Hong Qu Glutinous Rice Wine Research Articles

Microbial Communities and Flavor Compounds during the Fermentation of Traditional Hong Qu Glutinous Rice Wine.

As a traditional Chinese rice wine, Hong Qu glutinous rice wine (HQW) is popular among consumers due to its unique flavor. However, its quality changes during fermentation, and the potential relationships between flavor and microbes have not been systematically researched. In this work, physicochemical properties (pH, total sugar, alcohol, amino acid nitrogen), flavor compounds (organic acids, free amino acids, and volatile compounds), and microbial communities were investigated. The results revealed that Pantoea, Lactiplantibacillus, Lactobacillus, Leuconostoc, and Weissella predominated the bacterial genera, and Monascus was the predominant fungal genus. Organic acids, free amino acids, and key volatile compounds (esters and alcohols) significantly increased during fermentation. The correlations analysis showed that Lactiplantibacillus was closely associated with flavor compounds formation. This study deepens our understanding of the roles of microorganisms in flavor formation on traditional HQW fermentation.

Fungal community diversity and fermentation characteristics in regional varieties of traditional fermentation starters for Hong Qu glutinous rice wine

Hong Qu glutinous rice wine (HQ wine) is a traditional alcoholic beverage produced in China by fermenting cooked rice using a fermentation starter prepared with the fungus Monascus purpureus. This starter (Hong Qu, HQ) is made empirically by open spontaneous fermentation that is hard to control and standardize, resulting in inconsistent wine quality. This study investigates representative HQ samples from a large geographic region. It explores fungal microbiome compositions, identifies characteristic differences important for the production of various HQ wine styles, and reveals the key fungi responsible for HQ wine fermentation characteristics. The source of the HQ inoculum was found to be the main factor influencing fungal community composition and diversity, followed by processing technology and geographical distribution. Linear discriminant analysis effect size (LEfSe) uncovered 14 genera as potential biomarkers to distinguish regional varieties of HQ. Significant differences were also found in fermentation characteristics such as liquefying power (LP), saccharifying power (SP), fermenting power (FP), total acid content (TA) and liquor-producing power (LPP). The key fungi responsible for LP (5 genera), SP (3 genera), FP (1 genera), LPP (4 genera), and TA (4 genera) were determined using redundancy correlation analysis. Finally, Spearman’s correlation analysis indicated that LPP shows a strong positive correlation with FP and LP, while TA displays a strong negative correlation with FP. The results of this study may be utilized to prepare consistently high quality, next-generation HQ by better controlling fungal community structures, and to design fermentation processes for HQ wines with desirable oenological characteristics.

Comparison of microbial communities and amino acid metabolites in different traditional fermentation starters used during the fermentation of Hong Qu glutinous rice wine

Hong Qu glutinous rice wine (HQGRW) is produced from glutinous rice with the addition of the traditional fermentation starter Hong Qu (mainly Gutian Hong Qu and Wuyi Hong Qu) has been added. It is unpalatable and rejected by consumers because the bitter and umami tastes are too high. The objective of this study was to compare the dynamics of the microbial communities and amino acids especially those in the different traditional fermentation starters used during HQGRW fermentation, and elucidate the key microbes responsible for amino acids. Three widely-used types of Hong Qu starters were used which can make different bitterness and umami in our previous studies, namely, black Wuyi Hong Qu (WB), red Wuyi Hong Qu (WR), and Gutian Hong Qu (GT). The living dynamics of fungal and bacterial communities during the fermentation were determined by high-throughput sequencing and rRNA gene sequencing technology for the first time. The content of amino acids in the HQGRW were determined by reverse-phase high-performance liquid chromatography analysis. The results showed that there were differences between fungal communities during the fermentation process in Wuyi Hong Qu and Gutian Hong Qu starters and between bacterial communities during the fermentation process in the three types of starters. The amino acid content of the samples showed an increasing trend in each group. The total amino acids, as well as the bitter, sweet, umami, astringent amino acids, in the GT Hong Qu group increased more slowly during fermentation, as comparerd to those in WB and WR groups. Furthermore, Meyerozyma, Saccharomyces, Bacillus, Rhizopus, Pediococcus, Monascus, and Halomonas were strongly positively correlated with the content of bitter and umami amino acids (|r| > 0.6 with FDR adjusted P < 0.05) by Spearman’s correlation analysis. To conclude, these findings may contribute to a better understanding of the bitter and umami amino acid production mechanism during traditional fermentation and helpful in improving the taste of HQGRW.

