培养基组成对贝莱斯芽孢杆菌产抑真菌成分的影响

贝莱斯芽孢杆菌能够同时产生蛋白类和脂肽类等多种抑真菌的脂肽类物质,产物中各组分的具体组成和含量与培养基组成紧密相关。为获得最有利于主要抑菌成分产生的最佳条件,以炭黑曲霉为指示菌,考察了不同培养基组成和培养时间对贝莱斯芽孢杆菌发酵液抑菌活性和主要抑菌成分的影响。结果表明,培养基组成对抑菌成分的总产量及组分构成有显著影响:Landy培养基更利于菌体生长,但营养肉汤培养基(NB)则更有利于抑菌成分的合成;牛肉膏有利于抑菌成分的产生与积累,而蛋白胨则相反。同时,菌体在Landy培养基中所产抑菌物质主要是蛋白类,而在NB培养基中主要为脂肽类。经过优化组合,确定出最有利于总体抑菌物质合成的培养基组成为:葡萄糖20 g/L,NaCl 5 g/L,牛肉膏15 g/L,谷氨酸钠5 g/L,KH2PO420 g/L,MgSO4·7H2O 10 g/L,KCl 10 g/L,FeSO43 mg/L,MnSO4100 mg/L,CuSO 43.2 mg/L。此条件下的菌体生长量比Landy培养基提高了75.9%,发酵液的抑菌活性比用NB培养基提高了21.9%,而且脂肽类抑菌成分比例较高。     

凝结芽孢杆菌13002高密度培养

本实验旨在通过优化培养条件提高凝结芽孢杆菌13002发酵培养液中的菌体浓度,为生产高光学纯度L-乳酸奠定基础。通过单因素实验,最陡爬坡实验,Box-Behnken响应面实验确定凝结芽孢杆菌13002的最优培养基配方和最佳培养条件,并于自动发酵罐中进行高密度分批补料培养。结果表明,最优培养基配方为:葡萄糖20 g/L、细菌学蛋白胨10 g/L、酵母提取粉25 g/L、Na Cl 10 g/L,初始p H6.5。在此优化条件下,以6%的接种量,培养温度45℃,培养至16 h,以0.5 g/(L·h)速率添加补料至结束,最大菌体浓度达5.399 g/L,活菌总数为3.095×10~9CFU/m L,最终冻干后达0.730×10~(11)CFU/g。     

烟草粉螟高毒力Bt菌株的发酵条件

对烟草粉螟有较高毒力的苏云金杆菌Bt33、Bt53、BtHB3株菌株的发酵培养条件进行了试验。结果表明:3株菌株的最佳培养温度为30℃、pH值为7.5、转速为180rpm,Bt33、Bt53的最佳培养时间为40h,BtHB为36h,BtHB培养的最佳空气量为19/20[(培养容器容积-培养基体积)/培养容器容积],Bt33、Bt53均为9/10。     

酵母菌产酒精能力的葡萄糖消耗检测法

研究了1种检测酵母菌产酒精能力的新方法。利用酒精发酵液体培养基在厌氧条件下对产酒精酵母菌株YE-1、YE-2、YE-3进行酒精发酵培养,同时利用菌体发酵培养基在有氧条件下进行菌体培养实验作为对照。通过分光光度法检测培养基中葡萄糖的消耗量,根据所消耗的葡萄糖中用于酒精发酵的量,推算出3株酵母菌菌株在酒精发酵培养基中酒精含量分别为4.03 mg/mL、3.50 mg/mL和3.77 mg/mL,进而比较出各菌株产酒精能力的大小。     

响应面法优化胶质芽孢杆菌GM1增殖发酵培养基

在摇瓶培养条件下,优化提高胶质芽孢杆菌GM1菌体浓度的发酵培养基组分。在单因素的基础上选择糖蜜、复合氮源(硫酸氨与豆粕)以及初始pH为自变量,芽孢杆菌数为响应值,根据Box-Behnken实验设计原理以及Design Expert 8.0软件进行回归分析,确定了胶质芽孢杆菌GM1发酵培养基的最佳组成为:糖蜜3.2%、复合氮源0.4%,pH为8.0,在此条件下最大理论数值为3.03×108CFU.mL-1。经过3次平行实验验证,实际活菌体数为3.09×108CFU.mL-1,该实验值与预测值拟合较好。优化后的活菌浓度与优化前菌体浓度相比提高了40%,由此可知,响应面分析法优化微生物发酵条件是一种十分有效的方法。     

