Effects of growth conditions on the ion composition of Bifidobacterium bifidum subsp. pennsylvanicum

The cation content of Bifidobacterium bifidum subsp. pennsylvanicum was markedly influenced by the washing procedure of the cells, by the growth phase and the temperature, and by the composition of the culture medium. Optimal retention of cations was achieved by washing with 0.25 M MgCl₂ at 20 C. The intracellular Na⁺ concentration rose during growth in normal medium to a constant value in the stationary phase, the K⁺ concentration rose in the exponential phase, but fell in the stationary phase. Cells from 29-C cultures contained more Na⁺ and less K⁺ in the stationary phase than did cells from 37-C cultures, but the total cation content was the same at 29 and 37 C.Intracellular Na⁺ and K⁺ concentrations were dependent on the concentrations in the medium and on its osmolarity. The intracellular Na⁺/K⁺ ratio varied from 0.04 to 2.3. The concentrations of Na⁺, K⁺ and phosphate in the medium hardly affected growth. Mg²⁺-deficiency of the medium markedly decreased the concentration of Mg²⁺ within the cell; its concentration in the cell sap was greatly affected, but the amount of sedimentable, bound Mg²⁺ only slightly. The content of K⁺ within the cell decreased in Mg²⁺-deficient medium, but the concentration of Na⁺ did not. Omission of Tween 80 as well as its substitution by Tween 20 caused a decrease of intracellular K⁺. Cells from Tween 40 and Tween 60 cultures additionally contained markedly less Na⁺.The present investigations have been carried out with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Netherlands Foundation for Chemical Research (SON).     

Immunochemical studies on the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum

Antisera to lipoteichoic acid of Bifidobacterium bifidum subsp. pennsylvanicum were obtained by injecting lipoteichoic acid/methylated BSA complexes into rabbits. Precipitin tests showed that the glycerol phosphate backbone is primarily responsible for serological specificity while the polysaccharide part of the molecule plays a minor role. Whole cells of B. bifidum subsp. pennsylvanicum were capable of absorbing antibodies, indicating the presence of lipoteichoic acid (14% of the total content) at or near the bacterial surface. Cross-reactivity with strains of the genera Bifidobacterium and Lactobacillus was tested using absorption of antiserum by whole bacteria and reactivity of phenol extracts. The results indicated that lipoteichoic acid is a common antigen within the genus Bifidobacterium. The cross-reactivity with the lactobacilli tested was very low.     

Cell surface hydrophobicity ofBifidobacterium bifidum subsp.pennsylvanicum

The possible role of lipoteichoic acid with respect to cell surface properties ofBifidobacterium bifidum subsp.pennsylvanicum was studied. Standard suspensions of bacteria were mixed with octane or xylene.B. bifidum subsp.pennsylvanicum was shown to possess a strongly hydrophobic cell surface. Hydrophobicity of the bacteria could be reduced by treatment with trypsin, pepsin (at pH 4.5), HCl and penicillin. The latter treatment resulted in an increased excretion of lipoteichoic acid. Albumin was capable of inhibiting the adherence to octane when it was present in the assay buffer. The data suggest that both protein and lipoteichoic acid may be involved in cell surface hydrophobicity. A great divergence in cell surface properties was observed within the genusBifidobacterium.     

Structure of the lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum

The lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum were extracted from cytoplasmic membranes or from disintegrated bacteria with aqueous phenol and purified by gel chromatography. The lipoteichoic acid preparations contained phosphate, glycerol, galactose, glucose and fatty acids in a molar ratio of 1.0:1.0:1.3:1.2:0.3. Chemical analysis and NMR studies of the native preparations and of products from various acid and alkaline hydrolysis procedures gave evidence for the structure of two lipoteichoic acids. The lipid anchor appeared to be 3-O-(6'-(sn-glycero-1-phosphoryl)diacyl-beta-D-galactofuranosyl)-sn-1, 2-diacylglycerol. The polar part showed two structural features not previously described for lipoteichoic acids. A 1,2-(instead of the usual 1,3-) phosphodiester-linked sn-glycerol phosphate chain is only used substituted at the terminal glycerol unit with a linear polysaccharide, containing either beta(1----5)-linked D-galactofuranosyl groups or beta(1----6)-linked D-glucopyranosyl groups.     

