海藻酸钠固定细胞产D-阿洛酮糖的研究

D-阿洛酮糖3-差向异构酶(DPEase)是一种能催化D-果糖转化为D-阿洛酮糖的异构酶。本实验采用海藻酸钠作为载体,包埋重组大肠杆菌催化D-果糖生成D-阿洛酮糖。以固定化细胞的酶活活力回收率为指标,优化出最佳固定化条件为:海藻酸钠浓度3%,细胞包埋量60g/L,Ca Cl2浓度2%,固定化时间4h,0.01%浓度戊二醛溶液中交联4h。该条件下所得固定化细胞的酶活回收率高达76%,且具有较好的操作稳定性,重复操作8次后酶活回收率仍然保持61%。固定化后DPE细胞的最适酶反应温度提高了5℃、最适pH与游离细胞基本一致,耐热性明显提高,p H稳定性与游离细胞一致。     

D-阿洛酮糖合成及偶联酿酒酵母发酵的研究

该研究利用含有组成型启动子的质粒pET-20b载体,在大肠杆菌（Escherichia coli）BL21（DE3）中对3种D-阿洛酮糖3-差向异构酶进行异源表达,其后以D-果糖为底物进行静息细胞转化。结果表明,重组菌在摇床30 ℃、200 r/min转速下发酵48 h,能够利用D-果糖为底物生产D-阿洛酮糖,转化率分别达到27.56%、23.99%和25.98%。为降低D-果糖对D-阿洛酮糖纯化过程中的影响,在产糖的过程后偶联酿酒酵母（Saccharomyces cerevisiae）好氧发酵过程,消耗掉混合糖液中的D-果糖,结果显示转化24 h后D-果糖去除率达到94.22%,该研究为下游D-阿洛酮糖的分离和纯化提供了新的思路。     

枯草芽孢杆菌全细胞转化高效合成D-阿洛酮糖

D-阿洛酮糖是一种重要的稀少糖,在食品、化妆品和医药等领域都有着广泛的应用价值。目前,工业上生产D-阿洛酮糖以生物酶法为主。由于传统酶法存在步骤烦琐、成本高、产物纯化分离难等缺点,已难以满足工业生产需要。近年来,全细胞合成体系以其低成本、便操作、易分离等特点受到人们的关注。以一种来源于Caballeronia insecticola的D-阿洛酮糖3-差向异构酶(DAEase)为研究对象,实现了在生产安全菌株枯草芽孢杆菌WB800中的高效异源表达,并以D-果糖为底物全细胞催化合成D-阿洛酮糖。为了提高DAEase的表达量,通过设计构建含有不同组成型启动子的重组菌株对其表达进行了优化。并对全细胞反应体系的条件(温度、pH值、金属离子、细胞浓度)进行了优化,探究了不同底物浓度下D-果糖的转化效率。结果表明：启动子P_ylbP介导下的重组DAEase在枯草芽孢杆菌WB800内具有较高的表达水平,重组DAEase全细胞反应的较适温度为65℃,较适pH值为9.5,较适金属离子为5 mmol/L Mg²⁺。采用全细胞方法以500 g/L D-果糖为底物制备D...     

制备高含量低聚果糖联产D-阿洛酮糖的工艺研究

制备蔗糖源高含量低聚果糖主要利用色谱分离技术除去普通级低聚果糖产品中的葡萄糖和蔗糖，得到高含量低聚果糖组分和副产物组分。为进一步高值化利用副产物，赋予其功能性及扩大应用范围，在制备低聚果糖工艺技术基础上利用蔗糖转化酶、葡萄糖异构酶、D-阿洛酮糖3-差向异构酶、色谱分离依次处理副产物，并通过单因素试验优化D-阿洛酮糖3-差向异构酶异构反应及色谱分离纯化参数。结果得到：在反应温度45℃、p H5.75、每克底物55 U酶量、反应时间12 h的条件下，底物中果糖质量浓度在90%以上；色谱分离关键参数：洗脱液流量15 L/min，外加压力50 kPa。D-阿洛酮糖最高转化率为27.1%，成品糖浆中D-阿洛酮糖含量>91%，整体工艺总产率为91%。研究结果表明联产工艺技术可行性高，要进行工业化生产还需进一步优化。     

