酪蛋白水锯物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL.再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值.结果 表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物.当游离氨基减少量为154.65 μmol/g(蛋白)时,修饰产物的IC50值可降至0.6 μg/mL.毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化.研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高.     

乙醇-水相中酪蛋白水解物修饰及对2个性质的影响

利用Alcalase水解酪蛋白,制备水解度为11.6％、IC5值为42.8 mg/L的酪蛋白水解物.在乙醇-水体系中采用Alcalase催化类蛋白反应修饰酪蛋白水解物,固定反应时间6h,优化得到酶添加量、乙醇体积分数、底物质量浓度、反应温度分别为:8.36 kU/g,56.8％,568 g/L,37.5℃.制备不同反应程度的修饰产物,评估其ACE抑制活性及Zn2+螯合能力变化,发现修饰产物的ACE抑制活性得到改善,抑制最高达到62.5％(IC50达到27.7 mg/L),但是与反应程度有关;Zn2+螯合能力则由4.22 mg/g降低至1.97～3.86 mg/g.修饰产物的Zn2+螯合能力与类蛋白反应程度无关,与ACE抑制活性也不存在相关性.     

酪蛋白水解物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL。再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值。结果表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物。当游离氨基减少量为154.65μmol/g(蛋白)时,修饰产物的IC50值可降至0.6μg/mL。毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化。研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高。     

丙醇-水介质中酪蛋白水解物的酶修饰与ACE抑制活性变化

利用碱性蛋白酶水解酪蛋白,制备出水解度为12.6％、ACE抑制活性为48.2％的酪蛋白水解物.利用碱性蛋白酶、在丙醇-水介质中进行类蛋白反应修饰酪蛋白水解物,研究反应温度、酶添加量、底物质量浓度、丙醇浓度对修饰反应的影响.响应面法试验设计,得到最优条件为反应温度47℃、酶添加量8.3 kU/g,底物质量浓度56.8 g/100 mL,丙醇体积分数58.5％.利用此条件制备出的反应程度不同的5个修饰产物,ACE抑制活性分析结果显示,抑制活性最高可以达到63.8％.在相应条件下加入酪氨酸或苯丙氨酸,对比试验结果显示,添加苯丙氨酸或酪氨酸会导致修饰产物抑制活性增加或降低.     

酪蛋白水解物的类蛋白反应修饰及其产物ACE抑制活性特征

采用碱性蛋白酶水解酪蛋白,制备水解度为10.9%、IC50值为52.6μg/mL的酪蛋白水解物,并利用响应面法优化碱性蛋白酶催化的类蛋白反应修饰条件。修饰反应时间固定为6h时,适宜的条件为酶添加量3.1kU/g pro、底物质量浓度50g/100mL、反应温度25℃。制备9个修饰程度不同的修饰产物,结果显示：修饰产物ACE抑制活性均提高,并且活性最高的修饰产物的IC50降低至14.9μg/mL。该修饰产物离心分级后,上清液部分和沉淀部分的ACE抑制活性分别低于和高于修饰产物,表明沉淀部分是提高ACE抑制活性的主要原因;Tricine-SDS-PAGE电泳分析表明,修饰产物及沉淀部分有较大分子质量的肽分子生成;该修饰产物和上清液部分、沉淀部分的进一步酶水解处理则显示,酶水解会导致它们的ACE抑制活性降低,但是仍然高于最初的酪蛋白水解物。     

酪蛋白水解物的Plastein反应修饰及ACE抑制活性变化

采用中性蛋白酶水解酪蛋白,一定条件下制备水解度为13.0%、IC50质量浓度为40.4 mg/L的酪蛋白水解物。利用中性蛋白酶对所制备出的水解物进行plastein反应修饰,以反应体系的游离氨基量的变化为评价指标,通过单一因素试验研究酶添加量、底物质量分数、反应时间和反应温度对修饰反应的影响。结果表明,适宜的反应条件为中性蛋白酶添加量3 kU/g蛋白质、底物质量分数60%、反应时间6 h、反应温度20℃。制备5个不同反应程度的修饰产物,ACE抑制活性分析结果显示,修饰产物的IC50降至14.7~31.1 mg/L,表明中性蛋白酶催化的plastein反应修饰提高酪蛋白水解物的ACE抑制活性,且ACE抑制活性的提高程度与plastein反应程度有关。     

