www.nature.com/scientificreports/ core microbiota for AAF of Zhenjiar matic vinegar (Fig sb).These highly correlated oral co sted in Tabl ne 7).Ge cteinformatioppocesin3ndgmvironrmnanformationprocstgawcrecsentilohcmikc Discussion In this study.! ed to Lact 1d426 a po diver ed 1 60 in 、amic vineg e AA cally, and as10-. ng A ed to micr arker e micro sion and environmen A in inega (F lerant micro es such m obes dur that the grouping of AAF(day mity an ioh the cr le int ere s n v ork,A. cre d the the fer nted p might b ddine A.pasteur nd of ed with the co truc g A.pa mynaoh Chenjiang aromatic vinegar w. >www.nature.com/scientificreports/ Scientific Reports | 6:26818 | DOI: 10.1038/srep26818 7 core microbiota for AAF of Zhenjiang aromatic vinegar (Fig. 5b). These seven genera were highly correlated with dynamics of 69 flavours during AAF process, including 9 OAs, 16 AAs, and 44 VFs (|ρ|> 0.8). Therein, Acetobacter, Gluconacetobacer, Lactobacillus, and Enhydrobacter were mainly responsible for the change of OAs, while Acetobacter and Staphylococcus were mainly responsible for the change of AAs. These 7 genera were all con￾tributed to the change of VFs, in which Acetobacter, Lactobacillus, and Enhydrobacter were mainly responsible for that of volatile alcohols; Gluconacetobacer was mainly responsible for changes of volatile esters and heterocycles; and Staphylococcus was mainly responsible for volatile aldehydes. More detailed information about the func￾tional core microbiota is listed in Table S10. In addition, PICRUSt analysis revealed that the predicted functions of the core microbiota and non-core microbiota were all assigned to seven categories including metabolism, unclassified, genetic information processing, environmental information processing, organismal systems, cellular processes, and none (Fig. S7). Therein, metabolism was the main function (39.11%) of the microbial community in vinegar Pei, mainly including amino acid metabolism, carbohydrate metabolism, and biosynthesis of other secondary metabolites (Dataset S2). The core microbiota could contribute to 87.87% of metabolism function (Fig. S7). Genetic information processing and environmental information processing were essential to the micro￾bial community in vinegar Pei (occupied 21.49% and 13.33% respectively), which were also mainly carried out (88.60%) by core microbiota (Fig. S7). These suggested the core microbiota could perform the most function of the total microbial community in vinegar production. Discussion Microbiota inhabiting in vinegar Pei is of great importance for the quality and characteristics of cereal vine￾gars. Many molecular ecological approaches have been used to characterise the bacterial and fungal commu￾nity4–5,25–27. In this study, 151 bacterial genera and 202 fungal genera in vinegar Pei during AAF process were identified by next generation sequencing, revealing higher diversity and quantitative abundance than previous studies4–5,28–32. The majority of sequences in vinegar Pei were assigned to Acetobacter, Lactobacillus, Aspergillus, and Alternaria, which were consisted with the previous studies4,26. Acetobacter was increased during AAF process while Lactobacillus was gradually decreased. This succession tendency might be as a potential indicator to ensure the normal AAF process. Yeast community in vinegar Pei included 21 identified genera in this study, suggesting higher diversity than that in Tianjin duliu mature vinegar26 and traditional balsamic vinegar33. However, the abundance of yeast was only occupied 1.6% in fungal genera, and the conjecture was yeast autolysis occurred after alcohol fermentation34,35. During manufacturing, the AAF process is controlled empirically, and the com￾plexity of microbiota make it difficult to be used as a rational approach to monitor AAF process. Here, Miseq sequencing provided well depth to cover the complex microbiota in vinegar Pei. Based on structure of microbiota, the AAF process was divided into three distinct stages: I, day 0; II, days 1–9; and III, days 10–18. This division provided a succession profile of microbiota during AAF, which could be used to search for microbial markers characterising the AAF process and develop a microbiota-based strategy to monitor AAF process. Moreover, the correlation between the microbial succession and environmental factors showed that the gradient of titrat￾able acidity was the most important driver to promote succession of microbiota (Fig. 2c). Elevated levels of AA and LA in vinegar Pei resulted in a specially acidic stress, which selected most of acid-tolerant microbes such as Ga. europaeus29. Another important factor was the alcohol stress, which is a preferred carbon source for growth of functional microbes during vinegar production36. Eventually, a well-balanced and robust community is formed via long-time environmental selection. It is interesting that the grouping of AAF process based on microbial assembly (day 0, days 1–9, and days 10–18) is basically accordance with the grouping based on flavours (day 0, days 1–7, and days 8–18) (Figs 2 and 3b), suggesting the uniformity and high correlation between the evolution of microbiota and the change of flavours. There are few investigations of the correlation between microbiota and flavours in traditional fermented foods6 . Here, O2PLS approach was used to integrate the microbiota dataset and flavours dataset in order to dig into the association between microbiota and flavours in vinegar Pei during MSSF process. In this study, more bacterial genera showed a higher correlation with three flavour sets (|ρ|> 0.8) than fungal genera (Fig. 4), which indi￾cated bacterial community might be the main producer for vinegar flavours. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus, and Staphylococcus were selected as func￾tional core microbiota for AAF of Zhenjiang aromatic vinegar. Moreover, the function of core microbiota pre￾dicted by PICRUSt analysis accounted for more than 80% of functions of the microbial community in vinegar Pei. Among seven functional genera, Acetobacter and Lactobacillus are major functional microbes that have been studied extensively in vinegar industry4,29. In the further work, A. pasteurianus, a main species isolated from vin￾egar Pei, was added at the beginning of AAF to augment the flavours production of Zhenjiang aromatic vinegar. The result showed that the temperature of vinegar Pei in AAF process augmented with A. pasteurianus increased faster than that in the non-augmented AAF process (control) (Fig. S8a). The level of total acids in the AAF process of adding A. pasteurianus was higher than control, and the level of total acids at the 15th day of AAF process of adding A. pasteurianus was equivalent to the total acids at the 18th day of control AAF process (Fig. S8b), which suggested the fermented period might be shortened by adding A. pasteurianus. Moreover, variety of flavours such as AA, Glu, 2,3-butanediol, and ligustrazine (Table S11) were increased at the end of augmented AAF process compared with the control, which partly validated the correlation between Acetobacter and flavours. The structure and dynamics of microbiota after adding A. pasteurianus are being studied. According to our knowledge, this is the first report to systemic analyse the relationship between the structure (genotype) and function (phenotype) of microbial community in traditional fermented foods. Methods Study design and sampling. The framework of the experiment design was shown in Fig. S1a. The AAF of Zhenjiang aromatic vinegar was carried out from July to October, 2014 in Jiangsu Hengshun Vinegar Industry