Toxicity of dietary avidin and streptavidin to the cowpea bruchid, Callosobruchus maculatus (F.)

Avidin, a large protein from egg whites, powerfully binds biotin, a vitamin for many insects. When avidin was incorporated into the diets of larval Callosobruchus maculatus (F.), the cowpea bruchid, at relatively low levels (10–30 ppm), there was a marked dose-dependent increase in mortality, as well as a small increase in the developmental time of survivors. Avidin toxicity was prevented when biotin was added to the diet together with avidin. Sub-lethal doses of avidin caused reductions in fecundity. Avidin had no effect on the larval feeding rates during the first three instars, even when the larvae were consuming amounts of the protein that would later cause death. In the fourth instar there was a dose-dependent reduction in the rates of feeding as the avidin level in the food increased. Death of the C. maculatus larvae usually occurred at the pupal/adult stage still within the host seed. Streptavidin, a biotin-binding protein from a bacterial source, had effects similar to chicken egg avidin.     

Utilization of two improved enzyme immunoassays based on avidin-biotin interaction for the detection of Salmonella

Based on a strong interaction between avidin and biotin, two enzyme immunoassays have been modified and tested for the detection of Salmonella typhimurium in foodstuffs. In both assays, the antigen containing sample was first reacted with antibody to Salmonella which was precoated on a polystyrene microtiter plate. The bound antigen was then allowed to react with an appropriate amount of biotinylated antibody. In the first procedure, the presence of Salmonella was quantified by using peroxidase-labeled avidin. In the latter, avidin acted as a bridge between the biotinylated antibody and the biotinylated peroxidase. Samples containing 10(3) and 10(4) cells/ml of the Salmonella virulent strain, respectively, were detectable by these two methods. The results thus compared favorably with the detection limit of the standard ELISA (10(5) cells/ml). The superiority of the modified ELISA's utilizing biotin/avidin interactions was also demonstrated for the detection of Salmonella in artificially contaminated food samples inoculated with only 2-5 Salmonella cells followed by two incubation steps. No significant interference of E. coli (up to 5 x 10(6) cells/ml) was observed in the quantification of Salmonella cells.     

肠毒素大肠杆菌K88可视化快速检测方法的建立

本研究通过肠毒素大肠杆菌(E.coli)K88菌毛蛋白的适配体识别,结合纳米金标记和银增强信号放大技术,建立了一种快速、灵敏、特异的E.coli K88可视化快速检测方法。该检测方法是将能与E.coli K88特异性结合的生物素化的适配体1(aptamer 1),与目标菌E.coli K88以及纳米金-巯基化适配体2轭合物(aptamer 2-Au NPs)在一定条件下孵育,形成三明治式的aptamer 1-E.coli K88-aptamer 2-Au NPs复合物,随后通过生物素与亲和素的结合将复合物固定到修饰了链霉亲和素的微孔板上,最后运用银增强显色将反应信号放大。通过对检测方法条件的优化,本方法可特异、定量地检测E.coli K88,在1.0×10~1~1.0×10~5 cfu/孔目标菌范围内,其定量拟合线性曲线决定系数R2可达0.9903,且检测灵敏度达10 cfu/孔,而检测其它非目标菌株均为阴性。本方法为E.coli K88在临床样品中的可视化检测奠定了基础。     

Interventions for the reduction of Salmonella Typhimurium DT 104 and non-O157:H7 enterohemorrhagic Escherichia coli on beef surfaces

A study was conducted to determine if slaughter interventions currently used by the meat industry are effective against Salmonella Typhimurium definitive type 104 (DT 104) and two non-O157:H7 enterohemorrhagic Escherichia coli (EHEC). Three separate experiments were conducted by inoculating prerigor beef surfaces with a bovine fecal slurry containing Salmonella Typhimurium and Salmonella Typhimurium DT 104 (experiment 1), E. coli O157:H7 and E. coli O111:H8 (experiment 2), or E. coli O157:H7 and E. coli O26:H11 (experiment 3) and spray washing with water, hot water (72 degrees C), 2% acetic acid, 2% lactic acid, or 10% trisodium phosphate (15 s, 125 +/- 5 psi, 35 +/- 2 degrees C). Remaining bacterial populations were determined immediately after treatments (day 0), after 2 days of aerobic storage at 4 degrees C, and after 7, 21, and 35 days of vacuum-packaged storage at 4 degrees C. In addition to enumeration, confirmation of pathogen serotypes was performed for all treatments on all days. Of the interventions investigated, spray treatments with trisodium phosphate were the most effective, resulting in pathogen reductions of >3 log10 CFU/cm2, followed by 2% lactic acid and 2% acetic acid (>2 log10 CFU/cm2). Results also indicated that interventions used to reduce Salmonella Typhimurium on beef surfaces were equally effective against Salmonella Typhimurium DT 104 immediately after treatment and again after long-term, refrigerated, vacuum-packaged storage. Similarly, E. coli O111:H8 and E. coli O26:H11 associated with beef surfaces were reduced by the interventions to approximately the same extent as E. coli O157:H7 immediately after treatment and again after long-term, refrigerated, vacuum-packaged storage. It was also demonstrated that phenotypic characterization may not be sufficient to identify EHECs and that the organisms should be further confirmed with antibody- or genetic-based techniques. Based on these findings, interventions used by the meat industry to reduce Salmonella spp. and E. coli O157:H7 appear to be effective against DT 104 and other EHEC.     

