酶解大豆蛋白制备风味增强肽

利用不同蛋白酶酶解大豆蛋白质,大豆蛋白酶解过程中氨基酸的释放规律表明,氨基酸的释放与蛋白酶的种类和酶解时间有关.在酶解产物中,以蛋白酶Ⅰ酶解产物中鲜味氨基酸和含硫氨基酸含量最高,醇厚感强,更适合于酶解大豆蛋白制备风味基料.通过感官评定比较了酶解液及其Maillard反应产物的呈味差异,发现Maillard产物具有更好的醇厚感和鲜味.分析了酶解液中不同分子量级分Maillard反应产物的呈味特点,发现相对分子质量3 000～5 000的级分其Maillard产物能够明显地增强风味,包括鲜味、醇厚味以及后味.     

不同蛋白酶对蓝蛤酶解液风味特性的影响

为得到风味良好的蓝蛤酶解液,选用复合蛋白酶、木瓜蛋白酶、菠萝蛋白酶、碱性蛋白酶和中性蛋白酶进行单酶酶解,采用固相微萃取-气相色谱-质谱联用、电子鼻、电子舌及氨基酸自动分析仪等对不同酶解液的风味特征进行综合评价,利用滋味活性值评价滋味物质的呈味作用和强度。结果表明,复合蛋白酶酶解液的水解度较高,达到28.02%;不同酶解液的风味轮廓之间存在较大差异,电子鼻、电子舌能较好地区分不同酶解液的气味和滋味差异;鲜味和甜味是蓝蛤酶解液的主要呈味成分,复合蛋白酶组中游离氨基酸总量最高,碱性蛋白酶组中鲜味和甜味氨基酸比例最大。气相色谱-质谱联用分析共检出50 种挥发性物质,其中醛类和醇类物质最丰富,一些具有腥味的醛类在碱性蛋白酶组中相对含量最高,在复合蛋白酶和中性蛋白酶组中相对含量较低,具有增香效果的2-乙基呋喃和2-戊基呋喃在复合蛋白酶组中相对含量较高,复合蛋白酶酶解液的风味较佳。     

组合酶对复合骨素酶解液呈味物质的影响

为探究组合酶对牛骨素和鸡骨素的复合骨素酶解液呈味物质的影响,选取四种组合酶(木瓜蛋白酶+风味蛋白酶、菠萝蛋白酶+风味蛋白酶、碱性蛋白酶+风味蛋白酶、复合蛋白酶+风味蛋白酶)制备复合骨素酶解液,测定四种复合骨素酶解液的水解度、游离氨基酸、呈味核苷酸、味精当量(Equivalent umami concentration,EUC)、肽分子量分布等呈味物质指标,并进行对比分析。结果表明:碱性蛋白酶+风味蛋白酶(Alkaline proteinase+Flavourzyme,A+F)和复合蛋白酶+风味蛋白酶(Protamex+Flavourzyme,P+F)酶解液的水解度最大,分别为10.67%和11.27%;对呈味游离氨基酸组成分析发现,A+F酶解液鲜味氨基酸、苦味氨基酸、无味氨基酸含量最高,A+F和P+F酶解液总游离氨基酸含量最高;四种酶解液中肌苷酸较另两种核苷酸含量高,A+F酶解液总核苷酸含量最高;比较四种酶解液味精当量,A+F酶解液EUC值最大;A+F和P+F酶解液中分子量<1000 Da肽段含量最高,制备复合骨素酶解液的呈味效果更好;主成分分析表明A+F组合酶综合得分最高,A+F组合酶为美拉德反应提供丰富反应底物。     

不同蛋白酶制备鹅肉呈味肽的对比分析

以鹅肉为原料,采用中性蛋白酶、碱性蛋白酶、风味蛋白酶、木瓜蛋白酶制备呈味肽,对比分析4 种酶解液中的水解度、寡肽含量,采用氨基酸自动分析仪对酶解液游离氨基酸组成进行测定,并利用电子舌和感官评定方法对酶解液的鲜味等味道进行滋味评定。结果表明：45 ℃恒温水浴酶解6.5 h,加酶量1 200 U/g、pH 7.0、固液比1∶3（g/mL）的条件下,木瓜蛋白酶酶解液水解度最大且寡肽质量分数最高,其次是中性蛋白酶,鹅肉蛋白水解度达到29.69%,寡肽质量分数达到0.18%。此外,中性蛋白酶酶解液的风味最好。中性蛋白酶酶解后产生的游离氨基酸类型丰富,谷氨酸和丙氨酸的含量高,最终酶解液整体鲜味浓郁,并伴有酸味。因此,确定酶解鹅肉蛋白的最佳用酶为中性蛋白酶。     

