明胶水解物和酪蛋白水解物的成骨细胞增殖促进与抗凋亡作用

采用木瓜蛋白酶水解鲑鱼皮明胶和酪蛋白,得到3种水解程度的明胶水解物和酪蛋白水解物,然后通过细胞活力和流式细胞术分析,分别评价水解物对成骨细胞(h FOB.19)增殖和凋亡的影响。研究结果表明,明胶水解物和酪蛋白水解物对成骨细胞均具有明确的增殖促进作用,细胞活性分别增加103%~134%和100%~114%,水解物的水解度高,增殖促进作用大。明胶水解物和酪蛋白水解物对依托泊苷和氟化钠诱导的成骨细胞凋亡具有拮抗作用,凋亡细胞比例由31.7%下降至13.8%和22.6%(依托泊苷组),或者由19.5%下降至11.3%和17.5%(氟化钠组)。总体看,明胶水解物对成骨细胞的增殖促进和抗凋亡作用优于酪蛋白水解物。     

酪蛋白水解物对酸奶的影响研究

将酪蛋白水解物(含蛋白质7．6％)以2％(w／w)添加到奶液中混合发酵，做空白样对照。研究了二者发酵过程中各发酵参数的变化，并对二者的质构进行了分析比较。研究结果表明：酪蛋白水解物能明显促进酸奶发酵；促发酵作用随所添加的水解物水解程度提高而增强；添加酪蛋白水解物改变了酸奶发酵过程中的pH下降速度，在发酵中期二者的pH下降速度之间存在最大差距；质构分析表明添加2％酪蛋白水解物对酸奶整体质构有所改善。     

酪蛋白水解物替代乳清蛋白的加工性能评价研究

利用复合蛋白酶水解酪蛋白制备适度及深度水解酪蛋白产品,测定酪蛋白水解物的加工性能。结果表明,经过酶解后,适度水解酪蛋白溶解度接近90%,深度水解酪蛋白溶解度接近100%,显著高于酪蛋白和乳清蛋白。此外,适度水解酪蛋白吸油性、起泡性分别约为乳清蛋白的3倍和1.5倍。深度水解酪蛋白在起泡性和乳化性上也显著高于乳清蛋白。可见,两款酪蛋白水解物在起泡性、乳化性、吸油性、溶解性等方面均在一定程度上优于乳清蛋白,可广泛替代乳清蛋白在食品工业中大规模应用。     

酪蛋白类蛋白物的溶剂分级和酶水解对ACE抑制活性的影响

利用响应面法优化类蛋白反应条件修饰酪蛋白水解物制备酪蛋白类蛋白物.酪蛋白类蛋白物的ACE抑制活性高于酪蛋白水解物,IC50值从52.6 mg/L降低到14.9 mg/L.利用乙醇-水混合溶剂对酪蛋白水解物和类蛋白物进行分级,结果表明,极性最低的溶剂得到的上清液部分活性较高,而沉淀部分活性较低.4种蛋白酶水解酪蛋白类蛋白物的分级产物,导致活性下降,除碱性蛋白酶外,木瓜蛋白酶、胃蛋白酶和胰蛋白酶的水解产物ACE抑制活性为31.5％～46.8％,但是仍然高于酪蛋白水解物的ACE抑制活性(27.8％),表明类蛋白反应提高了酪蛋白水解物对一些蛋白酶的体外抵抗能力.     

酪蛋白水解物对酸奶的影响研究

将酪蛋白水解物(含蛋白质7.6%)以2%(w/w)添加到奶液中混合发酵,做空白样对照.研究了二者发酵过程中各发酵参数的变化,并对二者的质构进行了分析比较.研究结果表明酪蛋白水解物能明显促进酸奶发酵;促发酵作用随所添加的水解物水解程度提高而增强;添加酪蛋白水解物改变了酸奶发酵过程中的pH下降速度,在发酵中期二者的pH下降速度之间存在最大差距;质构分析表明添加2%酪蛋白水解物对酸奶整体质构有所改善.     

