长寿老人源双歧杆菌优良菌株的筛选

以人结肠腺癌细胞系HT-29细胞为试材,对来源于广西巴马百岁以上长寿老人肠道的24株双歧杆菌进行了体外黏附试验。结果发现,双歧杆菌均具有一定的黏附能力,其中TTF、Z2、TZ5和J-1菌株具有较高的黏附能力。进一步对4株初筛双歧杆菌耐胃酸、胆汁酸和合成B族维生素能力的试验发现,双歧杆菌TTF菌株不仅能合成较高的B1、B2、B6、B12等多种B族维生素,而且在pH3．0的条件下处理120min存活率达93．11％,同时在2％胆盐浓度下处理24h有较好的存活,具有显著的综合优势。     

Autoaggregation and surface hydrophobicity of bifidobacteria

Thirteen strains of four different Bifidobacterium spp. were observed for their autoaggregation ability and surface hydrophobicity, and correlation between these two traits was determined. Bifidobacteria were classified into high, medium and low autoaggregation strains according to autoaggregation ratio measured from changes in absorbance of media. High autoaggregation strains showed microscopic clustering of cells, whereas low and medium autoaggregation strains showed no such clustering. Autoaggregation ability decreased in high autoaggregation strains but increased in medium and low autoaggregation strains when the assay was performed at higher temperature (37°C compared with 25 and 10°C). Bacterial strains belonging to the high, medium or low autoaggregation group were correlated in terms of their surface hydrophobicity and evaluated based on changes in absorbance of the bacterial suspension before and after extraction with xylene. These results indicate that autoaggregation ability, together with surface hydrophobicity and microscopic image could be used for evaluating the adhesion ability of potential probiotic bifidobacterial strains. Moreover, a synergistic effect of pH and media may be involved in autoaggregation.     

Distribution of bifidobacteria in the gastrointestinal tract of calves

Development of gastrointestinal microflora of calves with special reference to bifidobacteria was investigated; fecal bacteria were enumerated in calves aged 3 days to 7 weeks. Bacteria were detected by using selective media, bifidobacteria using modified TPY agar with an addition of mupirocin and acetic acid and by fluorescence in situ hybridization (FISH). Bifidobacteria were dominant group of fecal flora of calves after 7 d of life, constituting 10 % of total bacterial counts. The highest bacterial concentrations were observed in rumen, cecum, and colon, the lowest in abomasum and duodenum. Bifidobacteria and lactobacilli exhibited the highest survival ability during stomach passage and dominated in all parts of the digestive tract. Bifidobacteria counts determined by FISH were significantly higher than those provided by cultivation. Modified TPY agar was highly selective and suitable for bifidobacteria isolation but FISH was shown to be a more precise method for their enumeration. Our results show that gastrointestinal microflora of calves in the milk-feeding period is similar to breast-fed infants with respect to the occurrence of bifidobacteria as a dominant bacterial group. The use of Bifidobacterium strains offers a promising way for providing beneficial effectors for calves in the milk-feeding period.     

Enrichment of bifidobacteria from human gut contents by oligofructose using continuous culture

Abstract Chemostat cultures of human faecal bacteria were used to determine the bifidogenic effect of oligofructose, a fermentable carbohydrate found in a number of plants. In single stage continuous culture, oligofructose preferentially enriched for bifidobacteria, in comparison to sucrose and inulin. This stimulatory effect was enhanced at a high dilution rate, high substrate concentration and low pH. These parameters are likely to approximate to those that occur in the proximal colon. Studies with a three-stage continuous culture model of the large intestine confirmed the bifidogenic effect of oligofructose. These in vitro data indicate that an increase in the concentration of fructose-based oligosaccharides in the diet may alter the balance of the gut microflora towards bifidobacteria, a purported health-promoting genus.     

肠道益生菌体外抑菌活性的研究

以6种常见肠道致病菌作为指示菌,研究了来自健康青年和儿童肠道的25株益生菌发酵浓缩液的体外抑菌效果.进一步研究了抑菌作用良好的LMa1和LMa3经pH调节、热处理、蛋白酶处理以及发酵过程中的抑菌活性变化.短链有机酸作为发酵终产物的主要成分之一,其抑菌效果也被测定.结果表明,抑菌物质包括短链有机酸（主要为乙酸和乳酸）和一类对热稳定、对部分蛋白酶敏感的物质,可能是某种细菌素.低pH是表现抑菌活性的必要条件,但仅有低pH（如低pH缓冲液）并无明显抑菌效果.     

