氨基甲酸乙酯水解酶异源表达及酶学性质分析

将合成的来源于长孢洛德酵母菌（Lodderomyces elongisporus）属的氨基甲酸乙酯水解酶基因在大肠杆菌中实现异源表达,并进行纯化及其酶学性质研究。摇床培养后酶的比活力为4.52 U/mg,经过镍离子亲和层析柱纯化至显示单一条带后酶的比活力为9.86U/mg。酶学性质研究表明,氨基甲酸乙酯水解酶的最适反应温度为30℃;最适pH值为7.0;金属离子Mg2+对该水解酶的酶活有微弱的促进作用,而Cu2+以及Fe3+对酶的活性有强烈的抑制作用;对低含量的乙醇溶液与NaCl溶液有一定的耐受性;并且该水解酶可以对不同底物的酰胺类化合物进行有效降解,以氨基甲酸乙酯（ethyl carbamate,EC）为底物进行反应时测得水解酶的动力学参数Km值为6.64 mmol/L,最大反应速率Vmax值为8.24 μmol/min。     

梅奇酵母氨基甲酸乙酯酶分离纯化与酶学性质分析及其对葡萄酒香气的影响

为了研究梅奇酵母所产生氨基甲酸乙酯（Ethyl Carbamate,EC）酶的性质,通过硫酸铵盐析、离子交换层析的方法对EC酶进行分离纯化,并对纯化后EC酶的酶学性质进行了分析。结果表明,纯化后EC酶对比纯化前的酶活提高了约1.2倍。纯化后EC酶最适反应温度为35 ℃,在25~45 ℃之间热稳定性较强;最适pH为5.5,在pH5~6时该酶稳定性强,即酸稳定性强,碱稳定性较差。当乙醇体积分数为12%~14%时,EC酶具有一定酶活。底物特异性结果表明,该EC酶对EC及其前体物质（尿素）具有较高的降解能力,且对于成品葡萄酒挥发性香气成分影响不显著。     

一株酸性糖化酶的分离纯化及酶学性质研究

从土壤中筛选得到一株产酸性糖化酶的黑曲霉ASP-S21,粗酶液通过（NH4）2s04沉淀、Sepharose HP阴离子交换层析、SephacrylS-200层析柱分离纯化,SDS-PAGE电泳测定其分子量为120kDa。该酶最适作用温度为65℃;酶的最适反应pH值为4．0;在pH2．2-7．6之间,具有较好的酸稳定性;酶的Km值为0．94mg／mL,Vmax=142．43mol（Glu）／min-L。Cu2＋和Co2＋对酶活有较强的促进作用,10mM的Cu2镟酶活力提高到129％,Fe^3＋对酶催化活力抑制作用较强。该酶且具有部分降解生淀粉的能力,在pH4．0,50℃反应1h,生淀粉酶活力为0．39U,RDA值为4．57％。得到的黑曲霉ASP-S21酸性糖化酶,产生的糖化酶活力高、耐酸稳定性好,酶争陛质符合淀粉糖化工业化过程中对酶的要求,具有良好的研究前景。     

高温甲壳素降解酶产生菌筛选及其酶学性质研究

采用平板透明圈法,结合摇瓶筛选得到一株高温甲壳素降解酶高产菌芽孢杆菌HU1,其甲壳素降解酶活力达到606U/mL.对其部分酶学性质进行了分析,结果表明:该酶的最适酶解温度为60℃,最适pH为6.5,对GlcNAcGlcNAc键具有较强的专一性,对CMC、纤维素、可溶性淀粉等无降解活性.一价的金属离子Na+、K+对该酶作用不明显;Mg2+、Zn2+、Ca2+对其有明显的促进作用;而Cu2+、Fe3+则具有显著的抑制作用.此外,酶活力还受DTT及SDS抑制,不受EDTA影响.     

葡萄酒中氨基甲酸乙酯形成及质量控制

氨基甲酸乙酯(Ethyl Carbamate,EC)是一种基因致癌物,葡萄酒及其他酒精饮料中含有微量的氨基甲酸乙酯.葡萄酒中氨基甲酸乙酯主要由酵母降解精氨酸产生的尿素和苹果酸-乳酸茵降解精氨酸产生的瓜氨酸与乙醇自发反应产生.阐述了氨基甲酸乙酯的致癌作用、葡萄酒中氨基甲酸乙酯的形成途径及影响其形成的因素;探讨了降低葡萄酒中氨基甲酸乙酯的措施,为我国葡萄酒生产中降低氨基甲酸乙酯含量提供了理论依据.     

