Correlation Between Structural Shifts Of Microbiota And Compositions Of Flavors During The Stage Of Acetic Acid Fermentation Of Zhenjiang Aromatic Vinegar

Traditional fermented vinegar is one typical kind of fermented food, which is produced by multispecies solid-state fermentation(MSSF), taking rice and sorghum as main materials. Although MSSF has been applied for centuries, the correlation between the microbial ecosystems and flavors are still not understood completely due to the enormous diversity and complexity of the microbial communities. Here the MSSF process of Zhenjiang aromatic vinegar(ZAV) was taken as a typical example system. Via metagenomics and metabolites analysis, the structural and functional dynamics of microbiota during acetic acid fermentation(AAF) was determined, and then the temporal correlation between microbiota and flavor compounds was explored using O2 PLS method. Main results of this thesis were summarized as follows:(1) Bacterial community of vinegar Pei during the acetic acid fermentation(AAF) of ZAV was investigated by Illumina sequencing. The richness and diversity of bacterial community were higher in the start of AAF, and then declined sharply once the AAF initiated, and then increased gradually after 5~7 days. A total of 151 bacterial genera were identified in vinegar Pei during AAF process. Lactobacillus was predominant in the early stage of AAF(days 0~9), while Acetobacter, Lactococcus, Gluconacetobacter, Enterococcus, Xanthomonas, and Bacillus were prevailing in the later stage of AAF(days 10~18). Moreover, lactic acid bacteria(LAB) included 19 species, in which Lactobacillus helveticus occupied 74%; while acetic acid bacteria(AAB) included 15 species, in which Acetobacter pasteurianus occupied 93%. Cluster analysis revealed the samples in early stage were clustered separately from the samples in late stage of AAF based on the assembly and variation of bacterial community, which indicated AAF process could be divided into three stages: I, day 0; II, days 1~9; and III, days 10~18. Furthermore, AMOVA showed the comparison of the divided three stages during AAF process was statistically significant. Moreover, the bacterial succession of Pei among different batches of AAF were highly similar(p>0.1), which suggested the MSSF process of ZAV was relative stable.(2) Fungal community of vinegar Pei during the AAF of ZAV was investigated by Illumina sequencing. The richness and diversity of fungal community were increased in the first 3 days of AAF. After day 7 the richness and diversity of fungal community were decreased until the end of AAF. A total of 202 fungal genera were identified in vinegar Pei during AAF process, in which Aspergillus and Alternaria was dominated in the AAF process. A total of 21 yeast genera were identified in vinegar Pei, including Cryptococcus, Debaryomyces, Saccharomyces, Candida, and so on. Cluster analysis revealed the samples in early stage were clustered separately from the samples in late stage of AAF based on the assembly and variation of fungal community, which indicated AAF process could be seperated into three stages: I, day 0; II, days 1~9; and III, days 10~18. Furthermore, AMOVA showed that the comparison of the divided three stages during AAF process was statistically significant. Moreover, the fungal succession of Pei among different batches of AAF were highly similar(p>0.1), which indicated the fungal community from different batches of AAF were uniformity.(3) The flavor compounds during AAF process were analyzed by HPLC and GC-MS. The content of total acidity was increasing with the progress of AAF while the p H value declined sharply in the first 3 days and then maintained 3.60. A total of 86 flavors were detected, including 9 organic acids, 18 free amino acids(AAs), and 60 volatile flavors(VFs). The total content of 9 organic acids increased from 1.90±0.93 g/100 g dry culture on day 0 to 12.99±0.53 g/100 g dry culture when the AAF ended. Therein acetic acid and lactic acid were the most abundant two kinds of characteristic organic acids, the relative content of which maintained 68%~91%. The total content of 18 free amino acids increased from 386.52±23.93 mg/100 g dry culture to 2031.65±103.44 mg/100 g dry culture during the AAF process, in which levels of glutamic acid, alanine, valine and leucine were highest, from 174.11±23.6 mg/100 g dry culture to 1132.59±30.38 mg/100 g dry culture. Volatile flavors were consisted of twenty-five esters, nine alcohols, nine carboxylic acids, seven aldehydes, four ketones, three heterocycles and other compounds. The content of volatile flavors except ethanol and acetic acid maintained in the range of 3.88±0.13~5.02±0.24 mg/100 g dry culture. Most of volatile flavors have special flavors, which make the vinegar flavor more harmonious. Based on the dynamics of flavor compounds, the AAF process could be seperated into three stages: i, day 0; ii, days 1~7; and iii, days 8~18, in which there were 60 flavor compounds correlated with the iii stage, including 9 OAs, 18 AAs, and 33 VFs. This indicated that the late stage of AAF was the main stage for production and accumulation of flavor compounds. Moreover, the dynamics of flavor compounds of Pei among different batches of AAF were highly similar(p>0.1), which suggested the dynamics of flavor compounds from different batches of AAF were relative uniformity.(4) The dynamics of abundant flavor compounds lead to change the environments of fermentation, which can influence on the succession of microbiota. The correlation between three important environmental factors(acidity, alcohol, and temperature) and the succession of microbial community were investigated. It was revealed that the acidic stress and alcohol stress were two best predictors of bacterial and fungal community composition, with the principal coordinate one(PC1) being significantly associated with the gradient of titratable acidity and alcohol during AAF process(Bacteria: titratable acidity(ρ=0.922, p<0.0001*), alcohol(ρ=-0.901, p<0.0001*); Fungi: titratable acidity(ρ=0.595, p<0.0001*), alcohol(ρ=-0.545, p<0.0001*), but the gradient of temperature was nearly not correlated with the bacterial and fungal community composition(Bacteria: ρ=-0.0974, p>0.1; Fungi: ρ=-0.0859, p>0.1).(5) The O2 PLS method was used to analyze the association between microbiota and flavor compounds during AAF. According to the VIP(pred) vector, total of 77 microbial genera(VIP(pred)>1.0) including 59 bacterial genera(VIP(pred)=1.00~1.63) and 18 fungal genera(VIP(pred)=1.00~1.46) have important effects on the flavor compounds. A total of 28 microbial genera(VIP(pred)>1.40) including 27 bacterial genera and one fungal genera have more important effects on the flavor compounds. These indicated the bacteria was the main force for producing flavor compounds. Based on correlation coefficient between microbiota and flavor compounds, a total of 94 genera including 61 bacteria and 33 fungi were correlated(|ρ|>0.7) with all flavor compounds. There were 23, 37 and 62 genera highly correlated with OAs, AAs and VFs respectively(|ρ|>0.8), and total 21 genera including 19 bacterial genera and 2 fungal genera were common to three flavor sets. In summary, the dynamics of flavors was mainly correlated with the bacterial community, which suggested that the succession of bacterial community is the essence of changes of flavor compounds.(6) Based on several conditions defined, a total of 7 genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were selected as functional core microbiota for acetic acid fermentation of Zhenjiang aromatic vinegar. The seven genera were highly correlated with dynamics of 69 flavors during AAF process, including 9 organic acids, 16 amino acids, and 44 volatile flavors(|ρ|>0.8). PICRUSt analysis revealed the predicted functions the core microbiota could contribute to 80% of functions of microbial community in vinegar Pei, which indicated the functional core microbiota were crucial to the fermentation of vinegar. Thus, the functional core microbiota identified in this study provide a new perspective to propose a rational approach to controlling and engineering the complex MSSF process at community level.