【作者】 王蔚新；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学， 2017， 博士

【摘要】 酸鱼作为一种风味独特的传统发酵水产品,不仅营养丰富,而且贮藏期长,具有良好的加工发展前景。本实验室前期已从酸鱼中分离纯化多种微生物,并阐明经微生物接种生产酸鱼能够加快蛋白降解并有效改善酸鱼风味。但微生物在酸鱼蛋白降解及风味物质形成过程中的作用及其机制还不清楚。本研究以酸鱼为研究对象,研究其滋味和关键风味物质,及其发酵过程中蛋白质、肽及氨基酸的变化;建立蛋白水解和风味研究模拟体系,研究不同菌株在蛋白质降解及代谢氨基酸生成重要风味物质过程中的作用。本课题对了解微生物在酸鱼蛋白降解和风味物质形成过程中的作用及其机制具有重要的学术意义,同时也为酸鱼工业化生产优化风味、稳定产品品质提供理论指导。本文首先通过分析游离氨基酸及分离纯化得到的小肽,对酸鱼进行滋味物质分析。结果表明,Asp和Glu两种鲜味氨基酸TAV值最高,并且对酸鱼鲜味具有良好的协同作用。酸鱼中分离得到27种小肽,主要为具有一定滋味和活性功能的二肽和三肽。利用GC-MS研究了贵州和广西两地酸鱼挥发性风味物质的组成和含量,结果表明,两产地酸鱼挥发物主要成分为萜类、酚类、醇类和酯类。利用相对气味活性值,确定两地酸鱼的关键气味成分主要为1-辛烯-3-醇、乙酸乙酯、乙酸-3-甲基丁酯、己酸乙酯、壬醛、桉叶油醇、芳樟醇等。分析传统发酵过程中酸鱼的pH、TCA-可溶性肽、蛋白质组成、SDS-PAGE图谱、水溶性含氮物分子量分布、游离氨基酸的组成及含量等的变化,结果表明,酸鱼发酵过程中,pH快速下降至4.5左右,TCA-可溶性肽逐步升高,肌浆蛋白和肌原纤维蛋白含量均显著降低,同时不溶性蛋白含量显著增加。SDS-PAGE图谱显示肌浆蛋白和肌原纤维蛋白发生变性和水解;水溶性含氮物发酵初期以氨基酸（<0.18KDa）和多肽（>10KDa）为主,随发酵进行,各肽段持续生成与降解变化,相对含量处于波动状态,后期氨基酸（<0.18KDa）和小肽（0.18-0.5 kDa）逐渐成为水溶性氮的主要部分;除Trp、Arg、His外,所有游离氨基酸含量在发酵过程中显著增加,发酵结束后主要游离氨基酸为Glu,His,Lys,Ala,Leu和Asp等。建立肌浆蛋白/肌原纤维蛋白水解模拟体系,接种各菌株胞外酶,通过比较非酸化和酸化条件下对照组与接种组培养前后SDS-PAGE电泳图谱及游离氨基氮的变化与差异,研究内源酶与微生物酶在酸鱼蛋白水解过程中的作用。结果表明,非酸化模拟体系,培养前后各组电泳图谱条带无显著变化;对照组游离氨基氮无显著变化,而各接种组显著增加。说明非酸化条件下,鱼肉组织蛋白酶因未被激活而不能发挥作用;各微生物酶虽然不能水解肌浆、肌原纤维蛋白中的大分子,但是均能水解一部分小分子蛋白或多肽。酸化模拟体系,培养前后各组电泳图谱条带均发生显著变化,但组间无明显差异;培养前后各组游离氨基氮均显著增加,但组间无显著差异。说明酸性条件下,鱼肉内源酶被激活,不仅可以初步水解肌浆蛋白和肌原纤维蛋白等大分子蛋白,而且还能进一步水解小分子蛋白和多肽,生成大量小肽和游离氨基酸,而几种微生物酶可能对肌浆蛋白和肌原纤维蛋白水解能力较弱,其作用被鱼肉内源酶所掩盖。可推断,酸鱼发酵初期,微生物可通过分泌大量蛋白酶来水解鱼肉中的小分子蛋白或多肽,从而获得氨基酸,进而满足生命需要。当发酵正常进行,随着鱼肉体系逐渐被乳酸菌产酸所酸化,内源酶被激活,大量水解蛋白质生成肽和氨基酸。因此酸鱼整个蛋白水解过程有酸、内源酶和微生物酶的共同参与。建立风味模拟体系,通过分析微生物数量、pH、SDS-PAGE图谱、游离氨基酸及挥发性风味物质变化,研究微生物在酸鱼氨基酸来源挥发性风味物质形成过程中的作用。结果表明,各菌种在模拟体系中生长良好,植物乳杆菌120和戊糖片球菌220均可通过乳酸发酵产酸降低体系pH,进而激活鱼肉内源酶,释放大量游离氨基酸,而木糖葡萄球菌135和酿酒酵母152因产酸能力较弱,不足以激活内源酶。不同菌种在模拟体系培养过程中产生挥发性风味物质能力不同。植物乳杆菌120和戊糖片球菌220均可通过异型乳酸发酵产生乙醇和乙酸,可降解亮氨酸和苯丙氨酸分别生成3-甲基-1-丁醇及苯乙醇。木糖葡萄球菌135在糖酵解过程中合成乙偶姻的能力较强,同时也能生成乙酸等有机酸,能降解亮氨酸生成3-甲基-1-丁醇,不能降解苯丙氨酸生成苯乙醇或能力很弱。酿酒酵母152能够通过酒精发酵途径产生乙醇和二氧化碳,可降解亮氨酸、异亮氨酸和缬氨酸、苯丙氨酸分别生成3-甲基-1-丁醇、2-甲基丁醇、2-甲基丙醇及苯乙醇和苯甲醇。混合接种各组挥发性风味物质的种类和总量比单一接种组并未见明显增加,某些物质含量出现减少甚至消失,如乙醇、CO₂、苯乙醇等,但同时也产生了几种单一接种时未出现的氨基酸来源风味物质,如2-甲基丙醇、3-甲基-1-丁酸和3-甲硫基丙醇。主成分分析表明,风味物质的形成与菌种有关。苯乙醇和3-甲基-1-丁醇是体系主要的代谢产物,其合成与植物乳杆菌120、戊糖片球菌220和酿酒酵母152密切相关。木糖葡萄球菌135代谢氨基酸能力较弱,对体系源于氨基酸的风味物质合成贡献不大。本文明确了传统酸鱼的滋味物质及关键风味物质,阐明了发酵过程中蛋白质、肽和游离氨基酸的变化,证实了酸鱼蛋白降解有酸、内源酶和微生物酶的共同参与,揭示了不同菌株利用氨基酸生成苯乙醇和3-甲基-1-丁醇等重要风味物质的能力存在差异。更多还原

【Abstract】 Suanyu,a traditional fermented aquatic product with unique flavor,is not only nutrition-rich,but also can be stored long.Therefore,Suanyu products have promising prospect in China.Our previous studies showed that fermentation with inoculation of some microorganisms isolated from Suanyu could speed up degradation of protein and improve the flavor quality of Suanyu.However,proteolysis and flavor formation mechanism during Suanyu fermentation are not clear.In this study,key odor compounds and taste compounds were investigated.Changes of proteins,peptides,and free amino acids were studied.Meanwhile,the