Characteristic Property of Low Bitterness in Protein Hydrolysates by a Novel Soybean Protease D3

ABSTRACT:  Enzymatic hydrolysis is 1 means of improving the functional properties of food protein; however, in most cases, bitter peptides are generated by such treatment, and the resulting product is therefore not acceptable as a food ingredient. We have already reported a novel cysteine protease, D3, purified from germinating soybean cotyledons. Because of its substrate specificities, most hydrophobic amino acid residues in the hydrolysate are presumed not to be located at the peptide termini. It was therefore expected that protein hydrolysate by protease D3 would taste less bitter than other enzymatic hydrolysates. The objective of this study was to demonstrate the low bitterness of protein hydrolysates by protease D3. For that purpose, soy protein and casein hydrolysates were prepared with treatment of protease D3, subtilisin, pepsin, trypsin, and thermolysin, respectively. The bitterness of these hydrolysates was evaluated by measuring points of subjective equality (PSE). The PSE value demonstrated that the protein hydrolysates by protease D3 were significantly less bitter than the other enzymatic hydrolysates, indicating that the products had a taste mild enough to be acceptable as a less-bitter peptide food ingredient. These results suggested that a prominent feature of protease D3 was its capacity to produce less-bitter peptides. Therefore, it is thought that protease D3 could be applied to produce protein hydrolysates for use as ingredients in a variety of food products.     

Physicochemical and Functional Properties of Soy Protein Substrates Modified by Low Levels of Protease Hydrolysis

ABSTRACT: Endo-protease treatments achieving low degrees of hydrolysis (DH 2% and 4%) were used to improve functional properties of hexane-extracted soy flour (HESF), extruded-expelled partially defatted soy flour (EESF), ethanol-washed soy protein concentrate (SPC), and soy protein isolate (SPI). These substrates had protein dispersibility indices ranging from 11% to 89%. Functional properties, including solubility profile (pH 3 to 7), emul-sification capacity and stability, foaming capacity and stability, and apparent viscosity were determined and related to surface hydrophobicity and peptide profiles of the hydrolysates. Protein solubilities of all substrates increased as DH increased. Emulsification capacity and hydrophobicity values of the enzyme-modified HESF and EESF decreased after hydrolysis, whereas these values increased for SPC and SPI. Emulsion stability was improved for all 4% DH hydrolysates. Hydrolyzed SPC had lower foaming capacity and stability. For substrates other than SPC, foaming properties were different depending on DH. Hydrolysis significantly decreased the apparent viscosities regardless of substrate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated differences in the molecular weight profiles of the hydrolysates. HESF and EESF, which had high proportions of native-state proteins, showed minor changes in the peptide profile due to hydrolysis compared with SPC and SPI.     

自然酸度下乳蛋白酶解动态分析及其物化特性研究

通过在自然变化pH值和人工调控恒定pH值两种酸度下,四种蛋白酶水解乳蛋白的动态分析及测定前种方法的酶解产物的主要理化特性,特别是苦味特性,以期筛选出低苦味水解酶及水解工艺.以蛋白水解度(DH)和10%三氯乙酸可溶性氮含量(TCA-SN)为指标,比较四种蛋白酶在两种工艺下水解的情况.以溶解度、黏度、苦味和蛋白水解电泳图谱指标研究了自然pH值条件下四种酶解物的理化特性.结果表明:四种蛋白酶分别在两种处理条件下的水解度和可溶性氮含量差异不大;pH未恒定条件下得到的酶解物与乳蛋白相比,在酸性范围内有较大的溶解度;蛋白浓度升高时,黏度变化不大;四种酶解物中Neutrase和木瓜蛋白酶的苦味最强,Protamex的苦味最小;SDS-PAGE电泳表明,两种中性蛋白酶主要水解乳中的酪蛋白.因此可用本研究选用的四种蛋白酶在pH值自然变化条件下酶解乳蛋白,从而简化酶解工艺,并且该酶解物都有较好的理化特性.     