TRYPSIN IMMOBILIZATION ON DERIVATIZED CELLULOSE BEADS BY BIOSPECIFIC AVIDIN‐BIOTIN INTERACTION AND CHARACTERIZATION OF THE IMMOBILIZED ACTIVITY1

Trypsin was immobilized on cellulose beads using the biospecific and high affinity avidin‐biotin interaction. Trypsin and cellulose beads were biotinylated with sulfosuccinimidyl‐6‐(biotinamido) hexanoate (NHS‐LC‐biotin). Avidin and biotinylated trypsin were sequentially adsorbed to the biotinylated cellulose beads. A similar procedure was carried out using controlled‐pore glass (CPG) beads. The properties of the two trypsin bioreactors were examined and compared. The substrate used for the assay of trypsin activity was p‐tosyl‐L‐arginine methyl ester and the extent of biotinylation of biotinylated trypsin and of immobilized biotin on cellulose beads and on CPG beads were determined using the 2‐[4′‐hydroxyazobenzene]benzoic acid dye‐binding method. Biotinylated trypsin in solution retained about 82% of the specific activity of native trypsin. Cellulose beads contained 0.184 μmol/mL (1.15 μmol/g) biotin and CPG beads, 0.329 μmol/mL (0.987 μmol/g). After regeneration, the biotin contents became slightly lower, namely, 0,159 μmol/mL for cellulose beads and 0.315 μmol/mL for CPG beads. The specific activities of trypsin immobilized on cellulose beads and CPG beads were 32 U/mL (202 U/g) and 43 U/mL (130 U/g), respectively. These studies indicate that cellulose beads can be biotinylated for use as bioselective support.     

嗜酸乳杆菌细菌素Lactobacillin XH2抑制大肠杆菌作用机理的探讨

将离子交换色谱纯化获得的抑菌效价为380.60 AU/mL的嗜酸乳杆菌细菌素Lactobacillin XH2作用于大肠杆菌（Escherichia coli）,从死亡率、细胞膜损伤及引起胞内物质外泄方面,探讨Lactobacillin XH2抑制大肠杆菌的机理。结果表明：Lactobacillin XH2作用于大肠杆菌后,在3 h内大肠杆菌死亡率达96%,菌体细胞变形并出现孔洞,菌体短小且不规整。进一步分析发现,细胞膜通透性增大,胞内K+快速大量泄露,膜电位紊乱失衡;胞内紫外吸收物质、乳酸脱氢酶等生命大分子物质大量流失;同时导致了大肠杆菌胞内三磷酸腺苷（adenosine triphosphate,ATP）外泄,细胞物质代谢、能量代谢失常,最终导致了菌体死亡。这些现象表明,孔道形成（pore formation）理论可能同样适用于Lactobacillin XH2对革兰氏阴性菌大肠杆菌的作用机理。     

The kinetics of the precipitation of chemically modified alpha S1-casein by calcium.

The aggregation of alpha S1-casein by Ca is viewed as resulting from a reduction in the net negative charge by the binding of positively charged Ca2+. This reduction in charge leads to the equivalent of isoelectric precipitation. A quantitative relationship between the rate of aggregation of the casein and the monomer net charge is predicted in this isoelectric precipitation model. Precipitation results are presented for alpha S1-casein labelled with dansyl chloride. Providing the changes in charge brought about by the modification are taken into account, the precipitation behaviour of these caseins can be reconciled with that of native alpha S1-casein. This emphasises the role of electrostatic repulsion in this process and lend further support to the isoelectric precipitation model.     

