发酵乳中ACE抑制肽生成的外部因素条件的研究

探讨了促进发酵乳中ACE抑制肽生成的外部因素条件。研究结果表明,菌株发酵凝乳(pH4.7～5.0)后,产生肽量迅速积累,ACE抑制活性大幅度增强,产生的肽量与ACE抑制活性具有正相关性。添加乳清蛋白和酪蛋白可提高发酵乳中的肽含量,添加酪蛋白组更为突出;添加乳清蛋白组肽粉ACE抑制活性减弱,而添加酪蛋白组肽粉ACE抑制活性增强。与发酵温度42℃相比,37℃发酵条件下有利于发酵乳ACE抑制肽生成;与4℃冷藏处理相比,37℃条件下保温培养对提高ACE抑制活性和肽含量更为显著(P<0.01),实验结果为发酵法生产ACE抑制肽提供了可靠的技术依据。     

复合酶解乳清蛋白制备降血压肽的研究

采用碱性蛋白酶、胰蛋白酶水解乳清蛋白制备ACE抑制肽,通过体外检测法测定其ACE抑制率。通过正交试验,得出最优水解条件,即碱性蛋白酶和胰蛋白酶[E1/E2]之比为5:4,温度为45℃,pH值为8.0,时间为150min,水解度11.92%的条件下,乳清蛋白肽对ACE的抑制能力最强,达到60.19%。     

乳清蛋白酶解ACE抑制肽分离纯化技术的研究

采用6000 u的超滤膜对乳清蛋白水解物中的ACE抑制肽进行了初步分离,确定了超滤的条件,并测定超滤前后水解物的ACE抑制率,结果表明通过超滤可以富积乳清蛋白水解物中的ACE抑制肽.采用阳离子交换树脂对水解物进行脱盐,通过测定水解物的氮回收率、脱盐率以及ACE抑制率,表明采用阳离子交换树脂脱盐率可以达到80%以上,氮的回收率达到90%以上,而水解物经过阳离子交换树脂基本上对其ACE抑制率没有影响.     

双酶水解乳清蛋白ACE抑制肽的制备工艺优化

以乳清蛋白为原料,采用胃蛋白酶和胰蛋白酶双酶先后水解乳清蛋白,通过单因素试验和正交试验优化胃蛋白酶和胰蛋白酶水解乳清蛋白制备血管紧张素转换酶(ACE)抑制肽的工艺,将水解物以3 kDa超滤膜过滤,研究表明,胃蛋白酶水解乳清蛋白最佳酶解工艺条件为水解温度37℃、底物质量浓度6 g/100 mL、酶与底物比3 728 U/g,此时乳清蛋白ACE抑制率为86%;胰蛋白酶水解乳清蛋白最佳酶解条件为温度55℃、底物质量浓度6 g/100 mL、酶与底物比3 480 U/g,此时ACE抑制率为72%。利用超滤离心管获得分子量小于3 kDa的乳清蛋白ACE抑制率96%。     

瑞士乳杆菌蛋白酶水解乳清蛋白制备A C E 抑制肽的条件优化

目的：本实验以WPC-80 乳清浓缩蛋白为原料,对瑞士乳杆菌蛋白酶水解乳清蛋白产生血管紧张素转移酶(ACE)抑制肽工艺条件进行研究。方法：通过单因素条件和响应面方法研究水解pH 值、水解温度、酶与底物比、底物浓度和水解时间对水解度和ACE 抑制率的影响。结果：研究发现,五个工艺条件对ACE 抑制肽的产生都有影响,通过响应面法分析,确定瑞士乳杆菌蛋白酶酶解乳清蛋白的最佳水解条件为：[E]/[S] 0.6%、底物浓度6%、pH9.18、温度38.90℃,时间8.0h,在此条件下水解,产物ACE 抑制率达到92.21% ,IC50 为0.375mg/ml,水解度为18.76%。结论：应用响应面方法优化水解工艺条件是可行的。     

固定化瑞士乳杆菌蛋白酶酶解乳清蛋白生产ACE抑制肽的条件优化

以乳清浓缩蛋白WPC-80为原料,研究固定化瑞士乳杆菌蛋白酶酶解WPC-80生产血管紧张素转化酶（angiotensinⅠ-converting enzyme,ACE）抑制肽的工艺条件。通过单因素试验和响应面方法研究了酶解温度、酶解pH值、底物与酶质量比（[S]/[E]）、酶解时间对固定化瑞士乳杆菌蛋白酶制备ACE抑制肽的影响,确定了酶解乳清蛋白制备ACE抑制肽的最佳工艺条件为：温度37 ℃、pH 7.5、[S]/[E]＝15%、酶解时间8 h。在此条件下,酶解产物的水解度为（6.05±0.36）%,ACE抑制率为（59.54±0.61）%。     

