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

Food Control 57 (2015) 216e224

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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 Xu-Cong Lv a, b, c, Zhi-Chao Chen a, Rui-Bo Jia b, c, Zhi-Bin Liu a, Wen Zhang a, Shao-Jun Chen c, Ping-Fan Rao a, Li Ni a, * a b c

Institute of Food Science and Technology, College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian 350108, China National Engineering Research Center of JUNCAO Technology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 January 2015 Received in revised form 25 March 2015 Accepted 31 March 2015 Available online 25 April 2015

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 culturedependent 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 eDGGE and culture-dependent technique. Comparative analysis revealed that some species (J. lividum, W. soli, B. aryabhattai and P. guilliermondii) could only be detected using PCReDGGE 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 culturedependent 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. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Microbial community dynamics Fuzhou Hong Qu glutinous rice wine PCRedenaturing gradient gel electrophoresis (PCReDGGE) Conventional plate count method Amplified ribosomal DNA restriction analysis (ARDRA) Species-specific PCR

1. Introduction

* Corresponding author. Institute of Food Science and Technology, Fuzhou University, Xue Yuan Road, University Town, Fuzhou, China. Tel./fax: þ86 591 2286 6378. E-mail addresses: [email protected], [email protected] (L. Ni). http://dx.doi.org/10.1016/j.foodcont.2015.03.054 0956-7135/© 2015 Elsevier Ltd. All rights reserved.

ng jiǔ in Fuzhou Hong Qu glutinous rice wine (also called q
ıng ho China) is known as the authentic Hong Qu glutinous rice wine mainly because it has a long brewing history, which can be traced back to the Spring and Autumn period of ancient China, Since 1995, the Fujian provincial government has honored Fuzhou Hong Qu

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glutinous rice wine as a precious gift for foreign guests to spread the Minyue culture (the culture originated in southeast area of China from the Spring and Autumn Period to Han Dynasty) and promoted the city brand of Fujian province. Nowadays, as one of the geographical indications protection products in China, Fuzhou Hong Qu glutinous rice wine top the list of the most popular Chinese rice wines because it possesses bright red color, low sugar and unique taste, and has multiple health-promoting functions due to the addition of Hong Qu (also called Chinese red yeast rice), which can significantly reduce total cholesterol, LDL cholesterol, and total triacylglycerol concentrations, and also possessing antioxidant activities (Taira, Miyagi, & Aniya, 2002; Yang, Tseng, Lee, & Mau, 2006). As one of the most typical representatives of Hong Qu glutinous rice wine, Fuzhou Hong Qu glutinous rice wine is brewed with the addition of two traditional wine fermentation starters d Hong Qu ng qu
) and Yao Qu (y
). It is difficult to control the (ho ao qu fermentation process due to the diverse microbial compositions of the traditional starters for Hong Qu glutinous rice wine. Our previous studies have demonstrated that traditional fermentation starters contained various types of microorganisms, such as filamentous fungi, yeasts and bacteria (Lv, Huang, Zhang, Rao, & Ni, 2012, 2013; Lv, Weng, Zhang, Rao, & Ni, 2012). Besides, traditional brewing under non-sterile and uncontrolled environmental conditions based on empirical knowledge often leads to the uncontrollability of fermentation process and the instability of the wine quality between different batches. What is more, some pathogens may involve in wine making process and pose a potential threat to consumer's health. Application of defined starter cultures is one of the approaches that could be used to standardize fermentations and ensure the stability in quality. Therefore, it is of primary importance to monitor the microbial dynamics during the traditional fermentation processes in order to provide high-quality and safe products for consumers. In previous study, we had used a combined approach of PCReDGGE and conventional culturedependent method to investigate the yeast diversity of traditional alcohol fermentation starters for Hong Qu glutinous rice wine brewing (Lv, Huang, Zhang, et al., 2013). And we also used a combined approach of PCReDGGE and 16S/18S ribosomal RNA gene clone libraries to investigate the bacterial and fungal dynamics during the traditional fermentation of Wuyi Hong Qu glutinous rice wine (Lv, Huang, Chen, et al., 2013; Lv et al., 2015). To the best of our knowledge, no study has been conducted to investigate the changes in the bacterial and fungal communities during the traditional brewing of Fuzhou Hong Qu glutinous rice wine. Therefore, the study focused on the microbial community and dynamics during the traditional brewing of Fuzhou Hong Qu glutinous rice wine using a combination of culture-dependent and culture-independent methods. This study would facilitate the development of defined starter cultures and the better control of Fuzhou Hong Qu glutinous rice wine brewing, and also allow for the successful understanding of the microbial mechanisms in traditional fermentation. 2. Materials and methods 2.1. The traditional brewing of Fuzhou Hong Qu glutinous rice wine Wine fermentation starters [Gutian Hong Qu and Yao Qu] were purchased from the wine factories in Gutian county of Fujian province in China. To initiate the brewing, 4.0 kg of glutinous rice was washed and soaked in water for 12 h at room temperature, and then steamed for 1 h at 100  C. After being cooled to room temperature, the steamed rice was mixed with traditional fermentation starter (400 g, of which Yao Qu 200 g and Gutian Hong Qu 200 g) and

