英文论文

Original PaperAnalysis of the fungi community inmultiple- and single-grains Zaopei from a Luzhou-flavor liquor distillery in western ChinaSi Shi1, Lei Zhang1, Zheng-yun Wu1, Wen-xue Zhang1 , Yu Deng2,Fang-da Zhong3 and Jia-min Li4(1) College of Light Textile and Food Science, Sichuan University, Chengdu, 610065,People’s Republic of China(2) Biogas Institute of Ministry of Agriculture, 610065 Chengdu, People’s Republic of China(3) Xijiu Co.Ltd of Maotai Group, 564622 Guizhou, People’s Republic of China(4) Sichuan Tuopai Liquor Co.Ltd, 629209 Shehong, People’s Republic of ChinaWen-xue ZhangEmail: zwxtl@Received: 29 September 2010 Accepted: 28 December 2010 Published online:5 January 2011AbstractThe 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.Keywords Zaopei– Multiple-grains – Single-grains – DGGE – FungiIntroductionChinese Luzhou-flavor liquor is the most accepted distilled spirits in China, and its processing technique is considered to be unique in the world. The production process of Luzhou-flavor liquor are characterized by recycling fermentation process, in which part of fresh grains are mixed with the fermented grains and steamed rice husks because of some starch remaining in the spent grains. And the following distillation of fermented grains is combined with the process of cooking newly added grains. This unique production method gives this Chinese liquor special feature described as highly flavored, sweet and refreshing. In addition to the unique processing technique, microbes growing in Zaopei are believed to play key roles in the fermentation and many researchers have chosen it as the research target (Zhang et al. 2009; Wu et al. 2009). To date, the presence of bacteria, moulds and yeasts have been revealed by both cultured and uncultured methods. For bacterial community, bacteria found in Zaopei include genera such as Bacillus, Lactobacillus, Flavobacteria and Streptococcus, among which the previous two are considered as the dominant genera (Wang et al. 2008a, b; Xiang et al. 2005a, b; Zhang et al. 2005). The fungal microbes.discovered in Zaopei mainly included the genera such as Aspergillus, Mucor, Rhizopus, Monascus, Trichoderma,Saccharomyces, Hansenula, Candida, Issatchenkia, Pichia, and Torulaspora, among which Saccharomyces and Aspergillus strains were usually found to be predominant (Shi 1986; Wu et al. 2006; Zhang et al. 2006, 2007; Zhou et al. 2010).Although many microorganisms in Zaopei have been identified, the results by cultured and molecular methods such as 16S rRNA or 18S rRNA gene library analysis can not be considered sufficient to achieve a confident investigation of microbial population in fermented samples. In most cases, they are time-consuming and can not directly reflect and present the relationship between the succession of microbes communities and environment factors during fermentation such as air conditions, raw materials compositions, fermentation location, and the type of liquor produced. Alternatively, the denaturing gradient gel electrophoresis (DGGE) has been demonstrated to be a rapid and valuable tool to analyze and directly monitor the succession of the microbial communities of Chinese liquor (Zhang et al. 2005, 2007). The PCR-DGGE is useful to testthe diversity of communities structure, showing phylogenetic affiliation of different samples and identifying species found in the DGGE profiles.In this study, we chose the fungi communities in multiple-grains and single-grains Zaopei from a Chinese Luzhou-flavor Liquor Distillery as the research target. The goal of our study was to employ PCR-DGGE technique to analyze the relationship between fungi communities and environment factors including the raw materials and fermentation location. In order to get a better understanding of dominant fungal communities between these two kinds of Zaopei, we also identified the intense bands from DGGE gels, combining with the traditional culturally identification.Materials and methodsSamples of ZaopeiZaopei fermented grains (Fig. 1) were taken and used for DNA extraction. The content of multiple-grains Zaopei was the mixture of sorghum, rice, glutinous rice, maize and wheat, and that for single-grains Zaopei only contained sorghum. These Zaopei samples respectively came from the two positions (top layer and bottom layer) of fermentation pits filled with multiple-grains or single-grains. The sample bags were sealed without air and stored at −20°C until analyzed.Fig. 1 The flow diagram of Luzhou-flavor liquor productionFungi isolation and identificationFor each