蛋白酶对文蛤肉水解效果的研究

本文探讨了木瓜蛋白酶、胰蛋白酶、精制中性蛋白酶、酸性蛋白酶、复合蛋白酶和风味蛋白酶等6种蛋白酶对文蛤肉的水解效果,以文蛤肉水解液的水解度、氮收率及水解得率为指标,并对酶解液进行感官鉴评和风味评分,结果表明精制中性蛋白酶、胰蛋白酶和风味蛋白酶为适用于文蛤肉水解的蛋白酶。     

Antioxidant and Angiotensin-Converting Enzyme Inhibitory Activities of Protein Hydrolysates Prepared from Threadfin Bream (Nemipterus spp.) Surimi By-products

Solid wastes from threadfin bream (Nemipterus spp.) surimi production composed of head and frame were hydrolyzed by various commercial proteases (Alcalase, Flavourzyme, Neutrase, Protamex, papain, and pepsin) to produce protein hydrolysates with bioactive properties. An Alcalase-hydrolyzed sample at 24.4% degree of hydrolysis (DH) displayed the highest antioxidant activity based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP) assay, and potassium ferricyanide method. In addition, it showed an inhibitory activity toward angiotensin-converting enzyme (ACE) of 25.5%. Antioxidant activity of threadfin bream by-product hydrolysates increased with hydrolysis time and reached the highest DPPH activity after 6 h, while that hydrolyzed for 3 h showed the highest reducing power based on FRAP and potassium ferricyanide assays. In addition, ACE inhibitory activity was found to be at an optimum after 3 h of hydrolysis. The hydrolysates (1 mg/mL) also retarded oxidation of a linoleic acid emulsion system to a similar extent as 0.1 mg/mL 3-tert-butyl-4-hydroxy anisole (BHA), indicating a potential use in the food system. Protein hydrolysates from threadfin bream surimi by-products could be tailor-made to possess both antioxidant and ACE inhibitory activity through controlling DH of Alcalase-catalyzed reactions.     

Rheological Behavior of Protein Hydrolysates from Papain-treated Male Gonad of Scallop (Patinopecten yessoensis)

ABSTRACT

In the present study, the rheological behavior of hydrolysates from scallop male gonad hydrolysates (SMGHs) was studied. The hydrolysates were obtained from scallop male gonads (SMGs) using papain. The changes in molecular mass of proteins in SMGHs were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the proteins of SMGs were significantly degraded after hydrolysis with papain. The degree of hydrolysis reached to 17.8% and 20.8% for SMGHs treated by papain at 300 and 3,000 U/g protein, respectively. The SMGHs exhibited a weak strain overshoot behavior as well as non-Newtonian shear thinning flow behavior. Moreover, the rheological properties of SMGHs possessed a papain dose-dependent manner. These results suggest that SMGHs could be applied as a potential gelling and thickening agent in food formulations.     

酶法提取鱿鱼皮胶原蛋白工艺条件的研究

研究了酶法提取鱿鱼皮胶原蛋白的工艺条件。根据5种蛋白酶水解液中羟脯氨酸（HYP）的含量,确定了胰蛋白酶和木瓜蛋白酶这2种提取率高的酶为水解用酶,采用L16（4^5）正交试验确定了这2种酶水解鱿鱼皮以制备胶原蛋白的最佳酶解条件。结果表明,胰蛋白酶和木瓜蛋白酶水解鱿鱼皮以制备胶原蛋白的最佳温度、加酶量、底物浓度、pH、时间分别为55℃、1200U·g^-1、1：20、pH8.0、4h和50℃、3200U·g^-1、1：20、pH6.0、6h。2种蛋白酶提取的胶原蛋白含量分别为11.08%和11.36%,提取率分别为95.16%和97.56%。     

Biochemical and Sensory Changes of Low-Salt Anchovy (Engraulis japonicus) Sauce Prepared by a Novel Technique

