代谢改造黑曲霉合成乙酰氨基葡萄糖

该研究以黑曲霉为底盘细胞,首先敲除N-乙酰氨基葡萄糖（N-acetylglucosamine,GlcNAc）摄取相关转运蛋白ngtA编码基因,获得GlcNAc的吸收利用缺陷型菌株,有利于胞外GlcNAc的积累。在此基础上,通过表达大肠杆菌来源的卤酸脱卤酶样磷酸酶编码基因yqaB,构建黑曲霉GlcNAc的完整合成途径,实现GlcNAc的合成,产量为1.78 g/L。通过共过表达氨基葡萄糖-6-磷酸乙酰转移酶基因gnaA和氨基葡萄糖-6-磷酸合成酶基因gfaA强化GlcNAc合成途径,GlcNAc的合成水平提升至3.64 g/L。为增加GlcNAc合成前体GlcNAc6P的胞内供给,利用RNA干扰技术对乙酰氨基葡萄糖-6-磷酸（GlcNAc6P）分解代谢途径中氨基葡萄糖-6-磷酸脱氨基酶基因nagB和乙酰氨基葡萄糖-6-磷酸脱乙酰酶基因nagA基因表达进行弱化表达,GlcNAc产量进一步提高至4.03 g/L。为减弱黑曲霉糖酵解途径与GlcNAc合成途径竞争共同前体物质果糖-6-磷酸,对磷酸果糖激酶基因pfkA进行弱化表达,GlcNAc的最终产量为4.61 g/L。     

产N-乙酰氨基葡萄糖的工程菌构建、发酵及应用研究进展

N-乙酰氨基葡萄糖（GlcNAc）是一种氨基葡萄糖（GlcN）衍生物,属于功能性氨糖类化合物,广泛应用于食品、医药及化妆品领域,市场前景非常广阔。传统的GlcNAc生产方法主要采用甲壳素水解法,该方法在原料供应、环境保护及产品安全方面存在许多潜在问题,构建高效生产GlcNAc的基因工程菌可以解决这些问题。该文着重综述了生产GlcNAc基因工程菌的构建及发酵方面的研究进展,并对GlcNAc的应用进展进行了介绍,并对如何利用工程菌提升GlcNAc产量进行了展望。     

Clostridium paraputrificum M-21β-N-乙酰氨基葡萄糖苷酶的表达、纯化与性质

以ClostridiumparaputrificumM21染色体DNA为模板,经PCR扩增获得编码β N 乙酰氨基葡萄糖苷酶的基因nag3A,与质粒pQE30T所构建的表达质粒pNAG3A在EscherichiacoliM15中表达良好.从重组E.coliM15中纯化得到的Nag3A以4 MU GlcNAc为底物时,最适作用温度和最适作用pH分别为50℃和7.0;在30℃以下和pH6～9之间该酶活性稳定.对4 MU GlcNAc的Km和Vmax分别为7.9μmol和21.8μmol/(min·mg).Nag3A可以水解几丁寡糖和几丁质,作用方式是从非还原端逐一水解β 1,4 D N 乙酰氨基葡萄糖苷键,产生N 乙酰氨基葡萄糖,对几丁二糖具有较高的水解活性.     

丙酮对黄粉虫N-乙酰- β-D-氨基葡萄糖苷酶活力的影响

以丙酮为效应物,研究其对黄粉虫（Tenebrio molitor Linnaeus）N-乙酰-β-D-氨基葡萄糖苷酶（NAGase）活力的影响,结果表明该酶的剩余活力随着丙酮浓度增大而呈指数下降。导致酶活力丧失50%（抑制半衰期,IC50）的丙酮浓度为7.2%,说明丙酮对黄粉虫NAGase有明显的失活作用。该酶的失活过程属于混合型,并进一步测定游离酶（E）和酶底物络合物（ES）与丙酮的结合常数（KI和KIS）,分别为5.32%和27.2%,KI〈KIS，说明底物存在对酶被丙酮的失活作用有一定的保护作用。     

N-乙酰氨基葡萄糖苷酶的异源表达及甘油糖苷的酶法合成

从杆菌状链霉菌中克隆了一个N-乙酰氨基葡萄糖苷酶（N-acetyl-glucosaminidase,NAGase）的编码基因sbnag2550,并将其在大肠杆菌BL21（DE3）中进行了异源表达。利用镍离子亲和层析得到纯酶后对SbNag2550的酶学性质进行了研究。该酶的最适温度为50 ℃,在45~60 ℃均表现出90%以上的酶活力。SbNag2550还表现出优良的热稳定性,在55 ℃孵育60 h仍能保留将近90%的酶活力。利用SbNag2550催化N-乙酰氨基葡萄糖（N-acetyl-glucosamine,NAG）和甘油发生逆水解反应,合成了甘油-N-乙酰氨基葡萄糖苷（glyceryl N-acetyl-glucosamine,GNAG）。在最适条件下,反应24 h时转化率达到28.20%,反应72 h时转化率为34.82%。本研究中首次通过酶法合成了GNAG,为其功能研究和应用开发奠定了基础。     