Human gut microbiota and bifidobacteria: from composition to functionality

The human gut is the home of an estimated 10(18) bacterial cells, many of which are uncharacterized or unculturable. Novel culture-independent approaches have revealed that the majority of the human gut microbiota consists of members of the phyla Bacteroidetes and Firmicutes. Nevertheless the role of bifidobacteria in gut ecology illustrates the importance of Actinomycetes and other Actinobacteria that may be underestimated. The human gut microbiota represents an extremely complex microbial community the collective genome of which, the microbiome, encodes functions that are believed to have a significant impact on human physiology. The microbiome is assumed to significantly enhance the metabolism of amino and glycan acids, the turnover of xenobiotics, methanogenesis and the biosynthesis of vitamins. Co-colonisation of the gut commensals Bifidobacterium longum and Bacteroides thetaiotaomicron in a murine model system revealed that the presence of bifidobacteria induced an expansion in the diversity of polysaccharides targeted for degradation by Bacteroides and also induced host genes involved in innate immunity. In addition, comparative analysis of individual human gut microbiomes has revealed various strategies that the microbiota use to adapt to the intestinal environment while also pointing to the existence of a distinct infant and adult-type microbiota.     

双歧杆菌对母乳低聚糖利用的研究进展

母乳低聚糖(human milk oligosaccharides,HMOs)是一类存在于人乳中复杂的混合低聚糖,是母乳中的重要成分,在婴幼儿生长发育中起到重要作用。母乳低聚糖可作为影响肠道微生物群组成的益生元,可选择性地促进母乳喂养婴儿肠道双歧杆菌的生长。婴儿肠道内相关的双歧杆菌具有糖苷酶和转运蛋白等分子工具,使其能够代谢HMOs,且代谢过程具有菌株特异性。本文对2′ 岩藻糖基乳糖、3′ 岩藻糖基乳糖、3′ 唾液酸乳糖、6′ 唾液酸乳糖、乳糖 N 四糖、乳糖 N 新四糖等常见HMOs的结构和特点进行总结,并讨论不同双歧杆菌对不同HMOs的利用特点和机制,为开发针对不同双歧杆菌的特异性益生元提供相应的理论指导。     

Growth of bifidobacteria and clostridia on human and cow milk saccharides

For healthy infants, which were born normally and fully breastfed, the dominant component of the intestinal microflora are bifidobacteria. However, infants born by caesarean section possess clostridia as a dominant intestinal bacterial group. The aim of the present study was to determine whether bifidobacteria and clostridia are able to grow on human milk oligosaccharides (HMOs) and other carbon sources - lactose, cow milk (CM) and human milk (HM). Both bifidobacteria and clostridia grew on lactose and in CM. Bifidobacteria grew in HM and on HMOs. In contrast, 3 out of 5 strains of clostridia were not able to grow in HM. No clostridial strain was able to utilise HMOs. While both bifidobacterial strains were resistant to lysozyme, 4 out of 5 strains of clostridia were lysozyme-susceptible. It seems that HMOs together with lysozyme may act as prebiotic-bifidogenic compounds inhibiting intestinal clostridia.     

Lessons from the genomes of bifidobacteria

The gut microbiota is a complex ecosystem composed of hundreds of different bacterial species that altogether play an important role in the physiology of their host. In the past few years the complete genome sequence of a number of bacterial strains isolated from the human gastrointestinal tract has been established including that of Bifidobacterium longum NCC2705 isolated from the feces of a healthy infant. Bifidobacteria are among the first species to colonise the human gastrointestinal tract and as such are believed to play an important role in gut homeostasis and normal development. The genome sequence of NCC2705 has revealed a number of features that suggest how this bacterium has adapted to its environment and that could help understanding how it interacts with its host. Here, we review general features of bifidobacteria and illustrate how genome-based approaches can help us better understand the biology of these organisms.     

Comparison of carbohydrate substrate preferences in eight species of bifidobacteria

Eight species of bifidobacteria were tested for their abilities to grow on a range of monosaccharides (glucose, arabinose, xylose, galactose and mannose). In contrast to the other sugars, glucose and galactose were utilized by all species and, in general, specific growth rates were highest on these sugars. Different substrate preferences were observed between species when the bacteria were grown in the presence of all five monosaccharides. For example, glucose and xylose were coutilized by Bifidobacterium longum, whereas glucose repressed uptake of all other sugars in B. bifidum and B. catenulatum. Galactose was the preferred substrate with B. pseudolongum. In B. angulatum, glucose and galactose were utilized simultaneously. B. breve did not grow on arabinose when this sugar provided the sole source of energy. However, glucose and arabinose were preferentially taken up during growth on sugar mixtures.     

Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract

In this review an overview of various molecular techniques and their application for the detection and identification of bifidobacteria and lactobacilli in the gastrointestinal (GI) tract is presented. The techniques include molecular typing techniques such as amplified ribosomal DNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD), pulsed field gel electrophoresis (PFGE), ribotyping and community profiling techniques such as PCR coupled to temperature and denaturing gradient gel electrophoresis (PCR-TGGE and PCR-DGGE, respectively). Special attention is given to oligonucleotide probes and primers that target the ribosomal RNA (rRNA) sequences and their use in PCR and different hybridisation techniques such as DNA microarrays and fluorescent in situ hybridisation (FISH). In addition, recent findings based on the molecular studies of bifidobacteria and lactobacilli in the GI-tract are reviewed.     

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.     

婴儿肠道双歧杆菌多样性的研究进展

双歧杆菌是婴儿肠道中最丰富的微生物,对婴儿肠道微生物的成熟和稳定有显著影响,与婴儿健康紧密相关。大量研究表明,婴儿肠道中最广泛存在的双歧杆菌有长双歧杆菌、短双歧杆菌和两歧双歧杆菌。母婴之间垂直传递可直接影响婴儿肠道中双歧杆菌各种属的早期定植和相对丰度变化,特别是分娩方式和喂养方式会显著影响婴儿肠道双歧杆菌。此外,胎龄和辅食等因素对婴儿肠道中双歧杆菌的组成与多样性也有一定影响。本文对婴儿肠道双歧杆菌的种属组成、相对丰度变化以及影响其多样性的因素进行了综述,为婴儿双歧杆菌群落的认识提供一定的理论依据。     

Genomics as a means to understand bacterial phylogeny and ecological adaptation: the case of bifidobacteria

The field of microbiology has in recent years been transformed by the ever increasing number of publicly available whole-genome sequences. This sequence information has significantly enhanced our understanding of the physiology, genetics and evolutionary development of bacteria. Among the latter group of microorganisms, bifidobacteria represent important human commensals because of their perceived contribution to maintaining a balanced gastrointestinal tract microbiota. In recent years bifidobacteria have drawn much scientific attention because of their use as live bacteria in numerous food preparations with various health-related claims. For this reason, these bacteria constitute a growing area of interest with respect to genomics, molecular biology and genetics. Recent genome sequencing of a number of bifidobacterial species has allowed access to the complete genetic make-up of these bacteria. In this review we will discuss how genomic data has allowed us to understand bifidobacterial evolution, while also revealing genetic functions that explains their presence in the particular ecological environment of the gastrointestinal tract. An erratum to this article can be found at     

Insights into the taxonomy, genetics and physiology of bifidobacteria

Despite the generally accepted importance of bifidobacteria as probiotic components of the human intestinal microflora and their use in health promoting foods, there is only limited information about their phylogenetic position, physiology and underlying genetics. In the last few years numerous molecular approaches have emerged for the identification and characterization of bifidobacterial strains. Their use, in conjunction with traditional culturing methods, has led to a polyphasic taxonomy which has significantly enhanced our knowledge of the role played by these bacteria in the human intestinal ecosystem. The recent adaptation of culture-independent molecular tools to the fingerprinting of intestinal and food communities offers an exciting opportunity for revealing a more detailed picture of the true complexity of these environments. Furthermore, the availability of bifidobacterial genome sequences has advanced knowledge on the genetics of bifidobacteria and the effects of their metabolic activities on the intestinal ecosystem. The release of a complete Bifidobacterium longum genome sequence and the recent initiative to sequence additional strains are expected to open up a new era of comparative genomics in bifidobacterial biology. Moreover, the use of genomotyping allows a global comparative analysis of gene content between different bifidobacterial isolates of a given species without the necessity of sequencing many strains. Genomotyping provides useful information about the degree of relatedness among various strains of Bifidobacterium species and consequently can be used in a polyphasic identification approach. This review will deal mainly with the molecular tools described for bifidobacterial identification and the first insights into the underlying genetics involved in bifidobacterial physiology as well as genome variability.     

Mining the microbiota of the neonatal gastrointestinal tract for conjugated linoleic acid-producing bifidobacteria

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml(-1) by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/microg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.     

