Chinese Liquor Production Research Articles

Schizosaccharomyces pombe can Reduce Acetic Acid Produced by Baijiu Spontaneous Fermentation Microbiota.

The spontaneous fermentation of alcoholic beverage is a bioprocess donated by microbiota with complex stress environments. Among various microbes, non-Saccharomyces yeasts have high stress tolerance and significantly affect the taste and quality of products in process. Although many researchers have focused on the influence of acid stress, the mechanism of non-Saccharomyces yeasts to tolerant stress remains unclear in microbiota. To bridge the gap, we constructed in situ and in vitro studies to explore the reduction pathway of acetic acid in non-Saccharomyces yeasts. In this study, we found Schizosaccharomyces pombe has special capacities to resist 10 g/L acetic acid in laboratory cultures and decrease the average concentration of acetic acid from 9.62 to 6.55 g/kg fermented grains in Chinese Maotai-flavor liquor (Baijiu) production. Moreover, Schi. pombe promoted metabolic level of mevalonate pathway (high expressions of gene ACCAT1, HMGCS1, and HMGCR1) to degrade a high concentration of acetic acid. Meanwhile, Schi. pombe also improved the concentration of mevalonic acid that is the precursor of terpenes to enhance the taste and quality of Baijiu. Overall, the synchronicity of reduction and generation in Schi. pombe advances the current knowledge to guide more suitable strategies for mechanism studies of non-Saccharomyces yeasts in fermented industries of alcoholic beverages.

Deciphering the crucial roles of transcriptional regulator GadR on gamma-aminobutyric acid production and acid resistance in Lactobacillus brevis

BackgroundIn lactic acid bacteria (LAB), acid stress leads to decreases of cell vitality and fermentation yield. Glutamate decarboxylase (GAD) system is regarded as one of the essential acid-resistance mechanisms in LAB. However, the regulation of GAD system is not well identified in the genus Lactobacillus. Although potential transcriptional regulator gene located upstream of GAD system genes was found in several Lactobacillus species, such as Lactobacillus (L.) brevis, the contribution of the regulator to acid resistance of the genus Lactobacillus has not been experimentally determined.ResultsThe potential transcriptional regulator gene gadR was disrupted by homologous recombination in L. brevis ATCC 367, leading to the decreased expression of gadC and gadB. The inactivation of GadR completely eliminated γ-aminobutyric acid (GABA) production and decreased the glutamate-dependent acid resistance. Moreover, expression of gadC and gadB in the presence of glutamate was increased and glutamate also stimulated the expression of gadR. In addition, L. brevis D17, a strain screened from acidic fermented grains of Chinese liquor production, had much higher expression level of gadR than the typical strain L. brevis ATCC 367. Under the pH-controlled and mixed-feed fermentation, L. brevis D17 achieved a titer of 177.74 g/L and a productivity of 4.94 g/L/h of GABA within 36 h. However, the L. brevis ATCC 367 only achieved a titer of 6.44 g/L and 0.18 g/L/h of GABA although the same fermentation control approach was employed.ConclusionsGadR is a positive transcriptional regulator controlling GABA conversion and acid resistance in L. brevis. L. brevis strains with hyper-expressing of gadR are excellent candidates for GABA production in industrial scale.

Effect of bioaugmentation on biochemical characterisation and microbial communities in Daqu using Bacillus, Saccharomycopsis and Absidia

SummaryDaqu, a saccharifying and fermenting agent for the production of Chinese vinegar and liquor, is manufactured through a spontaneous solid‐state fermentation process (SSF). To investigate the influence of bioaugmentation with native microorganisms on Daqu SSF process, physicochemical properties, microbial biomass, volatile compounds and microbial communities were analysed in laboratory‐scale Daqu. The results showed that the amylase activity of Daqu enhanced 6.35% ± 0.74% by inoculating Bacillus amyloliquefaciens, Saccharomycopsis fibuligera and Absidia corymbifera. Obviously higher microbial richness was found in the Daqu with bioaugmentation, although the microbial community structure remained relatively stable. Moreover, a total of twenty‐two volatile compounds were detected in the mature Daqu. Slightly increase in the content of alcohols was found in the enhanced group by fungal inoculation, including ethanol (46.58 ± 1.36 mg kg−1), isobutanol (0.19 ± 0.04 mg kg−1) and isoamyl alcohol (1.55 ± 0.13 mg kg−1). This study demonstrated that bioaugmentation had a positive effect on the amylase activity, main volatile compounds and microbial community richness.

