Optimization of Clostridium beijerinckii semi-solid fermentation of rape straw to produce butyric acid by genome analysis.

Butyric acid is a volatile saturated monocarboxylic acid, which is widely used in the chemical, food, pharmaceutical, energy, and animal feed industries. This study focuses on producing butyric acid from pre-treated rape straw using simultaneous enzymatic hydrolysis semi-solid fermentation (SEHSF). Clostridium beijerinckii BRM001 screened from pit mud of Chinese nongxiangxing baijiu was used. The genome of C. beijerinckii BRM001 was sequenced and annotated. Using rape straw as the sole carbon source, fermentation optimization was carried out based on the genomic analysis of BRM001. The optimized butyric acid yield was as high as 13.86 ± 0.77 g/L, which was 2.1 times higher than that of the initial screening. Furthermore, under optimal conditions, non-sterile SEHSF was carried out, and the yield of butyric acid was 13.42 ± 0.83 g/L in a 2.5-L fermentor. This study provides a new approach for butyric acid production which eliminates the need for detoxification of straw hydrolysate and makes full use of the value of fermentation waste residue without secondary pollution, making the whole process greener and more economical, which has a certain industrial potential.

Strengthening the microbial community and flavor structure of jiupei by simulating strong-aroma baijiu fermentation with Bacillus velezensis DQA21.

BACKGROUND: Bacillus velezensis DQA21 is a functional strain used in the fermentation process of strong-aroma baijiu; however, its specific role in the process is still unclear. RESULTS: In this study, specific roles of B. velezensis DQA21 in the fermentation process were explored. Bioaugmentation of B. velezensis DQA21 increased the diversity and abundance of the bacterial community during the first 32 days of fermentation and significantly inhibited the diversity and richness of the fungal community during days 12 to 32. According to cluster analysis, changes in the microbial community structure were observed during fermentation, and the fermentation process could be divided into two stages: stage I, days 0-12; and stage II, days 12-45. Additionally, the microbial community structures during the two fermentation stages were significantly different. Co-occurrence analysis showed that bioaugmentation with Bacillus increased the correlation between microorganisms in jiupei and had a significant impact on the overall network structure of the microbial community. In addition, Bacillus significantly increased the production of flavor substances in jiupei, causing the total esters, total alcohols, and total acids contents to increase by 19.1%, 81.1%, and 25.9% respectively. CONCLUSION: The results suggested that bioaugmentation with B. velezensis DQA21 had a strong impact on the microbial community structure in strong-aroma baijiu, enhancing the volatile flavor components. Additionally, the work also provides a better understanding on the effect of augmentation on the microbial community in jiupei, which could help better regulation of solid-state fermentation in strong-aroma baijiu. © 2024 Society of Chemical Industry.

Genome Analysis and Optimization of Caproic Acid Production of Clostridium butyricum GD1-1 Isolated from the Pit Mud of Nongxiangxing Baijiu.

Caproic acid is a precursor substance for the synthesis of ethyl caproate, the main flavor substance of nongxiangxing baijiu liquor. In this study, Clostridium butyricum GD1-1, a strain with high caproic acid concentration (3.86 g/l), was isolated from the storage pit mud of nongxiangxing baijiu for sequencing and analysis. The strain's genome was 3,840,048 bp in length with 4,050 open reading frames. In addition, virulence factor annotation analysis showed C. butyricum GD1-1 to be safe at the genetic level. However, the annotation results using the Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server predicted a deficiency in the strain's synthesis of alanine, methionine, and biotin. These results were confirmed by essential nutrient factor validation experiments. Furthermore, the optimized medium conditions for caproic acid concentration by strain GD1-1 were (g/l): glucose 30, NaCl 5, yeast extract 10, peptone 10, beef paste 10, sodium acetate 11, L-cysteine 0.6, biotin 0.004, starch 2, and 2.0% ethanol. The optimized fermentation conditions for caproic acid production by C. butyricum GD1-1 on a single-factor basis were: 5% inoculum volume, 35°C, pH 7, and 90% loading volume. Under optimal conditions, the caproic acid concentration of strain GD1-1 reached 5.42 g/l, which was 1.40 times higher than the initial concentration. C. butyricum GD1-1 could be further used in caproic acid production, NXXB pit mud strengthening and maintenance, and artificial pit mud preparation.

