In Vitro Fermentation of Breast Milk Oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study, Bifidobacterium infantis was shown to ferment purified HMO as a sole carbon source, while another gut commensal, Lactobacillus gasseri, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.     

短双歧杆菌对鼠伤寒沙门氏菌的抑制

【背景】鼠伤寒沙门氏菌是主要的肠道病原菌之一,利用益生菌治疗肠道病原菌感染已成为一种新型、绿色的微生态疗法。【目的】研究筛选出的短双歧杆菌无细胞发酵上清液(Cell-free supernatant,CFS)对鼠伤寒沙门氏菌的体外抑制作用及机制。【方法】采用微量稀释法测定短双歧杆菌YH68 CFS对鼠伤寒沙门氏菌的最小抑菌浓度(Minimum inhibitory concentration,MIC)和亚抑制浓度(Sub-inhibitory concentrations,SIC),并从鼠伤寒沙门氏菌的细胞形态、细胞膜通透性、膜完整性以及毒力基因表达的变化探讨YH68 CFS对鼠伤寒沙门氏菌的抑菌机理,同时检测YH68 CFS对鼠伤寒沙门氏菌粘附和侵袭肠上皮细胞HT29的影响。【结果】YH68 CFS (3×109 CFU/mL)对鼠伤寒沙门氏菌具有较好的抑制效果,抑菌圈直径为22.27±0.44 mm,最小抑菌浓度为250μL/mL,对鼠伤寒沙门氏菌的抑制机制是通过增加其细胞膜通透性破坏其完整性,形成难以修复的孔洞,最终达到抑菌的目的;亚抑制浓度为62.5μL/mL时YH68 CFS并不能影响鼠伤寒沙门氏菌的生长,但仍然能通过下调毒力基因表达的方式抑制其对肠上皮细胞的粘附和入侵。【结论】短双歧杆菌YH68对鼠伤寒沙门氏菌具有良好的抑菌作用,可作为治疗沙门氏菌感染的潜在益生菌。     

Structural Basis for Norovirus Inhibition by Human Milk Oligosaccharides

Histo-blood group antigens (HBGAs) are important binding factors for norovirus infections. We show that two human milk oligosaccharides, 2′-fucosyllactose (2′FL) and 3-fucosyllactose (3FL), could block norovirus from binding to surrogate HBGA samples. We found that 2′FL and 3FL bound at the equivalent HBGA pockets on the norovirus capsid using X-ray crystallography. Our data revealed that 2′FL and 3FL structurally mimic HBGAs. These results suggest that 2′FL and 3FL might act as naturally occurring decoys in humans.     

人乳寡糖的分离分析

糖是一类重要的生命活性物质,它不仅是细胞能量代谢的源泉,还常作为信号分子参与细胞的各种活动。人乳寡糖（human milk oligosaccharides,HMOs）在人乳干物质中的含量仅次于乳糖和脂肪,高于蛋白质,对婴儿的发育和健康具有重要作用。为了更好的理解人乳寡糖的生物功能和结构的关系,对其组成和结构开展分析研究是必不可少的。对人乳寡糖进行了简要的介绍,并就其预处理方法、分离分析和结构表征方法进行了综述,以期为人乳寡糖的深入研究提供参考。     

人乳寡糖的结构及其分离分析

母乳中存在的人乳寡糖（HMOs）是一类结构高度复杂的低聚糖，对婴儿的肠道菌群、免疫屏障、大脑发育发挥积极作用。由于母乳中基质复杂，寡糖的种类繁多，丰度跨度大，存在众多异构体，这都使得检测面临诸多挑战。现已有多种技术用于HMOs的分析，发现了200多种HMOs，液相色谱和毛细管电泳在分离HMOs方面效果显著，核磁共振、质谱、红外多光子解离光谱推动了对HMOs结构的全面解析。本文回顾了对HMOs实现高灵敏度和高特异性分析的多种技术方法，比较了不同技术的优缺点，还重点介绍了质谱以及不同技术联用在推动HMOs解析和测定方面的突破，为探究寡糖的结构-功能关系、深入理解HMOs的生物学功能提供了全面的技术支持。     

