Chinese Luzhou-flavor Liquor Research Articles

Differentiation and classification of Chinese Luzhou-flavor liquors with different geographical origins based on fingerprint and chemometric analysis.

In order to differentiate and characterize Chinese Luzhou-flavor liquor according to geographical origins, the volatile flavor compounds were analyzed for forty commercial Luzhou-flavor liquor samples from Sichuan, Jiangsu, and Hubei provinces. A total of 113 volatile flavor compounds were quantified; among them, 29 flavor compounds were quantified according to the internal standard method. The differences in flavor composition among different brands of Luzhou-flavor liquor were compared. A data matrix of 64 (flavor components) × 40 (samples) was studied and interpreted using chemometric analysis. The research object could be naturally clustered according to geographical origin (brand) based on the hierarchical cluster analysis (HCA), principal component analysis (PCA) and multivariate analysis of variance (MANOVA) methods. A 100% of predication ability was obtained by the application of K-nearest neighbor model (KNN) for study sample classification. The results demonstrate that the abundance of volatile flavor components in liquors combined with appropriate multivariate statistical methods could be used for the division and traceability of liquors from different geographic origins. PRACTICAL APPLICATION: This study can provide the basis for the identification of liquor authenticity and the traceability of liquor.

Isolation, Purification, and Identification of a Novel HX-Producing Strain and Optimization of Its Fermentation Medium

Hexanoic acid (HX) is a crucial flavor compound and precursor of ethyl caproate (EA), which determines the quality of Chinese Luzhou-flavor liquor (CLFL). The isolation, purification, identification, and optimization of fermentation conditions of HX-producing bacteria are essential for industrial CLFL production. In this study, one strain of HX-producing bacterium was isolated from six candidate bacterial strains and identified as Clostridium sartagoneforme. Then, the growth characteristics and HX production of C. sartagoneforme were investigated. Sodium acetate medium was identified as the optimal fermentation medium from four candidate media. C. sartagoneforme yielded 800.85 ± 12.87 mg/100mL HX in sodium acetate medium. Then, to further optimize the formula of the fermentation medium, the carbon and nitrogen sources and inorganic salt component of the fermentation medium were investigated using HX yields as an optimization index. Optimization was performed with a single-factor experiment and the Taguchi design method. The single-factor experiment showed that the highest HX outputs were obtained when the sodium acetate medium contained 2.5 g/L yeast extract, 1.8 g/L KCl, 20 g/L sodium acetate, 15 mL/L ethanol, and 1.5 g/L glucose. In the orthogonal experiment designed using the Taguchi design method, HX yields reached 2018.29 ± 46.37 mg/100mL in sodium acetate medium that contained 3.5 g/L yeast extract, 1.8 g/L KCl, 25 g/L sodium acetate, and 15 mL/L ethanol.

Transfer of Metal Elements in the Distillation Process of Chinese Luzhou-Flavor Liquor

Transfer of Metal Elements in the Distillation Process of Chinese Luzhou-Flavor Liquor

Analysis of bacterial diversity of Chinese Luzhou-flavor liquor brewed in different seasons by Illumina Miseq sequencing

Luzhou-flavor liquor is one of the most popular liquors in China, produced by the distillation of Zaopei, a mixture of fermented and fresh cereals. The quality and flavor of Luzhou-flavor liquor are closely related to the multitudinous bacteria in Zaopei. Little is known about the effect of brewing seasons, a main operating parameter, on the bacterial diversity of Zaopei. In this study Illumina Miseq sequencing platform was first applied to study the bacterial succession in Zaopei during liquor fermentation processes in winter and summer. The research results showed that 10 phyla and 13 families were detected in Zaopei. Differences in bacterial communities of summer and winter Zaopei were apparent. Proteobacteria was the most abundant phylum in winter Zaopei. However, bacteria belonging to Firmicutes were most frequently detected in summer Zaopei. The diverse genera decreased sharply at the first half of the 40-day fermentation stage, and then bacterial population were mainly represented by Acetobacter and Lactobacillus both in winter and summer Zaopei. Interestingly, the summer Zaopei contained significantly higher proportions of LAB and lower proportions of Acetobacter than winter Zaopei. Clustering analysis also revealed that bacterial community structure was distinct between the Zaopei fermented in the two seasons. Thermoactinomycetaceae, Prevotella, Alcaligenes, and Gluconacetobacter not previously identified in Zaopei were revealed in this study by high-throughput sequencing. This study might be helpful in achieving better control over the quality of Luzhou-flavor liquor brewed in different seasons.

