蛋公鸡和肉仔鸡蛋白消化酶活性及饲料氨基酸消化率的比较研究

试验选用成年海兰褐种公鸡和 4周龄艾维因商品代肉公鸡 ,用 Sibbald“TME”法测定豆粕、棉粕、菜粕氨基酸消化率和强饲 3种蛋白饲料后胰腺、小肠食糜蛋白消化酶比活 ,结果表明 :蛋公鸡和肉仔鸡饲料氨基酸消化率除豆粕组肉仔鸡显著高于蛋公鸡 (P<0 .0 5)外 ,棉粕、菜粕 2组没有显著差异 (P>0 .0 5)。蛋公鸡和肉仔鸡蛋白消化酶比活除菜粕组蛋公鸡胰腺胰蛋白酶原比活显著高于肉仔鸡外 (P<0 .0 5) ,豆粕、棉粕组蛋公鸡和肉仔鸡蛋白消化酶比活没有显著差异 (P>0 .0 5)。

甲胺磷对拟环纹豹蛛中肠蛋白消化酶活性影响的压电传感监测

利用压电体声波阻抗分析法实时动态监测了拟环纹豹蛛Pardosapseudoannulata中肠蛋白消化酶对酪蛋白的酶促水解过程及不同浓度梯度(0.008%、0.016%、0.024%和0.032%)的甲胺磷农药对酶活性的影响,并用紫外分光光度法进行了验证。结果表明,合适低剂量(0.008%)的甲胺磷农药可显著地增强拟环纹豹蛛蛋白消化酶的活性,较高剂量(0.032%)的农药却显著抑制蛋白消化酶的活性,且水解过程稳态频移值可准确地反映酶活性与农药浓度的关系。本工作为研究低剂量农药增强蜘蛛控虫力的机理及酶活性的快速检测提供了一种可行的新方法。

温度对条纹石鮨蛋白消化酶活性影响的初步研究

水温１５～２０℃时，条纹石鱼旨各消化器官的重量占鱼体重的百分比大小依次排列为比肝重＞比胃重＞比肠重，而在２５～３０℃时则排列为比胃重＞比肝重＞比肠重；水温１５～３０℃时，随着温度的上升，单位体重中酶蛋白所占的比例呈下降趋势；１５～２０℃时，消化道蛋白酶的活性随着水温的升高而显著增大，超过２０℃时，蛋白酶的活性又呈下降趋势；１５～２０℃时，各消化器官中蛋白酶的比活均上升９倍，２０～３０℃时，肝蛋白酶的比活继续增大，而胃及肠蛋白酶的比活则略有下降。

饲料中大豆异黄酮和大豆皂甙对牙鲆肝脏和肠道蛋白质消化酶活性和基因表达的影响

以鱼粉为蛋白源,小麦粉为糖源,鱼油为脂肪源配制基础饲料,分别添加不同浓度梯度(0.1%、0.4%和0.8%)的大豆异黄酮和大豆皂甙。以不添加大豆异黄酮和大豆皂甙的基础饲料作为对照组,共制成7种等氮(粗蛋白49.1%)、等能(总能20.1kJ/g)的实验饲料。在循环水系统中养殖初始体质量为(2.58±0.01)g的牙鲆(Paralichthys olivaceus)幼鱼,8周后观测牙鲆肝脏和肠道蛋白质消化酶活性和基因表达的影响。结果表明,牙鲆肠道胰蛋白酶的活性不受饲料中大豆异黄酮的显著影响(P>0.05),却随大豆皂甙添加量的升高而显著下降(P<0.05)。肠道糜蛋白酶的活性在饲料中大豆异黄酮添加量为0.1%时取得最大值(37.3±5.9)U/mg,显著高于其它处理组。饲料中添加不同水平的大豆异黄酮对肝脏中胰蛋白酶-1和糜蛋白酶-1基因表达量有显著(P<0.05)的影响,而添加大豆皂甙却对这些基因表达量的影响不显著。由此可见,大豆异黄酮直接作用于基因表达和酶活两个水平,而大豆皂甙只在酶活水平发挥显著的作用。同时,大豆异黄酮对牙鲆饲料利用率影响的双重性依赖于剂量的大小,其中的作用机制有待于进一步研究。

实现水稻育种新飞跃的基因工程

我国水稻虫害日益猖獗，但因水稻缺乏抗虫基因而只好依赖于化学杀虫剂来防治，这样就增加了稻作成本并污染了生态环境，因此，水稻抗虫基因工程育种备受重视。目前，水稻抗虫基因主要利用的是蛋白酶抑制剂基因、Bt毒蛋白基因和外源凝集素基因等。蛋白酶抑制剂可与昆虫消化道内的蛋白消化酶相互作用，形成酶一抑制剂复合物（E1），从而抑制蛋白消化酶的活性，同时，因复合物还能刺激消化酶的过量分泌，通过神经系统的反馈抑制，使昆虫产生厌食反应，最终因氨基酸饥饿导致昆虫不正常发育或死亡。蛋白酶抑制剂杀虫谱广，