Using a process spanning less than 10 days, a novel technique combining two-stage autolysis and external koji fermentation for the rapid preparation of low-salt anchovy sauce is presented. High-efficiency autolysis of anchovy was achieved by two-stage autolysis, and the second-stage autolysis exhibited a significantly positive effect on the improvement of hydrolysis yield and recovery of anchovy proteins. During the external koji fermentation of hydrolysates from two-stage autolysis, the optimum amounts of added water and salt were determined to be 20% and 8.0%, respectively. By investigating the effect of fermentation time on the biochemical and sensory changes of anchovy sauce, the optimal fermentation time was 8 days. The prepared anchovy sauce had an umami taste, was rich in free glutamate, and contained taurine and γ-aminobutyric acid. In addition, it contained 0.890 g/100 mL amino acid nitrogen (AAN), 1.886 g/100 mL total nitrogen, and 9.76% NaCl. Meanwhile, the recovery of total nitrogen (RTN) was 63.89%.     

南极磷虾木瓜蛋白酶酶解产物的营养学评价

分析了经1.5%木瓜蛋白酶55℃酶解3 h后的南极磷虾(Euphausia superba)蛋白酶解产物(上清和沉淀)的营养组成。结果显示:南极磷虾水解度为44.24%;上清冻干物(FDS)中粗蛋白和总糖含量分别为67.13%和13.91%,其必需氨基酸总量(ΣEAA)、鲜味氨基酸总量(ΣDAA)、牛磺酸Tau含量及F值(支链氨基酸/芳香族氨基酸)较酶解前分别提高了8.96%、6.06%、39.10%和16.67%,表明其氨基酸营养价值较酶解前要高。沉淀冻干物(FDP)中粗蛋白和总糖含量分别为44.49%和10.40%,而粗脂肪含量高达33.28%。FDP中总氨基酸含量(ΣAA)、ΣEAA、ΣDAA、Tau含量及F值均下降,但其ΣEAA在ΣAA中的比例高于40%,且8种EAA含量评分均高于FAO/WHO评分模式要求。脂肪酸分析结果表明,FDP中含有饱和脂肪酸(SFA)8种(44.81%),单不饱和脂肪酸(MUFA)6种(27.55%),多不饱和脂肪酸(PUFA)7种(27.64%),二十碳五烯酸EPA和二十二碳六烯酸DHA含量较丰富。研究认为在本实验条件下通过木瓜蛋白酶酶解南极磷虾得到的FDS和FDP具有较高的利用价值,可开发为人类重要的蛋白来源。     

紫菜活性肽复合酶法制备与清除羟自由基作用

以条斑紫菜为原料,利用胰蛋白酶和木瓜蛋白酶组成的复合酶水解紫菜蛋白制备生物活性肽并研究清除羟自由基作用。采用单因素试验考察胰蛋白酶与木瓜蛋白酶的比例、底物质量浓度、酶用量、水解温度、水解时间、pH对羟自由基清除率及水解度的影响,响应面法优化酶解条件,SephadexG-15凝胶层析法测定活性肽的分子质量分布。试验结果表明,复合酶最佳酶解工艺条件为胰蛋白酶与木瓜蛋白酶复合酶比例1.67,底物质量浓度20mg/mL,酶用量1.67%,pH 7.0,温度55℃,酶解4h,紫菜活性肽对羟自由基的清除率为80.6%,清除率为50%时的质量浓度为0.744mg/mL,分子质量大多分布于500～1500ku。     

Effect of Enzyme Type on the Antioxidant Activities and Functional Properties of Enzymatic Hydrolysates from Sea Cucumber (Cucumaria frondosa) Viscera

ABSTRACT

Effects of various proteases were obvious on the amino acid composition, antioxidant activities, and functional properties of enzymatic hydrolysates prepared from sea cucumber viscera (SCV). Alcalase, trypsin, and flavourzyme conferred better antioxidant activities on hydrolysates and endowed them with a higher degree of hydrolysis, coming to 19.08, 32.38, and 15.94%, respectively. For functional properties, neutrase- and flavourzyme-derived hydrolysates showed better foaming properties. Amino acid composition suggested that papain, neutrase, alcalase, and bromelain granted the hydrolysate’s high nutritional quality. The results indicated that SCV hydrolysates could be used in food systems to impart desirable characteristics to products.     