sample, homogenization and dilutions in sterile water were prepared and spread for cultivation on two different media: YPD-agar medium (1% yeast extract; 2% peptone; 2% glucose and 2% agar) and PDA–agar medium (20% potato; 2% glucose and 2% agar). Plates added withBenzylpenicillin sodium salt (1600U/ml) were incubated at 28°C for5 days for colony development. After viable counts, the morphologically different colonies from both YPD and PDA were purified by repetitive streaking on both mediums. All isolates were preserved on agar slants, stored at 4°C and subcultured every 3 months. The identification procedure was carried out according to the “Fungi identification manual” (Wei 1979).DNA extraction from samples of ZaopeiThe extraction of fungi DNA was carried out according to the manual of Yeast DNA Extraction Kit (Sangon, Shanghai, China). Samples were stored at –20°C.PCR primers and DNA amplificationIn this study, fungi community was amplified using a pair of universal primers U1f (5′-GTGAAATTGTTGAAAGGGAA-3′) and U2r-GC(5′-ACTCCTTGGTCCGTGTT-3′) with a GC clamp added to improve the sensitivity in the detection of mutations by DGGE (Ruan et al. 2009). The length of the target region is about 260 bp corresponding to positions 403 and 662 of the fungi 28S rDNA. Reactions were carried out in 50 μl volume containing template DNA 100 ng, 2 μl of each primer (10 μM) and 2 × PCR MasterMix (TIANGEN, Beijing, China). The final concentration of, PCR MasterMix is 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mm MgCl20.05 U/μl Taq-polymerase and 0.25 mM of each dNTP. A “touchdown” PCR program was performed to increase the specificity of amplification for environment samples and to reduce the spurious formation by products (Elena et al. 2007). The initial annealing temperature was 60°C for 1 min, which was reduced by 1°C every two cycles for 20 cycles. Finally, 10 cycles were performed at 50°C for 45 s. The denaturation and extension for each cycle were carried out at 94°C for 45s and 72°C for 1 min, respectively, while an initial 5 min denaturation at 94°C and a final extension at 72°C for 150 s. PCR products were routinely checked on 1.2%(w/v) agarose gels in 1 × TAE buffer.DGGE analysisDGGE was carried out on 50 μl volume of each PCR amplicon using the DCode TM Universal Mutation Detection System (BioRad, CA, USA). Electrophoresis was performed in 1 mm polyacrylamide gel (8% [w/v]acrylamide–bisacrylamide 37.5:1) containing a 30–60% urea–formamidedenaturing gradient (100% corresponds to 7 M urea and 40% [v/v] formamide), increasing in the electrophoretic run direction. The gel was subjected to constant voltage of 200 V for 4 h at 60°C. After electrophoresis, they were stained for 45 min in a SYBR Green I solution (AMRESCO, USA) and analyzed under UV illumination. Bands patterns of the DGGE profiles were cluster analyzed by the Quantity One software (version 4.5, BioRad) according to the manufacture’s instructions.Isolation and sequencing of DGGE bandsBands of interest from DGGE were excised, as described by Wang et al. (2009), reamplified and cloned into pUCm-T vertor (Sangon, Shanghai, China) according to the instructions. Selecting right competent cells with the correct inserts to sent for sequencing by Sangon. Searches in the GeneBank by BLAST program were performed to determine the closest known relatives.Nucleotide sequence accession numbersThe sequences obtained in this study have been submitted in the DDBJ of Japan under the accession numbers AB576177- AB576182.ResultsDGGE analysisThe DGGE fingerprints mainly consisted of 9 different bands as shown in Fig. 2. The dendrogram of these bands was divided into cluster 1 and 2 by cluster analysis (Fig. 3). These differences of cluster analysis were obviously caused by the bands position and density. Band 3 was visible in all these four samples. The greatest variability at species level was detected in multiple-grains samples. Excepting band 5, most bands existed in samples III and IV. The bands 3, 4, 6 and 8 of samples III were much more intense than that in the same positions of other samples. It indicated the microorganisms corresponding to the 4 bands were dominant in the top layer of multiple-grains sample. These results clearly showed the mixed fermented grains were more easily to cause the diversity of fungi community. Additionally, Band 1 was mainly detected in sample IV. This might suggest the importance of this microorganism in bottom layer fermentation, when there were not many fungi types compared with the top layer’s. Band 2 was only visible in samples