Emulsifying, rheological and physicochemical properties of exopolysaccharide produced by Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205

The rheological, emulsification and certain physicochemical properties of purified exopolysaccharides (EPS) of Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 were studied and compared with those of guar gum and xanthan gum. The two strains were grown in skim milk supplemented with 1.5% (w/v) casein hydrolysate at 37 ◦C for 24 h; they both produced heteropolysaccharides with different molecular mass and composition. The carbohydrate content of both polymers was more than 92% and no protein was detected. The EPS of B. longum subsp. infantis CCUG 52486 showed highly branched entangled porous structure under scanning electron microscopy. Higher intrinsic viscosity was observed for the EPS of B. longum subsp. infantis CCUG 52486 compared to the EPS of B. infantis NCIMB 702205 and guar gum. Both polymers showed pseudoplastic non-Newtonian fluid behaviour in an aqueous solution. The EPS of B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486 produced more stable emulsions with orange oil, sunflower seed oil, coconut oil and xylene compared to guar and xanthan gum. The EPS of B. longum subsp. infantis CCUG 52486 is the most promising one for applications in the food industry, as it had higher intrinsic viscosity, higher apparent viscosity in aqueous solution, porous dense entangled structure and good emulsification activity.

Screening human intestinal Bifidobacterium strains for growth, acidification, EPS production and viscosity potential in low-fat milk

Bifidobacterium strains of human origin were screened for their ability to grow in milk and produce exopolysaccharides (EPS). Bifidobacterium strains were grown in low-fat UHT milk and were evaluated for their growth, acidification properties, EPS production and ability to increase the viscosity of fermented milk. The strains that grew well in milk were strains of Bifidobacterium breve and Bifidobacterium longum and B. longum subsp. longum. Among the 22 strains, EPS was produced by Bifidobacterium bifidum ALM 35, B. breve NCIMB 8807 (UCC 2003), B. longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 at concentrations ranging from 25 to 140 . The molecular mass and the composition varied considerably, depending on the strain. Analysis of the correlation between the apparent viscosity of the fermented milk and pH indicated that the EPS produced during the acidification of milk possibly contributed to the viscosity of the milk products.

Effect of dairy-based protein sources and temperature on growth, acidification and exopolysaccharide production of Bifidobacterium strains in skim milk

The aim of the present study was to find out the best growing conditions for exopolysaccharide (EPS) producing bifidobacteria, which improve their functionality in yoghurt-like products. Two Bifidobacterium strains were used in this study, Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205. In the first part of the study the effect of casein hydrolysate, lactalbumin hydrolysate, whey protein concentrate and whey protein isolate, added at 1.5% w/v in skim milk, was evaluated in terms of cell growth and EPS production; skim milk supplemented with yeast extract served as the control. Among the various nitrogen sources, casein hydrolysate (CH) showed the highest cell growth and EPS production for both strains after 18 h incubation and therefore it was selected for subsequent work. Based on fermentation experiments using different levels of CH (from 0.5 to 2.5% w/v) it was deduced that 1.5% (w/v) CH resulted in the highest EPS production, yielding 102 and 285 mg L− 1 for B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486, respectively. The influence of temperature on growth and EPS production of both strains was further evaluated at 25, 30, 37 and 42 °C for up to 48 h in milk supplemented with 1.5% (w/v) CH. The temperature had a significant effect on growth, acidification and EPS production. The maximum growth and EPS production were recorded at 37 °C for both strains, whereas no EPS production was observed at 25 °C. Lower EPS production for both strains were observed at 42 °C, which is the common temperature used in yoghurt manufacturing compared to that at 37 °C. The results showed that the culture conditions have a clear effect on the growth, acidification and EPS production, and more specifically, that skim milk supplemented with 1.5% (w/v) CH could be used as a substrate for the growth of EPS-producing bifidobacteria, at 37 °C for 24 h, resulting in the production of a low fat yoghurt-like product with improved functionality.

Microbiological, chemical and rheological properties of low fat set yoghurt produced with exopolysaccharide (EPS) producing Bifidobacterium strains.

In this work, the microbiological and physicochemical differences of three types of low fat set yoghurts were studied, as well as the changes taking place during storage at 4 °C for 28 days. The first yoghurt was produced with yoghurt starters and exopolysaccharide (EPS) producing Bifidobacterium longum subsp. infantis CCUG 52486 (CCUGY), the second with yoghurt starters and Bifidobacterium infantis NCIMB 702205 (NCIMBY) and the third with just yoghurt starters (control yoghurt). No significant differences were observed in terms of cell concentrations; for all three yoghurts, similar final cell concentrations were obtained for the yoghurt starter cultures (~7.5 log cfu g−1) and the Bifidobacterium strains (~7.8 log cfu g−1). Both Bifidobacterium survived well during storage, as in both cases the cell viability decreased by less than 0.5 log cfu g−1after 28 days of storage. A decrease in pH followed by an increase in lactic acid was observed during storage for all three yoghurts, which was mostly attributed to the activity of the yoghurt starter cultures. The two yoghurts with the EPS producing Bifidobacterium strains exhibited lower syneresis than the control yoghurt. The lowest was shown by CCUGY, which also exhibited the highest storage modulus and firmness, and a well defined porous web-like structure in cryo-SEM. Examination of the micro-structure of the yoghurts using cryo-scanning electron microscopy (cryo-SEM) indicated that the above observations were due to the interaction between the EPS and the milk proteins. Overall, the results indicated that the EPS producing Bifidobacterium longum subsp. infantis CCUG 52486 is the most promising strain, and can be used with yoghurt starter cultures to manufacture low fat set yoghurt with probiotic activities and at the same time enhanced physicochemical and rheological properties.