不同蛋白酶制剂对牛肉的酶解效果研究

以复合蛋白酶、木瓜蛋白酶、胰蛋白酶和风味蛋白酶对牛肉进行预处理,测定酶解液的理化指标、多肽分子量分布、游离氨基酸等,筛选出适宜蛋白酶。结果表明,复合蛋白酶在可溶性氮、氨基酸态氮,水解度等指标上优于其他种类蛋白酶制剂。添加木瓜蛋白酶、复合蛋白酶的酶解液在2 KDa～5 KDa、小于1 KDa的多肽分布较高,这两部分多肽对食品风味具有重要作用。添加复合蛋白酶酶解液的氨基酸总量最多,其中鲜味氨基酸含量明显高于其他组别,苦味氨基酸含量低于其他组别。结合酶解液的感官评定结果,复合蛋白酶是比较适宜酶解牛肉的酶制剂。     

复配酶法制备南极磷虾鲜味酶解液的工艺优化

以脱脂南极磷虾粉为原料,采用复配酶法制备鲜味酶解液。以感官评价、多肽得率、水解度等评价手段,进行外源酶的筛选及单因素实验。为进一步优化酶解液滋味,以呈鲜味的氨基酸总量为指标进行酶解工艺优化。结果表明:最佳酶解条件为:胰蛋白酶和胃蛋白酶复配比1:100,总加酶量为2800 U/g,料液比1:4 g·mL?1,酶解pH7.0,酶解温度40 ℃,酶解时间3 h,该条件下获得的酶解产物中鲜味游离氨基酸含量为331.79 mg/100 mL。除色氨酸外,其余八种必需氨基酸含量占总氨基酸的63.66%,游离苦味氨基酸占总氨基酸的27.83%,游离鲜甜味氨基酸占总氨基酸26.22%,与鲜味形成具有关键作用的游离谷氨酸和游离天冬氨酸共占总游离氨基酸的10.26%。本研究可为南极磷虾鲜味调味料等产品的开发提供理论基础和技术指导。     

南美白对虾虾头制备鲜味水解物的研究

本文以25 ℃下自溶6 h后的南美白对虾虾头为原料,采用酶水解法进行鲜味水解物的制备。以鲜味滋味、蛋白质回收率、肽得率和水解度为评价指标,进行外源酶的选择和酶解工艺条件的逐步优化研究。研究表明,经四种单酶酶解,均明显提升酶解效率,其中菠萝蛋白酶(Bromelain)和风味蛋白酶(Flavorzyme)能有效提升酶解液的鲜味、降低苦涩味。制备具有良好鲜味特征水解物的条件为:自溶后的虾头,加入菠萝蛋白酶(420 U/g样品)和风味蛋白酶(12 U/g样品)两种酶(料液比1:1.5 (g/mL)),在pH7.5、50 ℃下酶解3 h,该工艺下蛋白质回收率、肽得率和水解度分别可达79.75%、71.71%和18.28%。获得水解物氨基酸组成中ΣEAA/ΣAA与ΣEAA:ΣNEAA分别为37%和1:1.7。65%的总蛋白氨基酸释放为游离氨基酸,其中,59%的总蛋白鲜甜味氨基酸释放为游离氨基酸,水解物营养价值高且具有良好鲜味。     

牡蛎复合酶解提高蚝油风味的工艺探讨

采用多种酶复配的方法对牡蛎进行酶解处理,以获得风味良好、且蛋白质回收率和水解程度都比较高的产品.研究结果表明采用含0.05%中性蛋白酶+0.1%碱性蛋白酶+0.1%风味蛋白酶+0.1%复合蛋白酶的复合酶,可以使蛋白质回收率达到85%、水解度达到43.5%,酶解后的游离氨基酸含量比酶解前提高122.2%,酶解液无腥味,且...     

