酪蛋白水解物中ACE抑制肽分离及氨基酸序列分析

以牛乳酪蛋白为底物,先经胃蛋白酶水解,再经胰蛋白酶水解,从水解物中分离获得血管紧张素转化酶（angiotensin converting enzyme,ACE）抑制肽并确定其氨基酸序列。酪蛋白的双酶水解物经超滤和葡聚糖凝胶色谱分离,分离得到3 个组分,收集高ACE活性抑制效果组分Ⅱ和Ⅲ。采用离子色谱法分析水解片段的氨基酸组成,液相色谱-质谱法分析水解片段的氨基酸序列,采用固相合成法制备获得的短肽片段,用分光光度法检测ACE活性抑制效果。结果表明,组分Ⅱ包含缬氨酸、丝氨酸、脯氨酸、亮氨酸、苯丙氨酸、谷氨酸、天冬氨酸、酪氨酸,共8 种氨基酸,组分Ⅲ包含痕量的丝氨酸和酪氨酸;将组分Ⅱ和Ⅲ经液相色谱-质谱分析,获得8 个片段,其中,αs2-f（56～57）∶YS、αs2-f（98～107）∶YQKFPQYLQY和κ-f（52～61）∶INNQFLPYPY具有ACE活性抑制作用,其IC50值分别为11.89、11.75 μg/mL和421 μg/mL。     

胃蛋白酶水解酪蛋白制备ACE抑制肽的条件

研究胃蛋白酶水解酪蛋白制备ACE 抑制肽的工艺条件。采用胃蛋白酶水解酪蛋白制备ACE 抑制肽,检测其水解产物的ACE 抑制率,以ACE 抑制率为指标,通过正交试验设计法,确定最终水解条件为水解温度37℃、pH3.0、酶与底物质量比1:100、底物质量分数7%,水解产物ACE 抑制率为92.5%,其IC50 值为0.24mg/mL。酪蛋白的水解度与ACE 抑制率之间并非简单的相关性。     

花生分离蛋白水解物中血管紧张素转化酶抑制肽的分离纯化与结构鉴定

利用碱性蛋白酶Alcalase对花生分离蛋白进行水解,制备花生分离蛋白水解物,并测定不同水解时间所得产物对血管紧张素转化酶(ACE)的抑制作用。未水解的花生分离蛋白没有ACE抑制活性,而利用碱性蛋白酶Alcalase水解所得的水解物具有很强的ACE抑制活性,水解30 min时水解物活性最高,其半抑制浓度为(IC50)0.56 mg/mL。通过超滤、Sephadex G-15凝胶过滤层析、反相高效液相色谱(RP-HPLC)、基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF-MS/MS)和氨基酸组成分析等分析手段从水解30 min所得的水解物中分离鉴定出两种新的ACE抑制肽,氨基酸序列为Gln-Gly-Gly-Ser-Gly-Met-Thr-Leu-Ala-Phe-Pro-Leu-Pro-Lys和Lys-Ile-Phe-Leu-Arg-Leu-Ser,其IC50值分别为10.6μmol/L和36.6μmol/L。     

酶解法制备绵羊乳酪蛋白ACE抑制肽的工艺优化及其抑制机制

为优化酶解法制备绵羊乳酪蛋白ACE抑制肽的工艺条件以及筛选和鉴定一种新的ACE抑制肽,选用5种蛋白酶水解酪蛋白,以水解度、分子质量分布和ACE抑制率为指标筛选最适蛋白酶,采用单因素和响应面试验优化工艺,采用三合一质谱仪(Orbitrap Fusion Lumos Tribrid MS)方法鉴定分子质量小于3 ku组分的氨基酸序列,筛选潜在ACE抑制肽,进行人工合成,测定其IC50值。采用Linewaver-Burk作图确定酶抑制动力学,结合分子对接解析肽段的抑制机制。结果表明:碱性蛋白酶水解酪蛋白的最佳条件为pH 6、底物含量8%、酶添加量4%、温度55 ℃、水解时间90 min,此时酪蛋白水解液ACE抑制率为99.1%。验证具有ACE抑制活性的肽段10条,筛选出一条新颖的降血压肽——LFRQFY(源自αs1-酪蛋白),其ACE抑制活性的IC50为(7.9±1.7)μmol/L,酶抑制动力学为混合抑制模式。分子对接结果表明:LFRQFY能与ACE的氨基酸残基Ala354(活性口袋S1)、His353(活性口袋S2)形成氢键,具有显著的体外降血压活性。     