The impact of host-microbe interactions on murine colonic secretomotor function

The overall objective of this thesis was to gain further insight into the mechanisms underlying commensal microbial influences on intestinal ion transport. In this regard, I examined the impact of commensal host-microbe interactions on colonic secretomotor function in mouse. I first examined the influence of two different probiotic (microorganisms which, when given in adequate amounts, can confer health benefits upon the host) strains, Bifidobacterium infantis 35624 and L. salivarius UCC118 on active colonic ion transport in the mouse, using the Ussing Chamber. I found that both probiotics appear to have converging effects on ion transport at a functional level. However, L. salivarius UCC118 may preferentially inhibit neurally-evoked ion transport. Next I examined the impact of the host microbiota itself on both baseline and stimulated colonic secretomotor function as well as probiotic induced changes in ion transport. I provide further evidence that the microbiota is capable of mediating alterations in colonic ion transport, and specifically suggests that it can influence cAMP-mediated responses. Finally, it has been well documented that many probiotics elicit their effects via secreted bioactives, therefore, I studied the effects of microbially produced GABA, contained in supernatants from the commensal microbe Lactobacillus brevis DPC6108, on colonic secretomotor function. In conclusion, I believe that commensal microbes have an important and strain specific functional influence on colonic ion transport and secretomotor function and these effects can be mediated via extracellular bioactives. Moreover, I believe that functional ex-vivo studies such as those carried out in this thesis have a critical role to play in our future understanding of host-microbe interactions in the gut.

Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKQ, which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

Effect of inulin on the growth and metabolism of a probiotic strain of Lactobacillus rhamnosus in co-culture with Streptococcus thermophilus

Metabolic studies are very important to improve quality of functional dairy products. For this purpose, the behaviors of pure cultures of Streptococcus thermophilus (St) and Lactobacillus rhamnosus (Lr) as well a co-culture of them (St-Lr) were investigated during skim milk fermentation, and the inulin effect as prebiotic was assessed. Lr was able to metabolize 6 g/100 g more galactose than St and St-Lr. Final lactic acid production by Lr was higher (9.8 g/L) compared to St (9.1 g/L) and St-Lr (9.1 g/L). Acetic acid concentration varied from 0.8 g/L (St-Lr) to 1.5 g/L (Lr) and that of ethanol from only 0.2 g/L (St-Lr) to 0.4 g/L (Lr), which suggests the occurrence in Lr of a NADH oxidase activity and citrate co-metabolization via pyruvate, both dissipating a part of the reducing power. Diacetyl and acetoin accumulated at the highest levels (18.4 and 0.8 mg/L, respectively) with St-Lr, which suggests possible synergistic interactions between these microorganisms as well as the Lr capability of co-metabolizing citrate in the presence of lactose. Inulin stimulated both biomass growth and levels of all end-products, as the likely result of fructose release from its partial hydrolysis and subsequent metabolization as an additional carbon and energy source. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

In vitro effects of phosphatidylcholine and transgalactooligosaccharides on the production of 1,2-sn-diacylglycerol by Bifidobacterium longum biovar infantis

Aims: To investigate the production of the tumour promoter 1,2-sn-diacylglycerol (DAG) by a human gut isolate of Bifidobacterium longum biovar infantis. Methods and Results: Bifidobacterium longum biovar infantis was grown in vitro using anaerobic static batch cultures in the presence of phosphatidylcholine (PC) and trans-galactooligosaccharides (TOS). Production of DAG was found to be dependent upon the presence of PC, while TOS had a reducing effect. Considerable differences in morphology, growth and metabolic end products from the micro-organism were observed under the different culture conditions. Conclusions: Our results have provided evidence that B. longum biovar infantis can produce DAG in vitro and that a prebiotic exerted a reducing effect upon this production. Significance and Impact of the Study: The results presented in this study demonstrate an ability of ostensibly beneficial member of the colonic environment to produce unwanted compounds under certain conditions. Therefore, it may be important that a combination of substrates and other factors are assessed when studying the behaviour of any bacterial group or species, especially when designing the dietary interventions.

