Deciphering the succession patterns of bacterial community and their correlations with environmental factors and flavor compounds during the fermentation of Zhejiang rosy vinegar

https://doi.org/10.1016/j.ijfoodmicro.2021.109070Get rights and content

Highlights

  • The main flavor compounds were acetic acid, lactic acid, ethyl acetate and acetoin.

  • Lactobacillus and Acetobacter respectively dominated the 0–30 days and 30–90 days.

  • Acetobacter and Lactobacillus were the major genera for the formation of flavor.

  • The abundant bacteria exhibited high correlations with other bacteria.

  • The environmental factors playing key roles in the fermentation.

Abstract

Zhejiang Rosy Vinegar (ZRV) is a traditional condiment in Southeast China, produced using semi-solid-state fermentation techniques under an open environment, yet little is known about the functional microbiota involved in the flavor formation of ZRV. In this study, 43 kinds of volatile flavor substances were identified by HS-SPME/GC–MS, mainly including ethyl acetate (relative content at the end of fermentation: 1104.1 mg/L), phenylethyl alcohol (417.6 mg/L) and acetoin (605.2 mg/L). The most abundant organic acid was acetic acid (59.6 g/L), which kept rising during the fermentation, followed by lactic acid (7.0 g/L), which showed a continuously downward trend. Amplicon sequencing analysis revealed that the richness and diversity of bacterial community were the highest at the beginning and then maintained decreasing during the fermentation. The predominant bacteria were scattered in Acetobacter (average relative abundance: 63.7%) and Lactobacillus (19.8%). Both sequencing and culture-dependent analysis showed Lactobacillus dominated the early stage (day 10 to 30), and Acetobacter kept highly abundant from day 40 to the end. Spearman correlation analysis displayed that the potential major groups involved in the formation of flavor compounds were Acetobacter and Lactobacillus, which were also showed strong relationships with other bacteria through co-occurrence network analysis (edges attached to Acetobacter: 61.7%; Lactobacillus: 14.0%). Moreover, structural equation model showed that the contents of ethanol, titratable acid and reducing sugar were the major environmental factors playing essential roles in influencing the succession of bacterial community and their metabolism during the fermentation. Overall, these findings illuminated the dynamic profiles of bacterial community and flavor compounds and the potential functional microbes, which were expected to help us understand the formation of flavor substances in ZRV.

Introduction

Zhejiang rosy vinegar (ZRV), a well-known traditional condiment in Zhejiang Province China, is produced using semi-solid-state fermentation technology, which is very popular in the Southeast area of China for its beautiful rosy color and perfect flavor (Shi et al., 2014). The fermentation process of the ZRV is shown in Fig. S1. Rice was soaked for 7 d, steamed for 30 min, naturally cooled at air temperature, and then transferred into a ceramic cylinder and saccharified by environmental microbial community. After saccharification, adding water to start the main fermentation stage. At the main fermentation stage, the saccharification, alcohol fermentation and acidogenic fermentation were carried out at the same time. After about 3 months of fermentation, the vinegar will be aged for 1 year prior to obtain the end product (Jiang et al., 2010; Jiang et al., 2013; Xia et al., 2016).

Compared with other cereal vinegars such as Zhenjiang aromatic vinegar (Wang et al., 2015a) and Shanxi aged vinegar (Nie et al., 2017), the fermentation process of ZRV is spontaneously activated by the microorganisms in the environment without adding any exogenous fermentation starter (Jiang et al., 2010; Jiang et al., 2013). Accordingly, the quality (flavor and physicochemical properties) of ZRV is greatly affected by different environmental factors, and in practical production, the fermentation is usually carried out from May to September according to the experience of craftspeople (Jiang et al., 2010). So far, the microbial community composition and their metabolic function during the fermentation process of ZRV have not been well characterized. The absence of this knowledge hinders the production control and the exploration of techniques to improve the quality of ZRV.

