Beneficial effects of dietary exogenous protease on the growth,intestinal health and immunity of GIFT (Oreochromis niloticus) fed plant‐based diets

This study investigated the effects of dietary exogenous protease on the growth performance, intestinal health, immune parameters and disease resistance of genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Five test diets with commercial protease at the levels of 0, 1.38, 2.76, 5.52 and 11.04 U/g (named PE0, PE1, PE2, PE5 and PE11, respectively) were administered to triplicate tanks with 30 fish for 60 days, and then, the fish were challenged with Streptococcus agalactiae for 14 days. The results indicated that weight gain increased as exogenous protease increased from 0 to 5.52 U protease/g diet and then decreased significantly (p < .05) with a further increase in exogenous protease supplementation (p < .05). The height of the villi in the proximal intestine and distal intestine, the width of the villi in three segments of the intestine, and the thickness of the muscle layer in the proximal intestine and mid‐intestine (p < .05) were increased in the fish fed the PE5 diet. Immune and antioxidant indices (except malondialdehyde), and survival after challenged with S. agalactiae were higher in fish fed PE5 diets than in those fed other diets (p < .05). In conclusion, 5.52 U/g protease supplementation in a plant‐based diet could promote the growth performance, intestinal physical barrier function, innate immunity and S. agalactiae resistance of GIFT.     

Effects of Manipulation of Proteases on Myofibril Protein Degradation of Tilapia Muscle in vitro and in vivo

This study investigated the role of three proteases in myofibril degradation. Tilapia muscle was soaked in MDL-28170, E-64, and AC-DEVD-CHO, specific inhibitors of calpain, cathepsin, and caspase, respectively, for 14 days to determine the in vivo degradation of myofibril. Desmin and troponin T were significantly inhibited after treatment with inhibitors compared to the control (p < .05). For in vitro analyses, myofibril was incubated with recombinant active μ-calpain, cathepsin B, and caspase-3. Desmin was significantly reduced when incubated with recombinant proteases (p < .05). However, troponin T showed significant degradation only in the μ-calpain and cathepsin B treatment groups (p < .05). Our results reveal μ-calpain and cathespin B have similar degradation patterns, while caspase-3 has partly similar degradation for myofibril in vivo and in vitro. This suggests that μ-calpain and cathespin B are the main proteases responsible for post-mortem myofibril degradation, while caspase-3 is minor.     

Cry1Ac毒素对小菜蛾幼虫中肠蛋白酶和羧酸酯酶活性的影响

为了明确Cry1Ac毒素对小菜蛾Plutella xylostella幼虫中肠蛋白酶和羧酸酯酶活性的影响,利用蛋白酶专性底物测定其中肠各蛋白酶活性在不同缓冲液中的最适pH,并用不同浓度Cry1Ac毒素饲喂小菜蛾幼虫后测定该毒素的致死中浓度LC_(50),观察中肠蛋白酶和羧酸酯酶活性的变化,分析Cry1Ac毒素与昆虫中肠酶活性间的关系。结果表明,小菜蛾幼虫中肠总蛋白酶在Tris-HCl、KH_2PO_4/NaOH和Glycine/NaOH三种缓冲液中最适pH均为11.0;强碱性胰蛋白酶的最适pH分别为10.0、10.5和10.5;弱碱性胰蛋白酶的最适pH均为8.5;胰凝乳蛋白酶的最适pH分别为8.5、9.0和9.0。Cry1Ac毒素对小菜蛾的致死中浓度LC_(50)为0.343μg/mL。用不同浓度Cry1Ac毒素饲喂小菜蛾幼虫后,其中肠各蛋白酶活性均随时间的变化而变化。弱碱性胰蛋白酶和胰凝乳蛋白酶的活性明显升高,且随浓度升高呈上升趋势;在取食36~72 h期间低浓度处理组的羧酸酯酶活性与对照组间无显著差异,而高浓度处理组羧酸酯酶活性显著高于对照组。表明Cry1Ac毒素能够显著影响小菜蛾的中肠蛋白酶和羧酸酯酶活性。     