L-鼠李树胶糖激酶在D-阿洛酮糖合成中的应用

D-阿洛酮糖-3-差向异构酶（D-psicose-3-epimerase,DPE）可以催化D-果糖和D-阿洛酮糖之间的可逆反应,但D-阿洛酮糖的产率较低。L-鼠李树胶糖激酶（L-rhamnulose kinase,RhaB）具有磷酸化酮糖类物质的活性,将其与DPE偶联,可以打破DPE的可逆平衡,从而提高D-阿洛酮糖的产率。在反应体系中引入多聚磷酸盐激酶（polyphosphate kinase,PPK）,可实现ATP的再生,降低反应的成本。本实验在大肠杆菌Rosetta（DE3）中分别过表达DPE、RhaB和PPK,优化其反应条件,计算产物得率。结果表明,RhaB的相对分子质量约为5.3×104,最适pH和温度为8.0和35 ℃。将DPE和RhaB偶联,最适条件如下：pH 8.5,温度35 ℃,最适金属离子Mg2+,DPE和RhaB酶量比（质量比）1∶2,产物得率为70%。将DPE、RhaB和PPK偶联,ATP浓度降为D-果糖的1/5,D-阿洛酮糖得率为50%。本研究为工业化生产D-阿洛酮糖提供理论依据。     

D-阿洛酮糖3-差向异构酶的异源表达和酶学性质

克隆了来源于Clostridium bolteae ATCC BAA-613的D-阿洛酮糖3-差向异构酶基因,利用重叠延伸PCR技术在Cb-dpe基因的上游加入了P43启动子,形成P43-Cb-dpe,再将P43-Cb-dpe连接到p MA5载体上构建出双启动子表达载体,并导入到Bacillus subtilis WB800中进行表达;与单启动子表达系统相比,双启动子表达载体能够显著提高Cb-dpe的表达量。对重组Cb-DPE酶进行了分离纯化和酶学性质的研究,结果表明:重组Cb-DPE的最适温度为55℃,最适pH为7.0,在温度30~40℃范围内和pH 6.5~7.5之间有良好的稳定性;Co~(2+)、Mn~(2+)可显著增强酶活;D-阿洛酮糖为底物时,K_m为26.68 mmol/L,小于果糖的61.80 mmol/L,说明该酶对D-阿洛酮糖的亲和性比对D-果糖的高。而在动力学参数方面,以D-阿洛酮糖为底物对应的催化效率K_(cat)/K_m为95.8 L/(mmol·min),大于以果糖作为底物时的54.1 L/(mmol·min)。     

生物转化生成D-阿洛酮糖的类球红细菌的筛选

对鱼塘淤泥、水样中富集分离的27株光合细菌的静止细胞反应产物,采用高效液相色谱进行分析,从中筛选到1株D-阿洛酮糖产率较高的菌株,在形态和常规生理生化方面鉴定的基础上,结合16S rDNA序列分析鉴定为类球红细菌(Rhodobacter sphaeroides),定名为类球红细菌SK011。SK011利用D-果糖(36 g/L,pH7.5)为底物进行静止细胞转化,45℃,5 h,D-阿洛酮糖产率达到6.54%。     

根癌农杆菌D-阿洛酮糖-3-差向异构酶基因克隆、 结构预测及原核表达

D-阿洛酮糖-3-差向异构酶能够催化D-果糖转化为D-阿洛酮糖。为实现D-阿洛酮糖-3-差向异构酶的异源表达,设计引物,克隆并分离其序列,通过生物信息学软件分析D-阿洛酮糖-3-差向异构酶DNA和蛋白质的结构特点。结果表明,该基因开放阅读框870bp,编码289个氨基酸;蛋白质二级结构中α-螺旋占38.41%,β-折叠占47.06%,无规则卷曲占14.53%;该蛋白为亲水性蛋白,不含信号肽,无跨膜区,定位于细胞膜;构建原核表达载体并导入E.coliBL21宿主中,表达的D-阿洛酮糖-3-差向异构酶分子质量约为33kDa,1mmol/LIPTG诱导E.coliBL21重组菌28h后,目的蛋白表达量和酶活分别为0.32g/L和3.8U/mL。根癌农杆菌D-阿洛酮糖-3-差向异构酶基因能够在大肠杆菌中实现表达。     

