Analysis of the Bacterial Community in Zaopei During Production of Chinese Luzhou‐flavor Liquor

Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene analysis were carried out to analyze the bacterial community in Zaopei during production of Chinese Luzhou‐flavor liquor. Primers PRBA338F and PRUN518R were used for DGGE. Polymerase chain reaction (PCR) for clone analysis was preformed with primers EU27F and 1490R. The results by DGGE showed that with increasing fermentation time the diversity of bacteria in Zaopei decreased and after one week, only one bacterium phylotype was dominant. Gene clone libraries (16S rRNA) containing 55 clonal sequences were constructed. The bacterial diversity shift observed by DGGE was also shown by the clone library analysis. Bacteria closely related to Lactobacillus acetotolerans appeared to play a key role during Chinese liquor fermentation.     

Monitoring of the prokaryotic diversity in pit mud from a Luzhou‐flavour liquor distillery and evaluation of two predominant archaea using qPCR assays

Pit mud plays a crucial role in the production of the Luzhou‐flavour liquor. Its quality directly determines liquor quality and yield. The aged pit mud produced good quality liquor but aging pit mud did not. The aim of this work was to investigate the prokaryotic diversity of the aged and aging pit mud from a Luzhou‐flavour liquor distillery using molecular methods. Two bacteria‐specific and two archaea‐specific 16S rRNA gene clone libraries were constructed and analysed using amplified ribosomal DNA restriction analysis. A total of 273 clones were studied, which resulted in 28 operational taxonomic units. Bacteroidetes and Firmicutes predominated in both the aged and aging pit mud, but Synergistetes and Actinobacteria were only detected in the aged pit mud. The family Methanosaeta dominated in the aged pit mud, while the Methanosarcina predominated in the aging pit mud. These results were confirmed using two genus‐specific quantitative real time PCR assays. This research distinguished microbial community structure in the aged and aging pit mud for the first time, and has laid an initial foundation for identifying good quality pit mud and for maintaining the quality of pit mud. Copyright © 2014 The Institute of Brewing & Distilling     

Comparison of bacterial community in matured and degenerated pit mud from Chinese Luzhou‐flavour liquor distillery in different regions

The bacterial community in the pit mud of a Luzhou‐flavour liquor distillery in different regions was analysed by combined polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative PCR (qPCR) in order to distinguish a matured and a degenerated pit mud, judged according to sensory and physicochemical characteristics. The phyla Firmicutes, Cloacimonetes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected. Firmicutes predominated in the pit mud. The diversity and homogeneity of the bacterial community in the matured pit mud were superior to those in the degenerated pit mud in the same distillery. There were significant differences in the bacterial community structure between the matured and degenerated pit mud. Moreover, the bacterial community in the degenerated pit mud samples was similar, which indicated that the bacterial community in the degenerated pit mud did not change within the two different regions. However, the bacterial community in matured pit mud samples was different, demonstrating that there were visible differences in the bacterial community between the samples of matured pit mud collected from the Luzhou‐flavour liquor distilleries in the two different regions. Notably, the quantity of Actinobacteria in the matured and the degenerated pit mud was found to be different by quantitative analysis. Potentially, the Actinobacteria could serve as an indicator bacteria to distinguish between matured and degenerated pit muds. Copyright © 2016 The Institute of Brewing & Distilling     

Spatial Distribution of Bacterial Communities and Related Biochemical Properties in Luzhou‐Flavor Liquor‐Fermented Grains

