羊栖菜多糖提取物对酪氨酸酶的抑制作用研究

目的:研究羊栖菜多糖提取物对酪氨酸酶活性的抑制作用和对结构的影响。方法:提取羊栖菜多糖并测定含量,以L-DOPA为底物,在475nm处测定吸光度,观察羊栖菜多糖提取物对酪氨酸酶二酚酶活力的影响;测定羊栖菜多糖作用下酪氨酸酶内源荧光和ANS结合的荧光的变化,以判断对酶结构的影响情况。结果:羊栖菜多糖提取物对蘑菇酪氨酸酶有明显的抑制作用,可以导致酪氨酸酶的二酚酶活力下降。羊栖菜多糖使二酚酶活力下降一半时的抑制剂浓度(IC50)为(2.16±0.37)mg/mL。羊栖菜多糖对酪氨酸酶的抑制作用是一个可逆过程,抑制类型为混合型抑制。通过动力学分析,测得羊栖菜多糖对酪氨酸酶的抑制常数(Ki)为(4.34±0.56)mg/mL。荧光测定结果表明,羊栖菜多糖的结合引起酪氨酸酶三级结构的明显改变。结论:羊栖菜多糖与酪氨酸酶的结合引起酶结构的变化,并以混合抑制的方式可逆抑制酪氨酸酶的二酚酶活性。本实验为进一步研究和设计新型酪氨酸酶抑制剂奠定科学基础。     

枸杞叶黄酮对胰脂肪酶活性的抑制作用

采用酶动力学法探讨枸杞叶黄酮对胰脂肪酶活性的抑制作用。采用紫外、荧光和红外光谱研究枸杞叶黄酮与胰脂肪酶相互作用的机理。结果表明:枸杞叶黄酮对胰脂肪酶活性有抑制作用,半抑制浓度(IC50)为(0.910±0.008) mg/mL,抑制类型为可逆非竞争抑制,抑制常数为6.04 mg/mL。紫外和荧光光谱显示:枸杞叶黄酮能使胰脂肪酶芳香氨基酸暴露,微环境和酶构象改变,内源荧光猝灭,猝灭类型为动态猝灭为主的混合型猝灭。热力学参数显示:枸杞叶黄酮与胰脂肪酶的相互作用力主要为疏水相互作用。红外光谱显示:枸杞叶黄酮对酶蛋白二级结构的影响,主要表现为复合物结构变得有序化,β-折叠向α-螺旋的转化。枸杞叶黄酮通过改变胰脂肪酶结构和微环境,进而抑制胰脂肪酶活性。     

鞣花酸抑制酪氨酸酶的动力学、荧光光谱分析及分子对接

以蘑菇酪氨酸酶为靶点,采用抑制动力学、荧光光谱分析结合分子对接模拟技术,系统研究鞣花酸(ellagic acid,EA)对酪氨酸酶的抑制作用及机理。体外研究与动力学结果表明,EA以可逆的混合型抑制方式显著抑制酪氨酸酶活性(IC₅₀=0.05 mg/mL),其结合常数K_IIS,表明EA与游离酶的结合比与酶-底物复合物的结合更紧密。荧光光谱猝灭分析表明,EA与酪氨酸酶存在静态猝灭作用,两者通过自发的吸热过程结合生成复合物,主要作用力为疏水作用力,只有1个或1类结合位点。同步和三维荧光光谱分析表明,EA使酪氨酸酶的微环境极性增大,疏水能力减弱,酪氨酸酶的Trp残基更靠近结合位点。分子对接模拟分析进一步印证上述实验结果,形象地表明EA对酪氨酸酶为混合型抑制,EA主要通过疏水作用力和氢键与游离酶或酶-底物复合物进行结合,最终导致酶活性降低。本研究对EA在食品工业中作为保鲜剂的各种应用具有一定参考意义。     

