双歧杆菌四联活菌体外模拟消化液耐受性的考察

考察双歧杆菌四联活菌中4种益生菌在模拟消化液中的耐受性。

按照现行版《中国药典》配制人工胃液与人工肠液,将4种益生菌分别加入其中,充分混匀,孵育0、15、30、60、90、120、180 min后,稀释、涂布与计数,绘制每种菌存活的衰减曲线,考察其存活率。

在人工胃液（pH 2.5～4.5）孵育3 h后,长双歧杆菌婴儿亚种（GMCC 0460.1）、嗜酸乳杆菌（GMCC 0460.2）、粪肠球菌（GMCC 0460.3）、蜡样芽胞杆菌（GMCC 0460.4）的存活率分别为53.23%～61.30%、75.31%～105.34%、90.76%～100.00%、81.02%～102.58%;在人工肠液（胆汁盐浓度0.03%～0.20%）孵育3 h后,长双歧杆菌婴儿亚种、嗜酸乳杆菌、粪肠球菌、蜡样芽胞杆菌的存活率分别为42.19%～71.49%、16.62%～45.03%、40.80%～69.50%、42.67%～95.43%。

4种益生菌在较长时间内耐受人工胃液（pH 2.5～4.5）和人工肠液（胆汁盐浓度0.03%～0.20%）,菌体存活量下降不到1个数量级,对胃酸和胆盐的耐受性较强。由于健康人体内胃肠的环境类似于上述环境,预示其在健康人体消化道内能较好保持菌体生存能力,发挥药效活性。

     

葛根芩连汤在模拟胃肠液中对微生物生长的影响

目的探究葛根芩连汤在模拟胃肠液中对微生物生长的影响。方法在模拟胃肠液中分别加入消化液10%量的金黄色葡萄球菌、大肠埃希菌、沙门菌、产气杆菌、黑曲霉、米曲霉、青霉和汉逊德巴利酵母,然后加入生药浓度为1 g/mL的葛根芩连汤水煎液,37℃恒温培养。分别在0、1、2和4 h吸取1 mL培养液做稀释平板计数。结果体外模拟肠液中,葛根芩连汤对金黄色葡萄球菌、大肠埃希菌、沙门菌、产气杆菌、黑曲霉、米曲霉和青霉有较强的抑制作用,而对汉逊德巴利酵母的生长则为先促进后抑制,并且在2 h时对黑曲霉、米曲霉、青霉和汉逊德巴利酵母的抑制效果最显著;在模拟胃液中,葛根芩连汤对金黄色葡萄球菌、大肠埃希菌、沙门菌、黑曲霉、青霉和汉逊德巴利酵母均有一定的抑制作用,而对产气杆菌无明显影响。结论葛根芩连汤在模拟胃肠液中对金黄色葡萄球菌、大肠埃希菌、沙门菌、黑曲霉、米曲霉、青霉和汉逊德巴利酵母等均具有一定的抑制作用,在模拟肠液中对产气杆菌抑制作用明显,而在模拟胃液中对其抑制效果不明显。     

Effect of Polyglycerol Esters of Fatty Acids on the Physicochemical Properties and Stability of β-Carotene Emulsions during Digestion in Simulated Gastric Fluid

