact of the intrinsic gut microbiota on the prebiotic effects of 2′-FL. Metagenomic analysis of feces collected from all donors showed that the homolog of the intracellular GH95 α-l-fucosidase gene was considerably enriched in two non-responders (individuals who showed no increase in Blautia proliferation), whereas the homologous genes encoding extracellular α-l-fucosidase were more abundant in responders, suggesting that lactose and fucose released into the environment could be substrates mediating the growth of Blautia. In vitro assays confirmed the ability of B. wexlerae to utilize the two carbohydrates but not 2′-FL. We also observed that B. wexlerae utilized fucose released from 2′-FL by Bifidobacterium bifidum, which possessed extracellular GH95 α-l-fucosidase, in co-cultures of these two organisms. Finally, increasing the proportion of extracellular GH95 by the addition of a B. bifidum strain led to Blautia proliferation by 2′-FL in fecal cultures of the two non-responders. These findings provided valuable perspectives on individualized nutritional approaches to properly control the gut microbiota. Future clinical trials are needed to obtain further insights into the characteristics of responders vs. non-responders.

The developing infant gut microbiome is highly sensitive to environmental exposures, enabling its evolution into an organ that supports the immune system, confers protection from infection, and facilitates optimal gut and central nervous system function. In this study, we focus on the impact of maternal psychosocial stress on the infant gut microbiome. Forty-seven mother–infant dyads were recruited at the HEAL Africa Hospital in Goma, Democratic Republic of Congo. Extensive medical, demographic, and psychosocial stress data were collected at birth, and infant stool samples were collected at six weeks, three months, and six months. A composite maternal psychosocial stress score was created, based on eight questionnaires to capture a diverse range of stress exposures. Full-length 16S rRNA gene sequences were generated. Infants of mothers with high composite stress scores showed lower levels of gut microbiome beta diversity at six weeks and three months, as well as higher levels of alpha diversity at six months compared to infants of low stress mothers. Longitudinal analyses showed that infants of high stress mothers had lower levels of health-promoting Lactobacillus gasseri and Bifidobacterium pseudocatenulatum at six weeks compared to infants of low stress mothers, but the differences largely disappeared by three to six months. Previous research has shown that L. gasseri can be used as a probiotic to reduce inflammation, stress, and fatigue, as well as to improve mental state, while B. pseudocatenulatum is important in modulating the gut–brain axis in early development and in preventing mood disorders. Our finding of reduced levels of these health-promoting bacteria in infants of high stress mothers suggests that the infant gut microbiome may help mediate the effect of maternal stress on infant health and development.

Introduction: Several studies indicated that depression is associated with liver injury. The role of probiotics in alleviating depression is focused on improving the abnormalities of the central nervous system through the gut-brain axis, while the effect on liver injury is still unclear. The aim of this study was to elucidate the potential link between the antidepressant effect of a potential probiotic strain Bifidobacterium pseudocatenulatum W112 and its effect on alleviating liver injury., Methods: The 4-week-old Kunming mice were exposed to chronic stress for 4 weeks to establish a depression model., Results: The depression-like behavior and related biomakers in chronic unpredictable mild stress (CUMS) mice were altered by supplemented with W112 for 2 weeks. Meanwhile, the modulation effect of W112 the gut microbiota in CUMS mice also result in an increase in the abundance of beneficial bacteria and a decrease in the abundance of harmful bacteria. Significantly, liver injury was observed in CUMS model mice. W112 improved liver injury by reducing AST/ALT in serum. Quantitative PCR results indicated that the mechanism of action of W112 in ameliorating liver injury was that the altered gut microbiota affected hepatic phospholipid metabolism and bile acid metabolism., Discussion: In short, W112 could significantly improve the depressive and liver injury symptoms caused by CUMS. The gut-liver-brain axis is a potential connecting pathway between the antidepressant effects of W112 and its alleviation of liver injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhao, Yuan, Zhang, Deng, Pan, Bai, Jia, Wang and Geng.)