酶水解大豆蛋白物理化学性质的综合表征

大豆分离蛋白分别用AS1.398中性蛋白酶和黑曲霉3.4310酸性蛋白酶在不同条件下水解后,测定其水解度(DH)、三氯乙酸(TCA)溶解度、等电点(IP)溶解度,以及不同分子量区段的相对含量等13个参数.通过主成分分析将这些参数线性组合为4个主成分,但是中性蛋白酶和酸性蛋白酶水解产物参数在主成分中的组合方式不同.以主成分为指标对全体样品进行聚类分析时,两种酶水解产物各自聚为一类;中性蛋白酶和酸性蛋白酶的水解产物单独进行聚类分析则分别得到5个和4个子类,其中包括水解条件差别较大的水解产物,说明水解工艺参数之间存在某种协同或互补效应.     

Evaluation of bitterness in enzymatic hydrolysates of soy protein isolate by taste dilution analysis

ABSTRACT:  Although enzymatic hydrolysates of soy protein isolate (SPI) have physiological functionality, partially hydrolyzed SPI exhibits bitter taste depending on proteases and degree of hydrolysis (DH). To determine proteolysis conditions for SPI, it is important to evaluate bitterness during enzymatic hydrolysis. Taste dilution analysis (TDA) has been developed for the screening technique of taste-active compounds in foods. The objectives of the present study were to evaluate bitterness of enzyme-hydrolyzed SPI by TDA and to compare bitterness of SPI hydrolysates with respect to kinds of proteases and DH. SPI was hydrolyzed at 50 °C and pH 6.8 to 7.1 to obtain various DH with commercial proteases (flavourzyme, alcalase, neutrase, protamex, papain, and bromelain) at E/S ratios of 0.5%, 1%, and 2%. The DH of enzymatic hydrolysates was measured by trinitrobenzenesulfonic acid method. The bitterness of enzymatic hydrolysates was evaluated by TDA, which is based on threshold detection in serially diluted samples. Taste dilution (TD) factor was defined as the dilution at which a taste difference between the diluted sample and 2 blanks could be detected. As DH increased, the bitterness increased for all proteases evaluated. Alcalase showed the highest TD factor at the same DH, followed by neutrase. Flavourzyme showed the lowest TD factor at the entire DH ranges. At the DH of 10%, TD factor of hydrolysate by flavourzyme was 0 whereas those by protamex and alcalase were 4 and 16, respectively. These results suggest that TDA could be applied for the alternative of bitterness evaluation to the hedonic scale sensory evaluation.     

Rheological properties of soy protein hydrolysates obtained from limited enzymatic hydrolysis

Soy protein products hexane-defatted soy flour, extruded-expelled soy flour, soy protein concentrate and soy protein isolate, were modified by using the enzyme bromelain to 2% and 4% degrees of hydrolysis (DH). Peptide profiles, water solubility, and rheological properties including dynamic shear, large deformation, and apparent viscosities of resulting hydrolysates were determined. Protein subunits profiles for the hydrolysed isolates and concentrates were extensively altered by the treatment while only minor changes were observed for the hydrolysed flours. Water solubility profiles of all hydrolysates in the pH range of 3.0-7.0 were enhanced by hydrolysis. For the unhydrolysed controls, the isolate had the highest storage modulus (G′), followed by the concentrate, the extruded-expelled flour and the hexane-defatted flour. The hydrolysates retained some of their gelling ability even though the losses in storage modulus (G′) were substantial. After heating step to 95 °C, the G′ values of all substrates at 25 °C decreased with increase in DH. Texture profile analyses of the soy protein gels were also lower in hardness after hydrolysis. The Power Law model provided excellent fit to hydrolysate dispersions flow (R²>0.99). Hydrolysis decreased the consistency coefficients of dispersion and increased flow behavior index resulting in thinner dispersions. These results suggest that limited protease hydrolysis of various soy protein meals with bromelain produce soy protein ingredients with modified rheological properties.     

Influence of the extent of enzymatic hydrolysis on the functional properties of protein hydrolysate from grass carp (Ctenopharyngodon idella) skin

Protein hydrolysates from grass carp skin were obtained by enzymatic hydrolysis using Alcalase^®. Hydrolysis was performed using the pH-stat method. The hydrolysis reaction was terminated by heating the mixture to 95 °C for 15 min. At 5.02%, 10.4%, and 14.9% degree of hydrolysis (DH), the hydrolysates were analyzed for functional properties. The protein hydrolysates had desirable essential amino acid profiles. Results demonstrated that the hydrolysates had better oil holding and emulsifying capacity at low DH. The water holding capacity increased with increased levels of hydrolysis. Enzymatic modification was responsible for the changes in protein functionality. These results suggest that grass carp fish skin hydrolysates could find potential use as functional food ingredients as emulsifiers and binder agents.     

