Investigation of the ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides from cheese

The ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides found in Cheddar cheese was investigated. The purified protease was incubated with α_s1-casein f1–9 and β-casein f193–209 in a model system (pH 6.8, 30°C) to determine hydrolysis. Residual substrate and hydrolysis products were determined by capillary electrophoresis. Both peptides were hydrolyzed by the protease during the 90-min assay. α_s1-Casein f1–9 was hydrolyzed into two products and β-casein f193–209 was degraded completely in 90 min to three hydrolytic products. This protease hydrolyzed bitter peptides that are known to accumulate in Cheddar and Gouda cheese during aging, suggesting a possibility to debitter Cheddar cheese.     

苦味传递机制与苦味肽研究进展

味觉是人类的重要生理功能之一。人类能够识别五种基本味:苦味、咸味、酸味、甜味和鲜味。由食物蛋白水解产生的苦味肽成为研究热点。本文主要从苦味的信号传递机制、苦味肽结构特点、苦味肽消除或降低的方法等三个主要方面综述了苦味肽的研究进展,以期为低苦味食品开发研究提供参考。     

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%.     

Partial characterization of peptides from emmentaler cheese

A novel isolation procedure for the identification of casein breakdown products in Swiss‐type cheese is described. The cheese extracts were cleaned‐up and fractionated on Waters Sep‐Pak C₁₈ and ion‐exchange (CM‐ and QMA‐Plus) cartridges. The advantages of this preparation technique are discussed. Peptides of different physico‐chemical nature were analyzed by RP‐HPLC. The sequence assays of characteristic segments of the peptide spectrum were carried out by HPLC‐analysis of the phenylthiocarbamyl amino acid derivatives and by automated Edman‐degradation. This experimental approach holds promise for the characterization of the cheese ripening process. Relationships between casein peptides and flavor development are proposed. The results obtained suggest that changes in casein peptides do not parallel the development of cheese flavor. The sequences of six predominant basic peptides are presented. It is the first report on the sequence of protein degradation products in Swiss cheese.     

乳清中酪蛋白糖巨肽的分离纯化

以酶凝干酪乳清为原料,通过沉淀、透析、超滤和离子交换层析等操作,分离提取酪蛋白糖巨肽,通过单因素分析和正交实验,优化出最佳提取工艺条件,同时采用SDS-聚丙烯酰胺凝胶电泳进行分析.结果表明:硫酸铵沉淀乳清中杂蛋白终饱和度为20%;超滤法纯化最佳工艺条件为温庹45℃,压力0.2 MPa,时间30 min;D201GF离子交换层析处理后制得的酪蛋白糖巨肽的纯度为83.97%;SDS-聚丙烯酰胺凝胶电泳结果显示,酪蛋白糖巨肽的相对分子量在15 ku左右.     

酪蛋白源ACE抑制肽的分离纯化

采用碱性蛋白酶酶解酪蛋白制备ACE抑制肽.通过正交实验得到最佳酶解条件为:温度50℃,pH值7.5,酶用量5%,底物质量分数5%,在此条件下,ACE抑制活性为47.23%.采用超滤对酶解液进行初步分离,得到分子量小于4 ku的组分,其ACE抑制活性为57.34%.用凝胶柱SephadexG一25进行进一步纯化,纯化后的组分ACE抑制活性最高可达62.78%,比原酶解物的ACE抑制活性高了24.77%,其相对分子质量在1 500 u以下.     

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.     

酪蛋白酶解产物中抗菌肽的初步研究

以干酪素酪蛋白为试验原料，采用胰蛋白酶在合适的条件下水解，经过不同分子质量的中空纤维超滤膜超滤，获得具有较强抑菌活性的分子量小于3ku的活性组分．结果表明该肽可抑制多种细菌的生长，尤其是对金黄色葡萄球菌和大肠杆菌具有很强的抗菌作用。     

Chemical characterization of bioactive peptides from in vivo digests of casein

The in vivo formation of biologically active caseinopeptides was studied. It was proved that bioactive peptides were released in the small intestine of minipigs in the course of luminal digestion of diets containing bovine casein. An opioid peptide and a phosphopeptide were isolated from jejunal chyme and were chemically characterized. The opioid peptide has been identified as a fragment of beta-casein (60-70). This peptide, named beta-casomorphin-11, displayed substantial opioid activity in an opiate receptor-binding assay. The caseinophosphopeptide has been shown to be a fragment of alpha s1-casein (66-74). Casein-derived phosphopeptides exhibit a potent ability to form soluble complexes with Ca and trace elements. Evidence exists that casomorphins and caseinophosphopeptides participate in the regulation of nutrient entry.     

Purification and identification of potentially bioactive peptides from enzyme-modified cheese.

