南极磷虾自溶过程中胰蛋白酶活性的检测

为探究南极磷虾(Eup Hausia superba)的自溶因素,将南极磷虾分别置于不同温度(30,40,50,60℃)、不同pH(6,7,8,9,10,11)、不同浓度NaCl溶液(0.2,0.4,0.6,0.8,1.0 mol/L)的条件下自溶3.5 h,提取粗酶液,以BApNA为底物,采用分光光度法检测粗酶液中胰蛋白酶活性的变化。结果显示:在温度为40℃、pH为8.0的条件下,胰蛋白酶活力达到最大值(0.076±0.002)U;在同等温度和pH条件下,随着Na~+浓度的升高,胰蛋白酶的活性逐渐被抑制,当Na~+浓度增大到1.0 mol/L时,胰蛋白酶活力为(0.004±0.547×10~(-4))U。试验结果表明,高温偏碱性的条件会加速南极磷虾的自溶,而高浓度的Na~+可以降低其自溶速率。     

大豆胰蛋白酶抑制剂对鲢丝氨酸蛋白酶的作用

研究了大豆胰蛋白酶抑制剂(STI)对鲢肌肉中肌原纤维结合型丝氨酸蛋白酶(MBSP)引起的肌原纤维蛋白降解作用的影响。大豆胰蛋白酶抑制剂经高度纯化后加入肌原纤维中以判断它对MBSP的抑制效果。在不添加STI的情况下,55℃加热30min即可观察到肌球蛋白重链的分解,延长加热时间则可检测到肌动蛋白及原肌球蛋白的降解。相反,在终浓度为0.75μg·mL-1的STI存在下,包括肌球蛋白重链在内的肌原纤维蛋白降解均能得到有效抑制,表明STI是MBSP的特异性抑制剂。由于肌球蛋白重链及肌动蛋白的完整性是构成鱼糜制品凝胶强度的主要因素,因此,STI的添加有可能提高鲢鱼糜制品的凝胶强度。     

南极磷虾粗酶液中蛋白酶的基础酶学性质研究及抑制剂的开发

本研究探讨了反应温度、pH、金属离子(Zn~(2+)、Mg~(2+)、Cu~(2+)、Fe~(3+)、Al~(3+)和Ca~(2+))对南极磷虾粗酶液中蛋白酶活性的影响,研究了苯甲基磺酰氟(PMSF)、EDTA·2Na、碘乙酰胺(IAM)和甲苯磺酰–苯丙氨酸氯甲基酮(TPCK)对蛋白酶活性的抑制效果。结果显示,南极磷虾粗酶液中蛋白酶的最适反应温度为40℃;最适反应pH值为8.0;当金属离子浓度为0.5 mmol/L时,Zn~(2+)、Mg~(2+)、Cu~(2+)、Fe~(3+)和Al~(3+)金属离子对蛋白酶酶活的抑制率分别为55.02%、55.02%、35.39%、20.67%和41.12%,而Ca~(2+)离子对蛋白酶活有明显的促进作用;PMSF和EDTA·2Na对蛋白酶酶活具有较为显著的抑制作用,PMSF的浓度为8.0 mmol/L时,抑制率为60%;EDTA·2Na浓度为0.6 mmol/L时,抑制率为86.67%;IAM在低浓度时有一定的抑制作用;TPCK低浓度时抑制效果不明显,浓度升高时有一定的抑制作用。在5~30℃的温度范围内,随着温度升高,EDTA·2Na对蛋白酶酶活的抑制效果逐渐增加。本研究阐明了影响南极磷虾粗酶液蛋白酶酶活的因素,开发了针对南极磷虾粗酶液中蛋白酶活性的抑制剂,为南极磷虾在食品领域的开发利用提供了基础理论数据。     

