Occurrence, Diversity, and Character of Bacillaceae in the Solid Fermentation Process of Strong Aromatic Liquors

Strong aromatic liquors, also known as strong aromatic Baijiu (SAB) in China, are manufactured by solid fermentation, with a multi-microbe mixing and cooperative fermentation process that uses Daqu as a brewing starter. Bacillaceae have a specific action in food fermentation, such as soybean and wine, and more recent studies have found Bacillaceae play important roles in the SAB making industry. This review describes the diversity, functionality, and influence of Bacillaceae in Daqu, pit mud, Zaopei, Huangshui within making processes of SAB. Furthermore, aromatic flavor components from the Bacillaceae metabolism of SAB are discussed in this review. Ultimately, the resulting improvements and deeper understanding will benefit practical efforts to apply representatives of Bacillaceae in improving the quality of SAB as well as biological control of the micro-ecological environment of brewing.


INTRODUCTION
Baijiu is a traditional distilling liquor made by solid fermentation that has been made for generations, stretching back over a 100 years. According to the different flavors, Baijiu can be divided into three most basic types (strong aromatic-flavor, sauce-flavor, light flavor) and nine derived types from above the three flavors (rice-flavor, jian-flavor, fuyu-flavor, te-flavor, fengflavor, dong-flavor, chi-flavor, sesame-flavor, laobaigan-flavor) (Zheng and Han, 2016). SAB is world famous for its special flavor, occupying about 70% Baijiu market in China, Wuliangye is the representative brand of SAB, and the annual production of SAB exceeded 10 million tons (Liu et al., 2017b;Xu et al., 2017). SAB is mainly made from multiple grains (sorghum, corn, rice, wheat, glutinous rice, and so on), with Daqu as a primary saccharification starter, and a solid state fermentation process was carried out in a fermented mud pit. Ultimately, alcohol and various aromatic materials are obtained by distilling (Figure 1). The production of SAB was carried out in 60 days or so, and complex metabolic reactions were detected during the long-term SAB fermentation (Hu et al., 2021b). Subsequently, the physical and chemical characteristics of Zaopei (mixture of steamed grains, rice husks, fermented grains, etc.) changed constantly, as a result, abundant compounds were produced , which were closely associated with the style of SAB. Owing to the natural fermentation style, numerous microbes are involved in the SAB making process (bacteria and fungi) (Ma et al., 2016;Wang et al., 2017;Zhang et al., 2017;Liu et al., 2018b;Qian et al., 2021), which mainly originated from Daqu, pit mud, the environment of the distillery and so on. The final SAB liquor structure is produced by the coaction of these microbes . Until now, studies on the microbes of SAB brewing focused on the microbial community structure and relevant flavor substances. Zou et al. (2018) discussed the diversity and function of the microbial community in SAB at the macro level. Several fungi and bacteria were are discussed in this work, however, a detailed introduction of the role for specific species of microbes throughout the SAB ecosystem is lacking, which is vital for brewing quality control. As important food-associated microorganisms, especially in SAB production, the function and roles of Bacillaceae were elaborated in the major fermentation phase, thus research in this field will benefit from SAB production by bioaugmentation (or other regulation strategies) of Bacillaceae in Daqu, pit mud, Zaopei, and Haungshui (a brown liquid byproduct was formed during SAB brewing, and deposited in the pit bottom) (Ding et al., 2015).
Bacillaceae is a gram-positive bacteria that plays an important role in the SAB making industry (Zheng et al., 2011;Ding et al., 2014;Wang et al., 2014;He et al., 2019b). Most of the Bacillaceae isolated from Daqu, pit mud, Zaopei, and Haungshui can produce various aromatic flavor components or flavor precursor, such as acetoin, 2,3-butanediol, four-carbon compound, pyrazines, and so on (Liu et al., 2017c;Zhang et al., 2021a). This feature could also be observed in the making process of various fermented foods. It is well known that the principal fragrance component is ethyl caproate in SAB which is associated with caproic acid bacteria (Luo et al., 2020;Cheng T. et al., 2021). Caproic acid bacteria is a group of microorganisms that can metabolize caproic acid, which mainly consists of Bacillus megaterium, Bacillus fusiformis, Bacillus licheniformis, and Clostridium, etc. (Zhao et al., 2012;Hu et al., 2015). Wang et al. (2015b) identified eight Bacillus from Daqu by employing the traditional method. The results showed that they belonged to five specials, Bacillus megaterium, Paenibacillus macerans, Bacillus pumilus, Bacillus atrophaeus, and Bacillus licheniformis, respectively. Liu et al. (2017c) identified three dominant Bacillus from a century mud pit of Luzhou Laojiao Liquor distillery, which showed Lysinibacillus sphaerieus, Brevibacillus brevis, and Paenibacillus larvae subsup. Pulvifaciens. The distribution of Bacillaceae have significant differences among Daqu, pit mud, Zaopei, and Huangshui in different geographical environments and seasons. A previous study suggested that the temperature variation and humidity may be important factors affecting the microbe community (Fu et al., 2011;Yin et al., 2014), as there are differences in these factors in various SAB distilleries ( Table 1).
As the dominant microorganism in the SAB making process, the review described the diversity of Bacillaceae, the function of Bacillaceae in Daqu, pit mud, Zaopei, and Huangshui. The effect on the flavor of SAB that contributes to Bacillaceae is also discussed. Through the analysis of this article, we hope to provide ideas for exploring the functional microorganisms in the SAB making industry, and accelerate a deeper understanding of the brewing mechanism of SAB, helping to improve the quality of SAB, and facilitating the better application of Bacillaceae in the food fermentation industry.