酪蛋白水解物对酸奶发酵的促进作用及其对酸奶质构的影响

将酪蛋白水解物(含蛋白质7.6%)以2%(w/w)添加到奶液中混合发酵,做空白样对照。研究了二者发酵过程中各发酵参数的变化,并对二者的质构进行了分析比较。研究结果表明,酪蛋白水解物能明显促进酸奶发酵;促发酵作用随所添加的水解物水解程度提高而增强;添加酪蛋白水解物改变了酸奶发酵过程中的pH下降速度:添加样在发酵前半期的pH下降速度高于空白样,而发酵后半期空白样的pH下降速度略快;发酵中期,二者的pH下降速度存在最大差距;质构分析表明,添加2%酪蛋白水解物对酸奶整体质构有明显改善。     

酪蛋白水解物对低脂发酵酸奶发酵速度及品质的影响

研究了不同添加量的酪蛋白水解物对低脂发酵酸奶的发酵速度、储存稳定性和质构特性的影响。结果表明,酪蛋白水解物具有明显促进低脂发酵酸奶发酵、缩短发酵时间的作用;可提高低脂发酵酸奶的储存稳定性,主要表现在提高表观黏度和降低储存过程中乳清析出;添加酪蛋白水解物可明显改善低脂发酵酸奶的质构特性。综合考虑,低脂发酵酸奶中添加酪蛋白水解物最适量范围为1.0%～1.5%。     

酪蛋白水解物对乳酸菌的促生长作用

研究了酪蛋白的木瓜蛋白酶水解物对乳酸菌(嗜热链球菌∶保加利亚杆菌=1∶1)的促生长作用.通过比较不同水解度的酪蛋白水解物的增值作用,发现酪蛋白经木瓜蛋白酶水解8 h后的水解物具有最强的促进乳酸菌生长作用.本试验进一步考察了该水解物对酸乳乳酸菌代谢产物(乳酸和胞外多糖)的产量及活菌数变化的影响.结果表明木瓜蛋白酶酪蛋白水...     

高活性酪蛋白抗氧化肽的制备方法研究

采用木瓜蛋白酶对酪蛋白进行水解,得到抗氧化活性较好的酪蛋白水解物,并且水解物在木瓜蛋白酶作用下进行类蛋白反应制备出高活性酪蛋白抗氧化肽。第一步制备酪蛋白水解时酶添加量为500 U/g酪蛋白、温度45℃、底物浓度5%、反应时间2 h。第二步类蛋白反应的最优条件为：酶添加量为500 U/g水解物、温度30℃,底物浓度50%、作用时间5.5 h。毛细管电泳结果确认,类蛋白反应修饰后抗氧化肽的组成情况发生变化。抗氧化活性分析结果表明,类蛋白反应修饰后的酪蛋白抗氧化肽对两种自由基的清除能力显著提高。     

酪蛋白水解物分别与维生素A、维生素C和维生素E协同保护乙醇氧化损伤的肝细胞

酪蛋白水解物可以治疗和修复乙醇氧化损伤的肝细胞。本文的研究目的在于评价维生素A、维生素C和维生素E分别与酪蛋白水解物(Casein Hydrolysate,CH)协同保护乙醇氧化损伤的肝细胞。已有研究表明,酪蛋白水解物对肝细胞HHL-5没有明显的毒性,甚至有着良好的促进增殖的结果。结果表明,300 mmo L/L的乙醇对细胞有着明显的损伤作用;酪蛋白水解物显著提高了乙醇损伤肝细胞HHL-5的细胞存活率。分别在维生素A(0.0344 mg/m L)、维生素C(0.0881 mg/m L)和维生素E(0.1077 mg/m L)的浓度下与酪蛋白水解物协同作用显著提高了乙醇损伤细胞的细胞存活率。通过CCK-8法和培养基内LDH含量的测定,选取最佳CH的协同浓度。通过胞内丙二醛含量的测定和胞内ROS含量的测定进行再次验证。2 mg/m L的酪蛋白水解物单独作用乙醇氧化损伤的细胞有着一定的修复作用,且该浓度下分别与维生素A、维生素C和维生素E协同保护作用显著增强(p0.05)。     

Preparation of casein phosphorylated peptides and casein non-phosphorylated peptides using alcalase

Casein was digested with a cheaper enzyme, alcalase, to produce casein phosphorylated peptides and casein non-phosphorylated peptides concurrently. The casein hydrolyzates were separated to the two kinds of peptides by using combined treatment of CaCl₂ and ethanol. Casein phosphorylated peptides and non-phosphorylated peptides constitute some peptides with molecular weight lower than 2509 Da and 2254 Da respectively as determined using size exclusion HPLC, particularly when a degree of hydrolysis of 20% for the casein hydrolyzates was achieved. At the end, the recovery of casein phosphorylated peptides reached 24%. Phosphorus component of casein phosphorylated peptides was found to be 3.08%. The nitrogen recovery of casein non-phosphorylated peptides was about 76%.     

