贻贝盐溶蛋白特性分析及其ACE抑制肽的酶法制备

以贻贝为原料提取盐溶性蛋白,首先研究盐溶性蛋白的分子质量分布、粒度分布和变性温度等性质,然后比较其3?种蛋白酶（胰蛋白酶、中性蛋白酶和胰酶）酶解产物的血管紧张素转化酶（angiotensin converting enzyme,ACE）抑制活性,筛选出ACE抑制活性较高的胰蛋白酶酶解物,其IC50值为215.96?μg/mL。利用质谱对胰蛋白酶酶解物中的多肽氨基酸序列进行鉴定,利用多肽序列与ACE的对接分数初步筛选出11?个分值大于180?分的多肽序列,通过氨基酸组成结合情况和两者间氢键等相互作用,进一步筛选活性肽并阐述抑制活性机理,最终推测出LYDIDVAK和WIAEEADK是活性较高的抑制肽。     

泥鳅ACE抑制肽的体外活性研究

以泥鳅蛋白为原料,酶解制备具有降血压活性的短肽,研究该肽的稳定性。在加热温度≤80℃条件下加热2h,泥鳅降血压肽(<2.5ku)的ACE抑制活性没有显著变化。当2.0≤pH≤8.0时,泥鳅降血压肽的ACE抑制活性变化不显著。冷冻干燥优于喷雾干燥。Mg2+对泥鳅降压肽的降压活性起到促进作用,而Zn2+和Mn2+抑制了肽的降压活性。葡萄糖对降压肽活性的影响程度明显大于蔗糖,随着浓度的升高,其ACE抑制活性逐渐降低,褐变程度加深;氯化钠对降压活性的影响不明显。在模拟胃肠道消化体系中,胃蛋白酶的消化显著提高了产物的降压活性;胰酶的消化降低了产物的降压活性。泥鳅多肽属于底物型ACE抑制肽。     

ACE抑制肽构效关系研究进展

血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制肽是一类从食源性蛋白质中分离得到的具有降高血压活性的多肽。由于其降血压效果好,而且没有降压药物的毒副作用从而引起了广泛关注。近年来,ACE抑制肽的构效关系研究成为研究重点。结构生物信息学研究表明,ACE抑制肽的ACE抑制能力不仅与其分子质量有关,而且与其氨基酸序列以及其立体空间构象之间存在高度相关性。ACE抑制肽的抑制类型与ACE抑制活性、构效关系也存在一定相关性。对ACE抑制肽构效关系进行深入研究将有助于指导开发高活性的功能性食品及降血压药物。     

豆粕血管紧张素转化酶抑制肽的结构鉴定及作用机制解析

以大豆粕为原材料,利用超声辅助酶解技术、超滤-?KTA层析相结合的方法分离纯化获取豆粕酶解产物中血管紧张素转化酶（angiotensin-converting enzyme,ACE）抑制肽,对其分子质量分布进行研究,后通过质谱分析与分子对接技术鉴定并筛选出ACE抑制活性肽的氨基酸序列,经固相合成肽序列,检测其ACE抑制肽的活性并基于分子对接技术探索其抑制机制。结果表明：经超声辅助酶解提取获得的豆粕肽分子质量主要分布在6 000 Da以下;根据分子对接的最低预测自由能筛选出的GVRP（－8.44 kcal/mol）和IIVTP（－9.04 kcal/mol）可以抑制ACE活性,半抑制浓度（50% inhibitory concentration,IC50）分别为（84±0.06）、（77±0.08）μmol/L;分子对接结果表明：GVRP、IIVTP能够与ACE的活性口袋S1、S1′、S2形成氢键相互作用,共有的过近接触（3.5 ?范围内）ACE氨基酸残基为His513、Ala354和Glu384。本研究基于串联质谱与分子对接技术,建立从混合多肽中快速鉴定、筛选活性多肽的方法,探究活性多肽与ACE稳定结合并体现其ACE活性的抑制机制,为后续的深入研究提供一定参考。     

鱼鳞明胶ACE抑制肽的制备及其活性研究

利用Alcalase 2.4L酶解鱼鳞明胶制备具有血管紧张素转化酶(ACE)抑制活性的降血压肽,通过超滤处理富集得到具有较高活性的ACE抑制肽,并对该组分进行分子量分布、氨基酸组成分析及抗消化性研究。结果表明:水解明胶6 h所得水解物的ACE抑制活性最高,其IC50为0.56 mg/mL,水解度为15.54%;超滤膜处理分离后,分子量小于5 kDa的多肽组分Ⅱ的ACE抑制活性最高,其回收率达90%以上;多肽中对ACE抑制活性贡献大的脯氨酸、羟脯氨酸、色氨酸等疏水性氨基酸的保存率高;体外消化实验显示,该抑制肽在胃肠道消化酶作用下能保持较好的ACE抑制活性。     

