酶法进行海洋低值蛋白资源高值化利用初探

首先分析了Ti鱼与四角哈两种低值海洋蛋白资源的营养成分，然后利用两株高产蛋白酶菌株通过液体发酵制备的蛋白酶制剂对两种蛋白资源进行了酶解，经过比较酶解率和肽含量，表明对于四角蛤，菌株SM98013产生的碱性蛋白酶和SM98011产生的中性蛋白酶都可高效酶解；而对Ti鱼，菌株SM98013产生的碱性蛋白酶的酶解效率较高。文中对海洋低值蛋白资源的高值化利用的途径进行了讨论。     

南美白对虾副产物酶解工艺优化及其蛋白肽活性评价

为高质化利用南美白对虾加工副产物蛋白资源,本研究以南美白对虾加工副产物虾头虾壳为原料,蛋白水解度为考察指标,采用单因素和响应面试验优化酶解工艺。检测虾蛋白肽分子量的分布范围,评价其抗氧化能力及免疫调节作用。结果表明,碱性蛋白酶与虾头内源酶自溶酶解具有较好的协同作用,最佳酶解工艺参数值为：酶添加量4%、酶解时间6.5 h、酶解温度40℃、料液比1∶5。经蛋白层析仪检测,分子质量分布在75～1 355 Da的蛋白肽占比达到76.35%。虾蛋白肽的羟自由基清除率IC₅₀为4.64 mg/mL、DPPH自由基清除率IC₅₀为3.08 mg/mL,提示其具有一定的抗氧化能力。虾蛋白肽中剂量组(10 mg/kg)能显著促进淋巴细胞增殖,并提高免疫低下模型小鼠的免疫器官指数和吞噬指数,表明虾蛋白肽具有显著的免疫调节作用。     

黄鳍金枪鱼酶解物免疫活性及其氨基酸分析

本文旨在研究黄鳍金枪鱼(Thunnus albacares)碎肉中4种不同蛋白酶酶解物的3k Da超滤组分对RAW264.7细胞的免疫活性。以RAW264.7细胞为实验模型, 采用噻唑蓝比色法检测细胞增殖活性, 中性红吞噬实验检测细胞吞噬能力, Griess试剂检测细胞培养上清液中NO含量, 并分析酶解物氨基酸组成和分子量分布。实验结果表明, 胰蛋白酶酶解物在12.5~200μg·mL^-1范围内无明显细胞毒性, 能增强RAW264.7细胞吞噬能力, 浓度为400μg·mL^-1时达到最大值132.50%(P<0.01), 浓度在50~200μg·mL^-1范围内能促进巨噬细胞NO释放(P<0.01), 且NO释放量与酶解液浓度存在一定的量效关系; 胰蛋白酶酶解物中必需氨基酸(essential amino acid, EAA)含量最高, 为51.09%, 其碱性氨基酸组氨酸、赖氨酸和精氨酸的含量分别为11.59%、11.00%和6.90%, 均高于其他蛋白酶酶解物, 并且分子量小于1k Da的小肽含量高达97.48%。研究结果表明, 4种蛋白酶酶解物中, 黄鳍金枪鱼碎肉胰蛋白酶酶解物的3k Da组分具有较明显的免疫活性, 这为其作为免疫活性肽的开发利用提供了理论依据。     

金枪鱼(Katsuwonus pelamis)碎肉蛋白降压肽的酶解制备及活性研究

以血管紧张素转换酶(ACE)抑制率为指标,通过正交试验L9(34)确定碱性蛋白酶(Alcalase)对金枪鱼(Katsuwonus pelamis)碎肉蛋白的最佳酶解条件,利用D101大孔树脂建立了酶解物的脱盐工艺,利用超滤、葡聚糖凝胶(G-25)色谱和反相-高效液相色谱(RP-HPLC)分离降压肽,并确定其纯度,利用氨基酸序列分析和ESI-MS鉴定纯化多肽的结构。结果表明,碱性蛋白酶酶解金枪鱼碎肉蛋白制备降压肽的最佳酶解条件是:pH 9.5,酶用量1.5%,酶解温度50°C,酶解时间5h;D101大孔吸附树脂脱盐工艺条件为:上样浓度10 mg/mL、上样流速1.5 BV/h、解吸剂为75%乙醇;酶解物经超滤和Sephadex G-25分离获得一个纯度较高的四肽,经氨基酸序列分析和ESI-MS鉴定结构为Phe-GlyGly-Val(FGGV),ESI-MS检测给出分子离子峰m/z 379.50([M+H]+)。利用碱性蛋白酶酶解并经超滤和色谱技术制备的金枪鱼碎肉蛋白降压肽具有良好的ACE抑制活性,可作为降压药物、保健食品或添加剂进行开发。     

