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。     

多酶级联系统制备蒜糖醇的细胞工厂构建及工艺优化

蒜糖醇是一种在食品、医药等领域具有较大应用前景的新型功能性甜味剂。为实现蒜糖醇的绿色生物合成,基于稀有糖转化策略通过共表达葡萄糖异构酶、D-阿洛酮糖3-差向异构酶及核糖醇脱氢酶,在大肠杆菌体内建立了利用廉价底物D-葡萄糖生产蒜糖醇的多酶级联系统;并通过偶联甲酸脱氢酶构建辅因子循环系统,提高胞内还原型辅酶Ⅰ的再生效率。为平衡多酶级联系统中的个体表达差异,以全细胞催化活性为指标,对细胞工厂的发酵条件和催化条件进行了优化。结果表明:重组菌在20℃、1.00mmol/L异丙基-β-D-硫代半乳糖苷的发酵条件下,诱导24h可得到较佳的蛋白质表达量;在40℃、50mmol/L Tris-HCl (pH值8.0)及5.0g/L甲酸钠的催化条件下,全细胞可在15h内转化25.00g/L的D-葡萄糖生成19.33g/L蒜糖醇。通过表达系统的优化进一步降低了底物和中间产物的残留量,蒜糖醇的产量提高至21.12g/L。研究旨在为全细胞生物转化法实现蒜糖醇的规模化合成提供理论依据,探索以预处理甘蔗糖蜜为底物生产蒜糖醇的可行性,希望对延长糖产业链,提高糖产品附加值起到积极作用。     

D-阿洛酮糖的分离纯化

通过生物法以D-果糖为原料生产D-阿洛酮糖,产物的分离纯化采用阴阳离子交换树脂脱色脱盐,再通过DTF-Ca2＋型离子交换树脂进行分离纯化。最佳分离条件：柱温60℃,10mL进样量,1mL/min流速。通过高效液相色谱测定,分离得到的D-阿洛酮糖纯度为98.3%。     

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5mmol/L Mg²⁺。采用全细胞方法以500g/L D-果糖为底物制备D-阿洛酮糖,4h内反应基本达到平衡,转化率为30.06%。研究旨在为D-阿洛酮糖工业规模化生产提供实验基础和理论依据。     

婴儿配方粉中克雷伯氏菌的检出及污染情况

对婴儿配方粉中肠杆菌科细菌进行筛查,检出婴儿配方粉中污染了肺炎克雷伯氏菌和产酸克雷伯氏菌,检测样本241份,污染几率分别为5.0%和2.5%。所采用的检验方法为VRBGA结合MIAC进行分离,采用API20E和VITEC2进行生化鉴定。     

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。     

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。     

氧化葡萄糖酸杆菌培养及转化1,2-丙二醇生产D-乳酸研究

对氧化葡萄糖酸杆菌培养及转化1,2-丙二醇生产D-乳酸进行研究.采用单因素实验优化了氧化葡萄糖酸杆菌的生长培养基,然后通过不同转化条件对该菌转化1,2.丙二醇生成D-乳酸的工艺进行了初步探讨.氧化葡萄糖酸杆菌生长的最佳碳源为6%山梨醇、最佳氮源为3%酵母粉;转化1,2-丙二醇的最优工艺为:10%静息细胞、20%(R,S)1,2-丙二醇维持pH6.0,250mL摇瓶装液30mL,28℃、220r/min培养30h.     

生物转化法产D-阿洛糖的分离纯化

利用重组菌E.coli BL21/p ET-22b-tl-rpib,将原料D-阿洛酮糖生物转化为具有抑癌效果的稀有糖D-阿洛糖。经液相色谱和串联质谱联用技术(LC-MS/MS)鉴定,反应混合液中25%的D-阿洛酮糖转化成了D-阿洛糖。等电聚焦法测定了重组蛋白的等电点为6.3,并成功运用到产物混合液的去蛋白沉淀中。经过预处理的混合糖液,通过DTFCa2+型离子交换树脂实现了分离纯化。最佳分离条件为:柱温60℃,进样量4m L,流速1m L/min。     

