【作者】 李浩；

【导师】 王筱兰；

【作者基本信息】 江西师范大学 ， 生物学， 2021， 硕士

【摘要】 曲霉型豆豉作为我国的传统豆豉之一,是一种药食同源的调味副食品,而豆豉的风味则是决定其品质的关键因素之一。然而,传统曲霉型豆豉粗放的生产方式难以保证豆豉品质的稳定,且存在较大食品安全隐患,故在开展良好风味豆豉的工业化生产之前,首先应对传统工艺下的曲霉型豆豉风味特征及菌群分布形成全面认识,而过去相关研究对其规律认识仍不太深入。因此,本文以传统曲霉型豆豉为研究对象,通过微生物可培养法、16S r RNA测序和宏基因鸟枪测序技术剖析不同发酵阶段豆豉的微生物群落组成;测定传统曲霉型豆豉的基本品质和风味成分(挥发性风味物质、游离氨基酸),评估豆豉的理化特征。结合以上研究数据,针对性地探讨传统曲霉型豆豉中微生物与风味形成的关联,并比较了可培养微生物与传统曲霉型豆豉的风味差异。具体过程与结果如下:就传统曲霉型豆豉的微生物来看,可培养微生物的丰度呈下降趋势,分离得到的18种微生物中,优势微生物以细菌为主,包括Bacillus velezensis、Bacillus licheniformis、Staphylococcus gallinarum、Staphylococcus saprophyticus、Pediococcus acidilactici。对后发酵阶段豆豉的可培养微生物进行扩增子测序发现,Staphylococcus是后发酵唯一全程优势属,平均相对丰度约为50%;以Weissella、Pediococcus及Lactobacillus为代表的乳酸细菌主要分布于豆豉后发酵初始阶段,Bacillus在发酵中后期的相对丰度略有上升,平均相对丰度为4%。就鸟枪法宏基因测序的结果来看,宏基因测序的物种注释结果与可培养法、16S r RNA的测序结果大体一致,细菌占据发酵菌群的绝对优势地位,真菌在整个发酵过程中的丰度极低,此外还检测到了少量病毒。发酵菌群的测序结果表明,氨基酸代谢和碳水化合物代谢最为活跃,并且在参与这两类代谢的微生物主要是乳酸细菌科(Staphylococcus、Pediococcus、Lactobacillus和Weissella),说明曲霉型豆豉在制曲阶段人工接种了曲霉等真菌,但是从整个发酵过程来看,细菌才是曲霉型豆豉风味形成的关键微生物群体。传统曲霉型豆豉的制曲和后发酵阶段平均温度约为30℃和45℃,制曲阶段为无盐发酵,而后发酵过程豆豉的含盐量为6%上下。测定结果表明,豆豉样品共检测到16种游离氨基酸,游离氨基酸的总含量伴随着发酵进程逐步增加,至后发酵第21天可达4.87 g/100g,而鲜甜味氨基酸的含量远高于苦味氨基酸;就挥发性成分来看,主体风味为烃类,酯类在前期占比较高,中后期醛类、吡嗪类和酸类等多类风味含量增加,其中含量较高的(相对含量>1%)风味物质包括乙酸、2-甲基丁酸、3-甲基丁酸、2-甲基丁醛、2-甲基丁酸甲酯、乙酸异戊酯、苯乙醛、可可醛、安息香醛、吡嗪、2-戊基呋喃和愈创木酚,上述挥发性物质均为发酵豆制品的特征风味物质。O2PLS模型建构[VIP(pred)>1.0]的结果表明,共有14个菌属为关键菌属,以Staphylococcus、Pediococcus、Lactobacillus、Weissella等属的重要性尤为突出,且这些属与游离氨基酸、有机酸、醇类、酯类、醛类、酮类、吡嗪类和酮类存在强烈相关。优势菌发酵豆豉的挥发性风味结果表明,Staphylococcus的主体挥发性风味成分有醛类、酮类、酸类;Bacillus则是酮类、吡嗪类、胺类;而Weissella、Pediococcus和Lactobacillus为酸类和醛类。本文通过从豆豉发酵阶段的风味和微生物两个方面切入,结合多种分析方法探讨风味形成与微生物内在关联,阐明了造成豆豉风味形成的重要源头即菌群组成变化,并结合豆豉优势微生物发酵培养后的风味测定完成初步评价。这将为更深入地探索曲霉型豆豉风味形成规律提供了研究思路,并为下一步复配发酵建立了试验基础以及菌株资源。更多还原

