4.1. Chemical and physical properties
In this study, combined with the methods of solid fermentation and liquid fermentation, semi-solid fermentation of light-flavor liquor was simulated with corn and sorghum as raw materials. The dynamics of microbial community in fermented grains during fermentation were studied by high-throughput sequencing.
The volatile compounds in fermented grains mainly include alcohols, aldehydes, acids and esters, etc., but ethyl acetate is the main the volatile components in the light flavor liquor. Among them, there are mainly two ways to form esters: the esterification of alcohol and acetic acid and the metabolism of ester yeasts. Due to acetic acid and ethanol are the precursor substances of esterification, a lot of ethanol and acetic acid are consumed during the esterification. As the fermentation progressed, the content of ethyl acetate in fermented grains increased continuously, reaching a maximum of 20 days, and then decreased continuously. It was speculated that in the early stage of fermentation, nutrients were abundant, and dominant microorganisms multiplied continuously, thereby continuously producing metabolites such as esters. In the late fermentation process, nutrients in the fermentation system were insufficient (Fig. 2), the microbial structure changes, while some of the microbials consume ethyl acetate in the system for survival [1, 30].
The fermentation process is a process in which starch macromolecules undergo three stages of saccharification, glycolysis, and esterification to produce aroma components such as alcohol and organic acids. Starch is the material basis for alcohol fermentation during fermentation. Starch is converted into other sugars, organic substances, etc. by saccharification of amylase and glucoamylase, thereby providing a material basis and energy source for the growth and metabolism of microorganisms, so that the fermentation process can be carried out normally. The change of starch content may be due to the rich nutrition in the early stage of fermentation, microorganisms in fermented grains made full use of the products of starch decomposition for metabolism, resulting in a rapid decline of starch. With the development of fermentation, the change of environmental factors, the increase of acids and esters, and the aggravation of anaerobic environment would affect the metabolism of microorganisms. Therefore, the rate of decrease of starch content in the late stage of fermentation decreased. During fermentation, saccharification proceeded simultaneously with fermentation, maintaining a state of relative dynamic equilibrium. Reducing sugar was obtained by amylase degradation of starch, which could provide energy to microorganisms while being used by yeast to produce alcohol. The trend of reducing sugar could be explained by the fact that the amylase hydrolyzed the starch to produce a large amount of reducing sugar due to the pre-fermentation period. Reducing sugar could not only provide energy for the growth of microorganisms, but also could be used by yeast for alcohol fermentation. In the early stage of fermentation, microorganisms were mainly in the stage of growth and reproduction, mainly using reducing sugar to provide energy, instead of yeast using reducing sugar for alcohol fermentation (Fig. 2 showed the low alcohol content in the early fermentation stage). At the time, the hydrolysis rate was greater than the utilization rate of reducing sugar, and therefore, the reducing sugar was in an accumulated rising state. After the yeast grew and reproduced rapidly, the alcohol fermentation rate was significantly accelerated, and the reducing sugar content was continuously decreased, then finally stabilized. During the early stage of fermentation, the starch was rapidly degraded, and the nutrition was extremely rich. Therefore, the yeast grew and multiplied, and the amount of ethanol began to increase. In the late stage of fermentation, it entered the esterification stage and consumed a part of alcohol for synthesizing aroma substances. In addition, in the middle and late stages of fermentation, when the amount of ethanol was accumulated to a certain extent, the diversification of the nutritional relationship would affect the growth and reproduction of the yeast to some degree, which would affect the convene of ethanol.
4.2. microbial community
Alpha values can be used to estimate the species diversity of microbial community species in the sample through several indices, including Observed Species, Chao1, ACE, Shannon and Simpson. The Chao1 index is used to measure the abundance index of the community. With the value increases, the abundance of the microbial community is correspondingly higher. The Simpson and Coverage indices reflect the diversity of the community [36]. The Alpha results indicated that the Coverage index of all samples was greater than 0.999, showing a good community diversity. In the analysis of high-throughput sequencing data, Shannon plots and Sobs plots characterize the microbial diversity detected by samples at different sequencing depths. When the rarefaction curves flatten out, it indicates that the sequencing depth is sufficient and the microbial diversity in the sample can be detected substantially. When the slope of the rarefaction curves is high, it indicates that the sequencing depth is not enough [37, 38]. If the sequencing depth continues to increase, more microbial diversity can still be detected. Moreover, the Shannon plots and Sobs plots also proved that the sequencing depth was enough to cover the entire microbial community.