ABSTRACT Bifidobacterium pseudocatenulatum is a member of the human gut microbiota, and specific variants of B. pseudocatenulatum have been associated with health benefits such as improving gut integrity and reducing inflammatory responses. Here, we aimed to assess the genomic diversity and predicted metabolic profiles of B. pseudocatenulatum cells found colonizing the gut of healthy Vietnamese adults and children. We found that the population of B. pseudocatenulatum from each individual was distinct and highly diverse, with intraclonal variation attributed largely to a gain or loss of carbohydrate-utilizing enzymes. The B. pseudocatenulatum genomes were enriched with glycosyl hydrolases predicted to target plant-based nondigestible carbohydrates (GH13, GH43) but not host-derived glycans. Notably, the exopolysaccharide biosynthesis region from organisms isolated from healthy children showed extensive genetic diversity and was subject to a high degree of genetic modification. Antimicrobial susceptibility profiling revealed that the Vietnamese B. pseudocatenulatum cells were uniformly susceptible to beta-lactams but exhibited variable resistance to azithromycin, tetracycline, ciprofloxacin, and metronidazole. The genomic presence of ermX and tet variants conferred resistance against azithromycin and tetracycline, respectively; ciprofloxacin resistance was associated with a mutation(s) in the quinolone resistance-determining region (GyrA, S115, and/or D119). Our work provides the first detailed genomic and antimicrobial resistance characterization of B. pseudocatenulatum found in the Vietnamese population, which can be exploited for the rational design of probiotics. IMPORTANCE Bifidobacterium pseudocatenulatum is a beneficial member of the human gut microbiota. The organism can modulate inflammation and has probiotic potential, but its characteristics are largely strain dependent and associated with distinct genomic and biochemical features. Population-specific beneficial microbes represent a promising avenue for the development of potential probiotics, as they may exhibit a more suitable profile in the target population. This study investigates the underexplored diversity of B. pseudocatenulatum in Vietnam and provides more understanding of its genomic diversity, metabolic potential, and antimicrobial susceptibility. Such data from indigenous populations are essential for selecting probiotic candidates that can be accelerated into further preclinical and clinical investigations.

Bifidobacteria have been investigated due to their mutualistic microbe–host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of Bifidobacterium pseudocatenulatum JCLA3. By multilocus analysis, the species of B. pseudocatenulatum JCLA3 was established as pseudocatenulatum. It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The ack gene was found as part of an operon formed by xfp and pta genes. Two genes of ldh were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were demonstrated by B. pseudocatenulatum JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5–5. The cbh gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. B. pseudocatenulatum JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of B. pseudocatenulatum JCLA3.

The genus Bifidobacterium has been widely used in functional foods for health promotion due to its beneficial effects on human health, especially in the gastrointestinal tract (GIT). In this study, we characterize the anti-inflammatory potential of the probiotic strain Bifidobacterium pseudocatenulatum G7, isolated from a healthy male adult. G7 secretion inhibited inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Moreover, oral administration of bacteria G7 alleviated the severity of colonic inflammation in dextran sulfate sodium (DSS)-treated colitis mice, which was evidenced by a decreased disease activity index (DAI) and enhanced structural integrity of the colon. The 16S rRNA gene sequencing result illustrated that the G7 alleviated DSS-induced gut microbiota dysbiosis, accompanied by the modulated bile acids and short-chain fatty acid (SCFA) levels. Overall, our results demonstrated the potential anti-inflammatory effects of Bifidobacterium pseudocatenulatum G7 on both in vitro and in vivo models, which provided a solid foundation for further development of a novel anti-inflammatory probiotic.

