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l-Arabinofuranosides with β-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a β-l-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 β-l-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-β1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-β1,3-Araf structures but not those containing Araf-β1,2-Araf and Araf-β1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two β-sandwich domains. A hairpin structure with two β-strands was observed in Bll3HypBA1, to extend from a β-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a β1,3-specific β-l-arabinofuranosidase. Key points: • β1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • β-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a β1,3-linkage-specific β-l-arabinofuranosidase. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is the second most common neurodegenerative disease but still lacks a preclinical strategy to identify it. The diagnostic value of intestinal mucosal α-synuclein (αSyn) in PD has not drawn a uniform conclusion. The relationship between the alteration of intestinal mucosal αSyn expression and mucosal microbiota is unclear. Nineteen PD patients and twenty-two healthy controls were enrolled in our study from whom were collected, using gastrointestinal endoscopes, duodenal and sigmoid mucosal samples for biopsy. Multiplex immunohistochemistry was performed to detect total, phosphorylate, and oligomer α-synuclein. Next-generation 16S rRNA amplicon sequencing was applied for taxonomic analysis. The results implied that oligomer α-synuclein (OSyn) in sigmoid mucosa of PD patients was transferred from the intestinal epithelial cell membrane to the cytoplasm, acinar lumen, and stroma. Its distribution feature was significantly different between the two groups, especially the ratio of OSyn/αSyn. The microbiota composition in mucosa also differed. The relative abundances of Kiloniellales, Flavobacteriaceae, and CAG56 were lower, while those of Proteobacteria, Gammaproteobacteria, Burkholderiales, Burkholdriaceae, Oxalobacteraceae, Ralstonia, Massilla, and Lactoccus were higher in duodenal mucosa of PD patients. The relative abundances of Thermoactinomycetales and Thermoactinomycetaceae were lower, while those of Prevotellaceae and Bifidobacterium longum were higher in patients' sigmoid mucosa. Further, the OSyn/αSyn level was positively correlated with the relative abundances of Proteobacteria, Gammaproteobacteria, Burkholderiales, Pseudomonadales, Burkholderiaceae, and Ralstonia in the duodenal mucosa, while it was negatively correlated with the Chao1 index and observed operational taxonomic units of microbiota in sigmoid mucosa. The intestinal mucosal microbiota composition of PD patients altered with the relative abundances of proinflammatory bacteria in the duodenal mucosa increased. The ratio of the OSyn/αSyn level in the sigmoid mucosa indicated a potential diagnostic value for PD, which also correlated with mucosal microbiota diversity and composition. Key points: • The distribution of OSyn in sigmoid mucosa differed between PD patients and healthy controls. • Significant alterations in the microbiome were found in PD patients' gut mucosa. • OSyn/αSyn level in sigmoid mucosa indicated a potential diagnostic value for PD. [ABSTRACT FROM AUTHOR]