Autolysis of Anchovy (Engraulis japonicus) Protein: Evaluation of Antioxidant Properties and Nutritional Quality of Resulting Hydrolysates

Protein hydrolysis of underutilized marine organisms is a method to improve added value. In the present article, anchovy protein was autolyzed at pH 8.5 and 50°C. After autolysis processing for 24 h, total nitrogen recovery reached 92.1%. The protein hydrolysates had antioxidant properties on reducing power, 1, 1-diphenyl-2-picryhydrazyl radical, and hydroxyl radical scavenging activity. More than 79% of the peptides in the hydrolysates had a molecular weight of lower than 3 kDa. Protein hydrolysates had balanced amino acid compositions, and total content of essential amino acids had no significant difference after hydrolysis. Protein hydrolysates from anchovy autolysis have potential in nutritional and functional foods.     

Optimization of Protein Recovery During Hydrolysis of Yellowfin Tuna (Thunnus albacares) Visceral Proteins

Protein hydrolysates were prepared from yellowfin tuna viscera using Alcalase®. Response surface methodology (RSM) was applied to study the effect of independent variables, enzyme activity, temperature, and time on the protein recovery (PR) as the response. The coefficient of determination (r² = 98%) was satisfactory, and the lack of fit test showed a non-significant value for the PR model equation, indicating that the regression equation was adequate for predicting the PR under tested combinations of values. Enzyme activity at 79.9 AU/kg protein, temperature at 59.9°C, and the time of 105 min were found to be the optimum conditions to reach the highest PR (85%). Considerable soluble protein was released with increasing degree of hydrolysis (DH). The yellowfin tuna visceral hydrolysates' amino acids profiles showed high essential amino acids content, although lysine, methionine, and phenylalanine were limited. In addition, the results showed that with increasing hydrolysis time, the amino acids increased. Yellowfin tuna visceral hydrolysates were rich in flavor amino acids which can be used as a taste enhancer.     

双酶法在鲨鱼肉水解液制备中的工艺改良研究

采用同时添加枯草杆菌中性蛋白酶与木瓜蛋白酶的改良酶解技术，对鲨鱼肉进行水解。正交试验结果表明，上述二酶同时水解的最适条件为：温度45℃，pH7．0，时间3h，酶浓度200U／ml（枯草杆菌中性蛋白酶及木瓜蛋白酶各100U／ml），原料：水＝1．5。蛋白质水解率达83．5％，所得水认的氨基酸含量为45．4mg/ml，其中必需氨基酸总量为19．8mg/ml。     

Antioxidant,ACE-Inhibitory,and Antimicrobial Activities of Peptide Fractions Obtained From Dried Giant Squid Tunics

ABSTRACT

Squid tunics were divided in two batches: freeze-dried and air-dried. Dried squid tunics were directly hydrolyzed with pepsin, Alcalase, and Esperase. Freeze-dried tunics showed better aptitude for hydrolysis than air-dried tunics. Pepsin showed the lowest efficiency in protein breakdown and gave hydrolysates with low antioxidant activity, whereas Alcalase and Esperase peptide fractions showed strong radical scavenging ability, ACE-inhibitory activity, and noticeable antimicrobial properties. B. cereus, B. coagulans, and D. hansenii were found to be the most sensitive bacteria. Dried squid tunics proved to be an alternative to a previously extracted gelatin for obtaining bioactive peptides.     

Nutritive value of squid and hydrolyzed protein supplement in shrimp feed

Though some protein sources like squid and protein hydrolysates are assumed as growth enhancers for shrimp, little is known about the biochemical basis of this phenomenon. Low, heat-dried squid (Dosidicus gigas) (SQ) and two commercial protein hydrolysates from fish (FH) and krill (Euphasia sp.) (KH) were assayed in feeding trials with Penaeus vannamei. Feeds were prepared with the tested proteins at 3%, 9%, and 15% of the total crude protein. A total of nine experimental feeds plus a commercial one as control (C32) were tried. Additionally, digestibility in vivo and in vitro was evaluated. Survival was not different among groups. Weight gain of shrimp and total and specific proteolytic activity for trypsin and chymotrypsin were affected by type and quantity of supplemented protein. In vivo and in vitro digestibilities were also influenced by the source and quantity of the protein supplement. Shrimp fed feed with FH at 3% protein supplementation grew more than those fed with higher supplementations. Groups fed SQ had similar results as those fed FH, and gained more weight when fed the lowest SQ quantity. SDS-PAGE showed a large concentration of small peptides in SQ, which may explain results similar to FH. KH enhanced shrimp growth at all supplementations and had a lower degree of hydrolysis (DH) than FH. SQ also demonstrated good growth performance, but better at the lower supplementation, probably because of the presence of small peptides and possibly free amino acids from protein hydrolyzed by endogenous enzymes in the squid mantle. We conclude that hydrolyzed protein is a good supplement for shrimp feeds, but it must meet specific requirements for adequate assimilation.     