葡萄酒中氨基甲酸乙酯的形成机理及影响因素

氨基甲酸乙酯是一种基因致癌物,在葡萄酒中主要由酵母降解精氨酸产生的尿素和苹果酸-乳酸菌降解精氨酸产生的瓜氨酸与乙醇自发反应产生。主要对葡萄酒中氨基甲酸乙酯的形成机制及其影响因素进行了综述,为探讨降低葡萄酒中氨基甲酸乙酯的措施提供理论依据。     

产氨基甲酸乙酯降解酶酵母菌的选育及产酶条件优化

氨基甲酸乙酯(EC)是一种强致癌物,近年来引起了广泛关注,研究通过筛选得到一株能降解氨基甲酸乙酯的酵母菌;通过形态学分析和ITS序列比对确定为Pichia fermentans酵母菌,再改用同一成分的液体培养基进行培养,经过离心和细胞破碎,得到粗酶液,测定酶活,通过对温度、pH、接种量的调节,得到最优产酶条件为pH 7左右,温度28℃,接种量2.5%。对酶学性质进行分析,得到该酶的最适pH为6~7,最适温度为30℃,在此温度下酶活性稳定,Fe~(2+)对此酶激活作用明显。     

Bacillus pumilus CN8菌株降解猪血Hb酶的分离纯化及酶学性质研究

Bacillus pumilus CN8菌株的发酵液,经离心分离得到粗酶液,再经硫酸铵盐析、透析,DEAE-Cellulose-52离子交换层析等步骤获得电泳纯的中性蛋白酶。得到比活达686.66U/mg的酶蛋白,纯化倍数为13.2,回收率为35.0%。SDS-PAGE测得其分子量大约为97000Da。该酶的最适作用pH为7.4,最适反应温度为42℃,在pH7.0~8.5范围内较稳定,在30~45℃比较稳定。经酶学性质测定,K+、Mg2+对酶活力具有保护作用,甘油对酶活具有抑制作用。     

可降解氨基甲酸乙酯酵母菌的筛选及降解条件优化

本实验旨在筛选得到一株可以有效降解氨基甲酸乙酯的酵母菌,并根据ITS DNA片段鉴定其种属。采用气相色谱-质谱联用(GC-MS)法,测定所筛选株菌的发酵产物中氨基甲酸乙酯的含量,确定一株降解能力较强的菌株,根据系统发育树确定酵母种属,并对该菌株的降解条件进行了优化。结果表明,降解能力较强的菌株为G1-3,为鲁考弗梅奇(Metschnikowia reukaufii)酵母,优化降解条件为接种量为3%,38℃,170 r/min,发酵4.5 d,最终氨基甲酸乙酯的降解量在65.57%左右。     

氨基甲酸乙酯降解酶的固定化研究

为了制备固定化酶,以体积分数5%的戊二醛溶液与壳聚糖交联8 h制备的载体对氨基甲酸乙酯(EC)降解酶粗酶液进行固定化,并对固定化EC降解酶的酶学性质展开分析。结果表明:固定化EC降解酶的最适温度和最适pH分别是42℃和3.6;该酶在乙醇体积分数7%～25%的范围内和使用次数在8次以内的酶活均保持在50%以上,说明该固定化酶的乙醇耐受范围广,催化活性高;而且分别在4℃和20℃下贮藏6周后的酶活损失率分别不足30%和40%。基于以上特性,为了考察实际应用效果,采用双水相萃取结合气相色谱-质谱联用(GC-MS)法和顶空固相微萃取结合气相色谱-质谱联用(HS-SPME-GC/MS)法分析了游离酶和固定酶对"美乐"葡萄酒中EC和挥发性风味物质的影响。结果表明,固定化EC降解酶的EC去除率达30.90%,是游离酶的81.53%;EC降解酶的处理方式对葡萄酒中挥发性物质的相对含量没有显著影响,只是引起了香气物质种类的减少和香气成分的改变,但固定化酶对挥发性风味物质的不利影响远低于游离酶。     

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.     

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.     

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.     

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.     

Influence des ions sur le pouvoir hydratant de l'ure e: e tude sur peau de porc ex vivo

This study deals with the influence of ions (NaCl and MgSO4) in a W/O emulsion containing 10% urea. Moisturization kinetics are assessed by corneometry on pig skin ex vivo. The formula's influence on urea penetration is measured by infrared spectrometry with an ATR device and the stripping method. Corneometry and spectroscopy were chosen to record simultaneously the hydratation levels and urea localization into superficial cell layers. Urea crystallization after evaporation of emulsions and aqueous solutions is described. Results show that urea does not hydrate nor penetrate when applied to the skin through an aqueous gel. In a W/O emulsion, sodium chloride increases the ability of urea to moisturize without improving penetration. In vitro urea crystallization is disturbed by sodium chloride or magnesium sulphate for solutions and emulsions. This stabilization by ions is correlated with good moisturization values. The stabilization of urea in the solute state provided by ions increases its water epidermal binding capacity without enhancing penetration.     

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.