Yeast dynamics and changes in volatile compounds during the fermentation of the traditional Chinese strong-flavor Daqu

Daqu is the major source of microorganisms, enzymes, flavour compounds, and their precursors in the Chinese liquor production process. In the present study, the yeast community and the volatile compounds of Chinese strong-flavor Daqu during its fermentation were investigated using PCR-DGGE and HS-SPME/GC–MS, respectively. The results indicated that the band number, dominance, diversity and similarity of the 26S rDNA were clearly distinct in the DGGE patterns, due to the significant diversity expressed by the disparate yeast communities in different phases. More than 14 yeast genera, such as Saccharomycopsis fibuligera, Hanseniaspora uvarum, Geotrichum bryndzae, Wickerhamomyces anomalus, Yarrowia lipolytica, Candida mucifera, Issatchenkia orientalis, Alternaria tenuissima, Kazachstania barnettii, Candida intermedia, Kazachstania barnettii, Pichia occidentalis, Saccharomyces cerevisiae, Pichia kudriavzevii and Saturnispora silvae were observed in the fermentation, among which the dominant strains were Pichia kudriavzevii and Saturnispora silvae. Furthermore, 43 volatile compounds, including 15 esters, 10 acids, 8 alcohols, 4 aldehydes, 2 alkenes, 1 ketone, 1 phenol, 1 furan and 1 pyrrole, were identified. The trajectory plots of the compounds acquired during consecutive stages of incubation revealed that different samples of Daqu correlated with specific groups of volatile compounds. These findings would help to understand the fermentation mechanism of Daqu production.

Effect of Fortified Daqu on the Microbial Community and Flavor in Chinese Strong-Flavor Liquor Brewing Process.

Daqu, an important fermentation starter for the production of Chinese liquor, as used in the current study included traditional Daqu and fortified Daqu inoculated with Bacillus velezensis and Bacillus subtilis. To evaluate the effect of fortified Daqu on strong-flavor liquor production, the differences of microbial communities among three inoculation patterns of fermented grains (FG) were analyzed by the Illumina MiSeq platform. A higher relative abundance of dominant genera including Bacillus, Lactococcus, Aspergillus, and Candida, and lower relative abundance of Lactobacillus, were observed in FG50, in which mixed Daqu (traditional: fortified Daqu = 1: 1, w/w, 50% fortified Daqu) was used as the starter. Then, volatile compounds of their distillations were also examined by HS-SPME-GC-MS. The results showed that the contents of skeleton flavor components, mainly including important esters and aromatic compounds, were higher in the corresponding liquor L50, which distillated from FG50. Moreover, most esters mainly positively correlated with Lactobacillus and Candida in the bottom layer of FG fermented with 50% fortified Daqu (FG50-B). Aromatic compounds were strongly positively correlated with Bacillus and Aspergillus in the middle layer of FG with 50% fortified Daqu used (FG50-M). In particular, hexyl hexanoate showed a positive correlation with higher abundances of Ruminococcus in the FG with addition of 100% fortified Daqu (FG100). This study observed microbial compositions in the FG with fortified Daqu addition, and it further revealed the correlations between pivotal microbes and main flavor compounds. These results may help to develop effective strategies to regulate microbes for the brewing process and further improve the flavors of Chinese liquor.

Complete Genome Sequence of Clostridium kluyveri JZZ Applied in Chinese Strong-Flavor Liquor Production.

