利用啤酒废酵母酶法制备蛋白肽工艺研究

以啤酒废酵母为原料,选用碱性蛋白酶(Alcalase)酶,制备酵母蛋白肽.分别从底物浓度、酶解温度、加酶量、酶解pH值和酶解时间等因素来研究Alcalasc酶对啤酒废酵母水解度的影响,并通过正交试验优化酶解条件,其最佳酶解条件为:酵母浓度20%、pH8.0、加酶量0.5%、酶解温度55℃、酶解时间16h.在此条件作用下,酵母的水解度(DH)可达38.2%,水解液中酵母蛋白肽含量可达5.32mg/mL.     

Preparation of casein phosphorylated peptides and casein non-phosphorylated peptides using alcalase

Casein was digested with a cheaper enzyme, alcalase, to produce casein phosphorylated peptides and casein non-phosphorylated peptides concurrently. The casein hydrolyzates were separated to the two kinds of peptides by using combined treatment of CaCl₂ and ethanol. Casein phosphorylated peptides and non-phosphorylated peptides constitute some peptides with molecular weight lower than 2509 Da and 2254 Da respectively as determined using size exclusion HPLC, particularly when a degree of hydrolysis of 20% for the casein hydrolyzates was achieved. At the end, the recovery of casein phosphorylated peptides reached 24%. Phosphorus component of casein phosphorylated peptides was found to be 3.08%. The nitrogen recovery of casein non-phosphorylated peptides was about 76%.     

Angiotensin-converting enzyme inhibitory peptides from goats' milk released by in vitro gastro-intestinal digestion

The aim of this study was to identify the angiotensin-converting enzyme (ACE) inhibitory peptides released after in vitro gastro-intestinal digestion of skimmed goats' milk. The experimental approach involved a recently developed harmonised static in vitro digestion model, with mass spectrometry (MS) to identify bioactive peptides. Peptides in the post-pancreatic digest were extracted by ultrafiltration and isolated by reversed-phase high-performance liquid chromatography followed by MS. Among the identified sequences, eighteen were identical to known bioactive peptides with ACE-inhibitory activity. Peptides with dipeptidyl peptidase IV-inhibitory and antioxidant activities were also identified. The antihypertensive tripeptides valine-proline-proline and isoleucine-proline-proline were released from goats’ milk protein during in vitro gastro-intestinal digestion at concentrations of 1829.8 ± 216.4 and 141.4 ± 15.1 μg L⁻¹, respectively. This research underlines the suitability of the harmonised digestive model system to study the release of short bioactive peptides during gastro-intestinal transit.     

Vicilin and the basic subunit of legumin are putative chickpea allergens

IgE-mediated reactions to food allergens constitute a major health problem in industrialized countries. Chickpea is consumed in Mediterranean countries, and reportedly associated with IgE-mediated hypersensitivity reactions. However, the nature of allergic reactions to chickpea has not been characterized.     

Angiotensin I-converting enzyme (ACE) inhibitory peptides derived from arachin by simulated gastric digestion

In silico analysis of the sequences of arachin, the major storage protein of peanut suggests that it is laden with antihypertensive peptides. Physiological proteases pepsin, trypsin, chymotrypsin and pancreatin were used to release these peptides. The degree of proteolysis and in vitro angiotensin I-converting enzyme (ACE) inhibition was maximum with pepsin. The ACE inhibitor index of human gastric juice catalysed digestion was similar to pepsin demonstrating that such peptides can be produced in vivo following ingestion of arachin. Three peptides purified from the simulated gastric fluid digests were synthesized. Among them, the pentapeptide, NAQRP was the most potent with an IC₅₀ of 32 ± 2 μM. Molecular docking simulation with human tACE indicate that in addition to a favourable C-terminal Pro residue, the length of the peptides advocate ACE inhibitor potency. These results further potentiate the use of arachin/peanut proteins as functional ingredients in auxiliary therapeutic foods toward blood pressure management.     

