白果活性蛋白的酶法水解及抗氧化活性研究

白果活性蛋白(GAP)是华中农业大学天然产物化学实验室首次从白果中分离得到的一种以清蛋白为主的且具有显著生物活性的蛋白,为扩大该蛋白在食品中的用途,提高其生物活性,研究了酶法水解制备白果肽段的方法及其抗氧化活性的变化。通过单因素分析、正交试验以及对试验结果的分析,确定了2709碱性蛋白酶水解白果活性蛋白的最佳水解条件为：底物浓度1%,pH值8.5,水解温度45℃,酶浓度7000 U/g,反应时间6 h。以Fenton体系和邻苯三酚体系检验其抗氧化活性,结果表明白果肽段较酶解前的活性蛋白,抗氧化活性有不同程度的提高,但两者均具有较强的抗氧化活性。     

红娘鱼降血压肽的酶解制备工艺优化及酶解产物的抗氧化活性研究

为有效利用红娘鱼制备降血压肽,以红娘鱼鱼糜为原料提取蛋白,并对其进行酶解制备降血压肽。以血管紧张素转换酶ACE抑制率和水解度为指标,通过响应面分析法对酶解红娘鱼鱼糜蛋白制备降血压肽的工艺条件进行优化,并对最优条件下制备的酶解产物进行分子量和抗氧化活性测定。结果表明,碱性蛋白酶是制备降血压肽的最适蛋白酶,响应面法优化制备降血压肽的最佳酶解条件为pH值9、酶与底物的比值(酶底比)1.4%、温度54℃、时间2 h,此条件下酶解制得的降血压多肽ACE抑制率理论值为88%,实际值为89.3%;经高效液相色谱(HPLC)分析可得酶解产物相对分子量<2 000 Da。通过测定酶解产物样品的1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率、羟自由基(·OH)清除率及还原力判定其体外抗氧化活性,结果表明酶解产物具有较强抗氧化活性。本研究结果为红娘鱼的高值化利用提供了数据支持和理论基础。     

基于抗氧化能力的牛血红蛋白的分步酶解工艺参数优化

为探究牛血加工利用新技术,提高牛血蛋白资源利用率。该研究采用分步酶解法提取牛血红蛋白的抗氧化肽粗提物,从6种蛋白酶中筛选出最适蛋白酶组合,通过单因素试验优化温度、pH值、料液比、酶添加量和酶解时间,并通过响应面设计进一步优化酶添加量和酶解时间,得到牛血红蛋白抗氧化肽粗提物的最佳酶解工艺。结果表明：牛血红蛋白分步酶解的最佳工艺为：料液比40 g/L,一次酶解：风味蛋白酶添加量3 800 U/g、时间130 min、温度50 ℃、pH值7.5;二次酶解：碱性蛋白酶添加量2 900 U/g、时间60 min、温度40 ℃、pH值9.0。此工艺条件下的牛血红蛋白抗氧化肽粗提物的ABTS自由基清除能力为（333.62±6.29）μmol /g,具有优良的抗氧化活性,可作为食源性抗氧化肽的来源。该研究为牛血等副产物的综合加工利用提供了新思路,同时为食源性抗氧化肽的研发提供理论基础。     

秋刀鱼制备黄嘌呤氧化酶抑制肽的工艺优化

为探讨秋刀鱼制备黄嘌呤氧化酶(XOD,xanthine oxidase)抑制肽的最佳工艺,该文以氮回收率和体外XOD抑制活性为指标,首先通过单因素试验确定中性蛋白酶和胰酶为水解秋刀鱼制备XOD抑制肽的最佳蛋白酶。在此基础上,采用响应面分析法研究加酶量、酶解时间和中性蛋白酶所占比例对酶解氮回收率及酶解产物XOD抑制活性的影响,进一步优化并最终确定了水解秋刀鱼制备XOD抑制肽的最优工艺:料液比1∶2(g/g),总加酶量0.3%(中性蛋白酶∶胰酶质量比=6∶4),在p H值7.0和55℃条件下酶解6 h,其理论氮回收率和酶解产物XOD抑制率分别为72.69%和30.32%,对应实际值分别为72.03%和30.96%,预测模型可靠性高,可用于秋刀鱼XOD抑制肽的酶法制备,为工业化利用秋刀鱼生产降尿酸肽提供一定的理论和技术指导。     