Stochastic Processes Drive the Assembly and Metabolite Profiles of Keystone Taxa during Chinese Strong-Flavor Baijiu Fermentation.

Multispecies communities participate in the fermentation of Chinese strong-flavor Baijiu (CSFB), and the metabolic activity of the dominant and keystone taxa is key to the flavor quality of the final product. However, their roles in metabolic function and assembly processes are still not fully understood. Here, we identified the variations in the metabolic profiles of dominant and keystone taxa and characterized their community assembly using 16S rRNA and internal transcribed spacer (ITS) gene amplicon and metatranscriptome sequencing. We demonstrate that CSFB fermentations with distinct metabolic profiles display distinct microbial community compositions and microbial network complexities and stabilities. We then identified the dominant taxa (Limosilactobacillus fermentum, Kazachstania africana, Saccharomyces cerevisiae, and Pichia kudriavzevii) and the keystone ecological cluster (module 0, affiliated mainly with Thermoascus aurantiacus, Weissella confusa, and Aspergillus amstelodami) that cause changes in metabolic profiles. Moreover, we highlight that the alpha diversity of keystone taxa contributes to changes in metabolic profiles, whereas dominant taxa exert their influence on metabolic profiles by virtue of their relative abundance. Additionally, our results based on the normalized stochasticity ratio (NST) index and the neutral model revealed that stochastic and deterministic processes together shaped CSFB microbial community assemblies. Stochasticity and environmental selection structure the keystone and dominant taxa differently. This study provides new insights into understanding the relationships between microbial communities and their metabolic functions. IMPORTANCE From an ecological perspective, keystone taxa in microbial networks with high connectivity have crucial roles in community assembly and function. We used CSFB fermentation as a model system to study the ecological functions of dominant and keystone taxa at the metabolic level. We show that both dominant taxa (e.g., those taxa that have the highest relative abundances) and keystone taxa (e.g., those taxa with the most cooccurrences) affected the resulting flavor profiles. Moreover, our findings established that stochastic processes were dominant in shaping the communities of keystone taxa during CSFB fermentation. This result is striking as it suggests that although the controlled conditions in the fermentor can determine the dominant taxa, the uncontrolled rare keystone taxa in the microbial community can alter the resulting flavor profiles. This important insight is vital for the development of potential manipulation strategies to improve the quality of CSFB through the regulation of keystone species.

Unique Probiotic Properties and Bioactive Metabolites of Saccharomyces boulardii.

Saccharomyces boulardii (S. boulardii) is a probiotic and is widely used to improve the nutritional and functional value of food. This study aimed to compare the probiotic properties of S. boulardii and Saccharomyces cerevisiae. A series of in vitro probiotic experiments was performed, including simulated gastrointestinal digestion, bile salt tolerance, hydrophobicity, self-aggregation, and antioxidant and antibacterial properties. Self-aggregation and hydrophobic properties of S. boulardii were relatively poor, but they showed high tolerance, antioxidant properties, and broad antibacterial properties. In addition, non-targeted metabolomics was used to comprehensively analyze the active metabolites of S. boulardii and the metabolic differences between S. boulardii and S. cerevisiae were compared. Saccharomyces boulardii produced many bioactive metabolites, which generally showed antioxidant, antibacterial, antitumor, anti-inflammatory, and other properties. In contrast to S. cerevisiae, S. boulardii produced phenyllactic acid and 2-hydroxyisocaproic acid. There were also significant differences in their metabolic pathways. These results may be of great significance in the medical and food industries and provide a basis for understanding the metabolism of S. boulardii. It also shows that metabolomics is an effective and novel method for screening microbial functional metabolites and identifying functional differences between similar microorganisms.

