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.     

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.     

Egg yolk protein modification by controlled enzymatic hydrolysis for improved functionalities

Delipidated egg yolk protein (EYP) is produced as a co-product of egg yolk lecithin extraction. This EYP is expected to have poor functionality because of protein denaturation caused by ethanol treatment during lecithin extraction. Two food grade endo-proteases were used to produce EYP hydrolysates (EYPh) with two degrees of hydrolysis (DH), 3% and 6%. Protein solubility was improved as DH increased. Solubility profiles for both EYP and EYPh were relatively less pH-dependent compared with soy protein. Except for foaming capacity, EYPh showed improvement in foam stability, foaming speed, and foam density. Emulsion stability was improved for all EYPhs. Treatments at DH of 6% showed significant increase in emulsification capacity. We have shown quantitatively how controlled enzymatic hydrolysis can be applied to ethanol-treated lipid-free EYP to increase protein solubility, and thus to improve foaming and emulsification properties.     

鳄鱼皮酶解产物功能特性及抗氧化活性

为了解鳄鱼皮酶解产物功能特性和抗氧化活性,采用2种商业蛋白酶(木瓜蛋白酶、碱性蛋白酶)在各自最适反应条件下分别酶解鳄鱼皮,研究水解度(DH)、酶种类及pH值对酶解产物功能特性及抗氧化活性的影响.结果显示:随着酶解时间延长,鳄鱼皮水解度逐渐增加,鳄鱼皮在碱性蛋白酶酶解作用下水解度较高,水解4h时可达12％;木瓜蛋白酶酶解产物与碱性蛋白酶酶解产物的溶解性差异不显著(P＞0.05).相同水解度下,碱性蛋白酶酶解产物的热稳定性在pH4时优于木瓜蛋白酶酶解产物.酶解时间在1h之内,木瓜蛋白酶酶解物亚铁离子螯合力明显增强;随着时间延长,酶解产物亚铁离子螯合能力变化不显著(P＞0.05).酶解3h后碱性蛋白酶酶解产物亚铁离子螯合能力高于木瓜蛋白酶酶解产物,但木瓜蛋白酶酶解产物具有较强的清除DPPH自由基能力.综上表明,碱性蛋白酶水解作用的鳄鱼皮水解度较高,其酶解产物乳化活性和热稳定性优于木瓜蛋白酶酶解产物;鳄鱼皮酶解产物抗氧化能力较强,有较高的开发利用价值.     

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.     

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.     

鲤鱼鱼肉蛋白酶水解物抗氧化性及功能特性

采用碱性蛋白酶对鲤鱼鱼肉蛋白进行酶解,制备不同水解度的水解物。测定水解物的抗氧化活性以及不同pH值条件下水解物的功能特性。结果表明:随着水解度的逐渐升高,水解物的抑制脂质氧化能力、D PP H自由基清除能力、还原能力以及金属离子(Cu2+和Fe2+)螯合能力逐渐增加(P<0.05);同时,水解物的溶解性、乳化性和起泡性都在pH值为4.0(等电点)时达到最低,而后溶解性和乳化性随着pH值升高而增大(P<0.05),而起泡性随着pH值的升高先上升后又下降。因此,鲤鱼鱼肉蛋白碱性蛋白酶水解物可以提高蛋白质的抗氧化活性和溶解性,但是较高的水解度会在一定程度上降低其乳化性和起泡性。     

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.     

不同蛋白酶水解高温花生粕和低温花生粕及其水解产物抗氧化活性的对比研究

由于高温花生粕中的花生蛋白在高温压榨过程中高度变性,因此在食品工业中蛋白利用率较低。本研究通过对比高温花生粕和低温花生粕经过不同商业蛋白酶(Alcalase 2.4 L,Neutrase,Papain,Protamex及Flavorzyme 500 MG)水解后水解产物特性的蛋白回收率、水解度、分子量分布及抗氧化活性,确定高温花生粕是否适合采用生物酶解的方式利用其中的蛋白质并筛选合适的蛋白酶。结果表明,高温花生粕经不同蛋白酶水解后,其蛋白质利用率均在60.61~67.86%,与低温花生粕相当;水解度及分子量分布方面,高温花生粕Flavorzyme水解产物的DH最高,高达44.92%,且含有较多的3 ku小分子肽及游离氨基酸;此外,高温花生粕不同酶水解产物的DPPH自由基清除活性均高于低温花生粕,这可能是由于高温花生粕水解产物中含有较多具有供电子的小分子肽、游离氨基酸以及高温压榨过程中生成的美拉德反应产物。     

