植物乳杆菌重组亚油酸异构酶的表达与纯化

对转化有植物乳杆菌亚油酸异构酶基因的大肠杆菌进行诱导表达,表达出大量重组亚油酸异构酶蛋白,但大部分目的蛋白形成了包涵体,用8mol/L尿素将包涵体蛋白溶解变性后,用金属螯合层析对重组蛋白进行了纯化.当咪唑溶液浓度为50mmol/L时,重组蛋白开始被洗脱下来;当咪唑溶液浓度为200mmol/L时,蛋白洗脱效果最佳;浓度为400mmol/L时,重组蛋白完全被洗脱下来.纯化后重组蛋白的收率为84.71%.     

重组羧肽酶B及其包涵体溶解性的研究

目的构建重组羧肽酶B(rCPB)的表达质粒和表达菌株,表达羧肽酶B(CPB),并对表达CPB包涵体的溶解性进行研究。方法构建CPB重组质粒pET-21a-CPB,将其导入表达菌株BL21(DE3)中,分别在25℃和37℃进行诱导表达;所得包涵体分别用不同浓度尿素添加不同还原剂溶解,以溶液中的蛋白质浓度判定其溶解性,利用非还原SDS-PAGE分析其溶解性提高的原因。结果诱导后生长温度不同对rCPB包涵体的纯度及后期处理均有影响;rCPB包涵体在10mol/L尿素溶液中的溶解度比在8mol/L尿素溶液中提高2￣3倍;添加0.75%β-巯基乙醇能显著改善rCPB包涵体的溶解效果。经非还原SDS-PAGE分析,添加β-巯基乙醇后,溶解rCPB聚体的含量减少。结论成功地表达了rCPB,并通过实验提高了rCPB包涵体的溶解度。     

重组N-乙酰鸟氨酸脱乙酰基酶包涵体蛋白的柱上复性

N-乙酰鸟氨酸脱乙酰基酶(NAO)具有广泛的底物选择性,可用于多种活性氨基酸的酶法拆分,具有广阔的工业应用前景。文中采用多种洗涤剂对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行洗涤去杂,并用DEAE Sepharose Fast Flow层析柱和三缓冲体系作为复性系统对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行复性实验。结果表明,4mol/L尿素与0.5%Triton X-100联合洗涤可以大量去除杂质;三缓冲体系能有效地实现重组N-乙酰鸟氨酸脱乙酰基酶包涵体的柱上复性。在流速为0.5 mL/min,尿素梯度为21 mL,尿素终浓度为1.8mol/L,蛋白质上样量为0.99 mg的实验条件下,蛋白回收率和复性酶比活分别达到52%和10.27 U/mg。     

重组羧肽酶B及其包涵体溶解性的研究

目的 构建重组羧肽酶B（rCPB）的表达质粒和表达菌株，表达羧肽酶B（CPB），并对表达CPB包涵体的溶解性进行研究。方法构建CPB重组质粒pET-21a—CPB，将其导入表达菌株BL21（DE3）中，分别在25℃和37℃进行诱导表达；所得包涵体分别用不同浓度尿素添加不同还原剂溶解，以溶液中的蛋白质浓度判定其溶解性，利用非还原SDS—PAGE分析其溶解性提高的原因。结果诱导后生长温度不同对rCPB包涵体的纯度及后期处理均有影响；rCPB包涵体在10mol／L尿素溶液中的溶解度比在8mol／L尿素溶液中提高2-3倍；添加0．75％β-巯基乙醇能显著改善rCPB包涵体的溶解效果。经非还原SDS—PAGE分析，添加β-巯基乙醇后，溶解rCPB聚体的含量减少。结论成功地表达了rCPB，并通过实验提高了rCPB包涵体的溶解度。     

天蚕素抗菌肽(Cecropin A)融合蛋白的表达及其分离纯化工艺研究

通过单因素实验确定了天蚕素抗菌肽融合蛋白表达的最佳条件,即温度为31℃,种子菌接种量为2.0%,培养基pH值为6.5,诱导时间为10 h,此时融合蛋白的表达量占细胞总蛋白的30.6%;开发融合蛋白纯化工艺,将工程菌细胞超声破碎后,所得包涵体使用8 mol/L尿素溶液溶解,泵入2 mol/L尿素溶液中搅拌使蛋白质复性,复性完成后将2 mol/L尿素溶液通过超滤置换成含0.5 mol/L NaCl、20 mmol/L的PB缓冲液,pH 7.2,并浓缩溶液体积至总体积的30%～40%,采用镍离子亲和层析法纯化Cecropin A融合蛋白,SDS-PAGE凝胶电泳结果显示目标融合蛋白纯度达到87%以上。     

原核表达抗克伦特罗重组单链抗体及其纯化研究

目的:原核表达抗克伦特罗重组单链抗体,对表达的包涵体蛋白进行提取和纯化,并鏊定重组抗体特性.方法:通过温度诱导大肠杆菌表达抗克伦特罗单链抗体,超声破碎菌体细胞,采用不同洗涤溶液提取包涵体.以不同变性剂溶解包涵体,Ni-NTA亲和柱纯化后,经脉冲稀释法将其复性.超滤及透析浓缩蛋白复性液,经Ni-NTA亲和柱二次纯化,得到纯化重组目的蛋白.通过SDS-PAGE电泳分析重组目的蛋白纯度,采用Western bloning和间接竞争EUSA对所制备的抗克伦特罗重组单链抗体特异性进行鉴定.结果:以大肠杆菌Es-cherichia coli BL21(DE3)为宿主菌诱导表达重组单链抗体,产率最高,占菌体蛋白的31%.以1 mol/L尿素洗涤包涵体,6 mol/L盐酸胍为变性剂,经Ni-NTA亲和柱纯化,所得复性重组单链抗体蛋白的纯度达96%.Western blotting分析显示,纯化并复性后的单链抗体可与鼠抗His标签蛋白单克隆抗体发生特异性的结合反应.间接竞争ELSIA结果表明,该重组抗体IC50值为3.35 ng/mL,与沙丁胺醇等结构类似物无交叉反应.结论:原核表达制备的抗克伦特罗重组单链抗体具有较好的抗原结合活性及特异性,为进一步开展克伦特罗的免疫快速检测研究奠定基础.     