Diverse CRISPR-Cas systems constitute an indispensable part of the bacterial adaptive immune system against viral infections. However, to escape from this immune system, bacteriophages have also evolved corresponding anti-defense measures. We investigated the diversity of CRISPR-Cas systems and the presence of prophages in the genomes of 66 Bifidobacterium pseudocatenulatum strains. Our findings revealed a high occurrence of complete CRISPR-Cas systems (62%, 41/66) in the B. pseudocatenulatum genomes. Subtypes I-C, I-U and II-A, were found to be widespread in this species. No significant association was found between the number of bacterial CRISPR spacers and its host’s age. This study on prophages within B. pseudocatenulatum genomes revealed that prophage genes related to distinct functional modules became degraded at different levels, indicating that these prophages were not likely to enter lytic cycle spontaneously. Further, the evolutionary analysis of prophages in this study revealed that they might be derived from different phage ancestors. Notably, self-targeting phenomenon within B. pseudocatenulatum and Anti-CRISPR (Acr) coding genes in prophages was observed. Overall, our results indicate that the competition between B. pseudocatenulatum and phages is a major driving factor for the genomic diversity of both partners.

Diverse CRISPR-Cas systems constitute an indispensable part of the bacterial adaptive immune system against viral infections. However, to escape from this immune system, bacteriophages have also evolved corresponding anti-defense measures. We investigated the diversity of CRISPR-Cas systems and the presence of prophages in the genomes of 66 Bifidobacterium pseudocatenulatum strains. Our findings revealed a high occurrence of complete CRISPR-Cas systems (62%, 41/66) in the B. pseudocatenulatum genomes. Subtypes I-C, I-U and II-A, were found to be widespread in this species. No significant association was found between the number of bacterial CRISPR spacers and its host's age. This study on prophages within B. pseudocatenulatum genomes revealed that prophage genes related to distinct functional modules became degraded at different levels, indicating that these prophages were not likely to enter lytic cycle spontaneously. Further, the evolutionary analysis of prophages in this study revealed that they might be derived from different phage ancestors. Notably, self-targeting phenomenon within B. pseudocatenulatum and Anti-CRISPR (Acr) coding genes in prophages was observed. Overall, our results indicate that the competition between B. pseudocatenulatum and phages is a major driving factor for the genomic diversity of both partners. [ABSTRACT FROM AUTHOR]

The interaction between wheat germ polysaccharide (WGP) and gut microbiota remains relatively less investigated. Thus, this study explored their interaction via in vitro batch fecal fermentation. WGP elevated dramatically the relative abundances of Bacteroides (especially Ba. xylanisolvens, Ba. uniformis, and Ba. intestinalis), Bifidobacterium (especially Bi. pseudocatenulatum) and Eubacterium, and decreased Alistipes, Klebsiella, Bilophila and Sutterella. Moreover, the metabolomics and Spearman correlation results showed that these alterations in gut microbiota gave rise to over 13-fold augmentation in the quantities of short-chain fatty acids (SCFAs) and indole-3-lactic acid, as well as 7.17- and 4.23-fold increase in acetylcholine and GABA, respectively, at 24 h of fermentation. Interestingly, PICRUSt analysis showed that WGP markedly reduced aging pathway, and enriched nervous system pathway. Therefore, the D-gal-induced aging mice model was used to further verify these effects. The results demonstrated that WGP had a protective effect on D-gal-induced behavioral deficits, particularly in locomotor activity, and spatial and recognition memory. WGP elevated dramatically the relative abundances of Bacteroides (especially Ba. sartorii and Ba. uniformis), Bifidobacterium (especially Bi. pseudocatenulatum) and Parabacteroides, and decreased Alistipes and Candidatus Arthromitus. These findings highlight the potential utility of WGP as a dietary supplement for retarding the aging process and mitigating age-associated learning and memory decline via the targeted enrichment of Bacteroides and Bifidobacterium and the related metabolites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Purpose: The relationships between gut microbiota and obesity-related co-morbidities have been increasingly recognized. Low-grade inflammation may be the main factor in the pathogenesis of such disorders. We investigated the effect of the potential probiotic Bifidobacterium pseudocatenulatum CECT 7765 on cardiometabolic risk factors, inflammatory cytokines and gut microbiota composition in obese children with insulin resistance. Methods: The study included 48 obese children (10–15 years old) with insulin resistance. They received dietary advice and were assigned to take the capsules with or without probiotic (109−10 CFU) daily for 13 weeks. Clinical, biochemical and gut microbiome measurements were made at baseline and at the end of the intervention. Results: There was a significant improvement in body mass index in all children after the intervention, suggesting that weight changes are related to the dietary advice. A significant decrease in circulating high-sensitive C-reactive protein (P = 0.026) and monocyte chemoattractant protein-1 (P = 0.032) and an increase in high-density lipoprotein cholesterol (P = 0.035) and omentin-1 (P = 0.023) in children receiving probiotic supplementation were observed compared to the control group. Regarding gut microbiota, probiotic administration significantly increased the proportion of the Rikenellaceae family members, particularly of the Alistipes genus. Conclusions: The beneficial effects of the intervention on inflammatory markers and lipid profile suggest that B. pseudocatenulatum CECT 7765 intake together with dietary recommendations can improve inflammatory status in children with obesity and insulin resistance. These effects are parallel to increases in bacterial groups associated with a lean phenotype. The modulation of gut microbiota with probiotic supplementation can be considered an effective tool to ameliorate some obesity-related disorders in children. [ABSTRACT FROM AUTHOR]