RDA analysis is mainly used to reflect the relationship between microbial community and environmental factors. Microbial community in the environment was affected by physical and chemical factors, which was an important reason for the variation of microbial abundance in fermented grains. The physical and chemical properties of fermented grains would change significantly during the fermentation process. Therefore, studying the interaction between microbial community structure and environmental factors could help to change the fermentation conditions to control the growth of beneficial microorganisms and inhibit the proliferation of harmful microorganisms. Wang studied the changes in physical and chemical properties of fermented grains during the fermentation process, but there is little research on the relationship between environmental factors and microbial community changes in the existing literature [16, 39].
Wang [40] conducted high-throughput sequencing of microorganisms in fermented grains of light-flavor liquor, and the research results showed that a total of 22 phyla were detected in bacterial community, mainly including Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. The results were completely consistent with this study (Fig. 4). Among them, Firmicutes and Proteobacteria took the dominated places in fermented grains. However, Wang [41] believed that the relative abundance of Firmicutes (91.73–99.73%) in the late stage of fermentation was due to the decrease of bacterial population diversity and the succession of a single Firmicutes fermentation model. Li et al. [20] also thought that Firmicutes and Proteobacteria were the main bacterial populations after analyzing fermented grains in the fermentation process of light-flavor liquor through pyrosequencing. In addition, a large number of studies showed that Firmicutes and Proteobacteria were also the main bacterial community in the fermentation of strong flavor liquor [42], Moutai flavor liquor [33] and sesame flavor liquor [43], indicating that these bacterial community were the key microorganisms in the fermentation of Chinese liquor.
In this study, high-throughput sequencing results of bacteria showed that there were 19 genera of bacteria with the relative abundance of more than 1.00%, among which Pediococcus, Weissella and Lactobacillus (a total of 91%) were the dominant genera (Fig. 5). Pediococcus was the dominant bacteria in the fermentation system, which kept the relative abundance at a high level during the whole fermentation process. This was significantly different from the composition and structure of Lactobacillus in the solid fermentation of light-flavor liquor. Wu et al. [44] analyzed the evolution of Lactobacillus in the solid fermentation of light-flavor liquor, and the DGGE analysis indicated that Lactobacillus was the dominant genus in fermented grains. The results of Wang [41] also confirmed that both the old and new factories were dominated by Lactobacillus (68.89–97.72%) in the middle and late stages of fermentation. Nevertheless, the results were not consistent with the results of this study. Correspondingly, the results in this study mainly included Pediococcus and Weissella. Among them, the Weissella could metabolize organic acids such as lactic acid and acetic acid to provide a synthetic precursor for flavor substances in Chinese liquor. The separation results of Ma [4] showed that the fermented grains were mainly composed of Pediococcus acidilactici, which showed 10% alcohol tolerance. Whereas, RDA results indicated that Pediococcus acidilactici might not contribute significantly to aroma components during the fermentation. Furthermore, it is also considered that most dominant lactobacillus in the fermentation process were lactobacillus of hetero fermentation, which could produce metabolic production of lactic acid, acetic acid, ethanol, etc. [29, 45, 46].
Moreover, Li [47] analyzed microorganisms in the fermentation process of Fen liquor based on high-throughput sequencing of ribosome and considered that the major bacteria were mainly Lactobacillaceae. Lei [27] showed that Leuconostocaceae, Lactobacillaceae and Acetobacteraceae were absolute dominant bacteria. Among them, Leuconostocaceae, Lactobacillaceae were also dominant bacteria in this study. This result was confirmed in this experiment (Fig. 6).
Ascomycota generally predominates in many fermented foods. Wang et al. [40] showed that there were 8 fungal types on phylum level in the fermented grains of light flavor liquor by high-throughput sequencing, among which Ascomycota was the dominant fungal phyla in fermented grains (85.94–99.04%). According to previous studies, Ascomycota was not only the dominant fungal community in fermented grains of light flavor liquor, but also the major fungus in a variety of fermented foods such as yellow wine [48], strong flavor liquor [43], Moutai-flavor liquor [22], vinegar [49], bread [50], etc. In addition, Li et al. believed that the structure of eukaryotic community in fermented grains was relatively stable on phylum level during the whole fermentation period. Since fermentation 3d, Ascomycota became the only dominant eukaryotic fungus in fermented grains [51], which was consistent with the results of this study (Fig. 6).
Li et al. [47] studied the microorganisms in the fermentation process of light-flavor liquor based on ribosome high-throughput sequencing analysis, concluded that the fungal population was mainly Saccharomycetaceae and Saccharomycopsidaceae, and the results were consistent with this study.
