牛乳酪蛋白源生物活性肽研究进展

乳酪蛋白源生物活性肽以其良好的消化性、低敏性及生物活性而日益受到重视。综述国内外在牛乳酪蛋白来源的阿片肽、阿片拮抗肽、ACE抑制肽、免疫调节肽和矿物元素结合肽等方面的研究,旨在为更好地利用牛乳酪蛋白源的生物活性肽提供参考。     

牦牛乳酪蛋白抗凝血肽制备工艺研究

酪蛋白为牛乳中主要的蛋白形式,不仅能为生物体生长发育提供必需的氨基酸,而且酶解后可得到一系列具有重要生理功能的生物活性肽,抗凝血肽就是其中一种,毒副作用小、抗凝血效果明显,可作为心脑血管疾病治疗的潜在药物。本试验以牦牛乳酪蛋白为原料,采用胰蛋白酶酶解酪蛋白制备抗凝血肽,通过筛选显著单因素和设计正交试验,确定了牦牛乳酪蛋白抗凝血肽最佳制备条件为:温度为30℃、pH为6.2、底物浓度为4%、酶底比为1.75%、反应时间为3h。在该条件下所制备的抗凝血肽的抗凝血效价单位数可达837.4U/mg。     

牛乳酪蛋白体外模拟消化液的ACE抑制活性及其肠道吸收

本文对牛乳酪蛋白进行体外模拟消化,以ACE抑制活性为指征,LC-MS/MS确定水解物肽谱,并采用大鼠肠道模型对其吸收进行初步研究。结果为人工胃液消化、人工肠液消化、胃肠联合消化三种消化方式的水解度均随时间的延长而增大,单胃和单肠消化的ACE抑制率均随水解时间的延长先快速增加,随后逐渐降低,而胃肠联合的ACE抑制率则是先降低再增加,胃肠联合消化产生水解度最高为25.51%,其ACE抑制率在2 h时达到最高值为68.03%;LC-MS/MS测定消化液的肽谱,分析得出模拟消化后可能产生的ACE抑制肽有IPP、RYLGY、LHLPLP、AYFYPEL、RPKHPIKHQ及WQVLPNAVPAK;使用FITC标记酪蛋白进行体外模拟消化,SDS-PAGE和Tricine-SDS-PAGE都表明FITC-酪蛋白经过胃肠消化后荧光标记稳定存在,且水解物的分子量在5 ku以下;大鼠肠道吸收模型发现标记后的牛乳酪蛋白经模拟消化后在肠道吸收率大小依次为十二指肠吸收空肠吸收回肠吸收结肠吸收,主要在十二指肠吸收。     

牦牛乳酪蛋白糖巨肽生产工艺研究

酪蛋白糖巨肽是κ-酪蛋白经酶解后生成的一类含有糖链的多肽,具有许多生理功能。本文研究了牦牛乳酪蛋白糖巨肽的生产工艺条件,通过正交实验得到了最佳工艺条件:底物浓度50g/L、酶底比为0.5/100、反应温度为45℃,酶解时间为3.5h。酪蛋白糖巨肽产品得率9.4%,纯度96.78%,蛋白质87.18%,唾液酸含量6.125mg/g。      

蛋白水解活性肽的研究现状

活性肽是蛋白质经酶水解而得的产物,不仅能提供给人类营养,同时具有防病治病,调节人体生理机能的功效,本文主要就肽在体内的消化吸收,蛋白水解物的酶法生产,水解物的去苦研究,及保健与功能食品的开发等方面进行综述。     

食物蛋白源降尿酸活性肽的研究进展

高尿酸血症是一种由嘌呤代谢紊乱引起的慢性疾病,已严重威胁到人类的健康。长期的高尿酸血症会导致痛风的发生。目前临床用于治疗高尿酸血症的药物如别嘌呤醇、秋水仙碱等存在毒副作用大等问题,因此开发高效、安全的天然降尿酸活性物质成为国内外学者研究的热点。食物蛋白源降尿酸活性肽是指利用食源性动、植物蛋白制得的具有降低生物体内血尿酸水平功效的生物活性肽。该文对目前国内外食物蛋白来源的降尿酸活性肽的制备、分离纯化、功能活性评价、结构鉴定予以综述,并对此研究领域的发展趋势予以展望,旨在为降尿酸功能性食品的研究及开发提供参考。     

酶法制备牦牛乳酪蛋白源血管紧张素转换酶抑制肽

应用6种商业蛋白酶水解牦牛乳酪蛋白,研究其水解产物的血管紧张素转换酶抑制活性,结合蛋白水解度及蛋白回收率的结果,蛋白酶C适用于制备牦牛乳酪蛋白源血管紧张素转换酶抑制肽。     

