Systematic Review of Actinomycetes in the Baijiu Fermentation Microbiome

Actinomycetes (a group of filamentous bacteria) are the dominant microbial order in the Daqu (DQ) fermentation starter and in the pit mud (PM) of the Baijiu fermentation microbiome. Actinomycetes produce many of the key enzymes and flavor components, and supply important precursors, which have a major influence on its characteristic aroma components, to other microorganisms during fermentation. This paper reviews the current progress on actinomycete research related to Baijiu fermentation, including the isolation and identification, distribution, interspecies interactions, systems biology, and main metabolites. The main metabolites and applications of the actinomycetes during Baijiu fermentation are also discussed.


Introduction
Baijiu is one of the six major distilled liquors in the world and has been produced for centuries in China [1,2]. Baijiu is colorless, clear, and has a unique flavor, which mainly arises from the co-fermentation of microorganisms naturally inoculated. This method results in a very wide range of fermenting organisms, from both the Daqu (DQ) saccharification starter, the pit mud (PM), and the fermented grains (FGs) [3][4][5]. Depending on its flavor characteristics caused by the different production processes, raw materials, and microbial communities, Baijiu can be classified into the four basic Baijiu aroma types-jiangxiangxing (JXXB), nongxiangxing (NXXB), qingxiangxing (QXXB), andmixiangxing (MXXB)-and eight Baijiu aroma types derived from the above four aroma types [1]. Baijiu production consists of four successive stages: (i) saccharification of the grain starch using DQ or Xiaoqu (XQ) as the starter culture; (ii) open solid-state fermentation; (iii) distillation to produce the final liquor product; and (iv) storage aging. The open saccharification fermentation results in a highly complex microbiome, with many interactions between species [6,7]. This huge microbial diversity is responsible for the unique flavor of Baijiu, which is distinct from those of other distilled liquors, such as whisky, vodka, and tequila, which are made by submerged fermentation [8]. In essence, the diversity and stability of microbial communities associated with high acid tolerance are directly influenced by factors of the natural environmental conditions (geographical limitations, temperature, humidity, pH, and climate), raw materials (sorghum or a mixture of wheat, barley, corn, rice, and sorghum), and complex production processes [9,10]. The high complexity of the naturally selected microbiome has the potential to produce distinct flavors containing different trace components and alcohol contents (35-60%), caused by their underlying metabolism and interactions.
Actinomycetes in Baijiu fermentation derive from the DQ or XQ starter, the PM, and the fermenting grains (FGs) [11,12]. They have diverse metabolic activities, including the hydrolysis of starch, cellulose, protein, and pectin in the raw material grains, and they produce various secondary metabolites, including flavor esters (ethyl caproate, ethyl butyrate, and ethyl lactate), and other flavor compounds (3-hydroxyl-2-butanone, 2,3-butanediol, and Table 1. Actinomycete species isolated and identified from Baijiu.