Saccharomycopsis fibuligera can hydrolyze starch [52], because of the property of secreting amylase, acid protease, and β-glucosidase, which is considered as the main amylolytic yeast during the fermentations of indigenous food involving cereals (like rice or sorghum) [53, 54]. Therefore, Saccharomycopsis fibuligera usually dominates as the main amylolytic microorganisms and plays a crucial role at the beginning of the alcoholic fermentation, mainly helping to reduce the starch or polysaccharides into fermentable, low molecular weight sugars, such as maltotriose, dextrin, and maltose, and these could be subsequently hydrolyzed to glucose, which is the precursor of ethanol and other flavor compounds [55]. Wang et al. [56] studied the fermentation capacity and flavor components of the species isolated from a Moutai-flavor Daqu. Furthermore, the results also demonstrated that Saccharomycopsis fibuligera was beneficial to produce high contents of ethyl acetate, ethyl palmitate and other aroma components.
It had been reported that Saccharomycopsis fibuligera was widely present in various starters. At present, the research on the Saccharomycopsis fibuligera in the domestic brewing industry is mainly concentrated in the fields of brewing liquor such as yellow rice wine, rice wine and light flavor liquor. In these studies, the Saccharomycopsis fibuligera was usually expressed in the form of dominant fungi in starters [57]. Among them, the Saccharomycopsis fibuligera occupied the dominant position of the fungus in Fen Daqu, while Saccharomycopsis cerevisiae is less [58]. Saccharomycopsis fibuligera could secrete α-amylase, glucoamylase, etc., which was the main starch-degrading bacteria in the fermentation process of starch as the main raw material [59].Therefore, we can speculate that the importance of Saccharomycopsis fibuligera in the brewing process of Chinese liquor is not the conversion of sugars into alcohol, but as the saccharification yeast involved in the hydrolysis of macromolecules such as starch in sorghum and other brewing materials. Thus, enough glucose is accumulated for growth and metabolism by other microorganisms, providing sufficient precursor material for ethanol production [20].
Studies had confirmed that during the period of Fen liquor brewing, the Saccharomycopsis fibuligera was more abundant in the early stage, and then gradually decreased. The result was also proved by this study (Fig. 8). The results could also be confirmed by RDA analysis (Fig. 10). In the late stage of fermentation, the alcohol content increased continuously, but the starch content continued to decrease. As a result, the Saccharomycopsis fibuligera was continuously decreased, but Saccharomyces cerevisiae became the dominant fungus in fermented grains and maintained the dominant position until the end of fermentation. This was similar to the change of yeast community in the brewing process of Fen liquor. In addition, studies had shown that Saccharomycopsis fibuligera had the property of producing highly active β-glucosidase, which could be applied to the liquor-making industry to improve the liquor quality [60].
Saccharomyces cerevisiae generally predominates during the alcoholic fermentations as it has a good capacity to grow in strict anaerobic conditions, whereas it is not an abundant yeast species in Daqu [61, 62]. Zheng et al. [6] studied the microbial diversity in Fen-Daqu by through PCR-DGGE analysis, but Saccharomyces cerevisiae was not detected. However, only one isolate of Saccharomyces cerevisiae was observed by culture-dependent approach. RDA analysis results showed a negative correlation between Saccharomyces cerevisiae and starch content and a little negative correlation with the content of reducing sugar. Due to the degradation of a large amount of starch in the early fermentation, the degradation products of reducing sugars such as glucose and fructose were produced. Chen [63] thought that excessive reducing sugar content would inhibit the proliferation of yeast. However, in the mixed fermentation process of molds and yeasts, the starch degraded to produce reducing sugar, and at the same time reducing sugar provided energy for the growth of the microorganisms and was continuously consumed, which would reduce the activity inhibition of reducing sugar on Saccharomyces cerevisiae and promote continuous fermentation. Furthermore, the research by Ma et al. [4] had demonstrated that Saccharomyces cerevisiae fermented despite low initial concentration, particularly because of the presence of competitive growth with fermentable sugars and higher alcohol tolerance.
In general, Saccharomyces cerevisiae is considered as the most critical microorganisms in the fermentation process because of its strong ability to competitively utilize fermentable glycogen and its alcohol tolerance and plays an important role in improving fermentation efficiency and promoting alcohol production. It also has the ability to replace the dominant position in the fermentation process [64]. In addition, compared with solid-state fermentation, the fungal diversity in the semi-solid fermentation process was less, especially some aroma-producing yeasts such as Pichia pastoris were not found in the fermentation process. Saccharomyces cerevisiae had a dominant position during the fermentation process. In addition, Ma et al. [4] also suggested that Saccharomyces cerevisiae was associated with some high alcohols and acids.