超声处理对菜籽蛋白酶解效果的影响

以双低菜籽饼粕蛋白为原料,以常规酶解为对照,以酶解产物的抑制活性为指标,分别考察了超声预处理蛋白酶、超声预处理蛋白原料和酶解过程前期施加超声3种超声处理方式对双低菜籽蛋白酶解制备ACEI 活性肽效果的影响。结果表明,3种超声辅助方式都能有效地促进菜籽饼粕蛋白的酶解,提高产物的ACE抑制活性,其中超声预处理蛋白原料效果最为明显。这种方式的最佳参数为：超声频率20 kHz、超声时间 30 min、超声功率1250 W、作用时间4 s、间歇时间2 s,该条件下酶解产物对ACE的抑制率从对照组的70.83%提高到92.97%,肽得率从29.6%提高到41.4%,半抑制浓度IC50从3.57 mg/mL降到2.48 mg/mL。这表明脉冲超声辅助酶解法是一种高效制备双低菜籽ACEI肽的方法。     

Isolation and characterization of a low molecular weight peptide contained in sourdough

To investigate a sourdough-specific peptide, low molecular weight peptides were extracted from sourdough. The peptide fraction was subjected to two kinds of chromatography to separate the peptides. Reverse-phase chromatography of the peptide fraction in the sourdough showed certain specific peptides. The specific peptide fraction was further separated by gel filtration chromatography. Liquid chromatography tandem mass spectrometry analysis identified one of the peptides as VPFGVG (six-mer). This sequence was estimated to occur at the 287-292 position of a low molecular weight glutenin subunit. The peptide (designed as SDP1) was produced by proteases derived from wheat flour. SDP1 showed angiotensin-converting enzyme (ACE) inhibitory activity, and the 50% inhibitory peptide concentration (IC50) was 336 microM. It is possible that the SDP1 peptide partially confers ACE inhibitory activity in sourdough.     

Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates

Sunflower protein isolates and the proteases pepsin and pancreatin were used for the production of protein hydrolysates that inhibit angiotensin-I converting enzyme (ACE). Hydrolysates obtained after 3 h of incubation with pepsin and 3 h with pancreatin were studied. An ACE inhibitory peptide with the sequence Phe-Val-Asn-Pro-Gln-Ala-Gly-Ser was obtained by G-50 gel filtration chromatography and high-performance liquid chromatography C18 reverse phase chromatography. This peptide corresponds to a fragment of helianthinin, the 11S globulin from sunflower seeds, which is the main storage protein in sunflower. These results show that sunflower seed proteins are a potential source of ACE inhibitory peptides when hydrolyzed with pepsin and pancreatin.     

Release of angiotensin I converting enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: from batch experiment to semicontinuous model

Gastrointestinal digestion is of major importance in the bioavailability of angiotensin I converting enzyme (ACE) inhibitory peptides, bioactive peptides with possible antihypertensive effects. In this study, the conditions of in vitro gastrointestinal digestion leading to the formation and degradation of ACE inhibitory peptides were investigated for pea and whey protein. In batch experiments, the digestion simulating the physiological conditions sufficed to achieve the highest ACE inhibitory activity, with IC(50) values of 0.076 mg/mL for pea and 0.048 mg/mL for whey protein. The degree of proteolysis did not correlate with the ACE inhibitory activity and was always higher for pea than whey. In a semicontinuous model of gastrointestinal digestion, response surface methodology studied the influence of temperature and incubation time in both the stomach and small intestine phases on the ACE inhibitory activity and degree of proteolysis. For pea protein, a linear model for the degree of proteolysis and a quadratic model for the ACE inhibitory activity could be constituted. Within the model, a maximal degree of proteolysis was observed at the highest temperature and the longest incubation time in the small intestine phase, while maximal ACE inhibitory activity was obtained at the longest incubation times in the stomach and small intestine phase. These results show that ACE inhibitory activity of pea and whey hydrolysates can be controlled by the conditions of in vitro gastrointestinal digestion.     