Aging is associated with gut microbiota alterations, characterized by changes in intestinal microbial diversity and composition. However, no study has yet focused on investigating age-related changes in the low-abundant but potentially beneficial subpopulations of gut lactic acid bacteria (LAB) and Bifidobacterium. Our study found that the subjects' age correlated negatively with the alpha diversity of the gut bifidobacterial microbiota, and such correlation was not observed in the gut LAB subpopulation. Principal coordinate analysis (PCoA) and analysis of distribution of operational taxonomic units (OTUs) revealed that the structure and composition of the gut bifidobacterial subpopulation of the longevous elderly group were rather different from that of the other three age groups. The same analyses were applied to identify age-dependent characteristics of the gut LAB subpopulation, and the results revealed that the gut LAB subpopulation of young adults was significantly different from that of all three elderly groups. Our study identified several potentially beneficial bacteria (e.g., Bifidobacterium breve and Bifidobacterium longum) that were enriched in the longevous elderly group (P < 0.05), and the relative abundance of Bifidobacterium adolescentis decreased significantly with the increase in age (P < 0.05). Although both bifidobacteria and LAB are generally considered as health-promoting taxa, their age-dependent distribution varied from each other, suggesting their different life stage changes and potentially different functional roles. This study provided novel species-level gut bifidobacterial and LAB microbiota profiles of a large cohort of subjects and identified several age-or longevity-associated features and biomarkers. Key points: • The alpha diversity of the gut bifidobacterial microbiota decreased with age, while LAB did not change. • The structure and composition of the gut bifidobacterial subpopulation of the longevous elderly group were rather different from that of the other three age groups. • Several potentially beneficial bacteria (e.g., Bifidobacterium breve and Bifidobacterium longum) that were enriched in the longevous elderly group. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effects of soy-based beverages manufactured with water-soluble soy extract, containing probiotic strains (Lactobacillus acidophilus LA-5 and Bifidobacterium longum BB-46) and/or acerola by-product (ABP) on pooled faecal microbiota obtained from lean and obese donors. Four fermented soy beverages (FSs) ("placebo" (FS-Pla), probiotic (FS-Pro), prebiotic (FS-Pre), and synbiotic (FS-Syn)) were subjected to in vitro digestion, followed by inoculation in the TIM-2 system, a dynamic in vitro model that mimics the conditions of the human colon. Short- and branched-chain fatty acids (SCFA and BCFA) and microbiota composition were determined. Upon colonic fermentation in the presence of the different FSs formulations, acetic and lactic acid production was higher than the control treatment for faecal microbiota from lean individuals (FMLI). Additionally, SCFA production by the FMLI was higher than for the faecal microbiota from obese individuals (FMOI). Bifidobacterium spp. and Lactobacillus spp. populations increased during simulated colonic fermentation in the presence of FS-Syn in the FMLI and FMOI. FS formulations also changed the composition of the FMOI, resulting in a profile more similar to the FMLI. The changes in the composition and the increase in SCFA production observed for the FMLI and FMOI during these in vitro fermentations suggest a potential modulation effect of these microbiotas by the consumption of functional FSs. Key points: • Soy beverages increased Bifidobacterium abundance in microbiota from obese individuals. • The synbiotic beverage increased Bifidobacterium abundance in microbiota from lean individuals. • The synbiotic beverage changed the microbiota from obese individuals, approaching the lean profiles. [ABSTRACT FROM AUTHOR]

Fucosyllactoses have gained much attention owing to their multiple functions, including prebiotic, immune, gut, and cognition benefits. In this study, human milk oligosaccharide (HMO) 2′-fucosyllactose (α-L-Fuc-(1,2)-D-Galβ-1,4-Glu, 2′FL) and its isomer 3′-fucosyllactose (α-L-Fuc-(1,3)-D-Galβ-1,4-Glu, 3′FL) with potential prebiotic effect were synthesized efficiently by a novel recombinant α-L-fucosidase. An α-L-fucosidase gene (PbFuc) from Pedobacter sp. CAU209 was successfully cloned and expressed in Escherichia coli (E. coli). The deduced amino acid sequence shared the highest identity of 36.8% with the amino sequences of other reported α-L-fucosidases. The purified α-L-fucosidase (PbFuc) had a molecular mass of 50 kDa. The enzyme exhibited specific activity (26.3 U/mg) towards 4-nitrophenyl-α-L-fucopyranoside (pNP-FUC), 3′FL (8.9 U/mg), and 2′FL (3.4 U/mg). It showed the highest activity at pH 5.0 and 35 °C, respectively. PbFuc catalyzed the synthesis of 3′FL and 2′FL through a transglycosylation reaction using pNP-FUC as donor and lactose as acceptor, and total conversion ratio was up to 85% at the optimized reaction conditions. The synthesized mixture of 2′FL and 3′FL promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus NRRL B-548, L. casei subsp. casei NRRL B-1922, L. casei subsp. casei AS 1.2435, and Bifidobacterium longum NRRL B-41409. However, the growths of E. coli ATCC 11775, S. enterica AS 1.1552, L. monocytogenes CICC 21635, and S. aureus AS 1.1861 were not stimulated by the mixture of 2′FL and 3′FL. Overall, our findings suggest that PbFuc possesses a great potential for the specific synthesis of fucosylated compounds. Key Points • A novel α-L-fucosidase (PbFuc) from Pedobacter sp. was cloned and expressed. • PbFuc showed the highest hydrolysis activity at pH 5.0 and 35 °C, respectively. • It was used for synthesis of 3′-fucosyllactose (3′FL) and 2′-fucosyllactose (2′FL). • The mixture of 3′FL and 2′FL promoted the growth of some Lactobacillus sp. and Bifidobacteria sp. [ABSTRACT FROM AUTHOR]