酪蛋白水解进程的研究

研究了牛乳酪蛋白的水解进程和水解产物的血管紧张素转换酶(angiotensin-I-convertingenzyme,ACE)抑制活性。研究结果表明,用胰蛋白酶对酪蛋白进行水解,在水解度相同时,所得的水解产物的ACE酶抑制活性也不一样;用高的酶/底物比水解酪蛋白,所得的水解物的ACE抑制活性较高,达88%～90%;水解时间和水解度与水解产物的ACE抑制活性之间没有相关性。     

丙醇-水介质中酪蛋白水解物的酶修饰与ACE抑制活性变化

利用碱性蛋白酶水解酪蛋白,制备出水解度为12.6％、ACE抑制活性为48.2％的酪蛋白水解物.利用碱性蛋白酶、在丙醇-水介质中进行类蛋白反应修饰酪蛋白水解物,研究反应温度、酶添加量、底物质量浓度、丙醇浓度对修饰反应的影响.响应面法试验设计,得到最优条件为反应温度47℃、酶添加量8.3 kU/g,底物质量浓度56.8 g/100 mL,丙醇体积分数58.5％.利用此条件制备出的反应程度不同的5个修饰产物,ACE抑制活性分析结果显示,抑制活性最高可以达到63.8％.在相应条件下加入酪氨酸或苯丙氨酸,对比试验结果显示,添加苯丙氨酸或酪氨酸会导致修饰产物抑制活性增加或降低.     

酶解牛乳酪蛋白制备ACE抑制肽的研究

目的利用酶技术制备酪蛋白源ACE抑制肽.方法以ACE抑制活性为指标,筛选最佳用酶,优化酶解反应条件,并研究酶解过程中水解度和游离氨基酸含量的变化.结果通过对5种蛋白酶的筛选,最终确定AS1.398中性蛋白酶为水解用酶,制备酪蛋白源ACE抑制肽,其最佳反应条件为pH 7、温度45℃、底物质量分数7.5%、酶用量([E]/[S])5%,水解6 h.在酶解过程中,随着时间的延长,水解度略有增加,而游离氨基酸含量大幅度增加.酪蛋白ACE抑制肽的半抑制浓度(IC50值)为0.68mg/mL.结论牛乳酪蛋白用蛋白酶水解可制备高活性的ACE抑制肽,是获得ACE抑制肽的良好来源.     

大豆粕碱性蛋白酶水解肽及ACE抑制活性的研究

豆粕是大豆经浸提脱脂后得到的副产品,含有大量的大豆蛋白,其水解物对血管紧张素转移酶(ACE)具有明显的抑制作用和降压效果。结果表明:以食品级豆粕为原料,经碱性蛋白酶水解后得到的ACE抑制肽在pH8.0,温度50℃,豆粕底物浓度40 g/L的条件下,酶解3 h得到的产物ACE抑制活性最高,为64.21%,对应水解度14.86%;进一步对ACE抑制肽进行高效液相色谱(HPLC)以及超高压液相色谱/四极杆-飞行时间串联质谱联用仪(LC-MS)的分析结果显示:活性物质的主要组分为三肽化合物,分子量为359,分子式为C15H30N6O4,由异亮氨酸、精氨酸和丙氨酸构成;其HPLC检测条件为色谱柱:Zorbax300SB-C18,5μm,4.6×150 mm;检测波长:228 nm,流动相:50%甲醇-超纯水,各含0.1%三氟乙酸;流速:0.7 mL/min;柱温:26℃;进样量:10μL。     

基于定量构效关系制备火麻仁蛋白ACE抑制肽

高血压是引发心血管疾病的主要因素，经常服用降压的药物会产生副作用，因而安全、高效的食源性降压肽成为关注点。本研究建立了血管紧张素转换酶(ACE)抑制肽的定量构效关系(QSAR)模型，确定ACE抑制肽的结构与其活性间的关系。将模型结果辅以计算机虚拟酶切精确掌握蛋白水解位点，筛选合适的蛋白酶水解火麻仁蛋白定向制备ACE抑制肽，并测定最终水解物的抑制效果。结果表明:基于二肽建立的SVM-AAindex模型具有最佳的预测性能(R2为0.81，RMSE为0.53)，且当二肽C端氨基酸为疏水性和芳香族氨基酸时具有较强的ACE抑制效果。蛋白质组分分析发现火麻仁蛋白含有34.60%的疏水性氨基酸和芳香族氨基酸是制备ACE抑制肽的良好来源。选择虚拟酶切产生较多短肽和生物活性肽的碱性蛋白酶和枯草杆菌蛋白酶对火麻仁蛋白进行定向水解，获得的最终水解液的ACE抑制IC50值分别为1.89和2.30 mg/mL。本研究建立了一种高效精准的制备火麻仁蛋白ACE抑制肽的方法，在降血压药物上具有很好的应用前景。     

