Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V,Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536

The consumption of probiotic-based products has risen greatly in recent decades. Due to their probiotic characteristics, microorganisms such as lactobacilli and bifidobacteria are in daily use in the production of food supplements. In the present study, three bifidobacterial strains (Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536) were tested for growth compatibility, resistance to antimicrobial agents, antibacterial activity against pathogens, resistance to gastric acidity, bile salt hydrolysis and adhesion to the human intestinal epithelial cell line HT29. All of these strains were resistant to gentamycin, but none showed in vitro growth incompatibility or the presence of known resistance determinants. B. breve M-16 V had the best probiotic characteristics and, indeed, was the only strain possessing antibacterial activity against Escherichia coli and Klebsiella pneumoniae. All strains were resistant to simulated gastric juice, while only B. longum subsp. longum BB536 and B. breve M-16 V showed a bile salt hydrolytic activity. Interestingly, a strong adhesion to HT29 cells was observed in all Bifidobacterium strains. In conclusion, B. breve M-16 V, B. longum subsp. longum BB536 and B. longum subsp. infantis M-63 showed several promising characteristics as probiotic strains.     

两歧双歧杆菌中serpin基因的克隆、表达与功能分析

从Bifidobacterium bifidum WBBI02基因组中克隆了serpin基因片段,构建了重组Serpin蛋白的原核表达体系,实现了Serpin的表达与纯化。纯化的Serpin蛋白进行了抑制肠道蛋白酶活性检测,以及对双歧杆菌粘附作用影响的显微观察研究。结果表明:WBBI02中长度为768 bp的serpin基因序列,与GENEBANK中Bifidobacterium longum NCC2705 serpin序列同源性为99. 9 %。原核表达载体pBX₂-WBBI02表达的Serpin能有效地抑制糜蛋白酶和胰弹性蛋白酶的活性,最高抑制率分别为90%和97%,显微观察结果证实Serpin能促进双歧杆菌对HT-29细胞的粘附。     

Diversity of Lactobacillus and Bifidobacterium in feces of herbivores, omnivores and carnivores

The Lactobacillus and Bifidobacterium population in the feces of 26 animals (16 species) were studied by culture-dependent and culture-independent techniques. Lactobacilli were detected from a few herbivores, all carnivores and some omnivores. Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus vaginalis and Lactobacillus ingluviei were the most dominant lactobacilli in carnivores. These species were, however, not predominant in herbivores and omnivores. Lactobacillus brevis, Lactobacillus casei, Lactobacillus parabuchneri, Lactobacillus plantarum, Lactobacillus sakei, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, usually present in raw plant material, were present in omnivores but not in carnivores. Bifidobacteria were detected in only four herbivores and two omnivores. Bifidobacterium pseudolongum was the only Bifidobacterium species detected in herbivores. Bifidobacteria detected in the two omnivores are phylogenetically not closely related to known species and are possible novel species in the genus.     

Galactokinase of Bifidobacterium bifidum

Crystalline galactokinase was obtained in good yield from Bifidobacterium bifidum grown on galactose medium. This preparation moved as a single protein band in analytical disc electrophoresis and sedimented as a single symmetrical peak under ultracentrifugation. The enzyme exhibited similar physicochemical properties to galactokinase purified from glucose-grown cells of B. bifidum. The enzyme has a molecular weight of 47,000. Only galactose and ATP were effective as substrate. Km values, optimal pH, cation requirement, inhibition by SH-reagent, heat stability and product inhibition were also investigated.     

Structural studies of cell wall polysaccharides from Bifidobacterium breve YIT 4010 and related Bifidobacterium species

The chemical compositions of the cell walls obtained from 8 strains in 5 species of Bifidobacterium were analyzed. These cell walls were shown to be composed of peptidoglycan and polysaccharide moieties. Some variations with respect to contents of neutral sugars and content of phosphorus were observed with some cell wall preparations from the same species. The neutral polysaccharides in cell walls of 4 strains of Bifidobacterium (B. bifidum YIT 4007, B. breve YIT 4010, B. infantis YIT 4025, and B. longum ATCC 15707) were purified and their chemical structures were analyzed. One of these polysaccharides, obtained from B. breve YIT 4010, was analyzed in detail by GLC, 1H- and 13C-NMR spectroscopic analyses, methylation, Smith degradation and acetolysis, and the results suggested the following structure for the repeating unit of the polysaccharide: (Formula: see text).     

