乳清蛋白生物活性肽及其应用前景

随着对生物活性肽的功能研究及分离、提取技术的成熟和研究与开发乳源活性肽的兴起,研究者发现乳清蛋白中含有多种生物活性的潜在肽段.综述了近年来乳清蛋白生物活性多肽的研究进展,介绍了其抗氧化活性、ACE抑制活性、降胆固醇活性及其他活性的作用机理和研究概况.随着研究水平的提高,乳清蛋白生物活性肽的功能特性必将得到充分的开发和利用.     

酶解牦牛乳清蛋白制备ACE抑制肽的工艺研究

目的:乳清蛋白是牦牛乳中的重要成分,其经酶解制备的乳源性ACE抑制肽以安全性高和无副作用等优点在高血压的防治中具有重要的研究和应用价值.本实验研究利用乳清蛋白制备ACE抑制肽的工艺技术.方法:本实验对从耗牛乳中提取的乳清蛋白,分别根据碱性蛋白酶、中性蛋白酶、木瓜蛋白酶、胰蛋白酶及胃蛋白酶各自的最适pH及温度,采用酶解法制备ACE抑制肽,以ACE抑制活性为指标初步筛选出最佳水解酶,并探究最佳酶水解乳清蛋白制备ACE抑制肽的最佳反应条件.结果:碱性蛋白酶为制备ACE抑制肽的最佳水解酶,英最佳反应条件为:pH8.5、温度60℃、E/S为5.5％、水解6h.制备的ACE抑制肽抑制率可达到85.2％.结论:碱性蛋白酶为最佳酶选,用碱性蛋白酶制备的ACE抑制肽体外抑制率较高,符合工业生产要求.这为进一步优化乳清蛋白降血压肽的制备工艺提供依据.     

食物蛋白源酪氨酸酶抑制肽的研究进展

黑色素过量生成和沉积会导致一系列的皮肤问题,特定食物蛋白肽可通过抑制酪氨酸酶活性减少黑色素的生成。基于此,系统总结了当前食物蛋白源酪氨酸酶抑制肽的来源及制备方法,重点介绍了目前用于评价活性肽抑制效果的模型方法,分析了肽链中氨基酸的种类及位置对肽活性的影响以及活性肽对酪氨酸酶活性的不同抑制机理,并在此基础上指出食物蛋白源酪氨酸酶抑制肽的发展趋势及未来面临的挑战,以期为酪氨酸酶抑制肽的研究及开发应用提供参考。     

血管紧张素转换酶(ACE)抑制肽的酶法制备

以扇贝裙边的酶解产物——蛋白多肽对ACE活性的抑制率作为控制水解程度的指标,确定制备ACE活性抑制肽的最佳酶种及酶解工艺技术条件.通过胰蛋白酶、胃蛋白酶、木瓜蛋白酶、复合风味酶、枯草杆菌蛋白酶5种酶酶解扇贝裙边得到的蛋白多肽对ACE抑制能力的比较发现,5种酶在不同酶解时间所得到的酶解蛋白多肽都具有一定的ACE抑制能力,其中胃蛋白酶酶解蛋白多肽的ACE抑制率最高,为91.33%;再经L9(3)4正交实验对胃蛋白酶酶解工艺条件进行优化,确定酶解反应最佳条件是酶量400 U.g-底1物,pH 2.5,温度32℃,底物浓度(原料∶水)1∶2,酶解时间3 h;试验结果还表明,单酶(胃蛋白酶)的酶解蛋白多肽比复合酶(复合风味酶)和双酶(胃蛋白酶 木瓜蛋白酶)的酶解蛋白多肽对ACE活性的抑制能力都强.     