Amino acid and microbial community dynamics during the fermentation of Hong Qu glutinous rice wine

Hong Qu glutinous rice wine (HQGRW) is typically very bitter after fermentation due to the presence of bitter amino acids (BAA). The amino acids are considered to primarily derive from the hydrolysis of protein from the raw material by microbial populations during fermentation, and temperature also has an important effect on bitter tastes. Here, the dynamics of fungal and bacterial communities during the traditional fermentation of HQGRW were investigated using high-throughput sequencing and RNA-based rRNA gene sequencing. Both principal component analysis (PCA) and hierarchical clustering analysis (HCA) revealed significant differences between the fungal and bacterial communities during fermentation at 20 °C and those performed at 25 °C and 30 °C. The growth of Saccharomyces and some LAB apparently inhibited the growth of several pernicious bacterial taxa including acetic acid bacteria. The amino acid contents of the samples all increased continuously under the different temperature conditions. Moreover, higher temperatures were associated with higher perceptual intensity of bitterness and contents of amino acids including bitter, sweet, umami, and astringent type amino acids as well as the total amino acid content during fermentation. Furthermore, the total BAA content was strongly and positively correlated with Pediococcus, Saccharomyces, Lactobacillus, Monascus, and Halomonas relative abundances, with correlations identified by |r| > 0.6 with P adjusted P < 0.05. In conclusion, these results contribute to a better understanding of the mechanisms underlying BAA production during the traditional fermentation of HQGRW and will help improve the quality and safety of these wines.

Dynamic changes of total acid and bacterial communities during the traditional fermentation of Hong Qu glutinous rice wine

Abstract Background Hong Qu glutinous rice wine (HQGRW) is brewed under non-aseptic fermentation conditions, so it usually has a relatively high total acid content. The aim of this study was to investigate the dynamics of the bacterial communities and total acid during the fermentation of HQGRW and elucidate the correlation between total acid and bacterial communities. Results The results showed that the period of rapid acid increase during fermentation occurred at the early stage of fermentation. There was a negative response between total acid increase and the rate of increase in alcohol during the early fermentation stage. Bacterial community analysis using high-throughput sequencing technology was found that the dominant bacterial communities changed during the traditional fermentation of HQGRW. Both principal component analysis (PCA) and hierarchical clustering analysis revealed that there was a great difference between the bacterial communities of Hong Qu starter and those identified during the fermentation process. Furthermore, the key bacteria likely to be associated with total acid were identified by Spearman's correlation analysis. Lactobacillus, unclassified Lactobacillaceae, and Pediococcus were found, which can make significant contributions to the total acid development (| r | > 0.6 with FDR adjusted P Conclusions This was the first study to investigate the correlation between bacterial communities and total acid during the fermentation of HQGRW. These findings may be helpful in the development of a set of fermentation techniques for controlling total acid. How to cite: Liang Z, Lin X, He Z, et al. Dynamic changes of total acid and bacterial communities during the traditional fermentation of Hong Qu glutinous rice wine. Electron J Biotechnol 2020;43. https://doi.org/10.1016/j.ejbt.2019.12.002

The dynamics of volatile compounds and their correlation with the microbial succession during the traditional solid-state fermentation of Gutian Hong Qu glutinous rice wine

Gutian Hong Qu glutinous rice wine (GHQW) is a representative alcoholic beverage in China. During its traditional brewing process, multiple strains are involved. However, the roles of these microorganisms, particularly their contributions to aroma formation, are poorly understood. Therefore, in this study, the dynamics of volatile components and microbial succession during the traditional fermentation process of GHQW were monitored, by using HS-SPME-GC/MS and high throughput 16 S rRNA and ITS sequencing techniques. Following a 32-day semi-open solid-state fermentation, massive changes in volatile profile and microbial communities were observed. Based on these, the potential correlations between volatile components and microorganisms during the fermentation were explored by using Pearson correlation analysis. It was revealed that 3 bacterial taxa, including Lactococcus lactis, Burkholderia gladioli and Cronobacter helveticus, and 5 fungal taxa, including Wickerhamomyces anomalus, Saccharomyces cerevisiae, Aspergillus vitricola, Aspergillus penicillioides and Monascus purpureus, showed strong connections with various volatile components. Thus these bacteria and fungi might play crucial roles in volatile components biosynthesis, and thus were preliminary considered as core functional microorganisms for GHQW brewing. The detailed information on the dynamic changes of volatile components and microbial communities throughout GHQW brewing can further expand our understanding of the formation mechanisms of GHQW aroma.

Changes in Cold and Hot Syndrome and Gastrointestinal Bacterial Community Structure in Mice by Intervention with Food of Different Nature.