人乳低寡糖的结构及其特殊功能

人乳低寡糖（HMO）由D-葡萄、D-半乳糖、N-乙酰葡萄糖胺、L-岩藻糖和N-乙酰神经氨酸等5种基本结构构成。基本结构又以不同比例和特殊连接方式形成9种核心结构,再经过多种变化构成人乳中100多种低寡糖。HMO有维护肠道生态平衡、抗御肠道感染、增强人体免疫力,预防癌症发生,防止慢发性结肠炎和促进新生儿大脑发育等功能。     

The presence of plasmids in Bifidobacterium breve

The presence of plasmids in Bifidobacterium breve was demonstrated by results of agarose gel electrophoresis, carbohydrate fermentation analysis, and DNA-DNA hybridization.     

Anti-obesity activities of fermented soygerm isoflavones by Bifidobacterium breve

Soygerm isoflavones were subjected to fermentation by Bifidobacterium breve. Most of isoflavone glycosides (daidzin, glycitin and genistin) in soygerms were deglycosylated to their corresponding isoflavone aglycones (daidzein, glycitein and genistein) within 24 h fermentation. Fermented isoflavones significantly inhibited pancreatic lipase activity in fermentation-time and dosage dependant manner. When fermented isoflavones were orally administered with olive oil to SD rats, the triglyceride (TG) level in plasma after 2 h of ingestion was significantly lower than the control of only olive oil administered group whereas no such significant decrease in plasma TG was observed in unfermented isoflavone administered group. This result indicates that oral administration of fermented isoflavones effectively suppressed absorption of excessive lipid into a body. Addition of either unfermented or fermented soygerm isoflavones effectively inhibited adipocyte differentiation from 3T3-L1 in a dose dependent manner. In conclusion, B. breve successfully converted soygerm isoflavones into their aglycones, and these aglycones were more effective in suppressing lipid absorption as well as adipocytes differentiation than their glycosides.     

Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.     

Comparative genomics of the Bifidobacterium breve taxon

Background

Bifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract (GIT) of a broad range of hosts, where their presence is positively correlated with the host’s health status. In this study, we assessed the genomes of thirteen representatives of Bifidobacterium breve, which is not only a frequently encountered component of the (adult and infant) human gut microbiota, but can also be isolated from human milk and vagina.

Results

In silico analysis of genome sequences from thirteen B. breve strains isolated from different environments (infant and adult faeces, human milk, human vagina) shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. These latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign DNA (i.e. CRISPR loci and restriction/modification systems), and genes that encode enzymes responsible for carbohydrate fermentation. Gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol.

Conclusions

Genome analysis of thirteen representatives of the B. breve species revealed that the deduced pan-genome exhibits an essentially close trend. For this reason our analyses suggest that this number of B. breve representatives is sufficient to fully describe the pan-genome of this species. Comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of B. breve.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-170) contains supplementary material, which is available to authorized users.     

Investigation of protein export in Bifidobacterium breve UCC2003

The molecular interactions between the bifidobacterial cell and its natural environment, namely, the gastrointestinal tract of its host, are particularly important in understanding the presumed positive effects of Bifidobacterium on the health status of the host. In this study an export-specific reporter system, designed for use in gram-positive organisms and based on the use of the staphylococcal nuclease (Nuc) as a reporter, was employed to identify exported proteins in Bifidobacterium breve UCC2003. A B. breve genomic library of translational fusions to the Nuc-encoding gene devoid of its own export signal was established in the shuttle vector pFUN (I. Poquet, S. D. Ehrlich, and A. Gruss, J. Bacteriol. 180:1904-1912, 1998) and screened for bifidobacterial export signals. Sequence analysis of the fusion proteins obtained that displayed a nuclease-producing phenotype in both Lactococcus lactis and B. breve predicted the presence of a classical signal peptide and/or single or multiple transmembrane domains, thus indicating that some of the export signals in B. breve are comparable to those used in L. lactis. Cell fractionation studies, zymograms, nuclease assays, and Western blotting were employed to confirm the function of the predicted signals and to determine the location and activity of the exported fusion proteins in B. breve and/or L. lactis.     