茶多酚对胰α-淀粉酶的抑制动力学研究

研究了绿茶茶多酚对胰α-淀粉酶活性的抑制作用。采用非连续测定和作图法研究,结果表明:茶多酚对胰α-淀粉酶具有明显的抑制作用,其半抑制浓度为0.82mg/mL;抑制类型为可逆性非竞争性抑制,其抑制常数Ki为1.08mg/mL。     

茶多酚对嗜水气单胞菌致病力的抑制作用

针对嗜水气单胞菌污染水产品所致动物感染和消费者食源性疾病等问题,筛选能够抑制嗜水气单胞菌致病力的天然产物茶多酚。溶血试验表明茶多酚对气溶素溶血活性具有直接抑制作用,通过荧光热漂移和七聚体形成试验分析其作用机制。结果表明,茶多酚虽在128μg/mL以下不抑制嗜水气单胞菌的生长,但能在1～8μg/mL直接抑制其主要毒力蛋白气溶素的溶血活性。荧光热漂移和七聚体试验发现,茶多酚通过与气溶素直接结合,降低了气溶素七聚体的形成并抑制其活性。细胞试验结果表明,4～8μg/mL茶多酚能对气溶素导致的人肺癌上皮细胞死亡具有显著的保护作用。此外,50 mg/kg茶多酚治疗可使斑点叉尾鮰嗜水气单胞菌感染模型的存活率提高至60%。综上,茶多酚通过直接与气溶素结合,抑制气溶素形成功能性七聚体而降低其活性,从而显著降低嗜水气单胞菌的体内、外致病力。研究结果提示茶多酚可作为抗生素替代或辅助药物用于治疗嗜水气单胞菌相关感染。     

红花黄酮提取物对酪氨酸酶的抑制作用

研究红花黄酮提取物对酪氨酸酶活性的抑制作用。以L-酪氨酸为底物,对红花黄酮提取物抑制酪氨酸酶活性进行动力学研究。结果表明：在试验浓度范围内,红花黄酮提取物对酪氨酸酶均具有抑制性,且当红花黄酮提取物浓度为3.0mg/mL时,抑制率达到最大值78.03%;IC50值为0.834mg/mL,抑制常数KI为0.036mg/mL,KIS为0.0064mg/mL。研究表明,红花黄酮提取物对酪氨酸酶有显著的抑制作用,酶抑制类型为可逆的混合型抑制。     

木犀草素对酪氨酸酶活性抑制及构象诱导作用

在37℃、pH 6.8的磷酸盐(100 mmol/L)缓冲体系中,采用酶抑制动力学方法,研究了木犀草素对酪氨酸酶的抑制作用、抑制类型和抑制动力学常数,并探讨了木犀草素对酪氨酸酶表面疏水性和二级结构的影响。结果表明,木犀草素是一种可逆的非竞争型酪氨酸酶抑制剂(半抑制率IC50为55.35±1.51μg/m,抑制常数Ki为63.57±2.12μg/mL);失活动力学时间进程分析表明木犀草素能快速与酪氨酸酶发生作用并迅速降低酶的活性;荧光光谱分析显示木犀草素能显著增强酪氨酸酶的表面疏水性;圆二色谱分析表明木犀草素诱导酪氨酸酶的构象伸展和解折叠,随着木犀草素浓度的增加,α-螺旋含量由31.3%逐渐增加到41.6%,由此可见木犀草素能够诱导酪氨酸酶的构象发生部分改变,使酶结构变化而不利于其形成活性中心,进而降低酪氨酸酶的活力。     

茶多酚对α-淀粉酶的抑制作用及分子机理

探究茶多酚对α-淀粉酶的抑制特性并分析其分子作用机制.采用抑制动力学的方法评价茶多酚对α-淀粉酶的抑制作用;通过荧光色谱法及圆二色谱法观察荼多酚对α-淀粉酶空间结构和稳定性的影响;利用分子对接技术,探究茶多酚与α-淀粉酶之间的分子相互作用.结果 表明,茶多酚对于α-淀粉酶的活性具有明显的抑制作用,竞争类型为非竞争性抑制...     