To reveal the effect of foods with different natures on cold or hot syndrome and gastrointestinal bacterial community structure in mice. Forty-five 6-week-old male ICR Kunming mice of clean grade were divided into 5 groups, 9 per group, including the control (CK), hot nature herbs (HM), Hong Qu glutinous rice wine (RW), tea rice wine (TW), and cold nature herbs (CM) groups. Distilled water or corresponding herbs were administered to mice (0.01 mL/g body weight) in the 5 groups by gastric infusion respectively, once daily for 28 d. Appearance, behavior, and serum biochemical indicators, including 5-hydroxytryptamine (5-HT), thyroid stimulating hormone (TSH), noradrenaline (NE), cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), the hot nature index, as well as the gastrointestinal bacterial community structure were analyzed in all groups after treatment. After supplementation for 28 d, CM and TW mice showed different degrees of cold syndrome, and HM and RW mice showed different degrees of hot syndrome. Compared with the HM and RW mice, the TSH, NE, cAMP levels and hot nature indices in the CM and TW mice were significantly decreased and 5-HT and cGMP levels were significantly increased (P<0.05). There was no obvious change in appearance or behavior in CK mice. Results of clustering analysis showed that the gastrointestinal bacterial community structures were highly similar in TW and CM mice as well as in RW and HM mice, and that they were from the same branch, respectively, when the distance was 0.02. The key microbes associated with cold syndrome were Lachnospiraceae uncultured, Lactococcus, etc., and the key microbes associated with hot syndrome were S24-7 norank, Ruminococcaceae uncultured, etc. CONCLUSION: The interventions with different nature foods could change cold or hot syndrome in mice, leading to changes in gastrointestinal bacterial community structure.

Microbial communities and volatile metabolites in different traditional fermentation starters used for Hong Qu glutinous rice wine

Hong Qu glutinous rice wine (HQGRW), as one of the most typical representatives of Chinese rice wine, is generally brewed from glutinous rice by adding two traditional wine fermentation starters—Hong Qu (HQ) and Bai Qu (BQ). The objective of this study was to determine the microbial communities and volatile metabolites of different traditional fermentation starters for HQGRW, and elucidate the potential correlation between microbiota and volatile metabolites. Both heatmap and principal component analysis (PCA) revealed the significant variances in volatile profiles among different wine starters. Microbiological analysis based on high-throughput sequencing (HTS) technology demonstrated that both of bacterial and fungal communities varied significantly in different starters. HQ was dominated mainly by bacteria of Bacillus ginsengihumi (20.17%), Pantoea sp. (10.39%), Elizabethkingia sp. (5.52%), Streptococcus sp. (5.03%) Brevundimonas sp. (3.03%), Rickettsia prowazekii (2.94%), Thermus thermophilus (2.54%), Bacillus amyloliquefaciens (1.48%), Bacillus aryabhattai (1.42%); fungi of Monascus purpureus (39.7%), Aspergillus niger (27.35%), Xeromyces bisporus (8.39%), Aspergillus penicillioides (6.89%), Aspergillus flavus (2.33%) and Pichia farinose (0.79%). By contrast, BQ contained much higher proportions of bacteria of Lactococcus lactis (10.45%), Lactobacillus brevis (9.99%), Pediococcus pentosaceus (8.29%), Weissella paramesenteroides (6.69%), Lactobacillus fermentum (4.83%), Gluconobacter thailandicus (3.93%), Lactobacillus alimentarius (3.59%), fungi of Rhizopus arrhizus (31.47%), Saccharomycopsis fibuligera (27.86%), Aspergillus niger (20.81%), Issatchenkia orientalis (3.79%), Saccharomycopsis malanga (3.15%), Clavispora lusitaniae (2.29%), Candida tropicalis (1.47%), Saccharomyces cerevisiae (1.11%) and Rhizopus microsporus (0.57%). Furthermore, core functional microbiota that might contribute to volatile flavour development was explored through Spearman's correlation-based network analysis. Lactobacillus brevis, Lactobacillus alimentarius, Lactobacillus plantarum and Aspergillus niger were found to be strongly associated with acid compounds (FDR adjusted P < 0.01), while Pichia sp., Candida sp., Monascus purpureus, Lactobacillus brevis and Lactobacillus alimentarius were positively correlated with concentrations of aromatic esters associated with fruity and floral notes (FDR adjusted P < 0.01), implying that these microorganisms might make significant contributions to the flavour of rice wine. These findings demonstrated that the aromatic quality of HQGRW may be critically influenced by the microbiota in traditional fermentation starters. To conclude, this study would contribute to the development of novel defined starter cultures for improving the aromatic quality of HQGRW.