Modification of the phosphoketolase assay for rapid identification of bifidobacteria

The phosphoketolase assay is commonly used as a definitive criterion for identification of bifidobacteria. A limitation of the assay is the time-consuming process of cell disruption, either by use of the French Pressure Cell or by sonication. We have replaced the time consuming cell disruption process with a more rapid cell membrane disruption process by pretreating cells with the detergent hexadecyltrimethylammonium bromide (cetrimonium bromide, CTAB). The effect of no pretreatment, sonication or the addition of CTAB (0.45 mg/ml) on color development in the phosphoketolase assay was tested using pure cultures of bifidobacteria and lactobacilli. No phosphoketolase activity was observed with bifidobacterial cultures without cell disruption or with lactobicilli that had undergone cell disruption. All bifidobacterial cultures gave a similar color formation whether sonication or CTAB addition was used to disrupt cells. Use of CTAB to disrupt cell membranes is an effective alternative to the time consuming traditional cell disruption procedures and increases the number of cultures that can be simultaneously assayed and presumptively identified using the phosphoketolase assay.     

Optimization of culture conditions of probiotic bifidobacteria for maximal adhesion to hexadecane

Culture growth conditions were optimized for adhesion to hexadecane of the probiotic Bifidobacterium bifidum HI 39 and HI 48. Among three growth media used, MILS lactose broth was the best medium to obtain maximum cell adhesion, followed by MRS and TPY lactose broth for B. bifidum HI 39 and HI 48. Increasing the incubation time from 6 to 18 h resulted in a gradual increase in percentage adhesion at 37 °C of both organisms in MILS, MRS and TPY media. Thereafter, incubation up to 48 h showed a marked reduction in adhesion of B. bifidum HI 39 and B. bifidum HI 48. When the test cultures were grown at pH values from 5.0 to 8.0 in MILS lactose broth at 37 °C for 18 h, there was a gradual enhancement in cell adhesion up to pH 7.0; but higher pH values retarded the bacterial adhesion. The study showed that the optimum conditions for adhesion to hexadecane of the selected bifidobacterial strains were pH 7.0 and incubation at 37 °C for 18 h in MILS broth.     

Biomarkers of human gastrointestinal tract regions

Dysregulation of intestinal epithelial cell performance is associated with an array of pathologies whose onset mechanisms are incompletely understood. While whole-genomics approaches have been valuable for studying the molecular basis of several intestinal diseases, a thorough analysis of gene expression along the healthy gastrointestinal tract is still lacking. The aim of this study was to map gene expression in gastrointestinal regions of healthy human adults and to implement a procedure for microarray data analysis that would allow its use as a reference when screening for pathological deviations. We analyzed the gene expression signature of antrum, duodenum, jejunum, ileum, and transverse colon biopsies using a biostatistical method based on a multivariate and univariate approach to identify region-selective genes. One hundred sixty-six genes were found responsible for distinguishing the five regions considered. Nineteen had never been described in the GI tract, including a semaphorin probably implicated in pathogen invasion and six novel genes. Moreover, by crossing these genes with those retrieved from an existing data set of gene expression in the intestine of ulcerative colitis and Crohn’s disease patients, we identified genes that might be biomarkers of Crohn’s and/or ulcerative colitis in ileum and/or colon. These include CLCA4 and SLC26A2, both implicated in ion transport. This study furnishes the first map of gene expression along the healthy human gastrointestinal tract. Furthermore, the approach implemented here, and validated by retrieving known gene profiles, allowed the identification of promising new leads in both healthy and disease states.     

Screening of Bifidobacterium strains isolated from human faeces for antagonistic activities against potentially bacterial pathogens

As probiotic bacteria, strains belonging to the genus Bifidobacterium colonise the gastro-intestinal tract of humans and animals at the time of birth, and they are found in young as well as in adult individuals in great numbers. Moreover, they can interact with the development of enteric infections by the production of antimicrobial metabolites. In this work 281 strains of bifidobacteria were anaerobically isolated from human faecal samples, supplied by volunteers of different ages (youngs, adults, elders), and preliminarly described by microscopic observation. All strains were screened by the fructose 6-phosphate phosphoketolase (F6PPK) test in order to confirm their classification within the genus Bifidobacterium. Selected strains were used to evaluate their antagonistic activities against Escherichia coli, Salmonella thyphimurium, Staphylococcus lentus, Enterococcus faecalis, Acinetobacter calcoaceticus, Sphingomonas paucimobilis, Listeria monocytogenes, Yersinia enterocolitica, Bacillus cereus, Clostridium sporogenes. Experiments were performed in vitro by different methods based on the observation of growth inhibition in Petri dishes. The strains that showed the highest inhibiting activities were compared by SDS-PAGE for total cell proteins, using type strains of human origin as references. Representative isolates were metabolically characterised by the BIOLOG system; a specific database was created with strains obtained from our collection and a statistical evaluation for metabolic patterns was carried out.