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transferred to a traditional Chinese wine jar. Finally, sterilized water was added up to 6 L and started the wine fermentation at 15e20  C for 42 days. Triplicate independent brewing was conducted. 2.2. Sample collection Wine mash samples (10 g) from each fermentation jar were aseptically collected at different phases of wine fermentation (1, 6, 11, 16, 21, 31 and 42 days), transferred to sterilized bottles, thoroughly mixed, and finally stored at 20  C before further analysis. 2.3. Enumeration and isolation of different groups of microorganisms Powdered fermentation starter samples (10 g) and wine mash samples (10 g) were homogenized by adding 90 ml of sterile tryptone salt (TS) solution [0.85% (wt/vol) NaCl; 0.1% tryptone (wt/ vol)] and mixing for 2 min using a Mix-1 stomacher blender (AES, Combourg). A 10-fold dilution series from 101 to 108 were made and plated in triplicate using spreading plate method on plates of PCA medium (Merck) for the viable mesophilic bacteria population, MRS agar medium (De Man Rogosa Sharpe, Merck) containing 10 mg/ml cycloheximide as selective agents for lactic acid bacteria population, Czapek-Dox agar (Difco, Detroit, Michigan, USA) supplemented with 10 mg/ml chloramphenicol as selective agents for molds (Pitt & Hocking, 1997), YM agar medium (Difco, Becton and Dickinson Company, Sparks, MD) supplemented with 0.005% (w/v) oxytetracycline and gentamicin sulfate (Oxoid Ltd, Hampshire) as selective agents for yeasts. Mesophilic bacteria were enumerated after incubation for 48 h at 37  C under aerobic conditions. Lactic acid bacteria were counted after incubation for 72 h at 30  C under anaerobic condition using AnaeroPack (Mitsubishi Gas Chemical, Tokyo). Yeasts and filamentous fungi were enumerated after incubation for 48 h at 28  C under aerobic conditions. For each sample, presumptive microbial colonies were picked up from the suitable diluted plates and purified at least two times by streaking. Selected strains were stored for a long term at 20  C in a 10% (w/v) dilution of the corresponding broth medium supplemented with 20% (w/v) glycerol. 2.4. Genotyping of bacteria and fungi based on amplified ribosomal DNA restriction analysis (ARDRA) Bacterial and fungal DNAs of the pure cultures were extracted according to Jeyaram, Mohendro Singh, Capece, and Romano (2008) and Zhu, Qu, and Zhu (1993), respectively. For the discrimination of bacterial isolates, the 16Se23S rDNA intergenic transcribed spacer of bacteria was amplified and digested separately with Hinf I, Hae III and Hind III (Invitrogen, USA) as previously described by Jeyaram et al. (2008). For the discrimination of fungal isolates, the ITS1-5.8S rDNA-ITS2 region was amplified with the primers ITS1/ITS4 as described previously (White, Bruns, Lee, & Taylor, 1990). The PCR products were digested with three restriction endonucleases (Hinf I, Hae III and Cfo I enzymes) according to the manufacturer's instructions (Invitrogen, USA), respectively. The restriction fragments were separated on a horizontal electrophoresis apparatus (Bio-Rad, USA) on 2.0% (w/v) agarose gels in 0.5 TBE buffer. The gels were stained with ethidium bromide and visualized under UV light. 2.5. Sequencing of the 16S rRNA gene and ITS1-5.8S rRNA gene intergenic region The 16S rRNA gene of bacteria was amplified with the PCR condition described in previous study (Lv, Huang, Zhang, et al.,

