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Food Control 57 (2015) 216e224

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Food Control

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Microbial community structure and dynamics during the traditionalbrewing of Fuzhou Hong Qu glutinous rice wine as determinedby 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 Institute of Food Science and Technology, College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian 350108, Chinab National Engineering Research Center of JUNCAO Technology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, Chinac College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China

a r t i c l e i n f o

Article history:Received 8 January 2015Received in revised form25 March 2015Accepted 31 March 2015Available online 25 April 2015

Keywords:Microbial community dynamicsFuzhou Hong Qu glutinous rice winePCRedenaturing gradient gelelectrophoresis (PCReDGGE)Conventional plate count methodAmplified ribosomal DNA restrictionanalysis (ARDRA)Species-specific PCR

* Corresponding author. Institute of Food Science aversity, Xue Yuan Road, University Town, Fuzhou, C6378.

E-mail addresses: fst.fzu@gmail.com, nili@fzu.edu.

http://dx.doi.org/10.1016/j.foodcont.2015.03.0540956-7135/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

The aim of this study was to investigate the bacterial and fungal community structure and dynamicsduring the traditional brewing of Fuzhou Hong Qu glutinous rice wine through culture-dependent andculture-independent methods. The culture-independent DGGE profiles revealed the presence of elevenbacterial species (Janthinobacterium lividum, Lactobacillus plantarum, Weissella soli, Leuconostoc mesen-teroides, Lactobacillus brevis, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis, Pediococcus acid-ilactici, 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). Re-sults 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 speciesduring the late brewing period. Some differences were also found between the profiles obtained by PCReDGGE 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 Aspergillusflavus) could only be detected by culture-dependent method. Therefore, the combination of culture-dependent and -independent approaches is quite necessary because they provided complementary in-formation about the composition of the microbial community during the traditional brewing of FuzhouHong Qu glutinous rice wine. This study provides the first detailed evaluation of fungal community anddynamics in Fuzhou Hong Qu glutinous rice wine traditional brewing process using culture-dependentand -independent methods, which would facilitate scientific understanding of the traditional brewingmechanism and be useful for the selection of the suitable and beneficial strains to improve the winequality.

© 2015 Elsevier Ltd. All rights reserved.

nd Technology, Fuzhou Uni-hina. Tel./fax: þ86 591 2286

cn (L. Ni).

1. Introduction

Fuzhou Hong Qu glutinous rice wine (also called q�ıng h�ong jiǔ inChina) is known as the authentic Hong Qu glutinous rice winemainly because it has a long brewing history, which can be tracedback to the Spring and Autumn period of ancient China, Since 1995,the Fujian provincial government has honored Fuzhou Hong Qu

X.-C. Lv et al. / Food Control 57 (2015) 216e224 217

glutinous rice wine as a precious gift for foreign guests to spreadthe Minyue culture (the culture originated in southeast area ofChina from the Spring and Autumn Period to Han Dynasty) andpromoted the city brand of Fujian province. Nowadays, as one of thegeographical indications protection products in China, FuzhouHongQu glutinous rice wine top the list of the most popular Chinese ricewines because it possesses bright red color, low sugar and uniquetaste, and has multiple health-promoting functions due to theaddition of Hong Qu (also called Chinese red yeast rice), which cansignificantly reduce total cholesterol, LDL cholesterol, and totaltriacylglycerol concentrations, and also possessing antioxidant ac-tivities (Taira, Miyagi, & Aniya, 2002; Yang, Tseng, Lee, & Mau,2006).

