【作者】 霍健聪；

【导师】 邓尚贵；

【作者基本信息】 西南大学 ， 农产品加工及贮藏工程， 2009， 博士

【摘要】 本研究以带鱼下脚料为原料,采用复合酶水解技术制备带鱼下脚料水解液,通过亚铁离子螯合修饰制备带鱼下脚料多肽亚铁螯合物,并对其抑菌特性初步研究,通过凝胶层析、高效液相及聚丙烯酰胺凝胶电泳进行对其有效抑菌成分进行分离纯化和鉴定,并对其抑菌特性、抗氧化特性和抑菌机理进行了系统研究。在带鱼下脚料蛋白分离特性研究中,主要进行了带鱼下脚料蛋白质的分离和氨基酸成分检测。结果表明:带鱼下脚料湿基蛋白质含量为16.44%,干基蛋白质含量为78.36%,蛋白质、矿物质及脂肪含量均较为丰富,极具开发潜力;带鱼下脚料蛋白中含有18种氨基酸,其中谷氨酸、天冬氨酸、甘氨酸和丙氨酸等呈味氨基酸含量占总氨基酸含量的57.70%;带鱼下脚料蛋白由碱溶性蛋白、碱不溶性基质蛋白、肌浆蛋白、肌原纤维蛋白和非蛋白氮构成,各蛋白组分分子量大小依次为:肌浆蛋白＞碱溶性蛋白＞肌原纤维蛋白＞非蛋白氮;带鱼下脚料中基质蛋白含量最高,为75.83%,其次是碱溶性蛋白质,占16.31%;肌原纤维蛋白含量为3.60%;肌浆蛋白含量最低,为2.15%;带鱼下脚料蛋白中氨基酸总量较高,但必需氨基酸尤其是赖氨酸含量不足,是一利低质蛋白质,表明带鱼下脚料不适于制作食品而适合制作天然海鲜调味料。通过对动物复合蛋白酶、风味酶以及由两者组成的复合酶最适作用条件的正交优化确立了最适水解条件,复合酶最适水解条件为:带鱼下脚料用最4.1g/100mL,温度40℃,复合比1:1,酶浓度6×10³IU/100mL,pH值7.0。同单种酶水解条件相比,采用复合酶水解时底物浓度提高,而酶浓度显著降低,复合酶水解效果明显优于单种酶;复合酶与带鱼下脚料的混浊液pH值接近于最适水解pH值,省略pH值调节环节;采用复合酶对未脱脂带鱼下脚料进行水解,水解度随时间的变化存在线性关系,可表达为y=5.345ln（x）+2.637,R²=0.990;采用复合酶对脱脂带鱼下脚料进行水解,水解度随时间的变化也存在线性关系,可表达为y=0.807x+1.125,R²=0.992。在以制备低水解度活性多肽目的前提下,采用未脱脂带鱼下脚料为原料达到相同水解度所需时间少于脱脂带鱼下脚料,采用未脱脂带鱼下脚料为原料可节约大量有机溶剂,安全性高,水解后带鱼脂肪以鱼油形式存在于水解液表面,分离十分简便,因此带鱼下脚料水解时无需进行脱脂。通过多肽亚铁螯合物制备条件研究发现:带鱼下脚料多肽亚铁螯合物制备中水解液水解度、最适pH值、反应时间、反应温度、抗氧化剂种类及用量等参数与前人研究成果既存在相似之处也存在不同点。本研究中多肽亚铁螯合物的最佳制备工艺为:水解度6%、pH值7.0、异抗坏血酸0.1%、反应时间20min和反应温度20℃,在上述条件下亚铁离子螯合率最高;通过对所制备的多肽亚铁螯合物进行紫外扫描以及螯合物定性检测发现:带鱼下脚料水解液经亚铁离子螯合修饰后生成了新的物质,即生成多肽亚铁螯合物,但所制备的多肽亚铁螯合物中含有少量未螯合的多肽,需要进一步分离纯化。通过对不同体积水解液制备所得多肽亚铁螯合物的抑菌活性研究表明:以50mL水解液经亚铁离子螯合后离心所得上清液制备的多肽亚铁螯合物抑菌活性最高;超滤实验表明由分子量大于5000Da多肽制备的多肽亚铁螯合物抑菌效果较好;实验以引起食品腐败的主要微生物为研究对象,首次对多肽亚铁螯合物的抗菌谱进行了研究,同时研究了pH值、温度、金属离子、蛋白酶、抑菌中浓度EC₅₀以及空间环境对多肽亚铁螯合物抑菌活性的影响。结果表明:多肽亚铁螯合物在中性偏酸性范围内（pH值6.0～7.0）抑菌效果较好;高温对多肽亚铁螯合物抑菌活性影响较大;食品中常见的钠离子对多肽亚铁螯合物抑菌活性影响较小,而高浓度钙离子则对其抑菌活性影响较大;蛋白酶对多肽亚铁螯合物抑菌活性影响显著,经蛋白酶处理后多肽亚铁螯合物抑菌活性基本完全丧失;抑菌中浓度EC₅₀实验表明,多肽亚铁螯合物对不同微生物的抑菌效果不同,其中对大肠杆菌、金黄色葡萄球菌、巨大芽孢杆菌和沙门氏菌均有较强的抑制作用,多肽亚铁螯合物抑菌中浓度EC₅₀较高,这与其未进行分离纯化有关;多肽亚铁螯合物暴露于开放空间后仅发生明显吸潮现象,但吸潮后仍不能被环境微生物利用,对环境中存在的多种微生物均有较强的抗降解能力。多肽亚铁螯合物通过葡聚糖凝胶Sephadex-25和Sephadex-75两次分离纯化后得到一种抑菌活性较高的多肽亚铁螯合纯化物,并经高效液相色谱梯度洗脱检测为单一组分;SDS-PAGE聚丙烯酰胺凝胶电泳表明多肽亚铁螯合纯化物分子量约为26kDa;同分离纯化前相比,经分离纯化后所得的多肽亚铁螯合纯化物抑菌活性显著提高,对大部分供试菌种的最低抑菌浓度MIC为1%,纯化物对大肠杆菌、金黄色葡萄球菌、巨大芽孢杆菌和沙门氏菌抑制效果较好,最低抑菌浓度均为0.5%;通过回归分析计算,当多肽亚铁螯合纯化物用量为500mg时,其半衰期t_0.5为38.18h;多肽亚铁螯合物不同纯化组分抑菌活性同其铁含量有关,实验证实铁含量越高,抑菌活性越弱,同时亚铁离子修饰对于带鱼下脚料水解多肽是否具有抑菌活性有直接关系;抗氧化实验证实:多肽亚铁螯合物对羟自由基有一定清除效果,而对过氧化氢清除效果不明显,对油脂也有一定抗氧化效果,且抗氧化效果接近维生素E;多肽亚铁螯合纯化物元素组成为:C,34.47%;H,5.92%;N,5.50%;O,49.86%;Fe,2.55%,元素组成符合多肽亚铁螯合物主要元素成分,多肽亚铁螯合纯化物为氨基酸和铁元素共同构成。红外光谱分析表明多肽亚铁螯合纯化物中的亚铁离子与氨基和羰基通过单齿共价键牢固结合在一起,并提出了多肽亚铁螯合纯化物的初步构象。利用摄影显微镜及透射电镜技术研究了多肽亚铁螯合纯化物对大肠杆菌（Escherichia coli）和枯草芽孢杆菌（Bacillus subtilis）的生长形态及细胞壁等微结构的影响,结果表明:多肽亚铁螯合物对微生物的作用可能是通过作用于革兰氏阳性菌和阴性菌外膜表面形成内外连通的孔道,造成菌体细胞内容物的外泄,细胞壁破裂,最终导致细胞死亡;同时由于多肽亚铁螯合物不同组分抑菌活性同其铁含量成负相关,推测多肽亚铁螯合物抑菌机理同乳铁蛋白类似,也有同微生物存在竞争性结合铁元素,从而抑制微生物正常生长繁殖的功效。多肽亚铁螯合物同时存在上述两利抑菌机理。更多还原

【Abstract】 In this paper,with the material of offal of Trichiurus haumela,hydrolysate was prepared by using complicated enzyme.Peptides iron（Ⅱ）chelate was produced with FeCl₂。