Deciphering the core microbes and their interactions in spontaneous Baijiu fermentation: A comprehensive review.

The spontaneous Baijiu fermentation system harbors a complex microbiome that is highly dynamic in time and space and varies depending on the Jiuqu starters and environmental factors. The intricate microbiota presents in the fermentation environment is responsible for carrying out various reactions. These reactions necessitate the interaction among the core microbes to influence the community function, ultimately shaping the distinct Baijiu styles through the process of spontaneous fermentation. Numerous studies have been conducted to enhance our understanding of the diversity, succession, and function of microbial communities with the aim of improving fermentation manipulation. However, a comprehensive and critical assessment of the core microbes and their interaction remains one of the significant challenges in the Baijiu fermentation industry. This paper focuses on the fermentation properties of core microbes. We discuss the state of the art of microbial traceability, highlighting the crucial role of environmental and starter microbiota in the Baijiu brewing microbiome. Also, we discuss the various interactions between microbes in the Baijiu production system and propose a potential conceptual framework that involves constructing predictive network models to simplify and quantify microbial interactions using co-culture models. This approach offers effective strategies for understanding the core microbes and their interactions, thus beneficial for the management of microbiota and the regulation of interactions in Baijiu fermentation processes.

Bacteria-induced amino acid metabolism involved in appearance characteristics of high-temperature Daqu.

BACKGROUND: Significant changes occurd in Daqu bricks on the 15th day of incubation, and brick color (yellow, brown, or dark) is generally used as a standard for quality evaluation by experienced workers. This study aimed to explore the basis behind the phenomenon through multi-omics studies. The physicochemical properties of different high-temperature Daqu were compared. Furthermore, PacBio sequencing and the ultra-high-performance liquid chromatographic-Q-exactive-mass spectrometric approach were employed to analyze the differences in the microbiome and metabolome among different Daqu samples. RESULTS: Bacillus was the biomarker of yellow Daqu, Thermoactinomyces and Thermoascus were the key genera in brown Daqu, and Burkholderiales, Sphingomonas, and Ralstonia were biomarkers in dark Daqu. The physicochemical characteristics (especially the color values) of different high-temperature Daqu showed strong correlations with the bacterial alpha diversity and the relative abundance of dominant bacterial genera. Amino acid metabolism pathways including tryptophan metabolism, ß-alanine metabolism, and arginine biosynthesis were the key factors resulting in the characteristic differences where Bacillus, Burkholderia, Ralstonia, and Sphingomonas were pivotal bacterial genera. The relative abundance of Bacillus had a positive correlation with the content of 3-hydroxykynurenamine, l-glutamic acid, and pantothenic acid, while it showed a negative correlation with indoleacetic acid, l-tryptophan, N-acetylserotonin, l-histidine, l-aspartic acid, phosphatidylserine, 5-methoxyindoleacetate, and L-serine. Burkholderia, Ralstonia, and Sphingomonas had the opposite effects. CONCLUSION: Microbes play different roles in amino acid metabolism pathways, producing different metabolites, contributing to the differences in Daqu appearance and quality. © 2022 Society of Chemical Industry.

Response of microbial community assembly and succession pattern to abiotic factors during the second round of light-flavor Baijiu fermentation.

The regulation of microbial communities is an important strategy for fermentation management. Dynamic microbiota during the Baijiu fermentation is shaped by a variety of abiotic factors. Therefore, this study aims to investigate the effects of the microbiota of the fermentation starter Daqu on the microbial assembly and the interaction of microbiota succession and abiotic factors during the second round (Ercha) of light-flavor Baijiu fermentation. The results revealed that Streptomyces, Bacillus, Lactobacillus, and Staphylococcus were the dominant bacterial genera in the initial fermentation, while Lactobacillus was dominant during the middle and later stages. Pichia and Saccharomycopsis were the dominant fungal genera during the whole fermentation process. A total of 54 volatile compounds were identified during the fermentation, among which 15 compounds, mainly including ethyl acetate, diethyl azelate, ethyl 2-hydroxyisocaproate, 3-furaldehyde, and ethylidene diacetate, were identified as important flavor metabolites. The SourceTracker software revealed that Daqu contributed 52.3 % of the bacterial community and 38.6 % of the fungal community to the fermentation. Ethanol, moisture, and pH were the major factors regulating the succession of dominant bacteria and fungi during the fermentation. The microbial succession and co-occurrence pattern driven by abiotic factors played a crucial role in shaping flavor profiles. These results provide guidance for controlling the fermentation process by optimizing operational parameters or bioaugmentation with specific microbes.