Methods for Identification of New Species of Microorganisms from Baijiu Production
Culture-independent methods, such as polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing technology, have been developed for identification of actinomycetes ( Table 2). The microbiomes of four different types of Baijiu were analyzed by these methods. Thermoactinomyces sanguinis could only be detected in the DQ and FG of JXXB, NXXB, QXXB, and JXXB by culture-independent methods [57][58][59][60]. Thermoactinomyces vulgaris, Olsenellauli, Olsenella profusa, Lancefieldella parvula, Corynebacterium tuberculostearicum, Corynebacterium minutissimum, Streptomyces coeruleorubidus, Streptomyces hainanensis, and Arthrobacter woluwensis were identified in NXXB fermentations by PCR-DGGE [61]. Arthrobacter stackebrandtii, Kocuria carniphila, Glutamicibacter creatinolyticus, Brevibacterium aurantiacum, Cellulosimicrobium funkei, Microbacterium oxydans, Corynebacterium glutamicum, Gordonia terrae, Dietzia maris, Acidipropionibacterium acidipropionici, Microbacterium hydrocarbonoxydans, Microbacterium schleiferi, and Gulosibacter molinativorax were first identified in the PM of NXXB by 16S rRNA gene sequencing [62]. Streptomyces albus, Kroppenstedtia eburnea, Saccharopolyspora rectivirgula, Brevibacterium celere, and Thermoactinomyces vulgaris were identified as the dominant microorganisms in the DQ of QXXB, according to a similar analysis of 16S rRNA [60]. In ZMXXB, Saccharopolyspora rectivirgula, Saccharopolyspora hordei, Saccharopolyspora rectivirgula, and Rothiakristinae were identified using 16S rDNA analysis [63]. Thermoactinomyces vulgaris, Thermoactinomyces intermedius, and Thermoactinomyces daqus were detected using ARDRA and each type represents an OTU (operational taxonomic unit) [64]. In addition, Laceyellatengchongensis, Laceyellasediminis, Laceyellasacchari, and Laceyella putidus were identified by a specific PCR assay using a new specific primer (109F/801R) [47].  Acomparison of actinomycetes identified by culture and culture-independent methods is shown in Table S2. Thermoactinomyces vulgaris, Streptomyces albus, Thermoactinomyces intermedius, Thermostaphylospora chromogena, Kroppenstedtia eburnea, and Streptomyces cacaoi were identified by both methods. Eighty species were detected and identified by cultureindependent methods, of which 55 species were detected without isolation. There are four major differences between the two methods: (i) the analyzed samples were from many different locations and sampling times; (ii) the isolation medium of actinomycetes were limited by traditional culture methods; (iii) metagenomics analysis of most samples by the cultured-independent methods could only assign the taxonomic classification of actinomycetes to genera; and (iv) low-abundance actinomycetes cannot be detected by targeted metagenomics such as 16S rRNA amplicon sequencing. To understand the diversity of actinomycetes within the Baijiu microbiome of a limited time, multi-omics analysis and more separation methods should be applied [75,76]. Further, combined with innovative techniques (high-throughput culture), uncultured actinomycetes should be targeted for isolation according to their special functional characteristics [77]. With the help of high-throughput analysis results, selective nutrient media, selective physicochemical conditions, density-based separation, inhibitors, and specific growth factors could be found to isolate the uncultured actinomycetes from the DQ, FGs, and PM of the different Baijiu ecosystems [77].

