鼠李糖乳杆菌JAAS8胞外多糖生物合成基因的克隆及生物信息学分析

根据已知乳杆菌胞外多糖生物合成基因的同源性,利用其保守区设计引物,扩增出鼠李糖乳杆菌JAAS8磷蛋白磷酸酶基因(wzb)序列,并通过染色体步移法克隆了鼠李糖乳杆菌 JAAS8 参与胞外多糖生物合成基因簇全部序列 (19.7kb),利用生物信息学方法预测了基因簇 16个阅读框的结构和功能。结果表明,该序列与已报道的乳杆菌胞外多糖生物合成基因具有高度同源性。welF、welG、welH、welI、welJ和welE为合成寡糖重复单元的糖基转移酶基因。rmlA、rmlB和rmlC基因与dTDP-L-鼠李糖前体的生物合成密切相关。wzd、wze、wzy、wzx、wzr和wzb基因预测蛋白主要参与胞外多糖合成过程中多糖链长检测、聚合和输出。     

干酪乳杆菌细菌素的抗菌机制分析

目的：分析和预测干酪乳杆菌细菌素基因及其编码蛋白的分子结构和理化性质,明确细菌素的抗菌机制。方法：利用生物信息学软件进行细菌素编码蛋白的结构预测和功能分析,采用氨基酸定点突变技术对预测的氨基酸位点进行点突变,检测突变体的抗菌活性。结果：干酪乳杆菌细菌素（LacA和LacB）属于热稳定、分泌信号肽的跨膜蛋白,LacA蛋白存在典型的抗菌结构域GxxxG,构建LacA蛋白中第40位V和第57位I的突变载体,两个位点中任何一个发生突变都明显影响细菌素的抗菌活性。结论：推测V40和I57是干酪乳杆菌细菌素发挥抑菌活性的关键氨基酸位点,为今后探索干酪乳杆菌细菌素（class IIb）抗菌机制提供理论依据。     

石榴查尔酮异构酶生物信息学分析

查尔酮异构酶（chalcone isomerase,CHI）是黄酮类化合物合成途径中的关键酶之一。为了进一步研究查尔酮异构酶的结构和功能,利用生物信息学方法对石榴查尔酮异构酶进行分析。结果表明,石榴查尔酮异构酶为亲水性蛋白,呈酸性,无跨膜区域,没有信号肽,亚细胞定位于线粒体,具有磷酸化位点,但不存在糖基化位点,氨基酸序列具有chalcone 3查尔酮超家族保守结构域;二级结构预测显示,α-螺旋和无规卷曲是其二级结构的主要构成部分;同源建模法构建其三级结构模型,结果表明该模型与二级结构预测结果相符合,模型质量评估结果较好。     

副干酪乳杆菌乳酸脱氢酶的生物信息学分析

目的：分析和预测副干酪乳杆菌(Lactobacillus paracasei)乳酸脱氢酶(lactate dehydrogenase,LDH)基因及其编码蛋白的结构和特性,以指导其生物学功能的实验研究。方法：利用美国国家生物技术信息中心(NCBI)和瑞士生物信息学研究所的蛋白分析专家系统(ExPASY)中有关基因和蛋白的序列和结构信息分析的各种工具,结合其他生物信息学分析软件,如ClustalX、VMD,从数据库中得到乳酸脱氢酶基因,分析、预测该基因编码的蛋白理化性质、翻译后的修饰位点、拓扑结构、二级结构、三级结构和功能域。并与其他微生物的乳酸脱氢酶蛋白序列进行比对,得出它们的保守序列。结果：该基因编码335个氨基酸,其蛋白理论分子质量为36608.8u,预测该蛋白有3个跨膜区。与干酪乳杆菌的乳酸脱氢酶进化关系最近。结论：应用生物信息方法可预测得到副干酪乳杆菌的乳酸脱氢酶的结构与功能方面的信息。     