Grain fermenting with separate layers in a fermentation pit is the typical and experiential brewing technology for Chinese Luzhou‐flavor liquor. However, it is still unclear to what extent the bacterial communities in the different layers of fermented grains (FG) effects the liquor's quality. In this study, the spatial distributions of bacterial communities in Luzhou‐flavor liquor FG (top, middle, and bottom layers) from 2 distinctive factories (Jiannanchun and Fenggu) were investigated using culture‐independent approaches (phospholipid fatty acid [PLFA] and polymerase chain reaction–denaturing gel electrophoresis [DGGE]). The relationship between bacterial community and biochemical properties was also assessed by Canonical correspondence analysis (CCA). No significant variation in moisture was observed in spatial samples, and the highest content of acidity and total ester was detected in the bottom layer (P < 0.05). A high level of ethanol was observed in the top and middle layers of Fenggu and Jiannanchun, respectively. Significant spatial distribution of the total PLFA was only shown in the 50‐y‐old pits (P < 0.05), and Gram negative bacteria was the prominent community. Bacterial 16S rDNA DGGE analysis revealed that the most abundant bacterial community was in the top layers of the FG both from Fenggu and Jiannanchun, with Lactobacillaceae accounting for 30% of the total DGGE bands and Lactobacillus acetotolerans was the dominant species. FG samples from the same pit had a highly similar bacterial community structure according to the hierarchal cluster tree. CCA suggested that the moisture, acidity, ethanol, and reducing sugar were the main factors affecting the distribution of L. acetotolerans. Our results will facilitate the knowledge about the spatial distribution of bacterial communities and the relationship with their living environment.     

Combining culture‐dependent and culture‐independent molecular methods for the isolation and purification of a potentially novel anaerobic species from pit mud in a Chinese liquor distillery

Strain S12–27–1‐3‐5 (a potentially novel anaerobic species) with a 16S rRNA sequence homology of <97% was isolated and purified from pit mud by combining culture‐dependent and culture‐independent molecular methods, such as cloning of 16S rRNA, amplified rRNA restriction analysis, and denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the 16S rRNA gene indicated that strain S12–27–1‐3‐5 was related to Aminobacterium mobile strain ILE‐3 DSM 12262^T and Aminobacterium colombiense strain DSM 12261^T (95 and 96% similarity value, respectively). The results verified that cloning of the 16S rRNA was efficient to identify whether a potentially new bacterial taxon existed in impure isolates and that the DGGE method was a powerful tool for screening the target bacteria and for identifying duplicate strains. Therefore, the application of the culture‐independent molecular methods for the isolation and purification of a potentially novel species was effective. Strain S12–27–1‐3‐5 (= DSM 27871 = JCM 19605 = CICC 10731^T) was an anaerobic amino acid‐degrading bacterium. The results of fermentation experiments demonstrated that strain S12–27–1‐3‐5 produced volatile fatty acids (VFAs) and the presence of Methanosarcina barkeri enhanced the generation of VFAs, which contribute to the aroma composition of Chinese liquor. This work could enrich the species resources and promote the development and utilization of an uncultured species. Copyright © 2016 The Institute of Brewing & Distilling     

Changes in Volatile Compounds of Chinese Luzhou‐Flavor Liquor during the Fermentation and Distillation Process

The aim of this study was to investigate the dynamic of volatile compounds in the Zaopei during the fermentation and distillation process by headspace solid‐phase microextraction‐gas chromatography mass spectrometry (HS‐SPME‐GCMS). Physicochemical properties analysis of Zaopei (fermented grains [FG], fermented grains mixed with sorghum [FGS], streamed grains [SG], and streamed grains mixed with Daqu [SGD]) showed distinct changes. A total number of 66 volatile compounds in the Zaopei were identified, in which butanoic acid, hexanoic acid, ethyl hexanoate, ethyl lactate, ethyl octanoate, hexyl hexanoate, ethyl hydrocinnamate, ethyl oleate, ethyl hexadecanoate, and ethyl linoleate were considered to be the dominant compounds due to their high concentrations. FG had the highest volatile compounds (112.43 mg/kg), which significantly decreased by 17.05% in the FGS, 67.12% in the SG, and 73.75% in the SGD. Furthermore, about 61.49% of volatile compounds of FGS were evaporated into raw liquor, whereas head, heart, and tail liquor accounted for 29.84%, 39.49%, and 30.67%, respectively. Each volatile class generally presented a decreasing trend, except for furans. Especially, the percentage of esters was 55.51% to 67.41% in the Zaopei, and reached 92.60% to 97.67% in the raw liquor. Principal component analysis based ordination of volatile compounds data segregated FGS and SGD samples. In addition, radar diagrams of the odor activity values suggested that intense flavor of fruit was weakened most from FG to SGD.     