巴斯德芽孢杆菌培养基及培养条件的优化

通过单因素和正交实验对巴斯德芽孢杆菌(Bacillus pasteurii)培养基组分及培养条件进行研究。结果表明,最适培养基配方(g/L)为甘露醇40、大豆蛋白胨25、氯化铵3、氯化钠10,pH为8.0;最适培养条件为温度30℃,摇床转速200r/min,接种量4.0%(V/V),装瓶量75mL/250mL;在优化条件下,菌体培养24h达到生长高峰期,活菌数达9.52×109cfu·mL-1,分别是普通LB培养基和牛肉膏蛋白胨培养基的1.71、1.55倍。     

IGF—I工程菌的高密度发酵与工艺优化

为研究表达重组IGF—I（人胰岛素样生长因子-I）工程菌的优化发酵工艺条件,考察了3种不同培养基以及诱导时机、诱导剂量和诱导时间对蛋白表达的影响,用5L自控发酵罐进行分批补料培养实验,确定高密度发酵工艺条件。结果表明,以2×YT＋0．5％葡萄糖为发酵培养基,经0．8mmol/LIPTG诱导5h,通过控制溶解氧以及pH反馈补料方式,发酵液最终菌体密度达到OD650 21．7（菌体50．1g／L）,目的蛋白表达量约占菌体总蛋白的30％。建立了该工程菌优化的高密度发酵工艺,为IGF—I的下游纯化和工业化生产奠定了基础。     

玉米赤霉烯酮降解菌ASAGW-10产芽孢条件研究

通过培养基筛选、单因素实验、正交实验获得芽孢率和芽孢量较高的培养基,优化发酵pH和温度,获得最优产芽孢培养条件,在20 L发酵罐中对发酵培养基和发酵条件进行验证,并对发酵pH的控制和补料发酵进行初步探索。获得的最佳培养基为:麸皮5 g/L、玉米粉3 g/L、NaCl 5g/L、KH_2PO_41 g/L、MnSO_4·H_2O 0.2 g/L、K_2HPO_41.5 g/L,在pH 6.0、37℃、200 r/min条件下震荡培养21 h,芽孢率达到91%,芽孢量达到5.2×10~(10)CFU/mL。在20 L发酵罐放大实验中通过恒定pH和批次补料发酵,芽孢率达到95%,芽孢量达到1.38×10~(11)CFU/mL。获得了玉米赤霉烯酮降解菌ASAGW-10菌剂的生产工艺,为后续开展降解菌性能和应用研究提供数据支撑。     

酿酒酵母超氧化物歧化酶的分离纯化研究

酿酒酵母CNU94经发酵培养后,离心收集菌体,将菌体用甲苯法破壁得到的粗酶液调节pH除杂蛋白,丙酮二次沉淀后,用DEAE32纤维素柱层析梯度洗脱,得到纯化的SOD,其比活为3500u/mg,收率为58.3%。PAGE电泳后的活性染色显示,酵母超氧化物歧化酶具4条明显的同功酶。该样品经KCN,H2O2,CHCl3乙醇抑制试验,酶类型为Cu/ZnSOD。     

不同金属离子对丁酸梭状芽孢杆菌Z-10菌体及芽孢的影响

研究了Ca2+、Mg2+、Mn2+、Zn2+、Fe2+和Cu2+6种金属离子对丁酸梭状芽孢杆菌Z-10菌体生长及芽孢形成的影响.实验结果表明,Ca2+、Mg2+、Mn2+3种离子对丁酸梭状芽孢杆菌芽孢的形成具有显著的促进作用,其中Mn2+对菌体生长的促进作用也非常显著;Fe2+可以一定程度的提高菌体的浓度,但对芽孢形成的促进作用不明显;低浓度的Cu2+对芽孢形成及菌体生长具有一定的促进作用,但浓度略高时即表现出更为显著的抑制作用;而Zn2+对丁酸梭状芽孢杆菌的菌体生长和芽孢形成都表现出明显的抑制作用.本实验最终确定在培养基中添加对菌体生长及芽孢形成均有促进作用的金属盐种类及浓度分剐为:MnSO4·H2O 1.0g/L、MgSO4·7H200.4g/L、CaCl2 0.8s/L、FeSO4·7H2O0.4g/L.     