磷脂酰-N-乙酰氨基葡萄糖苷的合成及脂质体制备

为了提升磷脂酰-N-乙酰氨基葡萄糖苷（PtdGlcNAc）的合成效率,选取不同的咪唑类离子液体,通过以磷脂酶D（phospholipase D,PLD）催化磷脂酰胆碱（phosphatidylcholine,PC）与N-乙酰-D-氨基葡萄糖（N-acetyl-beta-D-glucosamine,GlcNAc）的转酯反应,探究离子液体预处理对酶催化性能的影响。结果表明,经过1-丁基-3-甲基咪唑六氟磷酸盐（[BMIm][PF6]）预处理后,PLD的活性明显高于其他离子液体组和空白组。因此,采用[BMIm][PF6]作为PLD预处理的孵育溶液,通过优化实验,得到最佳反应条件为：1 U的加酶量,底物PC与GlcNAc的物质的量比为1∶60,环戊基甲醚作为有机相,离子液体与水体积比为4∶1,反应时间12 h,产率为79.7%,与未处理组的产率相比提升了56.9%。将合成的PtdGlcNAc制备成纳米脂质体,并对其结构进行了初步表征。该研究丰富了PLD反应体系的相关研究,为其他新型磷脂酰化合物的合成和应用提供了参考。     

沙蒿胶磁性微球固定化脂肪酶及部分理化性质的研究

以沙蒿多糖-壳聚糖复合磁性微球为载体,采用物理吸附法固定化脂肪酶,对固定化过程中对酶活力有影响的各种因素做了研究,同时对固定化酶的部分理化性质、最适pH、最适温度、酶的热稳定性以及表现米氏常数与游离酶做了比较.固定化酶的Km小于游离酶的Km,其最适pH和最适温度分别为8.0和50℃,而且固定化脂肪酶具有良好的热稳定性、可应用性和重复使用性.     

改造细胞质膜促进途径酶组装及N-乙酰氨基葡萄糖合成

功能膜微域（FMMs）可以作为内源性空间支架进行途径酶组装,适度地提高FMMs在质膜中的占比能够明显提高产物合成量,然而对FMMs进行改造后,细胞生长和产物的合成均受到抑制。在课题组前期研究的基础上,对细胞质膜进行理性改造以缓解FMMs过度改造对细胞造成的影响。首先通过过表达PlsX,PlsY和PlsC改造细胞质膜,之后,以枯草芽孢杆菌合成N-乙酰氨基葡萄糖（GlcNAc）为例,发现质膜改造的菌株中GlcNAc产量达到（5.18±0.16） g/L,与对照菌株相比产量提升了41.9%,同时,细胞质膜改造也明显降低FMMs过度修饰对菌株生长产生的不利影响。     

羟自由基与Neu5Gc抽氢反应的理论动力学研究

红肉风险物质N-羟乙酰神经氨酸（Neu5Gc）可诱发炎症，但与食品消费代谢和加工贮藏过程相关的羟自由基（OH·）互作机制未知。体外OH·模拟试验表明过氧化氢（H2O2）添加量2%，254 nm紫外灯15 处理60 min时对Neu5Gc标准品含量降低效果最佳为76.23%±2.17%。为探究该过程机制，首先在气相、水和苯相下以M062X/6-31+G（d, p）理论水平对OH·作用Neu5Gc分子中羟基氢和非羟基氢抽提反应的各驻点进行优化和焓值校正，M062X/def2TZVP水平计算能垒。其次，298 K~453 K内以过渡态理论和三参数阿伦尼乌斯方程分别获得反应速率和动力学参数。结果表明：气相和苯相下Neu5Gc中H24位抽氢能垒最低，分别为13.20 kJ/mol和19.54 kJ/mol；水相下H 29位能垒最低6.25 kJ/mol；气相下活化能最低为H 39位8.09 kJ/mol，苯相下最低为H 38位1.72 kJ/mol，水相最低为H 24位14.3 kJ/mol；273到453 K间，Neu5Gc分子中非羟基氢处抽氢速率最大可高于羟基氢108倍。综上，OH·主要通过对Neu5Gc非羟基氢原子的氢抽提反应进行相互作用。     

对硝基苯酚法对雅致放射毛霉脂肪酶特性的研究

以不同碳链长度的对硝基苯酚酯为底物对雅致放射毛霉（Actinomucor elegans AS3.27）脂肪酶的酶学特性和其发酵的腐乳在发酵过程中脂肪酶活力的变化进行研究,结果表明：分别以对硝基苯酚正辛酸酯（pNPC）和对硝基苯酚棕榈酸酯（pNPP）为反应底物所测得的脂肪酶酶学特性有所不同。以pNPC和pNPP为反应底物时,雅致放射毛霉脂肪酶的最适作用温度分别为30、35℃,最适作用pH为7.5、7.0,最适盐浓度都为0.20mol/L。以两种底物分别测定雅致放射毛霉接种的腐乳在发酵过程中的脂肪酶活力,均显示在后期发酵过程中脂肪酶活力呈现出不断下降的趋势,然而在第20～25d脂肪酶活力呈现出小幅度的反复。使用碱滴定法以聚乙二醇-橄榄油为底物进行验证,得到相同的趋势。     

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.     

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.     

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.     

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.     

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.