Characterization of the microbial community in three types of fermentation starters used for Chinese liquor production

This study examined and compared the microbial community in three typical fermentation starters (called as Daqu, Xiaoqu, and Fuqu in China) used for liquor production by analysing the 16S and 18S rRNA gene clone library. The results show that the microbial diversity in the three types of fermentation starters (JiuQu) differs significantly. The bacterial species in Daqu and Fuqu were mainly thermophilic or thermotolerant. In Daqu, the dominant bacterial species were Thermoactinomyces sanguinis (53.85%) and Pantoea agglomerans (19.23%), followed by uncultured bacteria (15.39%). The lactic acid bacterium Weissella cibaria (50%) and a member of Enterobacteriaceae, Enterobacter ludwigii (10%), were the dominant bacterial species in Xiaoqu. Low abundances of other bacteria, including Deinococcus radiodurans, Corynebacterium variabile and Acinetobacter baumannii, were reported for Xiaoqu. Enterococcus faecium, Clostridium beijerinckii and Bacillus cereus were observed in Fuqu and accounted for 46.67, 23.33 and 16.67% of the total bacteria identified, respectively. Fungal diversity was high in Daqu and consisted exclusively of thermophilic moulds, such as Aspergillus glaucus (62.5%), Thermomyces lanuginosus (12.5%) and Thermoascus crustaceus (12.5%). Only two fungal species were reported for Fuqu and Xiaoqu and both contained the mould Rhizopus oryzae. Saccharomyces cerevisiae and the non‐Saccharomyces yeast (Saccharomycopsis fibuligera) were also identified in Fuqu and Xiaoqu, respectively. This finding suggests that microbial community structure in JiuQu starters is the key factor to determine the variety of flavours. Copyright © 2015 The Institute of Brewing & Distilling     

Microbial succession and metabolite changes during the fermentation of Chinese light aroma‐style liquor

A combination of culture‐dependent and culture‐independent methods and SPME–GC–MS were used to monitor changes of bacterial and yeast communities, and flavour compounds during the fermentation process of Chinese light aroma‐style liquor. Bacillus and Lactobacillus were the main bacterial genera. Pichia anomala, Saccharomyces cerevisiae and Issatchenkia orientalis were the dominant yeast species. There was a close relationship between fermentation time and the shift of microbial community. Compared with the microbiota in the fermentation of other style liquors, higher bacterial diversity and different non‐Saccharomyces composition led to a variety of metabolites. Metabolite analysis showed that esters, acids, alcohols, aromatic compounds and phenols were the main flavour components and most of them were synthesised in the latter phase of fermentation. Principal component analysis further demonstrated that Bacillus and yeast were the most influential microorganisms in the first 10 days of fermentation, and lactic acid bacteria predominated in the later phase. Lactic acid bacteria regulated the composition of other bacteria and yeast, and synthesised flavour compounds to affect the organoleptic properties of liquor. S. cerevisiae and P. anomala were two important yeast species responsible for the characteristic aroma of liquor. These results present a comprehensive understanding of microbial interaction and potential starter cultures to produce desirable liquor quality. © 2018 The Institute of Brewing & Distilling     

PCR‐DGGE analysis on microbial communities in pit mud of cellars used for different periods of time

Polymerase chain reaction denaturing gradient gel electrophoresis (PCR‐DGGE) was used to analyse microbial community evolution in the pit mud of cellars used for different periods of time in production of Chinese Luzhou‐flavour liquor. The pit mud was collected from the cellars and the microbial DNA was extracted from the microbes in the pit mud. The Bf 968 primer was used for PCR‐DGGE to analyse the variable region 6 (V6) to variable region 8 (V8) of the microbial 16S rDNA. It was found that the band number, dominance, diversity and similarity of the 16S rDNA were clearly different in the DGGE patterns, because of the great diversity expressed by the different microbial communities in the different‐aged cellars. It is concluded that mutual collaboration and constraint exist between the different microbial communities in the different‐aged cellars, and this relationship leads to an evolutional change in the structures and in the numbers of the microbial communities in the pit mud of the cellars. Changes become more obvious with increasing age of the liquor cellars. Copyright © 2012 The Institute of Brewing & Distilling     