Exploring core functional microbiota responsible for the production of volatile flavour during the traditional brewing of Wuyi Hong Qu glutinous rice wine

The objective of this study was to explore the core functional microbiota for the production of volatile flavour during the traditional brewing of Wuyi Hong Qu glutinous rice wine, one of the most typical representatives of rice wine in China. Microbiological analysis based on high-throughput sequencing (HTS) technology demonstrated that bacteria of Lactobacillus, Bacillus, Leuconostoc, Lactococcus, Raoultella, Staphylococcus, Pediococcus, and Weissella, and fungi of Saccharomyces, Saccharomycopsis, Rhizopus, Monascus, Pichia, Wickerhamomyces, Candida, and Aspergillus were the predominant genera during the traditional fermentation process. Principal component analysis (PCA) based on the relative abundance showed that both of bacterial and fungal communities varied significantly in different fermentation phases. Some predominant microbial species or genera (including bacteria of Bacillus spp., Staphylococcus spp., Weissella spp., and P. acidilactici, and fungi of M. purpureus, R. oryzae, R. arrhizus var. arrhizus, and A. niger) were detected at the initial brewing stage, and their populations decreased as the fermentation progressed, while those of Lactobacillus, Gluconacetobacter, Leuconostoc, Pichia, Wickerhamomyces, and Saccharomyces increased to become the predominant genera at the final stage. A total of 79 volatile compounds were identified in traditional fermentation starters and during the traditional brewing process, mainly including esters, alcohols, acids, aldehydes, ketones, and phenols. Heatmaps and PCA also revealed the significant variances in the composition of volatile compounds among different samples. Furthermore, the potential correlations between microbiota succession and volatile flavour dynamics were explored through bidirectional orthogonal partial least squares (O2PLS) based correlation analysis. Three bacterial genera, namely, Gluconacetobacter, Lactobacillus, Lactococcus, and three fungal genera of Pichia, Wickerhamomyces, and Saccharomyces, were determined as the core functional microbiota for production of main volatile compounds in Wuyi Hong Qu glutinous rice wine. To conclude, information provided by this study is valuable to the development of effective strategies for the selection of beneficial bacterial and fungal strains to improve the quality of Wuyi Hong Qu glutinous rice wine.

Comparison study of the volatile profiles and microbial communities of Wuyi Qu and Gutian Qu, two major types of traditional fermentation starters of Hong Qu glutinous rice wine

Hong Qu, which mainly contains Monascus sp. and other microorganisms, as well as numerous microbial metabolites, is used as the fermentation starter of Hong Qu glutinous rice wine, a traditional alcoholic beverage. Two widely-used types of Hong Qu, namely Wuyi Qu (WYQ) and Gutian Qu (GTQ), were thoroughly compared for their fermentation properties, volatile profiles, and microbiota structures in this study. Significantly higher color value, glucoamylase and α-amylase activities were discovered in WYQ. And substantial variation in volatile components and microbial communities were also observed between them. It was identified that bacterial genus Burkholderia dominated GTQ (71.62%) and Bacillus dominated WYQ (44.73%), while Monascus purpureus was the most abundant fungal species in both types of starters (76.99%). In addition, 213 bacterial genera and 150 fungal species with low-abundance were also detected. Since the Linear Discriminant Analysis Effect Size algorithm, 14 genus-level bacterial taxa and 10 species-level fungal taxa could be utilized to distinguish these two types of starters. Moreover, the potential correlation of the volatile components and microbiota within WYQ and GTQ were further analyzed, by utilizing Partial Least Squares Discriminant Analysis. Ultimately, this study provides detailed insight into the volatile profiles and microbial communities presented in Hong Qu.

Evaluation of different PCR primers for denaturing gradient gel electrophoresis (DGGE) analysis of fungal community structure in traditional fermentation starters used for Hong Qu glutinous rice wine