超声协同等电点沉淀法提取螺旋藻藻胆蛋白工艺的优化

以螺旋藻粉为原料,通过正交试验,对超声波细胞破碎及等电点沉淀提取藻胆蛋白进行研究,并优化其工艺条件。结果表明,在藻液质量分数5%、630W(额定功率为900W)条件下超声20min,效果最佳;而采用冰醋酸作为等电点沉淀介质,在pH4.0 的条件下,从提取液中低温沉淀藻蛋白,其操作较为简便。由于冰醋酸溶液易挥发分离,简化了生产工艺。最后通过冷冻干燥,得到粗蛋白粉末。利用本法优化工艺后,提取液中藻蓝蛋白的含量高达4.36%;而经沉淀干燥得到的粗蛋白粉末得率为52.5%,其中藻蓝蛋白含量为7.7%、别藻蓝蛋白含量为7.9%、藻红蛋白含量为0.8%。     

高速剪切对大豆分离蛋白限制性酶修饰的影响

研究高速剪切预处理对大豆分离蛋白限制性酶修饰制备等电点处高分散性蛋白的影响。以酶修饰产物的水解度、分散性为指标进行综合评价,揭示水解度与分散性的关系。结果表明,以常规酶修饰为对照,高速剪切处理能有效地促进大豆分离蛋白的酶修饰,在0.5～2.0h 范围内,随水解时间的延长,水解度逐渐增大,分散性也随之增加。该处理方式的最适参数为底物质量浓度100g/L、剪切速率6000r/min、剪切时间4min,该条件下酶修饰物的水解度从1.29% 提高到4.16%,等电点处分散性由20.62% 提高到46.82%,分别提高了3.22 倍和2.27 倍(P ＜ 0.05),并将改性时间由4.5h 缩短至2.0h。     

制革中的电化学应用初探

在无膜、阳离子膜和阴离子膜的电解槽中,通85mA的直流电1h后,探讨直流电对酸皮等电点、酸皮的湿热稳定性、酸皮的铬吸收性、蓝皮的染料和加脂剂吸收性的影响。研究发现,在无膜和阳离子膜时,酸皮的等电点有所降低,有阴离子膜时,等电点上升;阴离子膜正极区,酸皮的耐湿热稳定性提高得最多;阳离子膜正极区,酸皮对铬的吸收最高;阳离子膜负极区染料的吸收率最高;无膜电解时,坯革对磺化油的吸收最好。     

响应面法优化玉米浸液蛋白提取工艺及单细胞蛋白发酵的研究

探索了用等电点法提取玉米浸液中蛋白质的方法,并利用提取粗蛋白后的上清液生产单细胞蛋白。利用单因素实验分析了pH、温度、时间对蛋白提取的影响,通过响应面法确定等电点提取蛋白的最佳工艺,并研究了用酿酒酵母、产朊假丝酵母发酵离心上清液生产单细胞蛋白的条件。结果表明:等电点法提取粗蛋白的优化条件为:pH7.6、提取温度26.4 ℃、提取时间20 min,提取率为77.5%。以提取粗蛋白后的上清液生产单细胞蛋白,产朊假丝酵母优于酿酒酵母,当上清液中干物质浓度为8%、pH为5.5时,产朊假丝酵母发酵得到的菌落数达到1.6×109 CFU/mL。     

不同等电点沉淀法和超速离心法提取牛奶乳清蛋白的双向电泳分析

为探索牛奶乳清蛋白提取的最适方法,以生鲜荷斯坦牛奶为对象,采用不同等电点（pH 4.6和pH 4.8）沉淀法和超速离心法提取牛奶乳清蛋白样品,并对提取效果进行双向凝胶电泳（two-dimensional electrophoresis,2-DE）图谱分析。依据凝胶图谱上蛋白斑点比较图谱质量,采用PDQuest 8.0凝胶图像分析软件进行凝胶图谱分析。结果显示：2 种方法均能有效提取牛奶乳清蛋白,且获得的2-DE凝胶图谱背景清晰、蛋白点个体独立、无明显的拖尾现象,且重复性强,但都存在一定的酪蛋白残留。与超速离心法相比,pH 4.6沉淀法和pH 4.8沉淀法提取乳清蛋白的2-DE凝胶图谱可检测到较多的蛋白质斑点。pH 4.6沉淀法提取乳清蛋白制备的2-DE凝胶图谱中蛋白点的表达丰度略高于pH 4.8沉淀法。研究表明：pH 4.6沉淀法提取乳清蛋白制备2-DE凝胶图谱略优于pH 4.8沉淀法和超速离心法。但2 种等电点沉淀法和超速离心法都可有效去除牛奶中的高丰度酪蛋白,提高低丰度蛋白的检出敏感性。     

The acylated protein derivatives of Canavalia ensiformis (jack bean): A study of functional characteristics