Distribution of Actinomycete Species from the Major Types of Baijiu
The distribution of actinomycete species from the major types of Baijiu, and from the DQ, PM, and FGs, are shown in Figure 1, and a more detailed distribution of Thermoactinomyces and actinomycetes in DQ is shown in Figure 2. The variation in the profile of the actinomycetes was unraveled among the niches of the different types of Baijiu. Thermoactinomyces, a thermophilic actinomycete, was found in the DQ and FG of JXXB from three Baijiu factories, and accounted for 34.4-66.1% of the microbiome during the DQ production process [78][79][80]. Actinomycetes, including the Actinopolysporaceae, Brevibacteriaceae, and Streptomycetaceae families, make up 3.3-9.1% of the DQ microbiome [81]. Actinomycetes were one of the main phyla (7.7%) and Thermoactinomyces was the dominant genus in white and yellow DQ [82]. Three types of high-temperature DQ contained four genera of actinomycetes, namely, Saccharopolyspora, Brevibacterium, Streptomyces, and Thermoactinomyces, which comprised 4.7, 1.2, 1.5, and 26.4% of the total sequences, respectively [83]. Thermoactinomyces and Saccharopolyspora accounted for 16.7 and 22.1%, respectively, of the genera in Northern JXXB DQ, under high-temperature conditions [84]. The abundance of actinomycetes in manually and mechanically produced high-temperature DQ was 1.94 and 3.08%, respectively, and increased to 14.19 and 5.06% during fermentation [85]. JXXB goes through multiple rounds of fermentation, where one round includes three distinct phases: stacking fermentation, anaerobic fermentation, and distillation. Thermoactinomyces and Streptomyces have very low abundances at the early stage of fermentation, from the first to the seventh rounds, but they are dominant at the middle stage of the seventh round [66,86]. Fermented grains undergo three processes: cooling, stacking, and cellar fermentation. In the early stage of stacking fermentation, the FG microbiome contained 11.5% Thermoactinomyces [6].
The relative abundance of actinomycetes increased in NXXB DQ from Day 5 to Day 20 of fermentation, then decreased over the next few weeks during fermentation and maturation [87]. Kroppenstedtia and Thermoactinomyces were dominant in medium-and high-temperature DQ, accounting for 45.1% and 12.3%, respectively [88]. Thermoactinomyces was dominant during storage of DQ, whereas Saccharopolypora and Micromonospora increased in abundance during the subsequent fermentation [89,90]. Saccharopolyspora and Thermoactinomyces were the dominant genera after 18 days of DQ fermentation, reaching maximum abundances of 33.8% and 17.8%, respectively. The relative abundance at the phylum level for actinomycetes was 16.4% and that of Thermoactinomyces in mature DQ was 16.3% (Yibin) and 13.2% (Luzhou) [91]. The relative abundance of Thermoactinomyces reached 78% for special-grade DQ and 33% for first-grade DQ during summer production [92].
Dynamic changes in actinomycete relative abundance were studied in the DQ, PM, and FGs of NXXB [93]. During the fermentation and maturation of DQ, the relative abundance of actinomycetes initially increased, then decreased, and the relative abundance of the different species also changed, including Thermoactinomyces, Saccharopolypora, and Micromonospora. Kroppenstedtia and Thermoactinomyces were dominant in medium-and high-temperature DQ, accounting for 45.1% and 12.3%, respectively [88]. Two genera of actinomycetes, Kroppenstedtia and Thermoactinomyces, dominated the DQ at a medium and high temperature, accounting for 45.11% and 12.29%, respectively. Saccharopolyspora and Thermoactinomyces were the dominant genera after 18 days of DQ fermentation, reaching maximum abundances of 33.8% and 17.8%, respectively [94]. Thermoactinomyces in mature DQ from different locations accounted for 16.26% (Yibin) and 13.21% (Luzhou) [91]. Among the different grades of DQ, the proportion of Thermoactinomyces in special-grade DQ reached 78% and that of first-grade DQ reached 33% [92]. In the PM of NXXB, the relative content of actinomycetes initially decreased and then tended towards stability with increased cellar age [95,96]. The relative abundance of actinomycete genera, such as Atopobium and Olsenella, gradually increased by more than 1% during aging of PM and then stabilized in mature PM [97][98][99][100]. Moreover, the abundance of actinomycetes in mature PM (1.68 × 10 10 copies per g) was 29 times that of aging PM (0.58 × 10 9 copies per g) [101], and the matured PM (2.23 × 10 9 ) was 24 times that of degraded PM (9.25 × 10 7 cells/g) [102]. In the same cellar, the diversity of the microbiome in mature pit mud was superior to that in degraded pit mud; the relative abundance of the core actinomycete species, such as Frankia casuarinae, Brachybacterium faecium, and Mycobacterium sinense increased, because of the long-term anaerobic conditions in PM [103].
A study of actinomycetes in different layers of a Baijiu cellar detected actinomycetes at a relative abundance of >1% in the microbiome of the middle and upper layers [100,104], whereas the relative abundance of the actinomycetes was up to 4.81% in the bottom layer [105].
In fengxiangxing Baijiu (FXXB) FGs, the actinomycete concentrations were, 440, 259, and 261 cells/g in the upper, middle, and lower layers [118], respectively, during two weeks of fermentation; actinomycetes exceeded 4% in the first three days, slowly decreased to 0.5% in the second week, and then dropped sharply and stabilized at 0.2% [119]. As discussed above, actinomycetes are a major constituent of the microbiomes of DQ, FGs, and PM; Thermoactinomyces and Saccharopolypora are the dominant genera in mature DQ in the major types of Baijiu [112,116,120,121]. Actinomycetes are vital for Baijiu production, contributing to the quality of the final product by producing important flavor compounds, including four esters and five alcohols [7,119]; actinomycetes in the FG are derived from the DQ and PM, making a major contribution to starch saccharification. The niche adaptation and distribution of actinomycetes in brewage environments changes with some environmental factors such as temperature, moisture, and nutrient availability. Baijiu product quality and safety are associated with the brewing microbial community, which also depends on the actinomycete species. An improved understanding of the dynamic changes in actinomycete relative abundance under different fermentation conditions will require further research.  [6,63,[71][72][73]79,80,[82][83][84][85]88,91,92,95,107,110,112,114,116,122].