Isolation, Identification, and Fermentation Medium Optimization of a Caproic Acid­Producing Enterococcus casseliflavus Strain from Pit Mud of Chinese Strong Flavor Baijiu Ecosystem.

Caproic acid is the precursor material of ethyl hexanoate, a representative flavor substance in strong flavor baijiu (SFB). Increasing the content of caproic acid in SFB helps to improve its quality. In the present study, caproic acid-producing bacteria from the pit mud of an SFB ecosystem were isolated, purified, and characterized. Strain BF-1 with the highest caproic acid yield (0.88 g/l) was selected. The morphological and molecular identification analysis showed that strain BF-1 was Enterococcus casseliflavus. The genome of E. casseliflavus BF-1 was sequenced and was found to be 2,968,377 bp in length with 3,270 open reading frames (ORFs). The caproic acid biosynthesis pathway in E. casseliflavus BF-1 was predicted based on the KAAS annotation. The virulence factors in the genome of strain BF-1 were annotated, which showed that E. casseliflavus BF-1 is safe at the genetic level. After adding essential nutrients based on the KAAS annotation, the optimum medium conditions for acid production by strain BF-1 were obtained by performing orthogonal experiments. The caproic acid yield of strain BF-1 reached 3.03 g/l, which was 3.44-fold higher than the initial yield. The optimized fer- mentation of caproic acid production by BF-1 was reported for the first time. The strain could be further used to regulate the ecosystem in baijiu production to improve its quality.

Wuliangye Baijiu but not ethanol reduces cardiovascular disease risks in a zebrafish thrombosis model.

Understanding how Baijiu facilitates blood circulation and prevents blood stasis is crucial for revealing the mechanism of Baijiu for cardiovascular disease (CVD) risk reduction. Here we established a zebrafish thrombosis model induced using arachidonic acid (AA) to quantitatively evaluate the antithrombotic effect of Wuliangye Baijiu. The prevention and reduction effects of aspirin, Wuliangye, and ethanol on thrombosis were compared using imaging and molecular characterization. Wuliangye Baijiu reduces thrombotic risks and oxidative stress in the AA-treated zebrafish, while ethanol with the same concentration has no similar effect. The prevention and reduction effects of Wuliangye on thrombosis are attributed to the change in the metabolic and signaling pathways related to platelet aggregation and adhesion, oxidative stress and inflammatory response.

Systematic analysis of Baobaoqu fermentation starter for Wuliangye Baijiu by the combination of metagenomics and metabolomics.

Baobaoqu (BBQ) is a traditional fermenting power, which is widely applied in Nong-flavor Baijiu brewing. There are two different types of BBQ (premium BBQ and normal BBQ) used in industrial manufacture, but the reasons for the significant differences between two kinds of BBQ have not been clearly illuminated. In this study, the combination of metagenomics and metabolomics was performed to compare the differences in the composition of microbial communities and the components of flavors between premium BBQ and normal BBQ. The results showed that the glycosidase-producing microorganisms are the biomarkers of premium BBQ, contributing a better ability of carbon source utilization than normal BBQ. In addition, several important flavors (ethyl hexanoate, phenylethanol, ethyl acetate) were rich in normal BBQ, which have a significant positive correlation with the biomarkers (Lactobacillus and Pichia kudriavzevii) of normal BBQ. It suggests that the microbial community has an advantage in utilizing raw materials in premium BBQ, while the community was inclined to form flavors in normal BBQ. The differences between two types of BBQ at the microbial and flavor level have theoretical and practical guiding significance in the application of premium and normal BBQ and in the further improvements of taste and quality of Baijiu.

Production of caproic acid by Rummeliibacillus suwonensis 3B-1 isolated from the pit mud of strong-flavor baijiu.

