Growth of Listeria monocytogenes at different pH values in uncultured whey or whey cultured with Penicillium camemberti

Wheys from making Camembert cheese were either uncultured or cultured with Penicillium camemberti, adjusted to pH 5.0, 5.2, 5.4, 5.6, 6.2, and 6.8, and filter sterilized. Whey samples were inoculated to contain 100 to 500 Listeria monocytogenes (strains Scott A, V7, CA, or OH) cfu/mL and incubated at 6 degrees C. Counts of L. monocytogenes were obtained by surface plating appropriate dilutions on Tryptose Agar. Listeria monocytogenes failed to grow at or below pH 5.4; except for strains Scott A and OH which grew in cultured whey at pH 5.4 and attained populations of 7.8 x 10(3) and 5.4 x 10(4) cfu/mL, respectively, after 35 d of storage. In uncultured whey at pH 5.6, 6.2, and 6.8, populations of L. monocytogenes increased from 7.20 to 7.81, 7.51 to 8.23, and 7.48 to 8.08 log10 cfu/mL, respectively, after 35 d of storage at 6 degrees C. In cultured whey at pH 5.6, 6.2, and 6.8, numbers of L. monocytogenes increased from 7.53 to 8.13, 7.82 to 8.55, and 7.95 to 8.80 log10 cfu/mL, respectively, after 35 d of storage. Generation times for L. monocytogenes at 6 degrees C in uncultured whey at pH 5.6, 6.2, and 6.8 ranged between 25.3 and 31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respectively, depending on the Listeria strain. In contrast, generation times were significantly (p less than 0.05) shorter in cultured whey and ranged between 16.6 and 27.4 h, 10.3 and 16.6 h, and 17.4 and 16.3 h at pH values of 5.6, 6.2, and 6.8, respectively.     

Study on antimicrobial activity of chitosan with different molecular weights

E. coli and Staphylococcus aureus are used to study the antimicrobial activity of chitosan of different molecular weights (MW). The effect of the concentration and MW of chitosan were investigated, respectively, and the antimicrobial mechanism was discussed. For chitosan with MW below 300 kDa, the antimicrobial effect on S. aureus was strengthened as the MW increased. In contrast, the effect on E. coli was weakened.     

Gelation behaviour of konjac glucomannan with different molecular weights

The deacetylation and gelation of konjac glucomannan (KGM) following alkali addition was investigated by Fourier transform infrared, while the rheological properties of KGM with different molecular weights were studied by dynamic viscoelastic measurements in shear mode and penetration force tests. It was found that gelation occurred after significant deacetylation had taken place. Rheometrical studies revealed that KGM with different molecular weights exhibited different gelation characteristics in small amplitude oscillatory shear flow. For the samples of fractionated KGM with lower molecular weights, a decrease in both the storage shear modulus (G') and loss shear modulus (G") was observed during gelation at temperatures above 75 degrees C. It is suggested that the decrease results from the wall slip between sample and measuring geometry owing to a rapid gelation process with syneresis and/or disentanglement of molecular chains adsorbed on the surface of parallel plates from those located in the bulk. Penetration force tests were employed to confirm the occurrence of slippage and thereby no decreases in rigidity of samples were observed during gelation. For the native KGM samples decreases in G' and G" during gelation were not observed, and it is suggested that this is due to the effect of the higher molecular weight and increased solution viscosity of these samples on the gelation kinetics.     

Influence of initial pH on hydrogen production from cheese whey

Batch experiments were conducted to investigate the effect of initial pH, between 5 and 10, on fermentative hydrogen production from crude cheese whey (87.5% (v/v) by Clostridium saccharoperbutylacetonicum). Hydrogen was produced over the range of pH studied. The hydrogen production rate and yield peaked at an initial pH 6 and then steadily decreased as the pH increased. The highest rate and yield were 28.3 ml h⁻¹ and 7.89 mmol g⁻¹ lactose, respectively. Sugar consumption was unaffected between pH 5 and 9 and remained at 97%. All final pHs were acidic and increased alongside the initial pH. There was no correlation between the initial pH and the fermentation time; the times were shorter (50–52 h) between pH 6 and 8, and longer (62–82 h) outside this range. A modified Gompertz equation adequately described fermentative hydrogen production from cheese whey. The respective maximum hydrogen production rate and hydrogen potential at an optimal pH of 6 were 47.07 ml h⁻¹ and 1432 ml. Lag phase times were much longer at acidic pHs than at alkaline pHs.     

Aging of tubulin at neutral pH

The aging of calf brain tubulin in neutral solution has been investigated using the techniques of sedimentation velocity and equilibrium, microtubule assembly, fluorescence spectroscopy, circular dichroism and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that tubulin incubated at 4 °C undergoes a slow association process leading to the generation of a 9 S component. The fraction of 9 S component increases progressively with incubation time and appears to follow mono-molecular kinetics. The generation of the 9 S species is paralleled closely by inhibition of microtubule assembly and loss of colchicine-binding ability. Fluorescence spectroscopy and circular dichroism spectra indicate that tryptophan moieties are perturbed during the aggregation process and that the tubulin dimer undergoes a conformational change. There is no protein degradation up to an incubation period of 50 hours. Sedimentation equilibrium experiments in 6 m-guanidine hydrochloride, both in the presence and absence of 2-mercaptoethanol, indicate that the aggregates are stabilized by disulfide bonds and hydrophobic interactions. At periods of incubation >50 hours, the protein starts to be degraded.     

