海洋来源生物活性肽的酶解工艺及其研究

小肽是动物降解蛋白质为氨基酸过程中的大部分中间产物,在水产动物饲料中添加小肽制品,可提高氨基酸利用率,减少疾病发生,充分发挥其生产性能,提高经济效益。研究制备的小肽是根据当地丰富的海洋渔业资源,利用海洋捕捞小杂鱼自身的内源酶系,酶解鱼肉蛋白而得到的。由于生化酶制备小肽成本较高,难以实现工业化大生产。试验通过对小杂鱼内源酶最佳酶解条件的研究,寻求低成本、高效益的小肽和氨基酸产品生产的最佳工艺条件。从试验结果得到主要工艺参数如下:CaCl2激活酶原的最适浓度0.03 mol/l;小肽产品生产的最佳工艺条件为:最佳酶解时间1 h,最适酶解温度55℃,最适酶解pH值7.0,最适固液比1:2;氨基酸产品生产的最佳工艺条件为:最佳酶解时间3.5 h,最适酶解温度55℃,最适酶解pH值6.5,最适固液比1: 2。     

胃蛋白酶、胰酶及酸度对植酸酶稳定性的影响

此文研究了大肠杆菌转基因植酸酶的最适pH值，不同酸碱环境下的稳定性及对胃蛋白酶、胰酶的耐受性等方面的性质。试验结果表明：大肠杆菌转基因植酸酶的最适pH值为4．5；在酸性环境下（pH3．0～5．5）具有良好的稳定性；对胃蛋白酶具有较强的耐受性，且优于对胰酶的耐受性。     

胰酶的制备与生物活性测定

胰酶是从动物胰脏中提取的一种混合酶制剂，含有胰蛋白酶、糜蛋白酶、肽酶、脂肪酶和淀粉酶等。呈白色或淡黄色无定形粉末，有特殊的肉臭味，有吸湿性，部分溶于水及低浓度的乙醇液中，不溶于高浓度的乙醇、丙酮和乙醚等有机溶剂中。水溶液pH2—3时稳定，pH6以上时不稳定，Ca^2＋可增加其稳定性，遇酸、热、重金属及鞣酸等蛋白质沉淀剂产生沉淀。     

中华根霉12~#产复合酶浸提条件及体外酶解饲料的研究

以麸皮和豆渣为培养基质,利用中华根霉12~#固态发酵生产饲用复合酶,对粗酶浸提条件进行了研究。以产酶种类和酶活力为指标,确定了提取溶剂为生理盐水,浸提时间为4 h,浸提温度为40℃,摇床转速150 r/min。以生成的还原糖量为指标,进行了复合酶降解豆粕粉和玉米粉的研究,结果表明,在40℃和pH值6.0条件下添加复合酶组的酶解还原糖得率均高于不加酶组,豆粕与复合酶液比例为14,酶解4 h时生成还原糖量最高;玉米粉与复合酶液比例为11,酶解6 h时生成还原糖量最高。试验结果表明,中华根霉12~#固态发酵生产的饲用复合酶可以用于降低饲料中的某些抗营养因子,改善饲料品质。     

不同厂家胰酶制备鸡胚成纤维细胞对比试验

本试验用半胚法制备鸡胚成纤维细胞,用胰酶对SPF鸡胚进行消化培养,并观察所得细胞的生长情况。在相同条件下设立两个对照组,观察胰酶在细胞制备过程中对试验的影响,并比较不同厂家(Gibco和Difco)生产的胰酶在相同条件下对鸡胚消化情况及所得细胞生长情况的影响。     

大豆虾粉肽适宜酶解好条件的研究

为了筛选生产大豆虾粉肽的适宜蛋白酶及酶解条件,试验采用单因素试验设计,以水解度为检测指标,按酶浓度、底物浓度、pH值、酶解温度和酶解时间逐一分别对3种微生物发酵产生的酸性蛋白酶、碱性蛋白酶和中性蛋白酶水解脱皮豆粕和虾粉混合蛋白的适宜酶解条件进行筛选.试验结果表明:酶解体系中的酶浓度、底物浓度、pH值、酶解温度和酶解时间均显著影响3种蛋白酶对脱皮豆粕和虾粉混合蛋白的酶解效果.在3种蛋白酶中,酸性蛋白酶的酶解效果最好,其次是碱性蛋白酶,中性蛋白酶的酶解效果最差.酸性蛋白酶水解脱皮豆粕和虾粉混合蛋白的适宜条件为:酶浓度为2000 U/g,底物浓度为9%,pH值3.5,酶解时间为3.5 h,温度50℃.     