Bacillaceae in Daqu
The volatile flavor characteristics of SAB are influenced by the microflora of raw materials and the environment. In the production of SAB, pit mud is the foundation of fermentation (Zheng et al., 2013;Zheng and Han, 2016), Daqu is the motive power of fermentation (Gou et al., 2015;Deng et al., 2020), and brewing technology is the core of fermentation. Numerous studies have shown that Bacillus contributes greatly to the flavor of SAB in Daqu, the secondary metabolites of Bacillus could interact with other microorganisms, and benefited from maintaining relative stability of the microecosystem of Daqu (He et al., 2019a;He G. et al., 2020). Daqu, as the saccharification starter of SAB, includes lots of microbes and enzymes, and Bacillaceae play a key role in the production of Daqu Hu et al., 2020;Xiang et al., 2020). For example, as the functional microbes in Daqu, Bacillus licheniformis could produce multiple enzymes, such as amylase, proteases, and lipases in Daqu (Yan et al., 2013b), besides, Bacillus licheniformis could also reduce the content of higher alcohols in SAB (too high to spoilt SAB taste, easy to cause headaches, easy to drunk) . Bacillus spp. could hydrolyze proteins in fermented foodstuffs, which benefits the flavor and flavor precursor formation in Daqu (Beaumont, 2002). Meanwhile, Bacillus spp. can also exhibit the capacity of inhibiting the growth of Streptomyces, and reduce geosmin to avoid soil odor in Daqu (Zhi et al., 2016). Zhai et al. (2020) analyzed. Daqu samples in the main product area of SAB (Yibin city) by high throughput sequencing, the result showed that microorganisms consist of Bacillus sp., lactic acid bacteria, Pedicoccus sp., Weissella sp., Leuconostoc sp., Thermoactinomyces sp. and Acetobacter sp.). Research has shown that Bacillus subtilis, Bacillus horneckiae, Bacillus megaterium, Bacillus licheniformis, and Brevibacillus make up common species of Bacillaceae in Daqu (Zhou et al., 2010;Xiang et al., 2020).
The functions of Bacillaceae within Daqu in SAB brewing were as follows: firstly, various hydrolases were secreted into Daqu, and the utilization ratio of raw material was elevated (Wang et al., 2017;Wu et al., 2020); secondly, flavor substances were produced, for example, Bacillus spp. could produce abundant organic acids in Daqu (e.g., malic, lactic, acetic, citric, succinic, propionic and butyric acids), and the organic acids were the main flavoring components of SAB and the precursors of ester production (Yan et al., 2013a), meanwhile, the study also found that Bacillus spp. could produce aromatic substances in Daqu (benzaldehyde, 4-methylphenol, benzeneethanol, ethyl phenylacetate, etc.) (He et al., 2019b). Thirdly, the study revealed ingredients that are good for health can be enriched in Daqu, for example, Bacillus licheniformis directly proliferated in Daqu,  which could significantly increase the content of pyrazines, terpene, and zearin, etc. Wang et al., 2015b;Zhou et al., 2016), substances that are good for human health (i.e., anti-cancer, anti-virus, anti-inflammatory, antioxidation). Recently, two strains of high yield of fibrinolysin (function as anti-thrombus) were also identified from Daqu (Bacillus velezensis, Bacillus licheniformis) (Zhang et al., 2021b). Fourthly, promoting the distillation of flavor components from Zaopei, and reducing the harmful components of microbial metabolite, Bacillus licheniformis could produce lichenin to enhance the volatilization of esters and other substances, and markedly decrease the volatilization of phenol and phenylethanol (Wang et al., 2015b).