酪蛋白水解物的中性蛋白酶修饰及其ACE抑制活性

采用碱性蛋白酶水解酪蛋白,制备水解度为13.5%、IC50为45.23μg/mL的酪蛋白水解物,然后利用中性蛋白酶对水解物进行类蛋白反应修饰,并研究酶添加量、底物浓度、反应温度和时间对修饰反应的影响。结果表明,修饰反应体系中水解反应占优势,表现为游离氨基含量增加;酶添加量、底物浓度、反应时间对修饰反应的影响显著,而反应温度的影响不大;在低酶添加量、高底物浓度和短反应时间下,修饰反应体系的游离氨基的增加幅度减少,水解反应相对降低。制备6个不同反应程度的修饰产物,ACE抑制活性分析结果显示,修饰产物的IC50降至15.56~19.98μg/mL,表明中性蛋白酶催化的类蛋白反应修饰可以提高酪蛋白水解物的ACE抑制活性。     

乙醇-水体系中酪蛋白水解物的类蛋白反应及ACE抑制活性的变化

采用Neutrase 0.8L蛋白酶水解酪蛋白,制备水解度为13.6%的酪蛋白水解产物,测得其对血管紧张素转化酶(ACE)的体外抑制活性IC50为(46.92±0.27)mg/L。在乙醇溶剂中,利用Neutrase 0.8L蛋白酶对水解物进行类蛋白反应修饰,并研究酶添加量、底物质量分数、反应温度、反应时间和乙醇浓度对修饰反应的影响。在优化条件下的类蛋白反应体系中,游离氨基浓度减少,说明合成反应占优势;酶添加量、底物质量分数、乙醇质量分数对修饰反应的影响显著,而反应时间和温度影响不大。通过单因素实验确定类蛋白反应的最适反应条件为:44%乙醇水溶液、反应温度为40℃,酶添加量为3 kU/g蛋白质、底物质量分数40%、反应时间6.0 h。此条件下,反应体系中游离氨基浓度变化达到202.19μmol/g蛋白质,修饰产物的IC50值降低至(25.96±0.29)mg/L,降低44.7%。     

花生分离蛋白水解物中血管紧张素转化酶抑制肽的分离纯化与结构鉴定

利用碱性蛋白酶Alcalase对花生分离蛋白进行水解,制备花生分离蛋白水解物,并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制作用。未水解的花生分离蛋白没有ACE抑制活性,而利用碱性蛋白酶Alcalase水解所得的水解物具有很强的ACE抑制活性,水解30 min时水解物活性最高,其半抑制浓度为(IC50)0.56 mg/mL。通过超滤、Sephadex G-15凝胶过滤层析、反相高效液相色谱(RP-HPLC)、基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF-MS/MS)和氨基酸组成分析等分析手段从水解30 min所得的水解物中分离鉴定出两种新的ACE抑制肽,氨基酸序列为Gln-Gly-Gly-Ser-Gly-Met-Thr-Leu-Ala-Phe-Pro-Leu-Pro-Lys和Lys-Ile-Phe-Leu-Arg-Leu-Ser,其IC50值分别为10.6μmol/L和36.6μmol/L。     

油菜蜂花粉ACE抑制活性酶解物的制备及分离

采用4 种蛋白酶水解油菜蜂花粉蛋白制备ACE 抑制活性物质,高效液相色谱法测定油菜蜂花粉蛋白水解物对ACE 的抑制率。结果表明：油菜蜂花粉蛋白酶水解物具有ACE 抑制活性,水解物对ACE 的抑制活性差异显著(P ＜ 0.05),其中碱性蛋白酶＞中性蛋白酶＞木瓜蛋白酶＞酸性蛋白酶,碱性蛋白酶水解物的IC50 为0.35mg/mL。4 种蛋白酶水解物经Bio P-2 凝胶分离后,ACE 抑制活性较强的组分主要集中在保留时间70～120min,在此区间碱性蛋白酶水解物分离组分对ACE 的抑制率达到90% 以上,分子质量在376.4～1355D 之间。     

Preparation of Alcalase-catalyzed casein plasteins in the presence of proline addition and the ACE-inhibitory activity of the plasteins in vitro