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial community structure. However, when DGGE is applied to evaluate the fungal community of traditional fermentation starters, the choice of hypervariable ribosomal RNA gene regions is still controversial. In the current study, several previously published fungal PCR primer sets were compared and evaluated using PCR-DGGE, with the purpose of screening a suitable primer set to study the fungal community of traditional fermentation starters for Hong Qu glutinous rice wine.Firstly, different primer sets were used to amplify different hypervariable regions from pure fungal cultures. Except NS1/FR1+ and ITS1fGC/ITS4, other primer sets (NL1+/LS2R, NL3A/NL4GC, FF390/FR1+, NS1/GCFung, NS3+/YM951r and ITS1fGC/ITS2r) amplified the target DNA sequences successfully. Secondly, the selected primer sets were further evaluated based on their resolution to distinguish different fungal cultures through DGGE fingerprints. Three primer sets (NL1+/LS2R, NS1/GCFung and ITS1fGC/ITS2r) were finally selected for investigating the fungal community structure of different traditional fermentation starters for Hong Qu glutinous rice wine. The internal transcribed spacer (ITS) region amplified by ITS1fGC/ITS2r, which is more hypervariable than the 18S rRNA gene and 26S rRNA gene, provides an excellent tool to separate amplification products of different fungal species. Results indicated that PCR-DGGE profile using ITS1fGC/ITS2r showed more abundant fungal species than that using NL1+/LS2R and NS1/GCFung. Therefore, ITS1fGC/ITS2r is the most suitable primer set for PCR-DGGE analysis of fungal community structure in traditional fermentation starters for Hong Qu glutinous rice wine. DGGE profiles based on ITS1fGC/ITS2r revealed the presence of twenty-four fungal species in traditional fermentation starter. A significant difference of fungal community can be observed directly from DGGE fingerprints and principal component analysis. The statistical analysis results based on the band intensities of fungal DGGE profile showed that Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Rhizopus oryzae, Monascus purpureus and Aspergillus niger were the dominant fungal species.In conclusion, the comparison of several primer sets for fungal PCR-DGGE would be useful to enrich our knowledge of the fungal community structures associated with traditional fermentation starters, which may facilitate the development of better starter cultures for manufacturing Chinese Hong Qu glutinous rice wine.

Development of Reverse Transcription Quantitative Real-Time PCR (RT-qPCR) Assays for Monitoring Saccharomycopsis fibuligera, Rhizopus oryzae, and Monascus purpureus During the Traditional Brewing of Hong Qu Glutinous Rice Wine

In this study, reverse transcription quantitative real-time PCR (RT-qPCR) assays using species-specific primers were developed for the quantification of the three major fungal species (Saccharomycopsis fibuligera, Rhizopus oryzae, and Monascus purpureus) during the traditional brewing of Hong Qu glutinous rice wine. Species-specific primers (Sfi-F/Sfi-R, Ro-F/Ro-R, and Mp-F/Mp-R) targeting the ITS-5.8S rRNA gene and the beta-tubulin gene sequences were designed and their specificity was evaluated by RT-qPCR with the total RNA from fungal species closely related to the traditional brewing of Hong Qu glutinous rice wine. Results of RT-qPCR using species-specific primers showed 100 % inclusivity (positive signal in the presence of target complementary DNA (cDNA)) and 100 % exclusivity (negative signals in the presence of non-target template). The specificity of each primer set was also tested when the target cDNA was mixed with non-target cDNA, and results showed that there was no significant difference (P > 0.05) in threshold cycle (Ct) values obtained from pure target cDNA and target cDNA mixed with non-target cDNA. The detection limits of the established qPCR assays were determined to be 101 gene copies/μL for S. fibuligera and 102 gene copies/μL for R. oryzae and M. purpureus, respectively. Good correlations were obtained for the tested species between 102 and 108 gene copies/μL by qPCR analysis. Finally, the established RT-qPCR assays were applied to quantify viable S. fibuligera, R. oryzae, and M. purpureus during the traditional brewing of Hong Qu glutinous rice wine. This study standardized the RT-qPCR assays for dominant fungi associated with the traditional brewing of Hong Qu glutinous rice wine, and it also proved the usefulness of RT-qPCR assays for the quantification of live cells in multi-species samples. Results would show great value in scientifically understanding of the brewing mechanism of Hong Qu glutinous rice wine.

Microbiota associated with the starter cultures and brewing process of traditional Hong Qu glutinous rice wine.

Hong Qu glutinous rice wine (produced mainly in Fujian province, China) is a traditional alcoholic beverage, which is prepared by fermenting cooked rice using a starter containing Monascus purpureus. In this review, the microbial diversity of fermentation starters from Fujian province, including fungi, bacteria, and yeast, is analyzed in comparison with those of "nuruk" (a traditional starter for making alcoholic beverages in Korea). The bacterial organization of Hong Qu starters was vastly variable in species composition and dominated by Bacillus sp. Lactic acid bacteria were also found in some starters. In case of fungi, Monascus sp. was dominant, whereas non-Saccharomyces yeast such as Saccharomycopsis fibuligera was detected. The microorganisms found in the nuruk starter are, in general, not significantly diverse compared with those found in the Hong Qu starter, with the exception of Monascus sp.; however, Hong Qu and nuruk both contain their own unique microbiota, which are quite diverse from each other.

