传统豆瓣酱后发酵过程中微生物群落结构及品质研究

【作者】 陈卓；

【导师】 苏伟；

【作者基本信息】 贵州大学 ， 食品加工与安全， 2024， 硕士

【摘要】 豆瓣酱作为我国标志性的调味品。目前市面上的豆瓣酱多是通过传统工艺,即瓣醅和椒醅混合后经过“日晒夜露”发酵而成。如今,豆瓣酱的研究焦点主要集中在瓣醅阶段中微生物和风味物质的相关性分析,而对于豆瓣酱后发酵过程中主要是针对其物理化学变化进行研究,对于其微生物和风味物质的相关性鲜有报道。本论文通过探索贵州本地豆瓣酱后发酵过程中微生物变化及其代谢差异,解析微生物群落演替和风味物质的变化,揭示豆瓣酱风味品质的形成机理及其与风味相关的核心微生物。主要研究结果如下:(1)通过对理化指标变化情况进行跟踪,发现豆瓣酱发酵过程中,水分含量一直呈下降趋势,满足国家特级豆瓣酱要求;盐度持续上升,在30 d和60 d含量增长较快;色差中L*、a*和b*在发酵过程中一直呈下降趋势;p H值逐渐降低,在第50 d趋于稳定;总酸含量持续升高,在30 d时候趋于稳定;氨基酸态氮持续上升,在50 d时趋于稳定;还原糖含量持续升高,40 d时达到最高值并开始下降。(2)采用顶空固相微萃取气相色谱质谱(HS-SPME-GC-MS)、液相色谱-质谱联用(LC-MS)技术测定豆瓣酱中代谢物。在豆瓣酱发酵过程中共检出67种挥发性风味物质(VFCs),其中酯类物质30种,酸类物质6种,醛类物质4种,醇类物质3种,吡嗪呋喃类物质6种,烯烃类物质7种,烷烃类物质7种,其它类物质4种。通过OPLS-DA对VFCs分析,共筛选出22种显著差异VFCs(Volatile flavor compounds)(VIP>1,P<0.05),其中主要为酯类物质。在豆瓣酱后发酵过程中有39种非挥发性风味物质被检出,其中共检测出8种有机酸、17种游离氨基酸、7种糖类物质和7种脂肪酸,通过OPLS-DA对非挥发性差异代谢物分析,共筛选出18个差异代谢物(VIP>1,P<0.05)。(3)采用高通量测序技术(High Throught Sequencing,HTS)对豆瓣酱后发酵过程中的微生物群落演替进行解析。从门水平上共鉴定出8个优势菌门(平均丰度>0.01),包含5个细菌门和3个真菌门,分别为毛霉菌门(Mucoromycota)、变形菌门(Proteobacteria)、担子菌门(Basidiomycota)、蓝藻细菌门(Cyanobacteria)、拟杆菌门(Bacteroida)、子囊菌门(Ascomycota)、厚壁菌门(Firmicutes)和放线菌门(Actinnobacteriota)。在属水平上共鉴定出26种优势菌属(平均相对丰度>0.01),其中包括16个细菌属和10个真菌属。为了准确评估微生物数目变化情况,对微生物进行绝对定量检测分析,发现豆瓣酱随着发酵时间进行微生物呈下降趋势,其中丰度最高的是Pectobacterium、Staphylococus、Weissella、Acetobacter、Pseudomonas、Pantoea和Bacillus。(4)基于Spearman相关性系数对优势微生物与VFCs进行相关性分析。结果显示:异戊酸甲酯和Staphylococcus、Pantoea、Firmicutes和Actinobacteriota呈正相关。此外,Aspergillus与辛酸乙酯呈强正相关,戊酸苯乙酯与Xanthomonas和Pantoea呈强正相关,异戊酸甲酯与Bacillus呈强负相关,通过研究发现Staphylococcus、Pantoea是影响豆瓣酱VFCs的关键微生物。总体来说,豆瓣酱发酵过程中细菌对VFCs影响较强。通过对非挥发性风味物质与优势微生物进行Spearman相关性分析,发现存在10种非挥发性风味物质和14种微生物的相关。其中,葡萄糖与5种微生物呈正相关,与3种微生物呈负相关(|r|>0.9,P<0.05)。Staphylococcus、Firmicutes、Penicillium、Wickerhamiella、Basidiomycota与葡萄糖呈强正相关,此外,豆瓣酱后发酵过程中赖氨酸、精氨酸、亮氨酸、高丝氨酸、丝氨酸等游离氨基酸和大多数微生物呈正相关,将理化指标与微生物进行Spearman相关性分析,发现共有23种微生物与6项理化指标之间存在相关性,除色差外其余理化指标与微生物相关性较强。综上所述,微生物群落对豆瓣酱中风味具有一定影响,细菌是主要的风味物质贡献者。更多还原