Interspecies Interactions of Actinomycetes and Other Microorganisms in Baijiu Fermentation
Baijiu flavor compounds are products of co-fermentation by multiple microorganisms. The interspecific interactions between actinomycetes and other microorganisms are closely related to the major flavor compounds in DQ, FGs, and PM [123,124]. Interspecies interactions between actinomycetes fall into four main categories (Figure 3). Actinomycetes produce various enzymes, such as cellulase, amylase, pectinase, and protease, which mediate these interactions, and the enzymolysis products can be assimilated by other microorganisms in the Baijiu fermentation microbiome [125]. For example, Streptomyces avicenniae hydrolyzes starch and produces melanin that scavenges free radicals from the cell surface of C. butyricum, promoting growth and caproic acid production [19,126] (Figure 3A). Some actinomycete metabolites are precursors for flavor component production by the key microorganisms Bacillus and caproic acid-producing bacteria (CPB) [18,37]. Acetic acid and lactic acid produced by Streptomyces and Micromonospora are precursors for yeast or Bacillus to produce ethyl acetate and ethyl lactate [37] (Figure 3B). Actinomycetes inoculated into CPB fermentation medium promote production of caproic acid and ethyl caproate [127]. Coculture of actinomycetes with lactic acid-producing bacteria (LPB) or acetic acid-producing bacteria (APB) promote the growth of the latter and production of ethyl lactate and ethyl acetate, which improves the Baijiu quality [18]. Actinomycetes produce antibiotics that inhibit pathogenic and functional bacteria, for example, 15 strains of Streptomyces could inhibit human pathogenic bacteria during DQ production [15]. These strains of Streptomyces both produce heptaene macrolide antibiotics, which inhibit the growth of yeasts, and degrade alcohols (3-octanol and 3-methyl butanol) and esters (ethyl octanoate and ethyl decanoate) [128] (Figure 3C). Non-protein and non-peptide antibiotics, or quinomycinA produced by actinomycetes, inhibit the biological activity of Bacillus subtilis [129,130]. In DQ production, Bacillus strains inhibit the growth of Streptomyces and degrade the geosmin produced by Streptomyces ( Figure 3D) [131]. Therefore, Bacillus amyloliquefaciens reduces the concentration of geosmin and the growth of Streptomyces strains, which relieves the inhibition of pyrazine compound production by Streptomyces and inhibits the formation of off-odors [132]. With further research, Bacillus subtilis may be able to downregulate the gene expression of the streptomycin Streptomyces griseus, which reduces the inhibiting effect of the latter [133]. The interaction mechanism between different actinomycetes and with other microorganisms is still poorly understood, but there is great potential for improving our under-standing of this using 'omics technology [75,134]. Moreover, genome-scale metabolic models (GSMMs), which can analyze and visualize the interaction mechanisms between actinomycetes and functional microorganisms in the Baijiu, should be constructed and applied [135].

Actinomycete 'Omics Research
The important metabolic pathways and functions of actinomycetes isolated from Baijiu fermentations have been analyzed by sequencing and annotation of the genomes of several actinomycete species (Table 3). Annotation of the Thermoactinomyces daqus H-18 genome revealed 1184 enzymatic reactions, 264 transporters, 867 compounds, 2361 transcription units, and 6 coding sequences (CDSs) of heat-shock proteins, which confer tolerance of high temperatures [45]. During growth at 60 • C in high-temperature DQ, actinomycete gene expression related to fatty acid biosynthesis increased six-fold [136]. The genes Clp, groEL, and pstB in thermophilic actinomycetes increase their survivability at a high temperature, allowing them to become dominant when increasing the DQ temperature [64]. Genome annotation of Streptomyces sp. FBKL4.005 revealed the genes coding for metabolic pathways associated with characteristic Baijiu flavors, sugar degradation, and streptomycin and neomycin production [29]. Comparative genomics has revealed the wide diversity of gene clusters in actinomycetes [137]. Genes coding for the geosmin biosynthetic pathway were all found in the core genome of Streptomyces [138]. In addition, metaproteomics and metabolomics were also used to investigate the functional changes in actinomycetes [68,139,140]. Actinomycete abundance had a negative correlation with lactic acid and a positive correlation with pH, determined by transcriptomic sequencing [141]. Actinomycete transcriptomic analysis revealed that aged PM has a higher content of seven key enzymes than degraded PM [103].
The combination or comparison of multiple 'omics studies has not yet been applied to actinomycetes from the Baijiu microbiome to analyze and amplify their genetic elements and metabolic pathway. The GSMMs of Baijiu actinomycetes have not yet been constructed and analyzed. 'Omics analysis, combined with the GSMMs of actinomycetes, should be performed to attain a comprehensive understanding of actinomycetes in future research. Gene engineering could be used to regulate and produce natural products from Baijiu actinomycetes.