It is necessary to point out that vinegars are not only a solution of acetic acid but also a complex matrix composed of alcohols, acids, aldehyde, ketones, etc. (Al-Dalali et al., 2020). The flavor of ZRV was originated from microbial, enzymatic, and chemical transformations. During the fermentation process, the decomposition of protein, fat, and starch produces a series of volatile and non-volatile compounds: acids, alcohols, esters, ketones, aldehydes and sugars (Zhao et al., 2020). Thus, it is essential to investigate the bacterial community structure and bacteria structural shifts during the fermentation. How community assembles and which species can coexist in the same locale are central ecological questions (Valyi et al., 2016). In recent years, great progress has been made in the study of bacterial compositions of other cereal vinegars. Focusing on microbial succession during fermentation processes, the culture-independent techniques including denaturing gradient gel electrophoresis (DGGE) (Haruta et al., 2006; Mamlouk et al., 2011; Wu et al., 2011; Xu et al., 2011) and high-throughput sequencing (Nie et al., 2013; Valera et al., 2015) are applied in the microbiota analyses of other vinegars.

With the development of culture-independent techniques there is increasing research to investigate food fermentation, concentrating on the dynamics of the microbiota (Gan et al., 2017; Lu et al., 2018; Randazzo et al., 2005) and the functionality of the microbial community (Bisanz et al., 2014; Jung et al., 2011; Weckx et al., 2011). Different environmental factors may lead to variation in microbial community composition (Nie et al., 2017). Several studies on bacterial community composition in other traditional Chinese vinegars have been reported, and results suggested that LAB and acetic acid bacteria (AAB) are dominant functional microorganisms (Nie et al., 2013; Wu et al., 2010; Wu et al., 2011; Wu et al., 2012; Xu et al., 2011). Compared with other vinegars such as apple cider vinegar (Trcek et al., 2016) and spirit vinegar (Trcek and Raspor, 1999), LAB and AAB are also the major bacteria during the fermentation process. Few studies focused on how environmental factors affect microbial succession and indirectly affect the kinds and concentrations of metabolites. More recently, the relationships between bacterial dynamics and flavor substance have been reported, and the results reflected a strong correlation between the dynamics of the bacterial community and aromatic metabolites (Nie et al., 2017; Wang et al., 2016; Zhang et al., 2020a). Therefore, bacterial co-occurrence, the correlation between environmental factors and bacterial community, and the correlations between bacterial community and metabolite changes should be further explored to unravel the fermentation mechanism of ZRV.

In this study, we applied 16S rRNA gene sequencing using the Illumina MiSeq platform to describe the composition and succession of microbial community during the ZRV fermentation. Structural equation modeling (SEM) was used to determine the direct and indirect impacts between environmental variables, bacterial community succession, and formation of flavor substances, which could help us better understand the potential major bacterial community for the production of flavor substances and provide a reference for improving the quality of ZRV.

Section snippets

Sample collection

ZRV samples were collected from a famous manufacturer located in Shaoxing city, Zhejiang province, China. The samples during the fermentation process were collected at 0, 10, 20,30, 40, 50, 60, 70, 80, and 90 d (Fig. S1), and 3 parallel samples from 3 different fermentation tanks were taken each time. The whole fermentation process was divided into prophase (0d ~ 30d), metaphase (40d ~ 50d) and anaphase (60d ~ 90d). Vinegar should be fully mixed before each sampling. The obtained samples were

Changes of physicochemical factors and flavor substance during the fermentation process

The physicochemical indexes and the fermentation liquid temperature during ZRV fermentation process were measured (Fig. 1). The reducing sugar content dropped sharply from about 50 g/L to 4 g/L in the first 10 days (Fig. 1B), the ethanol content increased rapidly from about 1% (v/v) to 6% (v/v) (Fig. 1C), the acidity increased slowly (Fig. 1D). After 10 days, the fermentation liquid enters a phase dominated by acetic acid fermentation, when the ascent rate of ethanol slows down due to the

Discussion

ZRV is a well-known traditional condiment in China and have a perfect rosy color and flavor because of its unique fermentation techniques. Preliminary studies had found the formation of rosy color of ZRV is due to Maillard reactions occurred between amino and carbonyl compounds (Yujian et al., 2019). In the whole fermentation process, no microbial agent was added, and all the microorganisms needed for brewing come from the environment of fermentation. The microorganisms in the fermentation

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The study was supported by the Basic Public Welfare Research Project of Zhejiang Province (LGG19C200001) and the Graduate Student's Research and Innovation Fund of Zhejiang Gongshang University (16020000359).

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