文蛤肉酶法水解条件的优化及最适蛋白酶的选择

以文蛤肉为原料,探讨了胰蛋白酶的加酶量、水解时间、水解温度、pH值及液固比对文蛤肉水解液总氨基氮含量和水解得率的影响规律,确定文蛤肉酶解的最优条件为酶解温度50℃,加酶量4000U/g原料,pH值为（8．00±0．05）,水解时间5h,液固比3：1。再以文蛤肉水解液的水解度、水解得率及风味评分值为指标对该酶解优化条件下获得的产品与精制中性蛋白酶水解优化条件下获得的产品进行比较。结果表明,胰蛋白酶为酶法水解文蛤肉最适宜的蛋白酶,其酶解所得文蛤肉水解液的水解度、水解得率及风味评分值分别为42．44％、82．86％及205．92。     

致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析

为进一步明确引起马铃薯晚疫病的致病因子,通过克隆马铃薯晚疫病致病疫霉菌分泌蛋白中的半胱氨酸蛋白酶家族基因,构建该基因的原核表达载体,在大肠杆菌BL21中诱导表达。本试验采用TA克隆法,提取来自云南马铃薯产区的致病疫霉菌XH05-5-4的总RNA,反转录cDNA,PCR扩增后将该基因片段连接到载体,转化大肠杆菌BL21并诱导表达。克隆获得半胱氨酸蛋白酶家族基因全长为1176 bp,包含1个最大的开放阅读框1077 bp,编码387个氨基酸。推测蛋白相对分子量41.671 kD,理论等电点4.88;经IPTG诱导和SDS-PAGE检测,所构建的原核表达载体表达的蛋白与预期蛋白相符合。生物活性测定表明：马铃薯叶片出现坏死,类似于晚疫病症状。利用TA克隆技术从致病疫霉菌XH05-5-4中克隆得到了1个半胱氨酸蛋白酶家族基因（登录号：KP938956命名：C1A-PH-SCH1）,通过同源性比对发现C1A-PH-SCH1为木瓜蛋白酶亚家族基因,成功构建了原核表达载体,并使其在大肠杆菌中得到了表达,对生物活性测定结果显示出现了类似于晚疫病症状的水渍坏死斑症状,推测C1A-PH-SCH1参与马铃薯晚疫病的发病过程。     

芝麻种子中内源性蛋白酶的热稳定性及其运用

近年来,芝麻的深加工偏重于制油,而忽略了对芝麻蛋白的高值利用。为了实现芝麻蛋白与油脂的高值综合利用,该研究将湿法脱皮芝麻进行干燥,经压榨制取芝麻油和压榨粕后,再利用内源性蛋白酶对芝麻压榨粕中的蛋白质进行水解,制取蛋白水解物。在此过程中,考察了干燥温度对湿法脱皮芝麻压榨出油率和内源性蛋白酶活性的影响。结果显示,在80～180 ℃干燥1 h时,脱皮芝麻出油率上升至88.55%～90.98%（带皮芝麻为81.76%）;对于内源性蛋白酶而言,在130 ℃时,仍可保留约57%的活性,表明芝麻种子中内源性蛋白酶具有良好的热稳定性。同时,通过将低变性芝麻粕（干燥条件为50 ℃、4 h）制浆,在pH 值为4.5、50 ℃下孵育6 h,经3 000 g离心5 min后,分离得到透明清液（蛋白水解物）,其清液中蛋白质的分布率为66%,且主要由38%小肽（< 1500 Da）和37%游离氨基酸组成,除此之外,还存在多种生物活性物质。该研究为芝麻内源性蛋白酶的实际应用以及芝麻蛋白新产品的开发提供了一种新思路。     

生姜蛋白酶和生姜汁对猪肉嫩化效果的比较

本文研究了生姜蛋白酶及生姜汁对猪肉的嫩化效果，对其用量、pH值、预处理温度进行了测试对比。结果表明：生姜蛋白酶及生姜汁对猪肉的嫩化效果十分显著(P≤O．01)，生姜蛋白酶最佳用量为O.01％，姜汁最佳用量5％，最佳pH都为7.O，最佳预处理温度生姜蛋白酶为300℃，生姜汁为40℃；并且生姜蛋白酶及生姜汁对猪肉的嫩化效果极为相似，可以证明，生姜汁对肉类的嫩化作用就是生姜汁中的生姜蛋白酶作用的结果。     

Effect of phytase supplementation of a plant‐based diet on phosphorus and nitrogen bioavailability in sea bream Sparus aurata

To assess to what extent addition of phytase to a plant‐based diet results in spatio‐temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant‐based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant‐based diets used in fish aquaculture.     