乳清蛋白水解制备功能性多肽的研究概况

乳清是生产干酪及干酪素过程中获得的大量液态副产物,含有丰富的营养成分,其主要成分乳清蛋白的营养价值极高。随着近年来小肽生物活性功能研究的开展,对于乳清蛋白水解物中功能性多肽的研究成为主要方向。近年来的研究中发现乳清蛋白中含有多种生物活性肽,包括ACE抑制肽、抑菌肽、抗病毒肽、抗氧化活性肽及其它活性肽。目前,国内外有关ACE抑制肽的研究较多,而对降胆固醇肽的研究多是以大豆蛋白为原料,而更具有研究价值的乳清蛋白源降胆固醇肽研究较少。本人以乳清蛋白为原料,对其酶解产物的降胆固醇活性肽进行分离制备,既充分利用了干酪的副产物,减少了资源浪费和环境污染,又能提供一种新型降胆固醇功能产品,为商业化开发乳清蛋白降胆固醇功能产品提供理论依据。本文结合本人对降胆固醇肽的研究,综述了乳清蛋白源功能性多肽的制备、分离纯化技术及其活性多肽的研究概况。     

产ACE抑制肽乳酸菌的筛选、益生特性评定及应用

目的:筛选出富产血管紧张素转化酶（Angiotensin converting enzyme,ACE）抑制肽的乳酸菌并评定其益生特性。方法:依据蛋白水解度及ACE抑制率对乳酸菌进行初筛,然后依据模拟胃肠消化后的ACE抑制率最终筛出2株乳酸菌ZJUIDS09和ZJUIDS11,进一步评价其耐酸、耐胆盐、抗生素耐药性和抑菌活性等益生特性,最后评定两株菌对荷斯坦脱脂乳、荷斯坦乳清、水牛脱脂乳和水牛乳清发酵后产物ACE抑制率。结果:筛选出菌株ZJUIDS09和ZJUIDS11,其发酵乳的蛋白水解度分别为5.79%±0.14%和5.75%±0.10%,ACE抑制率分别为87.39%±2.44%和90.41%±0.99%,IC₅₀值分别为0.31和0.25 mg/mL,经人工胃肠液消化后ACE抑制率分别为70.13%±0.15%和76.39%±2.91%。菌株ZJUIDS09和ZJUIDS11都具有良好的耐酸耐胆盐、抗菌和抗生素敏感性,经16S rDNA鉴定分别为罗伊氏乳杆菌（Lactobacillus reuteri）和瑞士乳杆菌（Lactobacillus helveticus）。通过对比同一蛋白浓度下不同底物（荷斯坦脱脂乳、荷斯坦乳清、水牛脱脂乳和水牛乳清）经菌株发酵后的ACE抑制率,确定菌株ZJUIDS09和ZJUIDS11的最佳发酵底物为荷斯坦脱脂乳。结论:本研究筛选的罗伊氏乳杆菌ZJUIDS09和瑞士乳杆菌ZJUIDS11有较强的产ACE抑制肽能力,具有开发降血压功能发酵乳制品的潜力。     

液态发酵法制备菜籽ACE抑制肽发酵条件优化

以菜籽粕为原料,通过枯草芽孢杆菌液态发酵生产菜籽ACE抑制肽。先以肽得率、ACE抑制率为指标通过单因素试验得到液态发酵的发酵条件,再以响应面法分析法,优化了枯草芽孢杆菌液态发酵的工艺条件,确定枯草芽孢杆菌液态发酵生产菜籽ACE抑制肽的最佳发酵工艺条件为发酵时间,发酵温度和接种量,最佳工艺条件分别为20 h、38℃和1×108个/mL。优化后的菜籽ACE抑制肽抑制率达到70.95%。     

德氏乳杆菌QS701产ACE抑制肽发酵条件的优化

通过单因素和正交实实验对德氏乳杆菌QS701产ACE抑制肽的发酵条件进行优化,以期提高ACE抑制肽的产量,并对该菌株的生长特征进行探讨。结果显示,影响ACE抑制活性的发酵条件的顺序为:发酵时间>发酵温度>接种量,在此实验中,德氏乳杆菌QS701的最佳发酵条件为:接种量3%、温度43℃、发酵时间72 h,在此条件下ACE抑制率可达到81.58%,所以将此发酵条件应用于ACE抑制肽的制备中。     