产酸克雷伯氏菌胞外多糖单糖组成的气相色谱分析

从青岛海藻化工厂海带浸泡液中分离得到产酸克雷伯氏菌(KlebsiellaoxytocaXCH-1)。在有氧条件下,该菌在生长过程中产生大量的胞外多糖,并且粘度很大。目前国内外尚未有关于该菌胞外多糖理化性质的研究报道。实验用KlebsiellaoxytocaXCH-1胞外多糖为化学均一物质。用气相色谱分析法分析KlebsiellaoxytocaXCH-1胞外多糖单糖组分,结果表明其主要由D-葡萄糖、D-半乳糖和L-鼠李糖组成。     

Efficient conversion of allitol to D-psicose by Bacillus pallidus Y25

An efficient method for conversion of allitol to D-psicose was achieved by a resting cell reaction of Bacillus pallidus Y25 for the first time. Notably, it was possible to produce D-allose and D-altrose from allitol directly via D-psicose by prolonging the reaction time. This method was applied for the preparation of D-psicose using the extract of Itea virginica as a starting material in this study. D-Psicose which is the absolutely key precursor for the production of other six carbon sugars could be obtained as the sole product at high yield.     

Rare Sugars and Antioxidants in Itea virginica,Itea oblonga Hand.-Mazz., and Itea yunnanensis Franch Leaves

Except Itea virginica, Itea ilicifilia, and Itea yunnanensis Franch, there had been few reports of other itea plants related to rare sugars. Likewise, little had been known about the antioxidants in itea plants. In this study, the rare sugars, phenolic profiles, as well as their antioxidant activities in the itea leaves, which were from Itea virginica, Itea oblonga Hand.-Mazz., and Itea yunnanensis Franch, were determined and compared. The first discovery of D-psicose and allitol in Itea oblonga Hand.-Mazz. further demonstrated the great potential application of itea plants in the production of rare sugars. Moreover, the leaves of Itea virginica and Itea oblonga Hand.-Mazz. showed good antioxidant activities in the assays of free radical scavenging activities and reducing power. The high performance liquid chromatography-tandem mass spectrometry experiment revealed the presence of several flavonoid glycosides as major antioxidant components in these leaves. These results indicated that the itea plants, especially Itea virginica, could be used for potential antioxidants in addition to functional sweeteners.     

Direct production of allitol from D-fructose by a coupling reaction using D-tagatose 3-epimerase, ribitol dehydrogenase and formate dehydrogenase

Allitol was produced from D-fructose via a new NADH-regenerating enzymatic reaction system using D-tagatose 3-epimerase (D-TE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH). D-fructose was epimerized to D-psicose by the D-TE of Pseudomonas cichorii ST-24 and the D-psicose was subsequently reduced to allitol by the RDH of an RDH-constitutive mutant, X-22, derived from Klebsiella pneumoniae IFO 3321. NADH regeneration for the reduction of D-psicose by the RDH was achieved by the irreversible formate dehydrogenase reaction, which allowed the D-psicose produced from d-fructose to be successively transformed to allitol with a production yield from D-fructose of almost 100%. The reactions progressed without any by-product formation. After separation of the product from the reaction mixture by a simple procedure, a crystal of allitol was obtained in a yield exceeding 90%. This crystal was characterized and determined to be allitol by HPLC analysis, its IR and NMR spectra, its melting point, and optical rotation measurement.     

新型甜味剂D-阿洛酮糖研究进展

稀有糖是在自然界中存在但含量极少的一类单糖及其衍生物。D-阿洛酮糖是一种重要的稀有糖,在食品、保健和医疗领域具有重要的应用价值。文中对D-阿洛酮糖的研究进展进行了综述,并展望了发展趋势。     

Chitosan productivity enhancement in Rhizopus oryzae YPF-61A by D-psicose

The effect of the rare sugar D-psicose on chitosan production by Rhizopus oryzae was studied. The fungus was not able to utilize D-psicose as a sole source of carbon, either for germination of the spores or for growth of the vegetative cells. In a medium containing a low amount of D-glucose, however, D-psicose supplementation between 5 and 12 g/l caused enhancement of the productivity of chitinous substances, especially chitosan, in the cell walls. Substantial changes in the chitosan molecule were not observed from FT-IR or 1H NMR data. It was inferred that a percentage age of the D-psicose added was transformed into another rare sugar tentatively identified as D-tagatose. It was concluded that D-psicose activates the synthesis of chitosan in the cell walls probably through its transformation into another sugar such as D-tagatose.     