Microbial characteristics and metabolite profiles of high-temperature Daqu in different maturation stages.

The maturation period of high-temperature Daqu (HTD) is usually 3-6 months, and the characteristics of HTD at different maturation stages were different. In this study, the microbial characteristics and metabolite profiles of HTD at different maturation stages were revealed with the combination of physicochemical detection, the third generation Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-ion mobility spectrometry (GC-IMS). Results showed that HTD matured for 6 months (Mix_m6) had higher saccharification power but less culturable thermotolerant bacteria and fungi than HTD matured for 3 months (Mix_m3). The average relative abundances of Thermoactinomyces, Paenibacillus, and Rasamsonia in Mix_m3 were higher than that in Mix_m6, while the average relative abundances of Bacillus, Pseudomonas, Thermoascus increased obviously with the prolongation of the maturation period. Streptomyces and Thermoactinomyces were biomarkers in Mix_m3, while Burkholderia and Pseudomonas were regarded as biomarkers in Mix_m6. Differences in microbiota structure led to different enrichment of metabolic pathways in HTD at different maturation stages, resulting in different flavor profiles, especially in ethyl acetate, 1-octen-3-one, (E)-3-Hexen-1, 2,3,5-trimethy-6-ethylpyrazine, pyrazine, tetramethyl content. The microbiota and metabolite characteristics of HTD comprehensively reflected the HTD quality in different maturation stages, which provided a reference for determining the optimal maturation time.

Integrated multi-omics approaches to understand microbiome assembly in Jiuqu, a mixed-culture starter.

The use of Jiuqu as a saccharifying and fermenting starter in the production of fermented foods is a very old biotechnological process that can be traced back to ancient times. Jiuqu harbors a hub of microbial communities, in which prokaryotes and eukaryotes cohabit, interact, and communicate. However, the spontaneous fermentation based on empirical processing hardly guarantees the stable assembly of the microbiome and a standardized quality of Jiuqu. This review describes the state of the art, limitations, and challenges towards the application of traditional and omics-based technology to study the Jiuqu microbiome and highlights the need for integrating meta-omics data. In addition, we review the varieties of Jiuqu and their production processes, with particular attention to factors shaping the microbiota of Jiuqu. Then, the potentials of integrated omics approaches used in Jiuqu research are examined in order to understand the assembly of the microbiome and improve the quality of the products. A variety of different approaches, including molecular and mass spectrometry-based techniques, have led to scientific advances in the analysis of the complex ecosystem of Jiuqu. To date, the extensive research on Jiuqu has mainly focused on the microbial community diversity, flavor profiles, and biochemical characteristics. An integrative approach to large-scale omics datasets and cultivated microbiota has great potential for understanding the interrelation of the Jiuqu microbiome. Further research on the Jiuqu microbiome may explain the inherent property of compositional stability and stable performance of a complex microbiota coping with environmental perturbations and provide important insights to reconstruct synthetic microbiota and develop modern intelligent manufacturing procedures for Jiuqu.

Insights into the bacterial, fungal, and phage communities and volatile profiles in different types of Daqu.