瑞士乳杆菌发酵乳制备ACE 抑制肽的条件优化

本研究以脱脂乳为主要原料,对瑞士乳杆菌发酵产生血管紧张素转化酶(angiotensin I-converting enzyme,ACE)抑制肽工艺条件进行研究。通过单因素试验和响应面试验设计,确定瑞士乳杆菌发酵脱脂乳生产ACE 抑制肽的最佳工艺条件为：脱脂乳浓度9.67%(m/V)、底物灭菌时间30min、接种量3%(V/V),温度38.82℃、发酵时间6.26h,测得ACE 抑制率为92.26%。研究结果证实利用发酵法生产乳源ACE 抑制活性肽对控制高血压具有重要意义。     

The impact of fermentation and in vitro digestion on the formation of angiotensin-I-converting enzyme inhibitory activity from pea and whey protein

Pea and whey protein were fermented by Lactobacillus helveticus and Saccharomyces cerevisiae in monoculture and in combination at 28 and 37 degrees C in order to release angiotensin-I-converting enzyme (ACE) inhibitory peptides. The fermentation products were subjected to in vitro gastrointestinal digestion, and the digests of nonfermented samples served as controls. After fermentation, the ACE inhibitory activity (%) increased by 18 to 30% for all treatments, except for the fermentations of whey protein with Saccharomyces cerevisiae at 28 degrees C, where no significant change was observed. After digestion, however, both fermented and nonfermented samples reached maximum ACE inhibitory activity. The whey digests tended to have lower (50%) inhibitory concentrations (IC50; 0.14 to 0.07 mg/ml), hence, higher ACE inhibitory activity, than the pea digests (0.23 to 0.11 mg/ml). The nonfermented whey protein digest showed the highest ACE inhibitory activity of all. For pea protein, the nonfermented sample had the lowest IC50 value. These results suggest that in vitro gastrointestinal digestion was the predominant factor controlling the formation of ACE inhibitory activity, hence, indicating its importance in the bioavailability of ACE inhibitory peptides.     

响应面法优化黄浆水发酵液制备工艺及其抗氧化活性研究

采用植物乳杆菌(Lactobacillus plantarum)、嗜酸乳杆菌(Lactobacillus acidophilus)和清酒乳杆菌(Lactobacillus sakei)3种乳酸菌对黄浆水进行组合发酵,以发酵液DPPH自由基清除率为评价指标,研究发酵温度、发酵时间、脱脂乳粉添加量、葡萄糖添加量和接种量对发酵液的影响。在单因素试验基础上,采用响应面法优化制备高抗氧化活性黄浆水发酵液的工艺条件。结果表明最佳发酵参数为:接种量1%、发酵温度37℃、发酵时间37.50 h、脱脂乳粉添加量8%、葡萄糖添加量5%。在此条件下制备的黄浆水发酵液DPPH自由基清除率为82.36%。抗氧化活性试验表明:黄浆水发酵液提取物抗氧化能力得到显著提升,其清除DPPH自由基、羟自由基和ABTS自由基的半抑制质量浓度(IC50)分别为2.03 mg/mL、1.12 mg/mL和0.30 mg/mL。     

ANGIOTENSIN I-CONVERTING ENZYME (ACE) INHIBITORY PEPTIDES FROM WHEY FERMENTED BY LACTOBACILLUS SPECIES

From 2% (w/w) whey powder in growth media, inhibitory peptides against angiotensin I-converting enzyme (ACE) were studied with nine Lactobacillus species. Lb. brevis, Lb. helveticus and Lb. paracasei were proved to be the most effective strains in liberating ACE inhibitory peptides from whey protein. The inhibition rates of these peptides against ACE ranging from 93.3 to 100%. Several distinct peaks were eluted when the whey proteins were fractionated on a Delta Pak C₁₈ column by reversed phase-high performance liquid chromatography (RP-HPLC). Among ACE inhibitory activities of 14 peptides purified by dialysis and by fractionation using RP-HPLC, two peptide fractions (H5 and H7) of Lb. helveticus showing IC₅₀ values of 5.3 and 7.8 were the most potent ACE inhibitors.
All of these peptides including some other peptides (H1 and B1), having strong inhibitory activities against ACE were pentapeptides positioning with Ala at their N-terminal and these petapeptides had mostly hydrophobic (Pro, Val and Leu) or aromatic (Phe) amino acids at the C-terminal.

PRACTICAL APPLICATIONS

There is a significant amount of research and interest in developing and charactering the peptides that inhibit angiotensin I-converting enzyme (ACE) activity as these natural products may have a role in blood pressure control in man. This study revealed that the identification of peptides, mostly composed of pentapeptides following fermentation of whey protein in growth medium with different strains have the ACE inhibitory activities. These peptides may have antihypertensive effect as natural and safe nutraceutical/functional ingredients, though the exact potency of the pentapeptides isolated in this experiment has not been determined.     

