【作者】 于志鹏；

【导师】 殷涌光；

【作者基本信息】 吉林大学 ， 农业机械化工程， 2014， 博士

【摘要】 食源性蛋白质的生理活性主要归功于蛋白质内部经裂解后释放出的片段，即为活性肽。活性肽可通过体外特异性酶或是体内胃肠道消化酶裂解方式制备获得，对人类健康有巨大贡献。我国禽蛋产量居世界之首，且蛋清中含有多种功能蛋白组分，包括卵清蛋白，卵转铁蛋白和半胱氨酸蛋白酶抑制剂等。蛋清蛋白的酶解产物具有多种生理活性，包括抑菌活性，抗黏活性，降血压活性和抗氧化活性等，突显蛋清蛋白活性肽对人类健康及疾病的预防和治疗方面的巨大潜力。本文通过色谱及组学技术分离纯化共获得19个活性肽片段，利用串联质谱鉴定19个活性肽的氨基酸序列，采用圆二色谱和傅立叶红外光谱探索其二级结构中α-螺旋，β-转角，β-折叠和无规卷曲等二级结构信息。研究分别对19个蛋清源活性肽的血管紧张素转化酶抑制活性、α葡萄糖苷酶抑制活性、α淀粉酶抑制活性和抗凝血酶活性进行了测定，筛选具有较高体外活性的活性肽，通过单次、短期和长期灌胃大鼠实验进行体内活性评价，借鉴基因组学技术分别从蛋白质水平和基因水平考察了蛋清源活性肽对大鼠体内目标蛋白和目标基因表达水平调控的影响，通过行为医学手段对长期服用活性肽大鼠的行为学进行了研究。首先，通过对蛋清蛋白质进行生物酶解及分离纯化，经质谱碎裂离子比对结合软件解析蛋清源活性肽组分中共有19个活性肽，其氨基酸序列分别为RVPSLM, TPSPR, DLQGK, AGLAPY, RVPSL，DHPFLF, HAEIN，QIGLF，HANENIF，VKELY，TNGIIR，KLPGF，EAGVD，EVSGL，NVLQPS，QITKPN，LEPINF，AEAGVD和ANENIF。19个活性肽的分子量分布范围在500-850u之间，研究发现19个活性肽二级结构中α-螺旋、β-折叠和β-转角含量均在6%以下。而后，建立血管紧张素转化酶抑制活性，α-淀粉酶抑制活性，α-葡萄糖苷酶抑制活性和凝血酶抑制活性的体外活性评价方法，筛选蛋清源活性肽的高活性组分。研究发现除了RVPSL和QIGLF具有较高血管紧张素转化酶抑制活性外，还发现了新的具有血管紧张素转化酶抑制活性的活性肽，其氨基酸序列为TNGIIR，半抑制浓度（IC50）为70μmol L-1；经α-淀粉酶抑制活性和α-葡萄糖苷酶抑制活性的测定，结果表明活性肽RVPSLM，TPSPR，KLPGF和NVLQPS具有较高的α-葡萄糖苷酶抑制活性，其对应的IC50分别为23.07μmol L-1，40.02μmol L-1,59.5μmol L-1和100.0μmol L-1，活性肽KLPGF具有α-淀粉酶抑制活性，其IC50为120.0±4.0μmol L-1；经凝血酶抑制活性筛选，表明RVPSL具有抗凝血酶活性开发的潜力。通过行为医学技术评价活性肽RVPSL，QIGLF和TNGIIR肽对长期灌胃大鼠的神经系统，焦虑以及学习记忆等行为的影响。通过高架十字迷宫焦虑模型发现蛋清源活性肽RVPSL和TNGIIR没有降低大鼠在开放臂活动的次数和时间，而QIGLF和卡托普利则显著降低大鼠在开放臂活动的时间和次数。结合Morris水迷宫和避暗学习记忆模型发现蛋清源活性肽RVPSL，QIGLF和TNGIIR对大鼠的学习记忆的能力均无明显的降低作用。最后，从大鼠的生理参数和蛋白表达水平以及基因表达水平三个方面，考察蛋清肽RVPSL，QIGLF和TNGIIR分别经单次灌胃，短期灌胃和长期灌胃原发性高血压大鼠后的作用效果。结果表明RVPSL和QIGLF的体内较低血压作用明显，并对大鼠的正常生长未造成影响，经长期灌胃RVPSL（50mg/kg bw）和QIGLF（50mg/kg bw）后大鼠的收缩压均降低30mmHg以上，而长期灌胃Captopril能够使大鼠的收缩压降低50mmHg；活性肽RVPSL和QIGLF对大鼠血清中Ang、Renin，AngⅡ和Aldosterone的蛋白质表达水平的影响结果表明活性肽RVPSL高剂量组原发性高血压大鼠血清中Renin，AngⅡ和Aldosterone的浓度及活性显著降低，可以证实活性肽RVPSL通过降低肾素活性和血管紧张素活性从而降低血管紧张素Ⅱ的生成量来实现调节血压的作用；通过探讨活性肽RVPSL对大鼠肾脏中目标基因表达的调节机制，表明RVPSL能够有效下调ACE和AT1表达水平，同时上调AT2的表达水平，同时也证明了AngⅡ的两个受体AT1和AT2在调节血压的作用上存在相反的生理功能。因此我们发现蛋清源活性肽RVPSL具有作为降血压功能成分的开发潜力。本研究为提高禽蛋资源附加值及开发功能肽活性成分提供理论和应用参考，并对活性肽的作用机理提供了佐证。蛋清源活性肽的开发不仅具有较高的学术价值，而且将显著提高禽蛋附加值，延伸了禽蛋加工产业链，创造更大的经济效益。更多还原

【Abstract】 Proteins are important components of foods and ingredients of food products. Inaddition to their nutritional role, proteins can act as functional food ingredients，including antihypertension, antioxidant, and anti-diabetic properties. Research carriedout to produce these foods has paid special attention to the study of the physiologicalrole played by dietary proteins. There are certain fragments within the sequence offood proteins that may show biological activity once released by hydrolysis. Thesefragments, known as bioactive peptides, can be produced in vivo by the action ofgastrointestinal enzymes and can also be obtained in vitro using specific enzymes, orduring the preparation of certain foods. Peptide research has contributed greatly tovarious areas of human health. The production of eggs in china is first in theworldwide. The egg