海地瓜血管紧张素转换酶抑制肽体外评价

目的:运用优化改进后的血管紧张素转化酶(ACE)抑制剂体外筛选方法,对海地瓜多肽进行初筛,找出ACE抑制活性最强的海地瓜多肽用于下一步研究。方法:对文献报道的ACE抑制剂体外筛选方法稍作改进,并将改进后的方法应用于海地瓜ACE抑制肽初筛。结果:ACE抑制剂筛选反应体系中加入抑制剂时,分解产物马尿酸峰面积下降,ACE活性明显受到抑制;改进后的ACE抑制剂筛选方法精密度、稳定性、重复性均符合要求;海地瓜多肽ACE抑制活性初筛结果显示,海地瓜多肽4的ACE抑制活性最强,可用于下一步分离纯化。结论:通过ACE抑制剂体外筛选方法,已初步筛选得到具有较强活性的海地瓜ACE抑制肽。     

金华火腿中生物活性肽的血管紧张素转化酶调节功能及其分离纯化

为了探究干腌火腿中生物活性肽的降血压功能,以金华火腿为原料,提取其生物活性肽成分,并通过分离纯化鉴定具有血管紧张素转化酶(ACE)调节功能的生物活性肽。结果表明:经Sephadex G-25分子排阻色谱分离后,金华火腿生物活性肽可分为A、B、C、D共4个组分,其中组分C的ACE抑制活性最强,达到31.8%。将组分C经离子交换柱分离后,金华火腿多肽可分为C1、C2、C3、C4共4个组分,其中C2组分的ACE抑制活性显著高于其它组分,活性高达49.0%(P＜0.05)。经Nano-LC-ESI-MS/MS鉴定得到C2中含有8条多肽,经ACE活性测定,序列IESDLERAEE在8条多肽中活性最强,ACE抑制活性达到59.1%。结论:金华火腿多肽具有良好的体外调节ACE活性的功能,为进一步开展其体内血压调节功能研究奠定了基础。     

酪蛋白磷酸肽副产物中ACEI肽的富集工艺研究

以工厂生产酪蛋白磷酸肽后的副产物(CPPBP)为原料,在研究其分离特性的基础上,初步去杂,并借助马尿酸法和HPLC法定位活性最高多肽的位置。接着,以高活力ACEI肽得率为指标,设计三因素三水平的正交实验优化富集工艺,最终研发一种二段量化富集ACEI肽的新工艺。该工艺第一段以CPPBP饱和多肽液为原料,设定沉淀条件为pH2、-16℃、沉淀1h,得到粗富集物(P粗)。第二段再以P粗饱和液为原料,在pH4、乙醇浓度70%、-20℃条件下,沉淀1h。最后产物得率为19.11%,经活性测定,ACE抑制率为46.29%。     

大孔吸附树脂分离纯化海蜇ACE抑制肽的工艺研究

本文主要从分子极性角度研究了大孔吸附树脂对具有血管紧张素转化酶(Angiotensin-I converting enzyme,ACE)抑制活性的海蜇多肽的分离纯化作用.取海蜇酶解产物作为研究对象,选用HP20SS、SP20SS、SP207三种不同型号的大孔吸附树脂分离纯化海蜇ACE抑制肽,以ACE抑制率为评价指标,对...     

小米米糠蛋白水解物及其膜分离组分的降血压相关活性研究

植物源蛋白肽具有较好的抗氧化和降血压活性,研究采用蛋白酶Alcalase水解小米米糠分离蛋白(MBPI)获得小米米糠蛋白水解物(MBPH),经膜分离技术得到成4种不同分子质量大小的多肽组分。通过氧自由基吸附能力(ORAC)、血管紧张素转化酶(ACE)抑制活性、肾素(renin)抑制活性和体内降血压活性,评价了小米米糠蛋白水解物及其不同分子质量组分抗氧化及降血压活性,鉴定了1 ku组分多肽的氨基酸序列。结果显示,低分子质量小米米糠蛋白水解物具有更好的氧自由基吸附能力和降血压活性,1 ku组分的ORAC值为1 455.41μmol TE/mg,ACE抑制率分为73.667%,肾素抑制率为74%,且在灌胃6 h后降低SHRs的收缩压约37.8 mm Hg;1 ku组分中鉴定出7个小肽序列VALVR、VLER、VVRP、WVGK、FGPK、VLLF和LFGK。研究认为小米米糠蛋白水解物,尤其是1 ku肽组分可能作为功能性成分用于降血压相关的功能食品和保健品的开发。     

Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension

Fermented milk is a potential source of various biologically active peptides with specific health benefits. Angiotensin converting enzyme inhibitory (ACE-I) peptides are one of the most studied bioactive peptides produced during milk fermentation. The presence of these peptides is reported in various fermented milk products such as, yoghurt, cheese, sour milk, etc., which are also available as commercial products. Many of the ACE-I peptides formed during milk fermentation are resistant to gastrointestinal digestion and inhibit angiotensin converting enzyme (ACE) in the rennin angiotension system (RAS). There are various factors, which affect the formation ACE-I peptides and their ability to reach the target tissue in active form, which includes type of starters (lactic acid bacteria (LAB), yeast, etc.), substrate composition (casein type, whey protein, etc.), composition of ACE-I peptide, pre and post-fermentation treatments, and its stability during gastrointestinal digestion. The antihypertensive effect of fermented milk products has also been proved by various in vitro and in vivo (animal and human trials) experiments. This paper reviews the literature on fermented milk products as a source of ACE-I peptides and various factors affecting the production and activity of ACE-I peptides.     

Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4.

Whey peptides in a yogurt-like product fermented by Lactobacillus helveticus CPN4 were fractionated by a Sep-pak C-18 cartridge followed by two-step reverse-phase HPLC. The antihypertensive activity was measured by systolic blood pressure in spontaneously hypertensive rats after oral administration of each fraction. Five major peptides in the final fraction were further purified by reverse-phase HPLC and were measured for these antihypertensive activities in spontaneously hypertensive rats. The only peptide in the final fraction that showed strong antihypertensive activity had a sequence of Tyr-Pro, which is found in alpha s1-casein (CN), beta-CN, and kappa-CN. The synthetic peptide Tyr-Pro yielded significant antihypertensive activity from 2 to 8 h after oral administration of 1 mg of peptide/kg of body weight, and the effect was maximal at 6 h after oral administration. The antihypertensive effect of the peptide was dependent on the peptide dosage from 0.1 to 10 mg of peptide/kg of body weight. The concentration of Tyr-Pro peptide increased during fermentation and reached about 8.1 micrograms/ml of whey in the pH 4.3 yogurt-like product. The antihypertensive peptide had a low inhibitory activity against angiotensin I-converting enzyme. The inhibition of 50% of the angiotensin I-converting enzyme (IC50) was 720 microM.     

Evaluation of the antihypertensive angiotensin-converting enzyme inhibitory (ACE-I) activity and other probiotic properties of lactic acid bacteria isolated from traditional Greek dairy products

The technological and probiotic potential of lactic acid bacteria isolated from artisanal Greek yoghurt and fermented milks were evaluated. Fifty-three strains were identified by rep-PCR and 16S rDNA sequencing to belong to different Lactobacillus or Enterococcus spp., as well as to Streptococcus thermophilus and Lactococcus lactis. Several strains exhibited promising technological and probiotic properties. Among them, we focused on the production of bioactive peptides with angiotensin-converting enzyme inhibitory (ACE-I) activity during milk fermentation. The majority of strains produced ACE-I peptides when grown in skimmed milk. ACE-I peptides were sometimes sequestered in the original fermented milk sample, but were released and detected following high performance liquid chromatography (HPLC) purification. Mass spectrometry analysis of major peptide peaks in HPLC fractions with ACE-I activity revealed that they derived from the N- or C-terminal of the isracidin peptide region of α_S1-casein and two internal peptide fragments, one from β-casein and one from κ-casein.     