甘薯叶提取物对酪氨酸酶的抑制作用

为探究甘薯叶提取物对酪氨酸酶活性的影响,利用不同极性溶剂萃取甘薯叶乙醇提取物,分别得到石油醚层、三氯甲烷层、乙酸乙酯层及正丁醇层萃取物,并采用多巴速率氧化法考察不同溶剂提取物对酪氨酸酶的抑制作用。结果表明,甘薯叶提取物中存在能抑制酪氨酸酶活性的物质,其中甘薯叶正丁醇萃取物对酪氨酸酶的抑制作用最明显,根据正丁醇萃取物抑制酪氨酸酶的Lineweaver-Burk双倒数拟合方程可得,甘薯叶提取物对酪氨酸酶的抑制作用是可逆过程,其抑制类型是混合型,KI与KIS分别为0.198、0.209 mg/mL,即甘薯叶提取物与游离酶的结合程度大于与酶-底物络合物的结合。     

对羟基肉桂酸对酪氨酸酶催化反应的抑制机理

本文研究了对羟基肉桂酸(HCA)对酪氨酸酶催化单酚底物L-酪氨酸和催化二酚底物L-多巴的抑制能力,并利用紫外-可见光谱、荧光光谱以及分子对接技术探究了其抑制机理。结果表明对羟基肉桂酸对酪氨酸酶催化单酚底物L-酪氨酸比催化二酚底物L-多巴具有更强的抑制作用,半抑制浓度分别为0.096 mmol/L和0.500 mmol/L;紫外-可见分析发现对羟基肉桂酸能与Cu2+发生螯合,使光谱发生明显红移。进一步通过荧光光谱分析得到,对羟基肉桂酸在酪氨酸酶溶液中并没有出现荧光淬灭反而随着对羟基肉桂酸浓度的增大荧光强度变强,说明对羟基肉桂酸被酪氨酸酶催化氧化成对应的醌类物质。利用分子对接技术揭示了对羟基肉桂酸通过氢键和疏水作用竞争性地占据了单酚和二酚底物的空间位置,并与酪氨酸酶中双核铜离子螯合,从而抑制酪氨酸酶催化L-酪氨酸和L-多巴氧化的活性机理。     

Reliable identification of grapevine rootstock varieties using RAPD PCR on woody samples

Since grapevine ( Vitis spp .) rootstock material is being traded increasingly as disbudded woody material a lack of distinctive morphological features on such material necessitates an alternative and reliable means of identification. Methods described here were developed for rapid and efficient extraction of DNA from woody samples rich in phenolic compounds and polysaccharides, and for subsequent identification of varieties by RAPD PCR. Using these methods, and with the application of only one selected RAPD primer, we were able to differentiate sixteen rootstock varieties, including the seven varieties most commonly used in Germany. Problems commonly encountered with reproducibility of RAPD patterns were avoided by choosing primers with a dinucleotide sequence and a high G/C content that allowed a rather high annealing temperature of 45°C. Methods described here should also be useful for other horticultural crops, especially those with woody tissues rich in phenolic compounds and polysaccharides.     

An internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics

An internet website (http://cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-ofcharge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.     

Chlorohydrins of bisphenol A diglycidyl ether (BADGE) and of bisphenol F diglycidyl ether (BFDGE) in canned foods and ready-to-drink coffees from the Japanese market

BADGE.2HCl and BFDGE.2HCl were determined in 28 samples of ready-to-drink canned coffee and 18 samples of canned vegetables (10 corn, 5 tomatoes and 3 others), all from the Japanese market. HPLC was used as the principal analytical method and GCMS for confirmation of relevant LC fractions. BADGE.2HCl was found to be present in one canned coffee and five samples of corn, BFDGE.2HCl in four samples of canned tomatoes and in one canned corn. No sample was found which exceeded the 1mg/kg limit of the EU for the BADGE chlorohydrins. However the highest concentration was found for the sum of BFDGE.2HCl and BFDGE.HCl.H₂O at a level of 1.5mg/kg. A Beilstein test confirmed that all cans containing foods contaminated with BADGE.2HCl or BFDGE.2HCl had at lest one part coated with a PVC organosol.     