低温下棕榈某些生理变化及低温锻炼对棕榈耐寒性的影响

以电导率为指标研究了低温对棕榈叶片细胞的膜透性的影响,同时测定了低温处理不同时间的棕榈叶片细胞内游离脯氨酸和可溶性蛋白含量的变化及超氧歧化酶（ＳＯＤ）活性的变化,结果表明,低温使棕榈叶片细胞的膜透性大幅度增加,游离脯氨酸和可溶性蛋白含量提高,而ＳＯＤ活性则表现为先增高继之下降的趋势,经过低温锻炼的棕榈在膜稳定性,保护性物质脯氨酸和可溶性蛋白含量上均高于未经过低温锻炼的棕榈,低温处理后经过锻炼的棕榈     

枯草芽孢杆菌发酵大豆过程中水提物的ACE抑制活性的变化

研究了枯草芽孢杆菌(Bacillus subtilis)SY发酵大豆过程中样品水提物的血管紧张素转换酶(ACE)抑制活性的变化,并从蛋白质变化的角度进行分析.结果表明:发酵前期样品的ACE抑制活性迅速增加,然后降低并保持平稳,发酵12,h样品的IC50值为0.312,mg/m L;中性蛋白酶活力在前36,h迅速增至718,U/g,多肽及游离氨基的含量也相应逐渐增加并趋于稳定.可见,大豆蛋白质的适度降解有利于产品保持良好的ACE抑制活性,在控制发酵进程的条件下,枯草芽孢杆菌SY有望应用于发酵大豆食品的生产来获得具有潜在调节血压作用的产品.     

油菜饼蛋白提取物对饲料中霉菌抑制作用的研究

通过硫酸铵沉淀、Sephadex G-100凝胶柱层析和HPLC分离等方法,从油菜饼的水溶性成分中分离得到蛋白质(肽)提取物.采用抑菌圈法测定该蛋白(肽)提取物的抑菌活性,结果表明对受试真菌有明显的抑制作用.对该蛋白(肽)提取物的稳定性测定表明,加热到90℃未丧失活性,对酸碱的适应范围也较大,在pH 7～8的时候抑菌效果最为明显,对蛋白酶K和胰蛋白酶敏感.通过与几种防腐剂对饲料中霉菌抑制作用的比较发现,油菜饼蛋白(肽)提取物的防霉性能优于恩拉鼎(Enramycin)预混剂、富马酸,仅次于复合型防霉剂霉必克,可以开发成为一种高效的植物源性饲料防腐剂.     

体外消化对大豆多肽性质和ACE抑制活性的影响

为了验证大豆多肽血管紧张素转换酶(ACE)抑制活性的生物可获得性,采用Alcalase蛋白酶催化大豆蛋白水解获得了3种水解程度不同的大豆多肽,再应用摸拟体外消化方法处理大豆蛋白及其水解产物,并进一步分析了摸拟消化前后大豆多肽结构、性质和ACE抑制活性的变化.结果显示:3个大豆多肽的相对分子质量均小于16000,且水解度越高,TCA可溶性蛋白含量就越高;经过摸拟体外消化,大豆蛋白出现降解,其分子大小和性质与大豆多肽趋于一致,大豆多肽则进一步降解;3种大豆多肽的ACE抑制活性在经过摸拟体外消化后显著下降,大豆蛋白的ACE抑制活性则显著上升,两者之间没有显著差异,表明虽然Alcalase对大豆蛋白的酶促处理能够释放降血压肽,但经过模拟体外消化后其ACE抑制活性又会下降,而直接口服大豆蛋白有可能获得同样的降血压效果.     

CPP生产副产物--CNPP酶膜耦合法 制备ACE抑制肽研究

采用酶解与膜分离耦合的方法,以酶膜耦合法生产酪蛋白非磷酸肽(CPP)得到的副产物——酪蛋白非磷酸肽(CNPP)为原料制备高活性ACE抑制肽.用Protease N、胰蛋白酶、Fla-vourzyme、AS1398、酸性蛋白酶等几种蛋白酶对经过酶膜耦合制备的酪蛋白非磷酸肽进行二次酶解,同时与1 K膜分离耦合,然后对二次酶膜耦合所得产物的ACE抑制活性进行比较.结果表明,Protease N比其他几种蛋白酶更适合用于酪蛋白非磷酸肽制备高活性的ACE抑制肽,其产物的蛋白质量浓度为1 g/L时的ACE抑制率达到55.60%.     