Characterization of interphase volatile compounds in Chinese Luzhou-flavor liquor fermentation cellar analyzed by head space-solid phase micro extraction coupled with gas chromatography mass spectrometry (HS-SPME/GC/MS)

This study aimed to investigate volatile compounds characteristics of Pit mud (PM), Zaopei (ZP), Huangshui (HS) in Chinese Luzhou-flavor liquor fermentation cellar by HS-SPME/GC/MS. Results indicated that pit age and spatial position had a stronger effect on volatiles than physicochemical properties. A total of 59 volatiles had been identified, and phenylethyl alcohol, butyric acid, pentanoic acid, hexanoic acid, heptanoic acid, ethyl lactate, ethyl hexanoate, ethyl hexadecanoate, ethyl linoleate and ethyl elaidate were dominant, accounting for roughly 66.30%. Additionally, the total level of volatiles significantly increased from upper layer (U) to under layer (D), except for 2-year-MPM and 40-year-MZP, and 40-year-DPM (834.14 mg/kg) had the highest content. PCA explanation plane primarily revealed the main differentiation based on their loading plot values. Results of PLS-DA showed 4-methylphenol and gamma-nonanolactone were correlated with 2-year-PM. In addition to ethyl phenylacetate, ethyl hexanoate, furfuryl hexanoate, ethyl nonanoate, perhydrofarnesyl acetone and 2-acetyl furan were associated with 40-year-PM. Isoamyl hexanoate, octyl octanoate, benzaldehyde and 2-nonenal were with 10-year-PM. 3-methyl-1-butanol, ethyl lactate, ethyl hydrocinnamate and 3-furancarboxaldehyde were related to 2-year-ZP, while ethyl decanoate and ethyl linolenate were relevant to 10-year-ZP. No specific compounds for 40-year-ZP and HS samples were found.

Proteomic and high-throughput analysis of protein expression and microbial diversity of microbes from 30- and 300-year pit muds of Chinese Luzhou-flavor liquor

Luzhou-flavor liquor is fermented based on the metabolism of special microbial communities in pit. In this study, total proteins and DNAs of microbes from 30- and 300-year pit muds were firstly extracted. Meanwhile, an efficient approach for protein extraction with increased protein content was optimized. iTRAQ-based proteomic was then applied to investigate the aroma-forming functional protein expression of microbes from the samples. Furthermore, high-throughput sequencing of 16S rDNA was employed to reveal microbial diversity. We comparatively identified 63 proteins of aroma-forming functional microbes in these samples, and found that 59 of these proteins were highly expressed in the 300-year pit mud. Those aroma-forming functional proteins were found to be involved in methanogenesis, as well as the formation of caproic acid and butyric acid during the liquor fermentation. High-throughput sequencing revealed that the microbes most commonly found in both samples were members of phylum Firmicutes (by 97% sequence similarity), both of which, along with another common Methanobacterium, were important components of aroma-forming functional colonies in the pit muds for the brewing of Chinese liquor. The findings in this study afford us new insight into the different protein expression levels and microbial communities in two pit muds.

Analysis of Clostridium cluster I community diversity in pit mud used in manufacture of Chinese Luzhou-flavor liquor

Chinese Luzhou-flavor liquor is prepared using distillation of mixed, fermented grains, such as sorghum and rice. Fermentation occurs in cellars lined with pit mud containing microbes. The identity and community diversity of bacteria in pit mud of fermentation cellars contributes to the aromatic compounds of the liquor. In order to determine the contribution of genus Clostridium to this fermentation, the community diversity of Clostridium cluster I in pit mud of different cellar ages (1, 50, 100, and 400 years) was investigated based on denaturing gradient gel electrophoresis followed by 16S rRNA sequencing. Differences in the community structure within pit mud depended on wall and bottom layer cellar locations. A better understanding of the role of Clostridium in Luzhou-flavor liquor manufacture will be useful.