'Lipoteichoic acid' of Bifidobacterium bifidum subspecies pennsylvanicum DSM 20239. A lipoglycan with monoglycerophosphate side chains

The lipid macroamphiphile of Bifidobacterium bifidum subsp. pennsylvanicum DSM 20239 was extracted with phenol/water and purified by treatment with nucleases and hydrophobic interaction chromatography. From analytical data, the results of Smith degradation, hydrolysis with HF and methylation studies, the following structure is proposed: (formula; see text) where n and m are approximately 7-10 and 8-15, respectively. The monoglycerophosphate residues have the sn-glycero-1-phosphate configuration; 20-50% of them are substituted with L-alanine in ester linkage. The lipid anchor is most likely a galactosyldiacylglycerol, part of which carries a third fatty acid. This is the first example among gram-positive bacteria of a glycerophosphate-containing lipid macroamphiphile that carries the glycerophosphate residues as monomeric side chains on a lipoglycan. Further, it contains L-alanine in place of the D-alanine found in lipoteichoic acids.     

两歧双歧杆菌86321生长特性的研究

目的研究两歧双歧杆菌86321的生长特性,为该菌生理功能研究和高效发酵剂的研制提供理论依据。方法通过生长曲线、产酸量、最适厌氧方式、最适pH、最适培养温度及最适接种量等一系列实验,对两歧双歧杆菌86321进行生长特性的研究。结果两歧双歧杆菌86321在BL培养基中培养时间可缩短至16 h,最高活菌数的lg值达到9.5;其最适厌氧方式为自然厌氧法或密封法,装液量视实际情况而定;在pH7.08.0生长良好,最适初始pH为8.0;在3742℃生长良好,最适温度为37℃;综合总菌量和生产成本,确定最适接种量为7%（v/v）。结论用BL培养基可以大大提高两歧双歧杆菌86321的产量。细菌产量的高低和发酵速度的快慢与菌种活力、厌氧方式、培养温度及pH等因素密切相关。     

Feruloyl oligosaccharides stimulate the growth of Bifidobacterium bifidum

Insoluble dietary fiber from wheat bran contains some feruloyl groups linked to the arabinose residues in the cell wall arabinoxylan. Treatment of wheat bran insoluble dietary fiber with xylanase from Bacillus subtilis yielded feruloyl oligosacchairdes, which were purified with Amberlite XAD-2. Saponification of the feruloyl oligosaccharides released ferulic acid and arabinoxylan oligosaccharides which consist of arabinose and xylose. The effect of the feruloyl oligosacchairdes on the growth of Bifidobacterium bifidum F-35 was investigated in vitro. The B. bifidum produced acid when cultivated anaerobically in TPY broth with 0.5% feruloyl oligosacchairdes as the carbohydrate source. The biomass yield of the B. bifidum increased with increasing the concentration of feruloyl oligosaccharides in TPY broth. The maximum cell growth was increased by 50% in TPY broth supplemented with 0.1% feruloyl oligosaccharides compared to TPY broth. These results indicated that the growth of B. bifidum F-35 was promoted by the feruloyl oligosaccharides from wheat bran insoluble dietary fiber, and not suppressed by the ferulic acid moiety of them.     

Lactoferrin-binding proteins in Bifidobacterium bifidum.

Lactoferrin is an iron-binding glycoprotein and its bacteriostatic and bactericidal effects on gram-positive and gram-negative bacteria are well known. On the other hand, it is known that certain kinds of lactic acid bacteria are resistant to its antibacterial effects. Moreover, it is reported that lactoferrin promotes the growth of bifidobacteria in in vitro and in vivo experiments. In our experiments, lactoferrin-binding protein was found both in the membrane and cytosolic fractions of Bifidobacterium bifidum Bb-11. The bifidobacteria were grown in anaerobic conditions with lactobacilli MRS broth containing cysteine, harvested by centrifugation, and processed by sonication. The lactoferrin-binding proteins on the PVDF-membrane transferred after SDS-PAGE were detected by far-Western (western-Western) method using biotinylated lactoferrin and streptavidin-labelled horse radish peroxidase. The molecular weights of the lactoferrin binding protein detected in the membrane fraction were estimated to be 69 kDa and those in cytosolic fractions were 20, 35, 50, and 66 kDa.     

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.     

Physiology and kinetics of Bifidobacterium bifidum during growth on different sugars

A strain of Bifidobacterium bifidum was grown on different sugars under pH-controlled conditions to estimate some kinetic parameters for growth and product formation. Glucose was the preferred sugar in terms of growth rate and yield, sugar utilisation rate and acetate formation rate, while lactose gave considerably lower values for these parameters. When present in a mixture with glucose, the rate of lactose utilisation was lower than when present on its own. Received: 24 February 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998     

Effects of Bifidobacterium bifidum on adaptive immune senescence in aging mice

Bifidobacteria are a natural component of the bacterial flora of the human body and have a symbiotic bacteria-host relationship with human beings. Aging is associated with reduced numbers of beneficial colonic Bifidobacteria and impaired immunity. The possible anti-senescence effects of Bifidobacteria are presently unknown. The aims of the present study were to investigate possible anti-senescence effects of B. bifidum on naturally senescent mice and to explore their mechanisms. After treatment with B. bifidum, mice were killed and samples collected. Cytokine production in serum and lymphocyte culture supernatant, anti-oxidation activity and gene expression were measured. B. bifidum significantly increased cytokine IL-2 and IFN-γ levels but decreased proinflammatory cytokines IL-6 and TNF-α concentrations. Moreover, B. bifidum improved anti-oxidation activity and reduced lipid peroxidation in thymus and spleen. In addition, B. bifidum down-regulated p16 expression in thymus and spleen. Taken together, the results indicate, for the first time, that B. bifidum delays senescence by several mechanisms, including enhancement of anti-oxidation activity in thymus and spleen, alteration of gene expression and improvement in immune function.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition.