不同蛋白能量比饲料对草鱼幼鱼消化酶活性的影响

以鱼粉和豆粕为蛋白源,鱼油和豆油等比例混合油为脂肪源,共配制4个蛋白水平(20%、25%、30%、35%),每一蛋白水平设4个脂肪水平(4.5%、8%、11.5%、15%),共16组饲料。每组设3个重复,在水温23~29℃下,连续投喂初始体重为(16.84±0.28)g的草鱼(Ctenopharyngodon idellus)10周,研究不同蛋白能量比饲料对草鱼幼鱼消化酶活性的影响。结果显示:肠道蛋白酶的活性随着饲料蛋白含量的升高而显著升高(P<0.05),饲料蛋白水平在20%~30%时,饲料中的脂肪水平对蛋白酶活性影响不显著(P>0.05),蛋白水平在35%时,高脂肪抑制了蛋白酶活性,肝胰脏的蛋白酶变化规律类似于肠道;肠道和肝胰脏淀粉酶活性在饲料蛋白含量相同的情况下,随着脂肪含量的升高呈现显著的下降趋势(P<0.05),但饲料中蛋白(30%,35%)和脂肪(8%,11.5%,15%)含量较高时,淀粉酶活性较稳定;肠道和肝胰脏的脂肪酶活性在饲料蛋白含量相同的情况下,随着脂肪含量的升高,呈显著上升的趋势(P<0.05),但在蛋白含量(30%,35%)和脂肪含量(8%、11.5%,15%)的6个试验组之间差异不显著(P>0.05);在脂肪含量分别为4.5%、8%和11.5%时,饲料的蛋白含量对肠道的脂肪酶活性无显著性影响(P>0.05),当脂肪含量为15%时,随着蛋白含量的升高,肠道的脂肪酶活性显著降低(P<0.05)。结果表明,草鱼饲料中蛋白含量为30%和脂肪为8%(即蛋白能量比21.7 mg/kJ)的试验组草鱼获得较高的消化酶活性。     

酶法水解牦牛血制备蛋白多肽粉的技术研究

摘 要：[目的][方法]利用中性蛋白酶将牦牛血红蛋白水解,探讨各因素对中性蛋白酶水解牦牛血红蛋白的影响以及水解度的关系,并对酶解液进行了活性炭脱色效果的研究。通过单因素和正交试验（L16(45)）,[结果]确定了中性蛋白酶水解血红蛋白的适宜条件为 pH 7.0,温度 45℃,酶底物浓度比 4000 U/g 蛋白质液,底物浓度 5%,酶解时间 7 h。通过正交试验,确定酶解液的最佳脱色工艺条件为活性炭用量 4%,脱色温度 75℃,pH 5.0,脱色时间 60 min。     

Effect of Setting Condition on the Gel-Forming Ability of Rohu

To investigate the setting condition of the gel-forming ability of rohu, optimum setting temperature for strong and weak gels of unwashed and washed rohu gel and optimum setting time for maximum proteolytic activity were investigated. Nine setting temperatures were studied for textural properties and trichloroacetic acid (TCA)-soluble peptide contents. Both unwashed gel (UW-gel) and washed gel (W-gel) showed similar optimum setting conditions for producing a strong gel that was set at 40°C for 30 min, followed by heating at 90°C for 20 min. They displayed different optimum setting conditions for weak gel. Weak gel from the degradation of UW-gel and W-gel formed at 65 and 60°C, respectively. The occurrence of protein degradation of W-gels during setting at 60°C suggested that washing did not remove the endogenous protease, and the degradation of unwashed and washed mince was due to water-soluble protease and myofibril-bound protease, respectively. Eight setting times for maximum proteolytic activity were shown by the TCA-soluble peptide contents, accompanying the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern. Both gels had similar results for setting for 120 min.     

Effects of dietary coated protease on growth performance,feed utilization,nutrient apparent digestibility,intestinal and hepatopancreas structure in juvenile Gibel carp (Carassius auratus gibelio)

This study was conducted to investigate the effects of dietary protease on growth performance, feed utilization, whole‐body proximate composition, nutrient digestibility, intestinal and hepatopancreas structure of juvenile Gibel carp, Carassius auratus gibelio (mean weight 8.08 ± 0.18 g). Six diets were prepared, including a positive control diet (dietary protein 350 g/kg, PC), one negative control diet (dietary protein 33 g/kg, NC) and four protease supplementations diets, which were 75, 150, 300 and 600 mg/kg protease NC diet. After 12 weeks of diet feeding in indoor recycle aquarium tanks, no significant difference (p > .05) was found on growth performance between fish fed diet with 75–600 mg/kg protease and the PC group. Compared with the fish fed the NC diet, the specific growth rate of fish fed 300 mg/kg protease increased significantly (p < .05), as well as protein efficiency ratios (p < .05), while feed conversion was the opposite (p < .05). The nutrient digestibility of crude protein and lipid was higher (p < .05) in fish fed 150 mg/kg protease diet than the PC diet. Whole‐body proximate composition of fish was not affected (p > .05) by the dietary treatment. Serum alkaline phosphatase and albumin were significantly affected by dietary protease (p < .05), while the content of total protein, glucose, triglyceride, total cholesterol, aspartate aminotransferase and alanine aminotransferase activities in serum was not affected (p > .05). Foregut muscular thickness was thinner (p < .05), when the fish fed diets supplementation of protease in 150 or 600 mg/kg diet than the NC diet. Protease activities in hepatopancreas and foregut were higher (p < .05), in the fish fed 150 or 300 mg/kg protease diet than the fish fed the PC diet, but those in the mid‐ and hindgut were not significantly affected (p > .05) by the dietary treatments. Based on the regression analysis of weight gain rate, the optimal dietary inclusion level of protease was 400 mg/kg in the diet for juvenile Carassius auratus gibelio.     