The effective composition of peptides iron（Ⅱ）chelate was purified and identified with Sephadex,HPLC and SDS-PAGE.Antibacterial character,antioxidation character and antibacterial mechanism were studied.The studies on material protein peculiarity and its protein fraction showed that the content of Trichiurus haumela offal protein was 16.44%（wet basis）,and 78.36%（dry basis）.Trichiurus haumela offal was rich in protein,mineral substance and fat,and it has great potential for development. Trichiurus haumela offal consisted of 18 amino acids,in which the content of Glu,Asp,Gly and Ala was up to 57.70%.Trichiurus haumela offal was composed of alkali-soluble protein,stroma protein, myogen,myofibrilar protein and non-protein nitrogen,meanwhile the molecule weight of these protein fraction was myogen＞alkali-soluble protein＞myofibrilar protein＞non-protein nitrogen. Stroma protein had the highest content,which was 75.83%,and the content of other protein were as follows:alkali-soluble protein 16.31%,myofibrilar protein 3.60%,myogen 2.15%.Trichiurus haumela offal was high in total amount of amino acid,but insufficient in essential amino acid, especially Lys,which demonstrated that Trichiurus haumela offal was a poor-quality protein and unfit for making food but seafood-flavor sauce.The optimal conditions of animal protemax,flavorease and comlicated enzymes of enzymes hydrolysis were established by orthogonal trial.The results showed that the optimal conditions were as follows:amount of Trichiurus haumela offal 4.1g/100mL,temperature 40℃,ratio of two enzymes 1:1,amount of enzyme 6×10³IU/l00mL,pH7.0.Compared with single enzyme,complicated enzymes has a notable reduction in dosage,but increase in concentration of substrate.The effect of complicated enzymes was better than single enzyme.The natural pH of mixture solution was close to the optimal pH,so the step of adjusting pH could be left out.The hydrolysis course for the complicated enzymes was described with 2 linear relationship being expressed as follows:y= 5.345ln（x）+2.637,R²=0.990（undefatted）;y=0.807x+1.125,R²=0.992（defatted）.In order to make bioactive peptide whose DH was usually lower than 20,it spent little time getting the hydrolysate of same DH with the undefatted material compared with defatted material.At the same time,after this process,the fat of Trichiurus haumela offal existed in the form of floating oil,and was easy to remove.So it was unnecessary to be defatted.During the research of preparation of peptide iron（Ⅱ）chelate,some parameters such as DH,pH, chelating time,chelating temperature,antioxidant category and dosage had both resemblances and differences compared with the other scholars.In this research,the optimum parameters were as follows:DH6%,pH 7.0,Vc 0.1%,chelating time 20min,chelating temperature 20℃.The Fe²⁺had the highest chelating ratio under the conditions mentioned above.The result of ultraviolet scanning showed that there was some new substance-peptides iron（Ⅱ）chelate.But the qualitative testing showed that this new material was not pure,and it needed to be purified to eliminate peptides that had not integrate with Fe²⁺.Antibacterial testing results showed that the antibacterial effect of peptides iron（Ⅱ）chelate was subjected to the volume of hydrolysate.It indicated that the peptides iron（Ⅱ）chelate made from clear solution of 50mL hydrolysate had the best antibacterial effect.Experiment on ultrafiltration showed that hydrolysate with molecular weight more than 5000Da had better antibacterial effect.In this paper,the antibiotic spectrum of peptides iron（Ⅱ）chelate was studied