油橄榄酒的酿造及香气成分分析

以制取橄榄油后的果汁为原料,通过调配、发酵等工艺制得油橄榄酒,进行理化指标及感官评价分析,并采用顶空固相微萃取结合气相色谱-质谱技术检测其香气成分,以期为油橄榄的综合开发利用,进一步提高其附加值提供理论依据。结果表明：产品酒精体积分数（6.42%）、总糖含量（7.84 g/L）、挥发酸含量（1.04 g/L）、总二氧化硫含量（103.05 mg/L）及干浸出物含量（27.66 g/L）等理化指标与感官评价结果均符合GB 15037-2006《葡萄酒、果酒通用分析方法》规定;油橄榄酒中共鉴定出69 种香气成分,总含量约23.44 mg/L,包括12 种醇类、25 种酯类、6 种有机酸、8 种羰基类（醛和酮）、14 种萜烯类和4 种酚类,通过香气成分的气味活性值初步确定主要香气成分有香叶醇、苯甲酸乙酯、辛酸乙酯和愈创木酚。     

粗壮脉纹孢菌降解豆皮粗纤维产可发酵糖培养基优化及单糖分析

研究考察了粗壮脉纹孢菌固态发酵豆皮酒糟培养基产纤维素酶的最优培养基组成,并用气相色谱法对该发酵条件下豆皮纤维素降解所产的可发酵糖进行定性检测。结果表明：产纤维素酶的最优培养基组成为豆皮64%、酒糟34%、(NH4)2SO4 2%,在此培养基中粗壮脉纹孢菌发酵产纤维素酶的酶活力为2.83 IU/g,较优化前的1.17 IU/g提高了141.88%,还原糖产量为18.01 g/100 g,较优化前9.91 g/100 g提高了81.74%。获得的可发酵糖的单糖组成主要为葡萄糖和木糖。     

Improved microcirculatory effect of D-allose on hepatic ischemia reperfusion following partial hepatectomy in cirrhotic rat liver

D-allose, one of the rare sugars produced from D-psicose, has been shown to be effective against reperfusion injury after ischemia and partial hepatectomy in cirrhotic rat liver by improving remnant liver blood flow and survival rates, and decreasing liver enzyme levels and liver tissue injury levels. These findings demand further study of the clinical implications of this sugar in view to the advancing fields of liver surgery and transplantation.     

糖水煎煮对人参化学成分的影响

目的：考察单糖、双糖、多糖水煎煮对人参化学成分的影响。方法：采用高效液相色谱法、苯酚-硫酸法及福林酚法分别测定糖水煎煮人参、单煮人参及人参生药中人参皂苷、水溶性糖和总酚等有效成分含量。结果：糖水煎煮后人参中稀有人参皂苷F1、F2、Rg3、化合物K含量及16 种皂苷单体加和值均显著增加;糖水煎煮人参中水溶性糖和总酚含量均显著高于人参生药组,且水溶性糖含量高于单煮人参组（除木糖醇组）,葡萄糖、麦芽糖、蔗糖、红糖、阿斯巴甜和硫酸软骨素组总酚含量显著高于单煮人参组（P＜0.05）。结论：糖水煎煮可提高人参中稀有皂苷、水溶性糖和总酚含量。     

老白干白酒中糖和糖醇类物质研究

该研究采用气相色谱-质谱（GC-MS）技术对衡水老白干中甜味突出酒样中的糖和糖醇类化合物进行检测。结果表明,该法对于检测酒样中糖和糖醇类化合物线性范围宽,达到了对酒样中糖和糖醇类化合物的分析要求,具有一定的准确性和精确性。共检测出11种物质,包括4种糖和7种糖醇类物质,并精确定量了其中的3种糖和6种糖醇类物质,其中葡萄糖（3.33 mg/L）和甘油（1.20 mg/L）含量较高,其他物质含量由高到低依次为果糖、木糖、阿拉伯醇、木糖醇、核糖醇、甘露糖醇、山梨糖醇。将老白干白酒与酱香型白酒中对应结果进行对比,发现由于两种香型白酒的制曲工艺以及核心发酵工艺不同,造成了发酵过程中的菌群有所不同,从而使得两者酒体中糖和糖醇类物质的含量有所差异。