鸡骨渣水解用酶的筛选

以鸡骨架高压蒸煮后的鸡骨渣为原料,研究不同蛋白酶对其酶解效果及酶解产物氨基酸的组成。结果表明：风味蛋白酶具有较强的水解能力,水解度(DH)达到23.15%;其酶解液澄清度高并具有较强的鸡鲜味;其酶解产物中7 种必需氨基酸含量为68.90%,谷氨酸、甘氨酸、天冬氨酸和精氨酸等风味氨基酸分别占氨基酸总量的5.09%、1.40%、2.59% 和10.45%。     

牡蛎复合酶解液的制备及其风味成分的测定

以酶解液的风味作为主要考察因素,对各种蛋白酶对牡蛎酶解的效果进行比较,确定了最佳的酶解时间为18h,最佳组合方式为0.1%木瓜蛋白酶+0.1%碱性蛋白酶+0.15%风味蛋白酶+0.15%菠萝蛋白酶.在此基础上利用复合酶水解制备牡蛎酶解液,并测定了酶解液的游离氨基酸组成,呈鲜味和甜味的氨基酸,总量达到61.19 mg/m...     

罗非鱼鱼皮鱼鳞蛋白的酶解及超滤分离

采用Alcalase2.4L、Protamex、Papain、PTN6.0S和Neutrase酶解罗非鱼鱼皮鱼鳞蛋白,探讨了超滤对酶解液分子质量分布和氨基酸组成的影响。结果表明,Alcalase2.4L、PTN6.0S酶解产物的蛋白质回收率、肽得率和水解度均较高,Alcalase2.4L∶PTN6.0S为1∶2添加时酶解效果最好,蛋白回收率为96.37%,水解度为13.24%,肽得率为83.13%。经超滤处理后,3000 u以下分子质量的肽段达到97.73%,比超滤前增加了5.37%,总氨基酸含量由84076.12 mg/100 g增加到97234.79 mg/100 g。     

Comparison of hydrolysis characteristics on defatted peanut meal proteins between a protease extract from Aspergillus oryzae and commercial proteases

A crude protease extract (CPE) was prepared from Aspergillus oryzae HN 3.042 in this work. Three commercial proteases (Alcalase 2.4L, Protamex and papain) and CPE were employed to hydrolyse defatted peanut meal (DPM). CPE was found to be the most effective protease with protein recovery of 80.6%. Moreover, CPE produced a higher degree of hydrolysis (DH, 43.4%) than the tested commercial proteases. The test of molecular weight distribution indicated that DPM proteins were mainly consisted of >10 KDa fraction (86.6%), whereas 3–6 KDa fraction was observed to be the main fraction of all the hydrolysates. CPE hydrolysate possessed a higher nutritional quality than DPM and other hydrolysates on the basis of FAO/WHO (1991) reference pattern. The sensory taste evaluation showed that CPE hydrolysate had better taste than other hydrolysates.     

Enzymatic hydrolysis of wheat gluten by proteases and properties of the resulting hydrolysates

The insolubility of gluten in aqueous solutions is one of the major limitations for its more extensive use in food processing. Wheat gluten was enzymatically hydrolyzed by several commercially available proteases (Alcalase 2.4L, PTN 6.0S, Pepsin, Pancreatin, Neutrase and Protamex™) with protein recovery of 81.3%, 42.5%, 53.3%, 61.6%, 46.3% and 43.8%, respectively. The hydrolytic efficiency of these proteases on wheat gluten was also compared. Alcalase served best for the preparation of wheat gluten hydrolysates with the maximum degree of hydrolysis (DH) 15.8%. Subsequently, the solubility of wheat gluten hydrolysates (WGHs) obtained with those enzymes was comparably evaluated. The products had excellent solubility (>60%) over a pH range of 2–12. The molecular weight distribution of WGHs was further determined by SDS-PAGE and size exclusion chromatography on Sephadex G-15. The results showed that with the increasing of DH values, there occurred a large amount of smaller polypeptides.     

Proteolysis characteristics of Actinomucor elegans and Rhizopus oligosporus extracellular proteases under acidic conditions

Enzymatic hydrolysis of soybean protein isolate by the extracellular proteases from Actinomucor elegans and Rhizopus oligosporus at pH 3.0, 3.5, 5.0, 5.5 and 6.0 was investigated. The activity of the A. elegans protease is lower than that of R. oligosporus, but both proteases exhibit considerable degradation of soybean protein at pH 5.5 and 6.0. The water‐soluble protein content and the degree of hydrolysis of the hydrolysates are increased significantly, and bitterness values are very low. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) reveals that these proteases have different cutting sites on peptide polymers. At pH 5.5, there is a lower content of total free amino acids (39.20 mg per 100 mL; 62.68% hydrophobic amino acids) in the R. oligosporus protease hydrolysate. In conclusion, treatment with R. oligosporus protease at pH 5.5 achieves efficient degradation of soybean protein, suggesting a promising industrial process for making bitterless protein hydrolysates.     

