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.

Expression of four beta-galactosidases from Bifidobacterium bifidum NCIMB41171 and their contribution on the hydrolysis and synthesis of galactooligosaccharides

This paper deals with two aspects tightly related to the enzymatic characteristics and expression of four beta-galactosidases (BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171. The growth patterns of this strain indicated a preference towards complex (i.e. lactose, galactooligosaccharides (GOSs)) rather than simple carbohydrates (i.e. glucose and galactose) and a collaborative action and synergistic relation of more than one beta-galactosidase isoenzyme for either lactose or GOS hydrolysis and subsequent assimilation. Native polyacrylamide gel electrophoresis analysis of protein extracts from cells growing on different carbohydrates (i.e. glucose, lactose or GOS) indicated that two lactose hydrolysing enzymes (BbgI and BbgIII) and one GOS hydrolysing enzyme (BbgII) were constitutively expressed, whereas a fourth lactose hydrolysing enzyme (BbgIV) was induced in the presence of lactose or different GOS fractions. Furthermore, the beta-galactosidase expression profiles of B. bifidum cells and the transgalactosylating properties of each individual isoenzyme, with lactose as substrate, clearly indicated that mainly three isoenzymes (BbgI, BbgIII and BbgIV) are implicated in GOS synthesis when whole B. bifidum cells are utilised. Two of the isoenzymes (BbgI and BbgIV) proved to have better transgalactosylating properties giving yields ranging from 42% to 47% whereas the rest (BbgI and BbgIII) showed lower yields (15% and 29%, respectively).

Comparative analysis of four beta-galactosidases from Bifidobacterium bifidum NCIMB41171: purification and biochemical characterisation

Four different beta-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4-5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4-6.8). Na+ and/or K+ ions were prerequisite for BbgI and BbgIV activity in Bis-Tris-buffered solutions, whereas Mg++ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg++, Mn++ and Ca++ ions exerting the most positive effect. Determination of the specificity constants (k(cat)/K-m) clearly indicated that BbgI (6.11 x 10(4) s(-1) M-1), BbgIII (2.36 x 10(4) s(-1) M-1) and especially BbgIV (4.01 x 10(5) s(-1) M-1) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for beta-D-(1 -> 6) galactobiose (5.59 x 10(4) s(-1) M-1) than lactose (1.48 x 10(3) s(-1) M-1). Activity measurements towards other substrates (e. g. beta-D-(1 -> 6) galactobiose, beta-D-(1 -> 4) galactobiose, beta-D-(1 -> 4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the beta-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.

A novel alpha-galactosidase from I'ifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression

A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of a parts per thousand 243 kDa and a subunit size of a parts per thousand 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (a parts per thousand 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) a parts per thousand yen3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).

In vitro evaluation of the fermentation properties and potential prebiotic activity of Agave fructans

Aims: This study was carried out to evaluate in vitro the fermentation properties and the potential prebiotic activity of Agave-fructans extracted from Agave tequilana (Predilife). Methods and Results: Five different commercial prebiotics were compared using 24-h pH-controlled anaerobic batch cultures inoculated with human faecal slurries. Measurement of prebiotic efficacy was obtained by comparing bacterial changes, and the production of short-chain fatty acids (SCFA) was also determined. Effects upon major groups of the microbiota were monitored over 24 h incubations by fluorescence in situ hybridization. SCFA were measured by HPLC. Fermentation of the Agave fructans (Predilife) resulted in a large increase in numbers of bifidobacteria and lactobacilli. Conclusions: Under the in vitro conditions used, this study has shown the differential impact of Predilife on the microbial ecology of the human gut. Significance and Impact of the Study: This is the first study reporting of a potential prebiotic mode of activity for Agave fructans investigated which significantly increased populations of bifidobacteria and lactobacilli compared to cellulose used as a control.

Cetobacterium somerae sp nov from human feces and emended description of the genus Cetobacterium

Phenotypic and phylogenetic studies were performed on four isolates of an unidentified gram-negative, microaerotolerant, non-spore-forming, rod-shaped bacterium isolated from the feces of children. The unknown organism was bile resistant and produced acetic acid as the major end product of metabolism of peptides and carbohydrates. It possessed a low DNA G + C content of 31 mol %. Comparative 16S rRNA gene sequencing demonstrated that the four isolates were phylogenetically identical (100% 16S rRNA sequence similarity) and represent a hitherto unknown sub-line within the genus Cetobacterium. The novel bacterium displayed approximately 5% sequence divergence with Cetobacterium ceti, and can be readily distinguished from the latter by physiological and biochemical criteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown fecal bacterium be classified in the genus Cetobacterium, as Cetobacterium somerae sp. nov. The proposed type strain of Cetobacterium somerae is WAL 14325(T) (ATCC BAA-474(T) = CCUG 46254T).