利用转基因植物表达猪疫苗的研究现状

利用转基因植物表达疫苗是植物基因工程技术与医学免疫学相结合而发展起来的新的研究方向。迄今为止，大部分植物表达疫苗的免疫原性都得到了证实。植物表达的疫苗与传统疫苗相比，具有生产成本低、安全性高、贮运简单、生物活性高和可直接食（饲）用等优点，其应用前景十分可观。植物表达的疫苗作为一种新型疫苗非常突出的一个特点是植物细胞壁具有“生物胶囊”作用，可以抵抗胃肠道中各种蛋白消化酶，为植物细胞中的抗原蛋白提供一定的保护。

Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

Abstract The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR （FQ-PCR） methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.

COMPARISON OF FOUR METHODS TO GENERATE IMMUNOREACTIVE FRAGMENTS OF A MURINE MONOCLONAL ANTIBODY OC859 AGAINST HUMAN OVARIAN EPITHELIAL CANCER ANTIGEN

In the present study, four different proteases (pepsin, papain, bromelain and ficin) were screened with a murine monoclonal antibody OC859 , in order to verify whether different digestion procedures could improve yield and stability of the F(ab’)2 or Fab fragments. The yields of F(ab’)2 or Fab fragments from digestion with pepsin, papain , bromelain and ficin were respectively 20. 3+/-2. 0%, 50. 5+/-5. 0%,74. 4+/-2. 7% and 82. 8 +/-10. 2% of the theoretical maximum. Immunoreactivity in a noncompetitive solid-phase radioimmunoassay (SPRIA) of the fragments generated by the four proteases were respectively 10+/-5%, 36+/-5%, 60+/-6% and 75+/-6%of the intact OC859 IgG. These results suggested that the fragmentation of OC859 with ficin gave a higher yield of superior immunoreactive fragments.

Inactivation of digestive proteases: Another aspect of gut bacteria that should be taken into more consideration

TO THE EDITOR Protein has been one of the main components of our diet, and a large amount of digestive proteases is released into the gut for their digestion. However, these proteases can digest not only the proteins we eat, but also the structural proteins built in our body. To protect against this damage, our body has taken a variety of measures. For instance, these digestive proteases are stored and secreted in the form of zymogen and only activated in gut lumen. These luminal digestive proteases are further prevented from direct contact with epithelial cells by the mucus layer that is incessantly secreted by the goblet cells in gut mucosa. In addition, large quantities of protease inhibitors are produced in the body to inactivate the digestive proteases that have entered the body. Despite these measures, the protection seems still weak and can be easily compromised. For instance,

Detection of anti-Helicobacter pyloriantibodies in serum and duodenal fluid in peptic gastroduodenal disease

AIM:To study the diagnosis of Helicobacter pylori(H pylori) infection through the determination of serum levels of anti- H pylori IgG and IgA antibodies,and the levels of anti-H pylori IgA antibodies in duodenal fluid. METHODS:Data were collected from 93 patients submitted to upper digestive endoscopy due to dyspeptic symptoms. The patients were either negative(group A)or positive (group B)to H pylori by means of both histological detection and urease tests.Before endoscopy,peripheral blood was collected for the investigation of anti-H pylori IgG and IgA antibodies.To perform the urease test,biopsies were obtained from the gastric antrum.For the histological evaluation,biopsies were collected from the gastric antrum (greater and lesser curvatures)and the gastric body. Following this,duodenal fluid was collected from the first and second portions of the duodenum.For the serological assaying of anti-Hpylori IgG and IgA,and anti-Hpylori IgA in duodenal fluids,the ELISA method was utilized. RESULTS:The concentration of serum IgG showed sensitivity of 64.0%,specificity of 83.7%,positive predictive value of 82.0%,negative predictive value of 66.6% and accuracy of 73.1% for the diagnosis of H pylori infection.For the same purpose,serum IgA showed sensitivity of 72.0%, specificity of 65.9%,positive predictive value of 72.0%, negative predictive value of 67.4% and accuracy of 69.8%. If the serological tests were considered together,i.e.when both were positive or negative,the accuracy was 80.0%, sensitivity was 86.6%,specificity was 74.2%,positive predictive value was 74.2% and negative predictive value was 86.6%.When values obtained in the test for detecting IgA in the duodenal fluid were analyzed,no significant difference(P=0.43)was observed between the values obtained from patients with or without H pylori infection. CONCLUSION:The results of serum IgG and IgA tests for H pylori detection when used simultaneously,are more efficient in accuracy,sensitivity and negative predictive value, than those when used alone.The concentration of IgA antibodies in duodenal fluid is not useful in identifying patients with or without H pylori.

