乳酸乳球菌细胞壁蛋白酶的分离纯化

本研究确定了分离纯化乳酸乳球菌细胞壁蛋白酶(CEP)的最佳技术路线.用裂解液(50mmol/L Tris-HCI,2mmol/L EDTA-Na2,100mmol/L NaCl,0.5%Tritonx-100,1mg/ml溶菌酶,pH8.5)悬浮菌体(20ml/g),37℃下保温3h,离心后取上清液即为粗酶液.粗酶液通过45%硫酸铵沉淀,DEAE-Sephadcx A-25和Sephacryl-S-300 HR两步层析,可以得到纯化的细胞壁蛋白酶.蛋白酶提纯倍数达到74.048%,最后回收率为14.865%,PAGE电泳检测为一条带,SDS-PAGE检测蛋白酶为单体结构,分子量大约为53kD.用纯化后CEP酶解乳清蛋白,酶解液ACE抑制率为45%.     

生姜蛋白酶的分离纯化及其酶学性质初步研究

以新鲜生姜为原料,以生姜蛋白酶酶活为考察指标,对生姜蛋白酶的提取及纯化工艺进行研究。结果表明:以pH6.0的磷酸氢二钠-磷酸二氢钠缓冲液为提取溶剂所得酶活力较高,最佳工艺为:以pH6.0磷酸氢二钠-磷酸二氢钠缓冲液25℃提取2次,料液比1∶1;应用硫酸铵盐析及透析袋透析对粗酶液进行纯化,粗酶液再以60%硫酸铵盐析24h,透析袋(14000)低温透析12h,冻干;酶学性质研究表明:生姜蛋白酶以酪蛋白为底物其最适pH值为6.0,30℃保温30min,酶活稳定。     

发酵乳杆菌细胞壁蛋白酶的分离纯化及酶学性质研究

研究分离纯化发酵乳杆菌细胞壁蛋白酶(cell envelope proteinase,CEP)的方法及其酶学性质。用50mmol/L Tris-HCl缓冲液(pH7.8)悬浮菌体,进行超声波破碎,细胞质量浓度为0.06g/mL,破碎功率330W,工作220次(工作时间3s,间隔时间5s),离心取上清液即为粗酶液。60%硫酸铵沉淀,Sephacryl S-300 HR凝胶层析,Native-PAGE割胶回收纯化发酵乳杆菌的CEP。用纯化的CEP 酶解脱脂乳,酶解液ACE(angiotensin I-converting enzyme)抑制率为50%。SDS-PAGE检测CEP分子质量为32.5kD,最适酶反应温度为41℃,最适酶反应pH值为8.0。Mg2+、Co2+、Ca2+对CEP活性有激活作用;Zn2+、Ni2+、PMSF、EDTA对CEP活性有抑制作用,说明CEP为丝氨酸蛋白酶且酶的活性中心结构的维持与金属离子有关。     

嗜酸乳杆菌细胞壁蛋白酶的分离纯化及酶学性质的研究

为优化CEP的分离纯化方法,采用聚乙二醇(PEG)-20000浓缩粗酶液,用超滤离心管过滤酶液,40%~60%硫酸铵盐析分级沉淀,Sephacryl-S-300HR纯化,并通过Native-PAGE切胶回收得到纯酶。所得CEP的分子质量为28.3ku,比酶活力62.066U/mg,最适温度32℃,最适pH6.5,耐热性比较强,耐酸性明显好于耐碱性。Co2+、Mg2+、Ca2+对CEP有激活作用;Ba2+、Mn2+、K+、Na+对CEP的活力影响不大;Zn2+、Ni2+则表现出较强的抑制CEP活性作用;EDTA对CEP活性有抑制作用,表明CEP是一种金属离子激活酶;PMSF对CEP有显著抑制作用,表明CEP是一种丝氨酸蛋白酶。用嗜酸乳杆菌CEP水解乳清蛋白,其酶解液对ACE的抑制率为56.31%,表明CEP水解乳清蛋白可以产生ACE抑制肽。     

灵芝菌粗酶液缓解糙米老化的条件优化

为研究灵芝菌粗酶液缓解糙米老化的最优条件,首先对灵芝菌发酵液的胞内与胞外粗酶液中α-淀粉酶、β-淀粉酶、纤维素酶和酸性蛋白酶酶活力进行测定。在单因素试验的基础上,以反应温度、反应时间和液料比为变量,以糙米回生值比率为响应值,通过响应面分析得到灵芝菌胞内粗酶液缓解糙米老化的最佳反应条件。结果表明：发酵过程中,灵芝菌胞外粗酶液各酶酶活力明显低于胞内,且发酵第4天时胞内各酶酶活力达到最高,其中α-淀粉酶、纤维素酶和酸性蛋白酶的酶活力分别为（69 496.21±1 250.72）、（75 517.73±2 142.82）、（7 515.79±204.08）U/g。灵芝菌胞内粗酶液缓解糙米老化最佳条件：反应温度32℃,反应时间2 h,液料比4∶1（mL/g）,所得回生值比率最高,可达24.0%。表明该灵芝菌可用于缓解糙米老化,为糙米进一步深化应用提供理论依据。     