Chinese strong-flavor liquor (CSFL), accounting for more than 70% of both Chinese liquor production and sales, was produced by complex fermentation with pit mud. Clostridium kluyveri, an important species coexisted with other microorganisms in fermentation pit mud (FPM), could produce caproic acid, which was subsequently converted to the key CSFL flavor substance ethyl caproate. In this study, we present the first complete genome sequence of C. kluyveri isolated from FPM. Clostridium kluyveri JZZ contains one circular chromosome and one circular plasmid with length of 4,454,353 and 58,581bp, respectively. 4158 protein-coding genes were predicted and 2792 genes could be assigned with COG categories. It possesses the pathway predicted for biosynthesis of caproic acid with ethanol. Compared to other two C. kluyveri genomes, JZZ consists of longer chromosome with multiple gene rearrangements, and contains more genes involved in defense mechanisms, as well as DNA replication, recombination, and repair. Meanwhile, JZZ contains fewer genes involved in secondary metabolites biosynthesis, transport, and catabolism, including genes encoding Polyketide Synthases/Non-ribosomal Peptide Synthetases. Additionally, JZZ possesses 960 unique genes with relatively aggregating in defense mechanisms and transcription. Our study will be available for further research about C. kluyveri isolated from FPM, and will also facilitate the genetic engineering to increase biofuel production and improve fragrance flavor of CSFL.

Comparison of Bacterial Diversity Between Two Traditional Starters and the Round-Koji-Maker Starter for Traditional Cantonese Chi-Flavor Liquor Brewing.

Xiaoqu is a traditional fermentation starter that is used for Chinese liquor production. Although microorganisms in the starters are closely associated with the quality and flavor of liquor, knowledge of the microbiota in xiaoqu is still far from complete, let alone the starters produced by new processes. Here, Illumina MiSeq high-throughput sequencing was applied to study bacterial composition in three types of xiaoqu used in Cantonese soybean-flavor (Chi-flavor) liquor, namely two traditional starters (Jiu Bing and Bing Wan) and a Round-Koji-maker starter (San qu) produced by the automatic starter-making disk machine. The results showed bacterial diversity in traditional starters was similar and higher than that in the Round-Koji-maker starter. Lactobacillus and Pediococcus were the dominant genera in all starters, while other different dominant genera also existed in different starters, which were Weissella, Acetobacter, and Gluconobacter for Jiu Bing, Weissella for Bing Wan, and Bacillus, Acetobacter, Acinetobacter and Klebsiella for San qu, respectively. Meanwhile, Cytophagaceae, one particular microbial family, and some pathogens including Klebsiella, Cronobacter, and Enterobacter were also found in San qu, indicating the automatic starter-making disk machine should be ameliorated before applied into industrial production. These results enriched our knowledge on xiaoqu-related microorganisms and might be helpful in industrial Chi-flavor liquor production and the development of fermentation technology.

Prokaryotic diversity and biochemical properties in aging artificial pit mud used for the production of Chinese strong flavor liquor

At present, artificial pit mud (APM) is widely used in Chinese liquor-making industry and plays a particular role in the production of Chinese strong flavor liquor (CSFL). However, APM frequently ages during fermentation, thus becoming unsuitable for sustainable use due to its low-quality. The reasons behind, and results of, APM aging during the production of CSFL are not yet understood. Sequencing the V4 region of the 16S rRNA gene shows that prokaryotic diversity is significantly decreased (Shannon's diversity index, P<0.01) and community composition is distinctly changed (from 1197 to 865 OTUs) in aging APM. On the phylum level, the increase of Firmicutes and decrease of Proteobacteria are the main consequences of APM aging during the production of CSFL. The counting of cultivatable bacteria confirmed that there was a large increase in Lactobacilli and aerobic spore-forming bacteria in aging low-quality APM (more than twofold). Unexpectedly, the total number of caproic acid-producing bacteria, mainly Clostridia, did not change significantly between the two kinds of APM. Furthermore, biochemical analysis indicates that the pH and the levels of NH4+ and K+ are decreased in aging low-quality APM (P<0.01). The results obtained in this study support the possibility that environmental factors (pH, nutrients) induce the decrease of prokaryotic diversity, and the changed community composition influences the environmental properties. Therefore, through interfering with the cycle, APM aging can be controlled potentially by adjustment of environmental factors and/or supplementation of diminished or missed microorganisms.