As one of the most typical representatives of Hong Qu glutinousrice wine, Fuzhou Hong Qu glutinous rice wine is brewed with theaddition of two traditional wine fermentation starters d Hong Qu(h�ong q�u) and Yao Qu (y�ao q�u). It is difficult to control thefermentation process due to the diverse microbial compositions ofthe traditional starters for Hong Qu glutinous rice wine. Our pre-vious studies have demonstrated that traditional fermentationstarters contained various types of microorganisms, such as fila-mentous fungi, yeasts and bacteria (Lv, Huang, Zhang, Rao, & Ni,2012, 2013; Lv, Weng, Zhang, Rao, & Ni, 2012). Besides, traditionalbrewing under non-sterile and uncontrolled environmental con-ditions based on empirical knowledge often leads to the uncon-trollability of fermentation process and the instability of the winequality between different batches. What is more, some pathogensmay involve in wine making process and pose a potential threat toconsumer's health. Application of defined starter cultures is one ofthe approaches that could be used to standardize fermentationsand ensure the stability in quality. Therefore, it is of primaryimportance to monitor the microbial dynamics during the tradi-tional fermentation processes in order to provide high-quality andsafe products for consumers. In previous study, we had used acombined approach of PCReDGGE and conventional culture-dependent method to investigate the yeast diversity of traditionalalcohol fermentation starters for Hong Qu glutinous rice winebrewing (Lv, Huang, Zhang, et al., 2013). And we also used a com-bined approach of PCReDGGE and 16S/18S ribosomal RNA geneclone libraries to investigate the bacterial and fungal dynamicsduring the traditional fermentation ofWuyi Hong Qu glutinous ricewine (Lv, Huang, Chen, et al., 2013; Lv et al., 2015). To the best of ourknowledge, no study has been conducted to investigate the changesin the bacterial and fungal communities during the traditionalbrewing of Fuzhou Hong Qu glutinous rice wine.

Therefore, the study focused on the microbial community anddynamics during the traditional brewing of Fuzhou Hong Quglutinous rice wine using a combination of culture-dependent andculture-independent methods. This study would facilitate thedevelopment of defined starter cultures and the better control ofFuzhouHong Qu glutinous rice wine brewing, and also allow for thesuccessful understanding of the microbial mechanisms in tradi-tional 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] werepurchased from the wine factories in Gutian county of Fujian prov-ince in China. To initiate the brewing, 4.0 kg of glutinous rice waswashed and soaked inwater for 12 h at room temperature, and thensteamed 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

transferred to a traditional Chinese wine jar. Finally, sterilized waterwas added up to 6 L and started the wine fermentation at 15e20 �Cfor 42 days. Triplicate independent brewing was conducted.

2.2. Sample collection

Wine mash samples (10 g) from each fermentation jar wereaseptically collected at different phases of wine fermentation (1, 6,11, 16, 21, 31 and 42 days), transferred to sterilized bottles, thor-oughly mixed, and finally stored at �20 �C before further analysis.

2.3. Enumeration and isolation of different groups ofmicroorganisms

Powdered fermentation starter samples (10 g) and wine mashsamples (10 g) were homogenized by adding 90 ml of steriletryptone 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 10�1 to 10�8 were madeand plated in triplicate using spreading plate method on plates ofPCAmedium (Merck) for the viable mesophilic bacteria population,MRS agar medium (De Man Rogosa Sharpe, Merck) containing10 mg/ml cycloheximide as selective agents for lactic acid bacteriapopulation, Czapek-Dox agar (Difco, Detroit, Michigan, USA) sup-plemented with 10 mg/ml chloramphenicol as selective agents formolds (Pitt & Hocking, 1997), YM agar medium (Difco, Becton andDickinson Company, Sparks, MD) supplemented with 0.005% (w/v)oxytetracycline and gentamicin sulfate (Oxoid Ltd, Hampshire) asselective agents for yeasts. Mesophilic bacteria were enumeratedafter incubation for 48 h at 37 �C under aerobic conditions. Lacticacid bacteria were counted after incubation for 72 h at 30 �C underanaerobic condition using AnaeroPack (Mitsubishi Gas Chemical,Tokyo). Yeasts and filamentous fungi were enumerated after incu-bation for 48 h at 28 �C under aerobic conditions. For each sample,presumptive microbial colonies were picked up from the suitablediluted plates and purified at least two times by streaking. Selectedstrains were stored for a long term at�20 �C in a 10% (w/v) dilutionof the corresponding broth medium supplemented with 20% (w/v)glycerol.