Clostridium butyricum has been grown in media devoid of biotin, to which long-chain fatty acids have been added to promote growth. We have shown previously that, under these conditions, exogenous fatty acids are extensively incorporated into the cellular phospholipids. Cells grown with elaidic acid, trans-9-18:1, have normal ratios of the glycerol acetal of plasmenylethanolamine (GAPlaE) to phosphatidylethanolamine (PE) plus plasmenylethanolamine (PlaE) compared with cells grown with biotin. When ethanol, cyclohexane, or n-octanol was added to elaidate-containing media, the ratio of GAPlaE to PE plus PlaE was significantly increased. Addition of dodecane and n-butanol did not affect this ratio. When cells were grown with oleic acid in the absence of biotin, the GAPlaE to PE plus PlaE ratio was increased 5.4-fold compared with elaidate-grown cells. In oleate-supplemented media, the addition of solvents or n-alcohols produced no further increase in this ratio. We conclude that these changes in lipid composition represent cellular responses to perturbation of the equilibria between the lamellar and nonlamellar liquid crystalline phases in the cell membrane.     

Molecular identification of monomeric aspartate racemase from Bifidobacterium bifidum.

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.     

Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus

A system was designed to investigate ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus. It involved the incubation of the organisms with labeled ferrous iron in the Norris medium at pH 5, in which the bacteria had grown. Iron uptakes were similar under aerobic and anaerobic conditions. Ferrous but not ferric iron was taken up by the organisms. Iron uptake showed saturation kinetics and a marked temperature dependence. 2,4-Dinitrophenol and thenoltrifluoroacetate but not azide or trypsin treatment inhibited iron uptake. Zinc inhibited iron uptake competitively. Iron uptake from used medium was much greater than that from fresh medium at the same pH. It is concluded that ferrous iron uptake by the microorganisms is a carrier-mediated active phenomenon, inhibited by zinc, which may involve a substance elaborated into the medium by the organism.     

Aspects of iron metabolism in Bifidobacterium bifidum var. pennsylvanicus

• 1.1. Btfidobacterium bifidum var. Pennsylvanias requires ferrous iron for growth, and cannot utilize ferric iron even in the presence of siderophores.
• 2.2. Acid production by the microorganisms is dependent in part on iron content of the medium.
• 3.3. Heme and heme-containing proteins inhibit the microbial growth, and it is proposed that this is in part responsible for the change in the infant's intestinal flora upon weaning.
• 4.4. Bacterial growth inhibition brought about by heme cannot be restored by heme biosynthesis intermediates, and known heme biosynthesis inhibitors have no effect on bacterial growth. The basis for heme-induced microbial growth inhibition remains unclear.

     

Growth of Bifidobacterium bifidum in whey-based media

Bifidobacteria play an important role in human health including the enhancement of resistance against infection in infants. To develop an inexpensive whey-based medium for Bifidobaterium bifidum, potential growth promoters — yeast extract, casein, bovine casein digest, tryptone, peptone and glucosamine — singly or in combinations, were evaluated for their bifidus growth-promoting activity. The effect of environmental conditions on growth in cheese whey was also evaluated. A whey-based medium for B. bifidum was formulated. Cheese whey supplemented with N-acetylglucosamine (1 mg/ml) and yeast extract (10 mg/ml) in the presence of sodium thioglycolate (0.1%) at pH 6.8 promoted the growth of B. bifidum at 37°C. Journal of Industrial Microbiology & Biotechnology (2000) 25, 177–179. Received 20 May 2000/ Accepted in revised form 20 July 2000     

分叉双歧杆菌对小鼠腹水瘤的抑制作用

本文观察了分叉双歧杆菌对小鼠腹腔移植的淋巴细胞腹水瘤(SRS)的抑制作用。结果发现,分叉双歧杆菌在SRS瘤细胞移植前或移植后应用,均能明显抑制瘤细胞的生长,特别在移植后应用,抗瘤效果更显著。主要表现为荷瘤小鼠存活时间延长、存活率提高。将该菌预先进行处理或不处理后加入体外培养的SRS瘤细胞中,发现该菌对离体的瘤细胞生长也有直接抑制作用。