Potential Applications of Actinomycetes in Baijiu Fermentation
Actinomycetes have useful regulatory functions in Baijiu fermentation, as discussed below. Actinomycetes produce highly active hydrolytic enzymes that enable full utilization of all the components of baijiu FGs [15,153], particularly cellulase, which degrades the abundant cellulose to produce short-chain fatty acids in FGs and distiller's grains [154]. Undesirable isopropanol and lactic acid produced in Baijiu fermentation are degraded by Arthrobacter protophormiae [38,155], and actinomycetes produce antibiotics that inhibit the growth of human pathogenic bacteria [15]. Geosmin produced by Streptomyces ameliorates the effect of excess acidity during fermentation and since actinomycetes are relatively heat tolerant, they can maintain their metabolic activity in high-temperature DQ and PM [128,156]. The hyphae of Thermoactinomyces facilitate the evaporation of water from DQ and help to soften the grains, which aids their starter function [128]. Actinomycetes, as dominant strains in PM, are regarded as indicators of PM aging, and are studied to distinguish between the PM of different maturities [4,96]. Actinomycetes can facilitate denitrification of the PM using sulfur and sulfides and inhibit degradation of the PM [157]. New DQ and PM inoculated with selected actinomycetes reach maturity relatively quickly and old PM can be maintained in good condition, thereby maintaining Baijiu quality [102]. As stated above, actinomycetes are important for DQ biocontrol, PM maintenance, the formation of Baijiu flavor, and resource utilization of distiller's grains. However, for the future practical application of actinomycetes, some progress needs to be made to promote caproic acid production, inhibit the growth of pathogenic bacteria, and degrade distiller's grains. Hence, actinomyces has the potential to improve the safety and quality of Baijiu, which provide a direction for applied value research.

Conclusions
Actinomycetes hydrolyze starch, protein, and cellulose to supply precursors for other microorganisms to produce flavor components during fermentation, as well as producing important Baijiu flavor compounds, such as ethyl caproate and ethyl butyrate. Actinomycete relative abundance and species distribution are used as an indicator of PM quality and are important microorganisms for inhibition of PM degradation. However, the metabolic mechanism, isolation methods, and underlying interactions between actinomycetes are poorly understood, and in-depth research on the multi-omics analysis of actinomycetes has not yet been reported. Therefore, innovative separation methods are needed to efficiently isolate unculturable actinomycetes from complex microbiomes. The omics-based approaches enhanced our understading of the diversity and functional dynamics of actinomycetes. Future studies should also consider the potential application of actinomycetes to improve the food safety of Baijiu during fermentation, by regulating harmful microorganisms, which would improve Baijiu quality. Future research should include multi-'omics studies and construction of actinomycete GSMMs, by combining bioinformatics tools, high-throughput culture methods, and genetic engineering. From the fundamental basis and new insights into actinomycetes that were provided here, through an in-depth theoretical study of Baijiu microbial populations, future studies can help improve the Baijiu product safety, sustainability, and brewage standards.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/foods11223551/s1, Table S1: Culture media used for isolation of actinomycetes, Table S2: Actinomycete species identified by culture and/or culture-independent methods.
Author Contributions: Conceptualization, C.C., H.L. and W.Z.; writing-original draft preparation, C.C. and W.Z.; writing-review and editing, C.C., H.Y. and W.Z.; visualization, C.C. and J.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by grants from the National Natural Science Foundation of China (31801522).

Conflicts of Interest:
The authors declare no conflict of interest. The company had no role in study design data collection and analysis, decision to publish, or preparation of the manuscript and did not affect the results and the conclusions. There are no potential conflicts of interest.