【Abstract】 Douban sauce,as an iconic condiment in China,is predominantly produced using traditional methods where the broad bean and chili sauce mixtures are fermented through a process of"sun drying and night dew."Currently,research on douban sauce primarily focuses on the correlation analysis between microorganisms and flavor substances during the broad bean fermentation stage.There is a lack of studies regarding the physical and chemical changes during the post-fermentation process of douban sauce and its correlation with microorganisms and flavor substances.This paper explores the microbial changes and metabolic differences during the post-fermentation process of local Guizhou douban sauce,analyzing the succession of microbial communities and changes in flavor substances.It elucidates the mechanism of flavor quality formation in douban sauce and its core microorganisms related to flavor.The main research findings are as follows:(1)By tracking the changes in physicochemical indicators,it was found that during the fermentation process of douban sauce,the moisture content showed a continuous decrease trend,meeting the requirements for national top-grade douban sauce;the salt content continued to increase,with a rapid growth in content at 30 and60 days;the color difference parameters L*,a*,and b*showed a continuous decrease trend during fermentation;the p H value gradually decreased and stabilized around day 50;the total acid content continued to increase and stabilized around 30 days;the amino acid nitrogen content increased continuously and stabilized around day 50;the reducing sugar content kept increasing,reaching its peak at 40 days and then started to decrease.(2)Metabolites in douban sauce were determined using headspace solid-phase microextraction gas chromatography-mass spectrometry(HS-SPME-GC-MS)and iquid chromatography-mass spectrometry(LC-MS)techniques.During the fermentation process of douban sauce,a total of 67 volatile flavor compounds(VFCs)were detected,including 30 esters,6 acids,4 aldehydes,3 alcohols,6 pyrazine and furan compounds,7 olefins,7 alkanes,and 4 other substances.Through OPLS-DA analysis of VFCs,22 significantly different VFCs(VIP>1,P<0.05)were selected,mainly esters.In the post-fermentation process of douban sauce,39 non-volatile flavor compounds were detected,including 8 organic acids,17 free amino acids,7sugar compounds,and 7 fatty acids.By OPLS-DA analysis of non-volatile differential metabolites,a total of 18 differential metabolites(VIP>1,P<0.05)were selected.(3)The microbial community dynamics during post-fermentation of douban sauce were analyzed using High Throughput Sequencing(HTS)technology.At the phylum level,a total of 8 dominant phyla(average relative abundance>0.01)were identified,including 5 bacterial phyla and 3 fungal phyla.These phyla are Mucoromycota,Proteobacteria,Basidiomycota,Cyanobacteria,Bacteroidetes,Ascomycota,Firmicutes,and Actinobacteriota.At the genus level,26 dominant genera(average relative abundance>0.01)were identified,including 16 bacterial genera and 10 fungal genera.To accurately assess changes in microbial abundance,absolute quantification analysis of the microbiota was conducted.It was observed that the microbial abundance in douban sauce decreased over the fermentation period,with Pectobacterium,Staphylococus,Weissella,Acetobacter,Pseudomonas,Pantoea,and Bacillus being the most abundant genera.Through Spearman correlation analysis,it was found that there is a certain correlation between the microbial community and VFCs during the post-fermentation process of Douban sauce.Specifically,isopentyl acetate is positively correlated with Staphylococcus,Pantoea,Firmicutes,and Actinobacteriota.Additionally,Aspergillus shows a strong positive correlation with ethyl caproate,ethyl phenylacetate,Xanthomonas and Pantoea,while isopentyl acetate has a strong negative correlation with Bacillus.Research has identified Staphylococcus,Pantoeaas key microorganisms influencing the VFCs in Douban sauce.In general,bacteria have a strong impact on VFCs during the fermentation process of Douban sauce.Spearman correlation analysis between non-volatile flavor substances and dominant microorganisms revealed correlations between 10 non-volatile flavor substances and14 microorganisms.Among these,glucose showed positive correlations with 5microorganisms and negative correlations with 3 microorganisms(|r|>0.9,P<0.05).Staphylococcus,Firmicutes,Penicillium,Wickerhamiella,and Basidiomycota are strongly positively correlated with glucose.Additionally,during the post-fermentation process of Douban sauce,free amino acids such as lysine,arginine,leucine,histidine,and serine are positively correlated with most microorganisms.Spearman correlation analysis between physicochemical indices and microorganisms revealed correlations between 23 microorganisms and 6 physicochemical indices,with strong correlations observed for all indices except color difference.In conclusion,the microbial community exerts a certain influence on the flavor of Douban sauce,with bacteria being the main contributors to flavor compounds.更多还原