Interphase microbial community characteristics in the fermentation cellar of Chinese Luzhou-flavor liquor determined by PLFA and DGGE profiles

In view of the equilibrium of fermentation rate in the cellar mainly depends on interphase microflora, it is essential to exploit interphase microbial community characteristics to understand the metabolic regulation mechanism. The objective of this study was to investigate interphase microbial community structure in the fermentation cellar of Chinese Luzhou-flavor liquor via combining phospholipid fatty acids (PLFAs) and denaturing gradient gel electrophoresis (DGGE). The total microbial biomass analyzed by PLFA in the pit mud (PM: 25.52–103.38nmol/g) and Zaopei (ZP: 29.96–64.50nmol/g) was obviously higher than Huangshui (HS: 4.35–7.82nmol/g), and was strongly correlated with pit age, which was validated by DGGE results. Among the specific microbial groups, ‘gram-positive bacteria, anaerobe bacteria and fungi’ dominated in the PM, while ‘gram-positive bacteria, aerobe bacteria and fungi’ predominated in the ZP and HS, respectively. Principal component analysis (PCA) based ordination of the relative PLFA abundance data segregated PM, ZP and HS. Eubacterial PCR-DGGE illustrated that a total of 31 bands belonged to five families (Lactobacillaceae, Clostridiaceae, Porphyromonadaceae, Synergistaceae and Acetobacteraceae) were identified, and 41.52% eubacteria were affiliated with Lactobacillaceae in the ZP, while 44.67% and 40.41% eubacteria were affiliated with Clostridiaceae in the PM and HS, respectively. Archaeal PCR-DGGE analysis demonstrated that Methanosaeta, Methanocorpusculum, Methanobrevibacter, Methanobacterium and Methanoculleus were the majority of archaea. Particularly, Methanosaeta, as acetrophic methanogens, increased gradually in the PM and HS with pit age, and decreased in the ZP, and no family Methanobacteriaceae was detected in the 30-year-PM. Furthermore, fungal community was dominated by genus Pichia.

Optimization of Caproic Acid Production from <em>Clostridium kluyveri</em> H588 and its Application in Chinese Luzhou-flavor Liquor Brewing

This study focused on the optimization of caproic acid production from Clostridium kluyveri H588 and its application in Luzhou-flavor liquor brewing. The critical variables that affected caproic acid production were identified by Plackette-Burman design (ethanol, sodium acetate, yeast extract and initial pH) and further optimized by using a four factor central composite design of response surface methodology. The results demonstrated that the optimum conditions for caproic acid production were determined to be ethanol concentration of 4.046% (v/v), sodium acetate concentration of 0.982% (w/v), yeast extract concentration of 0.145% (w/v) and initial pH of 6.41. Caproic acid production (214.23 mg/100 mL) in the optimized condition was in good agreement with the value predicted by the model equation (219.11 mg/100 mL), thereby assuring its effectiveness. Furthermore, the optimized condition of the lab-scale bioreactor was scaled-up to 5000 L fermentor and the obtained caproic acid broth was subjected to pit-filling fermentation. The result demonstrated this method of pit-filling fermentation was efficient in improving the quality of Luzhou-flavor liquor.

Development of a Rapid Discrimination Tool for Luzhou-flavor Pit Mud Classification by the Kohonen Artificial Neural Network Model

Understanding the volatile composition of pit mud (PM) samples and discriminating these samples has become a highly necessary task, owing to the fact that volatile profiling of PM can significantly affect Chinese Luzhou-flavor liquor quality. In this study, the volatile constitutions of 13 Luzhou-flavor liquor PM samples from four typical producing regions were investigated by gas chromatography–mass spectrometry (GC-MS). Owing to their high concentrations, compounds such as ethyl hexanoate, butyric acid, hexanoic acid, ethyl pentadecanoate, ethyl palmitate, ethyl (9E)-9-octadecenoate, ethyl (9E,12E)-9,12-octadecadienoate, and palmitic acid were considered to be predominant volatiles. A promising artificial neural network model, the Kohonen self-organizing map (SOM), was applied to rapidly discriminate the PM samples in terms of differences based on the quantitative information of volatile compounds. After Kohonen SOM training, 13 distinct clusters, corresponding to PM samples, were clearly visualized on a uniform distance matrix (U-matrix). The influence of volatile compounds on the classification of the PM could be displayed using component panels, which can give quantitative insight. GC-MS coupled with the Kohonen SOM model not only presented the volatile constitution of PM but also provided promising information for discrimination between different PM samples, even in other fermented foods.