European priorities for research to support legislation in the area of food contact materials and articles

A strong science base is required to underpin the planning and decision-making process involved in determining future European community legislation on materials and articles in contact with food. Significant progress has been made in the past 5 years in European funded work in this area, with many developments contributing to a much better understanding of the migration process, and better and simpler approaches to food control. In this paper this progress is reviewed against previously identified work-areas (identified in 1994) and conclusions are reached about future requirements for R&D to support legislation on food contact materials and articles over the next 5 or so years.     

Aroma characterization of various apricot varieties using headspace–solid phase microextraction combined with gas chromatography–mass spectrometry and gas chromatography–olfactometry

The characterization of the aromatic profile of several apricot cultivars with molecular tracers in order to obtain objective data concerning the aromatic quality of this fruit was undertaken using headspace–solid phase microextraction (HS–SPME). Six apricot cultivars were selected according to their organoleptic characteristics: Iranien, Orangered, Goldrich, Hargrand, Rouge du Roussillon and A4025. The aromatic intensity of these varieties measured by HS–SPME–Olfactometry were defined and classified according to the presence and the intensity of grassy, fruity and apricot like notes. In the six varieties, 23 common volatile compounds were identified by HS–SPME–GC–MS. Finally, 10 compounds, ethyl acetate, hexyl acetate, limonene, β-cyclocitral, γ-decalactone, 6-methyl-5-hepten-2-one, linalool, β-ionone, menthone and (E)-hexen-2-al were recognized by HS–SPME–GC–O as responsible of the aromatic notes involved in apricot aroma and considered as molecular tracers of apricot aromatic quality which could be utilized to discriminate apricot varieties.     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Tests of potential functional barriers for laminated multilayer food packages. Part I: Low molecular weight permeants

The advent of the functional barrier concept in food packaging has brought with it a requirement for fast tests of permeation through potential barrier materials. In such tests it would be convenient for both foodstuffs and materials below the functional barrier (sub-barrier materials) to be represented by standard simulants. By means of inverse gas chromatography, liquid paraffin spiked with appropriate permeants was considered as a potential simulant of sub-barrier materials based on polypropylene (PP) or similar polyolefins. Experiments were performed to characterize the kinetics of the permeation of low molecular weight model permeants (octene, toluene and isopropanol) from liquid paraffin, through a surrogate potential functional barrier (25 μm-thick oriented PP) into the food simulants olive oil and 3% (w/v) acetic acid. These permeation results were interpreted in terms of three permeation kinetic models regarding the solubility of a particular model permeant in the post-barrier medium (i.e. the food simulant). The results obtained justify the development and evaluation of liquid sub-barrier simulants that would allow flexible yet rigorous testing of new laminated multilayer packaging materials.     

Occurrence of bisphenol-F-diglycidyl ether (BFDGE) in fish canned in oil

The levels of bisphenol-F-diglycidyl ether (BFDGE) were quantified as part of a European survey on the migration of residues of epoxy resins into oil from canned fish. The contents of BFDGE in cans, lids and fish collected from all 15 Member States of the European Union and Switzerland were analysed in 382 samples. Cans and lids were separately extracted with acetonitrile. The extraction from fish was carried out with hexane followed by re-extraction with acetonitrile. The analysis was performed by reverse phase HPL C with fluorescence detection. BFDGE could be detected in 12% of the fish, 24% of the cans and 18% of the lids. Only 3% of the fish contained BFDGE in concentrations considerably above 1mg/kg. In addition to the presented data, a comparison was made with the levels of BADGE (bisphenol-A-diglycidyl ether)analysed in the same products in the context of a previous study.     

Contribution of European research to risk analysis

The European Commission's, Quality of Life Research Programme, Key Action 1—Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches; harmonization of risk assessment principles, methodologies, and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries; to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women; evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.