芽孢杆菌发酵液的抑菌活性及葡聚糖酶的基因克隆、表达分析

通过液培法和菌丝生长速率法,探究茯苓粉培养基（ABP培养基）诱导芽孢杆菌CmRh1产葡聚糖酶的发酵上清液对苹果炭疽病菌的抑制作用;采用PCR技术克隆葡聚糖内切酶基因,异源表达并初步验证其功能;采用在线生物信息学对芽孢杆菌葡聚糖酶的理化性质、信号肽、跨膜区、保守结构域、二级和三级结构、亚细胞定位等进行预测。结果表明：茯苓粉培养基（ABP培养基）诱导的芽孢杆菌CmRh1发酵上清液对苹果炭疽病菌具有一定的抑菌效果,抑菌率分别为16.07%和14.03%;从芽孢杆菌CmRh1菌株克隆到1个葡聚糖内切酶基因（Glu4）,开放阅读框（Open Reading Frame,ORF）为1500 bp,编码499 aa。生物信息学预测,Glu4是1个含有信号肽和1个跨膜结构域稳定的亲水性蛋白质,其分子量约为55 kDa,理论等电点为7.14;保守结构域预测,Glu4属于GH5型纤维素酶家族;二级结构元件主要包括α螺旋、延伸链、β转角和无规则卷曲,三级结构为典型的β-三明治结构,符合葡聚糖内切酶的结构特征;Glu4亚细胞定位于细胞外,是一个典型的分泌蛋白。SDSPAGE结合Western blot结果显示...     

几种植物淀粉合成酶的生物信息学分析

采用生物信息学方法比对分析了马铃薯、红薯、小麦、高粱、南瓜、水稻等6种植物的淀粉合成酶(SS)的核苷酸和氨基酸序列,对其甲基化位点、组成成分、理化性质、疏水性/亲水性、跨膜结构域、蛋白质二级结构、功能结构域进行预测和分析。结果表明:红薯、小麦、高粱和水稻都含有甲基化位点,马铃薯和南瓜不含甲基化位点。SS的平均全长为1 847 bp,开放阅读框的长度约为877 bp,起始密码子为ATG,终止密码子有3种;开放阅读框所编码的氨基酸残基平均数286,平均相对分子质量为31 915.88;平均等电点为8.264 5,中性溶液中平均带电荷为-0.598 1;平均亲水氨基酸63个、疏水氨基酸102个,SS为疏水性蛋白。6种植物的SS含有2个跨膜结构,其二级结构以无规则卷曲和延伸链为主要构件。通过进化树分析淀粉合成酶,小麦和高梁进化程度相似,和南瓜的进化相距较远。     

植物乳杆菌WU14 6-磷酸-β-葡萄糖苷酶的基因克隆、蛋白表达及生物信息学分析

植物乳杆菌是一类兼性异养,兼性厌氧的同型发酵乳酸菌,其在改善食品风味和发酵特性等方面发挥着重要作用,而这种功能特征与其具有糖苷酶活性密切相关。为了挖掘植物乳杆菌糖苷酶的应用价值,以植物乳杆菌WU14为研究对象,利用聚合酶链式反应(PCR)技术成功克隆了8个WU14的糖苷水解酶,生物信息学分析表明均为糖苷水解酶1家族(GH1)的6-磷酸-β-葡萄糖苷酶基因。鉴于植物乳杆菌中该类糖苷酶鲜有报道,采用原核异源表达展开研究。序列分析表明8个酶蛋白氨基酸序列具有GH1家族的6-磷酸-β-葡萄糖苷酶典型的2个保守催化位点,序列一致性在32%～74%之间。经SDS-PAGE,8个蛋白均在大肠杆菌中表达,其中BglAW14、BglCW14、BglFW14为部分可溶性表达,其余为蛋白表达,形成包涵体。生物信息学分析显示:8个基因的编码蛋白都无信号肽和跨膜结构,疏水性较强。由亚细胞定位预测可知,除BglEW14主要存在于分泌结构内和BglHW14主要存在于细胞膜外,其余6个基因都存于细胞质内。对8个6-磷酸-β-葡萄糖苷酶基因的克隆表达及生物学序列分析,为进一步探究植物乳杆菌来源的6-磷酸-β-葡萄糖苷酶的分子机理研究奠定了基础。     