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2013), and the ITS1-5.8S rRNA gene intergenic region gene of fungi was amplified by the method reported by Kurtzman and Robnett (1998). PCR products were then gel-purified with GFX™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences AB, Uppsala, Sweden), according to the manufacturer's instructions. Purified PCR products were directly sequenced using the ABI prism 3730 DNA analyzer (Applied Biosystems, USA). Finally, the DNA sequences were read and edited by Chromas 2.1. Blast searches of sequences were performed at the National Centre for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov/ BLAST/). 2.6. Microbial community analysis through PCReDGGE 2.6.1. Total DNA extraction from wine mash of traditional brewing process Fungal DNA extraction was carried out using a benzyl chloride method (Zhu et al., 1993). While bacterial DNA was extracted using Universal Genomic DNA Extraction Kit Ver: 3.0 (Takara, Dalian, China) after glass bead beating to disrupt the cell walls. The DNAs were then subjected to further purification by adsorption to a silica matrix using Fermentas (USA) DNA extraction kit according to the manufacturer's instruction. The yields and purity of the extracted DNA were analyzed spectrophotometrically at 260 and 280 nm using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The DNA extracts were stored in a freezer until analysis. 2.6.2. Nested PCReDGGE analysis For analysis of bacterial diversity, primer sets 27F (50 AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -GGCTACCTTGTTACGACTT-30 ) (Lane, 1991) were used to amplify the nearly complete 16S rRNA encoding gene under conventional PCR conditions in the first PCR step. Subsequently, the PCR product was diluted and amplified with DGGE primers 338f-GC/518r (Cocolin, Manzano, Aggio, Cantoni, & Comi, 2011) targeting the V3 region of the 16S rRNA gene to create a DNA fragment suitable for DGGE analysis. For analysis of fungal diversity, PCR amplification of the D1/D2 domain of the 26S rRNA gene was performed using the fungal universal primer set NL1/NL4 (Kurtzman & Robnett, 1998) in the first step, followed by nested PCR using the DGGE primer sets NL1GC/LS2 as described previously (Cocolin, Bisson, & Mills, 2000). The PCR products were analyzed by DGGE using a DCode apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Samples were applied to 8% (w/v) polyacrylamide gels in 0.5 TAE. Optimal separation was achieved by DGGE trial with a 40e60% ureaeformamide denaturing gradient (100% denaturant corresponding to 7 M urea and 40% (w/v) deionized formamide) using the primers 338f-GC/518r. For PCR amplicons obtained with the primers NL1GC/LS2, electrophoresis was performed in an 8% (w/v) polyacrylamide gel with a denaturing gradient ranging from 20% to 50%. Electrophoretic runs were carried out for 8 h at 120 V at 60  C, and then gels were stained with ethidium bromide solution (5 mg/ ml) for 30 min. The stained gels were photographed with a UV transillumination and analyzed using Fragment Analysis (Amersham Biosciences, Sweden). 2.6.3. Sequencing of the DGGE fragments and identification of the bands The major bands on the DGGE gels were excised and cloned into Escherichia coli plasmids with the pMD 19-T cloning kit (Takara, Dalian, China) following the manufacturer's instructions. The resultant clones were reamplified and checked again in DGGE gels, and finally sequenced in Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Sequences were compared with the

GenBank database using the BLAST program (http://www.ncbi.nlm. nih.gov/BLAST/) to identify the closest related species. 2.7. Species-specific PCR for identification of the Lactobacillus plantarum group and the Lactobacillus casei group Multiplex species-specific PCRs targeting the recA gene were performed for further discrimination when the results of 16S rDNA sequence analysis were not enough to identify strains at the species level. Strains belonging to the L. plantarum group, i.e. L. plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum, were distinguished using a multiplex PCR assay with the recA genebased primers paraF, pentF, planF and pREV as described by Torriani, Felis, and Dellaglio (2001). Strains belonging to the L. casei group, i.e. L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus, were distinguished using species-specific PCRs as described by Ward and Timmins (1999). 3. Results 3.1. Bacterial community and dynamics assessed by nested PCReDGGE The DGGE fingerprints based on primers 338f-GC/518r are presented in Fig. 1, and the sequencing results of the highlighted bands are listed in Table 1. Results showed that a total of fourteen bacterial species (Janthinobacterium lividum, Bacillus ginsengihumi, Bacillus subtilis, Janthinobacterium sp., Weissella soli, Bacillus aryabhattai, Bacillus megaterium, Bacillus amyloliquefaciens, Lactobacillus brevis, Pediococcus pentosaceus, L. plantarum, Leuconostoc mesenteroides, Lactococcus lactis subsp. lactis and Pediococcus acidilactici) were detected. The bacterial community of Gutian Hong Qu was mainly composed of J. lividum, B. ginsengihumi, B. subtilis and Janthinobacterium sp. The bacterial community of Yao Qu was composed of

Fig. 1. Bacterial DGGE profiles (40e60% denaturant gradient) of amplified 16S rDNA (V3 region) fragments from traditional fermentation starters (a) and traditional brewing of Fuzhou Hong Qu glutinous rice wine (b). Lanes 1, 6, 11, 16, 21, 31 and 42 refer to the fermented samples as described in Materials and methods. Q1: Gutian Hong Qu; Q2: Yao Qu. M1 and M2 represent DGGE ladders. The identities of the excised 16S rDNA (V3 region) fragments are described in Table 1.

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Table 1 Phylogenetic identification results of selected bands from the bacterial DGGE fingerprint in Fig. 1. Sample

Band no.

Closest relative (NCBI accession no.)