Streptococcus devriesei sp nov., from equine teeth

Phenotypic and phylogenetic studies were performed on four unidentified Gram-positive staining, catalase-negative, cc-hemolytic Streptococcus-like organisms recovered from the teeth of horses. SDS PAGE analysis of whole-cell proteins and comparative 16S rRNA gene sequencing demonstrated the four strains were highly related to each other but that they did not correspond to any recognised species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms form a hitherto unknown sub-line within the Streptococcus genus, displaying a close affinity with Streptococcus mutans, Streptococcus ferus and related organisms. Sequence divergence values of > 5% with thew and other reference streptococcal species however demonstrated the organisms from equine sources represent a novel species. Based on the phenotypic distinctiveness of the new bacterium and molecular chemical and molecular genetic evidence, it is proposed that the unknown species be classified as Streptococcus devriesei sp. nov. The type strain of Streptococcus devriesei is CCUG 47155(T) (= CIP 107809T).

A two-stage continuous culture system to study the effect of supplemental alpha-lactalbumin and glycomacropeptide on mixed cultures of human gut bacteria challenged with enteropathogenic Escherichia coli and Salmonella serotype Typhimurium

Aims: Certain milk factors may promote the growth of a gastrointestinal microflora predominated by bifidobacteria and may aid in overcoming enteric infections. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Methods and Results: Infant faecal specimens were used to ferment formulae supplemented with glycomacropeptide (GMP) and alpha-lactalbumin (alpha-la) in a two-stage compound continuous culture model. At steady state, all fermenter vessels were inoculated with 5 ml of 0.1 M phosphate-buffered saline (pH 7.2) containing 10(8) CFU ml(-1) of either enteropathogenic Escherichia coli 2348/69 (O127:H6) or Salmonella serotype Typhimurium (DSMZ 5569). Bacteriology was determined by independent fluorescence in situ hybridization. Vessels that contained breast milk (BM), as well as alpha-la and GMP supplemented formula had stable total counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and E. coli decreased significantly in all three groups prior to pathogen addition. Escherichia coli counts decreased in vessels containing BM and alpha-la while Salmonella decreased significantly in all vessels containing BM, alpha-la and GMP. Acetate was the predominant acid. Significance and Impact of the Study: Supplementation of infant formulae with appropriate milk proteins may be useful in mimicking the beneficial bacteriological effects of breast milk.

Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine

Aims: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. Methods and Results: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. Conclusions: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. Significance and impact of the Study: Selected strains could be used as adjunct starters to make high quality Feta cheese.

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

Aims: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. Materials and Methods: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. Results: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. Conclusions: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.

Inactivation of Escherichia coli by citral

Aims: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral, and (iii) possible synergistic effects of mild heat treatment and pulsed-electric fields (PEF) treatment combined with citral. Methods and Results: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 µl l-1of citral at pH 4.0 for 24 h at 20 ºC caused the inactivation of more than 5 log10 cycles of cells starting at an inoculum size of 106 or 107 CFU ml-1, whereas increasing the cell concentration to 109 CFU ml-1 caused less than 1 log10 cycle of inactivation. E. coli showed higher resistance to citral at pH 4.0 than pH 7.0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild-type strain. Occurrence of sublethal injury to both, the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally-injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. Conclusions: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Significance and Impact of Study: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

High hydrostatic pressure resistance of Campylobacter jejuni after different sublethal stresses

Aims: To study the development of resistance responses in Campylobacter jejuni to High Hydrostatic Pressure (HHP) treatments after the exposure to different stressful conditions that may be encountered in food processing environments, such as acid pH, elevated temperatures and cold storage. Methods and Results: C. jejuni cells in exponential and stationary growth phase were exposed to different sublethal stresses (acid, heat and cold shocks) prior to evaluate the development of resistance responses to HHP. For exponential-phase cells, neither of the conditions tested increased nor decreased HHP resistance of C. jejuni. For stationary-phase cells, acid and heat adaptation sensitized C. jejuni cells to the subsequent pressure treatment. On the contrary, cold-adapted stationary-phase cells developed resistance to HHP. Conclusions: Whereas C. jejuni can be classified as a stress sensitive microorganism, our findings have demonstrated that it can develop resistance responses under different stressing conditions. The resistance of stationary phase C. jejuni to HHP was increased after cells were exposed to cold temperatures. Significance and Impact of the Study: The results of this study contribute to a better knowledge of the physiology of C. jejuni and its survival to food preservation agents. Results here presented may help in the design of combined processes for food preservation based on HHP technology.

Characterization of some strains from human clinical sources which resemble “Leptotrichia sanguinegens”: description of Sneathia sanguinegens sp. nov., gen. nov.

Three strains of a gram-negative, blood or serum requiring, rod-shaped bacterium recovered from human clinical specimens were characterised by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed the unknown rod-shaped strains are members of the same species as some fastidious isolates recovered from human blood specimens and previously designated "Leptotrichia sanguinegens". Based on phylogenetic and phenotypic evidence, it is proposed that the isolates from human sources be classified in a new genus Sneathia, as Sneathia sanguinegens gen. nov., sp. nov. The type strain of Sneathia sanguinegens is CCUG 41628T.

Isolation and characterization of human colonic bacteria able to hydrolyse chlorogenic acid

Free hydroxycinnamates, including caffeic, ferulic and p-coumaric acids, exhibit antioxidant and anticarcinogenic properties both in vitro and in animal models. Given that the gut flora has a major role in human nutrition and health, some of the beneficial effects of phenolic acids may be ascribed to the microflora involved in metabolism.