微射流均质预处理提高大豆分离蛋白酶解效率及酶解产物乳化性能

为了探讨微射流均质预处理对大豆分离蛋白酶解效率及酶解产物乳化性能的影响,该文研究比较了微射流均质预处理前后大豆分离蛋白酶解产物的理化性质(水解度、亚基组成、蛋白溶解性、表面疏水性和分子量分布)和乳化性能(通过测定分析样品乳状液的平均粒径和微观结构评估样品的乳化性能)的变化。研究表明:大豆分离蛋白经过微射流均质预处理后采用木瓜蛋白酶水解,其酶解产物(水解度为1.7%)与对照大豆分离蛋白和未经预处理的酶解产物相比,在较低浓度下(30 g/L)制备出粒径细小的稳定乳状液(体积平均粒径≈1.6μm)。微射流均质预处理提高了大豆分离蛋白中α-7S和A-11S亚基的酶解敏感性,使酶解产物在水解度1.3%~1.7%范围内蛋白溶解性显著增加(P0.05),同时保持较高的表面疏水性值,与未经预处理的酶解产物相比形成了更多具有界面活性的可溶性多肽(分子量主要分布在11.3 k Da左右),在乳化过程中可有效防止乳液滴间发生桥联絮凝。因此微射流均质预处理是一种辅助提高大豆蛋白酶解效率和酶解产物乳化性能行之有效的方法。研究结果可为大豆蛋白深加工蛋白乳化剂提供理论和方法参考。     

超声辅助竹节虾头酶解及抗氧化肽分离研究

为了实现竹节虾加工副产物的高值化利用,以竹节虾加工废弃物中的虾头副产物为原料,以水解度和DPPH清除率作为评价指标,采用中性蛋白酶酶解,通过响应面法优化超声辅助酶解工艺,并依次通过超滤、凝胶层析色谱和反相高效液相色谱等分离方法,从竹节虾虾头酶解产物中分离制备抗氧化肽,采用超高压液相色谱串联质谱联用技术对肽的结构进行表征。结果表明,在中性蛋白酶添加量为3 000 U·g~(-1)、p H值7.0条件下,最佳超声辅助酶解工艺参数为超声时间41 min,超声温度55℃,超声频率22 k Hz,料液比1∶9(w/v),在此条件下获得的酶解产物DPPH清除率达69.50%。当水解时间为0.5~2.5 h时,超声辅助酶解的酶解产物水解度和DPPH清除率比非超声辅助酶解工艺分别高17.95%和18.83%,该工艺缩短了酶解时间,节约了能耗。酶解产物经超滤初步分离发现,相对分子质量在3k Da(SHP4)的组分具有显著的抗氧化活性。用凝胶层析法进一步分离纯化SHP4组分后得到4个峰,其中SHP4-II的DPPH清除率最高;SHP4-II通过反相高效液相纯化后也得到4个主要肽峰,在多肽含量为1.5 mg·m L~(-1)时,SHP4-II-4的DPPH清除率最高,达到85.69%,并且具有较好的分离度,质谱分析发现,该抗氧化肽的结构为Gly-Asn-Gly-Leu-Pro(455.99 Da)。本研究结果为虾肽抗氧化保健食品的研发提供了一定的科学依据。     

琥珀酰化和蛋白酶改性对小麦面筋蛋白功能性质的影响

对小麦面筋蛋白进行琥珀酰化和蛋白酶复合改性以提高其溶解性及其他特性。对复合改性面筋蛋白与原面筋蛋白、琥珀酰化面筋蛋白、碱性蛋白酶改性面筋蛋白进行了比较。结果表明:在pH 3~11、水解度4%~12%的范围内,复合改性面筋蛋白的溶解度亦随着水解度的增大而增大,比原面筋蛋白、酰化面筋蛋白、中性蛋白酶和碱性蛋白酶的改性产物都高。起泡性和起泡稳定性则先增加后降低,在水解度4%时具有较佳值,但在各水解度下较单一改性的面筋蛋白产物都要低。添加复合改性面筋蛋白面团黏弹性和面包口感较好,内部结构均匀、细腻。     

鸡肉蛋白热处理与酶解特性的关系研究

为阐明酶解前热处理对鸡肉蛋白酶解性质的影响，采用差示扫描量热法分析鸡肉蛋白的热性质，研究热处理温度对鸡肉蛋白巯基(SH)、二硫键(S-S)含量以及碱性蛋白酶(Alcalase)、木瓜蛋白酶(Papain)酶解过程中氨基酸、肽释放的影响。结果表明：鸡肉蛋白有4个吸热峰，对应温度为63.7℃、67.6℃、74.3℃和 77.9℃；热处理温度增加，鸡肉蛋白中SH含量逐渐降低，而S-S含量逐渐增加，游离SH与S-S还原折算的SH量之和在80℃前无明显变化，80℃后下降；酶解前热处理不利于鸡肉蛋白酶解过程中游离氨基酸、小分子量肽的释放和可溶性氮的回收，但有利于大分子量肽的生成，因此可根据酶解产物的应用目的选择热处理参数。     