Molecular profiling of Lactobacillus, Streptococcus, and Bifidobacterium species in feces of active racehorses

Diversity and compositions of the Lactobacillus, Streptococcus, and Bifidobacterium group in the feces of six healthy, actively racing horses (Thoroughbreds) were analyzed by using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR with primer sets specific for each group. PCR-DGGE analysis of the feces showed that Lactobacillus equi, Lactobacillus johnsonii, a phylogenetic relative of Lactobacillus salivarius, a phylogenetic relative of Lactobacillus gastricus, and Weissella confusa were predominant in almost all of the feces tested, and Streptococcus bovis/Streptococcus equinus was predominant in the Streptococcus group. The Bifidobacterium group was not detected by single-PCR but atypical species of the group were found in three of the six Thoroughbreds tested by nested-PCR. Calculation and estimation of lactic acid bacteria and bifidobacteria revealed that lactic acid bacteria were predominant in the feces and bifidobacteria were minor. These results indicate that the community of lactic acid bacteria and bifidobacteria in horse feces are unique because of the presence of specific species for horse feces and a minority of the Bifidobacterium group. Repeated tests of the feces from the same horse over 3 months showed that the diversity and composition of lactic acid bacteria and bifidobacteria in the feces was basically stable throughout the test period.     

Phylogenetic characterization of two novel species of the genus Bifidobacterium: Bifidobacterium saimiriisciurei sp. nov. and Bifidobacterium platyrrhinorum sp. nov.

Three bifidobacterial Gram-stain-positive, non-spore forming and fructose-6-phosphate phosphoketolase-positive strains, SMA1^T, SMB2 and SMA15^T were isolated from the faeces of two adult males of the squirrel monkey (Saimiri sciureus).On the basis of 16S rRNA gene sequence similarities, the type strain of Bifidobacterium primatium DSM 100687^T (99.3%; similarity) was the closest neighbour to strains SMA1^T and SMB2, whereas the type strain of Bifidobacterium stellenboschense DSM 23968^T (96.5%) was the closest neighbour to strain SMA15^T. The average nucleotide identity (ANI) values of SMA1^T and SAM15^T with the closely related type strains were 93.7% and 88.1%, respectively. The in silico DNA‒DNA hybridization values with the closest neighbours were 53.1% and 36.9%, respectively. GC contents of strains SMA1^T and SMA15^T were 63.6 and 66.4 mol%, respectively. Based on the phylogenetic, genotypic and phenotypic data obtained, the strains SMA1^T and SMA15^T clearly represent two novel taxa within the genus Bifidobacterium for which the names Bifidobacterium saimiriisciurei sp. nov. (type strain SMA1^T = BCRC 81223^T = NBRC 114049^T = DSM 106020^T) and Bifidobacterium platyrrhinorum sp. nov. (type strain SMA15^T = BCRC 81224^T = NBRC 114051^T = DSM 106029^T) are proposed.     

Carbohydrate preference, acid tolerance and bile tolerance in five strains of Bifidobacterium

AIMS: To assess the suitability of bifidobacteria for inclusion in synbiotic products on the basis of carbohydrate preference, acid and bile tolerance. METHODS AND RESULTS: Five strains of Bifidobacterium were analysed for their carbohydrate preference from 12 substrates. Maximum growth rates were used to compare substrate preferences. Galacto-oligosaccharides and isomalto-oligosaccharides were well utilized by all the test species. Most bacteria tested could also utilize at least one type of fructan molecule. To determine transit tolerance of potentially probiotic bifidobacteria, acid and bile resistance was tested. A wide range acid resistance was found. Bile tolerance also varied. CONCLUSIONS: GOS and IMO were generally well utilized by the tested species. Other substrates were used to different degrees by the different species. Most bifidobacteria are poorly resistant to strongly acidic conditions with the exception of Bifidobacterium lactis Bb12. Bile tolerances were widely variable and it was shown that caution should be exercised when using colorimetric methods to assess bile tolerance. SIGNIFICANCE AND IMPACT OF STUDY: The study allows the comparison of the properties of bifidobacteria, allowing a cost effective screen for the best species for use in synbiotic products to allow better survival and efficacy.     

Kinetics and metabolism of Bifidobacterium adolescentis MB 239 growing on glucose, galactose, lactose, and galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that beta(1-4) galactosides can be hydrolyzed before they are taken up.     

Kinetics and Metabolism of Bifidobacterium adolescentis MB 239 Growing on Glucose, Galactose, Lactose, and Galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β(1-4) galactosides can be hydrolyzed before they are taken up.     

水貂粪便中双歧杆菌的分离与鉴定

目的运用TPY培养基,从健康水貂的粪便中分离培养、筛选出2个肠道菌株。方法细菌培养、菌落形态观察、染色镜检、分离纯化、生化试验和药敏试验。结果分离培养出的2株菌株为双歧杆菌,其中1株为长双歧杆菌,另1株为青春双歧杆菌;双歧杆菌对氯霉素极其敏感,对阿米卡星耐药。结论本实验为毛皮特种经济动物微生态制剂的研究工作奠定了基础。     

Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum

To identify bacterial traits related to adhesion ability in human bifidobacteria, 13 strains of Bifidobacterium longum isolated from human gastric juice and intestine were studied. Strains were tested for their capability to adhere to Caco-2 cells and classified as adhesive (Adh+) or non-adhesive (Adh-). Adh+ and Adh- strains were then investigated for their autoaggregation ability and surface hydrophobicity. Comparing the properties of Adh+ and Adh-, we observed that strains were able to adhere to cell monolayers if they autoaggregate and manifest a good degree of hydrophobicity as determined by microbial adhesion to hydrocarbons. These two traits could be used for preliminary screening to identify potentially adherent isolates.     

Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.     

Effect of probiotics, Bifidobacterium breve and Lactobacillus casei, on bisphenol A exposure in rats

Bisphenol A (BPA), a putative endocrine disruptor, may be taken up by humans via the diet and have adverse effects on human health. In this study, we evaluated whether the probiotics, Bifidobacterium breve strain Yakult (BbY) and Lactobacillus casei strain Shirota (LcS), could exert a protective effect against dietary exposure to BPA. A group of rats fed on a diet containing 5% BbY or 5% LcS showed three advantageous effects compared to the control group; (i) the area under the blood concentration-time curve of BPA after its oral administration was significantly decreased, (ii) the amount of BPA excreted in the feces was significantly greater (2.4 times), and (iii) the percentage of BPA bound to the sediment fraction of the feces was significantly higher. These results suggest that BbY and LcS reduced the intestinal absorption by facilitating the excretion of BPA, and that these probiotics may suppress the adverse effects of BPA on human health.     

Characterization of Bifidobacterium apousia sp. nov., Bifidobacterium choladohabitans sp. nov., and Bifidobacterium polysaccharolyticum sp. nov., three novel species of the genus Bifidobacterium from honey bee gut

Bifidobacterium is one of the dominating bacterial genera in the honey bee gut, and they are the key degrader of diet polysaccharides for the host. Previous genomic analysis shows that they belong to separate phylogenetic clusters and exhibited different functional potentials in hemicellulose digestion. Here, three novel strains from the genus Bifidobacterium were isolated from the guts of the honey bee (Apis mellifera). Phylogenomic analysis showed that the isolates could be grouped into four phylogenetic clusters. The average nucleotide identity values between strains from different clusters are <95%, while strains in Cluster IV belong to the characterized species Bifidobacterium asteroides. Carbohydrate-active enzyme annotation confirmed that the metabolic capacity for carbohydrates varied between clusters of strains. Cells are Gram-positive rods; they grew both anaerobically and in a CO₂-enriched atmosphere. All strains grew at a temperature range of 20–42 °C, with optimum growth at 35 °C. The pH range for growth was 5–9. Strains from different phylogenetic clusters varied in multiple phenotypic and chemotaxonomic characterizations. Thus, we propose three novel species Bifidobacterium apousia sp. nov. whose type strain is W8102^T (=CGMCC 1.18893 ^T = JCM 34587 ^T), Bifidobacterium choladohabitans sp. nov., whose type strain is B14384H11^T (=CGMCC 1.18892 ^T = JCM 34586 ^T), and Bifidobacterium polysaccharolyticum sp. nov. whose type strain is W8117^T (=CGMCC 1.18894 ^T = JCM 34588 ^T).     

Rapid identification,differentiation, and proposed new taxonomic classification of Bifidobacterium lactis

Identification of Bifidobacterium lactis and Bifidobacterium animalis is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of B. lactis DSM10140(T), B. animalis ATCC 25527(T), and six potential B. lactis strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of B. animalis. All of the molecular techniques used (B. lactis or B. animalis PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that B. lactis and B. animalis form two main groups and suggest a revision of the strains assigned to B. animalis. We propose that B. lactis should be separated from B. animalis at the subspecies level.     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996     

Fatty acid composition of Bifidobacterium and Lactobacillus strains

Normal C(14), C(16), and C(18) saturated acids and C(16) and C(18) monoenoic acids are the main fatty acids of nine strains of Bifidobacterium. Their lactobacillic acid content was less than 5%. Lactobacillus strains contained the same fatty acids as main compounds except for octadecanoic acid, which was present only in very low amounts. Eight of nine Lactobacillus strains contained in the stationary phase more than 15% lactobacillic acid. No correlation was observed between the fatty acid composition and other physiological characteristics used in the literature for classification of strains of one genus. Aging of the culture, which involved a decrease of the pH, caused a lengthening of the chain length of the fatty acids of B. bifidum var. pennsylvanicus but only a conversion of octadecenoic to lactobacillic acid in the lactobacilli. Lowering of the temperature of cultivation decreased the chain length of the fatty acids of B. bifidum var. pennsylvanicus. L. lactis did not show any influence of the temperature on the chain length of the fatty acids. The percentage of unsaturated acids was temperature independent in both organisms.