Casein hydrolysates were prepared by hydrolysis of casein with alkaline protease Alcalase for 6 h and showed the highest ACE-inhibitory activity in vitro with an IC₅₀ value of 47.1 μg mL⁻¹. Casein hydrolysates prepared were subjected to Alcalase-catalyzed plastein reaction in the presence or absence of proline addition to prepare casein plasteins. Some optimal reaction conditions of plastein reaction in the presence of proline addition were studied using response surface methodology with the decrease in free amino groups in the casein plasteins as response. When the concentration of casein hydrolysates was fixed at 35% (w w⁻¹) and reaction time at 6 h, the optimal conditions were reaction temperature 48 °C, addition level of proline 0.54 mol/mol free amino groups of casein hydrolysates and addition level of Alcalase 9.5 kU g⁻¹ proteins. With these conditions, the maximal decrease in free amino groups in casein plasteins was 195.7 μmol g⁻¹ proteins. The ACE-inhibitory activities of twelve casein plasteins in vitro, prepared in the presence or absence of proline addition with different reaction extents, were evaluated and compared. The results showed that the ACE-inhibitory activity of the casein plasteins prepared in the presence of proline addition changed irregularly, different to that of the casein plasteins prepared in the absence of proline addition, and might relate to the different linking of proline to the peptides in casein hydrolysates during plastein reaction. When the casein plasteins prepared in the presence of proline addition had a decrease in free amino groups 195.7 μmol g⁻¹ proteins, the IC₅₀ value of the casein plasteins was lowered to 0.2 μg mL⁻¹.     

Charakterisierung pankreatischer Casein-Plasteine

Characterization of pancreatic casein plasteins. In the course of the plastein reaction hydrophobic peptides concentrate mainly in the aggregates (plasteins), whilst hydrophilic peptides remain in solution (supernatant). Liquid chromatographic and sequence analytical studies of pancreatic casein plasteins have shown that the aggregates consist mainly of the free amino acids tyrosine, phenylalanine and tryptophan. Plasteins contain, in addition, short-chain peptides, particularly from the C-terminal of β-casein. Characterization of the functional properties of the plasteins has shown clearly that aggregation of the short-chain peptides and free amino acids is brought by non-covalent, hydrophobic and ionogenic interactions. In the supernatants resulting from the plastein reaction caseinophosphopeptide sequences, in particular from α_s-casein, were determined.     

不同因素对酪蛋白酶解产物与锌盐螯合效果的影响

本研究采用酶解法制得酪蛋白酶解产物,分析了不同锌源、酶解产物浓度、酶解产物与Zn2+质量比、反应体系pH值、反应温度、螯合时间等因素对酶解产物与锌盐螯合效果的影响,确定了酪蛋白酶解产物与Zn2+螯合的适宜反应条件。研究表明,ZnSO4·7H2O与酪蛋白酶解产物的螯合能力显著高于醋酸锌或氯化锌与酪蛋白酶解产物的螯合能力(p<0.05)。当酶解产物浓度为0.04g/ml、酶解产物与Zn2+质量比为4:1、反应pH值为6.0、反应温度为40℃、螯合时间为20min时螯合效果较好。     

Plasteins. Their preparation, properties, and use in nutrition

The theoretical and practical aspects of the plastein reaction, which consist in the formation of a gel following the addition of an endopeptidase to a concentrated solution of a partial protein hydrolysate, are examined and the properties and possibilities of using plasteins in nutrition are discussed. It is shown that valuable protein food products can be obtained with the aid of the plastein reaction from proteins with an unbalanced amino acid composition and from chemically synthesized amino acids. Other applications of plasteins in nutrition are discussed and the studies carried out hitherto on the mechanism and driving forces of plastein formation are considered.     

Preparation and characteristics of yak casein hydrolysate–iron complex

The effect of mass ratio between yak casein hydrolysate and FeSO₄, reaction temperature, pH and holding time on ferrous‐binding capacity of yak casein hydrolysate was investigated. Iron‐releasing percentage and structural characteristics of the formed yak casein hydrolysate–iron complex were also examined. Results showed that casein hydrolysates with different hydrolysis degrees (DH) possessed different ferrous‐binding capacities with the highest of 7‐h hydrolysate. The optimal binding conditions between yak casein hydrolysate and iron are mass ratio of casein hydrolysate to FeSO₄ of 15:1 for 20 min at 40 °C and pH 6.0. The principal binding site for Fe²⁺ in yak casein hydrolysate consists of the carboxylate groups and amide groups. Compared with ferrous sulphate, yak casein hydrolysate–iron complex has higher iron‐releasing percentage at basic conditions, such as pH 7.0, 7.5 and 8.0. Ferrous‐iron enriched casein hydrolysate, i.e. yak casein hydrolysate–iron complex, may have great potential as an effective delivery vehicle of bioavailable iron.     