鹰嘴豆肽、大豆肽功能性质的研究

研究对比了鹰嘴豆肽和大豆肽的水解度、抗氧化性、吸油性、吸湿及保湿性等功能特性的差异,以及蛋白酶种类对肽产物功能性质的影响。结果表明,①酶Ⅱ(Protease from Bacillus sp.)制备的蛋白肽抗氧化性和吸油能力最好,且在低湿度下的吸湿性和高湿度下的保湿性也最好;酶Ⅲ(Papain from papaya latex)制备的蛋白肽水解度最高;酶Ⅰ(Protease fromAspergillus melleus)制备的蛋白肽在不同湿度条件下都有较好的吸湿性能。②大豆肽的水解度和抗氧化能力比鹰嘴豆肽好。③Desi肽吸油能力最强,在抗氧化性上仅次于大豆肽,高低湿度环境下都有很好的保湿能力,其中Desi肽Ⅱ这些特征最为明显。④Kabuli肽在不同湿度条件下的吸湿和保湿能力都较好。     

ACE inhibitory peptides and antioxidant peptides derived from in vitro digestion hydrolysate of hen egg white lysozyme

Lysozyme from hen egg white is a well-known antimicrobial protein with high ratio of hydrophobic and positively charged amino acid residues. In order to explore functional bioactivities of enzymatic hydrolysates of lysozyme, the protein was subjected to a simulated gastrointestinal digestion and the resulting hydrolysate (LPH2) showed a strong competitive angiotensin I-converting enzyme (ACE) inhibitory activity (IC(50)=12.6μg/ml) and a remarkable antioxidant activity. The LPH2 was fractionated using a 3kDa cut-off membrane and the obtained permeate LPH2-3kDa was analysed by MALDI-TOF-TOF MS. Using this technology, 38 different peptides were identified and some of these peptides were well fit with structure requirements of ACE inhibitory peptides and/or antioxidant peptides. The findings from this study suggest that the protein containing high proportion of hydrophobic and positively charged residues have the potential to generate multifunctional peptides, and these peptides would be beneficial ingredient to be used in functional foods.     

Iron-chelating activity of chickpea protein hydrolysate peptides

Chickpea-chelating peptides were purified and analysed for their iron-chelating activity. These peptides were purified after affinity and gel filtration chromatography from a chickpea protein hydrolysate produced with pepsin and pancreatin. Iron-chelating activity was higher in purified peptide fractions than in the original hydrolysate. Histidine contents were positively correlated with the iron-chelating activity. Hence fractions with histidine contents above 20% showed the highest chelating activity. These results show that iron-chelating peptides are generated after chickpea protein hydrolysis with pepsin plus pancreatin. These peptides, through metal chelation, may increase iron solubility and bioavailability and improve iron absorption.     

Production of dipeptidyl peptidase IV inhibitory peptides from defatted rice bran

The insulinotropic hormone glucagon-like peptide-1 is metabolised extremely rapidly by the ubiquitous enzyme dipeptidyl peptidase IV (DPP-IV). Therefore, human DPP-IV is a key regulator involved in the prevention and treatment of type 2 diabetes. To simplify the method of producing an inhibitory peptide against DPP-IV, we focused on rice bran (RB) as a source and subjected proteins from defatted RB to enzymatic proteolysis using 2 commercial enzymes. The RB peptides produced with Umamizyme G exhibited 10 times the inhibitory activity as those produced with Bioprase SP. The half-maximal inhibitory concentration (IC₅₀) value of the RB peptides was 2.3 ± 0.1 mg/ml. Leu-Pro and Ile-Pro were identified as the inhibitory peptides among the RB peptides produced with Umamizyme G. Ile-Pro was the strongest DPP-IV inhibitor among the 15 Xaa-Pro dipeptides and Pro-Ile tested. Ile-Pro competitively inhibited DPP-IV (K_i = 0.11 mM). Mass spectrometry indicated that the contents of Leu-Pro and Ile-Pro in the RB peptides were 2.91 ± 0.52 μg/mg.     

