【作者】 张海华；

【导师】 周惠明；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2011， 博士

【摘要】 谷氨酰胺肽是一类含有非氮端谷氨酰胺的肽的总称,这类肽主要功能是供给机体谷氨酰胺,谷氨酰胺肽是游离谷氨酰胺的稳态化替代品。谷氨酰胺肽的活性是以肽中非氮端谷氨酰胺的含量来表征的。游离谷氨酰胺是机体应激条件下的“条件必需氨基酸”,具有多种生理功能。但是游离谷氨酰胺在溶液中或遇热都不稳定且溶解度低,进而游离谷氨酰胺应用受到限制。当游离谷氨酰胺的氮端被取代时其稳定性和溶解性都会大大提高,因此获得谷氨酰胺肽是实现游离谷氨酰胺稳定化的一条有效途径。小麦面筋蛋白具有独特的氨基酸组成,其中谷氨酰胺的含量占到总氨基酸组成的30%左右,是谷氨酰胺肽的良好来源。因此研究以小麦面筋蛋白为原料,利用生物酶解技术高效制备谷氨酰胺肽,结合超滤、色谱与质谱等技术对谷氨酰胺肽组分进行分离鉴定,探讨谷氨酰胺肽作为游离谷氨酰胺替代品的体内体外生物活性,能够在获得谷氨酰胺替代品的同时为谷氨酰胺肽应用提供理论支持,还能够提高小麦面筋蛋白的附加值,因此具有一定的现实、经济意义。谷氨酰胺的不稳定性使得常规氨基酸分析方法不能定量结合态的谷氨酰胺,因此首先建立了一种经BTI(双(1,1 -三氟乙酸基)碘苯)衍生、酸水解、DNFB(2,4-二硝基氟苯)柱前衍生的UPLC快速、准确测定肽中非氮端谷氨酰胺含量(Gln%)的方法。进一步以Gln%为指标,采用响应面法对Alcalase(碱性蛋白酶)酶解小麦面筋蛋白制备谷氨酰胺肽工艺进行优化,固定底物浓度6 g/100mL、酶解时间1 h情况下得到最佳制备条件为:pH=7.5、T=50℃、酶用量0.033 AU/g Protein,在此条件下所得谷氨酰胺肽中Gln%=15.85 g/100g、水解度(DH)为11.8%、平均链长8个氨基酸,蛋白质回收率为83%。在优化结果基础上,采用微波、超声波、添加半胱氨酸、干热、湿热法对小麦面筋蛋白进行预处理,以期促进谷氨酰胺肽的释放。结果表明:添加半胱氨酸和湿热90℃前处理法能够有效促进谷氨酰胺肽的释放,尤其添加半胱氨酸法,当添加量为0.067 g/100mL时肽中谷氨酰胺含量达18.81 g/100g,比未处理提高了近20%。为进一步探讨各预处理方法对Alcalase酶解过程及Gln%的影响,对预处理前后小麦面筋蛋白的显微结构、二级结构、游离巯基含量、二硫键含量、疏水度、水合作用及溶解性进行了分析,发现微波处理后小麦面筋蛋白出现淀粉质纤维结构,蛋白片层经各预处理后都发生了不同程度的破裂;预处理方法对于蛋白二级结构的影响主要表现在β-折叠结构含量的增大上;疏水度和水合作用经各处理后也发生了不同程度的变化。在获得面筋蛋白结构等信息基础上,采用最小二乘回归法(PLSR)对预处理影响Alcalase酶解进程和Gln%的机理进行探讨,结果表明DH和Gln%与蛋白二级结构中的分子间β-折叠、水合β-折叠、1675 cm-1处β-折叠、总β-折叠、扩展结构、α-螺旋、β-转角结构含量和疏水度H0及游离巯基含量(-SH)具有较高相关性。谷氨酰胺肽粗品经超滤分级和DA201-C大孔树脂疏水分离,再经离子交换色谱和凝胶色谱联合使用,最终将谷氨酰胺肽中谷氨酰胺含量从18.8 g/100g提高至29.8 g/100g,进一步采用RP-HPLC实现了谷氨酰胺肽混合物的最终分离,得到四个主要色谱峰,经质谱结果分析得到四种肽序分别为:LLEQRFLVDYL、QQPDESQQ、ENSPQSGGWNQT、CLEYDWMDEQSDS。其中QQPDESQQ八肽中谷氨酰胺含量为60.94 g/100g。考虑到经济性与实用性,选取谷氨酰胺含量为20.1 g/100g的UF-E作为研究对象,对其理化性质与生理活性进行了整体研究。在理化性质方面,UF-E在pH 2～12范围内具有良好且稳定的溶解性,NSI均在80%以上;当UF-E浓度介于0～20 g/100g时其表观粘度基本恒定在1 mpa·s左右;DSC表明UF-E的变性峰值温度为(85±3)℃;UF-E能够抵制体内消化酶系统的消化作用;在50～90℃加热和121℃、30 min高压灭菌条件下,UF-E具有良好稳定性。体外活性中,以抑制亚油酸自氧化和螯合亚铁离子分别作为电子转移和质子转移两种氧化模式的代表,对UF-E的抗氧化性进行综合考察,结果表明UF-E具有良好的亚油酸氧化抑制作用,同时螯合亚铁离子的EC50为0.46 mg/mL。选取谷氨酰胺含量为20.1 g/100g的UF-E作为研究对象,采用灌胃投料方式研究其对负重游泳大鼠补充谷氨酰胺及多项生理指标的影响,并与游离Gln做对照,综合分析认为UF-E具有为机体补充谷氨酰胺和抗疲劳作用,且效果优于游离Gln。采用细胞学方法,以市售Ala-Gln二肽为对照,研究UF-E对正常大鼠脾细胞、人胃癌细胞(SGC7901)和小鼠淋巴瘤细胞(YAC-1)生长的影响,MTT测定结果和倒置显微镜观察结果表明:UF-E具有优于Ala-Gln的促进鼠脾细胞增殖和对人胃癌细胞和小鼠淋巴瘤细胞生长抑制作用,无论增殖还是抑制均呈显著剂量效应关系,当浓度为2 mmol Gln/L时,对正常鼠脾细胞增殖率和两种癌细胞的抑制率都达到最高,分别为575%和30%、80%。细胞实验结果初步说明UF-E对正常细胞和癌细胞生长抑制作用具有一定选择性。另外,还研究了合成八肽QQPDESQQ对人胃癌细胞的生长抑制作用,结果得出QQPDESQQ对人胃癌细胞的抑制具有显著剂量效应关系,说明QQPDESQQ是UF-E抑制人胃癌细胞生长的功效成分之一。更多还原

【Abstract】 Glutamine peptides are defined as a class of peptides contained non-N terminal glutamine, as steady carrier of free glutamine which can provide the glutamine supplement. Therefore the activity of glutamine peptides is presented by determining the non-N terminal glutamine content (Gln %). Glutamine is a conditional essential amino acid in stress situations and has multiply physiological function. Nevertheless the common usage of glutamine is restricted