酪蛋白水解物的酶法修饰与ACE抑制活性变化

利用枯草杆菌碱性蛋白酶水解酪蛋白制备酪蛋白水解物,其水解度为11.2%,IC50为47.1μg/mL。再应用相同的酶对酪蛋白水解物进行类蛋白反应修饰,考察底物浓度、温度和酶添加量对类蛋白反应的影响,并制备5个不同的修饰产物测定其ACE抑制活性和IC50值。结果表明,修饰产物的ACE抑制活性随修饰程度(游离氨基减少量)的增加而提高,并且都高于未经修饰的酪蛋白水解物。当游离氨基减少量为154.65μmol/g(蛋白)时,修饰产物的IC50值可降至0.6μg/mL。毛细管电泳分析结果显示类蛋白修饰后水解物的多肽组成情况发生明显变化。研究结果证明酪蛋白水解物的ACE抑制活性可以通过类蛋白反应的修饰作用而提高。     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Occurrence of bisphenol-F-diglycidyl ether (BFDGE) in fish canned in oil

The levels of bisphenol-F-diglycidyl ether (BFDGE) were quantified as part of a European survey on the migration of residues of epoxy resins into oil from canned fish. The contents of BFDGE in cans, lids and fish collected from all 15 Member States of the European Union and Switzerland were analysed in 382 samples. Cans and lids were separately extracted with acetonitrile. The extraction from fish was carried out with hexane followed by re-extraction with acetonitrile. The analysis was performed by reverse phase HPL C with fluorescence detection. BFDGE could be detected in 12% of the fish, 24% of the cans and 18% of the lids. Only 3% of the fish contained BFDGE in concentrations considerably above 1mg/kg. In addition to the presented data, a comparison was made with the levels of BADGE (bisphenol-A-diglycidyl ether)analysed in the same products in the context of a previous study.     

Contribution of European research to risk analysis

The European Commission's, Quality of Life Research Programme, Key Action 1—Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches; harmonization of risk assessment principles, methodologies, and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries; to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women; evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.     

低温带皮菜籽粕微粉的不同粒级部分的功能特性

为研究低温带皮菜籽粕微粉的不同粒级部分的功能特性,以经低温脱脂的带皮菜籽粕为原料,经微粉碎后筛分成212~425μm、150~212μm和106~150μm的3个不同粒级的微粉样品,检测这些样品的吸水性、吸油性、乳化性和乳化稳定性、蛋白质体外消化率。结果表明:1 3个不同粒级的微粉样品之间的粗纤维含量存在显著差异,表明三者的结构组成成分有一定差异。23个微粉样品的乳化活性和乳化稳定性随粒度级别的减小而显著增加(P0.01)。33个微粉样品的蛋白质体外消化率随粒度级别的减小而显著增加(P0.01)。4不同粒级带皮菜籽粕微粉样品的吸水性与吸油性受其结构组成物质不同和粒度的双重影响,与粒度的相关性不明显。     

Microbiology of food taints

Fresh and processed foods are often spoilt by the presence of undesirable flavours and odours caused by microbial action. The aim of this paper is to review the current knowledge of microbiologically induced taints that occur in a wide range of foodstuffs, including meats, poultry, fish, crustaceans, milk, dairy products, fruits, vegetables, cereals and cereal products. Examples have been chosen where the compounds responsible for the taint have been identified and sufficient data obtained to demonstrate the involvement of microorganisms. However, in some cases the full identity of the causative organism may not have been elucidated. The types of microorganisms covered by this review include bacteria, fungi, yeasts, actinomycetes and cyanobacteria. Although cyanobacteria do not in general infect foods, their presence in aqueous systems and water supplies can lead to off-flavours in aquatic organisms and processed foodstuffs. Several examples of each of these processes are discussed. Wherever possible, the likely biosynthetic pathway used by the microorganism to produce the offending compound in a foodstuff is indicated.     