2.4. Genotyping of bacteria and fungi based on amplified ribosomalDNA restriction analysis (ARDRA)

Bacterial and fungal DNAs of the pure cultures were extractedaccording to Jeyaram, Mohendro Singh, Capece, and Romano(2008) and Zhu, Qu, and Zhu (1993), respectively. For thediscrimination of bacterial isolates, the 16Se23S rDNA intergenictranscribed spacer of bacteria was amplified and digested sepa-rately with Hinf I, Hae III and Hind III (Invitrogen, USA) as previouslydescribed by Jeyaram et al. (2008). For the discrimination of fungalisolates, the ITS1-5.8S rDNA-ITS2 region was amplified with theprimers ITS1/ITS4 as described previously (White, Bruns, Lee, &Taylor, 1990). The PCR products were digested with three restric-tion endonucleases (Hinf I, Hae III and Cfo I enzymes) according tothe manufacturer's instructions (Invitrogen, USA), respectively. Therestriction fragments were separated on a horizontal electropho-resis apparatus (Bio-Rad, USA) on 2.0% (w/v) agarose gels in 0.5�TBE buffer. The gels were stained with ethidium bromide andvisualized under UV light.

2.5. Sequencing of the 16S rRNA gene and ITS1-5.8S rRNA geneintergenic region

The 16S rRNA gene of bacteria was amplified with the PCRcondition described in previous study (Lv, Huang, Zhang, et al.,

Fig. 1. Bacterial DGGE profiles (40e60% denaturant gradient) of amplified 16S rDNA(V3 region) fragments from traditional fermentation starters (a) and traditionalbrewing of Fuzhou Hong Qu glutinous rice wine (b). Lanes 1, 6, 11, 16, 21, 31 and 42 referto 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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2013), and the ITS1-5.8S rRNA gene intergenic region gene of fungiwas amplified by the method reported by Kurtzman and Robnett(1998). PCR products were then gel-purified with GFX™ PCR DNAand Gel Band Purification Kit (Amersham Biosciences AB, Uppsala,Sweden), according to themanufacturer's instructions. Purified PCRproducts were directly sequenced using the ABI prism 3730 DNAanalyzer (Applied Biosystems, USA). Finally, the DNA sequenceswere read and edited by Chromas 2.1. Blast searches of sequenceswere performed at the National Centre for Biotechnology Infor-mation (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 brewingprocess

Fungal DNA extraction was carried out using a benzyl chloridemethod (Zhu et al., 1993). While bacterial DNA was extracted us-ing Universal Genomic DNA Extraction Kit Ver: 3.0 (Takara, Dalian,China) after glass bead beating to disrupt the cell walls. The DNAswere then subjected to further purification by adsorption to asilica matrix using Fermentas (USA) DNA extraction kit accordingto the manufacturer's instruction. The yields and purity of theextracted DNA were analyzed spectrophotometrically at 260 and280 nm using a DU800 spectrophotometer (Beckman Coulter,Fullerton, CA, USA). The DNA extracts were stored in a freezer untilanalysis.

2.6.2. Nested PCReDGGE analysisFor analysis of bacterial diversity, primer sets 27F (50-

AGAGTTTGATCCTGGCTCAG-30) and 1492R (50-GGCTACCTTGTTAC-GACTT-30) (Lane, 1991) were used to amplify the nearly complete16S rRNA encoding gene under conventional PCR conditions in thefirst PCR step. Subsequently, the PCR product was diluted andamplified with DGGE primers 338f-GC/518r (Cocolin, Manzano,Aggio, Cantoni, & Comi, 2011) targeting the V3 region of the 16SrRNA gene to create a DNA fragment suitable for DGGE analysis. Foranalysis of fungal diversity, PCR amplification of the D1/D2 domainof the 26S rRNA gene was performed using the fungal universalprimer set NL1/NL4 (Kurtzman & Robnett, 1998) in the first step,followed by nested PCR using the DGGE primer sets NL1GC/LS2 asdescribed previously (Cocolin, Bisson, & Mills, 2000).

The PCR products were analyzed by DGGE using a DCodeapparatus (Bio-Rad Laboratories, Hercules, CA, USA). Samples wereapplied to 8% (w/v) polyacrylamide gels in 0.5� TAE. Optimalseparation was achieved by DGGE trial with a 40e60% ure-aeformamide denaturing gradient (100% denaturant correspond-ing to 7 M urea and 40% (w/v) deionized formamide) using theprimers 338f-GC/518r. For PCR amplicons obtained with theprimers NL1GC/LS2, electrophoresis was performed in an 8% (w/v)polyacrylamide gel with a denaturing gradient ranging from 20% to50%. 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 UVtransillumination and analyzed using Fragment Analysis (Amer-sham Biosciences, Sweden).