壳聚糖固定化胰蛋白酶制备牡蛎肽的研究

以壳聚糖为载体固定化胰蛋白酶,制备牡蛎肽,然后以肽得率为指标,分别研究了pH值,酶加量,酶解时间和温度等单因子对固定化胰蛋白酶酶解牡蛎蛋白质的影响。实验结果表明,酶解牡蛎蛋白的最佳工艺条件是:底物浓度为200μg/mL,加固定化胰蛋白酶量7mg/3mL,酶解时间3h,pH 8.0,温度40℃。在此条件下水解牡蛎蛋白,肽得率可达到20.39%。     

南极磷虾脱壳机工艺参数的试验研究

为了提高南极磷虾的利用价值,以自主设计的南极磷虾脱壳机为研究对象,对其中的关键组件和滚轴长度、滚轴间隙、进料速度等工艺参数进行船载试验;对脱壳后的南极磷虾虾仁进行感官评定和出成率测定,确定了滚轴间隙、滚轴长度、进料速度和磷虾放置时间的最优组合。结果显示:在滚轴长度1. 0 m(A2)、滚轴间隙1. 0 mm(B2)、进料速度125 kg/h(C3)、放置时间15 min(D1)的条件下,各项试验指标皆达最佳;实际生产中采用A2B2C3D1工艺组合,南极磷虾虾仁的出成率达到24%。研究表明:该设备可以提高南极磷虾脱壳生产效率,提升南极磷虾高值化加工的自动化技术水平,从而推动南极磷虾产业化进程。     

牛蛙肥大细胞中类胰蛋白酶的证实

类胰蛋白酶已被作为人类和某些哺乳动物组织中肥大细胞的标志.为检测蛙科动物肥大细胞胞浆中是否含有类胰蛋白酶,采用了小鼠抗人类肥大细胞类胰蛋白酶单克隆抗体AAl通过间接免疫过氧化物酶技术,对牛蛙的舌,肠和脾以及人食道鳞癌组织(用作阳性对照)石蜡切片中的肥大细胞进行染色.首次在Bouin氏液固定的牛蛙组织中证实了牛蛙肥大细胞胞浆中类胰蛋白酶的存在,且单克隆抗体AA1与牛蛙肥大细胞中的类胰蛋白酶可获得良好的交叉反应.类胰蛋白酶阳性细胞多位于牛蛙肠黏膜固有层,少量见于肠绒毛基底部及舌粘膜下腺体周围,而未见于脾组织中.人食道癌间质中可见多量类胰蛋白酶阳性细胞.研究证实,与人类肥大细胞相似的是牛蛙肥大细胞也含有这种特有的类胰蛋白酶,牛蛙有可能是用作肥大细胞生物学研究的理想实验动物.     

现场模拟光照条件对南极大磷虾冬季耗氧率的影响研究

为探究海上现场光照条件对冬季南极大磷虾(Euphausia superba)代谢的影响,基于海上暂养实验,采用黑暗组和光照组两种不同的实验条件,对9尾南极大磷虾个体进行了连续11 d的耗氧率研究。结果表明,光照条件下南极大磷虾的单位体质量耗氧率平均为(0.2704±0.0740)μL·(mg·h)^-1,黑暗条件下的单位体质量耗氧率平均为(0.2673±0.0840)μL·(mg·h)^-1。在光照与黑暗条件下南极大磷虾呼吸率无显著差异。通过对比发现,冬季南极大磷虾的耗氧率明显低于夏季的耗氧率[0.63~0.88μL·(mg·h)^-1],且改变一个自然日的光照条件对南极大磷虾耗氧率无显著影响,但会在一定程度上影响南极大磷虾呼吸率的稳定性。研究验证了冬季南极大磷虾处于一种低代谢模式状况,并证明了该模式的稳定性。相关结果可为揭示南极大磷虾越冬机制提供基础数据。     