The effects of six different polyglycerol esters of fatty acids (PGEs) and two different particle sizes produced using various processing parameters on the physicochemical properties and stability of the β-carotene emulsions during digestion in simulated gastric fluid (SGF) were investigated. β-Carotene emulsions were prepared by high-pressure homogenization using β-carotene (0.1% w/w) in soybean oil as the oil phase and 1% (w/w) PGE in Milli-Q water as the water phase. The particle size of β-carotene emulsions was measured by a laser diffraction technique, and the stability of emulsions was interpreted in terms of the increase in particle size and span value of emulsion droplets and the retention of β-carotene during digestion in SGF. The average particle size ranges of emulsions were 0.17 to 0.27 μm for fine emulsions and 1.16 to 1.59 μm for coarse emulsions. In the prepared β-carotene emulsions, the particle size decreased with increasing polymerization of the glycerol in PGEs, and the higher polymerization of the glycerol also increased the stability of emulsions during digestion in SGF. Although the β-carotene content in the emulsions significantly decreased with increasing digestion period, loss of β-carotene was more severe in unstable emulsions than in stable emulsions, suggesting that the particles incorporated into droplets could provide some protective barrier for decreasing the β-carotene degradation. Therefore, β-carotene emulsions stabilized by PGEs with high polymerization of the glycerol may be useful for further applications in food and drug formulations. Decaglycerol monooleate (MO750) was demonstrated to be the most effective emulsifier in stabilizing β-carotene emulsions in this study.     

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif

A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed β-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant β-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.     

Development of an enzyme activity screening system for β-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting

Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying β-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.     

β-Mannanolytic system of Aureobasidium pullulans

A xylanolytic yeast strain Aureobasidium pullulans NRRL Y 2311-1, was found to produce all enzymes required for complete degradation of galactomannan and galactoglucomannan. The enzymes differed in function and cellular localization: endo-β-1,4-mannanase was secreted into the culture fluid, β-mannosidase was strictly intracellular, and α-galactosidase and β-glucosidase were found both extracellularly and intracellularly. Among these enzyme components, only extracellular β-mannanase and intracellular β-mannosidase were inducible. The production of β-mannanase and β-mannosidase was 10- to 100-fold higher in galactomannan medium than in medium with one of the other carbon sources. β-mannanase and β-mannosidase were coinduced in glucose-grown cells by galactomannan, galactoglucomannan, and β-1,4-manno-oligosaccharides. The natural inducer of extracellular β-mannanase and intracellular β-mannosidase appeared to be β-1,4-mannobiose. Synthesis of both enzymes was completely repressed by glucose, mannose, or galactose. The synthetic glycoside methyl β-d-mannopyranoside served as a nonmetabolizable inducer of both β-mannosidase and β-mannanase. Received: 24 June 1996 / Accepted: 26 September 1996     

Differential expression of β-glucosidase in tomato — stress stimuli systems

In the present work the responses of β-glucosidase in leaves of tomato plants subjected to various stress factors of both pathogenic (fungi, bacteria, viruses) and abiotic origin (heat shock) were studied. Biochemical and cytochemical methods were applied. It was established that an increase of β-glucosidase activity is induced uniquely by fungal pathogens. The cytochemical tests confirm the finding. Hence, the conclusion can be drawn that β-glucosidase response is a specific character of fungal pathogenesis in tomato; probably, the enzyme is involved in plant — fungi recognition. The data are in accordance with our previous results on tobacco and wheat — stress stimuli systems.     

Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae)

The extracts obtained from 28 species of marine algae were evaluated for their antioxidant activity (AA) versus the positive controls butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). Most of the tested samples displayed antioxidant activity to various degrees. Among them, the extract of Symphyocladia latiuscula exhibited the strongest AA, which was comparable to BHT, GA, and AscA in radical scavenging activity, as shown in the DPPH (α,α-diphenyl-β-picrylhydrazyl) assay, and higher than those of the positive controls in β-carotene-linoleate assay system. In addition, the ethyl acetate-soluble fraction isolated from the crude extract of S. latiuscula exhibited the highest antioxidant activity in both assay systems. This fraction was further fractionated into seven subfractions (F1-F7) by vacuum liquid chromatography (VLC). F1 and F4 were found to be the most effective subfractions in scavenging DPPH radical assay and in the β-carotene-linoleate assay, respectively. The total phenolic content (TPC) and reducing power (RP) for all of the extracts, fractions, and subfractions (F1–F7) were also determined. The TPC of the 28 extracts ranged from 0.10 to 8.00 gallic acid equivalents (mg/g seaweed dry weight) while the RP ranged from 0.07 to 11.60 ascorbic acid equivalents (mg·g⁻¹ seaweed dry weight). Highly positive relationships between AA and TPC as well as between AA and RP were found for the extracts and fractions, while for the subfractions F1–F7 only weak or no such relations were found. The results obtained from this study indicate that further analysis is needed of those marine algal species that contain the most antioxidant activity in order to identify the active principles.     