酸性条件下毛霉和根霉蛋白酶的催化特性

研究比较了Actinomucor elegans蛋白酶和Rhizopus oligosporus蛋白酶在pH 3.0～6.0的条件下催化大豆分离蛋白(SPI)降解的特性,结果表明:在pH3.0和pH3.5的条件下,SDS-PAGE显示毛霉和根霉蛋白酶都能迅速地催化大豆7S和11S蛋白的降解,但此时水溶性蛋白的得率不高,均在35%以下;在pH5.0、5.5和6.0时,毛霉和根霉蛋白酶催化大豆分离蛋白降解的能力增强,水解度升高,水溶性蛋白的得率增加,但毛霉蛋白酶的水解产物水溶性蛋白得率始终高出约10%,SDS-PAGE显示毛霉蛋白酶催化下的大豆分离蛋白各个亚基降解消失的速度下降,同时伴随有新的条带产生,特别是碱性亚基几乎不能在4h内降解,根霉蛋白酶催化下的大豆分离蛋白各个亚基降解消失的速度在pH5.0和5.5时还比较强,但在pH6.0时明显下降,酸性亚基和碱性亚基均不能在4h内消失,表明在酸性条件下2个霉菌的蛋白酶其催化特性有明显的差异。     

PURIFICATION AND CHARACTERIZATION OF PROTEASES FROM HEPATOPANCREAS OF CRAWFISH (PROCAMBARVS CLARKII)1

Four trypsin‐like enzymes (CP‐I, II, III and IV in order of elution on DEAE‐Sepharose chromatography), purified from the hepatopancreas of crawfish, were inhibited by protease inhibitors such as phenyl methyl suifonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), aprotinin and tosyl lysine chloromethyl ketone (TICK). The molecular weights of CP‐I, II, III and IV were determined to be 35.0, 41.2, 37.9 and 39.5 kDa, respectively, using sodium dodecyl sulfate polyacryl‐amide gel electrophoresis (SDS‐PAGE), These proteases had optimal esterase activity at pH 8.0–8.5 and showed the highest activity at 60–70C. Crawfish proteases were rich in acidic amino acids. Activation energies for hydrolysis of tosyl arginine methyl ester (TAME) by these proteases were 6.98 – 8.34 kcal/mole. Unlike other serine proteases, the activities of CP‐I and CP‐II were activated by mercury while CP‐HI and IV were inhibited.     

THERMAL BEHAVIOR, SOLUBILITY AND STRUCTURAL PROPERTIES OF SOY CONCENTRATE HYDROLYZED BY NEW PLANT PROTEASES

A soy concentrate prepared by alcoholic extraction of defatted soy flour was hydrolyzed with three plant proteases: hieronymin and macrodontin, cysteine proteases, and pomiferin, a serine protease. A commercial microbial protease (alcalase) was included for comparative purposes. Working at optimal conditions for each enzyme, 5–15% degree of hydrolysis (DH) values were obtained. Hydrolysates exhibited a characteristic SDS-PAGE pattern: the plant proteases attacked the polypeptides of 7S and 11S proteins with different intensity and selectivity, especially the A and B polypeptides of the 11S protein. Intermediate molecular weight peptides (24 and 60 kDa) were produced as the hydrolysis progressed. Differential Scanning Calorimetry (DSC) thermograms of flour. concentrate and hydrolysates were analyzed to evaluate the thermal stability and denaturation enthalpies of the major proteins. An increase in the degree of protein denaturation resulting from enzymatic action and a lower thermal stability at low pH were detected. The surface hydrophobicity of all hydrolysates, unlike expected, did not increase. Solubility at pH 7.0 is closely related to the DH, independent of the protease used. Solubility at pH 4.5 appeared to be related to the extent of hydrolysis of polypeptide A by each protease.     