Caproic acid is the precursor of ethyl caproate, the main representative flavor substance of strong-flavor baijiu (SFB). Caproic acid-producing bacteria are considered to be the most important type of acid-producing microorganisms in the pit mud of the SFB ecosystem. In this study, the Rummeliibacillus suwonensis 3B-1 with a high yield of caproic acid (4.064 g/L) was screened from SFB pit mud. The genome of the R. suwonensis 3B-1 was sequenced, the total size was found to be 4117,671 bp and a calculated GC content of 35.86%. The caproic acid biosynthesis pathway was identified and analyzed, and it showed that 3B-1 could not only use ethanol, but it could also use glucose and other carbon sources as substrates to produce caproic acid. According to the genome analysis and with an optimized medium, the optimal conditions for caproic acid production were yeast powder at 3 g/L, sodium acetate at 15 g/L, and 1% biotin at 8 mL/100 mL. The yield of caproic acid reached 4.627 g/L, an increase of 13.9%, which was higher than that of general caproic acid bacteria. This is the first report of the synthesis of caproic acid by R. suwonensis. This strain could be used to produce caproic acid, an artificial pit mud preparation, and/or an enhanced inoculum in the production of SFB.

Moniliella aeria sp. nov., a novel yeast isolated from the air of a Wuliangye baijiu-making workshop.

During an investigation of the diversity of airborne yeasts in a famous Chinese baijiu fermentation workshop, two yeast strains were isolated from the air of the Wuliangye 501# baijiu-making workshop in Yibin, Sichuan Province, PR China and subjected to taxonomic analysis. The results of phylogenetic analysis of two regions of the rRNA gene cluster, the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, indicated that these strains represented a novel species of the genus Moniliella distinct from closely related species. The similarity between the novel species and the most closely related known species, Moniliella dehoogii, was 94.1 % for the D1/D2 LSU rRNA gene (30 substitutions and 12 indels out of 556 bp) and 95.6 % for the ITS region (9 substitutions and 6 indels out of 475 bp). In contrast with M. dehoogii, this novel species was able to assimilate d-ribose, l-arabinose, d-arabinose, cellobiose, d-glucono-1,5-lactone, dl-lactate, citrate, 1,2-propanediol, 2,3-butanediol and ethanol but was unable to ferment raffinose or assimilate inulin. On the basis of the results of phylogenetic analysis and the physiological characteristics, these investigated strains represent a novel species of the genus Moniliella, for which the name Moniliella aeria sp. nov. is proposed. Its holotype is CGMCC 20235T, and the MycoBank number is MB840188.

Effects of environmental factors on the microbial community changes during medium-high temperature Daqu manufacturing.

Environmental factors and microbial interaction influence the changes in microbial community and enzyme profiles during different stages of medium-high temperature Daqu manufacturing. In this study, the specific changes in microbial community and environmental factors at various stages of Daqu production were comprehensively analyzed using a combination of environmental factor analysis and high-throughput sequencing technology. Further, the effects of environmental factors and microbial interaction on microbial succession were investigated via correlation analysis; the enzymes related to saccharification and ethanol fermentation during Daqu production were explored. The study revealed that the whole process of Daqu production was divided into three stages based on temperature. Fungal were active in the early stage, while bacteria were more active in the middle to later stages. Bacillus, Weissella, Thermoactinomyces and Lactobacillus were the main bacterial genera, while Thermoascus, Thermomyces, Kodamaea, and Aspergillus were the dominant fungi. The effects of ambient humidity, CO2. and moisture on the microbial community were significant, and these environmental factors positively correlated with most fungi, but negatively correlated with Thermoactinomyces, Saccharopolyspora, and Acinetobacter. In addition, the enzymes encoded by fungi, related to saccharification and ethanol fermentation, might be consistent with the high abundance of Thermoascus and Thermomyces. Meanwhile, enzymes encoded by bacteria could mainly originate from Weissella.

Caproicibacterium lactatifermentans sp. nov., isolated from pit clay used for the production of Chinese strong aroma-type liquor.