2.6.3. Sequencing of the DGGE fragments and identification of thebands

The major bands on the DGGE gels were excised and cloned intoEscherichia coli plasmids with the pMD 19-T cloning kit (Takara,Dalian, China) following the manufacturer's instructions. Theresultant clones were reamplified and checked again in DGGE gels,and finally sequenced in Shanghai Sangon Biological EngineeringTechnology & 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 Lactobacillusplantarum group and the Lactobacillus casei group

Multiplex species-specific PCRs targeting the recA gene wereperformed for further discrimination when the results of 16S rDNAsequence analysis were not enough to identify strains at the specieslevel. Strains belonging to the L. plantarum group, i.e. L. plantarum,Lactobacillus pentosus and Lactobacillus paraplantarum, weredistinguished using a multiplex PCR assay with the recA gene-based primers paraF, pentF, planF and pREV as described byTorriani, Felis, and Dellaglio (2001). Strains belonging to the L. caseigroup, i.e. L. casei, Lactobacillus paracasei and Lactobacillus rham-nosus, were distinguished using species-specific PCRs as describedby Ward and Timmins (1999).

3. Results

3.1. Bacterial community and dynamics assessed by nestedPCReDGGE

The DGGE fingerprints based on primers 338f-GC/518r are pre-sented in Fig. 1, and the sequencing results of the highlighted bandsare listed in Table 1. Results showed that a total of fourteen bacterialspecies (Janthinobacterium lividum, Bacillus ginsengihumi, Bacillussubtilis, 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) weredetected. The bacterial community of Gutian Hong Qu was mainlycomposed of J. lividum, B. ginsengihumi, B. subtilis and Janthino-bacterium sp. The bacterial community of Yao Qu was composed of

Table 1Phylogenetic 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 Janthinobacterium lividum (JF970593) 100%a2 Janthinobacterium sp. (KJ867066) 100%

Uncultured bacterium clone (KJ417632) 100%a3 Bacillus ginsengihumi (HQ219845) 100%a4 Bacillus subtilis (JF719789) 100%

Yao Qu b1 Weissella soli (GU470977) 100%b2 Lactobacillus brevis (JN089401) 99%b3 Pediococcus pentosaceus (JN039354) 100%b4 Bacillus aryabhattai (JF951729) 100%

Bacillus megaterium (HE578782) 100%b5 Bacillus megaterium (JN106424) 99%

Bacillus aryabhattai (HQ857752) 99%b6 Pediococcus acidilactici (HQ603181) 99%b7 Bacillus amyloliquefaciens (CP002927) 100%

Fermented samples of different brewing stages c1 Lactobacillus plantarum (JN222930) 99%c2 Lactobacillus plantarum (JN222930) 99%c3 Janthinobacterium lividum (JF970593) 100%c4 Weissella soli (GU470977) 100%c5 Leuconostoc mesenteroides (GU458344) 100%c6 Lactobacillus brevis (JN089401) 99%c7 Pediococcus pentosaceus (JN039354) 100%c8 Pediococcus pentosaceus (JN039354) 99%c9 Bacillus aryabhattai (JF951729) 100%

Bacillus megaterium (HE578782) 100%c10 Lactococcus lactis subsp. lactis (FR873574) 100%c11 Bacillus subtilis (JF719789) 100%c12 Bacillus megaterium (JN106424) 99%