Purification and identification of antihypertensive peptides from seaweed pipefish (Syngnathus schlegeli) muscle protein hydrolysate

Angiotensin-I-converting enzyme (ACE-I) inhibitory peptides were purified from the seaweed pipefish muscle protein using papain, alcalase, neutrase, pronase, pepsin and trypsin. Among them, the alcalase hydrolysate exhibited the highest ACE-I inhibitory activity. The alcalase hydrolysate was separated into four fractions (Fr1, Fr2, Fr3, and Fr4) by fast protein liquid chromatography (FPLC) on a Hiprep 16/10 DEAE FF anion exchange column. Among four fractions, Fr3 has shown the highest ACE-I inhibitory activity and it was further purified into three fractions (Fr3-I, Fr3-II, and Fr3-III) using reverse-phase high performance liquid chromatography (RP-HPLC) on a Primesphere 10 C18 (20 × 250 mm) column. The Fr3-II has exhibited the highest ACE-I inhibition (IC₅₀, 0.62 mg/ml) than the Fr3-III (IC₅₀, 1.44 mg/ml). The amino acid sequences of the obtained peptides from Fr3-II and Fr3-III were identified as Thr-Phe-Pro-His-Gly-Pro (MW, 744 Da) and His-Trp-Thr-Thr-Gln-Arg (MW, 917 Da) respectively. Furthermore, cell viability assay showed that no cytotoxicity of alcalase hydrolysate on human lung fibroblasts cell line (MRC-5). These results suggest that peptides derived from seaweed pipefish can be developed as antihypertensive ingredients in functional foods.     

ACE-inhibitory activity of probiotic yoghurt

In this study, the in vitro angiotensin-converting enzyme (ACE)-inhibitory (ACE-I) activity of peptide fractions from different yoghurt batches was assessed. Inhibition of ACE activity resulted in an overall antihypertensive effect. Yoghurts were prepared either using a sole yoghurt culture including Lactobacillus delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342, or L. acidophilus L10, L. casei L26 and Bifidobacterium lactis B94 in addition to yoghurt culture. ACE-I activity was determined at weekly intervals during 28 days of cold storage. Peptide fractions showing high ACE-I activity were further purified using multiple-steps of RP-HPLC. All probiotic yoghurts showed appreciable ACE-I activity during initial stages of storage compared with the control yoghurt, with a significant (p<0.05) decrease afterwards. The ACE-I activity ranged from IC₅₀ of 103.30–27.79 μg mL⁻¹ with the greatest ACE inhibition achieved during first and third week of storage. The in vitro ACE-I activity could be related to the peptide liberation via degradation of caseins. In total, 8 ACE-I peptides were characterized originating from α_s2-casein (1), κ-casein (2) and β-casein, of which two well-known ACE-inhibiting peptides, namely Val–Pro–Pro (VPP) and Ile–Pro–Pro (IPP), were identified. These peptides are already used in commercial products.     

Bioactivity of hydrolysates obtained from bovine casein using artichoke (Cynara scolymus L.) proteases

The objective of this work was to obtain casein hydrolysates with aspartic proteinases present in extracts from the artichoke flower (Cynara scolymus L.) and evaluate their antioxidant, antimicrobial, and angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. The casein hydrolysates produced by the action of C. scolymus had elevated antihypertensive and antioxidant activity due to their high hydrophobic peptide content (93.84, 96.58, and 90.54% at 2, 4, and 16 h of hydrolysis, respectively). Hydrolysis time and molecular weight (<3 kDa) had a significant influence on the hypertensive and antioxidant activity of the hydrolysates, which were greater at hydrolysis times of 4 and 16 h and corresponding to the <3 kDa fractions. The <3 kDa fraction of the 16 h hydrolysate had an ACE inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 71.77 µg peptides per mL; DPPH and ABTS^•+ radical scavenging activities of 6.27 µM and 6.21 mM Trolox equivalents per mg of peptides, respectively; and iron (II) chelation activity with an IC₅₀ of 221.49 µg of peptides per mL. Antimicrobial activity against Enterococcus faecalis was also observed in the hydrolysates. From the peptide sequences identified in the hydrolysates, we detected 22 peptides (from the BIOPEP database) that were already in their bioactive form (AMKPWIQPK, AMKPWIQPKTKVIPYVRYL, ARHPHPHLSFM, DAQSAPLRVY, FFVAPFPEVFGK, GPVRGPFPII, KVLPVPQK, LLYQEPVLGPVRGPFPIIV, MAIPPKKNQDK, NLHLPLPLL, PAAVRSPAQILQ, RELEELNVPGEIVESLSSSEESITR, RPKHPIKHQ, RPKHPIKHQGLPQEVLNENLLRF, SDIPNPIGSENSEK, TPVVVPPFLQP, VENLHLPLPLL, VKEAMAPK, VLNENLLR, VYPFPGPIH, VYQHQKAMKPWIQPKTKVIPYVRY, VYQHQKAMKPWIQPKTKVIPYVRYL) and are reported to display antioxidant, antimicrobial, and ACE inhibitory activity. We also identified 12,116, 14,513, and 25,169 peptide sequences in the hydrolysates at 2, 4, and 16 h, respectively, that were contained in the primary sequence, and these are reported to display ACE inhibitory, antioxidant, dipeptidyl peptidase IV inhibition, antithrombotic, opioid, immunomodulation, antiamnesic, anticancer, chelating, and hemolytic bioactivity.     