酶解扇贝裙边制备复合氨基酸螯合钙的研究

扇贝裙边富含蛋白质、脂质等营养成分。为了高值化利用扇贝裙边,本研究选用中性蛋白酶、动物蛋白酶、风味蛋白酶、木瓜蛋白酶和酸性蛋白酶5种酶,以酶解液中游离氨基酸态氮为考察指标,对扇贝裙边进行酶解工艺条件探讨。首先,将5种酶制成复合蛋白酶进行正交实验,确定最佳酶解时间、温度、pH及加酶量,经检验,氨基酸转化率为77%;然后,通过实验确定CaCl2为最适钙源,以贝壳为原料,通过水飞法和酸法转化可制得贝壳源CaCl2。将扇贝裙边酶解液中复合氨基酸与来源于贝壳的钙螯合制备复合氨基酸螯合钙,以正交实验筛选出最佳螯合条件。经检验,该螯合反应螯合率达92%。     

Characterization of Collagen Enzymatic Hydrolysates Derived from Lizardfish (Synodus fuscus) Scales

Acid soluble collagen was extracted from the scales of lizardfish and characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis. After 8 h of collagen hydrolysis, hydrolysates had an estimated degree of hydrolysis (DH) from 4 ± 0.05 to 25 ± 0.63% (p < 0.05). All hydrolysates had angiotensin converting enzyme inhibitory and antioxidant activity. These activity levels showed little change after treatment with gastrointestinal proteases. Results indicated that the lizardfish by-products may be improved by enzymatic treatment with acid-soluble collagen from lizardfish scales.     

鲣鳔蛋白抗氧化酶解物制备工艺

为有效提高鲣鳔蛋白的附加值,研究以DPPH自由基清除率为抗氧化活性评价指标,采用蛋白酶酶解制备活性多肽的工艺,选用菠萝蛋白酶、复合蛋白酶、碱性蛋白酶、木瓜蛋白酶、胃蛋白酶、胰蛋白酶、中性蛋白酶7种酶在各自最适的条件下酶解,筛选出复合蛋白酶为最适用酶,通过单因素实验分别研究加酶量、溶液初始p H、酶解温度和时间对酶解物抗氧化活性的影响,在此基础上,根据响应面法优化鲣鳔抗氧化酶解物的制备工艺。结果显示,最佳酶解工艺条件为加酶量8.53 U/mg,p H 5.54,温度50.03°C,时间5.07 h。此外,利用超滤法对最佳条件下制备的酶解物进行初步分级,得到分子质量分别为大于10 000 u、3000~10 000 u和小于3000 u的3段组分,且这3段组分对DPPH自由基的半抑制浓度IC50值分别为0.64、0.52和0.37 mg/m L。研究表明,最优条件下制备的酶解物的DPPH清除率达72.00%,与模型预测值71.60%接近,且其中小于3000 u的组分具有较强的DPPH自由基清除活性。     