Bacillus aryabhattai (HQ857752) 99%c13 Bacillus aryabhattai (JN084155) 100%

Bacillus megaterium (JN084146.1) 100%c14 Pediococcus acidilactici (HQ603181) 99%

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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 significantchanges as the traditional brewing progressed. The early fermen-tation period (day 1) was characterized by Bacillus species,including B. aryabhattai and B. megaterium. However, B. aryabhattaivanished on the sixth day of the traditional fermentation process,and B. megaterium disappeared after eleven days of brewing, indi-cating the decrease of their proportion in the total population ofrice wine mash. P. pentosaceus and Lc. lactis subsp. lactis were thetwo predominant bacterial species that could be consistentlydetected after 6 days of fermentation. However, it was interestingto find that the band intensity of Lc. lactis subsp. lactis (band c10)decreased on the sixteenth day of the brewing process. On thecontrary, the band intensity of L. plantarum (band c1) and L. brevis(band c6) gradually increased from the eleventh day to the end oftraditional brewing, while P. pentosaceus (band c7 and c8) wasconsistently detected with high light intensity throughout thefermentation process but suddenly decreased at the end. All theseindicated that the dominance of bacterial species varied during thetraditional fermentation of Fuzhou Hong Qu glutinous rice wine.

3.2. Fungal community and dynamics assessed by nestedPCReDGGE

The fungal DGGE fingerprints based on primers NL1GC/LS2 areshown in Fig. 2, and the sequencing results of the highlighted bandsare listed in Table 2. A total of nine fungal species were detected, i.e.,Monascus purpureus, Rhizopus oryzae, Aspergillus oryzae, and sixyeast species including Saccharomyces cerevisiae,Wickerhamomycesanomalus, Pichia guilliermondii, Rhodotorula mucilaginosa, Candidaglabrata and Saccharomycopsis fibuligera. All of themwere detectedin the traditional fermentation starters (either Hong Qu or Yao Qu).

Among these, W. anomalus, R. mucilaginosa, C. glabrata, S. cerevisiaeandM. purpureuswere the dominant fungal species in Gutian HongQu. In the aspect of Yao Qu, the predominant fungal speciesincluded W. anomalus, P. guilliermondii, S. fibuligera, R. oryzae, andA. oryzae.

A significant transition of fungal community can be observedfrom Fig. 2. DGGE fingerprints illustrated a higher fungal diversityat the beginning of the traditional brewing process, which wascharacterized by seven fungal species, including S. fibuligera,R. oryzae,W. anomalus, P. guilliermondii, C. glabrata, S. cerevisiae andM. purpureus. However, fungal species diversity decreased as thefermentation progressed. P. guilliermondii vanished on the eleventhday. C. glabrata as well asM. purpureus disappeared after 21 days offermentation, indicating the decrease of their proportion in thetotal population of rice wine mash. The band intensity ofS. fibuligera, W. anomalus and R. oryzae decreased gradually towardthe end of the fermentation process, which was likely due to theincrease of ethanol concentration produced by S. cerevisiae. Asfermentation continued, S. cerevisiae increased gradually duringthe early period of traditional brewing, and eventually took overand dominated the fermentation at the end.

3.3. Bacterial and yeast compositions determined by conventionalplate count method

A total of 6 genera and 13 species of bacteria, including theBacillus (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 isolatesyielded similarity scores of over 99% to their corresponding species.However, 16S rDNA sequence analysis were not enough to identifyisolates belonging to the L. plantarum group and the L. casei group.In this study, species-specific multiplex PCR yielded amplicons

Fig. 2. Fungal DGGE profiles (20e50% denaturant gradient) of partial 26S rRNA genefragments obtained from traditional fermentation starters (a) and traditional brewingprocess (b), with primers NL1GC/LS2. Lanes corresponding to traditional fermentationstarters 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 inTable 2.

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

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

Sample Band no.

Hong Qu a1a2a3a4a5

Yao Qu b1b2b3b4b5

Fermented samples of different brewing stages c1c2c3c4c5c6c7

L. casei, L. paracasei and L. rhamnosus, respectively (as shown inFig. 4b). These strains yielded an amplicon of the expected size(290 bp) by species-specific PCR analysis using the species-specificprimer pairs para/Y2, but no amplification products were obtainedwhen using the other two species-specific primer pairs (casei/Y2and rham/Y2) (data not shown). The amplification profiles of spe-cific PCR using primers para/Y2 was consistent with the profile ofL. paracasei obtained by Ward and Timmins (1999), demonstratingthe presence of L. paracasei during the traditional fermentationprocess.