新疆传统发酵乳酪乳清中ACE抑制肽的超滤分离

本实验采用超滤法分离发酵乳中的ACE抑制肽,研究了超滤工艺的主要技术参数。结果表明:截留分子量为5000Da的超滤膜,在压力0.10MPa,温度37℃条件下,能得到具有较高ACE抑制活性的超滤液。     

酶解制备牦牛乳酪蛋白降血压肽的初步研究

目的:牦牛乳富含酪蛋白,其经酶解制备的降血压肽以安全性高、无副作用等优点已成为乳品开发和高血压防治药物研究的新热点.方法:本实验对从牦牛乳中提取的酪蛋白,依据复合蛋白酶、中性蛋白酶、碱性蛋白酶、木瓜蛋白酶及胰蛋白酶各自的最适pH及温度采用酶解方法制备降血压肽,并且以ACE抑制活性为指标初步筛选最佳水解酶.结果:五种酶ACE抑制肽抑制率分别为:复合蛋白酶为39.02%,中性蛋白酶为67.48%,碱性蛋白酶为56.10%,木瓜蛋白酶为71.54%,胰蛋白酶为8.94%.结论:说明,在最适pH及温度条件下,木瓜蛋白酶和中性蛋白酶为制备降血压肽的最佳酶选,这为后续进一步的制备降血压肽实验提供依据.     

响应面法优化酶解鱼鳞制备ACE抑制肽的研究

鱼鳞是鱼类加工的下脚料之一,含有丰富的蛋白质和矿质元素等营养物质,是非常好的可利用生物资源. 文中研究了碱性蛋白酶酶解鱼鳞制备ACE抑制肽的优化工艺. 以ACE抑制率为指标,在酶解温度、酶解pH、加酶量、底物质量浓度等条件下先进行单因素实验,在此基础上运用响应面法优化碱性蛋白酶酶解鱼鳞制备ACE抑制肽的工艺条件. 结果表明：在加酶量61%(约12 000 U/g)、pH 89、温度547 ℃的条件下酶解2 h,ACE抑制率理论值为8536%,实际酶解物的ACE抑制率为862%,相对误差为091%.     

Analysis of biomarkers for the cross-linkage of formaldehyde with bovine serum albumin peptides

Formaldehyde, a well-known environmental toxic hazard, has been found to produce endogenously via semicarbazide-sensitive amine oxidase-catalyzed oxidative deamination of methylamine. In diabetes, the activity of SSAO has been found to increase with a subsequent increase in endogenous formaldehyde production. It has been postulated that SSAO-induced production of formaldehyde may be involved in the alteration of protein structure, which may subsequently cause protein deposition associated with chronic pathological disorders. Formaldehyde has also been found to react （cross-link） with amino group of the N-terminal amino acid residue and with the side-chains of arginine, cysteine, histidine and lysine residues. Therefore, formaldehyde may be responsible, at least in part, for protein cross-linkage, oxidative stress and cytotoxicity. The cross-linking of formaldehyde with bovine serum albumin was studied using LC-MS and Mascot database. The peptides sequence for control BSA （untreated） digested with trypsin was matched in the online database search query by exporting the MS/MS data to online MASCOT database. In this way, a total of twenty-seven peptides were matched in the database search query. These twenty-seven peptides were then searched manually in all of the tryptic BSA samples treated with different concentrations of FA that were incubated in different time intervals. Six formaldehyde-treated BSA peptides （FKDLGEEHFK, HLVDEPQNLIK, KVPQVSTPTLVEVSR, RPCFSALTPDETYVPK, LVNELTEFAK, DAFLGSFLYEYSR） were found to be the possible markers for formaldehyde-protein/peptides adducts.     