Characterization of proteases from Planomicrobium sp. L-2 isolated from the gastrointestinal tract of Octopus variabilis (Sasaki)

A crude protease produced from Planomicrobium sp. L-2 is described, and its effectiveness as an additive in liquid detergent evaluated. We isolate the protease-producing Planomicrobium sp. L-2 from the gastrointestinal tract of Octopus variabilis. At least three caseinolytic protease clear bands were observed in zymogram analysis. The crude alkaline protease was highly tolerant of a pH range from 7.0 to 9.0, and temperatures to 50℃ after incubation for 1 h. Proteolytic enzymes were stable towards three surfactants （5% Tween 80, 1% Triton X-100 and 0.05% SDS） and an oxidizing agent （1% hydrogen peroxide）, in addition to being highly stable and compatible with popular commercial laundry powered detergent brands available in China. Our study demonstrates the potential these proteases have for development into novel classes of detergent additive. This study also suggests that the gastrointestinal tract of Octopus variabilis may be a rich source of commercially valuable strains of enzvme.

Proteinases from the digestive organs of black carp （Mylopharyngodon piceus）： Partial characterization and protein digestibility in vitro

A series of experiments based on electrophoretical and biochemical assays were conducted to partially characterize proteinases present in the hepatopancreas and intestine of black carp （Mylopharyngodon piceus）, and investigate enzymatic activity and protein digestibility in vitro. Casein digestion assays revealed the presence of acidic proteinases with optimum activity in the range of pH 2.0-2.5 and alkaline proteinases with significantly higher activities both in the range of pH 8.1-8.6 and near pH 9.5. The inhibition and substrate specificity assays showed that trypsin and chymotrypsin are the main active components of the alkaline proteinases. The SDS-substrate-PAGE showed that the crude extract of black carp intestine had eight types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa while the crude extract of black carp hepatopancreas had six types of alkaline proteinases with the molecular mass range of 27.5-78.5 kDa. These enzymes were characterized as trypsin （27.5 kDa, 30.1 kDa）, chymotrypsin （40.5 kDa, 42.5 kDa）, serine proteinases （32.1 kDa, 33.2 kDa） and non-serine proteinase （61.5 kDa, 78.5 kDa）.In vitro protein digestibility assays showed that black cardcan be able to utilize a wider range of proteins.

Enhanced Free Radical Scavenging and Inhibition of DPP-4 and ACE Activities by Compounds from Salmon Tissues Digested in Vitro with Gastrointestinal Proteases

Research on marine bioactive peptides has mainly focused on characterization of peptides in hydrolysates prepared with commercial industrial enzymes and the usefulness of such hydrolysates in health and functional foods. However, a relevant question is whether digestion of fish proteins with gastrointestinal proteases per se generates peptides that also can have health promoting properties and can reduce, e.g., diabetes 2, inflammation and hypertension either in relation to gastrointestinal digestion or as alternative to industrial proteases. The aim of the study was to investigate hydrolysates obtained from in vitro sequential digestion of salmon muscle and skin with gastrointestinal proteases including pepsin, pancreatic and pancreatic ＋ mucosal proteases for their ability to scavenge ABTS^＋ radicals and inhibit activity of angiotensin I-converting enzyme （ACE） and dipeptidyl peptidase 4 （DPP-4）. Furthermore, it was the aim to study the inhibitory mechanism and stability towards ACE and DPP-4 activity. Analysis of〈 10 kDa hydrolysates showed that gastrointestinal proteases generated peptides with clear radical scavenging activity and DPP-4 and ACE inhibiting activity as well. Hydrolysates from pepsin digestion exhibited the lowest ECso values for radical scavenging activity and ACE inhibition, whereas ECso increased in hydrolysates after subsequent digestion with pancreatic and mucosal proteases. Interestingly, ECso values for the DPP-4 inhibition were hardly affected by sequential digestion. Inhibition modes for the muscle hydrolysates were both competitive and non-competitive, but prolonged incubation showed that the inhibitory properties were unstable and therefore they were probably digested as competitive substrates by gastrointestinal proteases.

