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     

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     

Inhibition of growth of Escherichia coli by lactose and other galactosides

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a β-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in ΔpH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-β-d-thiogalactoside.     

Complete genome sequence of Bifidobacterium bifidum S17

Here, we report on the first completely annotated genome sequence of a Bifidobacterium bifidum strain. B. bifidum S17, isolated from feces of a breast-fed infant, was shown to strongly adhere to intestinal epithelial cells and has potent anti-inflammatory activity in vitro and in vivo. The genome sequence will provide new insights into the biology of this potential probiotic organism and allow for the characterization of the molecular mechanisms underlying its beneficial properties.     

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。     

Synthesis of β-mercaptoethyl-glycosides by enzymatic reverse hydrolysis and transglycosylation

Summary The synthetic potential ofAlmond β-glucosidase andJack bean α-mannosidase in the presence of high amounts of β-mercaptoethanol as glycosyl acceptor for the synthesis of β-mercaptoethyl-glycosides was studied. The regioselectivity, O-glycosylation and/or S-glycosylation, and the stereoselectivity were analyzed with the reverse hydrolysis and the transglycosylation methods. With both enzymes, high yields of condensation are obtained without the use of chemical protective groups.     

Optimal thermotolerance of Bifidobacterium bifidum in gellan-alginate microparticles

The purpose of this research was to encapsulate Bifidobacterium bifidum using gellan, sodium alginate and prebiotics as coating materials, and to maximize the thermotolerance of the probiotics with an optimal combination of the coating materials. The optimal ratio of the coating materials for the microparticles under heat treatments (75 degrees C, 1 min) was obtained by using the response surface method and the sequential quadratic programming technique. Optimization results indicated that 2% sodium alginate mixed with 1% gellan gum as coating materials would produce the highest thermotolerance in terms of B. bifidum count. The verification experiment yielded a result close to the predicted values, with no significant difference (P > 0.05). The results of heat treatments also demonstrated that the addition of gellan gum in the walls of probiotic microcapsules provided improved protection for B. bifidum. These probiotic counts remained at 10(5)-10(6) CFU/g for the microcapsules stored for 2 months, then treated in heat and in simulated gastric fluid.     

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.     

Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.     

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.     

Bile salt hydrolase and cholesterol removal effect by Bifidobacterium bifidum NRRL 1976

Summary Growing cells of Bifidobacterum bifidum NRRL 1976 exhibited an ability to remove cholesterol in the presence of bile salts. The cholesterol removal by Bifidobacterium bifidum was due to a co-precipitation together with unconjugated bile acids, which was linked to the bile salt hydrolase (BSH) activity of the cells at pH values lower than 5.0 and the cholesterol removed was partially recovered when the cells were washed with phosphate buffer at pH 7, while the remaining cholesterol was extracted from the cells. It is concluded that the removal of cholesterol from the growth medium by Bifidobacterium bifidum strain is due to both bacterial assimilation and precipitation of cholesterol.     

Kinetics and mathematical model of hydrolysis and transglycosylation catalysed by cellobiase

The kinetics of hydrolysis and transglycosylation reactions catalysed by cellobiase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus foetidus in the cellobiose-d-glucose reaction system have been studied. The formation of transglycosylation products was observed at cellobiose concentrations $\text{>10}{}^{\mathrm{?2}}\text{m}$, whereas at lower substrate concentrations the only reaction product was d-glucose. In the cellobiase-catalysed transglycosylation a (1→6)-β-linkage was formed after the transfer of a d-glucose residue to acceptor molecule. The basic transglycosylation products were isocellotriose and gentiobiose. A small amount of oligosaccharides with a higher degree of polymerization was also formed. The maximum content of transglycosylation products amounted to 25–30% of the total saccharide content in the system at the initial cellobiose concentration (0.1–0.3 m). The processes in the reaction system were inhibited by the substrate and product (d-glucose). A general scheme for cellobiose hydrolysis has been proposed and validated, allowing for the inhibition and transglycosylation effects. Based on this scheme, a mathematical model for cellobiose hydrolysis has been suggested to describe the kinetics of substrate consumption and product (d-glucose) accumulation, as well as the kinetics of formation and consumption of transglycosylation products throughout the course of enzymatic reaction with various initial amounts of cellobiose, starting from low concentrations up to 0.2–0.3 m (7–11% bv weight).     

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。     

Mechanisms of ferric and ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus

Iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out using ferric citrate at iron concentrations above 0.01 mM and pH 7, ferrous iron at concentrations less than 0.01 mM at pH 5. Two ferric iron transport systems were distinguished: the temperature-insensitive polymer, and the temperature-sensitive monomer uptake. Both showed a saturation phenomenon. The transport of ferrous iron at concentrations below 0.01 mM was temperature-dependent, and its affinity for iron was higher than that of a system operating at iron concentrations higher than 0.01 mM. The use of various metabolic inhibitors indicated that ferrous iron transport at pH 5 at both high and low iron concentrations was mediated by transport-type ATPase. Proton gradient dissipators abolished ferrous iron uptakes as well as the ferric monomer uptake. Uptake of the ferric polymer was insensitive to metabolic inhibitors. The functional significance of the various types of iron transport systems may be related to the nutritional immunity phenomenon.