酶解鹰嘴豆蛋白制备抗氧化肽工艺优化研究

为优化 Alcalase 蛋白酶酶解鹰嘴豆蛋白制备抗氧化肽的工艺条件,采用响应面分析法,以还原能力、超氧阴离子捕获率为响应值,研究了酶与底物的比值([E]/[S])、酶解温度和酶解时间对制备抗氧化肽工艺的影响.综合考虑成本和工艺要求等问题,最终确定酶解鹰嘴豆蛋白制备抗氧化肽的工艺条件为:底物浓度2%,pH值8.0,[E]/[S]2.72%,温度52℃,时间31 min.该条件下制备的鹰嘴豆酶解产物还原能力和超氧阴离子捕获率分别为0.667和61.55%,与理论预测值的相对误差在±1%以内,说明利用该文建立的模型在实践中进行预测是可行的.     

分步酶解制备花生短肽的研究

为了高效制备花生短肽,在花生蛋白Alcalase酶水解的研究基础上,进一步采用N120P酶水解花生蛋白并对各种影响因素进行了系统地研究;建立了短肽得率与各种影响因素的回归模型;确定出了N120P继续酶解花生蛋白Alcalase酶解物制备花生短肽的最佳工艺参数为pH值6.0,水解时间65min,水解温度57℃,酶与底物比2061U/g,.在此条件作用下,体系中短肽得率为89.01%,比Alcalase单独酶解提高10.86个百分点;水解度为23.76%,平均肽链长度为4.21.经高效液相色谱测定,大部分水解产物的相对分子质量小于1000.     

Enzymatic hydrolysis, grease permeation, and water barrier properties of zein isolate coated paper

An inexpensive zein-lipid mixture was isolated from yellow dent, dry-milled corn. Grease permeation through zein isolate applied to brown Kraft paper was found to be independent of loading levels at zein isolate levels above 30 mg/16 in.(2). The data shows that water vapor transmission rates depended on the amount of coating applied. Triacylglycerols were the most abundant lipid in milled corn but were absent in the zein isolate (perhaps due to hydrolysis by lipases). Zein from the paper was hydrolyzed enzymatically and the hydrolysis monitored by SDS-capillary electrophoresis. At an E:S ratio of 1:100 no further increase in the hydrolysate peak occurred after 10 and 30 min for alpha-chymotrypsin and pancreatin 8 x; however, zein and lipid were still present 1 h after hydrolysis by pancreatin 1 x.     

Reducing, radical scavenging, and chelation properties of in vitro digests of alcalase-treated zein hydrolysate

The objective of the study was to assess the antioxidant potential of alcalase-treated zein hydrolysate (ZH) during a two-stage (1 h of pepsin --> 0.5-2 h of pancreatin, 37 degrees C) in vitro digestion. Sephadex gel filtration and high-performance size exclusion chromatography were used to separate ZH into fractions. The amino acid composition, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+*)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH*) free radical scavenging activity, reducing power, and Cu (2+) chelation ability were tested to determine the antioxidant efficacy of ZH. Results showed that in vitro digests of ZH contained up to 16.5% free amino acids, with short peptides (<500 Da) making up the rest of the mass. The ABTS(+*) scavenging activity of ZH was decreased by 27% (P<0.05) after pepsin treatment but was fully recovered upon subsequent pancreatin digestion, while the DPPH* scavenging activity of ZH was substantially less than ABTS(+*) scavenging activity and showed a 7-fold reduction following pancreatin treatment. The reducing power of ZH increased 2-fold (P<0.05) following pancreatin digestion when compared with nondigested ZH. The ability of ZH to sequester Cu (2+) was reduced by pepsin digestion but was reestablished following pancreatin treatment. The antioxidant activity demonstrated by in vitro digests of ZH (1-8 mg/mL) was comparable to or exceeded (P<0.05) that of 0.1 mg/mL of ascorbic acid or BHA. The results suggested that dietary zein alcalase hydrolysate may have the benefit to promote the health of the human digestive tract.     