Effect of Gastrointestinal Protease Digestion on Bioactivity of Marine Peptides

Focus in nutritional science has turned towards components in, or added to, foods that may possess health beneficial activities beyond the classical nutritional value, namely functional food. Bioactive peptides are examples of such components. In vitro studies on bioactivities have mainly been executed without concerning subsequent digestion after intake and the aim of this work was hence to investigate how the in vitro antioxidative, antihypertensive and caspase activating activities of peptides are affected by digestion with gastrointestinal （GI） proteases. Five different fish protein hydrolysates were chosen to study the effect of in vitro digestion on bioactivity. The protein concentration decreased in all samples during digestion and the molecular weight distribution of the peptides shifted towards lower values. Thus, in vitro digestion with GI proteases resulted in a further degradation of the peptides obtained by hydrolysis. The antihypertensive effect increased in all samples after digestion with GI proteases whereas the antioxidative capacity decreased. The effect on the caspase activity depended on the proteases used in the preparation of hydrolysates. In conclusion, the caspase activity and antihypertensive activity are maintained during digestion with GI proteases, while the antioxidative capacity seems to be reduced.

养猪巧喂豆腐渣

1．熟喂。豆腐渣中的抗胰蛋白酶可降低体内蛋白消化酶的活性，阻碍机体对豆类蛋白质的消化、吸收。用豆腐渣喂猪，必须先加热煮沸10～15分钟，破坏其中的抗胰蛋白酶。

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion

The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is pre- requisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid （EDTA） digestion method to disas- sociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRPl-positive cells in cell suspensions prepared through cold digestion was con- sistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells.

Ultrasound speed and attenuation in progressive trypsin digested articular cartilage

Subtle changes of articular cartilage(AC) can lead to tissue degeneration and even osteoarthritis(OA).The early degeneration of AC is closely related to a change in proteoglycans(PG) content.The observation of PG is therefore an appropriate way of studying OA and evaluating the degree of AC degeneration.In this study,20 cartilage-bone samples were prepared from normal porcine femoral condyle cartilage and 10 samples were digested over 2 h using 0.25% trypsin solution.The dynamic process of PG-digestion was explored using a conventional A-mode ultrasound(US) experimental system with a 10 MHz center frequency.Quantitative acoustic parameters were calculated from ultrasonic radio-frequency echo signals and included US speed(USS),US amplitude attenuation coefficient(UAA) and broadband US attenuation coefficient(BUA).The experimental results showed that the conventional A-mode ultrasound is valuable for tracking the degree of PG-digestion.Histology also confirmed the validity of the ultrasound observations.For every AC sample,the degree of PG-digestion within a given time was different and was affected by individual differences.After two hours of degeneration,USS showed a mean decrease of 0.4%(P<0.05).UAA was significantly lower after a two-hour PG depletion period(from(2.45±0.23) to(2.28±0.41) dB mm?1).BUA showed no significant differences during this process.In conclusion,conventional ultrasound can provide useful information about trypsin-induced progressive PG depletion in AC and can reflect variations of PG content via the quantitative acoustic parameters USS and UAA.The results of this study may be used to identify an indirect indicator of cartilage matrix integrity and OA disease progression.

EFFECTS OF FIVE NEW COMPOUNDS ON THE LARVAL GROWTH AND DIGESTIVE PHYSIOLOGY OF THE ASIATIC CORN BORER, OSTRINIA FURNA CALIS LARVAE

Five new compounds were tested on the growth and antifeeding activity compared with toosendanin against fifth instar larvae Ostrinia furnacalis. The activities of two proteases, a weak alkaline trypsine like enzyme and a chymotrypsin like enzyme, in the midgut of Ostrinia furnacalis larvae were also measured. Experimental results suggest that when incorporated into an artificial diet at the concentration of 500mg/kg, the antifeeding activities of toosendanin,C 19 ,C 23 ,C 24 ,C 26 ,C 28 were 51 16%,57 61%,4 28%,51 08%,36 73% and 51 67%, respectively, C 19 ,C 24 ,C 28 had no significant difference with toosendanin. At 20mg/kg, the larval growth were remarkably suppressed by C 19 ,C 26 ,C 28 , the inhibition of C 28 was close to toosendanin in 48 h. The two proteases were activated by toosendanin and C 28 while they were inhibited in 48 h but activated in 24 h by C 19 ,C 24 and C 26 . In this paper, the related functions and mechanisms were discussed.

An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy. Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and de- creasing missed cleavages, which results in more peptides that are compatible with mass-spectrometry analysis. Compared to conventional solution digestion, immobilized protease digestion has the obvious advantages of short digestion time, no self-proteolysis, and reusability. We proposed a multiple-immobilized proteases-digestion strategy that combines the ad- vantages of the two digestion strategies mentioned above. Graphene-oxide （GO）-based immobilized trypsin and endoprotein- ase Glu-C were prepared by covalently attaching them onto the GO surface. The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoan- aerobacter tengcongensis. Compared to 12-hour solution digestion using trypsin or Glu-C, 14% and 7% improvement were obtained, respectively, in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-GIu-C. Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3% and 48.7% en- hancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone. The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO, which leads to fewer missed cleavages and more complete digestion. As a result, improved protein identification and sequence coverage can be expected.