Antihypertensive peptides inhibiting angiotensin I-converting enzyme have been isolated from enzymatic hydrolysates of various food materials, but no information is available on the isolation of antihypertensive peptides from enzyme-modified cheese. In this study, several bioactive peptides, mainly potential antihypertensive peptides from enzyme-modified cheese prepared by commercial and Lactobacillus casei enzymes, were purified and identified. Enzyme-modified cheese samples were prepared by combination of Neutrase (1883.0 U/ml), L. casei enzymes (amino peptidase activity 86.4 leucine aminopeptidase U/g), and Debitrase (22.0 leucine aminopeptidase U/g). The water-soluble fractions of the enzyme-modified cheeses that were prepared by different enzymes were subjected to reverse-phase HPLC on a Delta Pak C18 column. Each peak was purified on the same column using a binary gradient. One peak from the Neutrase digest, five peaks from the Neutrase-Debitrase digest, and two peaks from the Neutrase-Lactobacillus enzyme digest were purified and identified by API mass spectrometry. On the basis of their molecular masses, amino acid sequences of purified peptides were identified. beta-Casomorphin with a sequence like that of beta-casein (YPFPGPI f 60-66) was found after the Neutrase digest. All of the peptides purified from the digests with combination of Neutrase and Debitrase or Neutrase and L. casei enzymes contained active sites in their sequences. The presence of sites containing potential antihypertensive peptides suggests that the purified peptides may have antihypertensive properties. Thus, the enzyme-modified cheese process, mainly designed to produce flavor ingredients, may simultaneously produce bioactive peptides, which are considered to be of physiological importance.     

浅淡白酒中苦味的生成及解决措施

苦味在白酒中同臭味一样 ,不受人们的欢迎但在某些食品中又必需具有一定的苦味 ,如烟、茶、啤酒、葡萄酒、黄酒、咖啡、巧克力等。如果没有苦味 ,这些食物就要偏格 ,影响它们特有的风味。一般地说 ,苦味有两个特点 :一是苦味食品经长期食用 ,消费者久而惯之 ,对苦味的感觉就会迟钝。如饮用啤酒 ,最初感到很苦 ,日久则苦味大减 ,而且感觉不到苦。二是苦味反应慢 ,且有很强的持续性 ,不易消失 ,所以常常使人不快。如评酒时 ,都是说酒有后苦而不是前苦 ,就是这个原因。当其他味都消失时 ,苦味仍然存在 ,并感到比较突出。根据我们多年的体会 ,苦…     

苦瓜渣酶解制备多肽的研究

以苦瓜饮料加工所产生的苦瓜渣为原料酶解制备多肽,通过对比实验确定纤维素酶辅助酶解2h,在单因素实验的基础上,采用正交实验优化得出木瓜蛋白酶酶解条件为:[E/S]=3.0%、温度50℃、pH6.5、时间2.5h。葡聚糖凝胶G-25层析法测定结果显示,酶解液中多肽(<5000Da)主要含有三个组分,分子量分别为:1500、810、430Da。     

Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase

Peptides from hydrolysates of fish proteins and from cheeses were analysed for inhibition of prolyl endopeptidase (PE) isolated from porcine muscle. Muscles of cod, salmon, and trout were homogenised and incubated at pH 4.0 with pepsin and then at pH 7.5 with trypsin to obtain fish protein hydrolysates. Homogenates were incubated without exogenous enzymes at pH 4.0 and 7.5 to obtain fish protein autolysates. Water-soluble extracts from "rakfisk" (a Norwegian fermented/autolysed trout muscle dish) and water-soluble extracts from Cheddar, Norvegia, Jarlsberg, and Blue cheese were also prepared. Peptides in the supernatants obtained after heat-treatment of fish hydrolysates, autolysates and water-soluble extracts of rakfisk and cheeses at 95 degrees C for 15 min were analysed for inhibition of PE. Inhibition was also measured in peptide fractions separated by reversed-phase high-performance chromatography and by gel permeation chromatography. The peptide fractions from fish hydrolysates, fish autolysates, and water-soluble extracts of cheeses inhibited PE in hydrolysing Z-Gly-Pro-amidomethylcoumarin. Inhibition by peptides from rakfisk was negligible. Pepsin + trypsin hydrolysates from the three fish species contained PE inhibitory peptides with a broad range of apparent hydrophobicity and apparent molecular mass. Autolysates from muscles of the 3 fish species contained narrow peptide peaks of different molecular mass and different apparent hydrophobicity with strong PE inhibitory activity. The content of hydrophilic inhibitory peptides was lower in cheeses than in pepsin + trypsin hydrolysates of fish muscle.     

