乳酸乳球菌NICE系统食品级载体pRNB48的构建

乳酸菌食品级NICE系统是目前较理想的可控制蛋白质生产的食品级诱导表达系统。实验构建的食品级表达载体pRNB48含α-aga食品级选择标记、θ型复制子和nisA启动子,与宿主菌L.lactisNZ9000共同构成乳酸乳球菌食品级NICE系统,可用于目的基因在乳酸乳球菌中高效诱导表达。     

牛凝乳酶原基因在食品级乳酸乳球菌中的重组表达

将牛凝乳酶原基因连接pNZ8149载体,并转化乳酸乳球菌NZ3900,经乳酸链球菌素Nisin诱导,测得重组菌株胞内凝乳酶活力达到0.7 SU/mL,培养基中检测不到凝乳酶活力,实现了牛凝乳酶原基因在乳酸链球菌Nisin诱导基因表达系统（nisin controlled gene expression system,NICE）中活性表达。在此基础上,将分泌信号肽SPusp45连接于pNZ8149,构建了分泌型表达载体pNZ8149s,并实现牛凝乳酶原基因在NICE系统中分泌表达。当使用1 ng/mLNisin诱导5 h后,重组菌株胞内检测不到凝乳酶活力,培养基中凝乳酶活力为1.2 SU/mL,说明pNZ8149s能够促使凝乳酶原从乳酸乳球菌中分泌。该方法为重组牛凝乳酶在食品级菌株中重组表达提供了一种可行的方案。     

TGEV和PEDV融合S蛋白在乳酸乳球菌食品级细胞壁锚定高效诱导表达与免疫原性分析

近年来猪传染性胃肠炎和猪流行性腹泻在我国广泛流行,给养殖业带来了巨大的经济损失。目前对此两种疾病的防治手段还停留在使用常规疫苗。现有的疫苗免疫效果不够理想,急需寻求一种安全高效的防治手段。本文首先构建了猪传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)融合S基因,以乳酸乳球菌食品级细胞壁锚定高效诱导表达载体p RNV48为基础,构建了TGEV和PEDV融合S基因的表达质粒p RNV48-TPs。将得到的重组质粒用电穿孔方法转入乳酸乳球菌NZ9000感受态细胞中,获得了重组菌乳酸乳球菌Z9000/p RNV48-TPs。再利用nisin对重组菌进行诱导,提取细胞壁蛋白后采用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析目的蛋白的表达情况,然后分别对兔子进行注射免疫和口服免疫,并用Western Blot分析。通过乳酸乳球菌食品级细胞壁锚定高效诱导表达载体,诱导表达TGEV与PEDV融合S蛋白产生中和抗体,以期探讨口服疫苗的免疫反应机理,为由TGEV与PEDV研发的安全、高效的疫苗奠定良好的基础。     

乳酸乳球菌表达系统及其启动子的研究进展

乳酸乳球菌（Lactococcus lactis）是工业应用中一种重要的模式菌株，具有非致病性、分泌蛋白能力强、容易分离培养等特性，是异源蛋白表达和分泌的理想宿主。高效可控的启动子是实现外源蛋白高效表达的关键因素之一。根据诱导机制，启动子可分为组成型和诱导型。本文阐述了乳酸乳球菌表达系统及其启动子结构，总结了生物信息学预测启动子及筛选克隆新启动子的方法，并对乳酸乳球菌启动子未来的研究方向进行了展望，可为深入研究启动子的结构和功能并进一步在工业上生产外源蛋白提供参考。     

片球菌素BM-1免疫蛋白对甘露糖磷酸转移酶受体识别特异性研究

利用牛津杯法筛选到对片球菌素BM-1敏感的植物乳杆菌WQ0815和肠膜明串珠菌05-43。并利用Nisin诱导表达系统,在乳酸乳球菌NZ9000中诱导表达两敏感菌株的甘露糖磷酸转移酶系统IICD组分,抑菌实验表明不同来源的IICD组分皆可被片球菌素BM-1识别。进一步将片球菌素BM-1同源免疫蛋白分别与两种IICD组分同时在乳酸乳球菌中表达,抑菌实验表明:免疫蛋白可识别植物乳杆菌来源的IICD组分,不能识别肠膜明串珠菌来源的IICD组分。研究结果为IIa类细菌素的同源免疫蛋白对受体存在特异性识别提供了直接证据。     