Preparation and Functional Properties of Enzymatically Deamidated Soy Proteins

Heat-denatured soy protein was hydrolyzed by Alcalase to 2.0% or 4.0% degree of hydrolysis (DH), heated again at 100°C and deamidated with B. circulans peptidoglutaminase. The extent of deamidation was 6.0% and 8.2% for 2.0 DH hydrolysates and 12.8% and 16.0% for 4.0 DH hydrolysates heated for 15 and 30 min, respectively. Deamidation increased protein solubility and substantially enhanced emulsifying activity under mildly acidic (pH 4–6) as well as alkaline conditions. Deamidation improved emulsion stability and foaming power of heat-denatured hydrolysed soy proteins. Enzymatically deamidated soy protein hydrolysates had improved functional properties compared to nondeamidated hydrolysates and the native soy protein.     

Relevance of the Functional Properties of Enzymatic Plant Protein Hydrolysates in Food Systems

Proteins play a crucial role in determining texture and structure of many food products. Although some animal proteins (such as egg white) have excellent functional and organoleptic properties, unfortunately, they entail a higher production cost and environmental impact than plant proteins. It is rather unfortunate that plant protein functionality is often insufficient because of low solubility in aqueous media. Enzymatic hydrolysis strongly increases solubility of proteins and alters their functional properties. The latter is attributed to 3 major structural changes: a decrease in average molecular mass, a higher availability of hydrophobic regions, and the liberation of ionizable groups. We here review current knowledge on solubility, water‐ and fat‐holding capacity, gelation, foaming, and emulsifying properties of plant protein hydrolysates and discuss how these properties are affected by controlled enzymatic hydrolysis. In many cases, research in this field has been limited to fairly simple set‐ups where functionality has been assessed in model systems. To evolve toward a more widely applied industrial use of plant protein hydrolysates, a more thorough understanding of functional properties is required. The structure–function relationship of protein hydrolysates needs to be studied in depth. Finally, test model systems closer to real food processing conditions, and thus to real foods, would be helpful to evaluate whether plant protein hydrolysates could be a viable alternative for other functional protein sources.     

碱性蛋白酶限制性酶解对蓝圆鲹分离蛋白功能特性的影响

以蓝圆鲹（Decapterus maruadsi）分离蛋白为原料,采用碱性蛋白酶对其进行限制性酶解,研究水解度（degree of hydrolysis,DH）对分离蛋白酶解产物溶解性、持油力、乳化性与起泡性等功能特性的影响。结果表明,碱性蛋白酶酶解产物的相对分子质量显著下降。酶解可有效提高分离蛋白的溶解性,其溶解度随DH增加而增加。与对照组相比,分离蛋白经酶解后,产物乳化性与起泡性均显著提高,并呈现先升高后降低的趋势。不同DH酶解产物在pH值为4.0时的乳化性和起泡性最低;当pH值为10.0时,DH5（DH=5%）的乳化性最高（（100.9±0.7）m2/g）;当pH值为7.0时,DH5起泡性最高（（227.3±3.8）%）。除DH20外,其他组的持油力均有显著提高,其中DH5的持油力最高,达3.50 g/g（油/蛋白）。结果表明,一定程度的水解可以显著提高蓝圆鲹分离蛋白的功能特性。本研究为鱼蛋白在食品蛋白配料中的应用提供了一定理论参考。     

大豆浓缩蛋白的酶法改性及其在西式火腿中的应用

采用酶法对大豆浓缩蛋白进行改性。以水解度（ＤＨ）为３％和６％的酶法改性大豆浓缩蛋白的功能性质相当或超过大豆分离蛋白。将其应用于西式火腿的加工，提高了产品的出品率和质量。     

大豆蛋白酶法水解产物抗氧化活性的研究

人豆蛋白水解产物具有很强的抗氧化性质,山于人豆种植面积广、价格低廉,满足了人们崇尚天然、安全食品的需求,凶此_JF发抗氧化人豆肽具有较好的经济价值和社会意义。该文以人豆分离蛋白为原料,进行了人人豆蛋白酶觯工艺参数的优化研究,在此基础上,研究了人豆肽的分了最分布变化和人豆肽的抗氧化特性。探索人豆制备抗氧化肽的途径。     