金枪鱼(Thunnus sp.)胰腺酶解液对H2O2诱导的胰岛素瘤细胞(INS-1)氧化损伤的保护作用

本文首先通过MALDI-TOF/TOF-MS技术,测定金枪鱼(Thunnus sp.)胰腺酶解液中的多肽组分及其丰度,确定GQPVR、GLPPK和EHER是其中的优势肽;再利用Discovery Studio平台中的反向找靶和分子对接模块,发现这些优势肽均可以与Keap1蛋白结合,推测其可能具有抗氧化活性;最后,利用细胞实验对酶解液的抗氧化活性进行验证,发现其对H2O2氧化损伤的胰岛素瘤细胞(INS-1)具有剂量依赖型保护效果,高剂量和低剂量酶解液处理后细胞活力较模型组显著提高,凋亡率从79.17%分别下降到43.2%(P0.01)和28.9%(P0.01);胰岛素的分泌量从3.65mIU/L分别增加到5.75mIU/L(P0.01)和4.95mIU/L(P0.01)。DiscoveryStudio的抗氧化功能预测和细胞学实验相结合的研究方法,为酶解液的功能组分鉴定提供了新的思路。     

双酶分步水解金枪鱼(Eleotridae)碎肉制备高F值酶解液的工艺研究

以金枪鱼碎肉为原料, 采用双酶分步水解法制备高F值酶解液, 通过Box-Behnken试验设计, 分别确定两步酶解的最佳条件, 酶解液经活性炭静态吸附去除游离芳香族氨基酸, 对脱芳后的酶解液进行氨基酸组成分析并测定F值。结果表明, 胃蛋白酶为第一步水解用酶, 酶解的最佳工艺条件为酶用量650U/g, 料水比1∶7(g/mL), 温度35.9℃; 风味蛋白酶为第二步水解用酶, 酶解的最佳工艺条件为酶用量50700U/g, pH 6.51, 温度51℃, 最终水解度达到36.87%±0.54%; 酶解液在pH 3.0, 温度35℃条件下, 经5%(质量体积分数)的活性炭吸附时间3h后, 脱芳率达到63.18%, F值为30.33, 符合高F值肽的要求。     

响应面法优化罗非鱼(Oreochromis niloticus)鱼排蛋白分段酶解工艺

采用生物蛋白酶酶解罗非鱼鱼排蛋白,以水解度(DH)为指标,进行了单酶、复合酶酶解效果的比较分析,并利用响应面分析方法(RSM)对酶解参数进行优化。结果表明,采用风味蛋白酶和木瓜蛋白酶双酶组合分段酶解,固定总酶添加量为3.0%、双酶复合比2:1、酶解时间3h、分段酶解时间比0.6:1,酶解温度56℃,分段酶解pH为7.0和6.2时,酶解效果最佳,罗非鱼鱼排蛋白水解度达到32.49%,酶解产物中游离氨基酸含量达32.84mg/g鱼排,占罗非鱼鱼排总氨基酸含量的25.43%,其中呈味氨基酸含量达18.48%,必需氨基酸含量达67.96%。     

响应面法优化酶法制备藻蓝蛋白抗氧化肽的实验研究

为优化碱性蛋白酶酶解藻蓝蛋白制备抗氧化肽的工艺条件,采用响应面分析法,以超氧阴离子自由基清除率为响应值,研究了[E]/[S]、酶解温度和时间对制备抗氧化肽工艺的影响.结果表明,制备藻蓝蛋白肽的最佳酶解条件为:温度为44.9℃、时间为6.4 h和[E]/[S]为3.6%,此时对超氧阴离子自由基清除率达到75.39%.在该条件下制备的藻蓝蛋白酶解产物(0.5 g/L)还原能力的吸光度为1.03,并达到稳定.     

酶解蓝圆鲹蛋白制备降血压肽的工艺研究

以蓝圆(Decapterus maruadsi)鱼肉蛋白为原料,选用6种蛋白酶在各自适宜的条件下酶解制备鲹降血压肽,通过检测酶解产物对血管紧张素转化酶(ACE)的体外抑制活性,筛选出木瓜蛋白酶为制备蓝圆降血压肽的最佳用酶。考察了不同水解度的酶解产物对ACE抑制效果,当水解度为13.7%时,产物对ACE的最高抑制活性可达67.4％。在此基础上,分别了考察酶解时间、初始酶用量、初始底物浓度、酶解温度和 pH 值对酶解工艺的影响,鲹得到制备蓝圆降血压肽的适宜酶解工艺为：初始酶用量鲹为7000 U/g,初始底物浓度为25 g/L,酶解温度为45℃, pH为7.0,酶解240 min。采用超滤技术对酶解产物活性组分进行分离富集,发现产物中高活性部分均可富集于10 kDa和5kDa渗透液中,且高活性组分分子量主要集中在5 kDa以下,该组分对ACE的活性抑制率为88.47%。     

酶解马氏珠母贝肉制备降血压肽工艺条件优化

以马氏珠母贝（Pinctada martensii）肉为原料，选用6种蛋白酶在各自适宜的条件下进行酶解，采用HPLC法分析酶解产物对血管肾张素转换酶（ACE）的抑制活性（IP），筛选出碱性蛋白酶为其制备的最佳酶种类，并确定了水解度为21％时，产物具有较好的抑制活性。在此基础上，分别考察了加酶量，底物浓度，酶解温度及pH对水解度的影响。结果表明：当加酶量为0．175g／L，底物浓度为35g／L，温度为45℃，pH为9．5，水解时间240min为酶解最适宜工艺条件。通过SDS-聚丙烯酰胺凝胶电泳测定分子质量分布，表明酶解产物中主要为14．4ku以下的多肽分子。     