壳聚糖纯化黄芩黄酮工艺研究

目的：确定壳聚糖对黄芩黄酮浸提液纯化的优化工艺条件。方法：通过正交试验设计考察壳聚糖添加量、料液温度和pH值对黄芩黄酮浸提液纯化效果。结论：壳聚糖对黄芩黄酮浸提液具有较好的纯化作用,最佳工艺条件为A3B2C2,即药液温度50℃、壳聚糖加入量为2%、pH值为5。     

角蛋白酶酶活测定条件的研究

以提高羽毛粉做底物测定角蛋白酶酶活方法的准确性,试验研究了酶的制备方式、测定波长、试剂添加顺序、底物粒度大小、反应体系振荡频率、底物浓度、酶促反应温度、时间和pH值等因素对酶活的影响。结果表明:传统测定方法中使用的底物量并不符合酶促反应动力学中底物要饱和的要求,试剂添加顺序和底物粒度对酶活测定结果也有很大影响;羽毛粉测定角蛋白酶活性的最佳方法为:40 W超声波处理30 s的发酵液直接离心制取粗酶液,以45 mg过40目筛的羽毛粉做底物,按缓冲液(pH值8.8)、底物、酶的顺序加入试剂,于45℃水浴中反应,每隔15 min振荡一次,反应1 h后加入2 ml10%的终止剂TCA,于波长280 nm处进行测定。优化后条件下测得的角蛋白酶活性较优化前提高了约2.1倍。     

酶解法制备饲用羽毛肽粉

本研究以普通市售羽毛粉为原料,通过正交试验设计,分别研究胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶降解制备饲用羽毛肽粉的条件。综合分析实验结果,建立了3种混合酶降解制备饲用羽毛肽粉的最优工艺:在温度55℃,pH 8,混合酶浓度1/50 g/mL条件下反应24 h,终产品中小分子蛋白含量较无酶组约提高了200%。     

复配酶解法提取鸭硫酸软骨素工艺研究

为了探索硫酸软骨素的新型提取资源和更加环保的提取技术,试验以42日龄肉鸭胸软骨为研究对象,研究优化了复配酶解法提取鸭硫酸软骨素的工艺。试验采用单因素和响应面法,优化了鸭胸软骨除杂处理和酶法提取鸭硫酸软骨素的工艺。结果表明:采用甲醇、氯仿(V/V=2∶1)和1 mol/L NaCl溶液处理鸭胸软骨表面的油脂和杂蛋白效果最好;鸭胸软骨酶解液中杂蛋白的最佳沉降剂为9%TCA溶液;复合蛋白酶和胰蛋白酶复配提取硫酸软骨素效果最佳,配比为1∶0.5;鸭胸软骨粉复合酶解的最佳条件为:酶解温度48.53℃,酶解时间5.90 h,酶解pH 6.45。在该条件下,鸭硫酸软骨素的提取率达到46.02%,纯度达到96.39%。     

响应面法优化醇法提取黑豆种皮中花色苷工艺

试验采用乙醇浸提法提取黑豆花色苷,主要考察提取液浓度、浸提时间、料液比、温度和提取液p H值等因素对黑豆花色苷提取量的影响。并利用单因素试验和响应面分析法优化黑豆花色苷的提取工艺,经过优化的试验参数为pH值2.0,乙醇浓度50%,提取时间57 min,提取温度55.5℃,料液比1:9。经过3次平行试验得出的实际值为385.72 mg/100 g,与预测值386.38 mg/100 g基本吻合,说明该试验模型具有可行性。黑豆花色苷具有一定的抗氧化性,对DPPH自由基和超氧阴离子具有其较强的清除能力,其清除率分别为92.56%和85.76%。     

Purification of myeloperoxidase from equine polymorphonuclear leucocytes.

Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5).     

食盐助溶剂对解脂假丝酵母神经酰胺含量的影响

用食盐作为助溶剂采用细胞自溶法,对解脂假丝酵母33M candida lipolytica细胞壁进行破碎后提取神经酰胺,用SPE（固相萃取法）将神经酰胺与膜中其它脂类分离,分别应用TLC（薄层色谱）方法和HPLC-ELSD（高效液相色谱-蒸发光散射器）对其进行定性和定量分析,取得了良好效果。     

rBmTI-6, a Kunitz-BPTI domain protease inhibitor from the tick Boophilus microplus, its cloning, expression and biochemical characterization

Boophilus microplus is a rich source of trypsin inhibitors, numerous Kunitz-BPTI (bovine pancreatic trypsin inhibitor) inhibitors have been described from larvae and eggs, named BmTIs. Among them, were characterized inhibitors for trypsin, human neutrophil elastase, human plasma kallikrein and plasmin. BmTIs elicited a protective immunological response against B. microplus infestation in cattle. However, only a small amount of purified natural BmTIs can be obtained from larvae and eggs by chromatographic methods, thus if BmTIs are to be used as vaccine antigens (immunogens) the production of recombinant BmTIs (rBmTIs) is essential. In this work we describe the cloning, expression, purification and characterization of rBmTI-6. rBmTI-6 is a three-headed Kunitz-BPTI inhibitor, expressed in the Pichia pastoris system. Although rBmTI-6 was processed by proteases and glycosylated during the expression process, these post-translational modifications did not alter the ability of rBmTI-6 to inhibit protease activity. Purified rBmTI-6 inhibited trypsin and plasmin.     