Influence of a sugar moiety (rhamnosylglucoside) at 3-O position on the reactivity of quercetin with whey proteins

The reaction of whey proteins (WP) with quercetin and rutin (quercetin-3-O-rhamnosylglucoside) is influenced by the glycosidic bound sugar moiety. The protein derivatives formed showed a blocking of tryptophan (max. 49%), free amino (max. 32%) and thiol groups (max. 24%). The amount of quercetin and rutin bound covalently (up to 94 and 31 nmol mg⁻¹, respectively) was estimated by their characteristic absorbance between 300 and 340 nm. At least one molecule of the phenolic reactant was covalently bound to a β-lactoglobulin molecule (β-Lg). High molecular protein fractions were detected by SDS-PAGE (cross-linking with quercetin). All results confirm that quercetin is more reactive than rutin. The pH-dependent solubility of the derivatives decreased, although their hydrophilic character increased. The structural changes (circular dichroism (CD)) showed that especially rutin causes perturbation of the secondary (decrease of -helix elements accompanied by an increase in random coil) and tertiary structure. The in vitro proteolytic digestibility, especially of the rutin derivatives was elevated, due to an increase in denaturation of the derivatives.     

Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions

We have previously reported a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) in Zn(2+)-Phos-tag SDS-PAGE for advanced profiling of phosphoproteins with molecular masses of 10-200 kDa. In the current work, we describe characteristics of two neutral-pH gel systems, Bis-Tris-HCl and Tris-acetic acid (Tris-AcOH), based on comparative studies of the separation of a wide range of proteins with molecular masses from 10 to 350 kDa. For 10-200 kDa cellular proteins, the Bis-Tris-HCl system showed a higher resolving power in a 2-D fluorescence DIGE analysis of certain phosphoproteins, e.g. histone H3 (15 kDa) and elongation factor 2 (95 kDa). Furthermore, there was a large difference in the 1-D migration patterns of phosphorylated species of extracellular signal-regulated kinases 1 and 2 (ERK1/2, 44/42 kDa), which arise from changes in the phosphorylation status of the Thr-202 and Tyr-204, in the two buffer systems at the same concentration of Zn(2+)-Phos-tag. In contrast, shifts in the mobility of various phosphorylated species of a high-molecular-mass protein, ataxia telangiectasia-mutated kinase (ATM, 350 kDa), could only be detected in the Tris-AcOH system with a 3% w/v polyacrylamide gel strengthened with 0.5% w/v agarose.     

Coupled oxidation of NADPH with thiols at neutral pH.

NADPH and NADH are rapidly oxidized in neutral imidazole chloride buffer at 30 °C in the presence of mercaptoethanol or dithiothreitol. The product of the NADPH reaction has been determined to be enzymically active NADP⁺. Oxidation of the pyridine nucleotides is coupled to the autooxidation of the thiol and is inhibited by ethylenediamine tetraacetic acid, stimulated by metal ions (FeSO₄), and requires oxygen. The rapid oxidation of thiols and NADPH at neutral pH was found to occur only in imidazole and, to a lesser extent, in histidine buffer. Under the conditions employed, 300 μm dithiothreitol and 30 μm NADPH are oxidized in 30 min. Both NADPH and thiol oxidations are inhibited by catalase, whereas superoxide dismutase only inhibits the oxidation of NADPH. NADPH oxidation is also inhibited by the hydroxyl radical scavengers formate, mannitol, or benzoate. A reaction mechanism is proposed in which imidazole promotes the metal-catalyzed oxidation of thiols at neutral pH. The superoxide radical generated either by the thiol oxidation or directly oxidizes NADPH or forms hydrogen peroxide and hydroxyl radicals which can oxidize NADPH. Hydrogen peroxide is also involved in the autooxidation of the thiol.     

Outer membrane of Serratia marcescens: apparent molecular weights of heat-modifiable proteins in gels with different acrylamide concentrations.

The major proteins from the outer membrane of Serratia marcescens SM-6 are heat modifiable. The analysis of their apparent molecular weights in gels with different concentrations of acrylamide and the results obtained by radioactive labeling indicate that the major proteins are covalently linked to carbohydrate moieties.     

嗜碱菌Alkalimonas amylolytica N10在不同pH条件下的膜蛋白差异表达研究

为了探讨嗜碱菌的嗜碱机制,对一株新型专性嗜碱菌(Alkalimonas amylolyticaN10)在不同pH条件下的差异膜蛋白质组进行了初步的研究。在3种pH条件下(pH值分别为8.4、9.4和10.4)培养的该菌的膜蛋白,通过8%~20%的梯度SDS-PAGE进行分离。经胶图图像和统计计算,7条电泳条带的相对染色强度随培养液的pH值变化而改变,其中仅有一条条带强度随pH值增高而增加。这些条带经胰蛋白酶胶内消化和高效液相色谱-电喷雾离子阱串联质谱(LC-MS/MS)分析,共鉴定出了12种蛋白质。其中4种膜蛋白已有报道直接或间接参与细胞pH稳态的保持,其他几种蛋白则是首次发现其表达水平与生活环境的pH变化可能相关。     

Purification of glycomacropeptide from dialyzed and non-dialyzed sweet whey by anion-exchange chromatography at different pH values

Glycomacropeptide (GMP) was purified from sweet whey dialyzed in water by anion-exchange chromatography on DEAE-Sephacel at pH 2.0–4.5. The optimum pH range was 2.5–4.0. The yield of purified GMP increased and its sialic acid concentration decreased with increasing pH value. The GMP had an apparent isoelectric point <3.8. Dialysis of sweet whey was shown to be important to maximize the yield of GMP adsorbed to the anion-exchanger. Only highly sialylated GMP, accounting for approximately 55% of total sialic acid content, was adsorbed on the anion-exchanger from non-dialyzed sweet whey.