血管紧张素转换酶抑制肽定量构效关系研究进展

在血压调节方面,血管紧张素转换酶（ACE）的活性是重要的影响因子。ACE抑制肽具有抑制ACE活性、降低血压等作用。其毒副作用小、安全性高。该文主要对血压调节机制、ACE抑制肽的降压机理、食源性ACE抑制肽的制备以及定量构效关系进行综述介绍,对指导ACE抑制肽的分子设计过程给予理论分析,有望开发出高活性的功能性食品及降血压药物。     

Antihypertensive peptides derived from milk proteins

This paper reviews the angiotensin I-converting enzyme inhibitory peptides originated from milk proteins. Focus was put on the peptides derived from milk casein by the action of some proteolytic enzymes and fermented products by lactic acid bacteria. Some of the angiotensin I-converting enzyme inhibitory peptides exhibit significant antihypertensive effects in spontaneously hypertensive rats. However, there were some antihypertensive peptides with low inhibitory activity of this enzyme. Key factors needed for the peptide to demonstrate the antihypertensive effects are discussed. Fermented milk, which has inhibitory activity of the enzyme, showed the reduction of blood pressure of hypertensive subjects. The possibility of the bioactive peptides for functional foods are also discussed.     

Precooked sorghum flour as proper vehicle of ACE-I and DPP-IV inhibitory sorghum peptides

Sorghum flour was heat treated for producing an instant dispersion ingredient. The precooked sorghum flour was added with ACE-I and DPP-IV inhibitory sorghum peptides (3.0 g peptide 100 g⁻¹). The product was reconstituted in water, and peptide bioaccessibility was evaluated by equilibrium dialysis method after simulated gastrointestinal digestion. Total peptide dialysability of precooked sorghum flour added with sorghum peptides was higher than those obtained for precooked sorghum flour (315.9 ± 14.8 vs. 45.2 ± 5.6 µmol, respectively) (P < 0.05). The ACE-I and DPP-IV-IC₅₀ values of the bioaccessible peptides from the bioactive product were lower than those obtained for precooked sorghum flour ingredient (1.04 ± 0.12 vs. 1.82 ± 0.09 and 0.86 ± 0.02 vs. 2.12 ± 0.08 mg protein mL⁻¹, for ACE-I and DPP-IV, respectively) indicating a higher activity. Precooked sorghum flour was a good vehicle since it did not affect the bioaccessibility of ACE-I and DPP-IV inhibitory peptides provided by sorghum protein hydrolysate.     

Antihypertensive mechanism of a peptide-enriched soy sauce-like seasoning: the active constituents and its suppressive effect on renin-angiotensin-aldosterone system

We previously reported that a peptide-enriched soy sauce-like seasoning called fermented soybean seasoning (FSS) demonstrated antihypertensive effects both in spontaneously hypertensive rats (SHR) and Dahl salt-sensitive rats. Angiotensin I-converting enzyme (ACE) inhibitory substances (9 kinds of dipeptides and a nicotianamine) were identified from FSS. In the present study, we clarified the mechanisms underlying the antihypertensive effects of FSS in SHR. FSS was divided into the nicotianamine fraction and the peptide fraction. The peptide fraction was found to exert a more prevalent antihypertensive effect than the nicotianamine fraction in SHR. Among the peptides, we identified Gly-Tyr and Ser-Tyr as the 2 primary substances in FSS that contributed to the antihypertensive effect in SHR. These peptides were neither degraded by acid nor gastrointestinal proteases, and were absorbed into the circulating blood. FSS (2000 mg/kg) exerted ACE inhibitory activity in the lung of rats and provided a decrease (P= 0.0067) in the level of serum aldosterone after a single oral administration in SHR, resulting in the antihypertensive effect. The antihypertensive mechanism was found to be similar to therapeutic ACE inhibitors and other food-derived ACE inhibitory peptides, which are in wide use and are recognized as safe.