Microbial Community Composition Associated with Maotai Liquor Fermentation

The solid‐state fermentation state of Chinese Maotai liquor involves the interaction of several complex microbial communities leading to the generation of the most complex liquor fermentation system in the world and contributes to the unique flavor and aroma of the liquor. In this study, total DNA was extracted from 3 fermented grain samples (FG1, FG2, and FG3) and 12 environmental samples, including Daqu (DA1, DA2, DA3, and DA4), cellar mud (CS1, CS2, and CS3), soil (SL1 and SL2), air (A1 and A2), and sorghum (SH), and the 16S and 18S rRNA genes were amplified. The distribution of typical microorganisms in the samples was analyzed using nested PCR‐denaturing gradient gel electrophoresis, while quantitative PCR amplification of 16S rRNA and internal transcribed spacer genes was performed to estimate the microbial abundance present in each sample. The results indicated that Daqu was the primary source of bacteria, followed by the air, soil, and sorghum samples, while the majority of the fungi responsible for Maotai liquor fermentation were from Daqu and sorghum. Highest bacterial concentrations were found in fermented grains, followed by Daqu and sorghum, while the highest fungal concentrations were found in Daqu, followed by sorghum and an air sample from outside the liquor production area. The findings of this study may provide information regarding the mechanisms responsible for flavor development in Maotai liquor, and may be used to further optimize the traditional art of making liquor.     

Molecular Identification of Microbial Community in Surface and Undersurface Douchi During Postfermentation

To find the reason for fermentation failure of surface Douchi during postfermentation, the microbial communities in undersurface and surface samples were investigated using cell counting method and denaturing gradient gel electrophoresis (DGGE). The results showed that the microbial biomass in surface Douchi was obviously different from that in undersurface Douchi even sampled from the same fermentation tanks, and a 10‐ to 100‐fold reduction of microbial cell counts in undersurface had been observed. The bacterial DGGE profile and principal component analysis (PCA) results indicated that only Lactococcus lacts subsp. lactis and Bacillus thermoamylovorans were detected from surface Douchi, while Lactococcus lacts subsp. lactis, Staphylococcus lentus and 2 uncultured strains occupied the dominant positions in undersurface Douchi; when amplified using Bacillus‐specific primers, Bacillus thermoamylovorans, Bacillus subtilis, and Enterobacter sp. were found in undersurface Douchi, while only Bacillus thermoamylovorans were detected from surface Douchi; compared to the bacteria and Bacillus, the DGGE profiles and PCA plot of fungi indicated that the fungal community between surface and undersurface Douchi was similar and mainly composed by yeasts. In this study, we detected the microbial biomass and species in postfermentation stage of Douchi, and the various microbial diversity in undersurface and surface samples might be the cause of the fermentation failure in surface fermentation tanks.     

Bacterial and fungal diversity in the traditional Chinese liquor fermentation process

This study endeavored to investigate the variability of bacteria and fungi present during the fermentation process of the light-fragranced distilled liquor known as Fen liquor. To accomplish this, we used a combination of clone libraries of 16S rRNA genes, bar-coded pyrosequencing of the internal transcribed spacer region 1 (ITS1), and quantitative real-time PCR (qPCR). Fifteen families of bacteria and six families of fungi were detected. More than 91% of 16S rRNA gene sequences could be assigned to the family Lactobacillaceae, which were then classified to eight different operational taxonomic units (OTUs), based on a 3% cut-off. The most abundant OTU which contributed to 51% of the total 16S rRNA gene sequences was affiliated with Lactobacillus acetotolerans and had a significantly similar variation trend with the chemical constituents detected. Sixty percent of the fungal ITS1 region sequences were affiliated with the family Saccharomycetaceae. The most abundant OTU was very similar to Issatchenkia orientalis, which displayed notable similarities with respect to the change trends in both ethanol and organic acid contents. The sequences of the second most abundant OTU were closest to Saccharomyces cerevisiae, an important species in the process of ethanol production. Furthermore, about one fourth of the ITS1 region sequences belonged to the family Saccharomycopsidaceae. Conversely, very few sequences could be grouped together with filamentous fungi. The results of qPCR showed that the content of bacteria was increased while that of fungi was more stable in the fermentation process. It is very important to simultaneously investigate bacterial and fungal variations in food-fermentation processes.     