Maize genotypes classified as null at theglu locus have β-glucosidase activity and immunoreactive protein

Maize β-glucosidase (β-d-glucoside glucohydrolase; EC 3.2.1.21) was extracted from coleoptiles of 15 maize genotypes (3 normals, 10 nulls, and 2 hybrids) in two fractions, the soluble and the insoluble. The enzyme activity was measured spectrophotometrically in the soluble fraction and also studied on zymograms after native gel electrophoresis and isoelectric focusing. The enzyme was purified from a normal genotype by anion-exchange chromatography and preparative electrophoresis. Antisera were raised in four rabbits, and the soluble and the insoluble extracts of each genotype were analyzed for a cross-reacting material by ELISA and immunoblotting. The results showed that extracts from both the normal and the null genotypes had β-glucosidase activity, and the activity measured spectrophotometrically was 2- to 10-fold higher in normals than in nulls. Zymograms of the null genotypes were devoid of distinct bands that were present in those of normals and hybrids from crosses between normals and nulls. Zymograms of both the normal and the null genotypes had a diffuse, smeared zone of activity at the cathodic end of native gels. A cross-reacting antigen was present in extracts of both genotypes when assayed by ELISA and a 60-kD polypeptide (β-glucosidase monomer) was detected by four different monospecific β-glucosidase antisera on Western blots by immunostaining. Moreover, six of seven null genotypes had a larger amount of their 60-kD polypeptide in the insoluble fraction than in the soluble fraction. These data show that both the null and the normal genotypes have similar amounts of the enzyme protein, but the enzyme occurs mostly as insoluble or poorly soluble polymers in nulls, and the monogenic inheritance reported for the null alleles of theglu locus is likely to be for a factor encoded by another locus which affects directly or indirectly the solubility of the enzyme by increasing its polymerization into large quaternary structures.     

The use of fluorogenic substrates for measuring enzyme activity in peatlands

The fluorogenic model substrates, methylumbelliferyl [MUF]-β-D-glucoside, MUF-phosphate and MUF-sulphate, were used to investigate the activities of β-glucosidase, phosphatase and sulphatase, respectively, in Welsh peatland soils. The method was used to investigate depth dependent variations in enzyme activity in a riparian wetland, and flush channel wetland. The highest activities were found at depths of less than 10 cm, thus confirming other studies which suggest this upper depth to be the site of greatest microbial activity. The most serious limitation to the technique was found to be the (fluorescence) quenching effects of the phenolic materials that dominate peatland dissolved organic carbon. The problem necessitates the adoption of a time consuming quench correction procedure with every sample. Fluorogenic substrates have led to a greater understanding of the role of enzymes in other aquatic systems. It seems likely that they will prove of equal value in elucidating their role in nutrient cycling and the biogeochemistry of peatlands.     

Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood β-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q _O/q _O,max), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90–95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.     