Peptides with angiotensin I-converting enzyme (ACE) inhibitory activity from defibrinated,hydrolyzed bovine plasma

Defibrinated bovine plasma (DBP) was treated with the microbial protease Flavourzyme to obtain protein hydrolysates with various degrees of hydrolysis (DH). The angiotensin I-converting enzyme (ACE) inhibiting activity of the hydrolyzed protein was assessed with hippuryl-His-Leu as the substrate. The amount of hippuric acid released, due to uninhibited ACE activity, was determined by high-performance liquid chromatography. ACE inhibiting (ACEI) activity was found to increase with increasing DH; the 43% DH hydrolysate exhibited the highest activity and had an IC(50) of 1.08 mg/mL. Peptide fractions with high ACEI activity were isolated using size exclusion chromatography. The fraction that possessed the highest ACEI activity contained peptides with GYP, HL(I), HPY, HPGH, L(I)F, SPY, and YPH sequence motifs, as determined by reversed-phase liquid chromatography-tandem mass spectrometry using a novel immonium precursor-ion scanning technique. Some of these motifs correspond to sequences found in bovine serum albumin, a potential source of ACEI peptides in bovine plasma.     

Identification of angiotensin-I-converting enzyme inhibitory peptides derived from sodium caseinate hydrolysates produced by Lactobacillus helveticus NCC 2765

Angiotensin-I-converting enzyme (ACE) inhibitory activity was identified in milk proteins fermented with Lactobacillus (Lb.) helveticus NCC 2765 (Nestle Culture Collection, Vers-chez-les-Blanc, Switzerland). Hydrolyzing sodium caseinate for 1 and 2 h inhibited ACE activity, as measured by an in vitro ACE inhibition test. The hydrolysates with the highest ACE inhibitory potential were fractionated by gel permeation chromatography and their low molecular weight fractions collected. These fractions were subsequently subfractionated by reverse-phase high-pressure liquid chromatography. Several hydrophobic subfractions showed high ACE inhibitory potential, and their peptide composition was determined using an ion trap mass spectrometer equipped with an elctrospray ionization source. Analysis of the low molecular weight fraction identified 14 peptides with known antihypertensive activity and 1 with previously described opioid activity. On the basis of the peptide composition of active subfractions, two potentially active novel sequences were defined, and the following synthetic peptides were synthesized: FVAPFPEVFG (alphaS1 39-48), ENLLRFFVAPFPEVFG (alphaS1 33-48), NENLLRFFVAPFPEVFG (alphaS1 32-48), LNENLLRFFVAPFPEVFG (alphaS1 31-48), NLHLPLPLL (beta 147-155), ENLHLPLPLL (beta 146-155), and VENLHLPLPLL (beta 145-155). The ACE inhibitory potential of these synthetic peptides was assessed, and IC50 values were determined. NLHLPLPLL (beta 147-155), which was the only synthetic peptide also present in the sodium caseinate hydrolysates, and NENLLRFFVAPFPEVFG (alphaS1 32-48) showed the highest inhibition of ACE activity, with IC50 values of 15 and 55 microM, respectively. Furthermore, the stability of all synthetic peptides was assessed using an in vitro model simulating gastric digestion. The beta-casein-derived peptides remained intact following the successive hydrolysis by pepsin and pancreatin, whereas alphaS1-casein-derived peptides were degraded by pepsin.     