Daqu has a rich and diverse microbiota, giving them a suitable biotope for phages. However, the absolute abundances of bacteria and fungi, as well as the phage community characteristics in Daqu, remain unclear. In this study, the microbiota absolute abundance, indigenous phage composition and function, and volatile compound profiles of high-temperature Daqu (HTDQ), medium-temperature Daqu (MTDQ), and low-temperature Daqu (LTDQ) were investigated. Absolute microbiota quantification revealed that there were significant variations in microbial composition and absolute abundance across three types of Daqu. The absolute abundances of the top 30 bacterial genera in LTDQ, MTDQ, and HTDQ were 6.0 × 105, 5.3 × 104, 1.4 × 105 copies/ng DNA, while the top 30 fungal genera had 8.5 × 107, 2.1 × 106, and 6.2 × 105 copies/ng DNA, respectively. LTDQ were enriched in Pantoea, Staphylococcus, and Saccharomycopsis; MTDQ were dominated by Saccharopolyspora, Staphylococcus, Saccharomycopsis, and Aspergillus; HTDQ were dominated by Saccharopolyspora, Bacillus, Byssochlamys, and Saccharomycopsis. Volatile profile analysis revealed that LTDQ, MTDQ, and HTDQ comprised 68, 68, and 75 compounds, respectively, with 39 compounds shared by the three types. Fourteen volatile compounds were identified as highly discriminative features among three types of Daqu, which were closely related to Saccharopolyspora, Thermoactinomyces, Kroppenstedtia, Byssochlamys, and Thermomyces. Metaviromics indicated that Genomoviridae, Mimiviridae and Siphoviridae, and Parvoviridae were the dominant viruses in LTDQ, MTDQ, and HTDQ, respectively. The hosts of phages in Daqu mainly included Lactobacillus, Enterobacter, Pantoea, Bacillus, Pediococcus, and Staphylococcus. Phages may prey on numerous microbes living on Daqu via pathways such as genetic information processing, signaling and cellular processes, and replication and repair. This study highlights the use of absolute quantification to avoid misjudgment of differential taxa in comparative microbiome analysis and provides new insights into the phage community and function in Daqu.

Multidimensional profiling indicates the shifts and functionality of wheat-origin microbiota during high-temperature Daqu incubation.

Wheat-origin microbiota is a critical factor in the assembly of the microbial community during high-temperature Daqu incubation. However, the succession and functional mechanisms of these microbial communities in Daqu are still unclear. This study investigated the shifts in microbiota diversity from the wheat to the end of incubation by the third generation Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology. Results indicated that Staphylococcus, Pantoea, Alternaria, and Mycosphaerella were the dominant genera of wheat-origin microbiota while Bacillus and Thermoascus were the most predominant bacterial and fungal genera of Daqu microbiota, respectively. Metabolite detection revealed that volatile organic compounds (VOCs) changed obviously in different incubation stages and the 7th day to the 15th day of incubation was the critical period for the formation of VOCs. The content of non-VOCs, especially sugars, increased steeply in the first four days of incubation. The network analysis between microbes and metabolites showed that Thermoactinomyces and Staphylococcus had opposite correlations with most non-VOCs. Alternaria and Mycosphaerella had strong positive correlations with fructose. As key functional fungal genera in wheat-origin microbiota, Mycosphaerella, Aspergillus, and Alternaria participated in multiple metabolic pathways (gluconeogenesis I, sucrose degradation III, pentose phosphate pathway, 5-aminoimidazole ribonucleotide biosynthesis I, methyl ketone biosynthesis, and GDP-mannose biosynthesis) at the early stage of incubation, which played an important role in the formation of flavors and succession of microbiota. This work highlighted the shifts and functionality of wheat-origin microbiota in Daqu incubation, which can be a guideline to stabilize Daqu quality by wheat-origin microbiota control.

Comparative study on inhibitory effects of ferulic acid and p-coumaric acid on Salmonella Enteritidis biofilm formation.

Biofilm cells exhibit higher resistance than their planktonic counterparts to commonly used disinfectants in food industry. Phenolic acids are promising substitute offering less selective pressure than traditional antibiotics. This study aims to evaluate the inhibitory effects of ferulic acid (FA) and p-coumaric acid (p-CA) on Salmonella Enteritidis biofilm formation and explore the underlying inhibitory mechanisms. The minimal inhibitory concentration (MIC) of FA and p-CA were 1.0 and 0.5 mg/ml, respectively. The sub-inhibitory concentration (1/8 MIC) significantly decreased biofilm formation without growth inhibitory effects. The biomass and extracellular polymeric substances (EPS) of S. Enteritidis biofilm as well as the bacterial swimming and chemotaxis abilities were significantly decreased when exposed to sub-MIC concentrations of FA and p-CA. These two phenolic acids showed high affinity to proteins involved in flagella motility and repressed the S. Enteritidis biofilm formation-related gene expressions. Furthermore, these two phenolic acids maintained high antibiofilm efficiency in simulated food processing conditions. This study provided valuable information of multiple phenotypic and molecular responses of S. Enteritidis to these two phenolic acids.

Characteristics of the microbiota and metabolic profile of high-temperature Daqu with different grades.