Analysis of taste-active compounds in an enzymatic hydrolysate of deamidated wheat gluten

Hydrolyzed plant proteins are widely used as ingredients in culinary products for their glutamate-like ("umami") taste. Three hydrolysates were prepared from wheat gluten using different enzymatic approaches. Comparison of their taste profiles revealed the enzymatic hydrolysate of an acid-deamidated wheat gluten (WGH-3) to be the least bitter of all and to elicit an intense glutamate-like taste. Its umami taste intensity was similar to that of an enzymatic hydrolysate in which glutaminase had been employed to convert free glutamine to glutamic acid and which had a 3-fold higher concentration of free glutamate. Reconstitution studies based on the results of the chemical analysis of WGH-3 and sensory comparison of the model solution and WGH-3 indicated that other components in addition to glutamate and organic acids contribute to its glutamate-like taste. WGH-3 was fractionated by gel permeation chromatography and reversed phase high-performance liquid chromatography, and two fractions with a pronounced glutamate-like taste were obtained. In one of them four pyroglutamyl peptides were tentatively identified: pGlu-Pro-Ser, pGlu-Pro, pGlu-Pro-Glu, and pGlu-Pro-Gln. Apparently, these peptides were formed by cyclization of the N-terminal glutamine residues during the preparation of the hydrolysates.     

Preparation and Functional Properties of Gluten Hydrolysates with Wheat‐Bug (Eurygaster spp.) Protease

Suni‐bug (Eurygaster spp.) enzyme was partially purified from bug‐damaged wheat and used to prepare gluten hydrolysates at 3% and 5% degree of hydrolysis (DH). Functional properties of gluten and gluten hydrolysates were determined at 0.2% (w/v) protein concentration and pH 2–10. Gluten solubility after enzymatic hydrolysis increased significantly (P < 0.05) up to 89.1, 89.6, and 95.0% at pH 7, 8, and 10, respectively. Emulsion stability (ES) of gluten hydrolysates improved at neutral and alkaline pH (P < 0.05) and emulsifying capacity (EC) increased significantly (P < 0.05) except at pH 10. Foaming capacity (FC) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6, 7, 8; foam stability (FS) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6 and 7. Enzymatic modification of gluten by wheat‐bug enzyme resulted in hydrolysates with higher antioxidant activity compared to gluten. Significant correlations (P < 0.001) were found between solubility and EC, ES, FC, and FS values of gluten and its hydrolysates with 3% and 5% DH.     

Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis

Non-specific monoaminopeptidase (AP; E.C. 3.4.11) and X-prolyl dipeptidyl aminopeptidase (X-PDAP; E.C. 3.4.14.5), both from Aspergillus oryzae, demonstrate strong synergism in hydrolyzing proline-containing peptides. Incubation of AP alone with the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe does not generate free amino acids. However, when AP and X-PDAP are added in combination, complete and immediate hydrolysis of all peptide bonds, other than X-Pro bonds, is observed. In the enzymatic hydrolysis of casein, soy, and gluten, degree of hydrolysis (DH) values of 54, 54, and 47% were achieved, respectively, when subtilisin (E.C. 3.4.21.62) was supplemented with AP. Addition of a third enzyme, X-PDAP, resulted in significantly higher DH values of 69, 72, and 64%, respectively, establishing the utility of this synergism in protein hydrolysis.     

Hydrophobicity,Solubility, and Emulsifying Properties of Enzyme-Modified Rice Endosperm Protein

Rice endosperm protein was modified to enhance solubility and emulsifying properties by controlled enzymatic hydrolysis. The optimum degree of hydrolysis (DH) was determined for acid, neutral, and alkaline type proteases. Solubility and emulsifying properties of the hydrolysates were compared and correlated with DH and surface hydrophobicity. DH was positively associated with solubility of resulting protein hydrolysate regardless of the hydrolyzing enzyme, but enzyme specificity and DH interactively determined the emulsifying properties of the protein hydrolysate. The optimum DH was 6–10% for good emulsifying properties of rice protein, depending on enzyme specificity. High hydrophobic and sulfhydryl disulfide (SH-SS) interactions contributed to protein insolubility even at high DH. The exposure of buried hydrophobic regions of protein that accompanied high-temperature enzyme inactivation promoted aggregation and cross-linking of partially hydrolyzed proteins, thus decreasing the solubility and emulsifying properties of the resulting hydrolysate. Due to the highly insoluble nature of rice protein, surface hydrophobicity was not a reliable indicator for predicting protein solubility and emulsifying properties. Solubility and molecular flexibility are the essential factors in achieving good emulsifying properties of rice endosperm protein isolates.     