In vitro study on digestion of peptides in Emmental cheese: analytical evaluation and influence on angiotensin I converting enzyme inhibitory peptides

A simple in vitro protocol simulating gastrointestinal digestion of proteins and peptides to investigate the effect of digestive enzymes on the biological activity of peptides present in dairy products was developed. This protocol consisted in a 30 min incubation with pepsin followed by a 4 h incubation with trypsin or pancreatin. It was applied to an Emmental cheese water-soluble extract (WSE) and to a casein solution (as a control). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) allowed to monitor the digestion of proteins. Reversed-phase high-performance liquid chromatography (RP-HPLC) allowed to monitor the conversion of proteins and peptides into peptides and amino acids: it is proposed to use the mean retention time corresponding to the overall retention time distribution of molecules to assess the effect of digestive enzymes. The biological activity focused in this study was the angiotensin I converting enzyme (ACE) inhibitory activity. Digestion of Emmental WSE induced an increase of the ACE inhibition as compared to undigested WSE while a 10 kDa ultrafiltered WSE lost a part of its ACE inhibitory activity after digestion process. These results strongly suggest that digestive enzymes diminished the ACE inhibition by the peptides present in Emmental cheese WSE, while the digestion of peptides of high molecular weight would generate new ACE inhibitory peptides.     

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC₅₀ value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.     

Utilisation of chickpea protein isolates for production of peptides with angiotensin I‐converting enzyme (ACE)‐inhibitory activity

Chickpea protein isolates and the protease alcalase were used for the production of protein hydrolysates that inhibit angiotensin I‐converting enzyme (ACE). The highest degree of inhibition was found in a hydrolysate obtained by 30 min of treatment with alcalase. This hydrolysate was used as starting material for the purification of ACE‐inhibitory peptides. After Biogel P2 gel filtration chromatography and HPLC C₁₈ reverse phase chromatography, four peptides with ACE‐inhibitory activity were purified. Two of them were competitive inhibitors of ACE, while the other two were uncompetitive inhibitors. These results show that chickpea proteins are a good source of ACE‐inhibitory peptides when hydrolysed with the protease alcalase. © 2002 Society of Chemical Industry     

Production of angiotensin-converting enzyme inhibitory peptides in Iranian ultrafiltered white cheese prepared with Lactobacillus brevis KX572382

In this study, ultrafiltered (UF) Iranian white cheese made with adjunct cultures including six Lactobacillus isolates (Lactobacillus brevis, L. casei and L. plantarum) from traditional Iranian Motal cheese. The peptide extract (<5 kDa) of cheese samples were assessed for angiotensin-converting enzyme (ACE)-inhibitory activity during ripening (5 °C). Among the strains used, L. brevis KX572382 (M8) was selected because of the greater increase in (ACE)-inhibitory activity in the cheese (P < 0.05). The highest activity of M8 extract was observed on the 28th (71.72%) day of ripening (P < 0.05). Proteolytic activity assessment and RP-HPLC peptide profile of M8 water-soluble extracts (WSEs) indicated the effect of M8 on further protein degradation due to secondary proteolysis. A total of 7 different peptide sequences, previously known in the literature for their ACE-inhibitory activity, were tentatively identified by LC/ESI-MS in 28-day M8 peptide extract. Although the effect of M8 on pH and the proteolysis development in cheese was significant, no adverse effect was observed on the sensory properties. In conclusion, M8 strain can enhance the functional properties of Iranian UF white cheese.     

Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin.

The antibacterial properties of enzymatic hydrolysates of bovine lactoferrin were examined to determine whether active peptides are produced from this protein. Hydrolysates prepared by cleavage of lactoferrin with porcine pepsin, cod pepsin, or acid protease from Penicillium duponti showed strong activity against Escherichia coli O111, whereas hydrolysates produced by trypsin, papain, or other neutral proteases were much less active. Low molecular weight peptides generated by porcine pepsin cleavage of lactoferrin showed broad-spectrum antibacterial activity, inhibiting the growth of a number of Gram-negative and Gram-positive species, including strains that were resistant to native lactoferrin. The antibacterial potency of the hydrolysate was at least eightfold greater than that of undigested lactoferrin with all strains tested. The active peptides retained their activity in the presence of added iron, unlike native lactoferrin. The effect of the hydrolysate was bactericidal as indicated by a rapid loss of viability of E. coli O111. The lactoferrin hydrolysate described in the present study has commercial value as a natural preservative agent for use in foods and cosmetics, and as a functional component of new clinical foods for prevention or treatment of gastrointestinal disease.     