重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子

重寄生真菌盾壳霉Coniothyrium minitans是核盘菌Sclerotinia sclerotiorum的重要生防菌。为了探讨盾壳霉胞外蛋白酶在寄生核盘菌过程中的作用,采用明胶平板法对盾壳霉寄生核盘菌菌核产生的蛋白酶活性进行了检测,并进一步采用福林酚法定量测定蛋白酶活性,研究盾壳霉产生胞外蛋白酶的培养条件及影响蛋白酶活性的因子。试验结果表明,在被盾壳霉寄生的核盘菌菌核中检测到蛋白酶活性,表明蛋白酶可能参与盾壳霉重寄生作用。发现核盘菌菌核浸出液培养基适合盾壳霉产生胞外蛋白酶,摇培(20℃、200r/min)5d时蛋白酶活性最高,达到0.22U/mL。盾壳霉胞外蛋白酶酶促反应的最适温度为60℃,最适pH7.0。当温度不高于40℃时,蛋白酶酶活较稳定。5mmol/L的金属离子Mg²⁺、Zn²⁺、Ca²⁺、Cu²⁺、Mn²⁺、Li⁺和K⁺等对蛋白酶酶活没有显著影响(P>0.05),而Fe²⁺(5mmol/L)显著(P<0.05)提高了蛋白酶活性。盾壳霉蛋白酶对苯甲基磺酰氟(PMSF)敏感,说明盾壳霉产生的胞外蛋白酶可能主要是丝氨酸蛋白酶。这些结果为盾壳霉胞外蛋白酶的分离纯化和功能研究奠定了基础。     

玉米受伤诱导基因Wip1的启动子克隆及表达分析

【目的】植物蛋白酶抑制剂是抵抗动物摄食和病原侵染的重要防御蛋白。玉米Wip1(wound-induced protein)基因属于Bowman-Birk蛋白酶抑制剂(BBI)家族,了解Wip1(wound-induced protein)的启动子活性和Wip1组织表达特性、受胁迫诱导表达情况和在不同受伤时间下的表达模式,为抗虫基因在植物中应用提供新信息。【方法】以玉米叶片组织的cDNA为模板,采用克隆测序技术获得Wip1启动子序列和该基因的cDNA序列。将所得启动子与植物表达载体p1300连接,构建重组表达载体p1300-Wip1-GUS,并采用冻融法将其转入农杆菌LBA4404中。利用农杆菌介导的瞬时表达体系在玉米愈伤组织中对Wip1启动子的表达活性进行分析。利用热酚法提取玉米相应组织的总RNA,纯化后于含甲醛的变性胶中电泳分离,并将RNA转移到尼龙膜上,用[α-32P]dCTP标记探针的Northern blot技术对Wip1在玉米不同组织、不同胁迫条件、不同受伤时间下的表达模式进行分析。【结果】获得的启动子序列全长1 737 bp,cDNA全长512 bp。将含有重组质粒p1300-Wip1-GUS的农杆菌侵染玉米愈伤组织,3 d后,GUS染色显示侵染部位变蓝,说明所克隆的启动子在玉米愈伤中能启动GUS正常表达。基于Northern blot技术的植物组织表达结果显示,正常情况下,Wip1仅在胚芽鞘中有一定的表达,在受伤后的胚芽鞘、根、茎、叶、雌穗、雄穗、花丝及苞叶中均大量表达;在所设置的胁迫处理时间(2 h)内,Wip1不受缺氧、低温、脱水、PEG、ABA、NaCL和IAA胁迫诱导,仅受伤害胁迫诱导表达;Wip1在玉米叶片受伤胁迫下,玉米叶片受伤2 h时,检测到了Wip1表达,4—24 h时表达量迅速提高且处于持续增加趋势。【结论】玉米Wip1特异地受机械伤害诱导表达;Wip1启动子是一种有效的伤害诱导特异性启动子。