role of microorganisms in protein degradation and formation of volatile compounds derived from amino acids were discussed.The results are of definite academic significance to further understand the mechanism of microorganisms in protein degradation and aroma formation.In addition,this study would provide an evident theoretical foundation for flavor optimization and product quality stabilization in freshwater fish processing industry.Firstly,free amino acids and small peptides,as taste compouns,were analyzed.Results showed that Asp and Glu had the highest TAV,and they played a synergic effect on the umami taste.27 small peptides,mainly dipeptides and tripeptides,were isolated in WSN of Suanyu.Secondly,volatile compounds were detected by GC-MS in Suanyu from Guizhou and Guangxi.Results showed that the main volatile compounds of Suanyu from Guizhou and Guangxi were mainly terpenoids,phenols,alcohols,and esters successively.Key odor component were 1-octen-3-ol,ethyl acetate,3-methyl-butyl acetate,ethyl hexanoate,nonanal,eucalyptol,and linalool,determined by the comparison of relative odor activity Value(ROAV)of volatile compounds.Proteolytic changes in Suanyu were determined.The p H value gradualy decreased to about 4.5,accompanied with an increasing trend detected in trichloroacetic acid–soluble peptides concentration.A decrease in sarcoplasmic and myofibrillar protein fractions was accompanied by an increase in the insoluble fraction.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed most bands of sarcoplasmic,myofibrillar,and insoluble proteins lightened or disappeared due to proteolysis or denaturation.New bands emerged especially in insoluble proteins.In the beginning of fermentation,the main parts were free amino acids(<0.18KDa)and peptids(>10KDa).Fluctuation of molecular weight distribution of the water-soluble nitrogen in Suanyu was found during fermentation,and free amino acid <(0.18KDa)and samll peptids(0.18-0.5 kDa)became the main parts in the end product.Free amino acids analysis showed that nearly all amino acids increased,except Trp,Arg and His.Intense proteolysis in Suanyu brought numerous of small peptides and free amino acids,which contribute to the taste and flavor of Suanyu.With comparison and analysis of electrophoretograms and free amino nitrogen in neutral and acidic model system,the role of endogenous enzymes and microbial enzymes in proteolysis of Suanyu was investigated.In model system without acidification,no significant change was found in the electrophoresis pattern before or after incubation for all samples.Free amino nitrogen of the control showed no significant change,while that of inoculate samples changed obviously.It indicated that endogenous enzymes did not work without acid activation in neutral medium,whereas microbial enzymes could hydrolyze small molecular proteins or peptides.In model system with acidification,significant