A β-galactosidase (β-GalINF) was directly isolated from feces of an 8-month-old infant. Mass spectrum analysis showed β-GalINF with coverage over 50% to the β-galactosidase from Bifidobacterium longum EK3. Accordingly, the β-galINF was amplified from the feces metagenomic DNA by degenerate primers. After overexpressed in Escherichia coli, the β-GalINF was purified and biochemical characterized. β-GalINF existed as homotetramer and homodimer, whose activity (optimal at 50 °C, pH 6.5) was exhilaratingly increased to 484% by artificial intestinal juice. The Km and Vmax values for oNPG and lactose were 20.95 ± 2.76 mM, 5004.50 ± 318.8 μmol min−1 mg−1 and 140.2 ± 17.7 mM, 293.1 ± 14.7 μmol min−1 mg−1, respectively. The production rate of galacto-oligosaccharides by β-GalINF from 20% lactose at 50 °C was 33.4 ± 0.67%. These results suggested the β-GalINF with high hydrolytic and transgalactosylation activity from the infant intestinal has great potential as infant lactase preparation. Moreover, this study provided a new way for exploring undetected enzymes by uncultured-dependent methods. [ABSTRACT FROM AUTHOR]

In the present study, the modulatory effects of bifidobacterial spp. (Bifidobacterium breve NCIM 5671, Bifidobacterium longum NCIM 5672 and Bifidobacterium bifidum NCIM 5697) on adjuvant induced arthritis in rats were evaluated. Arthritis was induced in male Wistar rats by injecting 250 μg of Freund's adjuvant directly into the paw. Fifteen days before and 15 days after the induction of arthritis, suspended cultures of bifidobacteria (109 cfu/ml) were administered by oral gavage. Paw volume, bone mineral content, oxidative stress markers, antioxidant enzymes, cytokines, eicosanoids and expression of COX2, as well as bone hydrolytic enzymes, were assessed by RT PCR. Although piroxicam-treated groups (drug control) had better effects than bifidobacteria-treated groups, bifidobacteria probiotics administration exhibited significant (P < 0.05) prophylactic effects in terms of downregulating arthritis markers. Parameters including paw volume, bone mineral content, cytokines, and eicosanoids level were significantly (p < 0.05) modulated in bifidobacteria administered groups compared to arthritic control group. Among the three strains tested, B. breve NCIM 5671 exhibited superior prophylactic effects as assessed in the experimental rat model of arthritis. In conclusion, bifidobacteria probiotics administration can downregulate the markers of arthritis and hence can be a potential therapeutic regimen in the treatment of arthritis. [ABSTRACT FROM AUTHOR]

