基因改组选育高产杆菌肽地衣芽孢杆菌及改组菌株差异分析

为了提高地衣芽孢杆菌肽产量,采用全基因组改组技术进行菌株选育。以地衣芽孢杆菌TJ12为原始菌株,通过紫外诱变、亚硝基胍诱变、常压室温等离子体诱变构建了TJ12菌的突变体库。在优化其原生质体制备和再生条件的基础上,以其中4 株诱变菌株（U11、U23、H1、L71）作为亲本,采用聚乙二醇介导的方法进行3 轮多亲本的全基因组改组,同时,结合双亲灭活的筛选方法,最终选出1 株杆菌肽产量提高并能稳定遗传的优良菌株F3,对其摇瓶发酵36 h,杆菌肽A产量达760 mg/L,为野生菌株TJ12的1.7 倍。与野生菌株TJ12相比,改组菌株提前进入生长稳定期,两者发酵过程pH值几乎无差异;最终改组菌生长量虽低于野生菌,但菌株单位细胞还原糖产量及杆菌肽产量都高于野生菌株;其合成基因和调控基因表达量相对野生菌株都上调,其中合成基因上调幅度较大。推测改组菌株自身有了更强大的杆菌肽耐受机制,且合成基因相关部位可能发生了改变。     

基因组改组选育氨肽酶和自溶度高的植物乳杆菌

利用基因组改组方法筛选出促进干酪成熟的非发酵剂乳酸菌。采用紫外线和亚硝基胍两种传统的诱变方法对植物乳杆菌进行诱变,通过检测自溶度、氨肽酶、产酸能力指标获得6株氨肽酶活性和自溶度均有所提高的突变菌株。以获得的突变菌株为出发菌株,应用灭火双亲原生质体融合后致死损伤得到的互补获得活性融合子的方法,对其进行基因组改组,经自溶度、氨肽酶、产酸能力筛选,获得1株遗传性能稳定的菌株,其氨肽酶活力为34.01个酶活单位,比出发菌株提高了3.09倍,自溶度为56.45%,比出发菌株提高了3.16倍。     

利用全基因组改组技术提高SubtilosinA的产量

以Bacillus subtilis nja A1B2-2为出发菌株,在最佳的原生质体形成、再生和融合条件的基础上,将前期经过理化诱变筛选的四株高产菌株进行两轮基因组改组。结果表明,当以0.6mol/L Na Cl为高渗洗涤液,0.1mg/m L溶菌酶酶解40min后,涂布在以SMM为高渗体系的NB再生培养基上,原生质体形成率达到95.49%,再生率为89.25%。优化原生质体的融合条件,融合率达到了1.39×10-3。在此基础上将四株高产Subtilosin A菌株进行两轮全基因组改组,结合双亲灭活的筛选方法,挑选出一株遗传性状稳定的高产菌株R2-264,产量达17.59mg/L,比出发菌株提高了3.58倍。     

高产细菌素植物乳杆菌的诱变选育研究

为获得细菌素高产菌株,以植物乳杆菌(Lactobacillus plantarum)JLA-9为出发菌株,对其进行亚硝基胍(NTG)诱变、常压室温等离子体(ARTP)诱变,以及基因组改组。结果表明,亚硝基胍诱变的最佳浓度为4 mg/m L,经筛选得到两株突变株N4-26、N4-27,其抑菌效价分别为2531.93、3057.32 IU/m L;常压室温等离子体诱变最佳时间为10 s,经筛选得到两株突变株ARTP10-37、ARTP10-61,其抑菌效价分别为2974.27、3261.62 IU/m L。将上述抑菌效价提高的菌株经四轮基因组改组后,得到一株突变株F4-2,其抑菌效价达到7374.76 IU/m L,相对于原始菌株提高了2.35倍,且遗传稳定性良好。研究表明理化诱变结合基因组改组的方式是快速获得理想菌株的有效方法。     