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.

Clostridium swellfunianum sp. nov., a novel anaerobic bacterium isolated from the pit mud of Chinese Luzhou-flavor liquor production.

A novel Gram-positive, strictly anaerobic, spore-forming, rod-shaped bacterium, designated strain S11-3-10(T), was isolated from the pit mud used for Chinese Luzhou-flavor liquor production. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a monophyletic clade with the closely related type strains of Clostridium cluster I and was most closely related to Clostridium amylolyticum JCM 14823(T) (94.38%). The temperature, pH, and NaCl range for growth was determined to be 20-45 °C (optimum 37 °C), 4.0-10.0 (optimum pH 7.3), and 0-3.0% (w/v), respectively. The strain was able to tolerate up to 7.5 % (v/v) ethanol. Yeast extract or peptone was found to be required for growth. Acids were found to be produced from glucose, mannose and trehalose. The major end products from glucose fermentation were identified as ethanol, acetate and hydrogen. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids and polar lipids. The major fatty acids (>5%) were identified as iso-C(15:0), C(16:0), C(16:0)dma, C(14:0), anteiso-C(15:0) and iso-C(13:0). No respiratory quinone was detected. The diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid and the whole-cell sugars were found to include galactose and glucose as major components. The DNA G+C content was determined to be 36.4 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic evidence, the isolate is considered to represent a novel species of the genus Clostridium for which the name Clostridium swellfunianum sp. nov. is proposed. The type strain is S11-3-10(T) (=DSM 27788(T) = JCM 19606(T) = CICC 10730(T)).

Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR–DGGE

The aim of this study was to investigate and compare the microbial community structures of eubacteria and archaea in the pit mud of Chinese Luzhou-flavor liquor from the wall (C(w)) and bottom (C(b)) of cellar through nested PCR-denaturing gradient gel electrophoresis (DGGE). The Shannon-Wiener index (H) calculated from the DGGE profiles showed that the community diversities of eubacteria and archaea in samples from C(b) were almost higher than that from C(w). In addition, cluster analysis of the DGGE profiles revealed that some differences were found in the microbial community structure in samples from different locations. The closely relative microorganisms of all eubacterial 16S rRNA gene sequences fell into four phyla (Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria), including 12 genera and 2 uncultured eubacteria. Moreover, 37.1% eubacteria were affiliated with Clostridium. Particularly, genus Acinetobacter was absent in all samples from C(b) but present in all samples from C(w). The closely relative microorganisms of all archaeal 16S rRNA gene sequences fell into four genera, which included Methanobrevibacter, Methanoculleus, Methanobacterium and Methanosaeta, while the dominant archaea in samples from C(w) and C(b) were similar. Results presented in this study provide further understanding of the spatial differences in microbial community structure in the pit mud, and is of great importance for the production and quality improvement of Luzhou-flavor liquor.

Microbial Diversity and Succession in the Chinese Luzhou-Flavor Liquor Fermenting Cover Lees as Evaluated by SSU rRNA Profiles

The microbial diversity and the community succession in the fermenting cover lees of Chinese Luzhou-flavor liquor were investigated by small-subunit rRNA (SSU rRNA) culture independent method. All sequences retrieved from the 1, 7 and 60days fermented cover lees were respectively assigned into the genera of Streptococcus, Acetobacter, Arthrobacter, Bacillus, Staphylococcus, Serratia, Nocardia, Methanoculleus, Clostridium, Aneurinibacillus, Corynebacterium, Lactobacillus, Microbacterium, Trichosporon, Saccharomycopsis, Sagenomella, Talaromyces, Eurotium, Issatchenkia, Zygosaccharomyces, Saccharomyces and TM7 phylum. The fungal Issatchenkia, Saccharomycopsis and Talaromyces and the bacteria Staphylococcus and Lactobacillus were most abundant in the 1day fermented cover lees, the fungal Issatchenkia, Saccharomyces and Talaromyces and the bacteria Bacillus and Streptococcus were dominant in the 7days cover lees, the archaea Methanoculleus and the fungal Eurotium and Talaromyces were prevalent in the 60days cover lees. When the microbial community profiles in three samples were compared at species level, the prokaryotic community similarity coefficient was from 0.4042 to 0.5703 and descended to 0.2222, and that of eukaryotic community was from 0.3000 to 0.6000 and followed to 0.5215. These results suggested that microbial diversity variability and community succession have happened in the cover lees associated with fermentation proceeding and such variability and succession respond for the appearance of some unique flavor of Luzhou-flavor liquor.