发酵酸化技术对乳饼蛋白质降解及蛋白肽生物活性的影响

通过发酵酸化技术研制发酵型乳饼,以传统的直接酸化技术为对照,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）、液相色谱-质谱测定乳饼蛋白质降解情况和游离氨基酸含量,通过用液相色谱-串联质谱鉴定乳饼蛋白肽。并通过测定体外抗氧化活性、α-葡萄糖苷酶抑制率和血管紧张素转换酶（angiotensin-converting enzyme,ACE）抑制率表征乳饼蛋白肽的生物活性。SDS-PAGE结果表明,两种酸化技术均能促进乳饼蛋白质的降解,发酵酸化技术对乳清蛋白的降解程度更高,能促进游离氨基酸的释放。从发酵酸化乳饼中鉴定出潜在的ACE抑制肽23?条、抗氧化肽6?条和降糖肽5?条,而传统的直接酸化乳饼中分别为13、4?条和1?条。发酵酸化技术能显著提升乳饼蛋白肽的α-葡萄糖苷酶抑制活性和ACE抑制活性（P＜0.05）,其中分子质量＜3?kDa超滤组分对二者抑制率的IC50分别为1.250?mg/mL和0.416?mg/mL。发酵酸化技术能促进乳饼蛋白质降解,释放生物活性多肽,同时产生游离氨基酸,提升乳饼的生物学价值。     

血管紧张素转化酶抑制剂产生菌的筛选及发酵条件优化

以大豆分离蛋白为基质,以血管紧张素转化酶（ACE）抑制活性和多肽含量为评价指标,筛选高产ACE抑制剂的菌株,并以 ACE和蛋白水解度（DH）为考察指标对其产ACE抑制剂的发酵条件进行单因素优化。 通过微生物发酵法筛选出一株具有高ACE抑制 活性的枯草芽孢杆菌（Bacillus subtilis）BS90。 经单因素试验确定最优发酵条件为：采用液态发酵,大豆蛋白含量5%,初始pH值9.0,培 养温度35 ℃,培养时间40 h。 在此发酵条件下,DH和ACE抑制活性分别达到89.1%和81.8%,较优化前分别提高69.5%、13.4%。 为后续 ACE抑制肽的分离制备奠定了基础。     

Molecular Characterization and Identification of Bioactive Peptides Producing Lactobacillus sps. Based on 16S rRNA Gene Sequencing

In the present study, 3 bacterial cultures were isolated from faecal samples of human infant. The biochemical traits showed similarity with Lactobacillus sps and 16S rRNA sequence analyses, confirmed as Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus rhamnosus. The cultures were screened for their proteolytic activity and good ability to release peptides from milk proteins was found. Hence, these bacteria were used as a proteolytic starter culture for the fermentation of skim milk and whey for the liberation of small peptides. Bioactive nature of the peptides released from whey and skim milk was tested, and results demonstrated that peptides obtained after fermentation of whey and skim milk by Lactobacillus strains showed antimicrobial activity against all the pathogens causing food borne infections in humans. These peptides also indicated antioxidant as well as ACE (angiotensin-converting enzymes) inhibitory activity.     

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.     

Optimisation of hydrolysis conditions for the production of the angiotensin-I converting enzyme (ACE) inhibitory peptides from whey protein using response surface methodology

In the present research, the effect of process conditions on the angiotensin-I converting enzyme (ACE) inhibitory activity of whey protein concentrate hydrolysed with crude proteinases preparation from L. helveticus LB13 was investigated systematically using response surface methodology. It was shown that ACE inhibitory activity of the whey hydrolysates could be controlled by regulation of three process conditions (hydrolysis temperature, pH and enzyme to substrate (E/S) ratio). Hydrolysis conditions for optimal ACE inhibition were defined using a response surface model. E/S ratio at 0.60, pH at 9.18 and temperature at 38.9 °C were found to be the optimal conditions to obtain high ACE inhibitory activity close to 92.2% and DH of the whey protein was 18.8%.     

黑曲霉发酵豆粕制备抗氧化肽研究

研究黑曲霉发酵豆粕制备大豆多肽的工艺条件及多肽的抗氧化性能,并考察多肽的分子质量。结果表明：在接种量为2%、发酵液pH6.0、底物质量浓度9g/100mL、发酵时间34h 的条件下,所得发酵液中豆粕多肽的质量浓度最高,为3.38mg/mL。经凝胶层析分离后得到两个组分的大豆多肽(组分I 和组分II)。大豆多肽清除DPPH自由基和羟自由基活性及对脂质过氧化反应产物的抑制作用与其质量浓度均呈现良好的线性关系,且组分I 优于组分II。大豆多肽组分I 和组分II 的分子质量分别为675.58D 和1625.54D。