Influence of Degree of Protein Aggregation on Mass Transport Through Wheat Gluten Membranes and Their Digestibility—An In Vitro Study

The influence of the network structure of wheat gluten on the barrier properties against enzymes was investigated in vitro. The changes in the network structure were introduced by different temperature treatments. The modifications were assessed with solubility studies of wheat gluten proteins in sodium dodecyl sulfate (SDS). The physical barrier properties of wheat gluten membranes were investigated with transport studies examining the transfer of a model protein with no enzymatic activity (BSA) through gluten membranes. The protein network was an effective barrier for BSA, although lightly cross‐linked films were mechanically instable. Membrane breaks occurred in function of the cross‐linking density (percentage of SDS‐insoluble proteins) after only 24 hr for lightly cross‐linked films (≈30% SDS‐insoluble proteins), while highly cross‐linked membranes (≈80% SDS‐insoluble protein) were tight up to more than 33 days. The digestion experiments of the gluten films with pepsin showed that the hydrolysis of wheat gluten films with >72% of SDS‐insoluble protein was significantly retarded. In conclusion, technological treatments to increase the cross‐linking density of gluten have the potential to slow the digestion of cereal‐based foodstuff and to reduce the degradation rate of composite biomaterials.     

Inhibition of lipid oxidation in cooked beef patties by hydrolyzed potato protein is related to its reducing and radical scavenging ability

Protein hydrolysates were prepared by limited alcalase hydrolysis (0.5, 1, and 6 h, corresponding to degrees of hydrolysis of 0.72, 1.9, and 2.3, respectively) of heat-coagulated potato protein. The hydrolysates were characterized for peptide composition, ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activity, and Fe2+- and Cu2+-chelation capacity. Hydrolyzed and intact proteins were formulated (4%, w/w) into beef patties to determine in situ antioxidant efficacy. Thiobarbituric acid-reactive substances (TBARS) and peroxide value (PV) formed in cooked and PVC-packaged patties during storage (4 degrees C, 0-7 days) were analyzed. Hydrolysis increased the protein solubility by 14-19-fold and produced numerous short peptides (< 6 kDa). The FRAP values of the protein sample (23 micromol/g) increased markedly after hydrolysis but were similar between the three hydrolysates (597-643 micromol/g). Similarly, the ABTS radical-scavenging activity also was increased by hydrolysis and was the greatest for the 1-h hydrolysate. Hydrolysis increased the Cu2+-chelation activity but decreased the Fe2+-chelation ability of the protein. The production of PV in patties after 7 days of storage was lowered 44.9% and 74.5% (P < 0.05), and that of TBARS was reduced 40.9% and 50.3% (P < 0.05), by intact and hydrolyzed proteins, respectively.     

Quantitative analysis of cystine, methionine, lysine, and nine other amino acids by a single oxidation--4 hour hydrolysis method

The sulfur-containing amino acids cystine and methionine play important roles in animal, especially avian, nutrition. Because these sulfur-containing amino acids are destroyed to varying extents by 6N HCl hydrolysis, oxidation and hydrolysis of cystine to cysteic acid and methionine to methionine sulfone have been widely used for determination of cystine and methionine. Lysine is considered the next limiting amino acid after the sulfur amino acids in poultry nutrition; therefore, determination of the amino acid content of rations focuses first on these 3 amino acids. The objective of this investigation was to establish whether lysine and other amino acids could be accurately determined in proteinaceous materials which had undergone performic acid oxidation. To perform this evaluation, lysine was determined in a variety of protein-containing materials both with and without performic acid oxidation. Performic acid oxidation followed by 6N HCl hydrolysis at 145 degrees C for 4 h allows accurate measurement of 3 amino acids especially important to poultry nutrition, cystine, methionine, and lysine, in a single preoxidized hydrolysate; this method can be extended to another 9 protein amino acids.     

Gluten hydrolysis and depolymerization during sourdough fermentation

Hydrolysis and depolymerization of gluten proteins during sourdough fermentation were determined. Neutral and acidified doughs in which microbial growth and metabolism were inhibited were used as controls to take into account the proteolytic activity of cereal enzymes. Doughs were characterized with respect to cell counts, pH, and amino nitrogen concentrations as well as the quantity and size distribution of SDS-soluble proteins. Furthermore, sequential extractions of proteins and analysis by HPLC and SDS-PAGE were carried out. Sourdough fermentation resulted in a solubilization and depolymerization of the gluten macropolymer. This depolymerization of gluten proteins was also observed in acid aseptic doughs, but not in neutral aseptic doughs. Hydrolysis of glutenins and occurrence of hydrolysis products upon sourdough fermentation were observed by electrophoretic analysis. Comparison of sourdoughs with acid control doughs demonstrated that glutenin hydrolysis and gluten depolymerization in sourdough were mainly caused by pH-dependent activation of cereal enzymes.