乙醇体系中D-阿洛酮糖的结晶工艺优化

为开发一套D-阿洛酮糖的结晶工艺,本文以乙醇为结晶体系,分别对影响结晶过程的4个主要因素(包括D-阿洛酮糖溶液的密度、乙醇与D-阿洛酮糖的比例、结晶时间和结晶温度)进行了单因素实验,并在单因素实验的基础上,采用响应曲面法对结晶的工艺参数进行优化。结果表明,最优操作条件为:D-阿洛酮糖溶液密度为1.35 g/mL,乙醇与D-阿洛酮糖溶液比例为3.8:1,结晶时间为325 min,结晶温度为25℃。在此条件下,D-阿洛酮糖的初次结晶收率可达71.58%。以上结果可以得出结论:乙醇体系中可获得较高的D-阿洛酮糖结晶收率,本研究获得的模型可以用来优化D-阿洛酮糖在乙醇体系中的结晶过程。     

D-阿洛酮糖3-差向异构酶在大肠杆菌内的高效可溶性表达及发酵条件研究

将来源于Clostridium bolteae ATCCBAA-613的DAEase基因序列经密码子优化合成,以pCold TF为表达载体,冷休克启动子CspA低温诱导DAEase基因在大肠杆菌（Escherichia coli）BL21 （DE3）中表达,得到高效可溶性的重组Cb-DAEase并利用镍柱亲和层析分离纯化。结果表明,Cb-DAEase最适pH和温度为7.0和55 ℃,Co2+能够显著（P<0.05）增强酶活力。对培养条件进行优化得到,在7 g/L甘油、10 g/L酵母膏、1%接种量、0.25 mmol/LIPTG、诱导前培养5 h的条件下,Cb-DAEase活力达到（10.11±0.02）U/g,比优化前(1.38±0.01) U/g提高了7.33倍;以120 g/L的D-果糖为底物全细胞催化0.5 h后,D-阿洛酮糖产量为（11.47±0.04）g/L,比优化前（1.03±0.02）g/L提高了11.14倍。基于冷休克表达策略构建的重组菌经发酵优化后Cb-DAEase活力显著（P<0.05）提高,为高效制备D-阿洛酮糖提供了理论支持。     

功能性甜味剂D-阿洛酮糖研究进展

高糖摄入引起的糖尿病、高血压等疾病严重危害着人类的生命健康,这一形势也促使人们对天然、低热量糖替代品的开发日益关注.作为一种近年发现的具有特殊生理功效的稀少糖,D-阿洛酮糖具有与蔗糖相近的口感及容积特性,被称为食品中蔗糖"理想替代品".D-阿洛酮糖可以在D-阿洛酮糖3-差向异构酶的催化作用下进行生物合成,已被FDA批准...     

Bioproduction of D-allulose: Properties,applications, purification,and future perspectives

D-allulose is the C-3 epimer of D-fructose, which rarely exists in nature, and can be biosynthesized from D-fructose by the catalysis of D-psicose 3-epimerase. D-allulose is safe for human consumption and was recently approved by the United States Food and Drug Administration for food applications. It is not only able be used in food and dietary supplements as a low-calorie sweetener, but also modulates a variety of physiological functions. D-allulose has gained increasing attention owing to its excellent properties. This article presents a review of recent progress on the properties, applications, and bioproduction progress of D-allulose.     

In vitro digestibility of rare sugar (D-allulose) added pectin–soy protein gels

Confectionery gels are known to be high-caloric products due their high sugar content. Changing their formulations by substituting the sugar with alternative natural sweeteners and functionalising them, the addition of proteins has gained attention. Understanding the rate of digestion of these products is also important for selecting the appropriate formulation. In this study, in vitro gastric digestion behaviour of the gels formulated with D-allulose, a low-calorie rare sugar, soy protein isolate (SPI) (1%, 2.5%) and pectin (4%) were examined. Digestion decreased the hardness of the gels (P < 0.05), but, at 2.5% SPI concentration. Moisture content of the samples increased after digestion and presence of SPI induced higher water uptake. At the end of 2 h of digestion, 1% soy protein isolate containing gels had the highest brix values showing that after a certain concentration, soy protein isolate governed the system due to improved soy protein–pectin interaction or due to improved gelation with Maillard reaction. NMR relaxometry experiments further confirmed the changes in the gels with the increase in T₂ values. Power law model was fitted for the dissolution behaviour using the ^oBrix values of the digestion medium. Dissolution of sugar and the contribution of SPI to the gel network were clearly observed in SEM images. Results showed that these gels had the potential to slow down the emptying rate of stomach thus could lead to ‘fullness’ for a longer time.     

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.     

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.