by its aqueous instability and low solubility. While it is reported that the stability and solubility of glutamine could be improved if the N-terminal is substituted.Wheat gluten is rich in glutamine high to 30%, and is a good source for preparation of glutamine peptides. Therefore, research on glutamine peptides prepared by enzymatic hydrolyzing wheat gluten, furthermore on purification and identification with ultrafiltration, chromatography and mass spectra, followed on analysis of In vitro and In vivo activities of glutamine peptides, which can obtain the good carrier of free glutamine and can provide the theory basis for the common use of glutamine peptides, meanwhile, can increase the economic value of wheat gluten.A new method of quantitative analysis of non-N terminal glutamine in peptides was first investigated, since binding glutamine could not be detected by common amino acids analysis for aqueous instability of glutamine. The new method contained four steps: BTI derivation into DABA, HCl hydrolysis, DNFB pre-column derivation and UPLC determination. Preparation of glutamine peptides from Alcalase hydrolyzing wheat gluten was optimized by response surface method in the next place. The optimal conditions were pH 7.5, temperature 50℃and Alcalase doses of 0.033 AU/gProtein, when Gln % was indexed and substrate concentration and hydrolysis time was settled at 6 g/100mL and 1 h respectively. Under the optimal conditions, Gln % was 15.85 g/100g and DH was 11.8%, and the average chain length was 8 amino acids and protein recovery reached 83%.Pretreatments of microwave, ultrasonic, dry heat, aqueous heat and adding cysteine on wheat gluten were done in the aim of improving Gln%. Results showed that both adding cysteine and aqueous heat at 90℃can highly improve Gln%, especially with cysteine Gln % increased by 20% to 18.81 g/100g. The effect of pretreatments on Alcalase hydrolysis and Gln % was further investigated through comparative analysis of SEM, secondary structure of protein, -SH, -S-S, hydrophobicity, hydration and solubility of wheat gluten protein before and after pretreatments. The SEM showed the formation of amyloid fibrils especially after microwave treatment, and the sheets of protein torn by all the treatments. The effect of pretreatments on secondary structure of protein was mainly presented the increase ofβ-sheet content. Hydrophobicity and hydration varied as different pretreatments. Base on that structural information, the mechanism of pretreatments affected Alcalase hydrolysis and Gln % was deeply discussed by Partial Least Square Regression (PLSR) method. The results showed that the degree of hydrolysis and Gln % both were highly dependent on secondary structures of intramolecularβ-sheet, hydratedβ-sheet,β-sheet at 1675 cm-1, totalβ-sheet, extended structure,α-helices,β-turn