Characterization of the dominant bacterial communities of traditional fermentation starters for Hong Qu glutinous rice wine by means of MALDI-TOF mass spectrometry fingerprinting, 16S rRNA gene sequencing and species-specific PCRs

The main goal of this work was to investigate the bacterial flora present in the traditional fermentation starters using matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry fingerprinting combined with 16S rRNA gene sequencing and species-specific PCRs. A total of 1314 bacterial strains were obtained from ten kinds of representative wine fermentation starters (5 Hong Qu and 5 Yao Qu) and classified by MALDI-TOF mass spectrometry. Sixteen different bacterial species belonging to six different genera (Bacillus, Staphylococcus, Leuconostoc, Pediococcus, Lactobacillus and Lactococcus) were identified with high accuracy by comparing them with generated spectra of MALDI Biotyper database. Comparison of 16S rRNA gene phylogeny and MALDI-TOF mass spectrometry analysis showed that MALDI-TOF mass spectrometry fingerprinting was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate differentiation and classification of Bacillus species, especially for differentiating between Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus methylotrophicus. MALDI-TOF mass spectrometry was also found to provide valuable information at species levels for the genus of Lactobacillus. The identification revealed that most strains isolated from traditional fermentation starters were B. amyloliquefaciens (31.81%), followed by Bacillus megaterium (16.89%), Lc. lactis subsp. lactis (8.60%) and B. subtilis (8.14%). In the culture-dependent quantitative analysis, 69.01% of the detected isolates were Bacillus species, and 30.44% of the detected isolates belonged to the class of lactic acid bacteria (LAB), including Leu. mesenteroides, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, Pediococcus acidilactici, Lactobacillus coryniformis and Lc. lactis subsp. lactis. This study provides detailed evaluation of bacterial communities in the traditional fermentation starters for Hong Qu glutinous rice wine using a fast and cost-effective technique—MALDI-TOF mass spectrometry fingerprinting. Information provided by this study is fundamental and useful to develop effective strategies for the selection of beneficial bacterial strains and improve of quality of Hong Qu glutinous rice wine.

Unique sequence characteristics account for good DGGE separation of almost full-length 18S rDNAs.

A major limiting factor for DGGE-based microbial community studies is that the fragments should not be much longer than 500bp for successful analysis. However, relatively high-resolution was achieved based on DGGE of the long 18S rDNA fragment (>1500bp), which might be surprising due to the known decrease in DGGE resolution of DNA molecules with large melted regions. A unique sequence characteristic was found in a specific region (ca. 275bp, named the NS1-end region) of 18S rDNAs, and fungal communities separated from Hong Qu glutinous rice wine brewing system was used to reveal the relationship between high resolution capacity and the unique sequence characteristics. The results showed that DGGE separation of the long 18S rDNA fragments depended on their NS1-end regions. The region is composed of a sequence-variable and short-length GC-poor region (ca. 160bp) and a GC-rich region (ca. 110bp), which contribute to the high resolution capacity achieved for DGGE of the long 18S rDNA fragments. Thus DGGE of the long 18S rDNA fragment is recommended as a target fragment for studies of fungal communities whose 18S rDNAs possess similar sequence characteristics. Good resolution and almost full-length 18S rDNA sequences can thus be obtained to provide more accurate and reliable analysis of fungal communities. Since more sequences are obtained directly from the PCR product through the long rDNA fragment approach, this is a convenient and effective approach for sequence-based analysis without using other complementary methods such as an rDNA clone library method.

Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine

The main disadvantage of DNA-based quantification methods is their inability to distinguish between viable and non-viable cells, as dead cells may also retain a significant amount of DNA. To overcome this limitation, propidium monoazide (PMA) treatment was applied to effectively prevent amplification of DNA from dead cells. The objectives of this study were to develop and evaluate an accurate detection method that combines PMA staining with real-time qPCR to quantify viable dominant microorganisms during the traditional brewing of Hong Qu glutinous rice wine. Firstly, three methodological factors (concentration of PMA, incubation time and photoactivation time) were optimized for representative strains of different microbial species. For the tested microbial strains (Monascus purpureus, Saccharomyces cerevisiae and Lactobacillus plantarum), a treatment of 25 μM PMA for 20 min incubation followed by 10 min photoactivation was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction for the compromised cells. Compared with qPCR without PMA treatment, results of PMA-qPCR showed that the optimized PMA pretreatment can effectively exclude the dead microbial cells without any negative effects on the intact cells. The concentrations of viable cells calculated by PMA-qPCR were comparable (the same order of magnitude was obtained) with those obtained by culture-based method. Finally, the developed PMA-qPCR methods were successfully applied to monitor the total bacteria, LAB, fungi, yeasts, M. purpureus, S. cerevisiae and L. plantarum during the traditional brewing of Hong Qu glutinous rice wine. Both of the total bacterial and fungal populations increased significantly at the early brewing stage, but they decreased at the later brewing stage. PMA-qPCR results revealed that the predominant microorganisms varied in different brewing phases. L. plantarum and S. cerevisiae grew significantly and became the predominant bacterial and fungal species at the final stage of traditional brewing process, respectively. This study standardized the PMA-qPCR parameters for dominant microorganisms associated with the traditional brewing of Hong Qu glutinous rice wine, which would show great value in scientifically understanding of the brewing mechanism and provide useful information for the selection of beneficial microbial strains to improve wine quality.