Bacillaceae in Pit Mud
The SAB fermentation process was carried out in a pit cellar, in which mud functioned as a fermentation carrier of SAB. Pit mud contains a variety of important microorganisms (Tao et al., 2014;Liang et al., 2015;Liu et al., 2017a), and the special pit aroma in SAB is mainly derived from pit mud microbes (Zhang et al., 2017). The bacteria in pit mud consist of Bacillus, Clostridium, Pseudomonas, Sporolactobacillus, Lactobacillus, and actinomycetes, etc. (Yue et al., 2007;Zou et al., 2018;Liang et al., 2021). PCR-DGGE analysis revealed that eight families of bacteria species existed in pit mud, e.g., Clostridiaceae, Lactobacillaceae, Synergistaceae, Sphingomonadaceae, Ruminococcaceae, Clostridiales_Incertae Sedis XI, Lanchnospiraceae, and Plannococcaceae. They belong to three main microorganisms of Clostridiales, Lacotobacillaceae, and Bacillale, separately (Zheng et al., 2013). Fifteen Clostridium species and one Bacillus were isolated from one of the bestknown brands of SAB pit mud (Luzhou Lao Jiao liquor), all of them had the ability to produce organic acid . Recently, more bacteria of the Bacillaceae species were isolated from pit mud ( (Zhang et al., 2010Liu et al., 2013Liu et al., , 2017cLiu et al., , 2018cYe et al., 2013;Wang et al., 2018). The culturable bacteria's from pit mud were Bacillus species, and the predominant strains were Bacillus licheniformis and Bacillus subtilis (Zhang et al., 2010). Most of the Bacillaceae isolated from pit mud can produce acetoin, 2,3-butanediol, C4 compounds, pyrazines, volatile acid, etc., all the compounds listed above are important flavors, and are crucial to regulating the SAB flavor ; Table 2).
Bacillaceae is the most abundant genus in pit mud, which plays a key role in SAB production. It is involved in the formation of major flavors in SAB, for example, ethyl caproate, ethyl butyrate, and caproic acid, which have been correlated positively with Clostridium, Rummeliibacillus in pit mud . Amylase, glycosylase and debranched enzyme could be generated by Bacillus amylolitica, and butyric acid, acetoin, volatile acid could be synthesized by Clostridium and Bacillus in pit mud Wu et al., 2019).
Bacillaceae can be employed as an indicator of pit mud quality: when the total number of Bacillaceaes in the new pit mud is at a high level, with the improvement of pit mud quality, the relative abundance of Clostridium kluyver and other Clostridiums increased significantly . The relative abundance of Bacillus decreased inversely in aged pit mud (Yu and Liu, 2016). By depressing the reproduction of harmful bacterial, one study revealed that Clostridium butyricum produced a variety of enzymes, which could decompose polysaccharides into oligosaccharides, and produce multiple secondary metabolites, such as antibacterial peptide, butyric acid, acetic acid, and vitamins, and that they could inhibit the propagation of harmful bacteria .
Bacillaceae in pit mud not only metabolizes caproic acid, butyric acid, acetic acid and other important aromatic components of SAB, but also could metabolize some secondary metabolites, which interact and regulate the related microorganisms in pit mud, thus affecting the aroma components of SAB.

Bacillaceae in Zaopei
Research has shown that there are various Bacillaceae in Zaopei (Yao et al., 2010;Wang et al., 2011;Hu et al., 2021a). 16S rDNA analysis found multiple microbe species in Zaopei, Bacillus, and lactobacillus (Liu F. et al., 2020;Hu et al., 2021a;Li and Qiu, 2021). The distribution species of microbes in Zaopei may be related to the specific environment in a mud pit, such as sealed hypoxia, low pH, high content of ethanol, so the growth of Bacillaceae is suited to this specific micro-ecological environment. As fermentation progresses, the relative abundance of Bacillaceae increases . Zhang et al. (2010) investigated bacteria flora from Zaopei and pit mud in Guizhou province, and 477 strains were isolated, Bacillus Licheniformis was identified as the dominant group of Bacillus, accounting for 32.96% of the total number of Bacillus, meanwhile, a small number of uncertain strains of Brevi Bacillus were isolated from Zaopei.
Bacillaceae are essential for Zaopei. They function by firstly secreting multiple hydrolases in the early fermentation phase, characteristics that ensure full utilization of Daqu and Zaopei (Li and Qiu, 2021). For example, organic matter such as starch, protein, and purine can be decomposed by Clostridium (Fan et al., 2007;Hahnke et al., 2014;Zhang et al., 2014;Yang and Chen, 2021). As they are involved in the formation of flavor matter in SAL, Bacillus and Clostridium are responsible for the production of ethyl hexanoate, caproic acid, butyric acid, lactic acid, benzaldehyde, alcohols, fatty acids, phenol, and other compounds (Hu et al., 2015;Liu et al., 2018a;Chai et al., 2019; Table 2). In summary, Bacillaceae of Zaopei was very similar to pit mud, because they were in the same container during the braving process of SAB.
Bacillaceae are involved in Huangshui formation, which plays a key role in metabolizing nutrients, and could produce flavor components such as esters, acerbity, ketone, aldehyde . Unfortunately, up to now, only a few studies have reported on the function of bacterial in Huangshui, especially for investigating Bacillaceae. At present, Clostridium spp. and Bacillus spp. had found the capacity of cellulose degradation in Huangshui (Desvaux et al., 2000;Sasaki et al., 2012). Six strains of Bacillus were isolated from Huangshui, which could produce cellulase, including Bacillus cereus, Bacillus circulans, Bacillus megaterium, Bacillus endophyticus, Bacillus simplex, and Bacillus bataviensis, separately (Zeng et al., 2016). During the SAB production, cellulase producing bacteria could degrade the cellulose in mixed raw materials, and release the starch inside it, which is conducive to the action of the saccharifying enzyme, thus furthering the utilization rate of raw materials, improving the fermentation rate, and shortening the fermentation time (Hu D. et al., 2016). However, the structure and reaction mechanism of Bacillaceae cellulase are still unclear in Huangshui. Recently, a redundancy analysis of the microbe community structure and aroma components showed that SAB aroma components were positively correlated with Clostridia, but negatively correlated with Bacilli in Huangshui. Meanwhile, acidity was positively correlated with Bacilli and negatively correlated with Clostridia (Gao Z. et al., 2020).