Studies on the mechanism of the selenite-induced decrease in cell attachment

We previously reported that exposure of HeLa cells to selenite for 2 h results in a decrease in their ability to attach to fibronectin (Yan and Frenkel,Cancer Res. 52, 5803–5807 [1992]), as well as a decrease in the level of fibronectin receptor (α5β1 integrin) at the cell surface (Yan and Frenkel,Biol. Trace Element Res. 46, 79–89 [1994]). We have now found that after exposure to selenite, there was a decrease in the total cellular content of the receptor protein, as well as in the level of the mRNAs for both of the subunits. Exposure of cells to actinomycin D (an inhibitor of RNA synthesis) also resulted in a decrease in the level of these mRNAs, suggesting that the effect of selenite is the result of its known inhibitory effect on RNA synthesis (Frenkel,Toxicol. Lett. 25, 219–223 [1985]). Exposure of cells to actinomycin D for 2 h also resulted in a decrease in the ability of cells to attach to fibronectin. Furthermore, both selenite and actinomycin D caused a decrease in integrin mRNA levels and in cell attachment to fibronectin only when high-density cells were exposed to the agents. In contrast, when low-density cells were exposed,neither agent had any detectable effect on mRNA levels or on cell attachment. These results have suggested the following scheme for the mechanism of the inhibition of cell attachment by selenite: After exposure to selenite for 2 h, there is a significant inhibition of cellular RNA synthesis, which results in a general decrease in the cellular level of those mRNAs with relatively short half-lives, including in particular those of the fibronectin receptor. This leads to a decrease in the intracellular level of the receptor protein and, consequently, in its level at the cell surface, which in turn causes a decrease in the rate of cell attachment to fibronectin.     

Dose-dependent differential effect of hemin on protein synthesis and cell proliferation inLeishmania donovani promastigotes culturedin vitro

Leishmania donovani requires an exogenous source of heme for growth and transformation. Inin vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results ofin vitro cell culture experiments demonstrated that hemin (10 μM) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However,in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 μM) reduced the level of protein synthesis in general and of β-tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed duringin situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation inL. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.     

Potential Protection of Curcumin against Hypoxia-induced Decreases in Beta-III Tubulin Content in Rat Prefrontal Cortical Neurons

The central nervous system (CNS) is highly dependent on adequate supply of oxygen and is sensitive to hypoxia. It is known that hypoxia induces injuries on the brain tissue and the neuronal activity. Curcumin, a yellow pigment obtained from the rhizome of C. longa Linn., has been regarded as a multi-functional drug with antioxidative activity. In the present study, we first demonstrated a significant decrease in the content of β-III tubulin protein in rat prefrontal cortex (PFC) tissues induced by repeated hypoxia, but not in rat cerebellum tissue. These suggest a relatively higher sensitivity and probably a higher vulnerability of rat PFC tissue to hypoxia in vivo. We reconfirmed the effect of hypoxia to primary cultured neurons from rat PFC and found a significant decrease in the contents of β-III tubulin protein after chronic exposure to hypoxia. Moreover, we demonstrated that the hypoxia-induced decrease in β-III tubulin protein content could be restored by curcumin, suggesting a potential protection of curcumin against hypoxia-induced decreases in beta-III tubulin content in rat PFC neurons. Special issue article in honor of Dr. Ji-Sheng Han.     

Comparative characterization of growth and recombinant protein production among three insect cell lines with four kinds of serum free media

Three insect cell lines, Sf9, Sf21 and Tn5B1-4, and four different kinds of serum free media (SFM), Sf 900 II, EX-CELL 420, EX-CELL 405 and Express Five, were used to compare the nutrient consumption, byproduct formation, production of recombinant protein and protease activity in suspension cultures. The Sf 900 II SFM was appropriate for the cell growth and protein production of the Sf9 and Sf21 cell lines. When the Tn5B1-4 cell line was grown in the Express Five SFM, the specific growth rate was 1.6 fold higher than those of either the Sf9 or Sf21 cell lines. The glucose and glutamine consumption rates per cells, were 4 and 2.3. times higher than those of the Sf9 cell line, respectively. The overall yield coefficients of the lactate and ammonium ion were 2.8 and 1.5 times higher compared to those of the Sf9 cell line, respectively. The maximum specific β-galactosidase production rate was 4.5. fold that of the Sf9 cell line, a 3 times higher protease activity per cell.     

Effect of Azospirillum Lectins on the Activities of Wheat-root Hydrolytic Enzymes

This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast fractions of wheat-seedling roots. Lectin (40 μg mL⁻¹) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase. Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained by change in the antigenic properties of the mutant lectin.