Changes in microbial community during fermentation of high-temperature Daqu used in the production of Chinese ‘Baiyunbian’ liquor

Daqu is a fermentation starter used in the production of Chinese liquor. The present study investigated changes in the microbial community during the fermentation of Baiyunbian high-temperature Daqu during a cycle time of 140 days. Two methods were used, a culture-dependent method (viable cell counting) and a culture-independent method (high-throughput sequencing of bacterial 16S rRNA gene and fungal internal transcribed spacer region). It was found that thermotolerant microorganisms such as Bacillus, Thermomyces and Actinobacteria were dominant in all stages of Daqu fermentation, especially during the high-temperature period of days 7–38. In the initial 110 days of fermentation, operational taxonomic units and number of bacteria were higher than those of fungi, but the opposite was observed towards the end of fermentation (day 140). In mature Daqu, the predominant bacterial species were Bacillus and unclassified Thermoactinomycetaceae, followed by Thermoactinomyces, Kroppenstedtia and Saccharopolyspora; the predominant fungi were Aspergillus, unclassified Trichocomaceae, Thermomyces, Rhizopus, Monascus and Candida, among others. It is possible that the addition of mother Daqu, temperature and storage time played critical roles in the microbial composition of Daqu. Collectively, the above findings provide important information that can be used to optimize conditions for large-scale production of Daqu. Copyright © 2017 The Institute of Brewing & Distilling

Unraveling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput Amplicons and Metatranscriptomics Sequencing.

Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces) and lactic acid bacteria (genus Lactobacillus) classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol) production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic acid) in Chinese Maotai-flavor liquor production. Our findings provide insight into the effects of the core functional microbiota in soy sauce aroma type liquor production and the characteristics of the fermentation microbiota under different environmental conditions.

Biochemical characterisation and dominance of different hydrolases in different types of Daqu - a Chinese industrial fermentation starter.

Daqu is a fermentative saccharification agent that is used to initiate fermentation in the production of Chinese liquor and vinegar. This study investigated the differences of amylase, protease and esterase in dominance of different types of Daqu, which can be useful for quality control and flavor improvement of Daqu production by enzyme technology. Hydrolase activities in different Daqu samples were compared by principal component analysis (PCA). Based on protein electrophoresis and 1 H NMR spectroscopy, the protein patterns and metabolites in Daqu were further analysed. The results indicated that the highest amylase activities and diversities were found in low/medium-temperature of Daqu which had light-flavour and strong-flavour. Proteases play a significant role in determining the quality of high-temperature Daqu samples which had a sauce-flavour. Furthermore, the contributions of esterase to both strong and sauce flavour development in high-temperature Daqu are similar. Results from the present work showed that differences in amylase, protease and esterase play a leading role in different types of Daqu, which can be useful for quality control and technology development of Daqu. © 2017 Society of Chemical Industry.

Employment of Near Full-Length Ribosome Gene TA-Cloning and Primer-Blast to Detect Multiple Species in a Natural Complex Microbial Community Using Species-Specific Primers Designed with Their Genome Sequences.

It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences. The primers were intended to be specific at the species level within relevant microbial communities, i.e., a defined genomics background. The primers were tested with samples collected from the Daqu (also called fermentation starters) and pit mud of a traditional Chinese liquor production plant. Sixteen pairs of primers were found to be suitable for identification of individual species. Among them, seven pairs were chosen to measure the abundance of microbial species through quantitative PCR. The combination of near full-length ribosome RNA gene library sequencing and Primer-Blast may represent a broadly useful protocol to quantify multiple species in complex microbial population samples with species-specific primers.

Effect of bioaugmented inoculation on microbiota dynamics during solid-state fermentation of Daqu starter using autochthonous of Bacillus, Pediococcus, Wickerhamomyces and Saccharomycopsis