干酪乳杆菌胞壁蛋白酶提取条件优化及其水解特性研究

干酪乳杆菌细胞壁蛋白酶是其蛋白水解系统中一个关键的蛋白酶.采用Ca2+-free法研究干酪乳杆菌细胞壁蛋白酶的提取方法,确定其最佳实验条件.菌体用含有30 mmol/L CaCl2的50 mmol/L Tris-HCl (pH 7.0)缓冲液洗涤3次,然后用含有50.03 mmol/LEDTA-Na2的50 mmol/LTris-HCl(pH6.98)缓冲液悬浮,39.75℃保温,60 min后取出离心(4 500 r/min、20 min、4℃),上清液即粗酶液.用粗酶液与纯化后的酶分别水解各种酪蛋白,粗酶较纯化后的酶能产生更高的ACE抑制能力,而纯化后的酶水解β-酪蛋白产生的抗ACE活性能力最强.     

杜仲叶抗α-葡萄糖苷酶成分的研究

通过正交试验优化了杜仲叶中抗α-葡萄糖苷酶成分的提取条件,并利用葡聚糖凝胶对杜仲叶水提液进行纯化分离,分析了杜仲叶水提物纯化液的黄酮含量。研究结果表明,杜仲叶的提取条件为提取料液比为1∶10,提取温度为90℃,提取时间为40min。杜仲叶水提物纯化液具有明显的抗α-葡萄糖苷酶效果,且杜仲叶中总黄酮含量为3.31%,纯化液中黄酮的含量为18.12%,杜仲总黄酮粗品得率为7.32%。     

从猴头菇中提取纯化SOD

研究了从猴头菇中提取并纯化SOD的方法和最佳条件.采用乙醇-氯仿法通过均匀实验获得最佳提取条件为1g猴头菇加入到0.15mol·L-1的NaHCO3 5mL中,加入乙醇-氯仿(2:1,v/v)溶液6mL,在30℃下提取3.5h测得粗酶液活性为321.26U.采用丙酮二级纯化法,纯化所得SOD的酶比活为95.58U·mg-1.     

南美白对虾虾头蛋白酶提取工艺的研究

以南美白对虾虾头为原料,对提取条件进行优化设计.通过单因素实验研究pH、温度和料液比对内源蛋白酶活力的影响,然后设计正交实验确定内源蛋白酶的最佳提取条件,初步研究内源蛋白酶硫酸铵分级沉淀的最佳饱和度.结果表明:内源蛋白酶在pH为2和8时酶活力较高.通过正交实验得出酸性蛋白酶最佳提取条件为缓冲溶液pH3,料液比1:2,温度30℃;碱性蛋白酶最佳提取条件为缓冲溶液pH8,料液比1:3,温度50℃.酸性蛋白酶进行初步分离的结果是硫酸铵一级沉淀饱和度10%,硫酸铵二级沉淀饱和度50%;碱性蛋白酶进行初步分离的结果是硫酸铵一级沉淀饱和度20%,硫酸铵二级沉淀饱和度80%.     

鱿鱼内脏蛋白酶的提取工艺研究

本文以鱿鱼内脏为原料提取蛋白酶.考察了各因素对提取效果的影响,包括用于提取的NaCl溶液浓度、内脏和提取液的固液比、提取温度、提取时间等.此外,根据蛋白质等电点沉淀原理,调粗酶液的pH值去除杂蛋白;并进一步比较了各种絮凝剂去除粗酶提取液中杂蛋白的效果.结果表明:NaCl溶液浓度为0.5％,固液比为1.0∶1.5,常温下提取60 min可达到较好的提取效果,蛋白酶的回收率达到96.06％,比酶活达到1.33 U/mg.调粗酶液的pH值为4.5左右,杂蛋白去除效果较好;Fe2(SO4)3是较好的絮凝剂,向粗酶液中添加1％ Fe2(SO4)3溶液,当添加比为4:1(V/V,粗酶液Fe2(SO4)3溶液)时,进一步去除杂蛋白的效果较好,蛋白酶的回收率达80.33％,比酶活达到9.37 U/mg.     

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.     

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.     

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.     

Announcing a new international peer-reviewed journal:Food Science and Human Wellness now accepting manuscript submission in English

<正>We are pleased to announce the launch of a new international peer-reviewed journal-Food Science and Human Wellness,ISSN 2213-4530,which is an open access journal,produced and hosted by Elsevier B.V.on behalf of Beijing Academy of Food Sciences.Food Science and Human Wellness is an international peer-reviewed English journal that provides a forum for the dissemination of the