Abstract The oral delivery of probiotics to the colon is often challenging because probiotics tend to lose viability when exposed to the harsh conditions in the upper gastrointestinal tract, such as the highly acidic gastric fluids. Properly designed encapsulation technologies can be used to protect probiotics during their transit through the human gut. In this study, an anaerobic probiotic (Bifidobacterium pseudocatenulatum G7 or BPG7) was encapsulated within alginate microgels that also contained antacid agents to control their internal pH within the stomach. Probiotic-loaded microgels were exposed to a simulated gastrointestinal tract (GIT) model to establish the impact of gastric and small intestinal conditions on their physicochemical properties and cell viability. In the absence of antacids, no live probiotic cells were detected in the microgels after exposure to gastrointestinal conditions. Conversely, in the presence of antacids, there was only a 1.5 log CFU decrease in cell viability after incubation in simulated gastric fluids for 2 h. Subsequently, after the antacid microgels were incubated in simulated intestinal fluids, considerable amount of viable probiotic cells were still detected when CaCO 3 was used as an antacid but not when Mg(OH) 2 was used. Overall, these results indicated that alginate microgels containing CaCO 3 as an antacid were efficient at protecting the probiotic during passage through the upper GIT. This novel encapsulation technology utilizing all food grade materials may be useful for the oral delivery of probiotics to the gut in the form of functional foods or supplements. Graphical abstract Image 1 Highlights • Antacid-loaded alginate microgels were used to protect probiotics from acidic gastric conditions. • The effectiveness of two food-grade antacids were compared: CaCO 3 and Mg(OH) 2. • CaCO 3 was a more effective antacid than Mg(OH) 2 for protecting probiotics. • Probiotics survived throughout gastrointestinal tract when co-encapsulated with CaCO 3. [ABSTRACT FROM AUTHOR]