A comparison of micellar casein and β‐casein as sources of basic peptides through tryptic hydrolysis and their enrichment using two‐stage ultrafiltration

For the application of functional peptides in innovative products, these peptides have to be individually available. This may be accomplished by a well‐considered combination of a pure protein precursor, a selective cleaving enzyme, and an advanced separation technology. In this proof‐of‐principle approach, micellar casein and β‐casein were subjected to tryptic hydrolysis and the generated casein peptides were identified and characterised. The basic target peptides, β‐casein f(177‐183) and β‐casein f(170‐176), were then enriched. A permeate containing 35% of the target peptides was produced after two‐stage ultrafiltration of micellar casein tryptic hydrolysate. In comparison, enrichment up to 58% was achieved starting from β‐casein tryptic hydrolysate.     

Bioavailability of peptides from casein hydrolysate in vitro: Amino acid compositions of peptides affect the antioxidant efficacy and resistance to intestinal peptidases

Measurements of in vitro bioactivity to support health benefits of bioactive compounds should be accomplished with estimates of their bioavailability to bolster nutritional significance to health claims. In vitro bioavailability of casein and casein peptides (casein hydrolysate and four peptide fractions) measured by the amount of peptide nitrogen is discussed. Antioxidant activities during gastrointestinal digestion and Caco-2 cell absorption were investigated as indices of peptide degradation. The antioxidant capacities of Trolox equivalent and oxygen radicals were used for assessing antioxidant efficacy of surviving peptides. Results showed that casein hydrolysate improved bioavailability compared to casein. Amino acid composition of peptides affected the resistance of peptides to digestive enzymes and intestinal peptidases. The acidic peptide fractions had higher bioavailability and a higher residual ratio of antioxidant activity. The peptides in the digest and absorbate of acidic fraction F1 with the highest bioavailability (23.14%) and the residual ratio of antioxidant activity were identified, and 12 intact, absorbed peptides (IAP) were obtained. Eleven of twelve of the IAPs were from β-Casein, and their amino acid components were rich in acidic and hydrophobic amino acids. Identification of IAPs might provide insight into the mechanism of how peptide structure provides resistance against peptidases by Caco-2 cells.     

酪蛋白酶解物的分离及其抗氧化活性

采用胃蛋白酶和胰蛋白酶(P+T)先后连续水解酪蛋白,采用001阳离子交换树脂分离该酶解液得到多肽;以酪蛋白的单一胃蛋白酶(P)和单一胰蛋白酶(T)酶解液为对照,系统评价酪蛋白不同酶解液及分离所获得的酪蛋白多肽的抗氧化活性。结果表明,阳离子交换树脂对(P+T)酶解液具有较好的分离作用,洗脱曲线共有4个峰,分别为P1、P2、P3、P4,收集冻干制得4个多肽。抗氧化性评价显示,P1、P2、P3、P4的还原力和DPPH自由基清除能力远高于未经分离的P、T和(P+T)酶解液。多肽P4表现出极高的Fe2+螯合能力,螯合率高达94.95%;P、T和(P+T)酶解液的Fe2+螯合力相当,螯合率达31%~33%,而P1、P2、P3不具备Fe2+螯合能力。在脂质过氧化抑制方面,P酶解液表现出较好的抑制力,其次为T酶解液,多肽P1、P2、P3、P4及(P+T)不具有脂质过氧化抑制力。由此可见,(P+T)连续水解液经阳离子交换树脂分离可制备具有较高自由基清除力及Fe2+螯合力的肽段,研究结论可为酪蛋白抗氧化肽的制备及分离提供参考依据。     

酪蛋白酶解物中抗菌肽的分离及其分子量的测定

使用离子交换层析分离纯化酪蛋白酶解物中的抗菌肽并测定其分子量。选用Q-Sepharose Fast Flow为分离介质,对分离条件进行了研究,并对各洗脱组分进行抑菌活性测定,确定了适宜分离条件下酪蛋白抗菌肽的洗脱体积,同时测定了酪蛋白抗菌肽的分子量。结果表明,离子交换层析对酪蛋白酶解物中的抗菌肽的最佳梯度洗脱条件为:流动相A:pH10.5的0.05mol/L乙醇胺盐酸缓冲液;流动相Β:含1mol/L氯化钠的A相溶液,pH10.5;洗脱程序:7%B,1.55CV;13%B,1.40CV;25%B,1.25CV;100%B,1.55CV,洗脱流速:0.5mL/min,检测波长:280nm,酪蛋白抗菌肽的洗脱体积为76.57mL和89.48mL。酪蛋白抗菌肽的平均分子量为3137u。     