Purification of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide with an antihypertensive effect from loach (Misgurnus anguillicaudatus)

To isolate and characterize novel angiotensin I-converting enzyme (ACE) inhibitory peptide from loach (Misgurnus anguillicaudatus), six proteases, pepsin, α-chymotrypsin, bromelain, papain, alcalase, and Neutrase, were used to hydrolyze loach protein. The hydrolysate (LPH) generated by bromelain [ratio of enzyme to substrate, 3:1000 (w/w)] was found to have the highest ACE inhibitory activity (IC(50), 613.2 ± 8.3 μg/mL). Therefore, it was treated by ultrafiltration to afford fraction of LPH-IV (MW < 2.5 kDa) with an IC(50) of 231.2 ± 3.8 μg/mL, having higher activity than the other fractions. Then, LPH-IV was isolated and purified by consecutive purification steps of gel filtration chromatography and reverse-phase high-performance liquid chromatography to afford a purified peptide with an IC(50) of 18.2 ± 0.9 μg/mL, an increase of 33.7-fold in ACE inhibitory activity as compared with that of LPH. The purified peptide was identified as Ala-His-Leu-Leu (452 Da) by Q-TOF mass spectrometry and amino acid analyzer. An antihypertensive effect in spontaneously hypertensive rats revealed that oral administration of LPH-IV could decrease systolic blood pressure significantly.     

Antioxidant peptides with Angiotensin converting enzyme inhibitory activities and applications for Angiotensin converting enzyme purification

Five commercial peptides, namely, reduced glutathione (GSH), oxidized glutathione (GSSG), carnosine, homocarnosine, and anserine, were used to test angiotensin converting enzyme inhibitory (ACEI) activities using N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly (FAPGG) as a substrate. All of these peptides showed dose-dependent ACEI activities. Using 50% inhibition (IC(50)) of captopril as 0.00781 microM for the reference, the IC(50) values of GSH, carnosine, homocarnosine, and anserine were determined to be 32.4 microM, 5.216 mM, 6.147 mM, and 6.967 mM, respectively. GSH or carnosine showed mixed noncompetitive inhibition against ACE. When 0.0164 mM GSH or 0.4098 mM carnosine was added, the apparent inhibition constant (K(i)) was 49.7 microM or 3.899 mM, respectively. Commercial glutathione-Sepharose 4 fast flow, GSH-coupled CNBr-activated and GSH-coupled EAH-activated Sepharose gels were used for ACE purification. Commercial ACE could be adsorbed only by EAH-coupled GSH gels and eluted off the gels by increasing salt concentrations. These EAH-coupled GSH gels might be developed as affinity aids for ACE purification.     

Angiotensin I converting enzyme-inhibitory peptides from commercial wet- and dry-milled corn germ

Bioprocesses were developed to enhance the value of proteins from deoiled corn germ. Proteins were hydrolyzed with trypsin, thermolysin, GC 106, or Flavourzyme to generate the bioactive peptide sequences. At an enzyme to substrate ratio of 1:100, protein hydrolysis of wet-milled germ was greatest using thermolysin followed by trypsin, GC 106, and Flavourzyme. For the dry-milled corn germ, protein hydrolysis was greatest for GC 106 and least for Flavourzyme. Electrophoretic patterns indicated that the hydrolysis conditions used were adequate for generating low molecular weight peptides for both germs. Unhydrolyzed dry- and wet-milled corn germ did not appear to contain angiotensin I converting enzyme (ACE)-inhibitory peptides. After hydrolysis with trypsin, thermolysin, and GC 106 but not Flavourzyme, ACE inhibition was observed. ACE inhibition was greatest for the GC 106 hydrolysate for both wet- and dry-milled corn germ. Denaturing the protein with urea before hydrolysis, in general, increased the amount of ACE-inhibitory peptides found in the hydrolysate. Membrane fractionations of both the wet- and dry-milled hydrolysates indicated that most of the ACE-inhibitory peptides were in the <1 kDa fraction. Examination of the control total protein extracts (before treatment with proteases) from wet- and dry-milled germ revealed that neither had ACE-inhibitory properties. However, when both total corn germ control protein extracts were fractionated, the <1 kDa fraction of wet-milled corn germ proteins exhibited ACE inhibition, whereas the comparable low molecular weight fraction from dry-milled corn germ did not.     