Microbial community structure and dynamics during the traditional brewing of Fuzhou Hong Qu glutinous rice wine as determined by culture-dependent and culture-independent techniques

The aim of this study was to investigate the bacterial and fungal community structure and dynamics during the traditional brewing of Fuzhou Hong Qu glutinous rice wine through culture-dependent and culture-independent methods. The culture-independent DGGE profiles revealed the presence of eleven bacterial species (Janthinobacterium lividum, Lactobacillus plantarum, Weissella soli, Leuconostoc mesenteroides, Lactobacillus brevis, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis, Pediococcus acidilactici, Bacillus subtilis, Bacillus aryabhattai and Bacillus megaterium) and seven fungal species (Saccharomycopsis fibuligera, Pichia guilliermondii, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Rhizopus oryzae, Candida glabrata and Monascus purpureus) during the traditional brewing process. Furthermore, the microbial compositions of fermented samples were investigated using culture-dependent approach (conventional plate count technique combined with molecular identification). Results showed that the relative proportions of some fungal species (C. glabrata, W. anomalus, P. guilliermondii and S. fibuligera) and bacterial species (Lc. lactis subsp. lactis, Staphylococcus pasteuri, Bacillus sp., P. pentosaceus) detected at the early brewing stage decreased as the fermentation progressed, while S. cerevisiae, L. brevis, L. plantarum and Lactobacillus paracasei became the predominant species during the late brewing period. Some differences were also found between the profiles obtained by PCR–DGGE and culture-dependent technique. Comparative analysis revealed that some species (J. lividum, W. soli, B. aryabhattai and P. guilliermondii) could only be detected using PCR–DGGE technique. Conversely, some species (L. paracasei, S. pasteuri, Bacillus methylotrophicus, Mucor indicus and Aspergillus flavus) could only be detected by culture-dependent method. Therefore, the combination of culture-dependent and -independent approaches is quite necessary because they provided complementary information about the composition of the microbial community during the traditional brewing of Fuzhou Hong Qu glutinous rice wine. This study provides the first detailed evaluation of fungal community and dynamics in Fuzhou Hong Qu glutinous rice wine traditional brewing process using culture-dependent and -independent methods, which would facilitate scientific understanding of the traditional brewing mechanism and be useful for the selection of the suitable and beneficial strains to improve the wine quality.

Characterization of fungal community and dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine by means of multiple culture-independent methods

The objective of this study was to investigate the fungal community and dynamics during the traditional fermentation of Wuyi Hong Qu (black-skin-red-koji) glutinous rice wine using culture-independent approaches based on nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE), 18S ribosomal RNA (rRNA) gene clone libraries and species-specific quantitative PCR (qPCR). Both of nested PCR-DGGE and 18S rRNA gene clone libraries revealed that the predominant microorganisms varied in different brewing phases. The relative proportions of some fungal species (such as Monascus purpureus, Rhizopus oryzae, Pichia guilliermondii and Saccharomycopsis fibuligera) detected at the early brewing stage decreased as the fermentation progressed, while Saccharomyces cerevisiae became the dominant species at the later stage. The present study revealed that PCR-DGGE fingerprint and 18S rRNA gene clone libraries presented the similar results. Nevertheless, the differences of the fungal species detected by the two different molecular biological methods demonstrated that the combined approach of nested PCR-DGGE and 18S rRNA gene clone libraries would give a more comprehensive profile of the fungal dynamics than either alone. Finally, species-specific qPCR was also performed to quantify some dominant fungal species detected by nested PCR-DGGE and 18S rRNA gene clone libraries. Results showed that R. oryzae increased sharply and dominated during the early brewing period, but decreased continuously from day 10 to the end of the traditional brewing. S. fibuligera and P. guilliermondii were the dominant yeast species at early stage, while S. cerevisiae increased sharply during the early phase and became the primary species at the end of the traditional brewing process. This study provides the first detailed evaluation of fungal community and dynamics in Wuyi Hong Qu glutinous rice wine traditional brewing process using multiplex culture-independent methods, which would show great value in scientifically understanding of the brewing mechanism and be useful for the selection of the suitable and beneficial fungal strains to improve the wine quality.