罗非鱼副产物蛋白水解肽的呈味分析

为了探究罗非鱼副产物蛋白水解物中滋味活性物质的来源,该研究以罗非鱼的鱼皮、鱼头和鱼骨为原料,采用中性蛋白酶、菠萝蛋白酶、风味蛋白酶、复合蛋白酶、木瓜蛋白酶和碱性蛋白酶分别对三种副产物进行酶解,分析比较水解度、分子量、前体蛋白与滋味活性物质的关系。结果表明：三种罗非鱼副产物经过风味蛋白酶水解酶水解8 h后获得最高水解度,分别为14.29%、23.7%和31.86%。经过木瓜蛋白酶水解的副产物酶解液均呈现出显著苦味或酸味,而鱼头经过碱性蛋白酶酶解得到的水解产物具有鲜味,鱼骨则经过菠萝蛋白酶水解得到鲜味,鱼皮经过中性蛋白酶水解表现出鲜味和酸味,但鱼头却呈现出苦味。通过LC-MS/MS鉴定,胶原蛋白、肌原纤维蛋白以及肌浆蛋白是罗非鱼副产物酶解液滋味活性肽的重要味觉活性前体,这些滋味肽的分子量大部分小于1500 u,肽段中疏水性氨基酸残基对罗非鱼副产物滋味的形成具有重要作用,其中蛋氨酸在酶解液的鲜味的形成中起重要贡献。     

Antioxidant activities and functional properties of soy protein isolate hydrolysates obtained using microbial proteases

Soy protein isolate (SPI) hydrolysates were prepared using microbial proteases to produce peptides with antioxidant activity. The process parameters (substrate and enzyme concentrations), hydrolysis time, functional properties and the effects of ultrafiltration were further investigated. The results showed that the soy protein isolate exhibited a 7.0‐fold increase in antioxidant activity after hydrolysis. The hydrolysis parameters, defined by the experimental design, were a substrate concentration of 90 mg mL^?1 and the addition of 70.0 U of protease per mL of reaction. The maximum antioxidant activities were observed between 120 and 180 min of hydrolysis, where the degree of hydrolysis was approximately 20.0%. The hydrolysis increased solubility of the soy protein isolate; however, the hydrolysates exhibited a tendency to decrease in the interfacial activities and the heat stability. The SPI hydrolysates fractions obtained by ultrafiltration showed that the enzymatic hydrolysis resulted in samples with homogenous size and strong antioxidant activity.     

鳙鱼鱼肉蛋白的酶解及其对功能性质的影响

为了改善鳙鱼鱼肉蛋白(BCMP)的功能性质以扩大其在食品工业中的应用,以鳙鱼为原料制备了鳙鱼鱼肉蛋白,并利用碱性蛋白酶Alcalase2.4L对其进行水解,得到了3种不同水解度(DH4.5%、DH9.0%、DH13.5%)的酶解物。研究了BCMP及其酶解物的功能性质,包括溶解性、持水性、持油性、乳化性、起泡性。结果表明,与原鳙鱼鱼肉蛋白相比,酶解物的功能性质除持油性以外均有不同程度的提高。此外,DH4.5%的酶解物乳化性和起泡性最高,过度水解(DH9.0%、DH13.5%)反而造成乳化性和起泡性下降。     

Interaction and Functionality of Mixed Myofibrillar and Enzyme-hydrolyzed Soy Proteins

ABSTRACT Native and briefly heated (85 °C for 3 min) soy protein isolates (SPI) were partially hydrolyzed (4% DH) by Alcalase® and Flavourzyme™ before incorporation into a pork myofibril isolate (MPI) system. The hydrolysis of soy protein enhanced its interaction with MPI, leading to a decreased thermal stability of both soy and muscle proteins. Alcalase SPI hydrolysates, when compared with nonhydrolyzed SPI, improved viscoelastic properties and hardness of MPI gels, while Flavourzyme SPI hydrolysates had an adverse effect. Hydrolyzed SPI augmented emulsifying properties of MPI; the specific efficacy depended upon the type of enzymes used, the SPI:MPI ratio, and whether SPI was heated before hydrolysis.     