Two recently reported bacterial strains that were identified as the dominant caproate-producing bacteria in pit clay, were further characterized to determine their phylogeny and taxonomy. The two strains, designated as LBM19010T and JNU-WLY1368, were short rod-shaped, Gram-stain-positive, non-motile and strictly anaerobic. Analysis of the 16S rRNA gene sequences revealed that strains LBM19010T and JNU-WLY1368 shared a 16S rRNA gene sequence similarity of 99.93 % and belonged to a recent proposed genus Caproicibacterium in the family Oscillospiraceae. The proposed type strain, LBM19010T, showed the highest 16S rRNA gene sequence similarity to Caproicibacterium amylolyticum LBM18003T (96.34%), followed by Caproiciproducens galactitolivorans JCM 30532T (94.14 %). The pairwise average nucleotide identity and average amino acid identity values between strains LBM19010T and LBM18003T were 74.84 and 76.18 %, respectively. Growth of strain LBM19010T occurred at pH 4.5-7.5 (optimum, pH 5.0-5.5), 20-40 °C (optimum, 35 °C) and with 0-1 % (w/v) NaCl (optimum, 0 %). Strains LBM19010T and JNU-WLY1368 were both able to ferment several hexoses, disaccharides, starch and lactate but not pentoses. Caproate and butyrate were the major end-products from glucose. The predominant cellular fatty acids (>10 %) of strain LBM19010T were C16 : 0 (56.3 %), C14 : 0 DMA (19.5 %) and C14 : 0 (14.9 %). The identified polar lipids of strain LBM19010T were diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids and nine unidentified glycolipids. Based on phylogenetic, phenotypic and chemotaxonomic evidence, strains LBM19010T and JNU-WLY1368 belong to a novel species of the genus Caproicibacterium, for which the name Caproicibacterium lactatifermentans sp. nov. is proposed. The type strain is LBM19010T (=GDMCC 1.1627T=JCM 33782T).

Environmental factors and interactions among microorganisms drive microbial community succession during fermentation of Nongxiangxing daqu.

Nongxiangxing daqu (daqu), which is produced by the open fermentation of wheat, is the fermentation starter of baijiu. This work reports the occurrence and driving factors of microbial community succession (MCS), which determines daqu quality, during daqu fermentation. The co-occurrence networks of the 2 stages of the MCS showed that module 2 of stage P1 contained Mucoromycota and Actinobacteriota and was affected by temperature, humidity, CO2, and moisture; module 8 of stage P2 contained Mucoromycota and Saccharomycetes and was influenced by acidity and moisture. Twenty-two genera were thebiomarkers of the MCS. The MCS was driven by temperature, humidity, CO2, O2, acidity,moisture and interactions between the biomarkes and Lactobacillales, Saccharomycetales, and Acetobacter. The main driving factors of the bacterial community succession were acidity, moisture, and temperature, and that of the fungal community succession was moisture. These results guide the control of MCS during daqu fermentation.

Proteiniphilum propionicum sp. nov., a novel member of the phylum Bacteroidota isolated from pit clay used to produce Chinese liquor.