Predominant Bacteria Diversity in Chinese Traditional Sourdough

The purpose of this study was to identify the major bacteria in Chinese traditional sourdough (CTS). Five CTS samples (Hn‐87, Sx‐91, Gs‐107, Hf‐112, and Hr‐122) were collected from different Chinese steamed breads shops or private households. The total bacterial DNA was extracted from sourdough samples and sequenced using Illumina Hiseq 2000 system. Illumina tags were assigned to BLASTN server based on 16S rRNA libraries to reveal a genetic profile. Phylogenetic analysis revealed that the bacteria in traditional sourdough samples were dominated by the genera Leuconostoc and Lactobacillus. Beta diversity analysis, principal component analysis, and cluster analysis compared the bacterial differences in traditional sourdough samples. The results showed that Leuconostoc, Lactobacillus, and Weissella were the predominant genera among the 5 samples. This differentiated the sourdoughs into 3 typologies, namely, 1) Gs‐107 and Sx‐91, 2) Hr‐122 and Hn‐87, and 3) Hf‐112. This study identified 3 unique major bacteria genus in CTS bread ecosystems.     

Monitoring the microbial community during solid-state acetic acid fermentation of Zhenjiang aromatic vinegar

Zhenjiang aromatic vinegar is one of the most famous Chinese traditional vinegars. In this study, change of the microbial community during its fermentation process was investigated. DGGE results showed that microbial community was comparatively stable, and the diversity has a disciplinary series of changes during the fermentation process. It was suggested that domestication of microbes and unique cycle-inoculation style used in the fermentation of Zhenjiang aromatic vinegar were responsible for comparatively stable of the microbial community. Furthermore, two clone libraries were constructed. The results showed that bacteria presented in the fermentation belonged to genus Lactobacillus, Acetobacter, Gluconacetobacter, Staphylococcus, Enterobacter, Pseudomonas, Flavobacterium and Sinorhizobium, while the fungi were genus Saccharomyces. DGGE combined with clone library analysis was an effective and credible technique for analyzing the microbial community during the fermentation process of Zhenjiang aromatic vinegar. Real-time PCR results suggested that the biomass showed a “system microbes self-domestication” process in the first 5 days, then reached a higher level at the 7th day before gradually decreasing until the fermentation ended at the 20th day. This is the first report to study the changes of microbial community during fermentation process of Chinese traditional solid-state fermentation of vinegar.     

A Phylogenetic Analysis of Saccharomyces Species by the Sequence of 18S–28S rRNA Spacer Regions

Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces. The sequences of S. bayanus and S. pastorianus were quite similar, but not identical. Two phylogenetic trees constructed by the neighbor-joining method showed that all the species examined were distinguished from one another. The Saccharomyces sensu stricto species: S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus, were closely related and far from the Saccharomyces sensu lato species including S. barnetti, S. castellii, S. dairensis, S. exiguus, S. servazzii, S. spencerorum and S. unisporus, and an outlying species, S. kluyveri. © 1997 John Wiley & Sons, Ltd.     

Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must

The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.     

Identification of microorganisms producing lactic acid during solid‐state fermentation of Maotai flavour liquor

Lactic acid is the main acid produced during the Maotai liquor brewing process, influencing the quality of the base liquor and fermentation process. However, the microorganisms responsible for lactic acid production have not been identified. In this work, the dynamic changes in bacterial community structure in the Zaosha round (second sorghum feeding and fermentation) of the brewing process were analysed by 16S rRNA high‐throughput sequencing. Results show that lactic acid bacteria (LAB) and Bacillus spp. are the dominant bacteria in the brewing process, where Bacillus spp. are found in the early stage, whilst LAB are found throughout the brewing process. Furthermore, 10 types of LAB and five Bacillus spp. were isolated from Zaopei (a mixture of fermented grains including sorghum and wheat) by a culture‐dependent method. Lactobacillus panis accounts for 68% of the LAB, and Bacillus amyloliquefaciens for 54% of Bacillus spp. Solid‐state fermentation experiments were performed with L. panis and B. amyloliquefaciens and lactic acid production was consistent with the accumulation of lactic acid in Zaosha. The results showed that L. panis was the main producer of lactic acid in pits, while B. amyloliquefaciens plays an important role in the production of lactic acid in the early stages of fermentation. The approach used in this study may facilitate the identification of key microorganisms with specific functionality involved in other food and beverage fermentation processes. © 2018 The Institute of Brewing & Distilling     