【Abstract】 As one of the traditional Douchi in China,Aspergillus Douchi is a kind of flavoring non-staple food with the same origin of medicine and food,and the flavor of Douchi is one of the key factors to determine its quality.However,the traditional extensive production method of Aspergillus type Douchi is difficult to ensure the stability of Douchi quality,and there is a big food safety risk.Therefore,before the industrial production of good flavor Douchi,we should first form a comprehensive understanding of the flavor characteristics and flora distribution of Aspergillus type Douchi under the traditional technology,but the understanding of its law in the past is still not very deep.Therefore,the microbial community composition of traditional Aspergillus Douchi was analyzed by microbial culturability,16 S r RNA sequencing and macro gene shotgun sequencing;The basic quality and flavor components(volatile flavor compounds,free amino acids)of traditional Aspergillus Douchi were determined,and the physicochemical characteristics of Douchi were evaluated.Based on the above research data,the relationship between microorganisms and flavor formation in traditional Aspergillus Douchi was discussed,and the flavor differences between culturable microorganisms and traditional Aspergillus Douchi were compared.The process and results are as follows:In terms of traditional Aspergillus Douchi,the abundance of culturable microorganisms showed a downward trend.Among the 18 microorganisms isolated,the dominant microorganisms were bacteria,including Bacillus velezensis,Bacillus licheniformis,Staphylococcus gallinarum,Staphylococcus saprophyticus and Pediococcus acidilactici.The results showed that Staphylococcus was the only dominant genus in the whole process of post fermentation,and the average relative abundance was about 50%;The lactic acid bacteria represented by weissella,Pediococcus and lactobacillus were mainly distributed in the initial stage of post fermentation.The relative abundance of Bacillus increased slightly in the middle and late stage of fermentation,with an average relative abundance of 4%.According to the results of shotgun macrogene sequencing,the results of species annotation of macrogene sequencing were basically consistent with those of culturable method and 16 S r RNA sequencing.Bacteria occupied an absolute dominant position in the fermentation flora,fungi had a very low abundance in the whole fermentation process,and a small number of viruses were detected.The sequencing results of fermentation flora showed that amino acid metabolism and carbohydrate metabolism were the most active,and lactic acid bacteria(Staphylococcus,Pediococcus,Lactobacillus and weissella)were the main microorganisms involved in these two kinds of metabolism,which indicated that Aspergillus and other fungi were artificially inoculated in the starter making stage of Aspergillus Douchi,Bacteria are the key microbial community of Aspergillus Douchi flavor formation.The average temperature of traditional Aspergillus Douchi during koji making and post fermentation is about 30 ℃ and 45 ℃.The koji making stage is salt free fermentation,and the salt content of Douchi during post fermentation is about 6%.The results showed that 16 kinds of free amino acids were detected in Douchi,and the total content of free amino acids increased gradually with the fermentation process,reaching 4.87 g / 100 g on the 21 st day after fermentation,while the content of fresh sweet amino acids was much higher than that of bitter amino acids;In terms of volatile components,the main flavor compounds were hydrocarbons,esters accounted for a relatively high proportion in the early stage,and the contents of aldehydes,pyrazines and acids increased in the middle and late stage.The flavor compounds with higher content(relative content > 1%)included acetic acid,2-Methylbutyric acid,3-methylbutyric acid,2-methylbutyraldehyde,methyl 2-methylbutyrate,isoamyl acetate,phenylacetaldehyde,cocoa aldehyde,benzoic aldehyde,pyrazine,etc2-pentylfuran and guaiacol were characteristic flavor compounds of fermented soybean products.The results of O2 PLS model construction [VIP(PRED)> 1.0] showed that there were 14 bacteria genera as the key bacteria genera,and the importance of Staphylococcus,Pediococcus,Lactobacillus and weissella was particularly prominent,and these genera were strongly related to free amino acids,organic acids,alcohols,esters,aldehydes,ketones,pyrazines and ketones.The results showed that the main volatile flavor components of Staphylococcus were aldehydes,ketones and acids;Bacillus is ketone,pyrazine and amine;Weissella,Pediococcus and Lactobacillus are acids and aldehydes.In this paper,from the two aspects of flavor and microorganism in the fermentation stage of Douchi,combined with a variety of analysis methods to explore the internal relationship between flavor formation and microorganism,clarified the important source of the formation of Douchi flavor,that is,the change of flora composition,and combined with the flavor determination after fermentation and cultivation of Douchi dominant microorganism to complete the preliminary evaluation.This will provide a research idea for further exploring the flavor formation law of Aspergillus Douchi,and establish the experimental basis and strain resources for the next step of compound fermentation.更多还原