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1.
Inhibitory action of basic esterified milk whey proteins [methylated (Met) or ethylated (Et) beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA)], basic native proteins (chicken egg white lysozyme and calf thymus histone), and basic protein-like substances (L-polylysines) against the activity and replication of lactococcal bacteriophages (bIL66, bIL67, and bIL170) was tested. Chemical interactions of these proteins with phage DNA were determined as well as their protective effect on the growth of a laboratory plasmid-cured Lactococcus lactis subjected to an infection by the bacteriophages. All the proteins studied showed inhibitory activity against the three bacteriophages as tested by marked reduction of their lytic activities and decreasing the replication of studied phages. Histone and Met-BLG were more active toward bIL66 and bIL67, respectively, while both proteins were highly and equally active toward bIL170. Lysozyme showed lower antiviral activity. Antiviral activity of Et-BLG was a little bit lower than that observed in the case of the Met derivative. Esterified ALA also showed considerable but slightly lower antiviral activity as compared to other proteins. L-polylysines also showed an antiviral effect against the three bacteriophages studied, their influence being highly dependent on their molecular size. The best effective size of L-polylysines was in the range 15-70 kDa. Replication of bIL67 was inhibited by the presence of esterified ALA or BLG and native basic proteins. Complete inhibition of replication of bIL67 occurred when using polylysines with molecular masses in the ranges 4-15, 15-30, and 30-70 kDa, while protein-like substrates with lower molecular masses had only a slight effect. The presence of histone and Met-BLG at a concentration of 0.13 mg/mL in the incubation medium protected L. lactis against lysis when it was subjected to an infection by bIL67 (10(5) pfu/mL). The same action was achieved by l-polylysine (15-30 kDa) used at a concentration of 0.03 mg/mL in the incubation medium.  相似文献   

2.
The antiviral activity of methylated alpha-lactalbumin (Met-ALA), methylated and ethylated beta-lactoglobulins (Met- and Et-BLG) was evaluated against acyclovir (ACV)-sensitive and -resistant strains of herpes simplex virus type 1 (HSV-1) and compared to that of ACV and L-polylysines (4-15 kDa) using fixed or suspended Vero cell lines. Esterified whey proteins and their peptic hydrolyzates displayed protective action against HSV-1, which was relatively lower than that induced by ACV or L-polylysines. The higher activity of L-polylysines was maintained against an ACV-resistant strain of HSV-1, whereas ACV lost much of its activity. The mean 50% inhibitory concentration (IC50) was about 0.8-0.9 microg/mL for L-polylysines against ACV-sensitive and -resistant strains of HSV-1 when using two concentrations of virus (50% and 100% cytopathic effect, CPE). The IC50 values of ACV against the sensitive strain of HSV-1 were 3 and 15 microg/mL when using the low and high concentrations of virus, respectively. When using 50% CPE, IC50 values for esterified whey proteins ranged from 20 to 95 microg/mL, depending on the nature of the ester group, the degree of esterification, and the nature of the protein. Using the real-time PCR technique, it was shown that Met-ALA inhibited HSV-1 replication.  相似文献   

3.
该试验以JS25噬菌体为研究对象,在分析稳定性的基础上,确定其对宿主细胞的侵染时间及最佳感染复数,最后通过在即食食品中的抑菌效应确定对即食食品的生物防治效果。结果表明,JS25噬菌体最佳感染复数为0.1、侵染时间为20 min、最适温度及p H值分别为4~60℃和6~9;另外,JS25噬菌体的潜伏期为20 min,60 min后平均裂解量为24.55 PFU/cell;试验结果表明,JS25噬菌体在即食食品中有较高的稳定性,且随噬菌体初始效价的增加,对即食食品中金黄色葡萄球菌的抑制作用也逐渐增加;但是在不同即食食品中的生物防治效果存在差异,主要表现为当效价为108及1010 PFU/g时,能分别有效的抑制鲜牛奶和圆白菜在贮藏过程中的金黄色葡萄球菌数量,当效价大于109 PFU/g时,对肉干及火腿中的致病菌有良好的防治效果。  相似文献   