for the first time with some common food-spoiling microorganisms,the effect of pH value,temperature,metal ion, protease,EC₅₀and the effect when peptides iron（Ⅱ）chelate was exposed to atmosphere were also investigated.The results showed that the antibacterial activity was better at pH（6.0⁷.0）;peptides iron（Ⅱ）chelate had not good endurance to high temperature;the usual ion such Na⁺and Ca²⁺had different effect,Ca²⁺had greatly effect on peptides iron（Ⅱ）chelate,but not Na^-.All the proteases could deprive antibacterial effect of peptides iron（Ⅱ）chelate,especially pepsin.The experiment on EC₅₀indicated that antibacterial effect of peptides iron（Ⅱ）chelate according to microorganism strain,while peptides iron（Ⅱ）chelate had good antibacterial effect on Escherichia coli, Ataphylococcus aureus,Bacillus megaterium and Salmonella sp,though effective antibacterial dosage of peptides iron（Ⅱ）chelate was high,it was reasonable on account of not be purified.After being exposed to atmosphere for several days,peptides iron（Ⅱ）chelate could not been utilized by microorganism except imbibing because of its deliquescence.It indicated that peptides iron（Ⅱ） chelate had the ability of degradation-resistance to microorganism excited in atmosphere.Sephadex G-25 and sepahdex G-75 were employed to purify and separate peptides iron（Ⅱ） chelate and a basic constituent with high antibacterial activity was obtained.This constituent showed a singly active peak in HPLC.SDS-PAGE showed that the molecular weight of puridied peptides iron（Ⅱ）chelate was about 26kDa.After been purified,MIC of peptides iron（Ⅱ）chelate to Escherichia coli,Ataphylococcus aureus.Bacillus megaterium and Bacillus subtilis was 0.5%.Half life of peptides iron（Ⅱ）chelate was 38.18h with the dosage of 500mg by regression analysis.The antibacterial effect was concerned with content of Fe²⁺,and the experiment testified that the higher conten Fe²⁺,the better antibacterial effect,and modification of Fe^2-was critical to antibacterial effect of hydrolysate.Peptides iron（Ⅱ）chelate had effect of elimination on-OH and H₂O₂,also in oil anti-oxidation.The effect of anti-oxidation was close to Ve.The element construction of purified peptides iron（Ⅱ）chelate was as follows:C,34.47%;H,5.92%;N,5.50%;O,49.86%;Fe,2.55%. Purified chelate was composed of amino acid and Fe²⁺.IR spectrum showed that purified peptides iron（Ⅱ）chelate was strongly combined with monodentate covalent bond,and then primary structure of purified peptides iron（Ⅱ）chelate was put forward.After investigating the effects of purified peptides iron（Ⅱ）chelate on the growth and cellwall of Escherichia coli and Bacillus subtilis with camera microscope and transmission electron microscopy,the results showed that purified peptides iron（Ⅱ）chelate could construct channels on the cell wall of G⁺and G^-,then the substance of cell flowed out,cell wall collapsed,and the cell was dead.At the same time,the differences of antibacterial effect among constituents of peptides iron（Ⅱ） chelate had some connection with their Fe²⁺content,so peptides iron（Ⅱ）chelate could combine Fe²⁺ competitive with microorganism and play the role of inhibitor.There were two antibacterial mechanisms in purified peptides iron（Ⅱ）chelate.更多还原