This study investigated cloning and expression of enterovirus 71 viral capsid protein 1 (EV71‐VP1) in Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) M115. To achieve this, a codon‐optimized gene coding for EV71‐VP1 was analysed, designed, synthesized and cloned into a plasmid vector flanked by a transcriptional promoter and terminator sequences. The promoter was based on that of P919, a constitutive promoter of the gene encoding the large ribosomal protein of B. bifidum BGN4, while the terminator was based on that of the peptidase N gene of Lactococcus lactis. The construct was amplified in Escherichia coli XL1‐blue and then transferred into B. pseudocatenulatum M115 by electrotransformation. Western blot analysis revealed that the EV71‐VP1 was intracellularly expressed in B. pseudocatenulatum M115 under the control of the selected heterologous promoter. In addition, plasmid stability analysis showed the construct was maintained stably for more than 160 generations, enough for most future applications. The results derived from this study open the possibility to utilize the bacterium carrying a specific expression plasmid as cell factory for the production of proteins with high commercial and health‐promoting value. Significance and Impact of the Study: This study demonstrated the first successful expression of a codon‐optimized gene coding for enterovirus 71 viral capsid protein 1 (EV71‐VP1) in Bifidobacterium pseudocatenulatum M115, a novel probiotic strain isolated from human intestines. The EV71‐VP1 was constitutively expressed under the control of P919 promoter derived from B. bifidum BGN4 in the cytoplasm of bacterial cells supporting the use of heterologous promoter and terminator sequences for viral gene expression in Bifidobacterium species. Significance and Impact of the Study: This study demonstrated the first successful expression of a codon‐optimized gene coding for enterovirus 71 viral capsid protein 1 (EV71‐VP1) in Bifidobacterium pseudocatenulatum M115, a novel probiotic strain isolated from human intestines. The EV71‐VP1 was constitutively expressed under the control of P919 promoter derived from B. bifidum BGN4 in the cytoplasm of bacterial cells supporting the use of heterologous promoter and terminator sequences for viral gene expression in Bifidobacterium species. [ABSTRACT FROM AUTHOR]

Bacterial biofilm is an emerging form of life that involves cell populations living embedded in a self-produced matrix of extracellular polymeric substances (EPS). Currently, little is known about the molecular mechanisms of Bifidobacterium biofilm formation. We used the Bifidobacterium biofilm fermentation system to preparation of biofilms on wheat fibers, and multi-omics analysis of both B. pseudocatenulatum biofilms and planktonic cells were performed to identify genes and metabolites involved in biofilm formation. The average diameter of wheat fibers was around 50 μm, while the diameter of particle in wheat fibers culture of B. pseudocatenulatum was over 260 μm at 22 h with 78.96% biofilm formation rate (BR), and the field emission scanning electron microscopy (FESEM) results showed that biofilm cells on the surface of wheat fibers secreted EPS. Transcriptomic analysis indicated that genes associated with stress response ( groS , mntH , nth , pdtaR , pstA , pstC , radA , rbpA , whiB , ybjG ), quorum sensing ( dppC , livM , luxS , sapF ), polysaccharide metabolic process ( rfbX , galE , zwf , opcA, glgC , glgP , gtfA ) may be involved in biofilm formation. In addition, 17 weighted gene co-expression network analysis (WGCNA) modules were identified and two of them positively correlated to BR. Metabolomic analysis indicated that amino acids and amides; organic acids, alcohols and esters; and sugar (trehalose-6-phosphate, uridine diphosphategalactose, uridine diphosphate-N-acetylglucosamine) were main metabolites during biofilm formation. These results indicate that stress response, quorum sensing (QS), and EPS production are essential during B. pseudocatenulatum biofilm formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Liu, Wang, Zhang, Li, Lu and Zhu.)

Bifidobacterium pseudocatenulatum IPLA 36007 acts on isoflavone glycosides, releasing their corresponding aglycones. This strain-specific activity might be a key step in making isoflavones bioavailable and harnessing their oestrogenic activity. To investigate the molecular mechanisms involved in this activity, four glycosyl hydrolase-encoding genes in the IPLA 36007 genome ( AW18_01575 , AW18_09810 , AW18_08145 , and AW18_08090 ) were selected, synthesized with heterologous promoter and terminator signals (r-β- gluA , r-β- gluB , r-β- gluD and r-β- gluE , respectively), cloned into Escherichia coli , overexpressed as His-tagged proteins, and the enzymes purified and characterized. All four enzymes – GluA His , GluB His , GluD His and GluE His – proved to have β-glucosidase activity and deglycosylated (although at different rates) the isoflavone glycosides daidzin and genistin, releasing the aglycone moieties daidzein and genistein, respectively. GluD His and GluE His were also shown to hydrolyse β-glucosyl disaccharides such as cellobiose and gentiobiose, while GluA His and GluB His did not. Differences in activity were recorded for all four β-glucosidases at different pHs and temperatures under otherwise similar assay conditions, suggesting they have complementary activities under different environmental conditions. Two of the recombinant genes, r-β- gluA , and r-β- gluD , were cloned and expressed in the model lactic acid bacterium Lactococcus lactis , suggesting starter and probiotic organisms could be endowed with β-glucosidase activity. B. pseudocatenulatum IPLA 36007 contains additional β-glucosidases to those studied in this work, indicating a high level of redundancy for this enzymatic activity. Knowledge of glycoside-degrading enzymes should facilitate the development of novel, more effective or more selective prebiotics or functional foods for the promotion of bifidobacterial numbers in the human gut. It might also be of interest in the development of novel probiotics with specific health-promoting activities. [ABSTRACT FROM AUTHOR]