不同处理条件对苜蓿叶蛋白凝聚效果的研究

以现蕾期新鲜苜蓿Medicago sativa为材料,采用温度、pH值单因素处理及温度、加热时间、pH值和NaCl质量分数多因素处理,探讨不同方法对苜蓿叶蛋白凝聚效果的影响。结果表明,在单因素条件下,温度以75℃为宜,pH值4.0对苜蓿叶蛋白产量、叶蛋白纯度和粗蛋白提取率有显著影响。而在多因素条件下,叶蛋白的最适凝聚组合条件为温度85℃,时间2min,pH值4.0,以及NaCl质量分数0.4%。     

绿叶汁发酵液为添加剂改善玉米青贮品质的研究

本研究以饲料玉米(Zea mays)为原料,采用绿叶汁发酵液1.0%、1.5%、2.0%作青贮添加剂,并与传统青贮工艺添加1%、3% NaCl做比较,在实验室条件下贮藏60 d。测定其青贮品质(包括微生物群落结构、pH值、粗蛋白、干物质、纤维素含量、有机酸含量)及体外消化率,并与传统青贮工艺做比较。结果显示,添加绿叶汁发酵液可以明显改善青贮品质,pH值显著降低,干物质、粗蛋白及有机酸含量均有显著性增加(P<0.05)。经体外消化试验可知,与对照相比,添加绿叶汁发酵液可显著提高青贮饲料消化率。     

遗传算法优化超声辅助低共熔溶剂提取红枣总黄酮工艺研究

本文采用一种新型绿色溶剂提取红枣总黄酮,并对其提取工艺进行优化。通过单因素试验探究含水量、超声功率、提取时间、提取温度和料液比对红枣总黄酮得率的影响。在此基础上,采用遗传算法优化超声辅助低共熔溶剂提取红枣总黄酮工艺。结果表明：超声辅助低共熔溶剂提取红枣总黄酮最优的工艺参数为：含水量37%、超声功率167 W、提取时间30 min、提取温度54 ℃和料液比1∶26（g/mL）。在此条件下,所得红枣总黄酮得率为（29.33±0.37）mg/g。试验值和理论值的相对误差为1.24%。表明遗传算法可较好地模拟和预测不同提取条件下红枣总黄酮得率,且优化工艺参数是可行的。研究发现低共熔溶剂可作为一种新型、绿色溶剂用于高效提取红枣总黄酮。[关键词] 遗传算法|红枣|总黄酮|低共熔溶剂|工艺     

苦参碱的提取纯化工艺研究

为了优化苦参碱的提取工艺,以浸膏收率、苦参碱含量为评价指标,选择用水量、提取时间及提取次数为考察因素,利用L9（3^4）正交试验法优选出水煎煮法提取苦参碱的最佳工艺条件为用8倍量水提取3次,每次提取2h。采用乙醇沉淀及三氯甲烷萃取相结合的方法对苦参粗提物进行纯化,浓缩药液浓度为1．0g生药／mL时进行醇沉,醇沉后调节药液的pH值至9～11用三氯甲烷萃取3次。优选出的提取工艺稳定、可行,可用于苦参碱的工业化提取。     

响应面法优化产业化生产披萨干酪工艺参数

采用四因素五水平二次回归正交旋转组合试验设计,研究产业化生产披萨干酪的最佳优化工艺条件,选择发酵时间、凝乳时间、排乳清pH值以及NaCl添加量进行单因素试验,并验证回归模型的显著性.结果表明,产业化生产披萨干酪的最佳工艺条件为发酵时间27.5min、凝乳时间37.5min、排乳清pH6.3、NaC1添加量2.07％.此时最大得率24.78％.此时所制得的披萨干酪有良好的干酪味,质地紧密、光滑、富有弹性、颜色均匀,拉丝长度、融化性、起泡性好,与实验室制得的各指标相差不大,能满足披萨质量标准要求,适于大批量的生产,能够实现产品产业化、工业化.     

Effect of pH and ionic strength on mu- and m-calpain inhibition by calpastatin

The objectives of this study were to determine the extent to which pH and ionic strength influence mu- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, mu-calpain, and m-calpain were purified from at-death porcine semimembranosus. Mu-calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 microM (mu-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased mu-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of mu-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of mu-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on mu-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.