The superior grade Daqu (S_Daqu) and normal grade Daqu (N_Daqu) have obvious differences in flavor, fracture surface, appearance, etc., which can be accurately grouped by well-trained panel based on their sensory properties. However, the differences in microbial community diversity and metabolites between the S_Daqu and N_Daqu were still unclear. The culture-dependent method, the third generation Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology, and nuclear magnetic resonance (NMR) were combined to show the characteristics in microorganisms and metabolites. Results showed that the fungal counts were higher in N_Daqu while the richness of bacterial communities was higher in S_Daqu (P < 0.05). Lentibacillus, Burkholderia, Saccharopolyspora, Thermoascus, and Rasamsonia were the dominant genera of S_Daqu while Staphylococcus, Scopulibacillus, and Chromocleista were the dominant genera in N_Daqu. The content of differential acids, amino acids, and alcohols including fumarate, glucuronate, glycine, 4-carboxyglutamate, and myo-inositol in S_Daqu was higher than that in N_Daqu by 1H NMR coupled with multivariate statistical analysis. The network analysis regarding microbes and metabolites suggested that Saccharopolyspora showed a strong positive correlation with 4-carboxyglutamate while Thermoascus and Chromocleista were highly negatively correlated with alanine and isobutyrate, respectively. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) revealed that Macrococcus and Caulobacter were regarded as bacterial biomarkers in the S_Daqu while Chromocleista was the key fungal genera in N_Daqu. Functionality prediction indicated that the bacteria in S_Daqu were largely involved in more metabolic activities including biosynthesis, degradation, detoxification, and generation of precursor metabolite and energy.

Unraveling the microbial compositions, metabolic functions, and antibacterial properties of Huangshui, a byproduct of Baijiu fermentation.

Huangshui (HS) is a byproduct of Chinese Baijiu production that has tremendous potential to become a high-value product in the food industry. This study comprehensively analyzed the commonalities and differences in physicochemical properties, microbial structures, metabolomes, flavor profiles, and antibacterial properties among sources of HS. The HS samples were extracted from three different pits in three different workshops within an industrial Baijiu distillery. Lactobacillus was the dominant genus of bacteria in all of them, while Kazachstania, Aspergillus, and Pichia were the dominant genera of fungi. A total of 45 volatile compounds, including ethyl hexanoate, hexanoic acid, and 1-butanol, were detected in the HS by headspace solid-phase microextraction coupled with gas chromatography mass spectrometry, suggesting HS could be used to flavor liquor. Untargeted metabolomic analysis yielded 224 nonvolatile metabolites that were common in all HS samples. These metabolites included organic acids, carbohydrates, benzenoids, nucleic acids, and fatty acids, which are mainly involved in amino acid and sugar metabolism. While the compositions of the microbiome and metabolome among HS samples were affected by workshop factors, there were essentially no differences in the bactericidal properties of the HS samples among workshops. HS exhibited potent antibacterial activities against Escherichia coli and Staphylococcus aureus, and HS was effective in inactivating mature biofilms formed on polystyrene microplates, as confirmed by scanning electron micrographs. Our results provided a theoretical basis to increase the economic value of HS for applications in the food industry.

Characterization and comparison of the bacterial community on environmental surfaces through a fresh-cut vegetables processing line in China.

Zoning is typically adopted by food manufacturers and classified into three different zones including Zone 1 (food contact surface), Zone 2 (proximity to food and food contact surfaces) and Zone 3 (remote surfaces from processing). In this study, environmental surfaces belonging to these three zones were sampled during food processing in a fresh-cut vegetables (FVs) processing facility located in Beijing, China. Bacterial loads in terms of aerobic plate count and coliform count were evaluated by culture-dependent techniques. The profile and diversity of bacterial community in these three zones were investigated by high-throughput sequencing. Zone 1 showed similar microbiota and predominated by Pseudomonas and Acinetobacter, while the predominant genera on Zone 2 or 3 were quite varied among different processing steps. The SourceTracker analysis showed that most of the bacteria (above 80%) on surfaces were from unknown sources, while sorting and disinfection & cleaning steps contributed equally to the bacterial community of cutting areas. Microbial association network analysis revealed strong positive interactions of Comamonas and Janthinobacterium with Myroides and Serratia, respectively, whereas Pseudomonas showed no significant correlation with other genera. The function of microbial communities was predicted based on BugBase 16S rRNA database, and the results indicated that the proportions of potential biofilm-forming bacteria were above 70% in all samples, and species of Pseudomonas, Comamonas, Chryseobacterium and Janthinobacterium were opportunistic pathogens or spoilage bacteria. This study evaluated the bacteria risks including bacterial load, composition, as well as potential bacterial interaction, function and microbial transfer on food processing surfaces, which will facilitate our better understanding on the cross-contaminations and preventions of harmful microorganism in FVs processing industry.