Modification of the nitrogen solubility properties of soy protein isolate following proteolysis and transglutaminase cross-linking

The effect of (a) limited hydrolysis [0.5–2.0% degree of hydrolysis (DH)] with Alcalase™, (b) cross-linking with transglutaminase (TGase) and (c) combinations of these modifications on the nitrogen solubility (pH 3–8) of soy protein isolate (SPI) was investigated. Between pH 3.0 and 5.0, SPI hydrolysates, hydrolysates of cross-linked SPI and the cross-linked products of SPI hydrolysates displayed significant (P<0.05) increases in solubility compared to unmodified SPI. Cross-linking pre- or post hydrolysis did not alter the overall trend of increased (P<0.05) solubility relative to the unmodified control at low pH. At 2% DH, cross-linking pre- or post-hydrolysis resulted in greater solubility (P<0.05) than that observed in hydrolysates per se at low pH. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the 22 kDa 11S basic polypeptide was relatively resistant to Alcalase hydrolysis and that the 18 and 22 kDa 11S basic polypeptides were not susceptible to TGase cross-linking. The results demonstrate that a combination of enzymatic treatments and the order in which they are applied may have potential for creating novel food ingredients with improved functional properties, especially those properties that are dependant on high solubility at low pH.     

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.     

酶水解制备具有潜在免调节活性大豆肽的研究

由于已有的一些研究报道初步表明带正电荷的小分子肽具有免疫调节作用,本研究分别应用Alcalase、Flavourzyme、Protease A和Peptidase R四种商业酶,单独使用或将它们进行组合,分别以大豆分离蛋白（SPI）、可溶性大豆蛋白（SSP）和不溶性大豆蛋白（InSP）为底物,在不同的酶-底物比、底物浓度、pH和温度条件下进行水解大豆蛋白,测定了水解度、水解产物的分子量分布和带正电荷肽的相对含量.结果表明,Alcalase在相同条件下可以获得较高的水解度和较大的水解蛋白平均分子量;以InSP作为底物的水解产物中带正电荷肽的相对含量最高.水解条件对水解度有较明显的影响,但是平均分子量和正电荷肽相对含量的影响较弱。     

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.     

花生蛋白水解工艺的优化及水解产物分子量分布的研究

本实验主要研究了花生蛋白水解的最佳工艺及水解产物肽分子的分布。考察了酶的种类、加酶量、时间、温度和pH 值五个单因素对花生蛋白水解度(DH)的影响,采用三元二次旋转正交组合设计进行试验,建立了工艺参数间的数学模型,得出对水解率影响较大的三个因素的最佳范围：酶解温度44.9～47.6℃、pH7.93～8.15、酶解时间2.46～2.76h。得到花生蛋白最高水解度为19.61%。水解产物中肽分子主要分布在1000D 以下,含量高达84.73%。     

酶解大豆分离蛋白乳化特性的研究

利用枯草芽孢杆菌AS1．398中性蛋白酶对大豆分离蛋白进行水解，并利用浊度法测定了不同水解度、不同pH条件下酶解大豆分离蛋白的乳化特性，结果表明：AS1．398蛋白酶对大豆分离蛋白的最大水解度为36％，水解度为9％时乳化活性最大，水解度为3％是乳化稳定性最好。同一水解度时，pH越高，蛋白质的乳化特性越好。水解度为3％、9％、15％的大豆分离蛋白在pH等于或高于5．0时的乳化活性明显地高于原蛋白质，且水解度为3％时乳化稳定性也明显地高于原蛋白质。     

Hydrolysis degree,peptide profile and phenylalanine removal from whey protein concentrate hydrolysates obtained by various proteases

The action of various proteases was tested for preparing whey protein concentrate (WPC) hydrolysates with high degree of hydrolysis (DH), appropriate peptide profiles and reduced phenylalanine (Phe) content. The peptide profile analysis included the fractionation of hydrolysates by size‐exclusion HPLC. The rapid correct fraction area method was used to quantify the components of the chromatographic fractions. Activated carbon (AC) was used to remove Phe, and its efficiency was evaluated by measuring the amount of Phe by second‐derivative spectrophotometry. The results showed that the DH of WPC hydrolysates increased and that the protease from Aspergillus oryzae yielded the highest DH value. This protease also produced the best peptide profile, that is, the highest di‐ and tripeptide content (16.14%), the highest amounts of free amino acids (18.43%) and the lowest amount of large peptides (18.76%). The proteases from both A. oryzae and Bacillus subtilis produced the highest Phe removals (79.0 and 77.8%, respectively).     