废弃鱼骨中蛋白质的提取分离及酶解工艺研究

以蛋白提取率和多肽分子质量分布为评价指标,采用不同提取方法和不同蛋白酶对废弃鱼骨蛋白质的提取和酶解工艺进行了研究。结果表明,高压蒸煮法提取的鱼骨蛋白质中蛋白含量为86.15%,蛋白提取率为16.76%,明显高于恒温水浴和热回流提取法。中性蛋白酶酶解的多肽中蛋白含量为88.46%,多肽比例高达95%以上,明显高于其他三种酶。先利用高压提取法提取蛋白质,再利用中性蛋白酶酶解蛋白质制备多肽,可降低酶用量,该方法为多肽工业化生产提供参考及新思路。     

利用贝类废弃物制造水解动物蛋白

以扇贝加工废弃物—扇贝边为原料 ,采用枯草杆菌蛋白酶、胰蛋白酶、Flavourzyme对其进行深度水解 ;然后应用超滤技术对酶解液进行分离 ,除去未水解的蛋白质、细菌、微细的颗粒等物质 ,得到富含氨基酸和小分子肽的超滤透过液 ;再应用反渗透技术进行预浓缩 ,以除去其中过多水分 ;最后 ,对该浓缩液进行减压浓缩或喷雾干燥 ,即得到浓液状或粉状的水解动物蛋白产品。其中 ,粉状产品的粗蛋白含量为 6 6 .88% ,氨基酸态氮含量为 6 .96 % ,氨基酸态氮 /总氮 6 5 .0 5 % ,均优于国外同类产品     

Organic substances in sediment and settling matter during spring in a meromictic Lake Suigetsu

Vertical distribution of organic substances of settling matter and sediments was measured in a typically meromictic lake, Lake Suigetsu. Proteinous materials were rapidly eliminated in the early stage of settling in the lake water. No major difference was found in amino acid composition of the hydrolysates of all samples, indicating that there had occurred no selective decomposition of amino acids. In contrast, the decomposition of carbohydrates was sluggish. Water-extractable carbohydrates were abundant in the sediments. Dissolved carbohydrates and amino acids, formed in the sediments, were partly metabolized by photosynthetic sulfur bacteria,Chromatum sp. populated in the chemocline.     

深海适冷菌SM9913产生的低温蛋白酶

从1855m深的深海泥样中分离纯化得到200多株分泌蛋白酶的适冷菌，其中3株产低温蛋白酶，本文对其中一株Pseudomonas sp.SM9913(P.SM9913)生长的适冷性和它产生的蛋白酶的适低温特性进行了研究。该菌株能够在0℃正常生长，其最适生长温度为15℃，最高生长温度为35℃。为一株适冷菌。该菌株所产蛋白酶的比合成速率在10℃时最高，催化酪蛋白水解的最适温度为35℃，在0℃仍具有3％的酶活力。最适pH为8．0。该蛋白酶的热稳定性很低，在40℃保温10min即丧失85％的活力，40℃时的半衰期为6min，为一典型的低温酶。抑制剂试验表明，该蛋白酶为金属蛋白酶。     

Screening of protease-producing marine yeasts for production of the bioactive peptides

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.     

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp.SM9913

Pseudoalteromonas sp.SM9913 is a phychrotrophic bacterium isolated from the deep-sea sediment.The genes encoding chaperones DnaJ and DnaK of P.sp.SM9913 were cloned by normal PCR and TAIL-PCR(GenBank accession Nos DQ640312,DQ504163).The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria,and show some cold-adapted characteristics in their structures when compared with those from psychro-,meso-and themophilic bacteria.It is indicated that chaperones DnaJ and DnaK of P.sp.SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding,assembling and translocation of proteins at low temperature.This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.     

深海羽毛降解菌Bacillus sp. JM7极端碱耐受角蛋白酶的酶活性质研究

Bacillus sp. JM7, a strain isolated from the deep-sea of the South China Sea, was found to efficiently degrade 79.4% native chicken feather within 30 h. Scanning electron microscopy analysis showed that JM7 strain could gradually degrade feather by modifying the microstructure of feather keratin. A total of 25 protease genes were predicted from the draft genome of JM7 strain, among which a predicted subtilisin-like serine protease(designated as Ker02562) was further characterized for its keratinolytic activity. The recombinant Ker02562 functioned at a wide range of temperatures from 30℃ to 60℃, with an optimum at 40–50℃. Ker02562 was highly active at various pHs ranging from 5.0 to 13.0, with a maximum activity observed at pH 7.0–9.0. Remarkably, recombinant Ker02562 was stable in extreme alkaline environments(pH 10–13), which was much better than most other reported keratinases. Collectively, these favorable properties could make Bacillus sp. JM7 and Ker02562 attractive to be applied in the detergent formulation and feather bioconversion.