Type II arabinogalactan (AG) is a soluble prebiotic fiber stimulating the proliferation of bifidobacteria in the human gut. Larch AG, which is comprised of type II AG, is known to be utilized as an energy source for Bifidobacterium longum subsp. longum (B. longum). We have previously characterized GH43_24 exo-β-1,3-galactanase (Bl1,3Gal) for the degradation of type II AG main chains in B. longum JCM1217. In this study, we characterized GH30_5 exo-β-1,6-galactobiohydrolase (Bl1,6Gal) and GH43_22 α-L-arabinofuranosidase (BlArafA), which are degradative enzymes for type II AG side chains in cooperation with exo-β-1,3-galactanase. The recombinant exo-β-1,6-galactobiohydrolase specifically released β-1,6-galactobiose (β-1,6-Gal2) from the nonreducing terminal of β-1,6-galactooligosaccharides, and the recombinant α-L-arabinofuranosidase released arabinofuranose (Araf) from α-1,3-Araf-substituted β-1,6-galactooligosaccharides. β-1,6-Gal2 was additively released from larch AG by the combined use of type II AG degradative enzymes, including Bl1,3Gal, Bl1,6Gal, and BlArafA. The gene cluster encoding the type II AG degradative enzymes is conserved in all B. longum strains, but not in other bifidobacterial species. The degradative enzymes for type II AG side chains are thought to be important for the acquisition of type II AG in B. longum. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effects of three treatments, i.e., Bifidobacterium longum BB-46 (T1), B. longum BB-46 combined with the pectin (T2), and harsh extracted pectin from lemon (T3) on obesity-related microbiota using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). The effects of the treatments were assessed by the analysis of the intestinal microbial composition (using 16S rRNA gene amplicon sequencing) and the levels of short-chain fatty acids (SCFAs) and ammonium ions (NH4+). Treatments T2 and T3 stimulated members of the Ruminococcaceae and Succinivibrionaceae families, which were positively correlated with an increase in butyric and acetic acids. Proteolytic bacteria were reduced by the two treatments, concurrently with a decrease in NH4+. Treatment T1 stimulated the production of butyric acid in the simulated transverse and descending colon, reduction of NH4+ as well as the growth of genera Lactobacillus, Megamonas, and members of Lachnospiracea. The results indicate that both B. longum BB-46 and pectin can modulate the obesity-related microbiota; however, when the pectin is combined with B. longum BB-46, the predominant effect of the pectin can be observed. This study showed that the citric pectin is able to stimulate butyrate-producing bacteria as well as genera related with anti-inflammatory effects. However, prospective clinical studies are necessary to evaluate the anti/pro-obesogenic and inflammatory effects of this pectin for future prevention of obesity. [ABSTRACT FROM AUTHOR]

The acid tolerance is particularly important for bifidobacteria to function as probiotics because they usually encounter acidic environments in food products and gastrointestinal tract passage. In this study, two acid-resistant derivatives Bifidobacterium longum JDY1017dpH and Bifidobacterium breve BB8dpH, which displayed a stable acid-resistant phenotype, were generated. The relationship between acid tolerance and cell membrane was investigated by comparing the two acid-resistant derivatives and their parental strains grown in medium with and without Tween 80. The fold increase in acid tolerance of the two acid-resistant derivatives relative to their parental strains was much higher when cells were grown in medium with Tween 80 (10 ~ 10-fold) than without Tween 80 (181- and 245-fold). Moreover, when cells were grown in medium with Tween 80, the two acid-resistant derivatives exhibited more C18:1 and cycC19:0, higher mean fatty acid chain length, lower membrane fluidity, and higher expression of cfa gene encoding cyclopropane fatty acid synthase than their parental strains. No significant differences in cell membrane were observed between the two acid-resistant derivatives and their parental strains when cells were grown in medium without Tween 80. The present study revealed that, when cells were grown in medium with Tween 80, the significant fold increase in acid tolerance of the two acid-resistant derivatives was mainly ascribed to the pronounced changes in cell membrane compared with their parental strains. Results presented here could provide a basis for developing new strategies of cell membrane modification to enhance acid tolerance in bifidobacteria. [ABSTRACT FROM AUTHOR]

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities ( a) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a = 0.96 or 0.99 were then recovered by centrifugation, added to a sucrose-phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a = 0.96. Growing the cells in complex media adjusted at a = 0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose-phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures. [ABSTRACT FROM AUTHOR]

Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium-Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 °C but not at 42 °C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 10 integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705. [ABSTRACT FROM AUTHOR]

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m). Cell mass at the MBR reached 22.18 g L as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h and at the batch culture was μ = 1.13 h in the exponential period and μ = 0.31 h in the stationary period. In the fed-batch mode was μ = 1.102 h for 6 h with inoculation and declined to μ = 0.456 h with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h. The number of viable cells was 6.01 × 10 cfu L at the batch culture, but increased to 1.15 × 10 cfu L at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40 °C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30-40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR. [ABSTRACT FROM AUTHOR]