Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease involved in the degradation and inactivation of incretin hormones that act by stimulating glucose-dependent insulin secretion after meal ingestion. DPP-IV inhibitors have emerged as new and promising oral agents for the treatment of type 2 diabetes. The purpose of this study was to investigate the potential of β-lactoglobulin as natural source of DPP-IV inhibitory peptides. A whey protein concentrate rich in β-lactoglobulin was hydrolysed with trypsin and fractionated using a chromatographic separation at semipreparative scale. Two of the six collected fractions showed notable DPP-IV inhibitory activity. These fractions were analysed by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) to identify peptides responsible for the observed activity. The most potent fragment (IPAVF) corresponded to β-lactoglobulin f(78–82) which IC₅₀ value was 44.7 μM. The results suggest that peptides derived from β-lactoglobulin would be beneficial ingredients of foods against type 2 diabetes.     

乳清蛋白复合酶解条件优化及其水解产物对HMG-CoA还原酶活性的影响

采用胰蛋白酶、中性蛋白酶A.S1398组成复合酶联合水解乳清蛋白,建立了三因素(水解温度T℃,初始pH,胰蛋白酶活性占总活性的百分比)与乳清蛋白水解度(DH)之间的回归模型,通过模型优化了胰蛋白酶、中性蛋白酶A.S1398联合水解乳清蛋白的条件参数,并研究了在优化的水解条件下,乳清蛋白不同阶段水解产物在体外对HMG-CoA还原酶活性的影响。     

基于人工神经网络的碱性蛋白酶水解乳清蛋白预测模型的建立

乳清蛋白水解物的生物利用价值高,水解度是衡量水解物的重要指标之一。基于人工神经网络方法建立了碱性蛋白酶水解乳清蛋白的水解模型。利用Matlab的nnet工具包中的nftool建立神经网络模型。实验中设置了15个隐藏神经元,选用Levenberg-Marquardt训练算法迭代计算。实验结果表明:测试值与输出值的误差平均较小,拟合回归得到的整体R2=0.97995,测试集合的R2=0.96239,表明神经网络拟合效果和预测能力良好,能够较好地完成水解度预测的目标,选用的测试组的实验结果与神经网络输出值之间的误差均在±3%以内,说明此神经网络预测模型具有较高的预测能力和精度。      

Dual function peptides from pepsin hydrolysates of whey protein isolate

The aim of this study was to investigate the effect of pepsin hydrolysates of whey protein isolate (WPI) on vascular relaxation and emulsifying capacity. WPI was subjected to pepsin hydrolysis for 5 h. The chromatographic profiles of the samples showed the formation of a wide variety of peptides. Addition of WPI hydrolysates in phenylephrine-contracted rat aortic rings induced a similar concentration-dependent relaxation in both endothelium-intact and endothelium-denuded preparations. In endothelium-denuded vessels the maximum relaxation induced by WPI fractions increased along the time, reaching over 70% after 3 h-hydrolysis on. In addition, the vascular relaxation was not associated with an inhibition of the angiotensin-converting enzyme or activation of K⁺ channels. Hydrolysed fractions were further evaluated for the emulsifying capacity (EC) and all tested fractions were able to keep an EC over 60%. These results reinforce the potential of WPI pepsin-hydrolysates as an option in the search for dual function peptides from whey proteins.     

乳清蛋白源生物活性肽段序列及其功能

乳清蛋白经酶解产生的肽类除了具有极高的营养价值之外．而且还具有一定的潜在生物活性。目前发现的乳清蛋白源生物活性肽主要有阿片肽、抗高血压肽、抗茵肽和免疫调节肽。综述了上述4种肽的氨基酸序列及其主要功能；对酶解过程中应注意的因素，如酶的类型、水解度、预处理方式、酶的灭活方式、底物纯度和水解过程等理化参数进行了归纳总结。     

Preparation of ingredients containing an ACE-inhibitory peptide by tryptic hydrolysis of whey protein concentrates

This study describes the characterisation of whey protein hydrolysates obtained from tryptic hydrolysis to assess their application as ingredients with angiotensin-converting-enzyme (ACE) inhibitory action. The levels of α-lactalbumin (α-la) and β-lactoglobulin (β-lg) remaining after hydrolysis were quantified. Peptides were separated by RP-HPLC, and Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), the most potent β-lg-derived ACE-inhibitory peptide was monitored. A correlation curve was established for the production of this peptide as a function of hydrolysis time. Heat-induced gelation of hydrolysates was studied by small-deformation rheology. The gelation times and the strength of the final gels were highly dependent on the degree of hydrolysis. Smaller peptides liberated by hydrolysis contributed to the inability of whey protein hydrolysates to gel.     