Understanding the Shifts of Microbial Community and Metabolite Profile From Wheat to Mature Daqu.

Wheat-originated microbes play an important role in shaping the quality of high-temperature Daqu which is commonly used as a starter for producing sauce-flavor Baijiu. However, the shifts of microbiota from raw material to fresh Daqu and then to mature Daqu remain unclear. Hence, in the present study, the inner and outer of fresh and mature Daqu were collected to explore the correlation between microbiota and metabolites as well as the source of the microbiota in Daqu. Results indicated that the activities of amylase and protease between the inner and outer of fresh Daqu varied significantly while both parts became similar after maturation. The predominant bacteria shifted from Saccharopolyspora (outer) and Staphylococcus (inner) to Kroppenstedtia (both outer and inner), while the predominant fungi shifted from Thermoascus (both outer and inner) to Byssochlamys (outer) and Fusarium (inner). A combining analysis of headspace solid-phase micro extraction-gas chromatography-mass spectrometry, headspace gas chromatography-ion mobility spectrometry, and nuclear magnetic resonance was employed to detect the metabolites. The network analysis was conducted to perform the relationships between microbes and metabolites. The results showed that the bacteria, especially Saccharopolyspora, Bacillus, and Acinetobacter, had a strong correlation with the productions of esters, amino acids and their derivatives, and sugars and their derivatives, while most fungi such as Thermoascus, were negatively correlated with the phenylalanine, trimethylamine n-oxide, and isovalerate. SourceTracker analysis indicated that wheat was the important source of the Daqu microbiota, especially, the microorganisms in the inner of Daqu might be the drivers of the microbial succession during maturation. This study provided a comprehensive exploration to understand the microbial sources and shifts in high-temperature Daqu during maturation.

Occurrence of disinfectant-resistant bacteria in a fresh-cut vegetables processing facility and their role in protecting Salmonella enteritidis.

Chemical disinfectants are widely used to control foodborne pathogen contamination in fresh-cut vegetables (FVs) processing facilities. In this study, we investigated the disinfectant-resistant bacteria in a FVs processing facility and evaluate the effects of these bacteria on Salmonella enteritidis biofilm formation and disinfectant resistance. The disinfectant-resistance profiles were determined using 0.02% sodium hypochlorite (NaClO), 0.2% benzalkonium bromide (BAB) and 2% hydrogen peroxide (H2O2) solutions. The results showed the high occurrence of disinfectant resistant bacteria in the FVs processing environment, especially in the clean area. All isolates showed planktonic susceptibility to H2O2 and BAB, while the Gram-positive isolates were specifically resistant to NaClO. Isolates with biofilm-forming ability showed resistance to tested disinfectants. Disinfectant resistance of S. enteritidis was not significantly enhanced in most of the mixed-species biofilms, except for Bacillus paramycoides B5 which not only increased the biomass but also enhanced the survival ability of the Salmonella under NaClO treatment. Increased biomass and compact biofilm structures were observed in mixed-species biofilms by scanning electron microscopy (SEM). This study provides new insights into the disinfectant-resistant bacteria from food processing facilities and highlights their relevance for foodborne pathogen contamination.

Exploring the diversity and role of microbiota during material pretreatment of light-flavor Baijiu.