4.
为了揭示温度这一重要环境因子与罗非鱼免疫力及对海豚链球菌的易感性之间的关系,分别进行了不同温度下罗非鱼感染海豚链球菌的死亡率试验和不同温度对罗非鱼非特异免疫相关酶（超氧化物歧化酶,SOD;溶菌酶;碱性磷酸酶,AKP以及补体C3）活性影响的试验。试验结果表明,罗非鱼感染海豚链球菌的死亡率与水温的变化呈现明显的正相关;特别是在高温条件下,罗非鱼免疫力受到了明显的抑制,具体表现为SOD活性呈现先诱导后抑制的趋势,AKP活性在12h较28℃时显著上升（P〈0．05）,血清溶菌酶活性则受到抑制作用,而补体C3活性在12h和24h与对照组相比显著升高了10．99％和13．40％（P〈0．05）。试验结果显示高温能够引起罗非鱼免疫力低下,使鱼体对病原菌易感性增强,致使罗非鱼因感染海豚链球菌造成的死亡率显著升高,为研究罗非鱼海豚链球菌病爆发的环境机制提供了相关资料。  相似文献   

5.
The effect of heat treatment on the IgE binding ability of beta-lactoglobulin, as pure protein or in whole milk, was studied by inhibition of IgE antibody binding using FEIA-CAP inhibition. A slight but significant decreased IgE binding was seen between unheated and heat-treated beta-lactoglobulin solution at 74 degrees C (IC(50) = 2.03 and 3.59 microg/mL, respectively, p = 0.032). A more pronounced decrease was found at 90 degrees C with an IC(50) of 8.45 microg/mL (p = 0.014). The inhibition of IgE binding of milk after heat treatment at 90 degrees C was also significantly decreased (p = 0.007). However, at all heat treatments, a similar total amount of IgE antibodies could be inhibited at a sufficiently high concentration of beta-lactoglobulin. The inhibiting ability of beta-lactoglobulin was significantly impaired in some fermented acidified milk products such as yogurt as compared to that in nonfermented milk (p < 0.001). There was only a small difference of IgE binding between the native forms of genetic variants A and B.  相似文献   

6.
To investigate the effect of polysaccharide attachment to proteins on the production of IgG and IgE, the genetic attachment of polysaccharide to lysozymes (G49N and R21T) using the yeast expression system (Saccharomyces cerevisiae AH 22) and the Maillard-type polysaccharide attachment to native lysozyme and soybean P34 protein were attempted. The production of IgG and IgE was investigated by using mice immunized with the protein-polysaccharide conjugates or native proteins. The attachment of polysaccharide to lysozyme using the yeast expression system greatly suppressed the production level of IgG and IgE. The attachment of polysaccharide to native lysozyme and soybean P34 protein using the Maillard-type reaction was also found to be effective in reducing the production level of IgE compared to IgG.  相似文献   

7.
鸡γ-干扰素基因(ChIFN-γ)的瞬时表达及其抗病毒活性检测   总被引：1，自引：0，他引：1  
采用PCR技术从质粒pET-ChIFN-γ中扩增得到鸡(Gallus gallus)γ-干扰素(chicken interferon gamma,ChIFN-γ)基因,将其亚克隆到真核表达载体pCAGGS中.将鉴定正确的克隆命名为pCAGGS-ChIFN-γ,体外转染鸡胚成纤维(CEF)细胞,24 h后经间接免疫荧光(IFA)和免疫印迹(Western blot)检测,表达蛋白具有良好的免疫原性.然后利用表达绿色荧光蛋白(GFP)的重组水疱口炎病毒(VSV*GFP)在CEF细胞上检测表达的ChIFN-γ抗病毒活性(AVA),经检测其抗病毒活性为2×10~3AU/mL,并且其活性可被抗鸡IFN-γ的多克隆抗体阻断.  相似文献   