Porcine skeletal muscle troponin is a good source of peptides with Angiotensin-I converting enzyme inhibitory activity and antihypertensive effects in spontaneously hypertensive rats

In the search for novel peptides that inhibit the angiotensin I-converting enzyme (ACE), porcine skeletal troponin was hydrolyzed with pepsin, and the products were subjected to various types of chromatography to isolate active peptides. Glu-Lys-Glu-Arg-Glu-Arg-Gln (EKERERQ) and Lys-Arg-Gln-Lys-Tyr-Asp-Ile (KRQKYDI) were identified as active peptides, and their 50% inhibitory concentrations were found to be 552.5 and 26.2 microM, respectively. These are novel ACE inhibitory peptides, and the activity of KRQKYDI was the strongest among previously reported troponin-originated peptides. KRQKYDI was slowly hydrolyzed by treatment with ACE, and kinetic studies indicated that this peptide was a competitive inhibitor of the enzyme. When KRQKYDI was administered orally to spontaneously hypertensive rats (SHR) at a dose of 10 mg/kg, a temporary antihypertensive activity was observed at 3 and 6 h after administration.     

LC-MS/MS quantification of bioactive angiotensin I-converting enzyme inhibitory peptides in rye malt sourdoughs

This study quantified antiotensin I-converting enzyme (ACE) inhibitory peptides in rye malt sourdoughs supplemented with gluten proteins and fermented with six strains of Lactobacillus spp. Bioinformatic analysis of prolamins from barley, rye, and wheat demonstrated that the ACE inhibitory peptides LQP, LLP, VPP, and IPP are frequently encrypted in their primary sequence. These tripeptides were quantified by liquid chromatography-tandem mass spectrometry. Tripeptide levels in sourdoughs were generally higher as compared to the chemically acidified controls. Sourdoughs fermented with different strains showed different concentrations of LQP and LLP. These differences corresponded to strain-specific differences in PepO and PepN activities. The highest levels of peptides VPP, IPP, LQP, and LLP, 0.23, 0.71, 1.09, and 0.09 mmol (kg DM)(-1), respectively, were observed in rye malt: gluten sourdoughs fermented with Lactobacillus reuteri TMW 1.106 and added protease. These concentrations were 6-7 times higher as compared to sourdough without fungal protease and exceed the IC(50) by 100-1000-fold.     

发酵法制备蛤蜊ACE抑制肽的菌种筛选

为筛选适用于发酵蛤蜊制备降血压活性多肽的纳豆芽孢杆菌,本试验从7种日本纳豆中初筛6株具有较高蛋白酶活性的纳豆芽孢杆菌株用于发酵蛤蜊,以体外血管紧张素转化酶(ACE)抑制率及多肽含量为评价指标筛选目的菌株。结果表明,共筛选出3株目的菌株(GL-5、GL~(-1)2和GL-25),其发酵产物对ACE抑制率分别为71.55%、78.31%、75.08%,多肽含量分别可达8.12、9.62、8.79mg·m L~(-1)。其中,GL~(-1)2发酵产物经消化酶水解后仍保持68.31%的ACE抑制率,表明其具有较强的抗消化能力。本研究结果为蛤蜊高值化利用开发具有降血压作用的功能食品提供了一定的理论依据。     

Inhibition strength of short peptides derived from an ACE inhibitory peptide

A series of peptides, derived from an ACE inhibitory peptide (VTVNPYKWLP) found in our previous work, were synthesized. Their half maximal inhibition concentrations (IC(50)) for ACE inhibition have been determined. The effect of amino acid sequence on ACE inhibition was discussed on the basis of IC(50) of the synthetic peptides, and the following characteristics of the ACE inhibitory peptide have been clarified. First, the active portion of this peptide for ACE inhibition is KW. Second, the amino acid sequences near this dipeptide (KW) have a strong effect on the inhibitory activity. Especially, the proline residue in the C-terminal end strongly enhanced ACE inhibition. It should be noted that the IC(50) value of KWLP (5.5 μM) is the same as the ACE inhibitory peptide (VTVNPYKWLP) and that the IC(50) value of KW is 7.8 μM. The stability and absorption efficiency in vivo would be significantly improved by shortening the peptide length from 10 amino acids to four amino acids or two amino acids.     