white proteins include ovalbumin, ovotransferrin, ovomucoid,ovomucin, lysozyme, avidin, ovoinhibitor, ovomacroglobulin, and cystatin. Previousresearches suggested that hydrolysates of egg white proteins exhibited diverse ofphysiological properties, including novel antimicrobial activities, antiadhesiveproperties, immunomodulatory, anticancer, and antihypertensive activities, antioxidantproperties. Several biological activities have now been associated with hydrolysatesfrom egg white proteins, highlighting the importance of egg and egg components inhuman health and in disease prevention and treatment.Following our previous work,19bioactive peptides have been isolated and purifiedby proteomics technology including chromatographic, tandem mass spectrometry.Subsequently, The secondary structures of those peptides, i.e., α-helix, β-angle,β-sheet, random coil, and other information, were performed using circular dichroism(CD) and Fourier transform infrared spectroscopy (FTIR). In vitro angiotensinconverting enzyme inhibitory activity, α-glucosidase inhibitory activity, α-amylaseinhibitory activity and anti-thrombin activity of those peptides from egg whiteproteins were measured, aimed to screen bioactive peptides with high activity. After the single oral administration, short-term oral administration and long-termadministration with peptides from egg white proteins for rats, target proteinsconcerntration from serum and the gene expression levels of kidney were evaluatedby methods of Real-Time PCR and Enzyme-linked immune assay. At the same time,behavior of rats administrated with peptides from egg white proteins was studied bythe behavioral medicine assay.Firstly, egg white proteins were hydrolyzed and isolated, and the purified fractionof hydrolysates was characterized by tandem mass spectrometry. The result suggestedthat fraction of hydrolysates was identified and peptides sequence of the fraction wereRVPSLM, TPSPR, DLQGK, AGLAPY, RVPSL, DHPFLF, HAEIN, QIGLF,HANENIF, VKELY, TNGIIR, KLPGF, EAGVD, EVSGL, NVLQPS, QITKPN,LEPINF, AEAGVD and ANENIF, respectively. Those peptides consists of5-6aminoacid residues, and the molecular weight distribution of peptides range between500-850Da. Our result showed that α-helical, β-folding and β-angle components ofthose peptides were below6%by the Fourier transform infrared spectroscopy andcircular dichroism analysis.Subsequently, in vitro angiotensin converting enzyme inhibitory activity, α-amylaseinhibitory activity, α-glucosidase inhibitory activity and inhibiting the activity againstthrombin activity of thsoes peptides were performed aimed to obtain high activitypeptides. Research have shown that QIGLF and RVPSL exhibited high activityagainst angiotensin converting enzyme, and found a novel angiotensin convertingenzyme inhibitory peptides, i.e., TNGIIR, with IC5070μmol/L; α-amylase inhibitoryactivity and α-glucosidase