南极磷虾酶解产物中一种含氟多肽的研究

通过酶解法从南极磷虾肌肉酶解产物中分离、纯化含氟多肽,并采用LC-MS/MS和氨基酸序列分析对其结构进行研究。结果表明,采用胰蛋白酶酶解所得南极磷虾多肽粗提物的得率约为20%,其总氟含量为(731.73±1.01)μg·g^-1。粗提物精制后,制备得到纯度较高的6个多肽(分别命名为F1、F2、F3、F4、F5、F6),采用离子色谱法筛选出含氟量最高的多肽F6,测定其结合肽氟含量为(266.11±0.40)μg·g^-1。将F6的氨基酸序列片段通过NCBI-Nucleotide-Euphausiacea蛋白质数据库检索,按匹配值得分高于100分可筛选鉴定得到4种蛋白质[Clock,Tropomyosin,ATP synthase subunit 6(mitochondrion),Arginine kinase],通过对4种蛋白的分析发现,南极磷虾蛋白结合氟通过与Clock蛋白磷酸化结合的可能性最大。研究结果对今后探究南极磷虾中含氟蛋白的安全评价提供了技术支撑,对南极磷虾应用价值的进一步开发具有重要意义。     

壳聚糖固定化胰蛋白酶制备魁蚶肽的研究

以壳聚糖为载体、戊二醛为交联剂固定化胰蛋白酶,制备魁蚶肽.以肽得率为指标,研究了固定化胰蛋白酶量、时间、pH、温度和底物浓度等单因子对固定化胰蛋白酶酶解魁蚶蛋白质的影响.结果表明,酶解魁蚶蛋白的最佳工艺条件:固定化胰蛋白酶6 mg,水解反应时间4 h,温度50 ℃,pH 8.0,底物质量浓度98 μg/ml.在此条件下,魁蚶肽得率为23.93%.     

大黄鱼幽门腺胰蛋白酶的纯化及其特性研究

通过硫酸铵分级沉淀、Sephadex G-50凝胶层析制备大黄鱼幽门腺胰蛋白酶,并对其特性进行了研究。大黄鱼胰蛋白酶的最佳催化pH是8.0,在反应时间段内活性最高的温度为55℃。随着温度升高,大黄鱼胰蛋白酶稳定性显著下降。在55℃时的催化活性很高,但稳定性很差,15 min后酶即失活84.4%,1 h后活力下降则达95.6%。     

In vitro assay of plant protease inhibitors from four different sources on digestive proteases of rohu, Labeo rohita (Hamilton), fingerlings

The in vitro inhibitory effect of protease inhibitors from four seed extracts (soybean, grasspea, black gram and horse gram) on digestive proteases of rohu was assessed by enzyme inhibition assay and substrate sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. High proteolytic activity was detected in the intestinal extract of rohu (Labeo rohita) fingerlings at two different pH ranges (8–8.5 and 10–11). That protein digestion occurs mainly in the alkaline condition in this fish without a stomach is evident from very high trypsin activity (0.95±0.04 benzoyl‐dl ‐arginine‐p‐nitroanilide U mg protein⁻¹) in the intestine. In case of grass pea seed, more than 50% inhibition of alkaline protease activity was recorded when the ratio of inhibitor to enzyme was 9.41 μg U⁻¹. More than 40% inhibition of protease activity was recorded in case of horse gram seed when the ratio of inhibitor to enzyme was 5.51 μg U⁻¹. Black gram at 11.0 μg U⁻¹ and soybean seed proteins at 62.75 μg U⁻¹ resulted in 50% and more than 30% inhibition of digestive protease activity in rohu fingerlings respectively. A plot of the inhibition values obtained by changing the relative concentrations of enzyme/inhibitor resulted in different dose–response curves for different protein sources. The use of substrate gel electrophoresis allowed the visualization of the aforementioned differences in inhibition. Each seed extract produced a characteristic profile of protease inhibition. It is concluded that protease inhibitors present in plant protein sources adversely affect the digestive proteases in fish and hence there is a need to eliminate/reduce the amount of such inhibitors through proper processing before incorporation into aquafeeds.     