A novel, Gram-stain-negative, rod-shaped, strictly anaerobic bacterium of genus Proteiniphilum of the phylum Bacteroidota, named strain JNU-WLY501T, was isolated from pit clay used to produce strong aroma-type liquor in PR China. The genomic DNA G+C content and genome size of JNU-WLY501T were 41.4 % and 3.9 Mbp, respectively. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that JNU-WLY501T was closely related to Proteiniphilum acetatigenes DSM 18083T (95.7 %) and Proteiniphilum saccharofermentans M3/6T (94.9 %). The pairwise average nucleotide identity based on blast and average amino acid identity values of JNU-WLY501T compared with Proteiniphilum saccharofermentans M3/6T were 73.6 and 77.3 %, respectively, which both were lower than the threshold values for bacterial species delineation. The strain grew at 20-40 °C, with optimum growth at 37 °C. The pH range for growth was 5.4-9.1, with optimum growth at pH 7.5. The sodium chloride range for growth was 0.0-4.0 %, with optimum growth at 0 %. The strain did not use glucose, maltose, fructose or starch. Yeast extract, tryptone and peptone supported the growth of JNU-WLY501T, and the main fermentation products were acetate and propionate. The predominant cellular fatty acids (>5 %) of JNU-WLY501T were anteiso-C15 : 0 (30.6 %), anteiso-C17 : 0 (26.1 %), C16 : 0 (7.7 %), iso-C16 : 0 (5.0 %) and iso-C17 : 0 (5.0 %). The respiratory quinone of JNU-WLY501T was MK-5. On the basis of the morphological, physiological, biochemical, chemotaxonomic, genotypic and phylogenetic results, JNU-WLY501T represents a novel species of the genus Proteiniphilum, for which the name Proteiniphilum propionicum sp. nov. is proposed. The type strain is JNU-WLY501T (=GDMCC 1.2686T=JCM 34753T).

Tandem Solid-Phase Extraction Columns for Simultaneous Aroma Extraction and Fractionation of Wuliangye and Other Baijiu.

Wuliangye baijiu is one of the most famous baijiu in China, with a rich, harmonic aroma profile highly appreciated by consumers. Thousands of volatiles have been identified for the unique aroma profile. Among them, fatty acid esters have been identified as the main contributors to the aroma profile. In addition, many non-ester minor compounds, many of which are more polar than the esters, have been identified to contribute to the characteristic aroma unique to Wuliangye baijiu. The analysis of these minor compounds has been challenging due to the dominance of esters in the sample. Thus, it is desirable to fractionate the aroma extract into subgroups based on functional group or polarity to simplify the analysis. This study attempts a new approach to achieve simultaneous volatile extraction and fractionation using tandem LiChrolut EN and silica gel solid-phase extraction (SPE) columns. A baijiu sample (10 mL, diluted in 40 mL of water) was first passed through the LiChrolut EN (1.0 g) column. The loaded LiChrolut EN column was then dried with air and coupled with a silica gel (5.0 g) SPE column with anhydrous Na2SO4 (10.0 g) in between. The volatile compounds were eluted from the LiChrolut EN column and simultaneously fractionated on the silica gel column based on polarity. The simultaneous extraction and fractionation technique enabled the fractionations of all fatty acid esters into less polar fractions. Fatty acids, alcohols, pyrazines, furans, phenols, hydroxy esters, and other polar compounds were collected in more polar fractions. This technique was used to study the volatile compounds in Wuliangye, Moutai, and Fengjiu baijiu. In addition to fatty acid esters, many minor polar compounds, including 2,6-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, p-cresol, and 2-acetylpyrrole, were unequivocally identified in the samples. The procedure is fast and straightforward, with low solvent consumption.

Adaptability of a Caproate-Producing Bacterium Contributes to Its Dominance in an Anaerobic Fermentation System.

The transformation of diverse feedstocks into medium-chain fatty acids (MCFAs) by mixed cultures is a promising biorefinery route because of the high value of MCFAs. A particular concern is how to maintain the microbial consortia in mixed cultures to achieve stable MCFA production. The Chinese strong aroma-type liquor (Baijiu) fermentation system continually produces caproic acid for decades through a spontaneous inoculation of anaerobes from pit mud into fermented grains. Therefore, illuminating the dominant caproate-producing bacterium (CPB) in pit mud and how the CPB is sustained in the spontaneous fermentation system will help to reveal the microbiological mechanisms of stable caproate production. Here, we examined pit mud samples across four Chinese strong aroma-type Baijiu-producing areas and found that a caproate-producing Caproicibacterium sp. was widely distributed in these distilleries, with relative abundance ranging from 1.4 to 35.5% and an average abundance of 11.4%. Through controlling carbon source availability, we obtained different simplified caproate-producing consortia and found that the growth advantage of Caproicibacterium sp. was highly dependent on glucose. Then, two strains, named Caproicibacterium sp. strain LBM19010 and Caproicibacterium sp. strain JNU-WLY1368, were isolated from pit mud of two regions. The metabolic versatility of this species utilizing starch, maltose, glucose, and lactate reflected its adaptability to the fermentation environment where these carbon sources coexist. The simultaneous utilization of glucose and lactate contributed to the balance between cell growth and pH homeostasis. This study reveals that multiple adaptation strategies employed by the predominant CPB promotes its stability and dominance in a saccharide- and lactate-rich anaerobic habitat. IMPORTANCE The Chinese strong aroma-type liquor (Baijiu) fermentation environment is a typical medium-chain fatty acid-producing system with complex nutrients. Although several studies have revealed the correlation between microbial community composition and abiotic factors, the adaptation mechanisms of dominant species to abiotic environment are still unknown in this special anaerobic habitat. This study identified the predominant CPB in Chinese strong aroma-type Baijiu fermentation system. Metabolic versatility and flexibility of the dominant CPB with a small-size genome indicated that this bacterium can effectively exploit available carbon and nitrogen sources, which could be a key factor to promote its ecological success in a multispecies environment. The understanding of growth and metabolic features of the CPB responsible for its dominance in microbial community will not only contribute to the improvement of Chinese strong aroma-type Baijiu production but also expand its potential industrial applications in caproate production.

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.

Bacillus aquiflavi sp. nov., isolated from yellow water of strongly flavored Chinese baijiu.

A Gram-stain-positive, strictly aerobic and rod-shaped bacterium, designated as 3 H-10T, was isolated from a yellow water sample collected from the manufacturing process of strong flavor Chinese baijiu in Yibin region of Sichuan province (PR China). Oval endospores were formed at the subtermini of cells with swollen sporangia. The isolate was able to grow at temperatures of 20-45 °C (optimum growth at 37 °C), at pH 6.0-10.0 (optimum growth at pH 8.0) and in the presence of 0-2 % (w/v) NaCl (optimum growth with 0 % NaCl). Ribose was the major cell-wall sugar, and meso-diaminopimelic acid (meso-DAP) was the diagnostic amino acid. The main polar lipids of 3 H-10T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). MK-7 was predominant menaquinone and iso-C15 : 0 (60.7 %) was the major fatty acid. Comparisons of 16S rRNA gene sequence indicated that 3 H-10T was most closely related to Bacillus mesophilus SA4T (96.30 %), Bacillus ginsengihumi Gsoil 114T (96.27 %) and Bacillus shackletonii LMG 18435T (96.27 %). The average nucleotide identity (ANI) values between strain 3 H-10T and the three type strains mentioned above were 69.56, 70.19 and 70.67 %, respectively. The genomic DNA G+C content was 35.4 mol%. On the basis of its phenotypic, chemotaxonomic and phylogenetic properties, strain 3 H-10T represents a novel species of the genus Bacillus, for which the name Bacillus aquiflavi sp. nov. is proposed. The type strain is Bacillus aquiflavi 3 H-10T (=CICC 24755T=JCM 33703T).

Assessment of ß-glucans, phenols, flavor and volatile profiles of hulless barley wine originating from highland areas of China.

Low alcohol hulless barley wine (HW) is a popular beverage among the highland areas in China. It is known to have several health benefits due to the presence of ß-glucan and antioxidant compounds. Therefore, the total ß-glucan content, total phenols and flavonoids of HW samples from the highland areas of Sichuan province and Tibet were determined in this study. The results indicated that HW is abundant in both ß-glucan (54-76 mg/L) and phenolic compounds (131-178 mg/L). Moreover, this study also investigated the flavor and aroma characteristics of HW samples. A total of forty six volatile aroma substances were identified by GC-MS. The HWs could be classified into three distinct groups in terms of the region of origin according to the results of PCA based on the GC-MS data. These findings provide a useful foundation for further study of the health benefits and the flavor characteristics of HW in highland areas.