鱿鱼肝脏蛋白酶的鉴定及组织蛋白酶L的分离纯化

鱿鱼肝脏含有丰富的蛋白酶,为利用其内源蛋白酶进行可控的酶解,本研究以鲤鱼肌原纤维蛋白为底物对鱿鱼肝脏内源蛋白酶的种类和性质进行了研究。反应体系中添加E-64、1,10-菲啰啉和苯甲基磺酰氟(phenylmethylsulfonyl?fluoride,PMSF)后,肌球蛋白重链(myosin?heavy?chain,MHC)的降解得到了显著抑制,确定了鱿鱼肝脏含有金属类、半胱氨酸类、丝氨酸类3类蛋白酶。半胱氨酸类蛋白酶热稳定性最好,在50℃以上仍然具有较大活性,可将肌原纤维蛋白酶解成小分子质量的降解产物。利用特异性底物对半胱氨酸蛋白酶种类进行鉴定发现,该酶只酶解Z-Phe-Arg-MCA,添加亮抑酶肽后相对酶活性为0%,添加E-64相对酶活性仅存0.6%,初步确定鱿鱼肝脏中的半胱氨酸蛋白酶主要为组织蛋白酶L。最后,通过硫酸铵沉淀、离子交换层析、凝胶过滤对组织蛋白酶L进行分离纯化,在电泳上得到了分子质量约为25?kD单一条带。     

Actinomucor elegans、Aspegillus oryzae和Rhizopus oligosporus产蛋白酶条件及蛋白酶性质的比较

比较了腐乳生产菌株Actinomucor elegans、豆酱和酱油生产菌株Aspegillus oryzae以及天培生产菌株Rhizopus oligosporus产生蛋白酶的条件和所产蛋白酶的性质。结果表明,不同的菌株产酶条件及蛋白酶的性质有较大的差异:少孢根霉主要产生酸性蛋白酶,在pH2.5-4.0的酸性介质中、32℃条件下培养时产酶能力较强,所分泌的蛋白酶系在pH5.0时酶活力最高,在pH5.0附近最稳定;米曲霉可以产生酸性、中性及碱性蛋白酶,所产生的蛋白酶活力显著高于少孢根霉和毛霉,米曲霉在酸性条件下产酸性蛋白酶能力强,在中性条件下产中性蛋白酶能力强,在碱性条件下产碱性蛋白酶能力强,在28-32℃时产酶能力强,所分泌的蛋白酶系在pH5.0-9.0的广泛范围内有很强的活力,在pH6.0-8.0的范围内稳定性强;毛霉可以产生酸性、中性及碱性蛋白酶,但酶活力明显低于米曲霉,毛霉在中性偏酸性(pH5.5)的介质中产酸性蛋白酶的能力较强,但介质的酸碱度对毛霉产中性及碱性蛋白酶没有影响,在28℃时产酸性、中性和碱性蛋白酶的能力都比较强,毛霉所分泌的蛋白酶系在pH5.0-9.0的广泛pH范围内有活力,在pH5.0-6.0时酶活力最高,在pH5.0-7.0时稳定强。     

Characterization of ginger proteases and their potential as a rennin replacement

BACKGROUND: Ginger rhizome (Zingiber officinale Roscoe) contains ginger proteases and has proteolytic activity. Ginger proteases have been used for tenderizing meat but rarely for milk clotting. The purpose of this study was to purify ginger proteases and to research their biochemical characteristics. RESULTS: The milk clotting activity (MCA) and proteolytic activity (PA) of the proteases was stable after storage at 4 °C for 24 h. The MCA and PA of fresh ginger juice with 0.2% L ‐ascorbic acid remained stable for 6 days at 4 °C. When under storage at ?80 °C for 2 months, the MCA and PA of the fresh ginger juice and acetone precipitate were still high. Two peaks with protease activity were purified from a DEAE FF ion‐exchange column; the specific activity (units mg^?1 protein) of the MCA (MCSA) and PA (PSA) for the first peak was significantly higher than the second peak (P < 0.05). The protease activity of the ginger proteases was significantly inhibited by E‐64, leupeptin, and iodoacetic acid. Zymography results showed that two protease fractions purified from ginger juice with 62 and 82 kDa had a higher PA against α‐ and β‐casein than against κ‐casein. CONCLUSION: The ascorbic acid addition significantly stabilized the MCA and PA of ginger proteases. The protease inhibition test suggested that ginger proteases belonged to the cysteine type. The biochemical characteristics of ginger protease described in this paper can provide useful information for making new milk curd products. Copyright © 2009 Society of Chemical Industry