8.
The structural and antimicrobial functions of lysozyme reduced with food-compatible reducing agents-cysteine (Cys) and glutathione (GSH)-were investigated. The disulfide bonds were partially reduced by thiol-disulfide exchange reactions under heat-induced denaturing conditions from 55 to 90 degrees C. The results showed that treatment of lysozyme with Cys and GSH resulted in the introduction of new half-cystine residues (2-3 residues/mol of protein). The released SH groups, in turn, rendered the lysozyme molecule more flexible, being accompanied by a dramatic increase in the surface hydrophobicity and exposure of tryptophan residues. As a consequence, the resulting reduced lysozymes were more capable of binding to lipopolysaccharides (LPS) and permeabilizing the bacterial outer membrane, as evidenced by the liposome leakage experiment, than were native or heated lysozyme. Both reduced lysozymes displayed significantly higher antimicrobial activity than native or heated lysozyme against Salmonella enteritidis (SE) in sodium phosphate buffer (10 mM, pH 7.2) at 30 degrees C for 1 h. Their minimal inhibitory concentrations (MICs) against the tested bacteria were about 150- and 25-fold lower than their respective MICs of native or heated lysozyme. The results suggest that partially reduced lysozyme could be used as a potential antimicrobial agent for prevention of SE attack.  相似文献   

9.
Effect of denaturants on the emulsifying activity of proteins   总被引：1，自引：0，他引：1  
The relationship between protein flexibility and emulsifying activity was investigated by disrupting disulfide bonds and/or noncovalent interactions of the protein. Oil-in-water emulsions using model proteins (apomyoglobin, beta-casein, alpha-casein, lysozyme, bovine serum albumin, kappa-casein, and beta-lactoglobulin) were made in the presence of chemical denaturants (dithiothreitol and/or urea). In most cases, the presence of denaturants enhanced emulsifying activity. The effect was protein-specific and depended on the relative importance of disulfide bonds and noncovalent interactions in stabilizing the native conformation of each protein. Implications for the design of novel protein emulsifiers are discussed.  相似文献   

10.
Dairy industries are interested in knowing the heat treatment undergone by milk so as to control the quality of drinking milks or to control their heating systems. Among the different techniques available to characterize the heat treatment of milk, estimation of the denaturation of proteins has been widely used. However, because the concentration of the proteins in raw milk can fluctuate significantly, determining only the concentration of a native protein without knowing its concentration in the raw milk before undergoing heat treatment can lead to significant imprecision. The objective of this study was to develop, on Biacore 3000, a biosensor assay for determining the denaturation index of alpha-lactalbumin by quantifying separately the native and "heat-denatured" forms of alpha-lactalbumin with specific monoclonal antibodies. alpha-Lactalbumin denaturation index is independent of the concentration of alpha-lactalbumin in the original raw milk. The technique developed is discriminating, fast, repeatable, fully automated, and requires no pretreatment of the milk sample.  相似文献   

11.
An indirect enzyme-linked immunosorbent assay (ELISA) by inhibition was developed for quantifying lysozyme in hen egg white (HEW), a protein of value in not only the food and pharmaceutical industries but also for poultry research. Various experimental conditions (coating, antibodies dilutions, samples dilutions, preparations, blocking agents, and incubation times) were assayed to optimize this assay to the quantification of HEW in egg white samples. HEW samples were diluted 1:3000 to avoid matrix effects, possibly resulting from lysozyme interaction with other egg white proteins. Assay linearity for lysozyme ranged from 0.38 to 4.8 mug/mL, with intra- and interassay variations of 6.8% and 7.6%, respectively, and the lower detection limit was 0.264 mug/mL. We found that lysozyme concentrations in albumen from eggs laid by a hen cohort ranged from 2.2 to 4.5 mg/mL, thus underlining interhen variability. Overall, these data present an ELISA assay that is simple, quick, sensitive, accurate, and has been specifically designed to determine lysozyme concentrations in egg white samples.  相似文献   