contents, and on hydrophobicity and SH- content. Glutamine peptides were conteniously purified by ultrafiltration, DA201-C macroporous resin, the ion exchange chromatography and gel chromatography, and the final Gln % was increased to 29.8 g/100g. Thereafter, the glutamine peptides were separated into four peaks by RP-HPLC. The four peaks were collected and identified by mass spectrum, which were LLEQRFLVDYL, QQPDESQQ, ENSPQSGGWNQT, and CLEYDWMDEQSDS. Among these four peaks, QQPDESQQ has the highest Gln% of 60.94 g/100g.Considering economy and practical application, UF-E of 20.1 g/100g was selected and UF-E’s physicochemical and physiological activities were investigated. The results indicated that, UF-E had good solubility with NSI higher than 80% within pH 2-12; the stable viscosity of 1 mpa·s was obtained with UF-E concentration among 1-20 g/100g; the denatured temperature was (85±3)℃detected by DSC; UF-E can resist the digestion of gastrointestinal enzymes; UF-E was stable to the conditions of heating from 50℃to 90℃and of autoclaving at 121℃at 30 min, respectively. In the terms of physiological activity, the inhibition of linoleic acid autoxidation and chelating ferrous activity were detected, which are typically representative of electron transfer and proton transfer oxidation mode respectively. The results showed UF-E had better inhibition of linoleic acid autoxidation, and the EC50 of UF-E on chelating ferrous was 0.46 mg/mL.Effect of UF-E (Gln%=20.1 g/100g) on glutamine supplement and physiological functions of weight-loading swimming rats was researched through intragastric feeding administration, and free glutamine was selected as control. Results demonstrated that UF-E can effectively supply glutamine for sportive fatigue rats and can be a good substitute of free glutamine, and UF-E had better antifatigue effect than free glutamine.The effect of UF-E on proliferation of normal rat splenocyte, human gastric cancer cell (SGC7901) and mice lymphoma cell (YAC-1) was innovatively studied by MTT method and inverted microscope, which was compared with Ala-Gln. The results revealed that UF-E had dose-effect relationship with three kinds cell observed. At UF-E concentration of 2 mmol Gln/L, the proliferation rate was the highest to 575%, meanwhile the inhibition rates of SGC7901 and YAC-1 reached 30% and 80% respectively, when compared with drug blank group. Ala-Gln had somewhat inhibition on both SGC7901 and YAC-1, but with no obvious dose-effect relationship. In general, it can be speculated that UF-E was selective in inhibition of cells. The inhibition effect of QQPDESQQ on SGC7901 was also studied, and the results revealed that QQPDESQQ had obvious dose-effect relationship, and it could be further conluded that QQPDESQQ is one of functional components of UF-E on inhibiting SGC7901.更多还原