Biologically active casein peptides implicated in immunomodulation

Maternal milk should not only be considered as a nutrient, but also as a protecting agent against aggressions from the neonate's new environment. Breast-feeding facilitates transmission of a passive immunity by multifunctional factors which have a direct effect on the neonate's resistance to bacterial and viral infections. Among these factors are the main milk proteins, the caseins: during enzymic digestion of human and bovine caseins, immunomodulating peptides are released. Corresponding synthetic peptides stimulated in vitro phagocytic activity of murine and of human macrophages and exerted in vivo a protective effect against Klebsiella pneumoniae infection of mice. These data suggest that casein peptides may exert a stimulating function on the immune system of the newborn.     

山杏仁肽的体外抗氧化活性研究

分析了山杏仁蛋白的氨基酸组成,比较了分别使用蛋白酶M、AX、Alcalase和Papain酶解山杏仁蛋白所制备的山杏仁肽的抗氧化性质。结果显示:山杏仁蛋白中疏水性氨基酸占氨基酸组成的35.97%,谷氨酸含量超过了27%。使用蛋白酶M制备的山杏仁肽还原能力最好,并且与其质量浓度呈正相关性。使用Alcalase制备的山杏仁肽在清除二苯代苦味酰基自由基(DPPH.)方面能力最强,40 mg/mL时清除率达到91.97%。使用蛋白酶AX所制备的山杏仁肽在清除羟基自由基(.OH)及超氧阴离子自由基(O2-.)方面有着更强的能力,在山杏仁肽质量浓度为50 mg/mL时清除率分别达到78.91%和89.47%。     

Selection of marker peptides from casein phosphopeptide and application for quantification in infant formula

ABSTRACT

Casein phosphopeptides (CPPs) have been used worldwide as a nutritional supplement. However, the peptide components have been unknown; as a consequence, few quantification methods of CPP in infant formula were reported. This study introduced a quantification method based on peptide marker and corresponding peptide selection strategy using a simplified model with four commercial types of CPP. The peptides from four commercial CPPs were first identified. Due to the great variety of CPPs, two marker selection strategies were adopted: on one hand, universal marker peptide VLPVPQK can be used for the quantification of all four commercial CPPs, if the CPP can be obtained as a standard. On the other hand, the specific marker peptide LYQEPVLGPV can be used for identification and quantification of commercial CPP type K content in infant formula with a fixed calculation factor. In the simplified model, the combination use of the two markers can meet most of the requirements of CPP analysis in infant formula. The method validation revealed that this was suitable for the routine analysis laboratories without proteomics backgrounds. This selection strategy was suggested for the large-scale marker peptide selection with all commercial CPPs, which can give a comprehensive solution of CPP quantification in infant formula.

Abbreviations: CPP: Casein phosphopeptides; LC: Liquid chromatography; TQMS: Triple quadrupole mass spectrometry; MRM: Multiple reaction monitoring; RSD: Relative standard deviation; L*: [¹³C₆, ¹⁵N]-leucine; SSSEE: Peptides sequence of serine-serine-serine-glutamic acid-glutamic acid     

酪蛋白源ACE抑制肽的精制

采用离子交换树脂对酪蛋白源ACE抑制肽进行脱盐处理,结果表明,以10倍柱体积/h流速,经阴阳离子交换树脂后脱盐率达80%左右,氮回收率为83%,脱盐处理前后基本上对其ACE抑制活性和氨基酸含量没有影响。     

Ultrasound-assisted generation of ACE-inhibitory peptides from casein hydrolyzed with nanoencapsulated protease

BACKGROUND: Bioactive peptides generated from milk proteins are eminent ingredients for functional foods and nutraceuticals. Amongst several approaches to release these peptides, hydrolysis of milk proteins with proteolytic enzymes is a promising choice. It is, however, required to inactivate the enzyme after a predetermined time, which leads to impurity of the final product. Immobilization of enzyme molecules can overcome this problem as it simplifies enzyme separation from the reaction mixture. A fungal protease from Aspergillus oryzea was encapsulated within nanoparticles yielded via silicification of polyamidoamine dendrimer template generation 0. It was used to hydrolyze the dominant milk protein (casein) in the absence or presence of sonication. The production of angiotensin converting enzyme (ACE)‐inhibitory peptides was monitored during hydrolysis. RESULTS: Sonication did not affect maximum ACE‐inhibitory activity but shortened the process sixfold. Ultrafiltration permeate of the centrifugal supernatant of casein solution hydrolyzed during sonication inhibited ACE activity as efficiently as the supernatant obtained from it. CONCLUSION: The protease from Aspergillus oryzea encapsulated within nanospheres is suitable for generation of ACE‐inhibitory peptides from casein. The nanoncapsulation procedure is simple, rapid and efficient. This may enable the industrial production of functional products from milk. Copyright © 2011 Society of Chemical Industry