微生物发酵法制备乳清抗氧化肽的研究

通过筛选得到发酵法生产乳清抗氧化肽的最适乳酸菌菌株,同时以氧自由基清除率为指标研究了接种量、菌种比、发酵温度、初始pH值和时间对液态发酵法生产乳清多肽的影响,并采用正交试验确定了最佳发酵工艺条件.结果表明,接种量5%,菌种比1:1,发酵温度37℃,初始pH值6.4,时间16 h,此时测得乳清水解产物O2-清除率为39.45%,肽质量浓度为2.62 g/L.     

水牛奶乳清蛋白制备抗氧化活性肽工艺的研究

实验是以水牛奶为原料,分离纯化后得到乳清蛋白。利用碱性蛋白酶、中性蛋白酶、胰蛋白酶、木瓜蛋白酶和胃蛋白酶5种不同的蛋白酶对水牛奶乳清蛋白酶解以制备抗氧化活性多肽。酶筛选结果显示,中性蛋白酶是最适宜酶解水牛奶乳清蛋白制备抗氧化活性肽,其酶解液的还原能力和DPPH自由基清除率较其他4种酶高。探讨酶解反应时pH、温度、时间、酶浓度对酶解反应的水解度、酶解液的还原能力和DPPH自由基的清除率的影响,在单因素试验基础上,采用响应面法对酶解工艺进行优化。结果表明,中性蛋白酶酶解乳清蛋白的最佳工艺参数为:pH为7.4,温度为50.5℃,酶与底物浓度比为2.1%,酶解时间5.0h,此时2mg/mL酶解物的DPPH自由基清除率为32.58%。实测结果与预测值吻合效果良好。     

酶解乳清蛋白制备降胆固醇活性肽的工艺研究

利用生物酶技术酶解乳清蛋白,制备具有降胆固醇活性的多肽,通过响应面试验、二次旋转回归设计建立回归模型,以胆固醇胶束溶解度抑制率为评价指标,对乳清蛋白的酶解条件进行了优化。结果表明,乳清蛋白的最佳酶解条件为：酶解时间8.5 h、酶解温度55 ℃、酶解pH 8.0、加酶量4.7%、乳清蛋白含量5.8%,在此条件下,酶解乳清蛋白得到的活性肽的胆固醇胶束溶解度抑制率为18.21%。     

Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates

Inhibition of dipeptidyl peptidase-IV (DPP-IV) activity is a promising strategy for treatment of type 2 diabetes. In the current study, DPP-IV inhibitory peptides were identi?ed from mare whey protein hydrolysates obtained by papain. The results showed that all the mare whey protein hydrolysates obtained at various hydrolysis durations possessed more potent DPP-IV inhibitory activity compared with intact whey protein. The 4-h hydrolysates showed the greatest DPP-IV inhibitory activity with half-maximal inhibitory concentration of 0.18 mg/mL. The 2 novel peptides from 4-h hydrolysate fractions separated by successive chromatographic steps were characterized by liquid chromatography–electrospray ionization tandem mass spectrometry. The novel peptides Asn-Leu-Glu-Ile-Ile-Leu-Arg and Thr-Gln-Met-Val-Asp-Glu-Glu-Ile-Met-Glu-Lys-Phe-Arg, which corresponded to β-lactoglobulin 1 f(71–77) and β-lactoglobulin 1 f(143–155), demonstrated DPP-IV inhibitory activity with half-maximal inhibitory concentrations of 86.34 and 69.84 μM, respectively. The DPP-IV inhibitory activity of the 2 peptides was retained or even improved after simulated gastrointestinal digestion in vitro. Our findings indicate that mare whey protein-derived peptides may possess potential as functional food ingredients in the management of type 2 diabetes.     