Daqu, a traditional fermentation starter that is used for Chinese liquor and vinegar production, is still manufactured through a traditional spontaneous solid-state fermentation process with no selected microorganisms are intentionally inoculated. The aim of this work was to analyze the microbiota dynamics during the solid-state fermentation process of Daqu using a traditional and bioaugmented inoculation with autochthonous of Bacillus, Pediococcus, Saccharomycopsis and Wickerhamomyces at an industrial scale. Highly similar dynamics of physicochemical parameters, enzymatic activities and microbial communities were observed during the traditional and bioaugmented solid-state fermentation processes. Both in the two cases, groups of Streptophyta, Rickettsiales and Xanthomonadales only dominated the first two days, but Bacillales and Eurotiales became predominant members after 2 and 10 days fermentation, respectively. Phylotypes of Enterobacteriales, Lactobacillales, Saccharomycetales and Mucorales dominated the whole fermentation process. No significant difference (P > 0.05) in microbial structure was observed between the traditional and bioaugmented fermentation processes. However, slightly higher microbial richness was found during the bioaugmented fermentation process after 10 days fermentation. Our results reinforced the microbiota dynamic stability during the solid-state fermentation process of Daqu, and might aid in controlling the traditional Daqu manufacturing process.

Illuminating Anaerobic Microbial Community and Cooccurrence Patterns across a Quality Gradient in Chinese Liquor Fermentation Pit Muds.

Fermentation pit mud, an important reservoir of diverse anaerobic microorganisms, is essential for Chinese strong-aroma liquor production. Pit mud quality, according to its sensory characteristics, can be divided into three grades: degraded, normal, and high quality. However, the relationship between pit mud microbial community and pit mud quality is poorly understood, as are microbial associations within the pit mud ecosystem. Here, microbial communities at these grades were compared using Illumina MiSeq sequencing of the variable region V4 of the 16S rRNA gene. Our results revealed that the pit mud microbial community was correlated with its quality and environmental factors. Species richness, biodiversity, and relative and/or absolute abundances of Clostridia,Clostridium kluyveri, Bacteroidia, and Methanobacteria significantly increased, with corresponding increases in levels of pH, NH4 (+), and available phosphorus, from degraded to high-quality pit muds, while levels of Lactobacillus, dissolved organic carbon, and lactate significantly decreased, with normal samples in between. Furthermore, 271 pairs of significant and robust correlations (cooccurrence and negative) were identified from 76 genera using network analysis. Thirteen hubs of cooccurrence patterns, mainly under the Clostridia,Bacteroidia,Methanobacteria, and Methanomicrobia, may play important roles in pit mud ecosystem stability, which may be destroyed with rapidly increased levels of lactic acid bacteria (Lactobacillus,Pediococcus, and Streptococcus). This study may help clarify the relationships among microbial community, environmental conditions, and pit mud quality, allow the improvement of pit mud quality by using bioaugmentation and controlling environmental factors, and shed more light on the ecological rules guiding community assembly in pit mud.

Production of Butyrate from Lactate by a Newly Isolated Clostridium sp. BPY5.

Lactate-utilizing bacteria play important roles in the production of Chinese strong-flavored liquor (CSFL). However, the identity of these bacteria and their lactate-utilizing properties are largely unknown. Here, a lactate-utilizing, butyrate-producing bacterium BPY5 was isolated from an old fermentation pit for CSFL production. The isolate represented a novel species belonging to Clostridium cluster XIVa of family Lachnospiraceae based on phylogenetic analysis using 16S rRNA gene sequences. Strain BPY5 could ferment lactate into butyrate as the major metabolic product. Butyrate was significantly formed at initial lactate concentration from 66 to 104mM, but substantially declined when initial lactate exceeded 133mM. At initial lactate concentration of 66mM, lactate conversion was independent on initial pH from 5.5 to 7.0, but the conversion was completely inhibited when pH dropped below 4.8. Nevertheless, the inhibition on lactate conversion was largely relieved by the addition of acetate, suggesting that exogenous acetate could enhance lactate conversion at low pH condition. Additionally, lactate in CSFL-brewing wastewater was dramatically removed when inoculated with strain BPY5. These results implicate that the isolate may be applied for the industrial production of butyrate or the recovery of butyrate from lactate-containing wastewater.

Clostridium liquoris sp. nov., isolated from a fermentation pit used for the production of Chinese strong-flavoured liquor.