CONCLUSION AND PERSPECTIVES
At present, little information is available on the specific metabolite of each Bacillaceae species. This information is crucial for applying Bacillaceae in SAB making. Other approaches to food fermentation by the bioaugmentation of special Bacillaceae may provide references for future research ( Table 2). Some Bacillus species associated with SAB making showed the ability to produce various substances, such as hydrocarbons, organic acids, alcohols, aldoketones, esters, ethers, heterocycles, nitrogen-containing compounds, and phenolic compounds. All the metabolites listed above were identified by simulated fermentation (Table 2). Unfortunately, we still lack information about other Bacillus species, Clostridium species and metabolites, and further work is required.
In the process of making SAB, various microorganisms (associated with microbial proliferation and metabolism) interact, which contributes to the diversity of SAB flavor. The synergistic effect of diverse esters has been detected between pit mud microbes and Daqu microbes, for example, volatile acids and alcohols were provided by pit mud. Subsequently, esterifying reactions are achieved in SAB making by Daqu (Gao et al., 2021). As one of the main brewing microorganisms, Bacillaceae interact with other microorganisms, and this interaction effect has been studied by the method of single-strain bioaugmentation. However, there are at present few studies on the interaction among SAB brewing microorganisms. Previous research suggests that bioaugmentation of Bacillus velezensis and Bacillus subtilis in Daqu, which alter the microbial community and improve the flavor character of Daqu (He et al., 2019a). Bioaugmentation with Hydrogenispora could affect the abundance of Clostridium in pit mud . The study found that lactic acids were cut down by bioaugmentation with Bacillus during the SAB fermentation and the cause was Bacillus was negatively related to other bacterial species (Wang et al., 2017).
There are three main problems in the study of SAB Bacillaceae function: firstly, we lack studies on the interaction among SAB brewing microorganisms, to better understand the relationship between metabolic mechanism and the flavor production of brewing microorganisms, the interaction between Bacillaceae and other microbes should be further explored. Secondly, owing to the particularity of the mud pit environment, the anaerobic strains of Bacillaceae are difficult to isolate and culture from pit mud and Huangshui, so that functional properties of some Bacillaceae are difficult to determine. Thirdly, there are few studies on the isolation, culture, and flavor characteristics of a single strain of SAB Bacillaceae, especially the effect on solid-state fermentation. Fourthly, due to the different methods of metabolite detection on strains, as a result, different compounds were detected in different experiments with the same strain. Encouragingly, new technology can help solve the challenge, for instance, multi-omics approaches (metagenomes, metatranscriptomes, metaproteomes, and metabolomes) enable us to unravel the effects of Bacillaceae in the production of SAB. However, the high level of ethanol, acids, and humus in samples should be properly resolved. Isotope labeling and other biotechnology have been applied to study flavor formation pathways in Bacillaceae and will benefit from further exploration of the metabolic mechanism of flavor substances in SAB Bacillaceae. In the future, further study will promote the utilization of Bacillaceae in the brewing process, and improve the quality and stability of SAB.

AUTHOR CONTRIBUTIONS
WT, PH, and YY carried out the initial literature research and manuscript writing. ZQ, DH, and HL helped to provide expertise and insight relating to Baijiu microbiology. WT and XF revised the manuscript. All authors read and approved the final manuscript.