12.
An automated fluorescent microsphere-based flow cytometric triplex immunoassay, using the Luminex 100 flow analyzer with MultiAnalyte Profiling (xMAP) technology, was developed for the simultaneous detection of proteins from three vegetable sources as potential fraudulent adulterants in milk powder. In the final triplex inhibition immunoassay, soluble wheat proteins (SWP) and proteins from soy and pea were coupled to three different microsphere sets. A mixture of these microsphere sets was transferred to a microtiter plate well together with the sample and a mixture of three affinity-purified polyclonal antibodies raised against the proteins and labeled with a fluorophore (Alexa 532). After incubation for 1.5 h at room temperature in the dark, the fluorescence intensities on the microspheres were directly measured (no wash procedure) in the Luminex during 10 s per well (100 microspheres per set). The sensitivities of the three assays for plant protein extracts were determined as 0.5-0.6 microg/mL at 50% inhibition. For the detection of the vegetable proteins in milk powder, the samples were dissolved in buffer (0.1 g in 10 mL) and further diluted (20 times) to create a 50% inhibition at approximately 0.5% of the vegetable proteins in the total protein content of milk powder. With the help of calibration standards, prepared under conditions comparable to those for sample materials, the triplex immunoassay proved to be quantitative above 0.1%, although concentrations in high-heated milk powders were underestimated. Due to the xMAP technology, in which 100 different microsphere sets can be distinguished, this triplex immunoassay can easily be extended to detect other possible adulterants.  相似文献   

13.
Casein fractions have been shown to act as molecular chaperones and inhibit aggregation of whey proteins in dilute solutions (< or =1% w/v). We evaluated if this approach would stabilize protein solutions at higher concentration and thermal processing temperatures desired for beverage applications. Mixtures of beta-lactoglobulin (BLG) (6% w/v) with either beta-casein (BCN) (0.01-2% w/v) or alpha s-casein (ACN) (2% w/v) were adjusted to pH 6.0 and heated (70-90 degrees C) for 20 min, cooled, and then analyzed to determine the degree of aggregation. Aggregation was determined by solution turbidity as optical density (OD) at 400 or 600 nm. The addition of 0.05% (w/v) BCN or greater caused a drop in turbidity for solutions heated at 70-90 degrees C. In contrast, inhibition was observed in BLG-ACN mixtures at 70 degrees C but not at > or =75 degrees C. Moreover, prolonged heating (90 min) of BLG with 2% (w/v) BCN (pH 6.0) at 90 degrees C produced a clear solution while BLG-ACN solutions formed translucent gels after heating for 15 min. The weight-averaged molar mass and root-mean-square (rms) radius of soluble aggregates were determined by size exclusion chromatography in conjunction with multiangle laser light scattering (SEC-MALS). SEC-MALS confirmed the turbidity results by showing that the BLG-BCN mixture (8% w/v protein) produced aggregates with lower molar mass and smaller rms radius (majority 20-40 nm). These results showed that BCN is a feasible component to stabilize higher concentrations of whey proteins in beverages.  相似文献   

14.
The analysis of (R)-9- and (S)-9-hydroxy-10E,12Z-octadecadienoic acid as well as (R)-13- and (S)-13-hydroxy-9Z,11E-octadecadienoic acid (HODE) as free acids, esterified in triacylglycerols (storage lipids), and esterified in polar lipids (phospholipids, glycolipids, etc.) in barley, germinating barley, and finished malt was performed using [13-(18)O(1)]-(S)-13-HODE isotope dilution assays with GC-MS and straight- and chiral-phase HPLC. 9- and 13- HODE occur approximately racemically in barley, indicating an autoxidation. The enantiomeric excesses increase to 78% S for free 9-HODE and to 58% S for free 13-HODE in germinating barley as a result of lipoxygenase-2 (LOX-2) catalysis, but free HODEs are at low concentration. More than 90% of HODEs in barley and malt are esterified. In the storage lipids of green malt 53 mg/kg 9-HODE and 147 mg/kg 13-HODE were detected. This ratio of 30:70 reflects the regioselectivity of the LOX-2 enzyme in malt. In the polar lipids 45 mg/kg 9-HODE and 44 mg/kg 13-HODE were characterized. The latter indicate a hitherto unknown 9-lipoxygenase activity with polar lipids as substrates. During kilning the contents of most HODEs decreased significantly due to chemical and enzymatic degradation, whereas polar-esterified (R)-13-HODE increased (43%) in the finished malt.  相似文献   