不对称PCR技术结合免疫层析法快速检测产志贺毒素大肠埃希氏菌

产志贺毒素大肠埃希氏菌（Shiga toxin-producing Escherichia coli,STEC）可分泌志贺毒素,致病性强,建立简便快速的STEC检测方法对控制食源性疾病的发生和蔓延极为重要。使用生物素标记STEC的标志性毒力基因stx1与stx2的下游引物进行不对称聚合酶链式反应,获得生物素化的目标单链DNA与聚苯乙烯微球标记的目标基因的上游引物特异性结合形成结合物,结合物上的生物素因与试纸条检测线上链霉亲和素结合而使检测线显色。通过建立的方法可成功快速检测出携带不同毒力基因的STEC,同时利用该方法对23 株菌株进行stx基因的检测,结果显示该方法可准确检测到含有STEC标志性毒力基因stx的菌株,且表现出良好的特异性。     

生物膜干涉法检测花生蛋白与花生过敏患者血清IgE的结合能力

探索基于生物膜干涉技术对花生蛋白与花生过敏患者血清中免疫球蛋白E（immunoglobulin E,IgE）的结合能力进行检测的方法。利用链霉亲和素（streptavidin,SA）标记的传感器、生物素化的羊抗人IgE抗体、花生过敏患者血清池以及花生蛋白建立了一种测定花生蛋白与花生过敏患者血清IgE结合能力的新方法,优化检测条件为抗体1∶100稀释后线下固化20?min,血清1∶10稀释后过夜结合,完成传感器修饰。在线洗基线后用质量浓度为1?mg/mL的花生蛋白与传感器结合3?600?s,解离120?s。利用该法对不同热加工后花生蛋白与患者血清IgE的结合能力进行评估,并与常用的酶联免疫吸附实验（enzyme linked immunosorbent assay,ELISA）检测进行比较。结果表明,本方法可以直接评估过敏原蛋白与血清IgE的结合能力,与ELISA结果相关系数达到0.91。热加工中,油炸处理提高了花生蛋白的IgE结合能力,水煮和烘烤降低了花生蛋白的IgE结合能力,且去壳热加工比带壳热加工花生的蛋白的IgE结合能力更强。     

DETECTION OF FIMBRIAE (CFA/I) ON ENTEROTOXIGENIC ESCHERICHIA COLI USING ANTIBODIES

Fimbriae (CFA/I) were isolated after detachment from an enterotoxigenic Escherichia coli ( ETEC) strain H10407, purified by successive isoelectric point precipitation at pH 3.5, and injected into rabbits. The antisera containing the polyclonal antibodies against the fimbrial proteins (antigens) were then used to detect the presence of CFA/I on other E. coli cultures using an indirect ELISA system. The ELISA procedures for the CFA/I assay was as sensitive as the DNA hybridization for detecting the ETEC strains. Two bands (major and minor) were present when the isolated fimbrial proteins were subjected to SDS-PAGE. The major band had a molecular mass of 40.3 and the minor 36.4 kDa. Electron microscopy of the fimbriae appeared as thin thread rods with variable length (5–15 μm ).     

Autoinducer-2 activity of gram-negative foodborne pathogenic bacteria and its influence on biofilm formation

ABSTRACT:  This study evaluated whether autoinducer-2 (AI-2) activity would be associated with biofilm formation by Salmonella and Escherichia coli O157:H7 strains on food contact surfaces. In study I, a Salmonella Typhimurium DT104 strain and an E . coli O157:H7 strain, both AI-2 positive, were individually inoculated into 50 mL of Luria–Bertani (LB) or LB + 0.5% glucose (LBG) broth, without or with stainless steel or polypropylene ( Salmonella ) coupons. At 0, 14 ( Salmonella ), 24, 48, and 72 h of storage (25 °C), cells in suspension and detached cells from the coupons, obtained by vortexing, were enumerated on tryptic soy agar. In study II, a Salmonella Thompson AI-2-positive strain and an AI-2-negative strain, and an E . coli O157:H7 AI-2-positive strain and an AI-2-negative strain were inoculated into LB broth with stainless steel coupons. Cells were enumerated as in study I. In both studies, AI-2 activity was determined in cell-free supernatants. Cell numbers of S . Typhimurium DT104 on biofilms were higher ( P < 0.05) in LB than those in LBG, while the E . coli O157:H7 strain showed no difference ( P ≥ 0.05) in biofilm cell counts between LB and LBG after storage for 72 h. Both S . Typhimurium DT104 and E . coli O157:H7 strains produced higher ( P < 0.05) AI-2 activity in LBG than LB cell suspensions. Cell counts of AI-2-positive and-negative S . Thompson and E . coli O157:H7 strains were not different ( P ≥ 0.05) within suspensions or coupons (study II). The results indicated that, under the conditions of this study, AI-2 activity of the pathogen strains tested may not have a major influence on biofilm formation on food contact surfaces, which was similar between AI-2-positive and -negative strains.     