Strain BEY10T was isolated from an old fermentation pit, which had been used for the production of Chinese strong-flavoured liquor for over 20 years. The strain was strictly anaerobic, Gram-stain positive, rod-shaped, non-motile and spore-forming. Strain BEY10T grew at temperatures of 22-47 °C (optimum 37 °C), at pH 5.5-9.0 (optimum pH 7.5-8.5) and with NaCl concentrations of 0-4 % (w/v) (optimum 0 %). The isolate was able to utilize glucose, mannitol, lactose, xylose, maltose, glycerol, cellobiose and trehalose as carbon sources for growth. The major end-products from glucose fermentation were ethanol and butyric acid. The polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, phospholipids, a glycolipid and an aminolipid. The predominant fatty acids (>10 %) were C20 : 0, C18 : 0, C16 : 0, C12 : 0 and C14 : 0. The DNA G+C content was 34.4 mol%. Sequence analysis of the 16S rRNA gene indicated that strain BEY10T belongs to the genus Clostridium in the family Clostridiaceae. The closest phylogenetic neighbour is Clostridium lundense DSM 17049T, showing 97.6 % 16S rRNA gene sequence similarity with strain BEY10T. DNA-DNA relatedness values of strain BEY10T with Clostridium lundense DSM 17049T, Clostridium tetanomorphum DSM 4474T and Clostridium pascui DSM 10365T were 58.8 %, 57.9 % and 42.2 %, respectively. This characterization based on phylogenetic, phenotypic and chemotaxonomic evidence demonstrated that strain BEY10T represents a novel species of the genus Clostridium, for which the name Clostridium liquoris sp. nov. is proposed. The type strain is BEY10T ( = ACCC 00785T = DSM 100320T).

The synthesis of n-caproate from lactate: a new efficient process for medium-chain carboxylates production.

A unique microbiome that metabolizes lactate rather than ethanol for n-caproate production was obtained from a fermentation pit used for the production of Chinese strong-flavour liquor (CSFL). The microbiome was able to produce n-caproate at concentrations as high as 23.41 g/L at a maximum rate of 2.97 g/L/d in batch trials without in-line extraction. Compared with previous work using ethanol as the electron donor, the n-caproate concentration increased by 82.89%. High-throughput sequencing analysis showed that the microbiome was dominated by a Clostridium cluster IV, which accounted for 79.07% of total reads. A new process for n-caproate production was proposed, lactate oxidation coupled to chain elongation, which revealed new insight into the well-studied lactate conversion and carbon chain elongation. In addition, these findings indicated a new synthesis mechanism of n-caproate in CSFL. We believe that this efficient process will provide a promising opportunity for the innovation of waste recovery as well as for n-caproate biosynthesis.

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

Optimizing Semisimultaneous Saccharification and Fermentation for Ethanol Production from Chinese Distiller's Spent Grains

Spent grains are a main byproduct for Chinese liquor production. In this study, the semisimultaneous saccharification and fermentation (S-SSF) for ethanol production based on multi-enzyme degradation of the spent grains was investigated. After optimization with orthogonal design experiments, an enzyme combination composed of cellulase NS22086 at 0.30 mL/g, β-glucosidase NS22118 at 0.20 mL/g, xylanase NS22083 at 0.08 mL/g, complex enzyme NS22119 at 0.08 mL/g, complex enzyme NS22002 at 0.02 mL/g, and glucoamylase NS22035 at 0.01 mL/g was obtained. With 14% of the substrate concentration and the optimal enzyme combination, reducing sugar, glucose, and xylose at 109.97, 60.02, and 16.05 g./L, respectively, were attained in the saccharified solution, which are approximately fourfold of those obtained before optimizing and are among the highest values reported for different lignocellulosic materials. Experiments of batch and fed-batch S-SSF for ethanol production from the spent grains were then performed, and higher efficiency of saccharification and ethanol formation was observed in the fed-batch S-SSF, where an ethanol concentration of over 30 g/L was attained. Kinetic models satisfactorily describing the processes of S-SSF were developed and analyzed. This study contributes to the utilization of Chinese distiller's spent grains and other similar materials.

Performance of indigenous yeasts in the processing of Chinese strong-flavoured liquor during spontaneous mixed solid-state or submerged fermentation

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong-flavoured liquor, a comparison was conducted between solid-state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong-flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong-flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong-flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling