黄酒酵母ADH2、ALD2和ALD6基因敲除对黄酒中乙醛代谢的影响

以工业黄酒酵母N85尿嘧啶缺陷型单倍体2a-u3(MATa ura3)为出发菌株,研究分别敲除乙醛代谢相关酶的编码基因ADH2、ALD2和ALD6对乙醛代谢的影响。采用融合PCR技术分别构建乙醇脱氢酶编码基因ADH2敲除组件"ADH2S-URA3-ADH2X"和乙醛脱氢酶编码基因ALD2和ALD6敲除组件"ALD2S-URA3-ALD2X"和"ALD6S-URA3-ALD6X",转化N85尿嘧啶缺陷型单倍体菌株后,以尿嘧啶缺陷型的恢复为筛选标记,分别获得ADH2、ALD2和ALD6基因缺失菌株2a-A2(Δadh2)、2a-D2(Δald2)和2a-D6(Δald6)。分别将获得的工程菌与亲本菌株以及前期构建的丙酮酸脱羧酶基因PDC5缺失菌株2a-P5(Δpdc5)进行实验室规模的黄酒发酵实验。结果表明,与亲本菌株2a相比,工程菌2a-A2发酵液中乙醛含量降低了7.14%,而2a-P5发酵液中乙醛含量降低了32.68%,说明工业黄酒酵母乙醛的生成存在糖代谢途径,并且乙醛主要由丙酮酸脱羧而来;工程菌2a-D2和2a-D6发酵液中乙醛含量与亲本相比分别增加了144.91%和88.87%,说明乙醛脱氢酶是黄酒中控制乙醛降解的关键酶,而且ALD2基因在乙醛转化为乙酸过程中发挥更关键的作用。     

LEU2基因敲除对工业啤酒酵母高级醇生成量的影响

通过敲除啤酒酵母中β-丙基苹果酸脱氢酶基因(LEU2),研究该基因对工业啤酒酵母高级醇特别是异戊醇生成量的影响.通过醋酸锂一步转化法,将一段两端具有LEU2摹因序列同源区,中间为遗传霉素(G418)抗性基因的DNA片段转入酵母细胞中,与LEU2基因的ORF(open reading flames)进行同源重组,利用遗传霉素抗性进行筛选.将突变株与出发菌株进行发酵实验,测定其发酵性能和高级醇的生成量.筛选获得了LEU2摹因突变的工业啤酒酵母.在支链氨基酸含量较低的培养基中进行发酵测定,突变株发酵液中高级醇含量比出发菌株降低了9.97%,其中异戊醇的含量降低了11.82%,而酒精度、发酵速度等发酵性能没有明显变化.LEU2基因敲除可降低工业啤酒酵母在支链氨基酸含量较低的培养基中高级醇特别是异戊醇的生成量.     

热带假丝酵母ctfat1p基因在吸收脂肪族化合物中的功能研究

以热带假丝酵母(Candida tropicalis)1798中的ctfat1p基因为研究对象,利用重叠PCR将ctfat1p基因内约500 bp片段与G418抗性基因(kanr)相连接,经末端单酶切后电转化至C. tropicalis 1798感受态细胞中,通过一次同源单交换,将抗性基因kanr插入至ctfat1p基因内部,实现目的基因的敲除,并通过发酵实验分析Ctfat1p在热带假丝酵母脂肪酸跨膜转运过程中的功能。结果表明,经过G418抗性筛选和基因组PCR鉴定,成功获得ctfat1p基因缺失菌株C. tropicalis 1798 Δctfat1p;分析发现ctfat1p基因敲除对C. tropicalis 1798以油脂为底物培养12 h后重组菌OD600 nm值仅为野生型菌株的47.9%,表明ctfat1p基因敲除后影响C. tropicalis 1798对油脂吸收利用。通过基因敲除手段构建ctfat1p基因缺失菌株,可以减弱细胞对长链脂肪酸的摄取,验证了ctfat1p基因为热带假丝酵母油脂吸收和利用的关键基因。     

Cre／loxP系统介导删除酿酒酵母ALD4基因研究

设计与酿酒酵母编码乙脱醛氢酶ALD4因ORF两侧序列同源的删除组件引物,以质粒pUG6为模板进行PCR构建带有Kanr选择标记的删除组件.通过醋酸锂转化法将删除组件导入酿酒酵母菌,采用G418筛选阳性克隆子,将质粒pSH65转入阳性克隆子.半乳精诱导pSH65表达Cre酶切除Kanr基因,在ALD4基因ORF处留下1个loxP位点,通过2次的转化筛选验证获得ALD4双倍体基因缺陷型突变株Y01-ALD4.     

酸奶中保加利亚乳杆菌药物敏感性分析

从酸奶中分离保加利亚乳杆菌,对其遗传多样性和药物敏感性进行分析,并进一步对耐药菌株的抗性基因进行检测。利用改良MRS培养基,从国内不同品牌酸奶中分离获得18株保加利亚乳杆菌。18株分离菌先经RAPD分型后采用琼脂稀释法测定其对11种抗生素的药敏性,并通过PCR对耐药菌株中可能存在的抗性基因进行检测。结果显示,18株受试菌具有明显的遗传多样性和耐药表型多样性。18株菌全部对罗红霉素敏感,而全部对卡那霉素耐药;对氨苄青霉素、青霉素G、金霉素、氯霉素、四环素、林克霉素、链霉素、新霉素及庆大霉素等9种抗生素均表现出不同程度的耐药性。通过检测耐药菌株的抗性基因,从1株菌(B-8)中检出四环素抗性基因tet(M),从2株菌(B-8和B-41)中检出链霉素抗性基因ant(6),从4株菌(B-43、B-47、B-49和B-51)中检出卡那霉素抗性基因aph(3’)-Ⅲa。结果表明,供试18株保加利亚乳杆菌多重耐药现象比较严重。     

PCR法及环介导等温扩增法筛查食源微生物的耐药基因

耐药基因可通过垂直传播和水平传播等途径在微生物的种内和种间进行传递,食用受到耐药菌污染的食品,具有威胁人类健康的潜在可能。为建立耐药基因快速检测方法并应用于食源性微生物耐药基因的筛查,对食源性耐药菌在食品中的存在状况、耐药种类进行了探讨。采集了3个不同菜市场的9份食品样本,对食源菌进行了分离培养,使用四环素、链霉素、红霉素、氯霉素、甲氧苄胺嘧啶进行抗性菌筛选,并利用聚合酶链式反应(PCR)技术及环介导等温扩增(LAMP)法对8种抗性基因进行筛查,同时用实时荧光定量技术对结果进行了验证。9份食品样本中,菌落总数在105CFU/g至107CFU/g。红霉素抗性的菌落数最多,除1份样本外均在105CFU/g以上;氯霉素抗性菌菌落数最低,除1份样本之外均在103CFU/g以下。在对8种耐药基因筛查时发现,在四环素抗性菌中耐药基因tetA阳性率最高,为75.56%。采用LAMP法筛查耐药基因,阳性率高于PCR法。结果表明,具有抗药性的食源菌在样本中大量存在,这提示食品可以作为抗性菌存在和传播的重要介质。而LAMP作为一种简单、快速的基因检测技术可以用于耐药基因的快速筛查,为了解抗性菌在食品加工环境的污染和分布情况,以及消杀防控措施的采取和评估提供了基础信息。     

四川泡菜中乳酸菌链霉素抗性与抗性基因的检测（英文）

目的探明四川泡菜中链霉素抗性乳酸菌及抗性基因的种类。方法利用含有8μg/ml链霉素的MRS初步分离泡菜液中的抗性乳酸菌,通过16S r RNA分析确定抗性乳酸菌的分类地位后,测定同种不同菌株对链霉素的MIC,并与欧洲食品安全官方机构(EFSA)建议的最低临界值比较确定抗性菌株;通过PCR扩增链霉素抗性基因str A、str B,aad A、aad E、ant(6)、aac(6')-aph(2')和aph(3')-Ⅲa,确定抗性菌株的抗性基因。结果分离到67株链霉素敏感性或抗性菌株,这些菌株分别属于Pediococcus ethanolidurans(36),Lactococcus garvieae(14),Lactobacillus buchneri(12),Lactobacillus acetotolerans(2),Lactococcus lactis(1)和Staphylococcus.spp(2)。其中Lactococcus garvieae、Lactobacillus acetotolerans、Lactococcus lactis和Staphylococcus.spp全部为抗性菌株,Pediococcus ethanolidurans中有抗性菌株20株、Lactobacillus buchneri中有7株。在抗性基因检测中,除Lactobacillus acetotolerans抗性菌株没有检测到被检基因外,其他5个种的抗性菌株中均检测到部分或全部抗性基因。str A和aph(3')-Ⅲa基因在除Lactobacillus acetotolerans外的被检菌株中均有检出,其检出率分别为50%-100%和21.4%-100%;str B基因在抗性菌株Pediococcus ethanolidurans,Lactobacillus buchneri,Lactococcus garvieae和Lactococcus lactis检测率分别为70%、42.9%、28.6%和100%;aac(6')-aph(2')基因仅在3株Pediococcus ethanolidurans、1株Lactobacillus buchneri、1株Lactococcus garvieae和Staphylococcus spp中检测到。aad A、aad E and ant(6)在所有抗性菌株中都没有检测到。总体而言,str A、str B和aph(3')-Ⅲa基因在链霉素抗性菌株中的检出率高于其他抗性基因。结论当前的研究结果表明:四川泡菜中存在链霉素乳酸菌抗性菌株,这些抗性菌株对四川泡菜存在潜在安全风险。     