鳕鱼和鲅鱼鱼肉蛋白酶解产物功能特性及抗氧化性

采用风味蛋白酶对鳕鱼和鲅鱼鱼肉进行酶解,研究水解度、pH值及酶解时间对酶解产物功能特性和抗氧化活性的影响.结果表明:随着酶解时间延长,鳕鱼和鲅鱼鱼肉酶解产物的水解度、亚铁离子螯合力逐渐增加,DPPH自由基清除能力逐渐下降;不同pH值下,鳕鱼和鲅鱼鱼肉蛋白酶解产物均具有良好的溶解性和热稳定性;鲅鱼酶解产物的水解度、溶解性、热稳定性和亚铁离子螯合力显著高于鳕鱼酶解产物.     

响应面法优化软枣猕猴桃蛋白水解及多肽的抗氧化研究

以酶解产物清除羟基自由基能力为指标,选用碱性蛋白酶为水解酶,利用响应曲面法优化软枣猕猴桃蛋白最佳酶解工艺条件并制取抗氧化肽。考察其水解度和清除率的相关性。结果表明：碱性蛋白酶最佳酶解工艺为温度50℃、pH9、加酶量4000U/g、酶解时间3h,此时水解度达到最大值为25.08%。在此条件下将软枣猕猴桃蛋白分别水解1、2、3、4、5h得到肽混合物进行抗氧化活性分析,得到其对羟自由基清除率分别为18.69%、24.67%、28.04%、25.82%、26.65%。当酶解时间为3h时,此时抗氧化肽的羟自由基清除率最高。     

Effect of limited hydrolysis of soy protein on the interactions with polysaccharides at the air–water interface

The objective of the work was to study the effect of limited hydrolysis of soy protein on the interactions with polysaccharides with and without surface activity at the air–water interface at neutral pH where a limited incompatibility between macromolecules can occur. The surface pressure and phase angle as a function of time were evaluated with a drop tensiometer at 20 °C, pH 7 and ionic strength 0.05 M.Hydrolysates of 2% (H1) and 5.4% (H2) degree of hydrolysis (DH) with neutral protease from Aspergillus oryzae were obtained from a commercial soy protein isolate. The polysaccharides used were: hydroxypropylmethylcellulose (HPMC) as surface active polysaccharide; lambda carrageenan (λC) and locust bean gum (LB) as non-surface active polysaccharides.It was found that increasing DH decreased the surface pressure and increased film viscoelasticity (determined as the phase angle, θ) of soy protein hydrolysates and the nature of protein–polysaccharide interactions was strongly affected by DH. The presence of polysaccharides led to an increase of surface pressure of H1 but when added to H2, HPMC and λC decreased the surface pressure.The less hydrolyzed protein H1 gave rise to a higher surface pressure and film viscoelasticity in combination with the polysaccharides. This result points out that a limited protein hydrolysis was sufficient to improve the surface properties of soy proteins if used in combination with polysaccharides.Polysaccharides used in admixture with hydrolyzed soy proteins could control and improve the stability of foams and emulsions not only by increasing bulk viscosity but also by improving film viscoelasticity.     

The evaluation of proteases as coagulants for soy protein dispersions

The ability of different proteases to induce the gelation of soy protein isolate dispersions (5.33% w/w) was studied. The coagulation time and gel firmness were determined using dynamic viscoelastic measurements. Among the six protease tested, papain was the most effective coagulant in terms of gel strength and coagulation speed; the second was alcalase. Degree of hydrolysis (DH), pH and viscosity profiles of soy proteins were tested during the coagulation with different proteases. The result suggested that strong interactions, other than electrostatic interaction, existed between peptides in papain and alcalase induced coagulation. Thermal and pH stability tests indicated that papain was more stable than alcalase in the temperature (60–90 °C) and pH (5.8–7.0) ranges studied. Higher papain dosages within the range of 5–13.3 U/ml resulted in firmer soy protein gels, but concentrations higher than 13.3 U/ml produced weaker gels. With the addition of 0.625 mM cysteine, the soy protein coagulation ability of papain was improved. A soy protein gel formed with 0.025% papain in the presence of 0.625 mM cysteine had about the same strength as that induced by 0.133% papain without cysteine.