Similarity (%)

Hong Qu

a1 a2

Janthinobacterium lividum (JF970593) Janthinobacterium sp. (KJ867066) Uncultured bacterium clone (KJ417632) Bacillus ginsengihumi (HQ219845) Bacillus subtilis (JF719789) Weissella soli (GU470977) Lactobacillus brevis (JN089401) Pediococcus pentosaceus (JN039354) Bacillus aryabhattai (JF951729) Bacillus megaterium (HE578782) Bacillus megaterium (JN106424) Bacillus aryabhattai (HQ857752) Pediococcus acidilactici (HQ603181) Bacillus amyloliquefaciens (CP002927) Lactobacillus plantarum (JN222930) Lactobacillus plantarum (JN222930) Janthinobacterium lividum (JF970593) Weissella soli (GU470977) Leuconostoc mesenteroides (GU458344) Lactobacillus brevis (JN089401) Pediococcus pentosaceus (JN039354) Pediococcus pentosaceus (JN039354) Bacillus aryabhattai (JF951729) Bacillus megaterium (HE578782) Lactococcus lactis subsp. lactis (FR873574) Bacillus subtilis (JF719789) Bacillus megaterium (JN106424) Bacillus aryabhattai (HQ857752) Bacillus aryabhattai (JN084155) Bacillus megaterium (JN084146.1) Pediococcus acidilactici (HQ603181)

100% 100% 100% 100% 100% 100% 99% 100% 100% 100% 99% 99% 99% 100% 99% 99% 100% 100% 100% 99% 100% 99% 100% 100% 100% 100% 99% 99% 100% 100% 99%

Yao Qu

a3 a4 b1 b2 b3 b4 b5

Fermented samples of different brewing stages

b6 b7 c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14

seven species, including W. soli, L. brevis, P. pentosaceus, B. aryabhattai, B. megaterium, P. acidilactici and B. amyloliquefaciens. Bacterial DGGE fingerprints (Fig. 1) also showed significant changes as the traditional brewing progressed. The early fermentation period (day 1) was characterized by Bacillus species, including B. aryabhattai and B. megaterium. However, B. aryabhattai vanished on the sixth day of the traditional fermentation process, and B. megaterium disappeared after eleven days of brewing, indicating the decrease of their proportion in the total population of rice wine mash. P. pentosaceus and Lc. lactis subsp. lactis were the two predominant bacterial species that could be consistently detected after 6 days of fermentation. However, it was interesting to find that the band intensity of Lc. lactis subsp. lactis (band c10) decreased on the sixteenth day of the brewing process. On the contrary, the band intensity of L. plantarum (band c1) and L. brevis (band c6) gradually increased from the eleventh day to the end of traditional brewing, while P. pentosaceus (band c7 and c8) was consistently detected with high light intensity throughout the fermentation process but suddenly decreased at the end. All these indicated that the dominance of bacterial species varied during the traditional fermentation of Fuzhou Hong Qu glutinous rice wine.

Among these, W. anomalus, R. mucilaginosa, C. glabrata, S. cerevisiae and M. purpureus were the dominant fungal species in Gutian Hong Qu. In the aspect of Yao Qu, the predominant fungal species included W. anomalus, P. guilliermondii, S. fibuligera, R. oryzae, and A. oryzae. A significant transition of fungal community can be observed from Fig. 2. DGGE fingerprints illustrated a higher fungal diversity at the beginning of the traditional brewing process, which was characterized by seven fungal species, including S. fibuligera, R. oryzae, W. anomalus, P. guilliermondii, C. glabrata, S. cerevisiae and M. purpureus. However, fungal species diversity decreased as the fermentation progressed. P. guilliermondii vanished on the eleventh day. C. glabrata as well as M. purpureus disappeared after 21 days of fermentation, indicating the decrease of their proportion in the total population of rice wine mash. The band intensity of S. fibuligera, W. anomalus and R. oryzae decreased gradually toward the end of the fermentation process, which was likely due to the increase of ethanol concentration produced by S. cerevisiae. As fermentation continued, S. cerevisiae increased gradually during the early period of traditional brewing, and eventually took over and dominated the fermentation at the end.

3.2. Fungal community and dynamics assessed by nested PCReDGGE

3.3. Bacterial and yeast compositions determined by conventional plate count method

The fungal DGGE fingerprints based on primers NL1GC/LS2 are shown in Fig. 2, and the sequencing results of the highlighted bands are listed in Table 2. A total of nine fungal species were detected, i.e., Monascus purpureus, Rhizopus oryzae, Aspergillus oryzae, and six yeast species including Saccharomyces cerevisiae, Wickerhamomyces anomalus, Pichia guilliermondii, Rhodotorula mucilaginosa, Candida glabrata and Saccharomycopsis fibuligera. All of them were detected in the traditional fermentation starters (either Hong Qu or Yao Qu).