Comparison of dipeptidyl peptidase IV-inhibitory activity of peptides from bovine and caprine milk casein by in silico and in vitro analyses

The different potential for release of dipeptidyl peptidase IV (DPP-IV)-inhibitory peptides from bovine and caprine milk casein was assessed by in silico analysis and in vitro proteolytic assays. The former predicted a weakly higher potency for caprine casein than bovine as a precursor of DPP-IV-inhibitory peptides, although with markedly diverse potential for individual caseins. This was verified by protease hydrolysis; trypsin-treated casein hydrolysates (TCAHs) displayed the strongest bioactivity. Fractionation of caprine TCAHs revealed slightly, but significantly, higher inhibitory activity in peptides <5 kDa, and notably greater efficiency in the >5 kDa fraction, than their bovine counterparts. Through in silico trypsin hydrolysis and peptides synthesis, four novel caprine casein-derived DPP-IV-inhibitory peptides, including GPFPILV and HPINHR (half maximal inhibitory concentration = 163.7 ± 1.33 and 452.2 ± 7.15 μm, respectively), were found. This study corroborates the weak superiority of caprine casein over bovine in release of DPP-IV-inhibitory peptides, and identifies several novel caprine casein-derived DPP-IV-inhibitory peptides.     

Formation of bitter peptides in cheese and from casein]

Whereas a slightly bitter taste is desirable in certain foods, it is an off-flavour in cheese which may even lead to unfitness for consumption. Bitter principles from cheese have been found to be peptides with molecular weights ranging from 2000 to 3000. For the purpose of further characterization, bitter peptides were isolated from enzymatic casein hydrolysates as well as from bitter cheese and purified. 30 proteases from different origins proved to be able to form peptides with bitter taste of varying intensity from casein. Present experience shows that the formation of bitter peptides during casein hydrolysis can be inhibited only to a very small measure. Bitter peptides are extrmely resistant to proteases, which is probably attributable to their high contents of hydrophobic amino acids and hydrophobic bonds. The detection of only N- or C-terminal amino acid in each of 11 different bitter peptides shows that peptide chains are present and not cyclic peptides as repeatedly assumed. It must be aimed at avoiding the cheese defect "bitter" by using appropriate starter cultures and rennet substitutes as little disposed as possible to produce bitter peptides.     

生物活性肽的研究进展理论基础与展望

随着具各种生物活性的短肽的不断发现,其研究和开发日益受到各国科学家的关注。大量科学研究表明,通过选择适当的蛋白酶,水解的蛋白质可以得到大量的具有各种生物功能的生物活性肽,这些活性肽不仅具有极其广泛的活性和多样性,而且其来源丰富、成本低、安全性好。操作简单、便于工业化生产,因此已成为科学家们研究的新热点。例如：酪蛋白是哺乳动物乳中含量最丰富的蛋白质,长期以来,被人们视为一种营养蛋白,但近年来的研究结果表明,它是生物活性肽的重要来源,到目前为止,已经有数十种生物活性肽被水解和辨认出来。所以,生物活性肽非常好的研究与开发前景。     

分步酶解酪蛋白制备小分子ACE抑制肽

通过模拟胃肠道消化,采用单酶和复合酶分步水解酪蛋白获得小分子的血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制肽。首先通过胃蛋白酶水解条件的优化获得具有高ACE抑制活性肽。然后以此为底物通过胰蛋白酶和胰凝乳蛋白复合酶水解条件优化获得具有高ACE抑制活性的小分子肽。结果表明：第一步的胃蛋白酶水解最优条件为：[E]/[S]=6%、[S]=0.015g/mL、pH=1.8、t=37℃、t=2h,水解产物稀释10倍后ACE抑制率为84.5%,分子质量集中在2000D以下;第二步的复合酶水解最优条件为：m胰蛋白酶(6%):m胰凝乳蛋白酶(3%)=2:1、pH=7.8、t=48℃、t=5h,水解产物稀释10倍后ACE抑制率为85.9%,分子质量集中在500D以下。研究表明,通过分步酶解选择合适的酶解条件可以获具有较高ACE抑制活性的小分子肽。