植物乳杆菌LY-78乳酸脱氢酶基因的生物信息学分析

目的:探究植物乳杆菌LY-78菌株全部5个乳酸脱氢酶基因及其编码蛋白质的结构和功能。方法:应用多种生物信息学软件对植物乳杆菌LY-78菌株的5个乳酸脱氢酶基因及氨基酸序列进行结构分析和功能预测。结果:植物乳杆菌LY-78中5个乳酸脱氢酶的核苷酸及氨基酸序列均具有较高的保守性,均为位于细胞质的热稳定性、非分泌、非跨膜蛋白,除了ldh L3基因,其余4个基因均具有各自高度保守的功能位点和结构域,均存在典型的烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD~+)结合位点序列GXGXXG。结论:乳酸脱氢酶D1(D1-lactate dehydrogenase,D1-LDH)、D2-LDH、L1-LDH及L2-LDH均具有完整的功能位点和结构域,很可能具有真正的乳酸脱氢酶活性,L3-LDH由于缺失NAD~+结合结构域和功能位点,因而可能不具有乳酸脱氢酶活性,这些分析结果为今后植物乳杆菌乳酸脱氢酶基因改造和苯乳酸合成代谢机制研究提供了必要的理论基础。     

嗜水气单胞菌拮抗芽孢杆菌抗菌素相关基因分析

分别对抗鲟源嗜水气单胞菌的解淀粉芽孢杆菌G1的抗菌素相关基因进行PCR扩增与测序,并对其编码产物的氨基酸序列、跨膜螺旋信号、结构域与二级结构等进行预测与分析。结果表明:菌株G1仅含有伊枯草菌素合成必需基因,该基因与GenBank基因库中芽孢杆菌属其他细菌的伊枯草菌素A、杆菌抗霉素D、抗霉枯草菌素等伊枯草菌素家族基因自然聚类,与枯草芽孢杆菌MH25株和RB14株的伊枯草菌素A基因16S rRNA序列有98%的高度同源性,而且其编码产物的氨基酸序列与GenBank中芽孢杆菌属其他细菌的伊枯草菌素、伊枯草菌素A、脂肽类化合物Bacillorin、芽孢菌素D等抗菌物质的氨基酸序列有高度同源性,与枯草芽孢杆菌MH25的伊枯草菌素A合成酶B(GenBank登录号:ABY89499)的亲缘关系最近。此外,菌株G1伊枯草菌素合成必需基因的编码产物不具有明显的跨膜结构,但其结构域中存在AMP结合位点和PP结合位点,二级结构中存在α螺旋、伸展链、β转角和无规则卷曲。     

γ-PGA降解酶系基因pgdS和ggt的克隆及生物信息分析

以实验菌株枯草芽孢杆菌Bacillus subtilis 115基因组为模板,通过PCR技术成功克隆到γ-聚谷氨酸降解酶系基因pgd S和ggt,并进行测序,利用Ex PASy-Prot Param tool、Server 3.0 Signal P、TMHMM Server和Tmpred、PSORTB、Predict Protein、Swiss-Model Workspace软件,分别对蛋白质的理化性质、信号肽、跨膜区、亚细胞定位、二级结构、三维结构建模进行了分析和预测。结果表明:pgd S和ggt基因分别含1242个和1764个核苷酸,分别编码414个和588个氨基酸。Pgd S和GGT均为稳定型亲水性蛋白,都存在信号肽,其亚细胞分别定位于胞壁和胞外。GGT在N端存在一个强跨膜区。二级结构分析显示,Pgd S和GGT两种蛋白都以L(环)为主,分别占53.27%和52.47%。通过对γ-PGA降解酶系蛋白结构的分析,为日后有效控制γ-PGA合成及相关研究提供参考和理论依据。     

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.     

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.     

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.     

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.     

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.     

Analysis of benzo[a]pyrene in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples

A simple, rapid and inexpensive method has been developed for the determination of benzo[a     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.     

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.