A total of 6 genera and 13 species of bacteria, including the Bacillus (5 species), Lactobacillus (3 species), Pediococcus (2 species), Leuconostoc (1 species), Lactococcus (1 species) and Staphylococcus (1 species) genera, were identified (Fig. 3). All of these isolates yielded similarity scores of over 99% to their corresponding species. However, 16S rDNA sequence analysis were not enough to identify isolates belonging to the L. plantarum group and the L. casei group. In this study, species-specific multiplex PCR yielded amplicons

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Fig. 2. Fungal DGGE profiles (20e50% denaturant gradient) of partial 26S rRNA gene fragments obtained from traditional fermentation starters (a) and traditional brewing process (b), with primers NL1GC/LS2. Lanes corresponding to traditional fermentation starters and fermented samples of different brewing stages are marked at the top. Q1: Gutian Hong Qu; Q2: Yao Qu. The identities of the excised bands are described in Table 2.

about 318 bp (as shown in Fig. 4a), confirming the presence of L. plantarum during the traditional fermentation process (Torriani et al., 2001). Therefore, the L. plantarum group detected by conventional plate count method was finally identified belonging to L. plantarum. Similarly, species-specific PCRs were also conducted exclusively for the identification of the L. casei group isolates from traditional fermentation process using the primer pairs casei/Y2, para/Y2 and rham/Y2, which were specific to the L. casei group for

L. casei, L. paracasei and L. rhamnosus, respectively (as shown in Fig. 4b). These strains yielded an amplicon of the expected size (290 bp) by species-specific PCR analysis using the species-specific primer pairs para/Y2, but no amplification products were obtained when using the other two species-specific primer pairs (casei/Y2 and rham/Y2) (data not shown). The amplification profiles of specific PCR using primers para/Y2 was consistent with the profile of L. paracasei obtained by Ward and Timmins (1999), demonstrating the presence of L. paracasei during the traditional fermentation process. The relative abundance analysis of bacterial species revealed that Bacillus sp. and lactic acid bacteria (LAB) (including L. plantarum, L. brevis, Staphylococcus pasteuri, Lc. lactis subsp. lactis, L. paracasei, Leu. mesenteroides and P. pentosaceus) dominated during the traditional brewing process (Supplementary material S1 and S2). Significant changes of bacterial community during the traditional brewing process can be observed (Fig. 3). B. megaterium and B. ginsengihumi appeared frequently at the beginning of the traditional brewing process (day 1). However, they disappeared after 11 days' fermentation. In addition, five kinds of LAB (L. plantarum, L. brevis, Lc. lactis subsp. lactis, L. paracasei and Leu. mesenteroides) were found during the traditional fermentation process, although they were not detected in traditional wine starters by plate count method. The relative abundance of Lactobacillus spp. (L. plantarum, L. brevis and L. paracasei) increased rapidly during the middle fermentation period and became the three predominant bacterial species at the end of traditional brewing process. The fungal isolates from traditional starters and samples of traditional brewing process were grouped by ARDRA of the ITS-5.8S rDNA region and presented twelve restriction fragment patterns (Fig. 5), corresponding to 12 distinct species (99e100% homology). W. anomalus, R. mucilaginosa, S. cerevisiae, M. purpureus, R. oryzae, Aspergillus flavus and Aspergillus niger were the predominant fungal species identified in Gutian Hong Qu (Supplementary material S1 and S2). While Yao Qu was characterized by six fungal species, including S. cerevisiae, S. fibuligera, R. oryzae, A. oryzae, Emericella nidulans and Mucor indicus. Relative abundance analysis was only conducted for yeasts because it was difficult to count the colonyforming units of filamentous fungi using the plate count method (Fig. 6). Except R. mucilaginosa, all of the yeast species detected in Hong Qu and Yao Qu were also identified during the fermentation process. Non-Saccharomyces yeasts, including S. fibuligera, W. anomalus and C. glabrata, were primarily dominant during the early fermentation period. As the traditional fermentation

Table 2 Phylogenetic identification results of selected bands from the fungal DGGE fingerprint in Fig. 2. Sample

Band no.

Closest relative (NCBI accession no.)

Similarity (%)

Hong Qu

a1 a2 a3 a4 a5 b1 b2 b3 b4 b5 c1 c2 c3 c4 c5 c6 c7

Wickerhamomyces anomalus (FJ972217) Rhodotorula mucilaginosa (GU373744) Candida glabrata (HM591682) Saccharomyces cerevisiae (JN083825) Monascus purpureus (AF365024) Saccharomycopsis fibuligera (HM107786) Rhizopus oryzae (EU862181) Wickerhamomyces anomalus (FJ972217) Pichia guilliermondii (JF439367) Aspergillus oryzae (KJ650341) Saccharomycopsis fibuligera (HQ199205) Rhizopus oryzae (EU862181) Wickerhamomyces anomalus (HQ199214) Pichia guilliermondii (HQ199203) Candida glabrata (HQ199207) Saccharomyces cerevisiae (HQ199204) Monascus purpureus (AF365024)

100% 100% 100% 100% 99% 100% 99% 100% 100% 99% 100% 99% 100% 100% 100% 100% 99%

Yao Qu

Fermented samples of different brewing stages

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221

Fig. 3. The relative abundance (%) of bacterial species present in samples of wine starters and traditional fermentation process, identified with culture-dependent method.

progressed, S. cerevisiae eventually took over and dominated the traditional fermentation, and finally became the primary species during the late fermentation period. 4. Discussion In this study, both culture-dependent and culture-independent analyses were performed. Significant differences occurred between the results obtained with each method. Discrepancies between culturing and DGGE results have also been reported before (Kafili et al., 2009; Lv, Huang, Zhang, et al., 2013). It's worth noting that some species (such as Bacillus methylotrophicus, L. paracasei and S. pasteuri) detected through culture-dependent method may not