Analysis for organic residues from aids to polymerization used to make plastics intended for food contact

Polymers intended for food contact use have been analysed for organic residues which could be attributed to a range of substances employed as polymerization aids (e.g. initiators and catalysts). A wide range of polymers was extracted with solvents and the extracts analysed by gas chromatography-mass spectrometry (GC-MS). The overwhelming majority of substances identified were not derived from aids to polymerization but were oligomers, additives and adventitious contaminants. However, a small number of substances were identified as initiator residues. These included tetramethylsuccinonitrile (TMSN) which was observed in two polymers and it derived from recombination of two azobisisobutyronitrile (AIBN) initiator radicals. Methyl benzoate, benzoic acid, biphenyl and phenyl benzoate were detected in one poly(methyl methacrylate) sample and in two polyvinylchlorides and they are thought to be derived from benzoyl peroxide initiator. TMSN was subsequently targeted for analysis of poly-(methyl methacrylate) plastics using proton nuclear magnetic resonance spectrometry (¹     

Tests of potential functional barriers for laminated multilayer food packages. Part II: Medium molecular weight permeants

Experiments were performed to characterize the kinetics of the permeation of different medium molecular weight model permeants: bisphenol A, warfarin and anthracene, from liquid paraffin, through a surrogate potential functional barrier (25 microns-thick orientated polypropylene--OPP) into the food simulants olive oil and 3% (w/v) acetic acid. The characterization of permeation kinetics generally observed the permeation models previously reported to explain the experimental permeation results obtained for a low molecular weight group of model permeants. In general, the model permeants exhibited behaviour consistent with their relative molecular weights with respect to (a) the time taken to attain steady-state permeation into the food simulant in which they were more soluble, (b) their subsequent steady-state permeation rates, and (c) their partition between liquid paraffin and the OPP membrane.     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. I: Interlaboratory studies of methods of analysis

This paper describes the first part of a project undertaken to develop mussel reference materials for Paralytic Shellfish Poisoning (PSP) toxins. Two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin (STX) and decarbamoyl-saxitoxin (dc-STX) in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the second part of the project: the certification exercise. In the first study, 18 laboratories were asked to measure STX and dc-STX in rehydrated lyophilized mussel material and to identify as many other PSP toxins as possible with a method of their choice. In the second interlaboratory study, 15 laboratories were additionally asked to determine quantitatively STX and dc-STX in rehydrated lyophilized mussel and in a saxitoxin-enriched mussel material. The first study revealed that three out of four postcolumn derivatization methods and one pre-column derivatization method sufficed in principle to determine STX and dc-STX. Most participants (13 of 18) obtained acceptable calibration curves and recoveries. Saxitoxin was hardly detected in the rehydrated lyophilized mussels and results obtained for dc-STX yielded a CV of 58% at a mass fraction of 1.86 mg/kg. Most participants (14 out of 18) identified gonyautoxin-5 (GTX-5) in a hydrolysed extract provided. The first study led to provisional criteria for linearity, recovery and separation. The second study revealed that 6 out of 15 laboratories were able to meet these criteria. Results obtained for dc-STX yielded a CV of 19% at a mass fraction of 3.49mg/kg. Results obtained for STX in the saxitoxin-enriched material yielded a CV of 19% at a mass fraction of 0.34mg/kg. Saxitoxin could not be detected in the PSP-positive material. Hydrolysis was useful to confirm the identity of GTX5 and provided indicative information about C1 and C2 toxins in the PSP-positive material. The methods used in the second interlaboratory study showed sufficiently consistent analysis results to undertake a certification exercise to assign certified values for STX and dc-STX in lyophilized mussel.     

The Ministry of Environmental Protection Issued "Feasible Technology Guidelines for Pollution Prevention and Control in Wood Pulping Process of the Paper Industry (Trial)" and Two Other Guiding Technical Documents

On December 27t＂, 2013, the Ministry of Environmenta Protection announced that, in order to implement ＂The Environmental Protection Law of the People＇ s Republic of China＂, improve the working system in environmenta protection technologies, and promote technologica advancement in pollution prevention, the Ministry of Environmental Protection sponsored the formulation of three guiding technical documents including ＂Feasible Technology Guidelines for Pollution Prevention and Contro n Wood Pulping Process of the Paper Industry （Trial）＂     

1~(st) Intelli-Tissue~ EcoEc Tissue Machine Supplied by PMP Group Successfully Put into Operation in China

正On April 29th,2014,Intelli-Tissue EcoEc tissue machine supplied by PMP Group successfully put into operation at Hebei Xuesong Paper Co.,Ltd.,this is the first such kind of paper machine of PMP Group in China.