Transport and metabolism of glucose and arabinose in Bifidobacterium breve

Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using d-[U-¹⁴C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of d-[U-¹⁴C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DG had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DG by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.Abbreviations (2,4-DNP) 2,4-dinitrophenol - (2,4-DNP) carbonylcyanide m-chlorophenylhydrazone - (CCCP) (phosphoenolpyruvate phosphotransferase system) - PEP: PTS trichloroacetic acid - (TCA) 2-deoxy-d-glucose - (2-DG) 2-deoxy-d-glucose     

Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I,a Major Building Block of Human Milk Oligosaccharides,in Bifidobacterial Strains

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.Bifidobacteria are gram-positive anaerobic bacteria that naturally colonize the human intestinal tract and are believed to be beneficial to human health (21, 30). Breastfeeding has been shown to be associated with an infant fecal microbiota dominated by bifidobacteria, whereas the fecal microbiota of infants who are consuming alternative diets has been described as being mixed and adult-like (12, 21). It has been suggested that the selective growth of bifidobacteria observed in breast-fed newborns is related to the oligosaccharides and other factors that are contained in human milk (human milk oligosaccharides [HMOs]) (3, 4, 10, 11, 16, 17, 34). Kitaoka et al. (15) have recently found that bifidobacteria possess a unique metabolic pathway that is specific for lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc). LNB is a building block for the type 1 HMOs [such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and lacto-N-difucohexaose I (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc)], and GNB is a core structure of the mucin sugar that is present in the human intestine and milk (18, 27). The GNB/LNB pathway, as previously illustrated by Wada et al. (33), involves proteins/enzymes that are required for the uptake and degradation of disaccharides such as the GNB/LNB transporter (29, 32), galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP; LnpA) (15, 24) (renamed from lacto-N-biose phosphorylase after the finding of phosphorylases specific to GNB [23] and LNB [22]), N-acetylhexosamine 1-kinase (NahK) (25), UDP-glucose-hexose 1-phosphate uridylyltransferase (GalT), and UDP-galactose epimerase (GalE). Some bifidobacteria have been demonstrated to be enzymatically equipped to release LNB from HMOs that have a type 1 structure (lacto-N biosidase; LnbB) (33) or GNB from the core 1-type O-glycans in mucin glycoproteins (endo-α-N-acetylgalatosaminidase) (6, 13, 14). It has been suggested that the presence of the LnbB and GNB/LNB pathways in some bifidobacterial strains could provide a nutritional advantage for these organisms, thereby increasing their populations within the ecosystem of these breast-fed newborns (33).The species that predominantly colonize the infant intestine are the bifidobacterial species B. breve, B. longum subsp. infantis, B. longum subsp. longum, and B. bifidum (21, 28). On the other hand, strains of B. adolescentis, B. catenulatum, B. pseudocatenulatum, and B. longum subsp. longum are frequently isolated from the adult intestine (19), and strains of B. animalis subsp. animalis, B. animalis subsp. lactis, B. thermophilum and B. pseudolongum have been shown to naturally colonize the guts of animals (1, 2, 7, 8). However, it is unclear whether there is a relationship between the differential colonization of the bifidobacterial species and the presence of the GNB/LNB pathway. In the present study, we investigated the ability of individual bifidobacterial strains in the in vitro fermentation of LNB and in addition, we also tried to determine whether or not the GLNBP gene (lnpA), which is a key enzyme of the GNB/LNB pathway, was present.