Isoflavone content and its potential contribution to the antihypertensive activity in soybean Angiotensin I converting enzyme inhibitory peptides

A soybean angiotensin I converting enzyme (ACE) inhibitory peptide fraction was reported to have antihypertensive activity in a rat study. The purpose of the present study was to examine if the presence of isoflavones in the soybean ACE inhibitory peptide fraction may contribute to the blood-pressure-lowering property. The isoflavone concentration in soybean samples was analyzed on a C 18 reverse-phase column using a two-step gradient solvent system. Producing soybean hydrolysate led to a nearly 40% loss of isoflavones compared with the original soybean flour, but the isoflavone composition did not change and the dominant isoflavone chemicals remained as 6'-O-malonylgenistin and 6'-O-malonyldaidzin. Ion exchange chromatography affected significantly both the content and the composition of the isoflavones. The dominant isoflavones in the ion-exchanged fraction were aglycones and nonacylated isoflavones, accounting for 95.8% of the total amount of 987.7 microg/g. It was calculated that the isoflavone content in the soybean ACE inhibitory peptide fraction was 25 times less than the minimal effective isoflavone dose reported. In vitro study also showed that adding isoflavones into both soybean flour hydrolysate and soybean ACE inhibitory peptide samples to a concentration of as high as 31.5% (w/w) did not affect ACE inhibitory activity (IC 50 values). The findings along with previously published results indicated that the contribution of isoflavones in soybean ACE inhibitory peptide fraction to the blood-pressure-lowering property in a short-term feeding study might be negligible.     

红娘鱼降血压肽的酶解制备工艺优化及酶解产物的抗氧化活性研究

为有效利用红娘鱼制备降血压肽,以红娘鱼鱼糜为原料提取蛋白,并对其进行酶解制备降血压肽。以血管紧张素转换酶ACE抑制率和水解度为指标,通过响应面分析法对酶解红娘鱼鱼糜蛋白制备降血压肽的工艺条件进行优化,并对最优条件下制备的酶解产物进行分子量和抗氧化活性测定。结果表明,碱性蛋白酶是制备降血压肽的最适蛋白酶,响应面法优化制备降血压肽的最佳酶解条件为pH值9、酶与底物的比值(酶底比)1.4%、温度54℃、时间2 h,此条件下酶解制得的降血压多肽ACE抑制率理论值为88%,实际值为89.3%;经高效液相色谱(HPLC)分析可得酶解产物相对分子量<2 000 Da。通过测定酶解产物样品的1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率、羟自由基(·OH)清除率及还原力判定其体外抗氧化活性,结果表明酶解产物具有较强抗氧化活性。本研究结果为红娘鱼的高值化利用提供了数据支持和理论基础。     

酶解制备鱼鳞蛋白降血压肽的工艺优化

为有效利用鱼鳞制备降血压肽,以罗非鱼鱼鳞为原料,在121℃条件下进行热预处理15 min后,运用响应面分析法优化酶解制备鱼鳞蛋白ACE抑制肽的工艺条件。结果表明,以水解度和ACE抑制率为评价指标,筛选出碱性蛋白酶为最优酶。在单因素试验的基础上,根据Box-Behnken中心组合试验设计原理,最终确立最优的酶解工艺参数为:酶解时间2 h、酶解温度56.3℃、pH值8.0,酶底比1.1%,此条件下ACE抑制率理论值为87.95%,实际值为88.26%。最优条件下制得的酶解产物相对分子质量集中在300～3 000 Da之间,水解效果较好。本研究结果对酶解法制备鱼鳞蛋白降血压活性肽具有一定的实践参考价值。     

Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate.