对若干影响青竹梅果浆营养成份和抗氧化活性因素的考察

青竹梅果浆具有高酸度(5%)、高蛋白(1.6%)和低糖(1.5%)特征.果浆含16—17种氨基酸,其中天门冬氨酸含量很高,几乎占总氨基酸量的60—70%.发现果浆酸度对抗氧化活性有一定影响,但无线性关系,测试了外源柠檬酸的抗氧化活性,发现其抑制率仅占果浆总活性的三分之一.分析了在制备果浆中几种辅料的影响,其中只有 NaCl 有较大干扰.采用不同温度处理果浆未见抗氧化活性的显著变化,证明果浆热稳定性好,同时又观察室温与低温贮存八周果浆的变化,未见其抗氧化活性的明显衰减。     

基于分子对接技术从虾夷扇贝中筛选抗SARS-CoV-2活性肽研究

为从食品原料中筛选具有抗2019新型冠状病毒(SARS-CoV-2)能力的生物活性肽段,选用虾夷扇贝肌球蛋白为目标序列,利用计算机对虾夷扇贝肌球蛋白进行模拟酶解,对酶解所得的肽段进行毒性和生物活性预测。选取活性评分超过0.5且无毒性的肽段,以SARS-CoV-S/ACE2复合蛋白和COVID-19 Mpro水解酶为靶标进行分子对接,鉴定其病毒抗性。结果表明:肽段CSNAIPEL可以与SARS-CoV-S/ACE2复合蛋白上的GLN42和GLU329两个关键氨基酸结合,LibDock Score为136.03;肽段LPIY不仅能与SARS-CoV-S/ACE2复合蛋白上的ASP38和TYR491氨基酸结合,还能够与COVID-19 Mpro上的THR24、THR25和THR26氨基酸结合,LibDock Score 分别为142.85和168.04;肽段QRPR与COVID-19 Mpro 水解酶晶体上的THR24、THR25和THR26氨基酸结合,LibDock Score为154.93。研究表明,肽段CSNAIPEL、LPIY和QRPR三者表现出较好的抗SARS-CoV-2能力。本研究旨在为抗新型冠状病毒功能食品的研发提供新的思路。     

ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

Malnutrition affects up to one billion people in the world and is a major cause of mortality. In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death. The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure, cardiovascular functions and SARS infections. Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea.     

Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.     

Novel C-terminally amidated opioid peptide in human phaeochromocytoma tumour

As has often been observed in hypothalamic releasing factors and gastrointestinal hormones, the carboxy-terminal amide structure is a unique feature of peptides exhibiting hormonal or physiological activities. Although a variety of opioid peptides have hitherto been identified, such a C-terminal amidated species has never before been discovered in mammals. Here we present the first identification of a novel opioid octapeptide with a C-terminal amide structure, henceforth designated as 'adrenorphin', in human phaeochromocytoma tumour derived from adrenal medulla. The complete amino acid sequence of adrenorphin was determined by microsequencing and corresponds to the sequence of the first eight amino acids of peptide E which is derived from proenkephalin A. Adrenorphin has also been identified chromatographically in normal human and bovine adrenal medulla. Adrenorphin exhibits potent opioid activity in guinea pig ileum assay, suggesting a specialized physiological function.     

高分子量聚丙烯酸改性聚乙烯醇膜的耐水性能

将高分子量聚丙烯酸（PAA）与聚乙烯醇（PVA）混合,制备成PAA/PVA混合膜并对其进行热处理。考察了PAA分子量及其含量对混合膜耐水性能的影响,研究了热处理工艺条件对混合膜在沸水环境下的保留率。结果表明,随着PAA分子量和在混合膜中含量的增加,混合膜的耐沸水性能显著提高;当PAA加入量为30%（质量分数）,热处理温度和时间分别为160～180℃、3～5min时,混合膜的综合性能最佳;热处理后混合膜的玻璃化转变温度T_g提高到102℃,有效扩大了膜的使用温度范围。