南极磷虾拖网的性能优化分析

针对一顶中国目前正在使用的南极磷虾(Euphausia superba)拖网进行网具优化试验,设计了调整参数为浮沉比、上下手纲(等长)和叉纲的三因子L9(34)正交实验,并在3组L/S(水平扩张比)和5级拖速下完成了135次拖曳试验。结果表明:(1)随着拖速和L/S的增加,网具阻力增加,网口高度降低;(2)流速为3 kn情况下,L/S=0.45时,对阻力(Fs)和网具功率消耗(Ps)影响的主次顺序均为沉力(A)>叉纲(C)>手纲(B),对网口高度(Hs)影响的主次顺序为A>B>C,对能耗系数(Ce)影响的主次顺序为C>A>B,水动力性能的最优组合为A3B3C1;L/S=0.5时,对Fs、Hs和Ps影响的主次顺序均为C>A>B,对Ce影响的主次顺序为B>C>A,最优组合为A2B3C3;L/S=0.55时,对Fs和Ps影响的主次顺序均为C>A>B,对Hs影响的主次顺序为B>A>C,对Ce影响的主次顺序为B>C>A,最优组合为A2B3C3;(3)当L/S极低为0.2时,网口的水平扩张约为23 m,虽然此时网口高度有一定的提高,但网具的综合性能却显著下降,能耗系数增加;(4)增加沉力,适当放长手纲和叉纲可以有效提高已定型网具的性能。为确保在低拖速下达到网具的高扩张性能,应考虑改进网板性能,建议采用低拖速高扩张性网板。     

3种消毒剂对水生动物3种主要致病菌的载体定量杀菌试验

通过载体定量试验对四羟甲基硫酸磷(THPS)、溴硝醇(BNP)和甲醛溶液的杀菌效果进行测定。结果表明,THPS对于嗜水气单胞菌BSK-10菌株的平均杀灭率在浓度为0.500μl/L时,在THPS与消毒剂接触5 min内即100%被杀灭。而甲醛溶液浓度为0.500μl/L时同样接触5 min杀灭率未达100%;THPS对于爱德华氏菌Et菌株的平均杀灭率与菌株BSK-10相近;而对于哈维氏弧菌GYC1108-1株的平均杀灭率在浓度为1.00μl/L时,在菌与消毒剂接触5 min内即100%被杀灭。而甲醛溶液浓度为0.500μl/L时同样接触5 min杀灭率未达100%。BNP对于菌株BSK-10的平均杀灭率在浓度为16 mg/L时,在菌与消毒剂接触5 min内即100%被杀灭;而对于菌株GYC1108-1的平均杀灭率在浓度为8 mg/L时,在菌与消毒剂接触5 min内即100%被杀灭;BNP对于菌株Et的平均杀灭率与菌株GYC1108-1相近。试验表明THPS相对BNP和甲醛溶液的杀菌效果好,鉴于甲醛溶液本身对水体和水生动物的有害影响,可采用四羟甲基硫酸磷取代甲醛溶液。     

Chemical composition of dried eyeballs from <Emphasis Type="Italic">Euphausia superba</Emphasis> and <Emphasis Type="Italic">Euphausia pacifica</Emphasis>

ABSTRACT:   Dried krill eyeballs were obtained from Euphausia superba and Euphausia pacifica by using a patented industry method and their chemical compositions were examined. Crude protein content was 77.7% and 80.8% of the dry matter of E. superba and E. pacifica , respectively. The dominating amino acids in both krill were glutamic acid, aspartic acid, glycine, arginine, leucine and tyrosine. Crude fat content was 10.9% and 5.4% of dry matter of E. superba and E. pacifica , respectively. The main lipid class of the extracted lipids was phospholipids at 88.5% in E. superba and 96.4% in E. pacifica . The dominating fatty acids in both krill were 22:6 ( n- 3), 20:5 ( n- 3), 16:0 and 18:1 ( n- 9). Astaxanthin (3, 3'-dihydroxy-β, β-carotene-4, 4'-dione) content of E. superba and E. pacifica was 566 mg/100 g and 252 mg/100 g of dry matter, respectively. Retinol of E. superba and E. pacifica was 153.0 mg/100 g (510 000 IU/100 g) and 57.6 mg/100 g (192 000 IU/100 g) of dry matter, respectively. The lipophilic extract of E. superba by using n -hexane contained 1923 mg/100 g of astaxanthin at approximately four times higher than the dried eyeballs.     

Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2–9.0) and 55 °C. CaCl₂ did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated ( P <0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.     

双氟沙星与异育银鲫血浆蛋白的结合率

为了研究双氟沙星(difloxacin,DIF)与异育银鲫(Carassais auratus gibebio)血浆蛋白的结合性质,采用平衡透析法测定了DIF与异育银鲫血浆蛋白的体外结合率,并研究了温度、血浆浓度及药物浓度对其的影响。结果显示:DIF血浆蛋白结合率在4℃、15℃、20℃、25℃、30℃、37℃条件下变化差异不显著(P>0.05);药物血浆蛋白结合率显著高于二倍稀释血浆的结合率,也显著高于四倍稀释血浆的结合率(P<0.05)。当DIF浓度在0.5～5μg/mL范围之间时,药物与血浆蛋白结合率在32.4%～19.6%之间,并随着药物浓度的升高而降低,最大结合能力(β)和解离常数(Kdp)分别为3.5×10-7 mol/g和4.04×10-6 mol/L;当DIF质量浓度为5～40μg/mL范围内时,血浆蛋白结合率的变化差异不显著(P>0.05)。此外,游离药物与总药物浓度呈线性关系,关系式为y=0.77x-0.18。结果表明,DIF与异育银鲫血浆蛋白结合率在本实验条件下不受温度影响,且具有浓度依赖性;在0.5～5μg/mL范围内结合率为非线性,而在5～40μg/mL范围内为线性。本研究旨在为DIF药物代谢动力学理论研究及其临床用药提供理论参考。     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.     

香菇多糖提取物体外抗IHNV的机制

本研究以虹鳟性腺细胞(rainbow trout gonad cell,RTG)为细胞模型,采用MTT法体外测定香菇多糖提取物的细胞毒性及其对传染性造血器官坏死病毒(IHNV)直接灭活、预防与生物合成的影响,探讨香菇多糖提取物抗IHNV的作用机制。结果显示:香菇多糖提取物对RTG细胞没有毒性作用。浓度为100μg/m L时,香菇多糖提取物对IHNV的直接灭活能力为52.48%,预防效果为50.38%,抑制IHNV生物合成的能力达84.41%。结果表明:香菇多糖提取物具有一定的抗IHNV作用,该作用主要与抑制IHNV在细胞内的生物合成有关。     

响应面法提取南极磷虾磷脂及其分子种组成的测定

南极磷虾(Euphausia superba)生物储量巨大,含有丰富的磷脂酰胆碱(phosphatidylcholine,PC),而PC是生物生长的必需磷脂。现今南极磷虾的研究主要针对磷虾油和蛋白,尚未见有PC分子组成的系统研究报道。该研究使用超声辅助Bligh Dyer脂质提取法提取南极磷虾中磷脂,并采用Box-Benhnken设计原理优化提取工艺,以正离子模式下胆碱碎片(m/z 184)为特异性碎片,利用鸟枪脂质组学法鉴定南极磷虾组织中的PC分子组成。结果表明,南极磷虾磷脂的最佳提取条件为超声时间21.0 min、超声温度30℃、溶剂用量3.1 mL,此条件下磷脂实际提取率的平均值为(7.59±0.03)%。利用鸟枪脂质组学法鉴定出南极磷虾中共含有36种PC分子,其中EPA/DHA脂肪酸链含量占47.75%,质量分数为269.82μg·g~(-1)。研究表明南极磷虾组织中含有丰富的EPA和DHA,而磷脂状态存在的ω-3脂肪酸具有较高的利用效率,这为开发南极磷虾类营养保健产品提供数据支持和理论基础。     

Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein⁻¹ min⁻¹) followed by silver carp (1.084 ± 0.061 U mg  protein⁻¹ min⁻¹), and catla (0.193 ± 0.006 U mg  protein⁻¹ min⁻¹). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell‐shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone (TLCK) and N‐tosyl‐L‐phenylalanychloromethane (TPCK) indicated the presence of trypsin‐like and chymotrypsin‐like enzymes in all these three carps. SDS‐PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS‐PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH‐stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.