International Journal of Food Microbiology 232 (2016) 15–21 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro New insight into microbial diversity and functions in traditional Vietnamese alcoholic fermentation Vu Nguyen Thanh a,⁎, Nguyen Thanh Thuy a, Nguyen Thuy Chi a, Dinh Duc Hien a, Bui Thi Viet Ha b, Dao Thi Luong b, Pham Duc Ngoc b, Pham Van Ty b a b Food Industries Research Institute, 301 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam Hanoi University of Science, 334 Nguyen Trai, Thanh Xuan, Hanoi, Viet Nam a r t i c l e i n f o Article history: Received 20 January 2016 Received in revised form 13 April 2016 Accepted 20 May 2016 Available online 24 May 2016 Keywords: Traditional alcoholic fermentation Amylolytic starter Microbial succession Rhizopus Saccharomycopsis fibuligera Amylase a b s t r a c t The roles of microorganisms in traditional alcoholic fermentation are often assumed based on abundance in the starter and activity in pure culture There is a serious lack of hard evidence on the behavior and activity of individual microbial species during the actual fermentation process In this study, microbial succession and metabolite changes during days of traditional Vietnamese alcoholic fermentation were monitored Special attention was devoted to starch degradation In total, 22 microbial species, including species of filamentous fungi (Rhizopus microsporus, Rhizopus arrhizus, Mucor indicus, Mucor circinelloides, Cunninghamella elegans, Aspergillus niger), yeast-like fungus (Saccharomycopsis fibuligera), yeasts (Saccharomyces cerevisiae, Clavispora lusitaniae, Wickerhamomyces anomalus, Lindnera fabianii, Pichia kudriavzevii, Candida rugosa, Candida tropicalis), and bacteria (Stenotrophomonas maltophilia, Lactobacillus brevis, Lactobacillus helveticus, Acinetobacter baumannii, Staphylococcus hominis, Bacillus megaterium, Enterobacter asburiae, Pediococcus pentosaceus) were identified Despite the presence of a complex microbiota in the starter, the fermentation process is consistent and involves a limited number of functional species Rapid change in microbial composition of fermentation mash was observed and it was correlated with ethanol content Microbial biomass reached maximum during first days of solid state fermentation Acidification of the medium took place in day 1, starch degradation in days 2, 3, 4, and alcohol accumulation from day Although Sm fibuligera dominated by cell count amongst potential starch degraders, zymography indicated that it did not produce amylase in the fermentation mash In mixed culture with Rhizopus, amylase production by Sm fibuligera is regulated by the moisture content of the substrate Rhizopus was identified as the main starch degrader and S cerevisiae as the main ethanol producer Bacterial load was high but unstable in species composition and dominated by acid producers M indicus, Sm fibuligera, W anomalus and bacteria were regarded as satellite microorganisms Their possible influence on organoleptic quality of fermentation product was discussed © 2016 Elsevier B.V All rights reserved Introduction In Vietnam, traditional alcoholic fermentation of rice has centuries of history Before the introduction of beer, this had been the main way for obtaining alcoholic drinks (Peters, 2012) Even now, traditional fermentation of alcohol from rice still makes up 80% of the total national distilled liquor production Alcohol is being produced by a vast network of small breweries across the country (Da, 2009) Being one of the countries where rice cultivation originated, Vietnam possesses a rich assortment of rice varieties and so the rice liquors produced from (Da, 2009) Also, with 54 distinct ethnic groups, each with its own language, lifestyle, and cultural heritage, there is high variation in methods for alcohol production Despite of the differences, breweries share the same production principle where starter (banh men) containing a relatively ⁎ Corresponding author http://dx.doi.org/10.1016/j.ijfoodmicro.2016.05.024 0168-1605/© 2016 Elsevier B.V All rights reserved stable microbial community is used Banh men provides microorganisms necessary for breaking down of starch and subsequent fermentation of the released monomers to ethanol (Lee and Fujio, 1999; Aidoo et al., 2006; Thanh et al., 2008) Banh men is made from uncooked dough of rice, water, a variety of oriental herbs, and a small amount of the starter from previous batch By loopback inoculation, banh men is passed from generation to generation and is regarded as technological and cultural heritage Due to economic and cultural importance, traditional alcoholic fermentation receives strong interest from scientific and technological communities Studies on traditional Vietnamese alcoholic fermentation could be categorized into two main groups: (i) microbiological characterization of banh men; and (ii) selection of active strains from banh men for process improvement Microbial composition of banh men is relatively well understood Typically, each gram of the starter contains 103–106 CFU of mold, 106–107 CFU of