超声辅助酶解鲣加工副产物制备蛋白胨

为了提高水产加工副产物的利用率和附加值,以鲣加工副产物为原料,采用胰蛋白酶结合超声处理制备蛋白胨,并分析了鲣加工副产物超声辅助酶解过程中蛋白酶活性、水解度、氮回收率、TCA-可溶性氮回收率、酶解产物分子量分布等指标的变化,探究了超声辅助酶解的作用时间、温度以及超声功率等因素对鲣加工副产物酶解产物的影响,通过单因素实验优化确定了蛋白胨制备的最佳酶解工艺条件。结果显示,超声处理造成胰蛋白酶酶活发生显著变化,在功率为120 W的超声场下,胰蛋白酶酶活在30 min内呈上升趋势,但在超声场处理30 min后,胰蛋白酶的酶活呈明显下降趋势,至4 h后酶活变化趋势趋于平缓。超声功率和酶解温度都会影响胰蛋白酶对鲣加工副产物的酶解效率,在超声功率240 W和酶解温度50°C条件下,氮回收率比无超声处理组提高了18.66%,水解度提高了30.43%,TCA-可溶性氮回收率提高了33.20%,且超声处理有效提高了酶解液中小分子肽的含量。超声辅助酶解鲣加工副产物的最佳工艺条件为温度55°C,超声功率240 W,酶解时间4 h,此时氮回收率为81.40%,TCA-可溶性氮回收率为40.75%。     

Enzymatic Hydrolysis of Catfish (Pangasius hypophthalmus) By-Product: Kinetic Analysis of Key Process Parameters and Characteristics of the Hydrolysates Obtained

In this study, kinetic evaluation of enzymatic hydrolysis of catfish (Pangasius hypophthalmus) by-product meat was conducted. The effects of key factors such as type of proteases (neutrase, papain, and bromelain), enzyme to substrate ratio (E/S), substrate concentration, and extent of hydrolysis on process efficiency, properties and functionalities of hydrolysates were evaluated. The results demonstrated that, for catfish shredded meat, bromelain and papain had higher affinity than neutrase. Kinetics parameters V_m and K_m of neutrase, papain, and bromelain were 4.486, 9.060, and 18.249 μM/min and 35.433, 6.582, and 7.034 g/l, respectively. Optimal E/S ratios of neutrase, papain, and bromelain were estimated to be 0.92, 2.76, and 3.31 mg/g. At optimal E/S, the highest possible substrate concentrations were found to be 108.4 g/l for neutrase, 36.2 g/l for papain, and 135.8 g/l for bromelain. Both kinetics study and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that bromelain and papain had higher proteolytic activity toward catfish protein than neutrase. A significant increase in antioxidant activity of hydrolysates was observed with neutrase and papain hydrolysate, but not with bromelain. The results could be helpful for industrial processors to develop an efficient process for catfish by-product valorization.     

Influence of Degree of Hydrolysis on Chemical Composition,Functional Properties,and Antioxidant Activities of Chinese Sturgeon (Acipenser sinensis) Hydrolysates Obtained by Using Alcalase 2.4L

Protein hydrolysates from Chinese sturgeon were prepared using Alcalase 2.4L enzyme. Under the optimum conditions (enzyme–substrate ratio of 3.5%, pH of 8.5, and temperature of 55°C), the degree of hydrolysis (DH) was 13.8%, 16.7%, and 19.1% after 1, 3, and 6 h, respectively. The contents of crude protein and amino acid increased at DH of 19.1% to 86.97% and 78.29%, respectively. There was an obvious increase in the low-molecular-weight peptides, which could enhance the hydrolysate’s functional properties such as solubility, representing more than 90% at different pH levels. The obtained protein hydrolysates revealed good emulsification properties and high oil absorption. Furthermore, good antioxidant activities such as 1, 1-diphenyl-2-picrylhydrazyl (DPPH) inhibition, ferric reducing power, and ferrous ion (Fe²⁺⁾ chelating ability were attained depending on the solution concentration. The findings indicate that the functional and antioxidant properties of protein hydrolysates could be useful in many applications of the food industry.     

Purification and Identification of an Antioxidative Peptide from Digestive Enzyme Hydrolysis of Cutlassfish Muscle

ABSTRACT

The major objective of this study was to investigate the pepsin digests of cutlassfish muscle for their antioxidant activity. The peptide isolated after 3 h of pepsin hydrolysis demonstrated the highest scavenging activity for both 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and alkyl radicals. It also had the highest yield of the isolates with the highest protein content, which was likely related to its higher degree of hydrolysis than the other hydrolysates. The antioxidant peptide purified from the 3 h pepsin hydrolysate indicated the amino acid sequence, Phe-Ser-Gly-Gly-Glu. This study suggests that cutlassfish muscle is an excellent antioxidant source and could be effectively used as a food ingredient and in pharmaceuticals.