Evidence of immunomodulatory effects of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52486

Bifidobacterium longum bv. infantis CCUG 52486 was originally isolated from healthy elderly subjects and demonstrated to have particular ecological fitness and anti-pathogenic effects. Bifidobacteria are commonly associated with immunomodulatory properties, especially in older people, but this strain has not been investigated for effects on immune function. This study aimed to explore the immunomodulatory effects of this novel probiotic, compared with three commercial strains, B. longum SP 07/3, L. rhamnosus GG (L.GG) and L. casei Shirota (LcS). Human peripheral blood mononuclear cells (PBMCs) were isolated from fasting blood of young or older volunteers and exposed to probiotic strains or Con A. NK activity and activation, and cytokine release were enhanced by all probiotics with strain-specificities. The effect of B. infantis on NK activity was influenced by ageing. Except for L.GG, probiotics increased IFN-γ production to a much greater degree in young subjects, and increased IL-6 production to a much greater degree in older subjects. Based on IL-10/IL-12 ratios, B. infantis resulted in the most anti-inflammatory profile of all of the probiotics. These results suggest that B. infantis CCUG 52486 has strong immunomodulatory potential compared with well-known commercial strains, and that the immune response to probiotics may be influenced by ageing.

Molecular cloning and comparative analysis of four beta-galactosidase genes from Bifidobacterium bifidum NCIMB41171

Bifidobacterium bifidum NCIMB41171 carries four genes encoding different beta-galactosidases. One of them, named bbgIII, consisted of an open reading frame of 1,935 amino acid (a.a.) residues encoding a protein with a multidomain structure, commonly identified on cell wall bound enzymes, having a signal peptide, a membrane anchor, FIVAR domains, immunoglobulin Ig-like and discoidin-like domains. The other three genes, termed bbgI, bbgII and bbgIV, encoded proteins of 1,291, 689 and 1,052 a.a. residues, respectively, which were most probably intracellularly located. Two cases of protein evolution between strains of the same species were identified when the a.a. sequences of the BbgI and BbgIII were compared with homologous proteins from B. bifidum DSM20215. The homologous proteins were found to be differentiated at the C-terminal a.a. part either due to a single nucleotide insertion or to a whole DNA sequence insertion, respectively. The bbgIV gene was located in a gene organisation surrounded by divergently transcribed genes putatively for sugar transport (galactoside-symporter) and gene regulation (LacI-transcriptional regulator), a structure that was found to be highly conserved in B. longum, B. adolescentis and B. infantis, suggesting optimal organisation shared amongst those species.

Examination of faecal Bifidobacterium populations in breast- and formula-fed infants during the first 18 months of life.

Bifidobacteria in the infant faecal microbiota have been the focus of much interest, especially during the exclusive milk-feeding period and in relation to the fortification of infant formulae to better mimic breast milk. However, longitudinal studies examining the diversity and dynamics of the Bifidobacterium population of infants are lacking, particularly in relation to the effects of weaning. Using a polyphasic strategy, the Bifidobacterium populations of breast- and formula-fed infants were examined during the first 18 months of life. Bifidobacterium-specific denaturing gradient gel electrophoresis demonstrated that breast-fed infants harboured greater diversity than formula-fed infants and the diversity of the infants' Bifidobacterium populations increased with weaning. Twenty-seven distinctive banding profiles were observed from ∼1100 infant isolates using ribosomal intergenic spacer analysis, 14 biotypes of which were confirmed to be members of the genus Bifidobacterium. Two profiles (H, Bifidobacterium longum subsp. infantis; and I, Bifidobacterium bifidum) were common culturable biotypes, seen in 9/10 infants, while profile E (Bifidobacterium breve) was common among breast-fed infants. Overall, inter- and intra-individual differences were observed in the Bifidobacterium populations of infants between 1 and 18 months of age, although weaning was associated with increased diversity of the infant Bifidobacterium populations. Breast-fed infants generally harboured a more complex Bifidobacterium microbiota than formula-fed infants.

Papel de cepas de Streptococcus mutans e Bifidobacterium spp. na etiologia ou proteção contra doenças bucais

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genome analysis and characterisation of the exopolysaccharide produced by Bifidobacterium longum subsp. longum 35624™

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

Bifidobacterium breve UCC2003 metabolises the human milk oligosaccharides lacto-N-tetraose and lacto-N-neo-tetraose through overlapping, yet distinct pathways

In this study, we demonstrate that the prototype B. breve strain UCC2003 possesses specific metabolic pathways for the utilisation of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), which represent the central moieties of Type I and Type II human milk oligosaccharides (HMOs), respectively. Using a combination of experimental approaches, the enzymatic machinery involved in the metabolism of LNT and LNnT was identified and characterised. Homologs of the key genetic loci involved in the utilisation of these HMO substrates were identified in B. breve, B. bifidum, B. longum subsp. infantis and B. longum subsp. longum using bioinformatic analyses, and were shown to be variably present among other members of the Bifidobacterium genus, with a distinct pattern of conservation among human-associated bifidobacterial species.