III, it could possibly become the special indicator to distinguish different samples. Themicroorganisms corresponding to bands 3, 7 and 9 might have better adaptability in different materials for the existence in the two kinds of Zaopei. These two kinds of Zaopei samples in cluster 1 and 2 explained the evolutionary distance of 0.37 and 0.45, respectively. The distance between the two clusters was only 0.23. This illustrated the different fermented materials had bigger impact on fungi communities than fermentation location, and different location in multiple-grains Zaopei was more influential compared with single-grains Zaopei.Fig. 2 The DGGE fingerprint of fungi community in both multiple-grains and single-grains Zaopei (I. the bottom layer of single-grains Zaopei; II. the top layer of single-grains Zaopei; III. the top layer of multiple-grains Zaopei; IV. the bottom layer of multiple-grains Zaopei)Fig. 3 Cluster analysis of DGGE banding patterns of fungi communities of different ZaopeiIdentification analysisThe obtained sequences were analyzed by the BLAST program at the NCBI web site to search similar ones. The identification obtained by sequencing the bands cut from the gel is reported in Table 1. As shown in Fig. 2, the top layer of multiple-grains Zaopei sample presented the larger number of intense bands. The closest relative corresponding to band 2 was Candida etchellsi and appeared only in sample III. Bands 6 and 8, representing Debaryomyces hansenii and Pichia farinose respectively, were detected in multiple-grains Zaopei (samples III and IV), both had larger number in sample III. Band 7 was visible in all these samples except sample I and its closest relative was Candida lactis-condensi. Band 1 corresponding to Thermomyces lanuginosus occurred in multiple-grains Zaopei, and Thermoascus aurantiacus corresponding to band 9 was mainly present in the top layer of Zaopei (samples II and III). Unfortunately, the sequences obtained for bands 3, 4 and 5 were not possible to identify the corresponding species. Since the quality and the length of the sequences obtained were not satisfactory, this caused much trouble in the process of reamplify and clone.Table 1 The identities of bands obtained from DGGEThe numbers of I, II, III and IV represent what samples the bands derive from. (I. the bottom layer of single-grains Zaopei; II. the top layer of single-grains Zaopei; III. the top layer of multiple-grains Zaopei; IV. the bottom layer of multiple-grains Zaopei)These results indicated that the highest diversity in terms of types and quantities of fungi community in the top layer of multiple-grains Zaopei and the genera of Debaryomyces, Candida and Pichia became dominant.Comparison between DGGE method and thetraditional culture methodFungi isolates were separated directly from the Zaopei samples according to the traditional methods. As summarized in Table 2, six different genera were detected and identified. In contrast, more than six genera of fungi were detected by DGGE technique. Nevertheless, the results from both methods suggested the genera of Debaryomyces, Pichia and Candida were the dominant community in multiple-grains Zaopei. A few other fungi communities such as Citeromyces and Aspergillus were discovered by using the traditional methods. Overall, the combination of both methods could make the investigation more comprehensive and objective. Yet the molecular method of DGGE could avoid the mistakes in the identification of species due to the problems related to biochemical tests, and more fungi communities especially the uncultured ones could be detected more easily.DiscussionTo test the applicability of the method for investigating the differences between multiple-Zaopei and single-grains Zaopei, four different samples were analyzed. The results obtained are shown in Fig. 2 and 3, and the identification is reported in Table 1and 2. It is important to underline that the application of DGGE technique allows a better understanding of the ecology of Zaopei. Since the results are simple to interpret, and the DGGE fingerprint can directly show the differently structural characteristics and the community diversity. Despite the evidence of different cases of co-migration (Liang et al. 2008), it has to emphasize the dominant strains during fermentation can be easily detected and identified.In this study, we observed the obvio us differences of fungi community’s structure and diversity between multiple-grains and single-grains Zaopei. By the cluster analysis of bands patterns, we knew more clearly that