15.
Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.  相似文献   

16.
The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.  相似文献   

17.
仿刺参肠多糖对免疫功能的影响及抗肿瘤研究   总被引：1，自引：0，他引：1  
多糖能参与机体的免疫调节,具有抗肿瘤等多种功能。本实验采用灌胃不同剂量仿刺参(Apostichopus japonicus)肠多糖的方法,研究了多糖对小鼠(Mus musculus)免疫功能的影响及其对小鼠的抗肿瘤作用。结果显示,仿刺参肠多糖对接种的H22肿瘤具有剂量依赖性的抑制作用,高剂量组(400mg/kg/d)抑瘤效果最好;胸腺指数高剂量肿瘤组最高(P<0.05),而其他各组差异不明显,脾脏指数在肿瘤组内随着剂量的增大表现为先升高再降低,而空白组呈一直上升的趋势;高剂量多糖可以显著提高荷瘤小鼠和空白小鼠的白介素2(IL-2)含量(P<0.05),对荷瘤小鼠的肿瘤坏死因子(TNF-α)含量也具有显著提升作用(P<0.05),但对空白组小鼠的TNF-α含量影响不显著;荷瘤小鼠的自然杀伤(NK)细胞活力普遍低于空白小鼠,随灌胃剂量的提升空白小鼠NK细胞活力具有上升趋势,荷瘤小鼠的NK细胞活力在中剂量组达到最高后开始下降。实验表明,仿刺参肠多糖可以促进小鼠的免疫功能,并且对小鼠体内肿瘤在一定剂量范围内具有浓度依赖性的抑制作用。  相似文献   

18.
Barley (Hordeum vulgare L.) malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low molecular weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins. It is now reported that some proteins that were concentrated using an "affinity" method inhibited the activity of a malt serine endoproteinase. Two-dimensional electrophoretic and in vitro analyses showed that the inhibited enzyme was serine endoproteinase 1 (SEP-1) and that the inhibition could be quantified using a semipurified preparation of this enzyme. Amino acid sequencing and MALDI-TOF MS were used to identify the components of the partially purified inhibiting fractions. Only the "trypsin/alpha-amylase inhibitors" or chloroform/methanol (CM) proteins, most of which had truncated N and C termini, and one fragment of beta-amylase were present in the inhibitory fractions. When a CM protein fraction was prepared from barley according to traditional methods, some of its component proteins inhibited the activity of SEP-1 and some did not. This is the first report of the purification and identification of barley malt proteins that can inhibit an endogenous serine proteinase. It shows that some of the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests.  相似文献   

19.
Proteolysis of milk proteins can be attributed to both native proteases and the proteases produced by psychrotrophic bacteria during storage of fresh raw milk. These proteases cause beneficial or detrimental changes, depending on the specific milk product. Plasmin, the major native protease in milk, is important for cheese ripening. Milk storage and cheese-making conditions can affect the level of plasmin in the casein and whey fractions of milk. A microbial protease from a psychrotrophic microorganism can indirectly increase plasmin levels in the casein curd. This relationship between the plasmin system and microbial proteases in milk provides a means to control levels of plasmin to benefit the quality of dairy products. This paper is a short review of both the plasmin system and microbial proteases, focusing on their characteristics and relationship and how the quality of dairy products is affected by their proteolysis of milk proteins.  相似文献   

20.
应用噬菌体表面展示技术,从生长抑素免疫的小鼠脾脏中提取总RNA,用RT-PCR技术反转录成cDNA,通过PCR扩增出抗体重链可变区VH基因和轻链可变区VL基因,再用编码(G1y4Ser)3的Linker在体外将VH和VL连接成单链抗体(ScFv)基因,克降到噬菌粒载体pCANTAB5E中,电转化至感受态的大肠杆菌TG1,经辅助噬菌体M13K07超感染,形成噬菌体单链抗体库,有效库容为9.3×107。为利用亲和富集筛选技术,获得具有与SS结合活性的完整重组噬菌粒克隆奠定了基础。  相似文献   

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