产胞外葡萄糖氧化酶菌株的分离鉴定及其诱变育种研究

从来自全国各地120余份土样中筛选得到1株胞外葡萄糖氧化酶生产菌株1504,经培养特征、形态特征及分子生物学鉴定确定菌株1504为黑曲霉。以黑曲霉1504为出发菌株,采用紫外诱变、亚硝酸钠化学诱变以及紫外-亚硝酸钠复合诱变3种方法对其进行诱变育种,通过3步筛选的方法筛选高产胞外葡萄糖氧化酶的突变菌株。对突变株进行摇瓶发酵试验,最终选育出1株胞外葡萄糖氧化酶产酶活力较高的突变菌株UNⅡ021,该菌株产胞外葡萄糖氧化酶活力达到186.32 U/mL,为原始菌株的3.8倍,经5代传代试验表明,其产酶能力稳定。     

葡聚糖酶产生菌的诱变选育

利用紫外线诱变育种方法分离对前期分离到一株葡聚糖酶野生菌株P5进行诱变。通过平板初筛、摇瓶复筛,从诱变菌株中筛选出一株高产菌株,其发酵酶活力可以达到5.85U/mL;在此基础上,对诱变菌株的发酵产酶工艺进行了初步的优化,其最适碳源为淀粉,最适氮源为花生饼粉,正交试验结果表明,淀粉含量为5%、花生饼粉含量为4%、NaH2PO4含量为0.2%时,该菌株的产酶量最高,酶活力达到21.23U/mL。     

基因组改组技术改善植物乳杆菌C88的益生作用

采用基因组改组技术得到融合子菌株,并筛选益生性最优的融合子。采用基因组改组方法,结合紫外和亚硝基胍诱变,对突变菌株进行原生质体融合,经体外实验对融合子的耐酸性、耐高胆盐浓度、黏附相关性和抗氧化能力进行分析,筛选并获得益生性提高的融合子。筛选获得的两株融合子为F1-9和F1-17,均能够在低p H值和高胆盐浓度环境中生长;与原始菌株相比,融合子在表面疏水性、自聚性和与致病菌共聚性方面均有显著提高;在抗氧化方面较原始菌株也有所提高。融合子突变株连续传代培养,遗传稳定性良好。基因组改组技术能够使菌株的益生性得到明显提高,并且能够稳定遗传。     

基因组改组:几株同源酱油曲霉的多亲株电融合育种

酱油酿造中原料的全氮利用率是一个主要的生产指标，通过育种提高生产菌株的中性蛋白酶活是一个有效的方法。利用一株用曲精中分离、纯化的酱油曲霉，制备其分生孢子的原生质体，并优化了原生质体制备的条件。将分生孢子的原生质体进行紫外诱变，得到7株酶活提高幅度较大的紫外诱变株U，以此作为候选株文库，采用基因组改组（Genome shuffling），进行多亲株灭活电融合，获得9株酶活进一步提高的菌株。     

常压室温等离子体诱变筛选高乳糖酶活力酵母的研究

探讨常压室温等离子体(ARTP)诱变筛选高乳糖酶活力乳酸克鲁维酵母的条件。以ARTP诱变育种方法为诱变手段,对乳酸克鲁维酵母进行不同时长(10 s依次增加到300 s)的诱变处理,并结合高通量筛选方法快速筛选出33株乳糖酶活力提高50%以上的突变菌株。通过复筛最终得到4株高乳糖酶活力的突变菌株,采用三角瓶摇床培养,结果表明:筛选得到的突变菌株的乳糖酶活力均大于原始菌株,其中最大乳糖酶活力提高到0.257 U/mL,是原始菌株(0.090 U/mL)的2.8倍;4株突变菌株生长速度比原始菌株显著提高。ARTP诱变育种技术结合高通量筛选方法是1种快速、有效的新型微生物诱变育种方法。     

米曲霉孢子原生质体复合诱变及高活力蛋白酶菌株选育

以沪酿3.042米曲霉的孢子原生质体为诱变对象,经溶菌酶2%+蜗牛酶2%+纤维素酶2%,33℃水浴酶解时间5h的孢子原生质体最佳制备条件作用后,对其进行紫外线-氯化锂,NTG复合诱变,并利用酪蛋白平板初筛和固体发酵测定中性蛋白酶活力复筛,筛选了8株高产中性蛋白酶突变株群,为后续的细胞融合、基因组改组等实验提供了优良的候选文库。其中最高产菌株UN97,经37℃培养40h产酶活力为6834U/g(干基),为原菌株的1.62倍,传代培养8次,遗传性能稳定。     

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