Dietary habits contribute to the composition and function of the gut microbiota. Different dietary structures, including vegan, vegetarian, and omnivorous diets, affect intestinal Bifidobacteria; however, the relationship between Bifidobacterial function and host metabolism in subjects with different dietary patterns is unclear. Here, we analyzed five metagenomics studies and six 16S sequencing studies, including 206 vegetarians (VG), 249 omnivores (O), and 270 vegans (V), through an unbiased theme-level meta-analysis framework and discovered that diet significantly affects the composition and functionality of intestinal Bifidobacteria. The relative abundance of Bifidobacterium pseudocatenulatum was significantly higher in V than in O and Bifidobacterium longum, Bifidobacterium adolescentis, and B. pseudocatenulatum differed significantly in carbohydrate transport and metabolism in subjects with different diet types. Diets high in fiber were associated with B. longum with increased capacity for carbohydrate catabolism and genes encoding GH29 and GH43&#95;27 were significantly enriched in V. Bifidobacterium adolescentis and B. pseudocatenulatum, associated with O, had a higher prevalence of the genes related to carbohydrate transport and metabolism, which showed the enrichment of GH26 and GH27 families. The same Bifidobacterium species has different functions in subjects with different diet types, resulting in different physiological significance. The diversification and functionalities of Bifidobacterial species in the gut microbiome can be influenced by the host diet and this aspect should be considered when studying host-microbe associations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Aim: Dietary plant fibers affect gut microbiota composition; however, the underlying microbial degradation pathways are not fully understood. We previously discovered 3- O -α-D-galactosyl-α-L-arabinofuranosidase (GAfase), a glycoside hydrolase family 39 enzyme involved in the assimilation of side chains of arabinogalactan protein (AGP), from Bifidobacterium longum subsp. longum ( B. longum ) JCM7052. Although GAfase homologs are not highly prevalent in the Bifidobacterium genus, several Bifidobacterium strains possess the homologs. To explore the differences in substrate specificity among the homologs, a homolog of B. longum GAfase in Bifidobacterium pseudocatenulatum MCC10289 (MCC10289&#95;0425) was characterized. Methods: Gum arabic, larch, wheat AGP, and sugar beet arabinan were used to determine the substrate specificity of the MCC10289&#95;0425 protein. An amino acid replacement was introduced into GAfase to identify a critical residue that governs the differentiation of substrate specificity. The growth of several Bifidobacterium strains on β-L-arabinopyranosyl disaccharide and larch AGP was examined. Results: MCC10289&#95;0425 was identified to be an unprecedented 3- O -β-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase) with low GAfase activity. A single amino acid replacement (Asn 119 to Tyr) at the catalytic site converted GAfase into AAfase. AAfase releases sugar source from AGP, thereby allowing B. pseudocatenulatum growth. Conclusion: Bifidobacteria have evolved several homologous enzymes with overlapping but distinct substrate specificities depending on the species. They have acquired different fitness abilities to respond to diverse plant polysaccharide structures., Competing Interests: Horigome A, Xiao JZ, and Odamaki T are employees of Morinaga Milk Industry Co., Ltd. The authors declared that there are no other conflicts of interest., (© The Author(s) 2023.)