碱性蛋白酶钝化胰蛋白酶抑制剂的研究

以碱性蛋白酶水解无糖豆粉溶液 ,检测水解前后胰蛋白酶抑制剂活性的变化 ,采用四因素五水平饱和最优试验设计 ,当作用时间为 1h时 ,最佳酶解钝化条件为 :pH值 8 88～ 9 0 8;温度4 3 2 2～ 4 4 90℃ ;酶用量 11 5uL/g;底物浓度 5 99%～ 6 72 %。     

碱性蛋白酶水解螺旋1藻蛋白质的研究

利用碱性蛋白酶对螺旋2藻蛋白进行水争,用正交方法选择最适温度、酶与底物比（E/S）、pH值等反应条件,以蛋白质收率为衡量指标,优化出最佳水解工艺条件,并研究水解时间对水解效果的影响。     

Profiles,antioxidative and ACE inhibitory activity of peptides released from fermented buttermilk before and after simulated gastrointestinal digestion

Bioactive peptides, released from buttermilk by fermentation and/or gastrointestinal proteases, may have health promoting effects. Thus, a comprehensive analysis of the peptide fraction of fermented buttermilk, before and after different phases of simulated gastrointestinal digestion, was performed using ultra high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS). Results showed that digestion simulation substantially changed the peptide profile of fermented buttermilk. A total of 81, 120 and 46 peptides were identified in fermented buttermilk, its gastric and intestinal digests, respectively. These peptides released mostly from β-casein followed by αs1-casein, κ-casein and β-lactoglobulin. In addition, 14 peptides released from milk fat globule membrane proteins (lactadherin, butyrophilin and GlyCAM-1). Bioactivity, mainly angiotensin converting enzyme (ACE) inhibitory activity, has been reported before for only 54 of the detected peptides. Radical scavenging, ferric reducing and ACE inhibitory activities of fermented buttermilk peptides increased significantly after digestion, indicating promotion in fermented buttermilk-peptide bioactivity by gastrointestinal digestion.     

Novel antibacterial lactoferrin peptides generated by rennet digestion and autofocusing technique

Bovine lactoferrin was hydrolysed with a range of proteolytic enzymes including calf rennet, fungal rennin, and porcine pepsin. Lactoferrin hydrolysates were assessed for their antibacterial activities against Escherichia coli and Bacillus subtilis. At pH 3, calf rennet lactoferrin hydrolysate before (LFH) showed the highest antimicrobial activity, then pepsin LFH, while fungal rennin LFH was the least active. The calf rennet and pepsin LFH were fractionated using autofocusing and chromatographic techniques. The activity-guided fractionation of calf rennet LFH identified a potent antimicrobial peptide of 11-residues, lactoferricin B (Lf-cin B), and three other novel antibacterial peptides. The 11-residues Lf-cin B was the most potent antibacterial peptide and was isolated from both rennet and pepsin LFH. Pepsin LFH had a main antimicrobial peptide of 25-residues, which was not detected in calf rennet LFH. It could be concluded that calf rennet LFH had stronger antibacterial properties than porcine pepsin LFH. Besides, autofocusing could be used for scaling up the isolation of the potent rennet LFH peptides that would have a widespread commercial use as a natural food preservative substituting porcine pepsin digest, especially in Islamic communities.     

凝血酶抑制肽研究进展

凝血酶是凝血过程的关键酶,以凝血酶作为靶点,用凝血酶抑制剂来阻碍凝血过程的发生是抗凝血常用方法之一。活性肽具有诸多生物功能,其抗凝血活性目前也受到越来越多的关注。该文综述了近年来有关凝血酶抑制肽的研究成果,重点介绍食物源凝血酶抑制肽的来源和结构信息,并对酶法制备抗凝活性肽的应用前景作出了展望。