Improvement of functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle proteins hydrolyzed by Bacillus mojavensis A21 proteases

Functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle protein hydrolysates, with different degrees of hydrolysis (DH from 7.3% to 18.8%), obtained by treatment with Bacillus mojavensis A21 alkaline proteases were investigated. Protein contents for all freeze-dried cuttlefish muscle protein hydrolysates (CMPHs) ranged from 80% to 86%. For the functional properties, hydrolysis by A21 proteases increased (p < 0.05) protein solubility to above 78% over a wide pH range (2.0–11.0). However, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity and foaming stability) decreased with the increase of the DH. All CMPHs exhibited significant metal chelating activity and DPPH free radical-scavenging activity, and inhibited linoleic acid peroxidation. Antioxidant properties of protein hydrolysates increased with protein hydrolysis and the highest activities were obtained at DH of 16%. The IC₅₀ values for DPPH radical-scavenging and metal chelating activities were found to be 0.52 ± 0.01 mg/ml and 0.67 ± 0.13 mg/ml. The obtained results suggested that functional properties and antioxidant activities of cuttlefish muscle protein hydrolysates were influenced by the degree of hydrolysis.The composition of amino acids of undigested and hydrolyzed proteins was determined. CMPHs have a high percentage of essential amino acids such as arginine, lysine, histidine and leucine. They have a high nutritional value and could be used as supplement to poorly balanced dietary proteins.     

脱色猪血红蛋白酶解产物品质及营养特性研究

研究了脱色猪血红蛋白酶解产物的功能性、风味和营养特性。在pH3.0～9.0,产物具有良好的溶解性;在pH值为7.0左右时,显示出良好的乳化性和乳化稳定性;深度酶解和脱色导致产物的持油性较差。脱色酶解产物没有苦味,其疏水值与Q准则相互吻合。酶解和脱色可以提高猪血红蛋白的营养特性。脱色猪血红蛋白酶解产物可作为食品原料加以利用。     

Physicochemical and antioxidant properties of buckwheat (Fagopyrum esculentum Moench) protein hydrolysates

The enzymatic hydrolysis of common buckwheat (Fagopyrum esculentum Moench) protein isolate (BPI) by Alcalase and some physiochemical and antioxidant properties of the resulting hydrolysates were characterised. The hydrolysis resulted in remarkable decrease in the globulins or protein aggregates and concomitant increase in peptide fragments. The surface hydrophobicity of the hydrolysates decreased with increasing degree of hydrolysis (DH) and reached a minimum at DH 15%, but increased at further hydrolysis, whereas their amino acid compositions were unchanged. The polyphenol content of the hydrolysates gradually decreased with DH increasing from 0% to 15%, while it on the contrary increased upon further hydrolysis. The hydrolysates exhibited excellent antioxidant activities, including DPPH radical scavenging ability, reducing power and ability to inhibit linoleic acid peroxidation. The antioxidant activities of these hydrolysates were closely related to their polyphenol contents. The results indicated that polyphenol-rich buckwheat proteins are unique protein materials for the production of the hydrolysates with good nutritional and antioxidant properties.     

Valorisation of tuna viscera by endogenous enzymatic treatment

Fish processing industry generates a considerable amount of by-products which represent an environmental problem. Only a small portion of these residues is used for the production of low marketable products. Therefore, industrially scalable processes yielding value-added products would be highly desirable. Different to others studies, this work deals with potential valorisation of bullet tuna (Auxis rochei) viscera using its endogenous enzymes without previous separation stage for the production of bioactive hydrolysates. Functional and bioactive properties of hydrolysates produced at different degree of hydrolysis (DH 3%, 6% and 9%) were evaluated. The endogenous enzyme hydrolysates (EH) obtained by the proposed low cost treatment were compared with hydrolysates produced with a well-known commercial enzyme: subtilisin. Regarding functional properties, EH presented similar or even better solubility, emulsifying and oil binding capacities than subtilisin hydrolysate (SH). EH also showed very interesting antioxidative properties, particularly metal reducing and radical scavenging activity. Additionally, the ACE inhibitory activity of EH at low degree of hydrolysis was comparable to SH. According to these results, bullet tuna viscera protein can be value-added by endogenous enzyme hydrolysis.