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.     

Effect of antimicrobial proteins from porcine leukocytes on Staphylococcus aureus and Escherichia coli in comminuted meats

Study is aimed to elucidate the effect of porcine leukocyte antimicrobial protein on the proliferation of Staphylococcus aureus and Escherichia coli inoculated in ground meats. The antimicrobial proteins were isolated from porcine blood using 0.2 M sodium acetate extraction and cation-exchange column chromatography. Antimicrobial protein preparation consisted predominantly of 7.5 and 6 kDa molecules. Immunoblotting demonstrated that the 7.5 kDa molecule displayed defensin immuno-reactivity, a unique blood granulocyte antimicrobial protein. Both 7.5 and 6 kDa molecules could decrease apparent proliferation of test cultures. Sterilization (121?°C, 30 min) and proteolytic enzymes (pepsin, trypsin, and chymotrypsin) could significantly decrease bacteriocidal activities of antimicrobial protein preparation. Furthermore, antimicrobial protein preparation enhanced exposure of intercellular nucleotide of test cultures. Antimicrobial protein preparation inhibition of S. aureus and E. coli growth was concentration and time dependence. Adding 160 μg/g antimicrobial protein preparation to ground ham meat and sausage mince could significantly (P<0.05) hurdle viable colony formation of test cultures at 6 and 12 h, respectively. Taken together, porcine leukocyte antimicrobial protein preparation has potential to inhibit proliferation of S. aureus and E. coli inoculated in ground meat via induction of bacterolysis, suggesting this antimicrobial protein preparation be an alternative natural bio-preservation source for ground meat products.     

Improved L-threonine production of Escherichia coli mutant by optimization of culture conditions

L-threonine production was investigated in a minimal salt medium using L-threonine-overproducing Escherichia coli MT201, derived from E. coli K-12. It was observed that dry cell weight reached 12.5 g/l with 15.9 g/lL-threonine. To increase dry cell weight and L-threonine production, the fermentation process was optimized. When biotin was added as growth factor, L-threonine production reached 52.0 g/l from 15.9 g/l without biotin. Dry cell weight and L-threonine production were further increased by continuous feeding of the feed media with an optimized L-methionine concentration (5.0 g/l). However, high-cell-density culture caused oxygen-limited condition, which resulted in the accumulation of organic acids. To overcome this problem, oxygen-enriched air was supplied to the fermentor with the minimal salt medium. Under these optimal conditions, we achieved an L-threonine production of 80.2 g/l in the minimal salt medium.     

Expression of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403 in methylotrophic yeast Pichia pastoris

The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 produced intracellularly in Escherichia coli was able to attach to the surface of cells of Lactobacillus casei NRRL B-441, Bacillus subtilis 168, E. coli XL1-blue and Saccharomyces cerevisiae IFO0216. Therefore, this domain is a suitable fusion partner for the adhesion of proteins to cell surfaces. The production of cell-surface adhesive proteins using this domain in Pichia pastoris is particularly attractive, because this organism has better capability to allow the correct folding of the recombinant proteins than prokaryotic hosts. However, when this domain is produced in this yeast, its cell-surface binding activity may be limited by glycosylation. In this study, therefore, we constructed a CPH mutant (CPHM) devoid of the potential N-glycosylation sites by site-directed mutagenesis. CPHM was successfully expressed extracellularly in P. pastoris (GS115) using the methanol inducible AOX1 promoter with an alpha-mating factor signal sequence, whereas the native CPH was not produced in this host. Western blot analysis revealed that the apparent molecular size of CPHM was 18 kDa greater than that of CPH produced in E. coli (32 kDa), which is attributed to O-glycosylation. However, CPHM produced in P. pastoris was capable of binding to the cell surfaces despite its modification by the yeast, and its dissociation rate constant from the surface of L. casei NRRL B-441 cells was 3.5-fold lower than that of CPH produced in E. coli. These results demonstrate the applicability of the constructed domain (CPHM) for the production of cell-surface adhesive proteins in P. pastoris.     