Baijiu (Chinese liquor) is a type of traditional distilled alcoholic beverage produced through spontaneous solid-state fermentation with sorghum as the primary material. Material processing, including sorghum soaking, steaming and cooling which is carried out in an open environment, is an integral part of Baijiu manufacturing. However, the microbiota involved in material pretreatment and its associate with the alcoholic fermentation is unclear. This research is aimed to exploring the diversity and role of microbiota during material pretreatment of light-flavor Baijiu. Results showed that Cyanobacteria, Epicoccum, and Cladosporium predominated in the sorghum at the beginning of soaking. Lactobacillus and Pichia became the predominant bacterial and fungal genera by the end of soaking. With the dynamics of microbiota, the pH declined sharply and the categories and concentration of volatile flavors such as alcohols, esters, acids, phenols, ketones, and aldehydes increased. Correlation analysis indicated that Lactobacillus and Pichia showed positive correlation with various flavors during soaking. Furthermore, SourceTracker analysis revealed that the microbiota involved during cooling processing was an important source of the Lactobacillus during fermentation of light-flavor Baijiu. This study illustrates the role of microbiota during material pretreatment and the association with alcoholic fermentation, which contributes to reveal the mechanism of Baijiu manufacturing.

Characterization of bacteria and yeasts isolated from traditional fermentation starter (Fen-Daqu) through a 1H NMR-based metabolomics approach.

Daqu is a traditional fermentation starter for the production of baijiu and vinegar. It is an important saccharifying and fermenting agent associated with alcoholic fermentation and also a determining factor for the flavour development of these products. Bacterial and yeast isolates from a traditional fermentation starter (Fen-Daqu) were examined for their amylolytic activity, ethanol tolerance and metabolite production during sorghum-based laboratory-scale alcoholic fermentation. The selected strains (Bacillus licheniformis, Pediococcus pentosaceus, Lactobacillus plantarum, Pichia kudriavzevii, Wickerhamomyces anomalus, Saccharomyces cerevisiae, and Saccharomycopsis fibuligera) were blended in different combinations, omitting one particular strain in each mixture. 1H nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical analysis was used to investigate the influence of the selected strains on the metabolic changes observed under the different laboratory-controlled fermentation conditions. Principal component analysis showed differences in the metabolites produced by different mixtures of pure cultures. S. cerevisiae was found to be superior to other species with respect to ethanol production. S. fibuligera and B. licheniformis converted starch or polysaccharides to soluble sugars. Lactic acid bacteria had high amylolytic and proteolytic activities, thereby contributing to increased saccharification and protein degradation. W. anomalus was found to have a positive effect on the flavour of the Daqu-derived product. This study highlights the specific functions of S. cerevisiae, S. fibuligera, B. licheniformis, W. anomalus and lactic acid bacteria in the production of light-flavour baijiu (fen-jiu). Our results show that all investigated species deliver an important contribution to the functionality of the fermentation starter Daqu.

Effect of the environment microbiota on the flavour of light-flavour Baijiu during spontaneous fermentation.

Light-flavour Baijiu is a type of Chinese liquor with a pure and mild flavour produced by traditional spontaneous solid-state fermentation. The flavour of this liquor has been found to vary in the different periods of annual production. To explore the factors affecting flavour, the microbiota of the surrounding environment, starter and fermentation process in different periods were investigated. Results showed that the ester content and acidity of light-flavour Baijiu were significantly lower when annual production was resumed after a summer break. HCA plot of volatile flavour profile and bacterial PCoA results indicated that the differences occurred at later stages, mainly due to different structures of Lactobacillus. Correlation analysis by O2PLS indicated that Lactobacillus positively correlated with esters. Species-level analysis showed that the lack of L. acetotolerans on the surface of the jar might cause a lag in fermentation and lower ester content. Thereafter, L. acetotolerans was revived during fermentation and enriched on the surface of the jar, which promoted ester formation. As important sources of L. acetotolerans, the air and fermentation jars played a critical role during fermentation. Therefore, this systematic study on environmental microbial ecology is valuable for quality control and to explore environmental microbiota functions during spontaneous fermentation.

A Comprehensive Review of Spirit Drink Safety Standards and Regulations from an International Perspective.

Standards and regulations related to spirit drinks have been established by different countries and international organizations to ensure the safety and quality of spirits. Here, we introduce the principles of food safety and quality standards for alcoholic beverages and then compare the key indicators used in the distinct standards of the Codex Alimentarius Commission, the European Union, the People's Republic of China, the United States, Canada, and Australia. We also discuss in detail the "maximum level" of the following main contaminants of spirit drinks: methanol, higher alcohols, ethyl carbamate, hydrocyanic acid, heavy metals, mycotoxins, phthalates, and aldehydes. Furthermore, the control measures used for potential hazards are introduced. Harmonization of the current requirements based on comprehensive scope analysis and the risk assessment approach will enhance both the trade and quality of distilled spirits. This review article provides valuable information that will enable producers, traders, governments, and researchers to increase their knowledge of spirit drink safety requirements, control measures, and research trends.