The relative abundance analysis of bacterial species revealedthat Bacillus sp. and lactic acid bacteria (LAB) (includingL. plantarum, L. brevis, Staphylococcus pasteuri, Lc. lactis subsp. lactis,L. paracasei, Leu. mesenteroides and P. pentosaceus) dominatedduring the traditional brewing process (Supplementary material S1and S2). Significant changes of bacterial community during thetraditional brewing process can be observed (Fig. 3). B. megateriumand B. ginsengihumi appeared frequently at the beginning of thetraditional brewing process (day 1). However, they disappearedafter 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 fermentationprocess, although they were not detected in traditional winestarters by plate count method. The relative abundance of Lacto-bacillus spp. (L. plantarum, L. brevis and L. paracasei) increasedrapidly during the middle fermentation period and became thethree predominant bacterial species at the end of traditionalbrewing process.

The fungal isolates from traditional starters and samples oftraditional brewing process were grouped by ARDRA of the ITS-5.8SrDNA 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 nigerwere the predominant fungalspecies identified in Gutian Hong Qu (Supplementary material S1and S2). While Yao Qu was characterized by six fungal species,including S. cerevisiae, S. fibuligera, R. oryzae, A. oryzae, Emericellanidulans and Mucor indicus. Relative abundance analysis was onlyconducted for yeasts because it was difficult to count the colony-forming units of filamentous fungi using the plate count method(Fig. 6). Except R. mucilaginosa, all of the yeast species detected inHong Qu and Yao Qu were also identified during the fermentationprocess. Non-Saccharomyces yeasts, including S. fibuligera,W. anomalus and C. glabrata, were primarily dominant during theearly fermentation period. As the traditional fermentation

Fig. 2.

Closest relative (NCBI accession no.) Similarity (%)

Wickerhamomyces anomalus (FJ972217) 100%Rhodotorula mucilaginosa (GU373744) 100%Candida glabrata (HM591682) 100%Saccharomyces cerevisiae (JN083825) 100%Monascus purpureus (AF365024) 99%Saccharomycopsis fibuligera (HM107786) 100%Rhizopus oryzae (EU862181) 99%Wickerhamomyces anomalus (FJ972217) 100%Pichia guilliermondii (JF439367) 100%Aspergillus oryzae (KJ650341) 99%Saccharomycopsis fibuligera (HQ199205) 100%Rhizopus oryzae (EU862181) 99%Wickerhamomyces anomalus (HQ199214) 100%Pichia guilliermondii (HQ199203) 100%Candida glabrata (HQ199207) 100%Saccharomyces cerevisiae (HQ199204) 100%Monascus purpureus (AF365024) 99%

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

X.-C. Lv et al. / Food Control 57 (2015) 216e224 221

progressed, S. cerevisiae eventually took over and dominated thetraditional fermentation, and finally became the primary speciesduring the late fermentation period.

4. Discussion

In this study, both culture-dependent and culture-independentanalyses were performed. Significant differences occurred betweenthe results obtained with each method. Discrepancies betweenculturing and DGGE results have also been reported before (Kafiliet al., 2009; Lv, Huang, Zhang, et al., 2013). It's worth noting thatsome species (such as Bacillus methylotrophicus, L. paracasei andS. pasteuri) detected through culture-dependent method may not

Fig. 4. Species-specific PCRs for identification of isolates belonging to the L. plantarumgroup and the L. casei group. Panel a, species identification of the L. plantarum groupisolates from traditional fermentation process by species-specific multiplex PCR (tar-geting recA gene). Panel b, species identification of the L. casei group isolates fromtraditional 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 productsfrom different species, and different efficiencies of genomic DNAextraction from different species (Abriouel et al., 2006; Katano &Fukui, 2003; Sekiguchi, Tomioka, Nakahara, & Uchiyama, 2001).Therefore, it is recommended that the DGGE method should besupplemented with culture-dependent method to reduce omis-sions and generate a better view of microbial diversity in naturallyfermented foods (Cocolin, Campolongo, Alessandria, Dolci, &Rantsiou 2011; Nielsen et al., 2007). In addition, an obviousadvantage of culture-dependent methodology is that it allows acollection of pure cultures, which can be used for further selectionof suitable strains to design mixed starters capable of providingbeneficial contributions to wine quality.