Protein concentrate was prepared from jack bean (JNP) and it was modified by acylation using acetic (JAP) and succinic anhydrides (JSP). Proximate analyses revealed that moisture and ash content increased following acetylation and succinylation, while both acetylation and succinylation reduced percentage crude fat and protein. Acetylation and succinylation reduced protein solubility in the acidic pH range below the isoelectric point (4.5) of the protein concentrate but improved the solubility of the unmodified protein concentrate at the isoelectric point and pH range alkaline to the isoelectric point. Both acetylation and succinylation increased the water absorption capacity of unmodified protein concentrates at all levels of ionic strength investigated (0.1-1.0 mol/l). Acetylation improved oil absorption capacity whereas the tendency to absorb oil reduced after succinylation. Maximal emulsifying activity of native and modified proteins were obtained at pH 10. Emulsion stability of acylated proteins was higher than those of native proteins in the range of pH 4-10 but lower when the pH was 2. Foam capacity and stability of both native and modified proteins increased with increase in protein concentration. Foam capacity of modified proteins increased progressively with increase in pH from 2 to 10. Also, acylated protein derivatives had improved foam capacity over the native protein except at pH 2. Gelation capacity of both native and modified proteins was maximal at the region of isoelectric point.     

Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 1. Forces driving adsorption

Genetically modified Bt crops express insecticidal Cry proteins (Bt toxins) that may enter agricultural soils. A mechanistic understanding of Cry protein adsorption to soils is critical for risk assessment, as this process governs Cry protein fate and bioavailability. We used quartz crystal microbalance and optical waveguide lightmode spectroscopy to elucidate the driving forces of the adsorption of monomeric Cry1Ab to negatively charged quartz (SiO(2)) and positively charged poly-L-lysine (PLL) at pH 5-8 and constant ionic strength of 50 mM (NaCl). Bovine serum albumin and hen egg white lysozyme were used as reference proteins because of their known adsorption behavior. Electrostatics governed Cry1Ab adsorption; as pH increased above the isoelectric point of Cry1Ab, the initial rate and the extent of adsorption decreased on SiO(2) and increased on PLL. Reversible adsorption to SiO(2) suggested weak Cry1Ab-SiO(2) electrostatic interactions and no irreversible conformational changes of Cry1Ab at the surface. High conformational stability of Cry1Ab was further supported by supply rate-independent extent of adsorption of Cry1Ab to apolar gold. Some evidence is presented that the nonuniform surface charge distribution of Cry1Ab resulted in patch-controlled electrostatic attraction with sorbents that carried the same net charge as Cry1Ab. A more detailed discussion of this mechanism is given in a companion paper.     

Purification and characterization of extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas sp. RY-1

A novel bacterial strain capable of growing in a medium containing a medium-chain-length polyhydroxyalkanoate (MCL-PHA) as the sole carbon source was isolated from a soil sample. The isolate, which was identified as Pseudomonas sp. RY-1, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated in a medium containing a MCL-PHA, such as polyhydroxyoctanoate (PHO) or polyhydroxynonanoate (PHN). The extracellular MCL-PHA depolymerase of this organism was purified to electrophoretic homogeneity. The enzyme was a tetramer with identical subunits and a total molecular mass of 115 kDa. The isoelectric point of this enzyme was estimated to be 5.9 by isoelectric focusing. The maximal activity was observed at pH 8.5 and 35 degrees C. The enzyme was insensitive to phenylmethylsulfonyl fluoride and dithiothreitol, unlike other short-chain-length (SCL) PHA depolymerases. The K(m) values for PHO and PHN were 0.86 and 1.47 mg/ml, respectively. The enzyme could not hydrolyze SCL-PHAs and p-nitrophenyl esters of fatty acids.     

Functional properties of raw and heat processed cashew nut (Anacardium occidentale, L.) kernel protein isolates

The functional properties viz. solubility, water and oil absorption, emulsifying and foaming capacities of the protein isolates prepared from raw and heat processed cashew nut kernels were evaluated. Protein solubility vs. pH profile showed the isoelectric point at pH 5 for both isolates. The isolate prepared from raw cashew nuts showed superior solubility at and above isoelectric point pH. The water and oil absorption capacities of the proteins were slightly improved by heat treatment of cashew nut kernels. The emulsifying capacity of the isolates showed solubility dependent behavior and was better for raw cashew nut protein isolate at pH 5 and above. However, heat treated cashew nut protein isolate presented better foaming capacity at pH 7 and 8 but both isolates showed extremely low foam stability as compared to that of egg albumin.     

THE ISOELECTRIC FOCUSING OF BEER PROTEINS IN POLYACRYLAMIDE GEL

A technique has been developed for fractionating beer proteins by isoelectric focusing in polyacrylamide gel which enables protein species with isoelectric points differing by as little as 0·1 pH unit to be separated as clearly defined bands. The isoelectric profile of an all-malt beer was found to be similar to that of the parent wort but significant changes occurred when a beer was stabilized in the laboratory by three different treatments.