changes were found in the electrophoresis pattern and free amino nitrogen during the incubation of all samples,but no intergroup difference was discovered.This illustrated that endogenous enzymes played a major role in sarcoplasmic and myofibrillar protein hydrolysis,while the proteolysis performance of microbial enzymes were negligible in acidic medium.Presumably,microorganisms hydrolyzed small molecular proteins and peptides by secreting proteases in early fermentation period before acidification.As fermentation proceeds normally,endogenous enzymes were activated by acids produced by lactic acid bacteria,whereupon a large amount of proteins were hydrolyzed to peptides and amino acids.Hence,acid,endogenous enzymes and microbial enzymes participate together in proteolysis of Suanyu protein.The role of microorganisms in formation of volatile compounds derived from Suanyu proteolysis was investigated by establishing flavor model system.All the strains grew well in the model system.Lactobacillus plantarum 120 and Pediococcus pentosaceus 220 could reduce the system’s acidity through lactic fermentation,activate endogenous enzymes,and release a large number of free amino acids.Staphylococcus xylosus 135 and saccharomyces cerevisiae 152 could not activate endogenous enzymes as a result of their weak acid-producing ability.Production ability of volatile compounds depended on strains.Lactobacillus plantarum 120 and Pediococcus pentosaceus 220 could produce ethanol and acetic acid by heterolactic fermentation,and metabolize Leu and Phe to 3-methyl-1-butanol and phenethyl alcohol.Staphylococcus xylosus 135 had a strong ability to synthesize acetoin,and transfer Leu into 3-methyl-1-butanol.But phenethyl alcohol was not detected.Saccharomyces cerevisiae 152 could generate ethanol and carbon dioxide by alcoholic fermentation,and transfer Leu,Ile,Val,and Phe into 3-methyl-1-butanol,2-methylbutanol,2-methyl propanol,and phenethyl alcohol and phenylcarbinol,separately.No significant increase in the varieties of quantities of volatile compounds of groups with combined inoculation compared with that of single ones was found.Certain compounds decreased or even disappeared,such as ethanol,carbon dioxide,phenethyl alcohol,and so on.But meanwhile,some compounds,which were not found in single inoculation samples,derived from amino acids were detected,such as 2-methyl propanol,3-methyl-1-butyric acid and 3-Methylthiopropanol.The results of principal component analysis indicated that the formation of flavor compounds is related to the strains inoculated.Phenethyl alcohol and 3-methyl-1-butanol were the major products in systems,which production might be closely associated with Lactobacillus plantarum 120,Pediococcus pentosaceus 220 and saccharomyces cerevisiae 152.While Staphylococcus xylosus 135 did not contribute significantly to the formation of flavor compounds derived from amino acid.更多还原