Fig. 4. Species-specific PCRs for identification of isolates belonging to the L. plantarum group and the L. casei group. Panel a, species identification of the L. plantarum group isolates from traditional fermentation process by species-specific multiplex PCR (targeting recA gene). Panel b, species identification of the L. casei group isolates from traditional fermentation process by species-specific PCRs.

be recognized by PCReDGGE assay (Supplementary material S2). This discrepancy can be introduced by DGGE in a number of ways, including the selective amplification, co-migration of PCR products from different species, and different efficiencies of genomic DNA extraction from different species (Abriouel et al., 2006; Katano & Fukui, 2003; Sekiguchi, Tomioka, Nakahara, & Uchiyama, 2001). Therefore, it is recommended that the DGGE method should be supplemented with culture-dependent method to reduce omissions and generate a better view of microbial diversity in naturally fermented foods (Cocolin, Campolongo, Alessandria, Dolci, & Rantsiou 2011; Nielsen et al., 2007). In addition, an obvious advantage of culture-dependent methodology is that it allows a collection of pure cultures, which can be used for further selection of suitable strains to design mixed starters capable of providing beneficial contributions to wine quality. In the present study, bacterial and fungal isolates from traditional fermentation starters and traditional fermentation process were discriminated and grouped by amplified ribosomal DNA restriction analysis (ARDRA), which was found to be a useful, simple and rapid method compared to current molecular and phenotypic techniques. 16Se23S rRNA internal transcribed spacer and ITS15.8S rRNA-ITS2 gene region have more sequence variability and as a result can effectively differentiate the closely related bacteria and fungi. Representative isolates from each group were chosen for full-length sequencing of their 16S rDNA or ITS-5.8S rRNA gene regions. However, due to high genetic homology inside certain bacterial genera, the 16S rRNA gene may not be especially useful for the discrimination of closely-related species (Berthier & Ehrlich, 1998), such as the L. plantarum group (i.e. L. plantarum, L. pentosus and L. paraplantarum) and the L. casei group (i.e. L. casei, L. paracasei and L. rhamnosus). Therefore, species-specific PCRs were performed for further discrimination when 16S rDNA sequence analysis was not enough to identify species belonging to the L. plantarum group and the L. casei group. Microbial dynamics obtained from bacterial DGGE fingerprints and the conventional culturing method combined with molecular classification identification showed that Bacillus sp. and LAB dominated in traditional fermentation starters and throughout the fermentation process, but they varied in different brewing phases. It has been reported by earlier researchers that the bacterial microflora of rice wine starters and rice wine traditional fermentation were highly variable in species composition dominated by Bacillus species and LAB (Sujaya, Nocianitri, & Asano, 2010; Thanh,

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Fig. 5. Grouping fungal isolates from traditional fermentation starters and samples of traditional fermentation process by ARDRA of ITS-5.8S rDNA PCR products using three restriction enzymes (Hae III, Hinf I and Cfo I). M-DNA Ladder marker, with fragments of 2000, 1000, 750, 500, 250 and 100 bp. Profile A1-Pichia anomala (AY349448); A2-Candida glabrata (JN093145); A3-Saccharomyces cerevisiae (EU789403); A4-Monascus purpureus (AY498579); A5-Rhizopus oryzae (EU484274); A6-Saccharomycopsis fibuligera (EU798695); A7-Emericella nidulans (EF652458); A8-Aspergillus flavus (EF661566); A9-Aspergillus oryzae (EU409806); A10-Mucor indicus (DQ118994); A11-Rhodotorula mucilaginosa (AF444655); A12-Aspergillus niger (FJ717685).

Maile, & Tuan, 2008). Members of the Bacillus spp. might be beneficial because of their ability to secrete a wide range of hydrolytic enzymes, including amylases, proteases and fibrinolytic enzyme. These hydrolytic enzymes may play an important role in the traditional fermentation process (Amoa-Awua & Jakobsen, 1995). In addition, Bacillus spp. can produce nitrogenous flavor compounds such as diverse pyrazines (Li et al., 2011; Zhang, Wu, Zhang, Hu, & Zhang, 2011; Zheng, Tabrizi, Nout, & Han, 2011), but their potential contribution to the final flavor of Fuzhou Hong Qu glutinous rice wine is still unknown and requires further study. The predominance of LAB in fermented foods is commonly due to their ability to tolerate low pH (Abriouel et al., 2006), produce a variety of antimicrobial substances such as bacteriocin, and suppress the growth of pathogens and the toxigenic and spoilage organisms (Castellano, Belfiore, Fadda, & Vignolo, 2008; Jones, Hussein, Zagorec, Brightwell, & Tagg, 2008). This is probably the reason why potential foodborne pathogens were not detected during the traditional fermentation of Fuzhou Hong Qu glutinous rice wine in the present study. Lactobacillus species, the predominant LAB species during the traditional brewing process, may play an important role during liquor manufacture because lactic acid