蚕丝蛋白水解液中多肽相对分子质量分布的测定

蚕丝经脱胶后用硫酸进行水解处理,然后经过一系列的中和、过滤、沉淀脱色等过程制备得到丝素肽。水解过程采用正交实验优化水解工艺条件。然后利用高效液相色谱法,测定了蚕丝蛋白水解液中多肽组分相对分子质量的分布情况。根据胰岛素、酵母核糖核酸、杆菌肽和还原性谷胱甘肽四种标准品的相对分子质量对数与色谱峰的保留时间的关系,绘制出标准相对分子质量分布的标准曲线。结果表明:蚕丝蛋白在固液比为1∶40,硫酸浓度为30%,水解温度为100℃,水解时间8h的条件下进行水解其丝素肽得率最高。蚕丝蛋白水解液中多肽组分的相对分子质量基本都分布在3500u以下,且用此方法得到的色谱峰的分离效果也很好。该方法简便快速且灵敏度高,结果的准确性好,为丝素肽的研究提供了更有效的方法。      

Antibacterial activity of peptides and folding variants from milk proteins

Several milk proteins, such as immunoglobulins, lactoperoxidase, lysozyme and lactoferrin, have long been recognised for promoting health benefits in both newborns and adults. However, it has been shown that not only intact milk proteins but also derivatives thereof can participate in the host defence by exerting many kinds of biological functions. This review focuses on the use of technological processes to induce protein modifications aimed at the enhancement or generation of antibacterial activity. This includes the release of antibacterial peptides by enzymatic digestion, and proteins that exert antibacterial activity by acquiring a new conformation, such as the folding variant of α-lactalbumin or the lysozyme molecule. Current knowledge of antibacterial peptides is evaluated, paying special attention to the formation of these peptides by different proteolytic enzymes, their antibacterial effect, and, where relevant, the methods designed for their production. Finally, the contributions that demonstrate the in vivo health benefits of these compounds are also considered.     

Calcium binding of peptides derived from enzymatic hydrolysates of whey protein concentrate

This study was carried out to investigate the peptides derived from enzymatic hydrolysates of whey protein concentrate. The physiological activity of peptides in whey protein may be used in food additives to promote the absorption of calcium and prevent bone disorders. The whey protein was hydrolysed by trypsin, and the separation of peptides, the properties of hydrolysates and the analysis of the ability to inhibit the formation of calcium phosphates were then investigated. Calcium-binding peptides were produced by tryptic hydrolysis of whey protein concentrate and further purified by precipitation and chromatography on DEAE-cellulose. The hydrolysates were loaded onto an ion-exchange column, followed by stepwise elution with 0, 0.25, 0.5, and 0.75 m NaCl in equilibration buffer to separate the peptides. Trypsin hydrolysates were shown to peak with 0.25 m NaCl and 0.5 m NaCl. The results of SDS-PAGE analysis showed that the peptides with a small molecular weight of about 1.4 to 3.4 kDa were present in the fraction resulting from 0.25 m and 0.5 m NaCl stepwise elution by ion-exchange chromatography of tryptic hydrolysates. The results of this study show that the whey protein hydrolysates produced by the action of trypsin have the ability to inhibit the formation of calcium phosphates.     

Modeling of lactic acid rejection from lactose in acidified cheese whey by nanofiltration

The continuously increasing demand of lactic acid opens a window for the integration of membrane technology in the dairy industry, improving the sustainability by avoiding the use of large amounts of chemicals and waste generation. Lactic acid recovery from fermentation broth without precipitation has been studied by numerous processes. In this work, a commercial membrane with high lactose rejection and a moderate lactic acid rejection, enabling a permselectivity up to 40%, is sought to perform the simultaneous removal of lactic acid and lactose separation from the acidified sweet whey from mozzarella cheese production in a single stage. The AFC30 membrane of the thin film composite nanofiltration (NF) type was selected because of its high negative charge, low isoelectric point, and divalent ion rejection, as well as a lactose rejection higher than 98% and a lactic acid rejection lower than 37%, at pH 3.5, to minimize the need of additional separation steps. The experimental lactic acid rejection was evaluated at varying feed concentration, pressure, temperature, and flow rate. As the dissociation degree of lactic acid is negligible in industrially simulated conditions, the performance of this NF membrane was validated by the irreversible thermodynamic Kedem-Katchalsky and Spiegler-Kedem models, with the best prediction in the latter case, with the parameter values: L_p = 3.24 ± 0.87 L × m⁻² × h⁻¹ × bar⁻¹ and = 15.06 ± 3.17 L × m⁻² × h⁻¹, and σ = 0.45 ± 0.03. The results obtained in this work open the way for the up-scaling of membrane technology on the valorization of dairy effluents by simplifying the operation process and the model prediction and the choice of the membrane.     