Characterization of the microbial community in different types of Daqu samples as revealed by 16S rRNA and 26S rRNA gene clone libraries.

Daqu is a fermentative saccharification agent that is used to initiate fermentation in the production of Chinese liquor and vinegar. Different types of Daqu can be distinguished based on the maximum fermentation temperature, location of production, and raw materials used. We aimed to characterize and distinguish the different types of Daqu using a culture-independent cloning method. The lowest microbial diversity was found in Daqu produced at high-temperature. Principal component analysis (PCA) was used to compare the bacterial composition of Daqu from different regions (i.e., northern Daqu and southern Daqu). Staphylococcus gallinarum and Staphylococcus saprophyticus were found in southern Daqu, and were absent in northern Daqu. The fungi Saccharomycopsis fibuligera and Lichtheimia ramosa dominated in low/medium-temperature Daqu, whereas Thermomyces lanuginosus occurred in high-temperature Daqu. Our study identified potential biomarkers for the different types of Daqu, which can be useful for quality control and technology development of liquor or vinegar production.

Microbiota dynamics related to environmental conditions during the fermentative production of Fen-Daqu, a Chinese industrial fermentation starter.

Chinese Daqu is used as a starter for liquor and vinegar fermentations. It is produced by solid state fermentation of cereal-pulse mixtures. A succession of fungi, lactic acid bacteria and Bacillus spp. was observed during the production of Daqu. Mesophilic bacteria followed by fungi, dominated the first phase of fermentation. Next, lactic acid bacteria increased in relative abundance, resulting in an increase of the acidity of Daqu. At the final stages of fermentation, Bacillus spp. and thermophilic fungi became the dominant groups, possibly due to their tolerance to low water activity and high temperature. Both culture-dependent and culture-independent analyses confirmed that Bacillus spp. were ubiquitous throughout the process. Yeast species such as Wickerhamomyces anomalus, Saccharomycopsis fibuligera and Pichia kudriavzevii were present throughout almost the entire fermentation process, but the zygomycetous fungus Lichtheimia corymbifera proliferated only during the final stages of fermentation. Canonical correspondence analysis (CCA) revealed the significance of acidity, moisture content and temperature in correlation with the composition of the microbial communities at different stages.

Monitoring the ecology of Bacillus during Daqu incubation, a fermentation starter, using culture-dependent and culture-independent methods.

Daqu, a traditional fermentation starter, has been used to produce attractively flavored foods such as vinegar and Chinese liquor for thousands of years. Although Bacillus spp. are one of the dominant microorganisms in Daqu, more precise information is needed to reveal why and how Bacillus became dominant in Daqu, and next, to assess the impact of Bacillus sp. on Daqu and its derived products. We combined culture-dependent and culture-independent methods to study the ecology of Bacillus during Daqu incubation. Throughout the incubation, 67 presumptive Bacillus spp. isolates were obtained, 52 of which were confirmed by 16S rDNA sequencing. The identified organisms belonged to 8 Bacillus species: B. licheniformis, B. subtilis, B. amyloliquefaciens, B. cereus, B. circulans, B. megaterium, B. pumilus, and B. anthracis. A primer set specific for Bacillus and related genera was used in a selective PCR study, followed by a nested DGGE PCR targeting the V9 region of the 16S rDNA. Species identified from the PCR-DGGE fingerprints were related to B. licheniformis, B. subtilis, B. amyloliquefaciens, B. pumilus, B. benzoevorans, and B. foraminis. The predominant species was found to be B. licheniformis. Certain B. licheniformis strains exhibited potent antimicrobial activities. The greatest species diversity occurred at the Liangmei stage of Daqu incubation. To date, we lack sufficient knowledge of Bacillus distribution in Daqu. Elucidating the ecology of Bacillus during Daqu incubation would enable the impact of Bacillus on Daqu to be accessed, and the quality and stabilization of Daqu-derived products to be optimized.