was identified to be a precursor of ethyl lactate, one of the major flavor compounds in strong aromatic liquor (Fan & Qian, 2005). It was also reported as dominant function bacteria in several typical Daqu starters for Chinese liquor (Xiu, Guo, & Zhang, 2012). Lc. lactis was firstly detected in Hong Qu glutinous rice wine. As one of the homofermentative LAB species, Lc. lactis has been isolated from sour cassava (Brauman, Keleke, Malonga, Miambi, & Ampe, 1996) and Obushera (Muyanja, Narvhus, Treimo, & Langsrud, 2003). Leu. mesenteroide, one of the predominant bacterial species during the middle fermentation period, has been also isolated during the fermentation of shochu, wine, and alcoholic beverages, such as Mecal, Pulque and Boza (Endo & Okada, 2005; Escalante et al., 2008; ez-Zapata, Rojas-Herrera, Rodríguez-Luna, & LarraldeNarva Corona, 2010). It was reported by other authors that Leuconostoc spp. are indigenous to plant material and often dominate the initial stages of fermentation before being succeeded by more acid tolerant Lactobacillus spp. (Ampe, Omar, Moizan, Wacher, & Guyot, 1999; Stiles & Holzapfel, 1997). Lactobacillus spp. (L. plantarum, L. brevis and L. paracasei), often exist in the starters used for alcoholic beverages and fermented foods (Jin, Kim, Jin, Eom, & Han, 2008; Tamang et al., 2007; Thanh et al., 2008), became the

Fig. 6. The relative abundance (%) of yeast species present in samples of wine starters and traditional fermentation process as revealed by culture-dependent method. Quantitative analysis was only conducted for yeasts because it was difficult to count the colony-forming units of filamentous fungi using the plate count method.

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predominant bacterial species at the end of traditional fermentation process, where they replaced the less acid tolerant heterofermentative LAB that mainly initiate fermentation but begin to reduce in numbers as the pH decreases (Hammes, Haller, & Ganzle, 2003; Li, 2004; Plengvidhya, Breidt, Lu, & Fleming, 2007). Many traditional alcoholic products are co-fermented by LAB and yeasts. Yeasts stimulate LAB growth by supplying essential metabolites including pyruvate, amino acids and vitamins (Jespersen, 2003). In the current study, a total of five yeasts (including S. cerevisiae, P. guilliermondii, S. fibuligera, W. anomalus and C. glabrata) were detected involving in the traditional fermentation process. Non-Saccharomyces yeasts, including P. guilliermondii, S. fibuligera, W. anomalus and C. glabrata, dominated in the earlier stages of traditional brewing process. P. guilliermondii, which was also found in Wheat Qu for Shaoxing rice wine (Xie et al., 2007) and Manipur ‘Hamei’ (a traditional starter used for rice wine production in India) (Jeyaram et al., 2008), could produce volatile phenols (4-ethylguaiacol (Suezawa & Suzuki, 2007), 4-ethylphenol (Dias et al., 2003) and important esters in the initial stage of wine fermentation (Moreira et al., 2011)). However, strains of P. guilliermondii have also been reported to produce a high concentration of isobutyl alcohol, isoamyl alcohol and unpleasant odor consisting of isobutyric acid, isovaleric acid and 2-phenylethanol (Wah, Walaisri, Assavanig, Niamsiri, & Lertsiri, 2013). S. fibuligera, the most abundant yeast species detected in Yao Qu and at the beginning of traditional brewing (day 1) in the current study, has a strong capability to produce various enzymes, particularly a-amylase, glucoamylase, acid proteases and b-glucosidase (Chi et al., 2009). In the brewing of rice wine, S. fibuligera can metabolize the native starch into maltose, dextrin, and glucose, indicating this species may play an important role during the initial stage of alcoholic fermentation. It had also been reported to be the principal amylolytic microorganism in traditional alcohol starters such as Loog-pang, Marcha and banh men (Limtong, Sintara, Suwannarit, & Lotong, 2002; Thanh et al., 2008; Tsuyoshi et al., 2005). S. cerevisiae, the most effective ethanol producer known so far, was detected in a low frequency at early stage of traditional brewing. However, it took over the fermentation processes, due to its efficient fermentation catabolism and ethanol tolerance in the stressful conditions of the fermenting mash. M. purpureus and R. oryzae were the predominant filamentous fungi existed in traditional wine starters and during the traditional brewing of Fuzhou Hong Qu glutinous rice wine. R. oryzae, a strong amylase producer and frequently found in amylolytic fermentation starters for rice wine brewing (Dung, Rombouts, & Nout, 2007; Thanh et al., 2008; Wang, Shi, & Gong, 2008; Xie et al., 2007), was the most frequent species detected in Yao Qu in our previous study (Lv, Weng, et al., 2012; Lv, Huang, et al., 2012). Previous study by others also pointed out that R. oryzae can produce volatile compounds, such as ethanol, 2-methyl-1-butanol and 3-methyl-1butanol (Christen, Bramorski, Revah, & Soccol, 2000; Feng, Larsen, & Schnürer, 2007), which were the main volatile compounds in Chinese yellow rice wine. As the most dominant fungal species in Hong Qu, Monascus species can esterify mixed acids and ethanol to form esters such as ethyl caproate that contribute to the aroma of the Chinese liquor (Du, Tang, Gu, Liu, & Ren, 2005), and also produce citric acid, acetic acid and succinic acid, as well as substances with physiological activity that facilitates the formation of unique flavor and functional components (Taira et al., 2002; Yang et al., 2006). A. oryzae, well known for its capacity to secrete large amounts of hydrolytic enzymes (such as proteases, amylase, glutaminase and metallopeptidase), has been safely used in the production of sake, soybean paste, soy sauce and Chinese rice wine (Bourdichon et al., 2012; Zhang, Guan, Cao, Xie, & Lu, 2012).