超声处理对牛乳酪蛋白结构及抗原性的影响

利用超声波处理牛乳致敏蛋白α-酪蛋白（casein,CN）和β-CN,结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、圆二色光谱、荧光光谱及酶联免疫吸附测定等方法分析α-CN和β-CN结构和抗原性的变化。结果表明：随着超声波功率的增大,α-CN的分子质量无显著性变化,β-CN在600 W时发生聚集,两种酪蛋白羰基含量上升,自由巯基含量先下降后回升,在500 W时达到最低,疏水性则呈现与自由巯基含量相反的规律;二级结构中,α-螺旋结构经超声处理后其相对含量减少,无规卷曲相对含量随着超声功率先增加后降低,功率为500 W时相对含量最高,β-折叠的相对含量变化趋势则与无规卷曲相反,这表明超声波处理破坏了酪蛋白的高级结构;随着超声功率的增强,抗原性呈现先升高后降低的趋势,其中500 W时抗原性最高,与酪蛋白疏水性及无规卷曲结构相对含量呈正相关,与β-折叠结构的相对含量呈现负相关。超声处理酪蛋白在影响蛋白构象及结构的同时改变其抗原性,且抗原性与疏水性及无规卷曲相对含量相关。     

具有铅离子螯合作用的亚麻籽蛋白源半胱氨酸肽的制备及结构鉴定

研究发现含有半胱氨酸残基即巯基的植物蛋白肽具有铅离子螯合能力,可以降低铅中毒人群体内铅的浓度,缓解中毒症状,减轻铅损伤。本研究以脱脂亚麻籽饼为原料,从中提取亚麻籽蛋白并进行酶解,优化酶解工艺得到高巯基含量的酶解物,采用共价色谱法对亚麻籽蛋白酶解物中半胱氨酸肽进行富集,并鉴定其结构,最后通过分子对接技术探究其与铅离子的螯合机制,旨在获得具有铅离子螯合作用的半胱氨酸肽。结果发现,采用 protease M 酶,在酶解温度 50 ℃、酶解 pH 3.5、酶添加量 900 U/g、酶解时间 4 h 条件下,得到巯基含量为 13.37 μmol/g 的亚麻籽蛋白酶解肽,铅离子螯合量为 11.40 mg/g;通过共价色谱法对其进行富集得到巯基含量为 44.8 μmol/g、铅离子螯合量为 24.98 mg/g 的亚麻籽蛋白源半胱氨酸肽,比亚麻籽蛋白提高了 6.74 倍和 3.92 倍;对亚麻籽蛋白源半胱氨酸肽进行结构鉴定,得到 3 种含有半胱氨酸的肽段,分别为 WAIINLQHSGEGLCGRVV、CTGLLEAVAAALMMNIYIVGLNQLTDIE、QGGQGQQQQCEKQIQEQDYLRSCQQFLWEKVQKGGRS,且 3 种肽段都可以与铅离子稳定螯合,其中半胱氨酸中的巯基在螯合过程中起重要作用。以上结果表明,对亚麻籽蛋白酶解工艺进行优化并富集,可以得到高巯基含量且能与铅离子螯合的半胱氨酸肽,提高亚麻籽榨油后副产物的利用率,并为亚麻籽蛋白源半胱氨酸肽缓解铅中毒的研究提供理论依据。     

牛乳清蛋白源脂肪酶抑制肽的制备及影响因素研究

以胰脂肪酶和α-淀粉酶的抑制率为指标,研究了牛乳清蛋白源活性肽(APWP)的制备工艺、影响因素及其模拟胃肠道消化.结果表明:APWP的最佳制备条件为在60 mg/mL的乳清蛋白溶液中,加入750 U/g碱性蛋白酶,45℃酶解4 h,所得每毫克活性肽对胰脂肪酶和α-淀粉酶抑制率分别为0.78±0.01％和0.27±0.0...     

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。     

牛乳中酪蛋白的结构特性及其应用

酪蛋白及其制品具有较高的营养价值和优良的功能特性,本文概述了牛乳中酪蛋白的组成及其结构特性,并针对其各种特性概述了在食品工业中的应用情况。     

酪蛋白抗氧化肽的制备及初步分离

采用凝乳酶对牛乳酪蛋白酶解,以水解液对DPPH的清除率为指标,进行了酶解工艺优化。酶解液经超滤,Sephadex G-15凝胶层析柱和DEAE-52离子交换层析分离,得到了初步纯化。     

酪蛋白抗菌肽的酶法制备

对胃蛋白酶水解酪蛋白制备酪蛋白抗菌肽进行研究.结果表明:酪蛋白水解物中,分子量3386 Da的组分具有最强的抑菌活性和良好的热稳定性,胃蛋白酶水解酪蛋白释放抗菌肽的适宜条件为:干酪素浓度10 mg/mL、酶浓度2.0%、温度40 ℃、pH 1.5、水解时间3.5 h.     