inhibitory activity of those peptides were measured, resultsshowed that bioactive peptide RVPSLM, TPSPR, KLPGF and NVLQPS performedhigher α-glucosidase inhibitory activity, and their values of IC50responsed to23.07μmol/L,40.02μmol/L,59.5μmol/L and100.0μmol/L, respectively. Peptide KLPGFshowed high α-amylase inhibitory activity with an IC50of120.0±4.0μmol/L. resultsalso suggested that peptide RVPSL was potential to exhibit high anti-thrombinactivity.In addition, the present study was designed to evaluate the effect of RVPSL,QIGLF and TNGIIR orally administered on the anxiety-related behavior. Our workassessed the anxiolytic-like effects of those peptides after an oral administration in spontaneously hypertensive rats faced to the same behavioral situations using elevatedplus maze. The results showed that compared to negative control, the number ofentries into the open arms significantly decreased by QIGLF and Captopril treatment,but the number of entries did not decreased by TNGIIR and RVPSL treatment.Followed by the Morris water maze test and avoiding darkness test, the resultssuggested that egg whited derive peptides, i.e., RVPSL，QIGLF, and TNGIIR, did notaffect the ability of learning and memory for rats.Finally, the research focused on the antihypertensive effect of peptides (i.e.,RVPSL，QIGLF, and TNGIIR) on mRNA Expression and physiological parameter ofspontaneously hypertensive rats (SHRs), after sigle oral administration, short-termadministration, and long-term administration, respectively. In addition, Kidney mRNAlevels of renin, ACE, and AT1and AT2receptors were detected by real-time PCR;besides, Ang I, Ang II, renin, and aldosterone concentrations of serum were alsomeasured. Our results indicated that blood pressure of treatment group administratedwith RVPSL (50mg/kg bw) and QIGLF (50mg/kg bw) to spontaneous hypertensiverats for long-term decreased significantly by30mmHg compared to the negativegroup, but which was still difference from that of captopril treatment group (withdecrease by50mmHg). At the same time, Serum Ang II, renin, and aldosteroneconcentrations of group treated with RVPSL were reduced compared to that of thenegative group. Subsequently, Kidney mRNA levels of renin, ACE, and AT1receptorof RVPSL treated group decreased significantly compared to that of negative group.our results indicated that the peptide RVPSL affected the expression of major RAScomponents by downregulating the ACE and AT1receptor while upregulating the AT2receptor in the kidney or serum of SHRs. Our findings suggested that RVPSL mightbe of potential use in the treatment or prevention of hypertension.This study was able to investigate the in vivo bioactivity of the peptide from egg protein and was helpful to clarify the mechanism of bioactive peptides. The researchpossesses highly academic value and will enhance economic returns for enterpriseseventually.更多还原