重组人EC-SOD包涵体的复性

目的将E.coli中表达的人胞外超氧化物歧化酶(hEC-SOD)包涵体进行复性.方法分离包涵体,用8mol/L尿素溶解后进行透析复性.结果包涵体蛋白可以部分复性,比活达到900U/mg protein.结论透析复性法可以复性EC-SOD包涵体.     

重组人EC—SOD包涵体的复性

目的将E．coil中表达的人胞外超氧化物歧化酶(hEO-SOD)包涵体进行复性。方法分离包涵体，用8mol/L尿素溶解后进行透析复性。结果包涵体蛋白可以部分复性，比活达到900U／mg protein。结论透析复性法可以复性EC-SOD包涵体。     

重组大肠杆菌制备副溶血弧菌噬菌体内溶素Lys qdvp001 CHAP域及诱导条件初步优化

目的:为了利用噬菌体内溶素防控副溶血弧菌,通过克隆表达工程菌pET30a-CHAP制备目的蛋白,并将以包涵体存在的目的蛋白复性得到有活性的噬菌体内溶素蛋白。方法:首先克隆工程菌pET30a-CHAP,经 IPTG的诱导表达后,检测菌液上清中内溶素含量判断表达形式,再进行诱导条件初步优化。通过将表达的pET30a-CHAP包涵体蛋白先用洗涤剂洗涤去除杂蛋白,然后用尿素变性剂溶解,并用Ni2+ SepharoseTM 6 Fast Flow亲和层析柱进行层析纯化,用EDTA、氧化型谷胱甘肽、还原型谷胱甘肽等为折叠复性促进剂,经过透析制备可溶性的 pET30a-CHAP蛋白,再检测目的蛋白的抑菌活性。结果:本实验确定了重组菌内溶素的表达形式为没有活性的包涵体;初步确定最佳诱导条件为诱导温度为16 ℃,IPTG终浓度0.5 mmol/L,诱导时间为7 h;复性后的内溶素具有抑菌活性。结论:本研究为利用内溶素防控副溶血弧菌以及其他革兰氏阴性细菌提供了制备方法。     

重组干扰素-tau在大肠杆菌中的高效表达

目的研究重组干扰素-tau的纯化和复性。方法将构建好的重组质粒pBV220转化E.coli BL21,发酵培养后迅速升温至42℃诱导干扰素-tau以包涵体的形式高效表达。裂菌后利用8mol/L尿素溶解目的蛋白质,经凝胶过滤色谱层析纯化后,再透析复性。结果干扰素-tau的表达量占菌体总蛋白质量的20%以上,纯化后纯度可达9 6%以上,活性得到有效恢复。结论建立了干扰素-tau纯化及复性的方法,获得了高表达、高纯度、有活性的干扰素-tau。     

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.     

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.     

低温带皮菜籽粕微粉的不同粒级部分的功能特性

为研究低温带皮菜籽粕微粉的不同粒级部分的功能特性,以经低温脱脂的带皮菜籽粕为原料,经微粉碎后筛分成212~425μm、150~212μm和106~150μm的3个不同粒级的微粉样品,检测这些样品的吸水性、吸油性、乳化性和乳化稳定性、蛋白质体外消化率。结果表明:1 3个不同粒级的微粉样品之间的粗纤维含量存在显著差异,表明三者的结构组成成分有一定差异。23个微粉样品的乳化活性和乳化稳定性随粒度级别的减小而显著增加(P0.01)。33个微粉样品的蛋白质体外消化率随粒度级别的减小而显著增加(P0.01)。4不同粒级带皮菜籽粕微粉样品的吸水性与吸油性受其结构组成物质不同和粒度的双重影响,与粒度的相关性不明显。     

Microbiology of food taints

Fresh and processed foods are often spoilt by the presence of undesirable flavours and odours caused by microbial action. The aim of this paper is to review the current knowledge of microbiologically induced taints that occur in a wide range of foodstuffs, including meats, poultry, fish, crustaceans, milk, dairy products, fruits, vegetables, cereals and cereal products. Examples have been chosen where the compounds responsible for the taint have been identified and sufficient data obtained to demonstrate the involvement of microorganisms. However, in some cases the full identity of the causative organism may not have been elucidated. The types of microorganisms covered by this review include bacteria, fungi, yeasts, actinomycetes and cyanobacteria. Although cyanobacteria do not in general infect foods, their presence in aqueous systems and water supplies can lead to off-flavours in aquatic organisms and processed foodstuffs. Several examples of each of these processes are discussed. Wherever possible, the likely biosynthetic pathway used by the microorganism to produce the offending compound in a foodstuff is indicated.     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. I: Interlaboratory studies of methods of analysis

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four postcolumn derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.