In the present study, bacterial and fungal isolates from tradi-tional fermentation starters and traditional fermentation processwere discriminated and grouped by amplified ribosomal DNA re-striction analysis (ARDRA), which was found to be a useful, simpleand rapid method compared to current molecular and phenotypictechniques. 16Se23S rRNA internal transcribed spacer and ITS1-5.8S rRNA-ITS2 gene region have more sequence variability andas a result can effectively differentiate the closely related bacteriaand fungi. Representative isolates from each group were chosen forfull-length sequencing of their 16S rDNA or ITS-5.8S rRNA generegions. However, due to high genetic homology inside certainbacterial genera, the 16S rRNA genemay not be especially useful forthe 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 PCRswere performed for further discrimination when 16S rDNAsequence analysis was not enough to identify species belonging tothe L. plantarum group and the L. casei group.

Microbial dynamics obtained from bacterial DGGE fingerprintsand the conventional culturing method combined with molecularclassification identification showed that Bacillus sp. and LABdominated in traditional fermentation starters and throughout thefermentation process, but they varied in different brewing phases.It has been reported by earlier researchers that the bacterialmicroflora of rice wine starters and rice wine traditional fermen-tation were highly variable in species composition dominated byBacillus species and LAB (Sujaya, Nocianitri, & Asano, 2010; Thanh,

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 re-striction 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-Candidaglabrata (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).

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Maile, & Tuan, 2008). Members of the Bacillus spp. might bebeneficial because of their ability to secrete a wide range of hy-drolytic enzymes, including amylases, proteases and fibrinolyticenzyme. These hydrolytic enzymes may play an important role inthe traditional fermentation process (Amoa-Awua & Jakobsen,1995). In addition, Bacillus spp. can produce nitrogenous flavorcompounds such as diverse pyrazines (Li et al., 2011; Zhang, Wu,Zhang, Hu, & Zhang, 2011; Zheng, Tabrizi, Nout, & Han, 2011), buttheir potential contribution to the final flavor of Fuzhou Hong Quglutinous rice wine is still unknown and requires further study.

The predominance of LAB in fermented foods is commonly dueto their ability to tolerate low pH (Abriouel et al., 2006), produce avariety of antimicrobial substances such as bacteriocin, and sup-press the growth of pathogens and the toxigenic and spoilage or-ganisms (Castellano, Belfiore, Fadda, & Vignolo, 2008; Jones,Hussein, Zagorec, Brightwell, & Tagg, 2008). This is probably thereason why potential foodborne pathogens were not detectedduring the traditional fermentation of Fuzhou Hong Qu glutinousrice wine in the present study. Lactobacillus species, the predomi-nant LAB species during the traditional brewing process, may playan important role during liquor manufacture because lactic acid

Fig. 6. The relative abundance (%) of yeast species present in samples of wine starters and tranalysis was only conducted for yeasts because it was difficult to count the colony-forming

was identified to be a precursor of ethyl lactate, one of the majorflavor compounds in strong aromatic liquor (Fan & Qian, 2005). Itwas also reported as dominant function bacteria in several typicalDaqu starters for Chinese liquor (Xiu, Guo, & Zhang, 2012). Lc. lactiswas firstly detected in Hong Qu glutinous rice wine. As one of thehomofermentative LAB species, Lc. lactis has been isolated fromsour cassava (Brauman, Keleke, Malonga, Miambi, & Ampe, 1996)and Obushera (Muyanja, Narvhus, Treimo, & Langsrud, 2003). Leu.mesenteroide, one of the predominant bacterial species during themiddle fermentation period, has been also isolated during thefermentation of shochu, wine, and alcoholic beverages, such asMecal, Pulque and Boza (Endo& Okada, 2005; Escalante et al., 2008;Narv�aez-Zapata, Rojas-Herrera, Rodríguez-Luna, & Larralde-Corona, 2010). It was reported by other authors that Leuconostocspp. are indigenous to plant material and often dominate the initialstages of fermentation before being succeeded by more acidtolerant 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 alco-holic beverages and fermented foods (Jin, Kim, Jin, Eom, & Han,2008; Tamang et al., 2007; Thanh et al., 2008), became the

aditional fermentation process as revealed by culture-dependent method. Quantitativeunits of filamentous fungi using the plate count method.