Characterization of digestive enzymes protease and alpha‐amylase activities in the thick‐lipped grey mullet (Chelon labrosus,Risso 1827)

Activities of pepsin, trypsin, chymotrypsin, alpha‐amylase and lipase, as well as their optima and stability to pH and temperature, were determined in digestive extracts of thick‐lipped grey mullet, Chelon labrosus, of three different sizes: Group 1 (45.2 ± 3.0 g), Group 2 (180.9 ± 4.2 g), and Group 3 (328.5 ± 43.3 g). SDS‐PAGE zymograms were also used to assess the role of serine proteases in the digestive tract of C. labrosus. On the other hand, possible changes in the digestive enzyme profile of C. labrosus during development were observed, with a comparatively lower pepsin activity, higher activities of alkaline proteases and alpha‐amylase and no lipase activity recorded in pre‐adult specimens. It is suggested that these variations are linked to the changes in diet composition with age, moving from a partly carnivorous to a more herbivorous feeding habit.     

Some properties of the intestinal proteases of the rabbitfish,Siganus canaliculatus (Park)

Some properties of the intestinal proteases of the rabbitfish were examined. At 25°C, both trypsin and chymotrypsin showed pH optima of 8.0. Leucine aminopeptidase, however, displayed maximum activity in the pH range, 7.0–9.0. Leucine aminopeptidase had the highest optimum temperature (60°C), and chymotrypsin, the lowest (30°C). The optimum temperature of trypsin was 55°C. The activation energy, E_a, was found to be 8.24 for trypsin and 8.50 kcal mol^–1 for chymotrypsin. The E_a for leucine aminopeptidase was 6.29 kcal mol^–1 above 40°C and 1.73 kcal mol^–1 below 40°C. Substrate concentration-velocity plots showed that all three enzymes followed Michaelis-Menten kinetics; the K_m and V_max were estimated for the three enzymes. The effects of various protease inhibitors on enzyme activity were also examined and confirmed the protease classes to which each enzyme belonged. The three proteases examined have similar properties to proteases in other fishes.     

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.     

Effect of plant protease inhibitors on digestive proteases in two fish species, Lutjanus argentiventris and L. novemfasciatus

This work provides a comparative study of the inhibitory effect of several plant protein sources on digestive proteases of two snappers: yellow snapper (Lutjanus argentiventris) and dog snapper (Lutjanus novemfasciatus). Seed extracts did not affect gastric proteases whereas they significantly inhibit intestinal proteases. Inhibition of alkaline proteases showed that pancreatic proteases of L. argentiventris were more sensitive to seed protease inhibitors than those of L. novemfasciatus. Legume seeds showed the highest inhibitory capacity on alkaline proteases causing inhibition higher than 50% in total proteolytic activity. Protease inhibition on digestive extracts was assessed using different relative concentration of seed extracts and represented by constructing dose response curves. In order to reduce the inhibitory effect, seed extracts were acid-treated before the inhibition assay. Results showed that acid treatment did not affect the inhibitory capacity of seeds on alkaline proteases in both species. However, when the action of gastric enzymes was simulated on seed extracts, the inhibitory capacity was reduced significantly, mainly in the case of L. novemfasciatus. The responses of fish enzymes to heat-treated seed extracts were also tested. Only higher temperatures were capable of reducing the inhibitory capacity of seed, with the specific response to the snapper species. The use of biochemical assays allows us to quantify the action of inhibitors on total proteolytic activity. In addition, zymograms obtained by substrate-SDS-PAGE provided qualitative information about the number and type of proteases affected by each inhibitor. Each seed extract produces a characteristic profile of inhibition on alkaline protease. The results obtained are important for future formulation of feeds for these snapper species.     

利用大西洋鲑内源酶酶解加工废弃物制取抗氧化肽的研究

大西洋鲑(Salmo salar)加工废弃物中含有丰富的蛋白质,具有较高的开发利用价值,直接遗弃会造成资源浪费和环境污染,探索利用自身内源蛋白酶酶解制取抗氧化肽具有重要的意义。对大西洋鲑内脏所含内源蛋白酶的性质进行了初步研究,测得主要含有3种内源蛋白酶,最适p H为2.0、6.0、9.0,p H 9.0的碱性蛋白酶具有最高的酶比活力。以水解度和总抗氧化活性为指标,通过单因素试验确定大西洋鲑内源蛋白酶处理鲑鱼的加工废弃物干粉料的最适条件,再用正交实验L16(45)对其酶解条件进行优化,确定影响酶解液总抗氧化活性的主要影响因素(P﹤0.05)为温度、p H和酶解时间,比较后得到最佳酶解条件为:温度35℃、加酶量0.7 m L/50 m L、料液比0.7 g/50 m L、p H 9.0、酶解时间7 h,该条件下酶解液的总抗氧化活性为258.29 U,大西洋鲑加工废弃物干粉料水解度为15.00%。研究表明,采用大西洋鲑内源蛋白酶酶解鲑鱼加工下脚料并制取抗氧化肽,具有一定的可行性。