Investigation of gliadin binding to different selected proteins using a biotin-streptavidin system

Using a biotin streptavidin system 12 out of 19 different test proteins were found to bind to gliadin, with casein, fibrinogen, and fibronectin showing the strongest association. Binding was demonstrable using different gliadin subfractions and enzymatic digests. There was no consistent effect of several carbohydrates on binding. Binding of most proteins to gliadin was diminished slightly by removal of Ca²⁺ and strongly by dioxane, ethylene glycol, Triton X-100, and Tween 20. The results suggest a non-selective association between gliadin and the proteins investigated, mainly due to hydrophobic interactions, which might be of significance in intestinal pathobiochemistry, e. g. disease.

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Untersuchung der Bindung von Gliadin an unterschiedliche ausgewählte Proteine in einem Biotin-Streptavidin-System

Zusammenfassung Unter Verwendung eines Biotin-Streptavidin-Systems konnte bei 12 von 19 Testproteinen eine Bindung an Gliadin festgestellt werden, wobei Casein, Fibrinogen und Fibronectin die stärkste Assoziation zeigten. Die Bindung war an unterschiedlichen Gliadinsubfraktionen und-digestaten nachweisbar. Verschiedene Kohlenhydrate hatten keinen beständigen Effekt auf die Bindung. Die Bindung der meisten Proteine an Gliadin wurde durch Entfernung von Ca²⁺ leicht und durch Dioxan, Ethylenglycol, Triton X-100 und Tween 20 stark verringert. Die Ergebnisse weisen auf eine unselektive Bindung im wesentlichen infolge hydrophober Interaktionen zwischen Gliadin und den untersuchten Proteinen hin, die auch für die intestinale Pathobiochemie (Zöliakie) bedeutsam sein könnte.

     

PCR 检测乳品中大肠杆菌的研究

用大肠杆菌人工污染乳样品直接或8h 增菌后提取DNA 模板,以大肠杆菌丙氨酸消旋酶基因alr 保守区设计引物,经过PCR 扩增凝胶电泳检测,不增菌条件下全脂乳和脱脂乳的检出限为104CFU/ml,增菌后检出限为1CFU/ml。与国标鉴别培养法比较,PCR 法时间缩短、灵敏度和准确度提高,该法更适宜乳品生产经营的快速实时检测。     

Impact of food processing on the structural and allergenic properties of egg white

BackgroundEgg is one of the most nutritious foods that is easily available and it has become a favorite source of major nutrients like lipids and proteins around the world. However, eggs can trigger severe allergic reactions, especially in infants and children. The reactions are mostly IgE-mediated with a range of symptoms related to nose and throat and further can lead to life-threatening anaphylaxis. A total of ten allergens have been recognized to date and researchers are actively working on understanding their structure-function relationship which could help reduce the allergy incidences. Major egg allergens are abundant in egg white which include ovalbumin (OA), ovomucoid (OVM), ovotransferrin (OVT) and lysozyme (Lys). In addition to allergens, avidin present in egg white is also extensively studied due to its anti-nutritional properties. Avidin is known to form a complex with biotin which makes it unavailable for absorption.Scope and approachThis review focuses on the effects of thermal and non-thermal processing methods on the structure and functional properties of various egg proteins including a wide range of allergens and anti-nutrients.Key findings and conclusionsNovel processing techniques including various non-thermal techniques show promising results in reducing the allergic reactions of egg. Anti-nutrients like avidin can be partly inactivated under combined high-pressure processing and heat treatment.     

Proteome analysis of virulence factor regulated by autoinducer-2-like activity in Escherichia coli O157:H7

Many pathogenic bacteria, including Escherichia coli O157:H7, can control gene expression in a cell density-dependent manner by producing small signaling molecules (autoinducers) in a process known as quorum sensing. In this study, the effects of the autoinducer-2-like activity on the expression of proteins, including virulence factors, in E. coli O157:H7 were characterized by proteomic analysis. Compared with the control, E. coli O157:H7 strains in the presence of autoinducer-2-like activity exhibited elevated virulence by more rapidly forming cell aggregates on epithelial cells and rapidly killing the nematode Caenorhabditis elegans, the surrogate host. Two-dimensional gel electrophoresis revealed 18 proteins that were upregulated by autoinducer-2-like activity and 4 proteins that were down-regulated. These proteins were further characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and are involved in the metabolic process, adaptation and protection, cell motility, secretion, envelope biogenesis, and protein translation. These results indicate that the newly identified proteins are associated with the control of virulence in E. coli O157:H7 and that these proteins can be potential targets for the development of antibiotics and other antimicrobial agents.