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predominant bacterial species at the end of traditional fermenta-tion process, where they replaced the less acid tolerant hetero-fermentative LAB that mainly initiate fermentation but begin toreduce 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 LABand yeasts. Yeasts stimulate LAB growth by supplying essentialmetabolites 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. anomalusand C. glabrata) were detected involving in the traditionalfermentation process. Non-Saccharomyces yeasts, includingP. guilliermondii, S. fibuligera, W. anomalus and C. glabrata, domi-nated in the earlier stages of traditional brewing process.P. guilliermondii, which was also found in Wheat Qu for Shaoxingrice wine (Xie et al., 2007) and Manipur ‘Hamei’ (a traditionalstarter used for ricewine production in India) (Jeyaram et al., 2008),could produce volatile phenols (4-ethylguaiacol (Suezawa &Suzuki, 2007), 4-ethylphenol (Dias et al., 2003) and important es-ters in the initial stage of wine fermentation (Moreira et al., 2011)).However, strains of P. guilliermondii have also been reported toproduce a high concentration of isobutyl alcohol, isoamyl alcoholand unpleasant odor consisting of isobutyric acid, isovaleric acidand 2-phenylethanol (Wah, Walaisri, Assavanig, Niamsiri, &Lertsiri, 2013). S. fibuligera, the most abundant yeast speciesdetected in Yao Qu and at the beginning of traditional brewing (day1) in the current study, has a strong capability to produce variousenzymes, particularly a-amylase, glucoamylase, acid proteases andb-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 roleduring the initial stage of alcoholic fermentation. It had also beenreported to be the principal amylolytic microorganism in tradi-tional 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 pro-ducer known so far, was detected in a low frequency at early stageof traditional brewing. However, it took over the fermentationprocesses, due to its efficient fermentation catabolism and ethanoltolerance in the stressful conditions of the fermenting mash.

M. purpureus and R. oryzae were the predominant filamentousfungi existed in traditional wine starters and during the traditionalbrewing of Fuzhou Hong Qu glutinous rice wine. R. oryzae, a strongamylase producer and frequently found in amylolytic fermentationstarters 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 previousstudy (Lv, Weng, et al., 2012; Lv, Huang, et al., 2012). Previous studyby others also pointed out that R. oryzae can produce volatilecompounds, such as ethanol, 2-methyl-1-butanol and 3-methyl-1-butanol (Christen, Bramorski, Revah, & Soccol, 2000; Feng, Larsen,& Schnürer, 2007), which were the main volatile compounds inChinese yellow rice wine. As the most dominant fungal species inHong Qu, Monascus species can esterify mixed acids and ethanolto form esters such as ethyl caproate that contribute to the aromaof the Chinese liquor (Du, Tang, Gu, Liu, & Ren, 2005), and alsoproduce citric acid, acetic acid and succinic acid, as well as sub-stances with physiological activity that facilitates the formationof unique flavor and functional components (Taira et al., 2002;Yang et al., 2006). A. oryzae, well known for its capacity tosecrete large amounts of hydrolytic enzymes (such as proteases,amylase, glutaminase and metallopeptidase), has been safelyused in the production of sake, soybean paste, soy sauce andChinese rice wine (Bourdichon et al., 2012; Zhang, Guan, Cao, Xie,& Lu, 2012).

5. Conclusion

In the present study, the dynamic changes and diversity of mi-crobial community succession in the traditional brewing of FuzhouHong Qu glutinous rice wine were systematically investigated forthe first time using a combination of culture-dependent andculture-independent methods. Moreover, results would be usefulfor optimization of microbial composition and quality control ofwine starters to upgrade Fuzhou Hong Qu glutinous rice wine.Further studies are required to understand the microbial in-teractions of LAB, yeasts and filamentous fungi to clarify the factorsthat contribute to the distinctive flavor of Fuzhou Hong Qu gluti-nous rice wine.

Acknowledgments

This work was financially supported by grants from the NationalNatural 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 Agri-culture 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.

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