Microbial succession in the traditional Chinese Luzhou-flavor liquor fermentation process as evaluated by SSU rRNA profiles

The community succession of microbes inhabited in the fermenting lees of Luzhou-flavor liquor was investigated based on small-subunit rRNA culture independent method. All sequences recovered from fermenting lees respectively fell into the genera of Lactobacillus, Streptococcus, Bacillus, Staphylococcus, Clostridium, Pelobacter, Actobacter, Serratia, Burkholderia, Rhodoccous, Corynebacterium, Arthrobacter, Microbacterium, Curtobacterium, Leptotrichia, Methanocuuleus, Saccharomyces, Zygosaccharomyces, Saccharomycopsis, Pichia, Talaromyces, Aspergillus, Eurotium, Fomitopsis and Trichosporon. The fungal Pichia, Saccharomycopsis and Talaromyces were most abundant in the lees fermented for 1 day, the fungal Eurotium and the bacteria Burkholderia, Streptococcus and Lactobacillus were dominant in the lees fermented for 7 days, only the bacteria Lactobacillus, Burkholderia were prevalent in the lees fermented for 60 days. Most genera almost existed in the fermenting lees, while their distributions were significantly different in 1, 7 and 60 days fermented lees. The prokaryotic community similarity coefficient was from 0.5000 to 0.5455 and followed to 0.1523, and that of eukaryotic community was from 0.5466 to 0.5259 and to 0.3750 when compared at species level. These results suggested that many microbes in lees have community successions associated with fermenting and that such successions maybe contribute the fermentation process of Luzhou-flavor liquor and is main reasons that the characteristic flavor factors are produced.

Ethanol/Moisture Contents in Fermented Grains and their Effects on the Yield and Quality of Chinese Luzhou-Flavor Liquor

The ethanol/moisture contents in fermented grains were measured and systematic experiments were carried out to investigate the optimal operation condition for distillation of Chinese Luzhou-flavor liquor. The results show that from the top surface (0 m depth) of fermentation tank to the bottom (2 m depth), the moisture content in the grains increases linearly from 60.67 wt.% to 70.44 wt.%, while the ethanol content presents a parabolic distribution with maximal content of 6.29 wt.% at 1.5 m depth. It is found that for efficient distillation of fermented grains, there exists an optimal steam flowrate at which the maximum yield of distilled liquor will be achieved. For the grains with given moisture content, the optimal steam flowrate keeps nearly constant with different ethanol contents, while for the grains with given ethanol content, the optimal steam flowrate increases as the moisture content become increased. Combined with the gas chromatography analysis, it is also presented that the distilled liquor obtained from the grains with higher ethanol content and/or lower moisture content will contain higher concentration of alcohol-soluble flavor compounds, so that the distilled liquor has better quality. The findings in this study provide important reference for the optimization of distillation process of Chinese Luzhou-flavor liquor.

Analysis of the fungi community in multiple- and single-grains Zaopei from a Luzhou-flavor liquor distillery in western China

The fungi communities of both multiple-grains and single-grains Zaopei were analyzed by denaturing gradient gel electrophoresis (DGGE) and traditional identification methods. The results of the DGGE fingerprint and the dendrogram based on banding patterns showed the diversified community structure and the phylogenetic affiliation of different Zaopei samples. The results indicated that the fungi community was affected by both raw materials and fermentation location. The genera of Debaryomyces, Pichia and Candida were dominant communities in multiple-grains Zaopei suggested by the results of both DGGE and the traditional methods, the DGGE result suggested Candida dominant in single-grains Zaopei was much different from the results by traditional method. Additionally, DGGE results showed the existence of thermophilic fungi (Thermomyces lanuginosus and Thermoascus aurantiacus) which were not detected by the traditional method. This work may contribute to further understanding of the brewing in Chinese Luzhou-flavor liquor.

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.

Analysis of the Bacterial Community inZaopeiDuring Production of ChineseLuzhou-flavorLiquor

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.