Developing New Sacchariferous Starters for Liquor Production Based on Functional Strains Isolated from the Pits of Several Famous Luzhou‐flavor Liquor Brewers

Two new sacchariferous starters were developed based on functional strains isolated from the pits of several famous Chinese Luzhou‐flavor liquor brewers and a genetically engineered mold strain previously constructed in this laboratory. Compared with traditional Daqu, the new starters, especially with the genetically engineered strain, possessed higher abilities of saccharification and fermentation as well as a higher alcohol yield ratio and percent conversion. Applying these new starters in liquor production has advantages that include easy preparation, convenient operation and time‐ savings and thus can greatly elevate the efficiency of liquor production. The taste and flavor of the liquors obtained with the new starters were comparable to those produced with traditional Daqu. This study further contributes to the investigation of the mechanisms of traditional liquor production and the improvement of process control. It also demonstrates the possibility of producing high quality traditional or new‐style liquors in different locations.     

Bacterial Community of Koumiss from Mongolia Investigated by Culture and Culture-Independent Methods

The bacterial community of fermented horse milk (koumiss) from Mongolia was studied using three methods: cultivation, direct identification by 16S rRNA clone library and denaturing gradient gel electrophoresis (DGGE). Ninety-eight strains were isolated by traditional cultivation and 61 of those were randomly selected for further identification by 16S rRNA gene sequencing. The strains were dominated by lactic acid bacteria (LAB; six different lactobacilli), Acinetobacter, Bacillus and Psychrobacter. Construction of the clone library analysis revealed that 16S sequences of 220 clones, genus Lactobacillus was dominant, but Streptococcus thermophilus, Acetobacter pasteurianus and uncultured clones were also detected. Ten unique bands were sequenced from the DGGE and revealed: Lactococcus lactis, Lactococcus lactis subsp. lactis, Clostridium acidurici, Acinetobacter johnsonii, Dickeya sp., Enterobacter sp., Pseudomonas sp., Raoultella sp., and Ruminococcus sp.. In vitro growth inhibition of three human pathogens, Escherichia coli, Enterobacter sakazakii and Staphylococcus aureus by 14 culturable bacteria displayed that only three of the isolates tested inhibit growth of E. sakazakii while most of the other bacteria delayed growth of the target bacteria.     

Optimization of Calvatia gigantea mycelia production from distillery wastewater

The focus of wastewater management has evolved from treatment technology into resource recovery, which enables one to minimize contaminants and to generate value‐added products. Calvatia gigantea is used not only as a source of food, but has also been used as a traditional Chinese medicine for thousands of years. In this study, the mycelial production of C. gigantea was studied under submerged fermentation conditions using non‐pretreated distilled wastewater from Chinese liquor production. The fermentation medium composition was optimized using response surface methodology involving a Box–Behnken design. Fermentation conditions were optimized using an orthogonal experimental design. The optimized medium composition was the non‐pretreated distilled wastewater of Chinese liquor supplemented with cornflour at 2.35 g/100 mL, (NH₄)₂SO₄ at 1.11 g/100 mL and CuSO₄ at 0.12 g/100 mL. The optimized fermentation conditions were a rotation speed of 150 rpm, an inoculum size of 10% (v/v), a fermentation temperature of 26 °C and a fermentation time 4.5 days. A maximum mycelial biomass yield of 2.75 g/100 mL was achieved using the optimized medium under the optimized conditions. Results from this study suggest that this is a feasible technology for the mycelial production of C. gigantea using the non‐pretreated distilled wastewater from Chinese liquor production. Copyright © 2015 The Institute of Brewing & Distilling