aclose mutual relationship amongst the fermentation environment in different Zaopei, and it in turn had an impact on the evolution of microorganisms. The fungi type and quantity of multiple-grains Zaopei were more abundant than those of single-grains Zaopei, and the communities in the top were richer than those in the bottom. These results suggested the main fungi communities had more dependence on oxygen, and a variety of mixed grains might provide adequate nutrition for fungi growth and metabolism. Theoretically speaking, molecular methods could detect more genera and species including cultured and uncultured microorganisms. However, compared with the species results by traditional methods, the bands types of fungi communities on the gels was not so abundant in this study. This which might be caused by primers choice, because different DNA templates might not have the same ability to combine with one pair of primers, it was necessary to design some other primers in order to investigate the fungi communities as possible as it could (Zhang et al. 2007).Some have reported the genus Aspergillus was the main microbes responsible for starch saccharification in Zaopei (Luo 2003; Shi 1986; Xiong 1994). This is in agreement with our study results by traditional methods. However, we also detected two kinds of thermophilic fungi (Thermomyces lanuginosus and Thermoascus aurantiacus, respectively) in the samples by molecular methods, which have seldom been reported for Chinese liquor so far. Some researchers found that the Thermomyces lanuginosus can produce the protease such as xylanase and α-amylase with thermal stability (Li et al. 1997, 2004; Liu and Li 1998), and it may provide more sugar sources after biochemical reactions for alcohol production. Therefore, the thermophilic fungi detected in our study may have special function in degrading biopolymers, and we should continue to study it in depth. Additionally, the genera of Candida, Debaryomyces and Pichia were discovered as the dominant microbes in this study, which was close to the results by Zhou et al. (2010). Those strains in the report got adapted to anaerobic conditions and grew well as pH value was 2.7–3.1 with comparatively low nutritional needs. The properties of those strains might be closely associated with the production process ofmultiple-grains Luzhou-flavor liquor. The genus Candida commonly produces polyalcohol and furanone in the fermentation and it may do the main work in the formation of aromatic compounds; the genus Debaryomyces is considered as non-beneficial microorganism for liquor production because it consumes the sugar without alcohol fermentation and the reasons for its existence in the Zaopei should be studied more in order to improve the liquor quality (Wei 1979). The genera of Candida etchellsi and Pichia farinosa were mainly detected in the top layer of multiple-grains sample. It suggested their specifical function in forming flavor compounds orstarch saccharification in the spent grains. This might explain the top layer liquor always had higher quality and output. In general, the genus Saccharomyces is dominant microorganism during ethanol fermentation and plays key roles (Shi 1986). However, our study failed to detect it. These results seemed to suggest that Saccharomyces may be not necessary for the fermentation of Chinese Luzhou-flavour liquor. Perhaps, initial templates DNA ratio and templates competition may affect the detection of microorganisms present at low abundance in complex samples (Luciana et al. 2006).These preliminary results suggest that DGGE analysis is a suitable tool to provide an overview of the fungi community and to differentiate some of species involved in Zaopei. Because the relationship between bacteria communities and fermentation environment in Zaopei is also very important, more research is still needed in order to obtain a satisfactory and whole characterization of microorganisms in different Zaopei.Acknowledgments This work was financially supported by the Specialized Project (No.2010ZZ6004-01) of Science and Technology Bureau of Guizhou Province, China and by the Program (08ZC0880) of Science and Technology Bureau of Sichuan Province, China.ReferencesElena DM, Danilo E, Salvatore C (2007) Yeast dynamics during spontaneous wine fermentation of the catalanesca grape. Int J Food Microbiol 117:201–210Li DC, Yang YJ, Peng YL et al (1997) Purification and properties of a thermostable α-amylase from the thermophilic fungus Thermomyces lanuginosus. 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