Bovine skin gelatin was hydrolyzed with sequential protease treatments in the order of Alcalase, Pronase E, and collagenase using a three-step ultrafiltration membrane reactor. The molecular weight distributions of the first, second, and third hydrolysates were 4.8-6.6, 3.4-6.6, and 0.9-1.9 kDa, respectively. The angiotensin I converting enzyme (ACE) inhibitory activity of the third hydrolysate (IC(50) = 0.689 mg/mL) was higher than that of the first and second hydrolysates. Two different peptides showing strong ACE inhibitory activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Val and showed IC(50) values of 2.55 and 4.67 microM, respectively.     

Inhibitory profile of nonapeptide derived from porcine troponin C against angiotensin I-converting enzyme

A novel angiotensin I-converting enzyme (ACE) inhibitory peptide (RMLGQTPTK; 9mer) from porcine skeletal troponin C was investigated for its inhibitory profile. This peptide was noncompetitive and as hydrophobic as the known ACE inhibitory peptides. Aminopeptidase M quickly hydrolyzed 9mer, resulting in production of MLGQTPTK and LGQTPTK with inhibitory activities similar to those of 9mer. The main hydrolysis product of 9mer with carboxypeptidase A and B was RMLGQTPT showing very weak activity. Most products derived from 9mer hydrolysis by ACE, aminopeptidase, or carboxypeptidase showed weak but definite ACE inhibitory activities. Thus, 9mer was estimated to be a wholly efficient inhibitor with these fragment peptides.     

A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate

Alaska pollack frame protein, which is normally discarded as an industrial byproduct in the processing of fish in plants, was hydrolyzed with pepsin. This was fractionated into five major types of Alaska pollack frame protein hydrolysates (APH-I, 10-30 kDa; APH-II, 5-10 kDa; APH-III, 3-5 kDa; APH-IV, 1-3 kDa; and APH-V, below 1 kDa) using an ultrafiltration membrane bioreactor system. Angiotensin I converting enzyme (ACE) inhibitory activities of the fractionated hydrolysates were investigated, and the fraction that exhibited the highest ACE inhibitory activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. Finally, we purified a novel ACE inhibitory peptide with an IC50 value of 14.7 microM, and the sequence of the peptide was Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala. In addition, the ACE inhibition pattern of the peptide was found to be noncompetitive.     

Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats

Angiotensin converting enzyme (ACE) inhibitory peptides prepared from soy protein by the action of alcalase enzyme was tested for its hypotensive effect on spontaneously hypertensive rats (SHR). Captopril, an ACE inhibitor used widely for hypertension treatment, was also applied in comparison. A significant (p < 0.05) decrease in systolic blood pressure of SHR was observed when soy ACE inhibitory peptides were orally administrated at three different dose levels (100, 500, and 1000 mg/kg of body weight/day), whereas little change occurred in the blood pressure of normotensive rats even at the highest dose. After a month-long feeding, blood pressure readings of SHR fell by approximately 38 mmHg from the original level at the lowest dose; a steadily and progressively hypotensive effect existed for these soy ACE inhibitory peptides administration groups. An obvious fluctuation was observed at the third week, although Captopril had a stronger hypotensive effect. The ACE activity of serum, aorta and lung, and lipid content of serum of SHR upon administration of soy ACE inhibitory peptides did not show a significant difference from that of the control group, whereas the serum ACE activity increased and the aorta ACE activity decreased significantly (p < 0.05) for the Captopril group. Serum Na(+) concentration decreased significantly in both the peptides-treated groups and the Captopril-treated group in comparison with the control group, whereas no lowering effect was observed for serum K(+) and serum Ca(2+) concentrations. These results suggested that the hypotensive effect of ACE inhibitory peptides derived from soy protein could be at least partly attributed to the action on salt/water balance.