乳酸乳球菌镉抗性菌株的鉴定及质粒消除的研究

为构建乳酸菌食品级载体/受体系统.从分离自内蒙古传统乳制品103株乳源性乳酸菌中筛选出7株高浓度镉抗性菌株.特异性16SrDNA PCR扩增产物的序列测定结果表明.其中两株为乳酸乳球菌.PCR扩增镉抗性基因部分序列证实了这两株乳酸乳球菌含有镉抗性基因,为镉抗性阳性菌株.通过物理和化学方法消除乳酸乳球菌内源质粒,确定了镉抗性质粒,并获得一系列衍生菌株,包括无质粒菌株.为进一步研究各种质粒的功能及基因表达提供了理想的宿主,也为乳酸菌食品级载体/受体系统的构建奠定了基础.     

构建食品级表达纳豆激酶的乳酸乳球菌重组菌株

构建整合型表达载体pFY008,以HisH基因作为同源重组的靶位点,通过同源重组将含有纳豆激酶原基因的表达盒PnisA-aprN整合到乳酸菌的基因组上,实现纳豆激酶在乳酸乳球菌中食品级的表达。构建的食品级基因工程菌安全性高、稳定性好,并赋予了其溶栓的功能。     

不同来源食品级乳酸菌及双歧杆菌对乳酸链球菌素敏感性的研究

目的:研究不同来源的食品级乳酸菌和双歧杆菌对乳酸链球菌素(nisin)的敏感性,为构建以nisin作为食品级筛选标记的受体乳酸菌表达系统以及筛选食品发酵、保鲜与贮藏过程中的nisin抗性乳酸菌提供科学依据.方法:采用试管稀释法研究不同来源的食品级的18株乳酸球菌、15株乳酸杆菌和5株双歧杆菌对nisin的敏感性.结果:大部分乳球菌和部分乳杆菌对nisin不敏感,但乳酸乳球菌(Lactococcus lactis)ML0230、99210,肠膜明串珠菌葡聚糖亚种(Leuconostoc mesenteroides subsp dextranicum)AS 1.2141T,嗜酸乳杆菌(Lactobacillus acidophilus)La1,德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckii subsp bulgaricus)S-1、DR、LD、AS 1.2625T,长双歧杆菌(Bifidobacterium longum)Blm对nisin非常敏感.在nisin质量浓度为5 μg/mL(IU/mL)时即可抑制其生长.结论:初步筛选出17株以nisin作为食品级筛选标记的受体乳酸菌,同时筛选出21株食品发酵、保鲜与贮藏过程中的nisin抗性乳酸菌.     

嗜酸乳杆菌β-半乳糖苷酶基因的克隆及其作为食品级筛选标记的研究

为研究嗜酸乳杆菌β-半乳糖苷酶基因(lacZ)作为食品级筛选标记的活性及筛选稳定性,作者从嗜酸乳杆菌基因组中克隆β-半乳糖苷酶基因并在乳酸乳球菌中表达,通过X-gal显色筛选阳性克隆;用X-gal显色筛选方法将重组乳酸乳球菌传60代,并测定β-半乳糖苷酶酶活和比活力。结果显示:通过表达有活性的β-半乳糖苷酶和X-gal显色可成功筛选出阳性克隆菌株;对借助X-gal传60代后的重组乳酸乳球菌进行β-半乳糖苷酶比活力检测,与X-gal筛选的第二代相比没有显著差异(P=0.5920.05),与红霉素筛选比较也没有显著差异(P=0.8820.05)。由此可见,β-半乳糖苷酶基因作为筛选标记具有较好的活性和稳定性,为以β-半乳糖苷酶基因作为筛选标记的食品级载体的研究奠定了基础。     

乳酸链球菌素和乳酸乳球菌在食品工业中的应用

乳酸乳链球菌和乳酸乳球菌均为革兰氏阳性细菌,乳酸链球菌产生的乳酸链球菌素由于其特殊的抗菌性能及安全无毒的特性被广泛应用于各类食品的防腐保鲜。乳酸乳球菌作为一种安全且低免疫原性的基因工程菌,可用于表达一些酶或其他生产营养物质的蛋白等以提高食品的安全性及其营养价值,还可用于表达抗原作为口服疫苗。综述乳酸链球菌素和乳酸乳球菌在食品工业中的应用。     

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.