海洋生物活性肽的制备及其研究状况

对两种制备海洋生物活性肽的方法进行比较,认为从海 洋生物中直接提取天然存在的生物活性肽很难实现规模 化工业大生产;蛋白酶解制备法可实现规模化大生产,为 海洋蛋白质资源的充分利用、高值化提供有效的途径。同 时,也对两种制备方法的海洋生物活性肽的研究进展进 行了探讨。     

Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages.Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.     

Bovine leukemia virus alters growth properties and casein synthesis in mammary epithelial cells

Bovine leukemia virus (BLV) is widespread in US dairy herds, yet only about 1% of infected cattle develop bovine leukosis and are culled from the herd. A major concern is whether BLV infection of dairy cows alters milk yield. Although several studies have examined the effect of BLV on milk production in vivo, the results were inconclusive. No in vitro studies have been done. The discovery of BLV in mammary epithelial cells (MEC) of infected cows raises the possibility that the virus could affect these cells directly. The purpose of this study was to use an in vitro system to determine if BLV could alter milk yield by altering cell number and/or milk production per cell. A short-term cell line established from the MEC of a BLV-negative cow, and a proven casein-producer mouse cell line, Comma D, were stably transfected with a plasmid containing the entire BLV genome. Untransfected parental lines served as negative controls. The BLV-containing bovine MEC line has a reduced population-doubling time, higher saturation density, and increased longevity. The Comma D line is an already-transformed cell line, and growth properties did not change after transfection with BLV. Under appropriate differentiation conditions, both the bovine and mouse MEC transfected with BLV displayed decreased casein production and mRNA synthesis compared with control cell lines without BLV. Our results suggest that effects of BLV infection on milk production may not be related solely to overall animal health but may also be mediated directly at a cellular level.     

Supramolecular structure of the casein micelle

The supramolecular structure of colloidal casein micelles in milk was investigated by using a sample preparation protocol based on adsorption of proteins onto a poly-l-lysine and parlodion-coated copper grid, staining of proteins and calcium phosphate by uranyl oxalate, instantaneous freezing, and drying under a high vacuum. High-resolution transmission electron microscopy stereo-images were obtained showing the interior structure of casein micelles. On the basis of our interpretation of these images, an interlocked lattice model was developed in which both casein-calcium phosphate aggregates and casein polymer chains act together to maintain casein micelle integrity. The caseins form linear and branched chains (2 to 5 proteins long) interlocked by the casein-stabilized calcium phosphate nanoclusters. This model suggests that stabilization of calcium phosphate nanoclusters by phosphoserine domains of α_s1-, α_s2-, or β-casein, or their combination, would orient their hydrophobic domains outward, allowing interaction and binding to other casein molecules. Other interactions between the caseins, such as calcium bridging, could also occur and further stabilize the supramolecule. The combination of having an interlocked lattice structure and multiple interactions results in an open, sponge-like colloidal supramolecule that is resistant to spatial changes and disintegration. Hydrophobic interactions between caseins surrounding a calcium phosphate nanocluster would prevent complete dissociation of casein micelles when the calcium phosphate nanoclusters are solubilized. Likewise, calcium bridging and other electrostatic interactions between caseins would prevent dissociation of the casein micelles into casein-calcium phosphate nanocluster aggregates when milk is cooled or urea is added to milk, and hydrophobic interactions are reduced. The appearance of both polymer chains and small aggregate particles during milk synthesis would also be expected based on this interlocked lattice model of casein micelles, and its supramolecule structure thus exhibits the principles of self-aggregation, interdependence, and diversity observed in nature.     

Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides

Yak (Bos grunniens) milk casein derived from Qula, a kind of acid curd cheese from northwestern China, was hydrolysed with alcalase. The hydrolysates collected at different hydrolysis times (0 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min) were assayed for the inhibitory activity of angiotensin-I-converting enzyme (ACE), and the one obtained at 240 min hydrolysis showed the highest ACE inhibitory activity. The active hydrolysate was further consecutively separated by ultrafiltration with 10 kDa and then with 6 kDa molecular weight cut-off membranes into different parts, and the 6 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Pro–Pro–Glu–Ile–Asn (PPEIN)(κ-CN; f156–160) and Pro–Leu–Pro–Leu–Leu (PLPLL) (β-CN; f136–140), respectively. The molecular weight and IC₅₀ value of the peptides were 550 Da and 566.4 Da, and 0.29 ± 0.01 mg/ml and 0.25 ± 0.01 mg/ml, respectively.     

生物酶法耦合膜技术制备活性短肽的工艺研究

主要探讨生物酶法耦合膜技术生产活性短肽的工艺中各级膜工作过程和其他流程试验,采用正交试验确定了最佳酶解条件:加酶量2%、料液比1∶4、酶解时间3h。鱼初加工的下脚料,鱼头、鱼尾、鱼皮、鱼鳞等经过该工艺过程,最终得到了活性短肽产品,86%以上肽为易于人体吸收的生物活性短肽。