Bacterial community dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine as determined by culture-independent methods

Wuyi Hong Qu (black-skin-red-koji) glutinous rice wine, as one of the most typical representatives of Hong Qu glutinous rice wine in Fujian province of China, is brewed under non-sterile and uncontrolled fermentation condition based on empirical knowledge, causing uncontrollability of fermentation process and instability of the final quality. The objective of this study was to investigate the bacterial dynamics during the traditional fermentation of Wuyi Hong Qu glutinous rice wine using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S ribosomal RNA (rRNA) gene clone libraries analysis.The DGGE profile indicated that the dominant bacterial species in the traditional wine fermentation starters were Pediococcus pentosaceus, Pediococcus acidilactici and Bacillus sp. (including Bacillus aryabhattai or Bacillus megaterium and Bacillus amyloliquefaciens). Bacterial dynamic obtained from the PCR-DGGE revealed the presence of Bacillus sp. and LAB (including Lactobacillus plantarum group, Lactobacillus brevis, P. acidilactici and P. pentosaceus) during the traditional fermentation process, but they varied in different brewing phases. The relative proportions of some bacterial species (such as Bacillus sp., P. acidilactici, L. brevis and P. pentosaceus) detected at early fermentation stage decreased as the fermentation progressed. While L. plantarum group was consistently detected with high light band intensity throughout the fermentation process. 16S rRNA gene clone libraries revealed that the two different molecular biological methods gave similar results, but clone library analysis was more representative of the bacterial community to some extent. For example, Leuconostoc mesenteroides was detected by 16S rRNA gene clone library but not discovered by bacterial DGGE profile throughout the whole fermentation process. Therefore, the combined approach of nested PCR-DGGE and 16S rRNA gene clone libraries would give a more comprehensive profile of the bacterial dynamics than either alone. Finally, species-specific multiplex PCR was also performed to confirm the L. plantarum group. Result showed that only L. plantarum can be detected from the total bacterial DNA extracted from samples of different fermentation phases.This is the first report to reveal the dynamics of bacterial species involved in Wuyi Hong Qu glutinous rice wine brewing process using culture-independent methods. It might be useful to control wine production systems and improve wine quality.

Yeast diversity of traditional alcohol fermentation starters for Hong Qu glutinous rice wine brewing, revealed by culture-dependent and culture-independent methods

Hong Qu glutinous rice wine, one of the most popular traditional rice wines in China, is brewed from glutinous rice with the addition of two traditional fermentation starters – Hong Qu (hóng qū) and Yao Qu (yào qū). The aim of this study was to investigate the yeast flora present in these fermentation starters using a combination of culture-dependent and -independent molecular biological methods.Molecular identification of a total of 500 yeast isolate from 10 kinds of representative wine fermentation starters (5 Hong Qu and 5 Yao Qu) was achieved by a combination of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of the internal transcribed spacer region ITS1-5.8S-ITS2 and sequencing of the D1/D2 domain of the 26S rRNA gene. The PCR–RFLP method generated 13 different ITS/RFLP profiles. Sequencing identification allowed identifying 13 different ITS/RFLP profiles into 12 different species of yeast belong to eight different genera (Pichia, Saccharomyces, Candida, Cryptococcus, Rhodotorula, Sporobolomyces, Rhodosporidium and Saccharomycopsis). Species diversity was thus considerable: Saccharomycopsis fibuligera was the most commonly isolated specie, followed by Saccharomyces cerevisiae. On the other hand, the yeast diversity associated with wine starters for Hong Qu glutinous rice wine was also investigated through culture-independent method using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) patterns and sequencing of the DNA bands. The results revealed by culture-independent method were almost the same as that of culture-dependent methodology. However, several yeast species can not be detected by traditional microbiological procedures but can be detected using PCR–DGGE analysis. Similarly, some species could only be detected by culture-dependent method. For example, Sporobolomyces nylandii was detected existing in wine starter samples (Q7 and Q10) through culture-dependent method, but not detected in wine starters for Hong Qu glutinous rice wine through fungal PCR–DGGE assay. Therefore, it is recommended that polyphonic approaches based on culture-dependent and culture independent methods seem the best strategy to get a more comprehensive picture of the microbial diversity in traditional wine starters. These results enrich our knowledge of rice wine-related yeasts, and can be used to promote the development of the traditional brewing industry.