酪蛋白的胰蛋白酶水解物分离及其抗氧化性研究

采用胰蛋白酶水解酪蛋白,使用D201阴离子交换树脂对酪蛋白酶解液进行分离,并收集5个多肽片段（P1、P2、P3、P4、P5）,评价酪蛋白酶解液及分离所获得的酪蛋白多肽的抗氧化性。结果表明,酪蛋白多肽的还原力和DPPH自由基清除力均显著高于酪蛋白酶解液（P＜0.05）;多肽P2的还原力最高,为0.234,然后是多肽P3,为0.057;多肽P4的DPPH自由基清除活力最高,达到（23.8±0.18）%,然后是多肽P3,为（4.5±0.16）%;多肽P5的Fe2+螯合率最高,为（99.80±0.12）%,多肽P3的Fe2+螯合率最低,为（2.50±0.14）%,而酶解液的Fe2+螯合率为（31.27±5.80）%;多肽P2和P3不具备脂质过氧化抑制力。多肽P1、P4、P5具有较好的抗氧化性。     

Preparation and characterisation of an easily absorbabl Mg‐casein hydrolysate complex produced through enzymatic hydrolysis and ultrafiltration

In this study, we synthesised a Mg‐casein hydrolysate complex that allowed the effective absorption of Mg. The type of enzyme (papain, alcalase 2.4 L, pepsin, trypsin) and the enzyme/substrate ratio for casein hydrolysis was optimised. When the enzyme/substrate ratio was 30%, the alcalase 2.4 L‐hydrolysate showed the highest Mg‐chelation efficiency, of 96.1%. To characterise and enhance the function of casein hydrolysate, we fractionated the casein hydrolysate according to molecular weight using ultrafiltration. The Mg‐chelation efficiency was increased with the decrease in the molecular‐weight range of the hydrolysate fractions. The smallest casein hydrolysate (fraction 5, 1 kDa<) is used for preparation of Mg‐casein hydrolysate complex. Synthesised Mg‐casein hydrolysate complex (fraction 5) exhibited 100% Mg solubility and 39.5% Mg bioavailability. These results indicated that the Mg‐casein hydrolysate remained a stable chelate during simulated gastro‐intestinal digestion in vitro. The Mg‐casein hydrolysate complex exhibited excellent antioxidant activity as well as Mg binding.     

The effect of enzymatic modification on lysinoalanine formation in field-bean protein isolate and beta-casein

Lysinoalanine (LAL) was determined in alkali-treated partial hydrolysates (PH) of casein, peptides isolated from these PH and in PH of field-bean protein to clarify whether intermolecular or intramolecular LAL bridges are preferentially formed. Furthermore, the formation of LAS in plasteins was studied as a contribution to plastein research. The formation of LAL in the peptide mixtures of beta-casein and the decrease of the LAL content in the PH (as compared to intact proteins) indicates that the formation of LAL favours the intramolecular cross-linking of polypeptide chains. The LAL content decreases as the degree of hydrolysis of the PH of the field-bean protein isolate increases, and depends upon the protease used in the production of the hydrolysates. The LAL contents of the alkali-treated plasteins are less than those of the initial hydrolysates. The decrease of the LAL content is directly proportional to the hydrolysis proceeding during the plastein reaction.     

Ace Inhibition and Enzymatic Resistance In Vitro of a Casein Hydrolysate Subjected to Plastein Reaction in the Presence of Extrinsic Proline and Ethanol- or Methanol-Water Fractionation

Casein was hydrolyzed by alcalase to a degree of hydrolysis of 10.9% to obtain a hydrolysate having ACE-inhibition in vitro with an IC₅₀ value of 52.6 μg/mL. The prepared hydrolysate was modified by alcalase-catalyzed plastein reaction with extrinsic proline added at 0.4 mol/mol free amino groups (on the basis of the hydrolysate), and fractionated by ethanol- or methanol-water solvents in proportions of 3:7, 5:5, or 7:3 (v/v), respectively. With the decrease of free amino groups of the modified hydrolysate as the response, the optimized plastein reaction conditions were alcalase addition of 3.1 kU/g peptides, substrate concentration of 50% (w/v), and reaction temperature of 25°C. Four modified hydrolysates prepared with different reaction times exhibited higher ACE-inhibitory activities than the original hydrolysate. The evaluation results showed that solvent fractionation of the modified hydrolysate with the maximum activity (IC₅₀ = 13.0 μg/mL) yielded the separated soluble fraction's higher activity but the precipitate fraction's lower one. Further enzymatic digestion of the modified hydrolysate with the maximum activity and its two fractionated products by four proteases in vitro caused damage to the activities, but the residual activities of the final digests were higher than that of the original hydrolysate, indicating that the plastein reaction could confer casein hydrolysate protease resistance.