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5. Conclusion In the present study, the dynamic changes and diversity of microbial community succession in the traditional brewing of Fuzhou Hong Qu glutinous rice wine were systematically investigated for the first time using a combination of culture-dependent and culture-independent methods. Moreover, results would be useful for optimization of microbial composition and quality control of wine starters to upgrade Fuzhou Hong Qu glutinous rice wine. Further studies are required to understand the microbial interactions of LAB, yeasts and filamentous fungi to clarify the factors that contribute to the distinctive flavor of Fuzhou Hong Qu glutinous rice wine. Acknowledgments This work was financially supported by grants from the National Natural Science Foundation of China (No. 31371820, 31171733), Projects of Science and Technology of Fujian Education Dept., China (JA14107) and Scientific Research Start-up Funding of Fujian Agriculture and Forestry University (grant no. KXML2012A). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodcont.2015.03.054. References ~ amero, M., Keleke, S., & Ga lvez, A. Abriouel, H., Ben Omar, N., Lucas, R., Martínez-Can (2006). Culture-independent analysis of the microbial composition of the Afgue  by using three different DNA rican traditional fermented foods poto and de extraction methods. International Journal of Food Microbiology, 111, 228e233. Amoa-Awua, W. K. A., & Jakobsen, M. (1995). The role of Bacillus species in the fermentation of cassava. Journal of Applied Bacteriology, 79, 250e256. Ampe, F., Omar, N. B., Moizan, C., Wacher, C., & Guyot, J. P. (1999). Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations. Applied and Environmental Microbiology, 65, 5464e5473. Berthier, F., & Ehrlich, S. D. (1998). Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region. FEMS Microbiology Letters, 161, 97e106. Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J. C., Gerds, M. L., Hammes, W. P., et al. (2012). Food fermentations: microorganisms with technological beneficial use. International Journal of Food Microbiology, 154, 87e97. Brauman, A., Keleke, S., Malonga, M., Miambi, E., & Ampe, F. (1996). Microbiological and biochemical characterization of cassava retting, a traditional lactic acid fermentation for foo-foo (cassava flour) production. Applied and Environmental Microbiology, 62, 2854e2858. Castellano, P., Belfiore, C., Fadda, S., & Vignolo, G. (2008). A review of bacteriocinogenic lactic acid bacteria used as bioprotective cultures in fresh meat produced in Argentina. Meat Science, 79, 483e499. Chi, Z. M., Chi, Z., Liu, G., Wang, F., Ju, L., & Zhang, T. (2009). Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnology Advances, 27, 423e431. Christen, P., Bramorski, A., Revah, S., & Soccol, C. R. (2000). Characterization of volatile compounds produced by Rhizopus strains grown on agro-industrial solid wastes. Bioresource Technology, 71, 211e215. Cocolin, L., Bisson, L. F., & Mills, D. A. (2000). Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiology Letters, 189, 81e87. Cocolin, L., Campolongo, S., Alessandria, V., Dolci, P., & Rantsiou, K. (2011). Culture independent analyses and wine fermentation: an overview of achievements 10 years after first application. Annals of Microbiology, 61, 17e23. Cocolin, L., Manzano, M., Aggio, D., Cantoni, C., & Comi, G. (2011). A novel polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) for the identification of Micrococcaceae strains involved in meat fermentations. Its application to naturally fermented Italian sausages. Meat Science, 58(1), 59e64. Dias, L., Dias, S., Sancho, T., Malferito-Ferreira, M., Lourero, V., & Loureiro, V. (2003). Identification of yeasts isolated from wine-related environments and capable of producing 4-ethylphenol. Food Microbiology, 20, 567e574. Du, L.-Q., Tang, C., Gu, J.-Q., Liu, L., & Ren, X.-Z. (2005). Exploration to the esterification property of esterase produced by Monascus spp. China Brewing, 142, 23e24 (in Chinese).

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