奶牛场采奶环节大肠埃希氏菌分离鉴定及对抗生素和消毒剂的抗性分析

为了解奶牛场采奶过程中大肠埃希氏菌污染状况、菌株所携带毒力基因及其对抗生素和消毒剂抗性.选择成都市某奶牛场随机采集655份样品,对致病性大肠埃希氏菌进行分离鉴定,并检测分离株携带的毒力基因、耐药基因以及抗消毒剂基因,筛选出毒力强菌株,分别检测菌株对抗生素和消毒剂的敏感性.结果 表明,一共分离得到249株大肠埃希氏菌,鉴...     

四川泡菜中乳酸菌链霉素抗性与抗性基因的检测

目的 探明四川泡菜中链霉素抗性乳酸菌及抗性基因的种类。方法 利用含有8 μg/ml链霉素的MRS初步分离泡菜液中的抗性乳酸菌,通过16S rRNA分析确定抗性乳酸菌的分类地位后,测定同种不同菌株对链霉素的MIC,并与欧洲食品安全官方机构（EFSA）建议的最低临界值比较确定抗性菌株;通过PCR扩增链霉素抗性基因strA、strB, aadA、aadE、ant(6)、aac(6')-aph(2')和aph(3')-Ⅲa,确定抗性菌株的抗性基因。结果 分离到67株链霉素敏感性或抗性菌株,这些菌株分别属于Pediococcus ethanolidurans (36), Lactococcus garvieae (14), Lactobacillus buchneri (12), Lactobacillus acetotolerans (2), Lactococcus lactis (1)和Staphylococcus.spp (2)。其中Lactococcus garvieae、Lactobacillus acetotolerans、Lactococcus lactis和Staphylococcus.spp全部为抗性菌株,Pediococcus ethanolidurans中有抗性菌株20株、Lactobacillus buchneri 中有7株。在抗性基因检测中,除Lactobacillus acetotolerans抗性菌株没有检测到被检基因外,其他5个种的抗性菌株中均检测到部分或全部抗性基因。strA 和aph(3')-Ⅲa基因在除Lactobacillus acetotolerans外的被检菌株中均有检出,其检出率分别为50%-100%和21.4%-100%;strB基因在抗性菌株Pediococcus ethanolidurans, Lactobacillus buchneri, Lactococcus garvieae和Lactococcus lactis检测率分别为70%、42.9%、28.6%和100%;aac(6')-aph(2')基因仅在3株Pediococcus ethanolidurans、1株Lactobacillus buchneri、1株Lactococcus garvieae和Staphylococcus spp中检测到。aadA、aadE and ant (6)在所有抗性菌株中都没有检测到。总体而言,strA、strB和aph(3')-Ⅲa基因在链霉素抗性菌株中的检出率高于其他抗性基因。结论 当前的研究结果表明：四川泡菜中存在链霉素乳酸菌抗性菌株,这些抗性菌株对四川泡菜存在潜在安全风险。     

采用同源重组技术构建金黄色葡萄球菌sae基因缺失突变株

针对金黄色葡萄球菌sae基因前后两段序列设计两对引物,PCR扩增出sae基因上下游同源臂序列,克隆到穿梭载体pBT2中;两段序列之间用来自质粒p646的Em抗性基因片段连接,作为筛选标记,从而构建同源重组穿梭质粒pBT2△sae;将pBT2△sae电转化到金黄色葡萄球菌菌株RN4220中,40℃经过七轮培养,进行抗性培养基筛选和PCR验证,以及RT-PCR观察基因表达水平.获得一株金黄色葡萄球菌sae基因缺失突变株RN△sae,为进一步研究sae基因的调控机制等提供有用的实验材料.     

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.     

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.