酶解小黑豆乳清多肽抗肿瘤作用的研究

研究以小黑豆乳清为原料,比较木瓜蛋白酶、537酸性蛋白酶、ProteAX蛋白酶三种蛋白酶水解多肽产物的分子量分布及其对小鼠H22肿瘤细胞生长抑制作用。结果表明,ProteAX酶解多肽的分子量分布明显不同于另外两种多肽,且对小鼠H22肿瘤细胞生长抑制率最高,当ProteAX酶解多肽浓度为40mg/mL时,可达51.36%。用ProteAX酶解多肽喂食用氨基比林-亚硝酸钠（AP-NaNO2）诱导的肝损伤模型小鼠,结果显示,与模型组相比,该多肽可以显著降低肝脏MDA和血清ALT、AST活性（p<0.05）,并提高肝脏GSH-Px活性（p<0.05）。肝脏组织切片结果显示,模型组小鼠大部分肝细胞形态不正常,细胞损伤严重,出现明显的癌前期病变;而小黑豆乳清多肽高剂量组动物的肝细胞形态基本正常,接近正常肝组织;提示ProteAX蛋白酶解多肽可以抑制AP-NaNO2诱导的肝细胞损伤和癌前期病变。      

MALDI-based identification of stable hazelnut protein derived tryptic marker peptides

Food allergy is an important health problem especially in industrialised countries. Tree nuts, among which are hazelnuts (Corylus avellana), are typically causing serious and life-threatening symptoms in sensitive subjects. Hazelnut is used as a food ingredient in pastry, confectionary products, ice cream and meat products, therefore undeclared hazelnut can be often present as a cross-contaminant representing a threat for allergic consumers. Mass spectrometric techniques are used for the detection of food allergens in processed foods, but limited information regarding stable tryptic peptide markers for hazelnut is available. The aim of this study was to detect stable peptide markers from modified hazelnut protein through the Maillard reaction and oxidation in a buffered solution. Peptides ³⁹⁵Gly-Arg⁴⁰³ from Cor a 11 and ²⁰⁹Gln-Arg²¹⁷, ³⁵¹Ile-Arg³⁶³, ⁴⁶⁴Ala-Arg⁴⁷⁸ and ⁴⁰¹Val-Arg⁴¹⁷ from Cor a 9 hazelnut allergens proved to be the most stable and could be detected and confirmed with high scores in most of the modified samples. The identified peptides can be further used as analytical targets for the development of more robust quantitative methods for hazelnut detection in processed foods.     

乳清蛋白肽美拉德反应产物的制备及其抗氧化特性的评价

以乳杆菌A-2发酵乳清蛋白与乳糖发生美拉德反应,制备得乳清蛋白肽美拉德反应产物,对其进行分离纯化并研究抗氧化活性。将乳杆菌A-2接种于复原乳清粉培养基经热加工得发酵产物,经Sephadex G 15层析柱分离,采用SDS-PAGE图谱证实乳杆菌A-2发酵乳清粉在干热处理条件下确实发生了美拉德反应;以抗坏血酸(维生素C)和丁基羟基茴香醚(BHA)为阳性对照,用ABTS法和ORAC法来测定和评价乳杆菌9029乳清蛋白发酵产物的抗氧化作用。在反应体系中,H-DPWF-1、BHA和维生素C的ABTS清除率分别是77.38%、86.47%和73.95%,H-DPWF-1清除ABTS自由基的能力的介于维生素C和BHA之间;样品H-DPWF-1的相对ORAC值为3.43mmol TE/g,维生素C的相对ORAC值为9.67mol TE/g,即H-DPWF-1清除AAPH的能力为维生素C的1/3。结果显示H-DPWF-1具有较强的抗氧化活性,具有广阔应用前景和潜力。     

乳清多肽的分离纯化及体外抗氧化活性研究

采用乳酸菌发酵乳清制备乳清多肽,利用超滤和凝胶Sephadex G-25分离纯化乳清多肽,并研究了超滤后乳清多肽对羟自由基、氧自由基和DPPH自由基的清除作用。结果表明,超滤后乳清多肽主要包含两个级分,分子量分别为2 031 u和328 u,对羟自由基、氧自由基和DPPH自由基具有清除作用。