yeast and 103–106 CFU of lactic acid bacteria (Lee and Fujio, 1999; Dung et al., 2007) Banh men contains 16 V.N Thanh et al / International Journal of Food Microbiology 232 (2016) 15–21 a complex and relatively stable microbiota, including Amylomyces rouxii, Rhizopus microsporus, Mucor indicus, Mucor circinelloides, Saccharomycopsis fibuligera, Hyphopichia burtonii, Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia anomala, Candida tropicalis, Pichia ranongensis, Clavispora lusitaniae, Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus brevis, Weissella confusa, and Weissella paramesenteroides (Lee and Fujio, 1999; Dung et al., 2007; Thanh et al., 2008) Strains of A rouxii and S cerevisiae have been selected for the production of defined granulated starters for controlled production of rice wine (Dung et al., 2005) There is also a number of other attempts to improve fermentation process that has been published locally in the forms of technological reports Until now, the implementation of modified processes has proven to be difficult and commercial production of rice alcohol in Vietnam still relies on the starter produced in the traditional way There is a major gap in understanding of the role and behavior of individual species in actual fermentation process Most often, strains are selected from banh men based on amylase activity and alcohol production capability However, banh men production and alcoholic fermentation using banh men are two independent processes and the populations thrived from the first process may not be functioning in the second one In this article we describe microbial succession and metabolite changes during the course of rice fermentation using banh men Special attention was devoted to starch degradation and the group of amylase producers Materials and methods 2.1 Rice fermentation Rice fermentation was carried out in the laboratory using traditional procedures Three fermentation experiments were conducted separately using one of the three starter samples collected at My Duc, Phuc Tho and Son Tay districts of Hanoi, Vietnam Khang Dan rice variety was cooked using a kitchen rice cooker For each fermentation experiment, kg of cooked rice was sprinkled over with 60 g of pulverized banh men, mixed well and dispensed on a sterile tray with a layer of ca cm The tray was covered with a sterile cotton blanket and incubated at 28 °C After day of solid state fermentation, the fermentation mass was transferred into a cylindrical ceramic jar and litter of sterile water was added The jar was closed with a ceramic lid and alcoholic fermentation was carried out for days at 28 °C Samples were collected at different intervals (see Fig 2) for microbiological and biochemical analyses In order to ensure uniformity, samples were collected at different positions and mixed before analysis For solid state fermentation samples, an equal amount (by weight) of sterile distilled water was added Data were presented as mean values obtained from three experiments 2.2 Microbial isolation and enumeration For microbial enumeration, serial dilutions of samples were made using sterile physiological solution containing 0.9% NaCl For isolation of yeast and fungi, the diluted samples were plated on malt-glucose agar (malt, 10 g/L; glucose, 10 g/L; agar, 15 g/L; chloramphenicol, 100 mg/L) The plates were incubated at 28 °C for days Fast growing mucoraceous fungi were counted after 1–2 days, and yeast after days For isolation of bacteria, plate count agar with bromocresol purple (yeast extract, 2.5 g/L; peptone, 5.0 g/L; glucose, 1.0 g/L; L-cysteine, 0.1 g/L; Tween-80, 1.0 g/L; bromocresol purple, 0.04 g/L; agar, 15 g/L) was used The plates were incubated at 37 °C in a candle extinction jar for 24 h Acidification of the medium was indicated by the formation of yellow halos around colonies Microbial colonies with distinctive appearances were picked from each day of fermentation (8 points) and with one representative per fermentation experiment (3 batches) The isolates were purified and maintained in cultures 2.3 Identification of microorganisms For identification of microorganisms, the isolates were initially divided into genetic groups by microsatellite PCR fingerprinting and then the representatives from each groups were identified by rRNA gene sequencing The identification was extrapolated to the rest of the isolates of the same genetic group For DNA extraction, one loop-full of cells was transferred to a micro-tube containing mL 2× SSC (15 mM sodium citrate, 150 mM NaCl, pH 7.0) and heated at 99 °C for 10 using a PHMT Thermo-Shaker dry heater block (Grant-bio, England) Cells were collected by centrifugation at 10,000 g for To the cell pellet, ca 100 μL glass beads (0.2–0.5 mm in diameter) (Roth, Germany), 100 μL phenol/chloroform and 150 μL water were added Cells were disrupted using a Mini-Beadbeater-8 (Biospec, USA) for 45 s The tube was centrifuged at 14,000 g for 10 and the upper layer was transferred to a new micro-tube The DNA solution was further purified using a Silica Bead DNA Gel Extraction kit (Thermo Scientific) according to the manufacturer's instructions For microsatellite PCR fingerprinting, primer (GAC)5 was used PCR was carried out in a C1000 Touch Thermal Cycler (BioRad, USA) using following thermal program: 94 °C for min; 30 cycles of 94 °C for 40 s, 52 °C for 40 s and 72 °C for 30 s; 72 °C for 10 PCR products were separated in 1% agarose gel in ×0.5 TAE (20 mM Tris, 10 mM acetic acid, 0.5 mM EDTA) For identification of yeast, 26S rRNA gene (D1/D2) was amplified and sequenced using primer pair NL1 (5′GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′GGTCCGTGTTTCAAGACGG-3′) (Kurtzman and Robnett, 1998) For fungi, the ITS region was amplified and sequenced using primer pair ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′TCCTCCGCTTATTGATATGC-3′) (Esteve-Zarzoso et al., 1999) For identification of bacteria, the 16S rRNA gene was amplified and sequenced using the universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) (Lane, 1991) The PCR conditions were as follows: 94 °C for min; 30 cycles of 94 °C for 30 s, 52 °C for 40 s and 72 °C for 60 s; 72 °C for 10 PCR products were sequenced using the service provided by Macrogen Inc (Korea) Species identification was based on the similarity of the obtained sequences with the reference sequences at EzTaxon (http://www ezbiocloud.net/eztaxon) (Kim et al., 2012) and GenBank (http://www ncbi.nlm.nih.gov/nuccore/) databases 2.4 Chemical and biochemical analyses The pH of fermentation mash was measured using a SevenEasy bench-top pH meter (Mettler-Toledo, Austria) For determination of ethanol content, samples were centrifuged at 10,000 g for 10 at °C and solid content discarded Ethanol concentration was measured based on boiling temperature using a traditional ebulliometer (Dujardin-Salleron, France) For amylase extraction, to the sample an equal amount (by weight) of 100 mM sodium acetate buffer, pH 5.0 was added The mixture was shaken at 200 rpm in an orbital shaker for h at 30 °C and then enzyme extract was obtained by centrifugation at 10,000 g for 10 at °C For amylase assay, soluble starch (Sigma) was used as the substrate Enzymatic hydrolysis was carried out in 50 mM sodium acetate buffer, pH 5.0 at 50 °C for 20 The amount of reducing sugar produced was measured using Nelson-Somogyi method (Nelson, 1944; Somogyi, 1952) One unit of amylase was defined as the amount of the enzyme that produced μM of reducing sugar (calculated as glucose) per minute 2.5 Zymography of enzyme extracts Zymography was performed based on conventional SDS-PAGE (Sambrook et al., 1989) using 10% polyacrylamide gel containing 1% starch Samples were loaded without the heat-treatment step After electrophoresis, the gel was rinsed twice in 2% Triton X-100 for V.N Thanh et al / International Journal of Food Microbiology 232 (2016) 15–21 30 at °C Then, it was rinsed twice in 100 mM sodium acetate buffer, pH 5.0 for 15 at °C To perform starch hydrolysis reaction, the gel was incubated in 100 mM sodium acetate buffer, pH 5.0 at 50 °C for 30 For visualization, the gel was stained in 100 mL Lugol's solution (I2, g/L; KI, 10 g/L) Active amylase fractions appeared as transparent bands on the dark-blue background 2.6 Influence of moisture content on amylase production For preparation of rice substrates with different moisture contents, rice (100 g) was soaked with different amounts (from 20 to 150 mL) of distilled water in 1000 mL Erlenmeyer flasks for h The flasks were autoclaved (without post-autoclave drying) at 121 °C for 15 After cooling, pure or mixed cultures of Sm fibuligera BMQ908 and R microsporus BMF40 were inoculated The initial moisture contents were determined using a KERN DBS (Kern & Sohn GmbH, Germany) infra-red moisture meter After 48 h of solid state fermentation at 28 °C, amylase was extracted and analyzed as described above Results and discussion 3.1 Microorganisms associated with traditional alcoholic fermentation In total, 130 microbial strains were isolated from different stages of rice fermentation Amongst them, 41 were fungi, predominantly of Mucoraceae, 16 yeast-like fungus Sm fibuligera, 29 yeasts, mainly of S cerevisiae, and 44 bacteria (see Table 1) Grouping and identification a larger number of strains based on morphological and physiological characteristics are often difficult and time consuming Different species may share similar phenotypic characteristics and strains of the same species may show high variation depending on the culture age and sexual activity By using microsatellite primed PCR we could successfully differentiate a large number of strains A typical grouping of fungi based on microsatellite PCR is presented in Fig The primer (GAC)5 also provided good resolution for yeasts and bacteria The most frequent fungus was R microsporus, which accounted for 17 strains amongst 41 fungal strains identified, and followed by R 17 arrhizus (9 strains), M indicus (7 strains), M circinelloides (3 strains), Cn elegans (3 strains) (see Table 1) The fermentation mash nearly lacked of Aspergillus, Trichoderma, and Penicillum, the most common saprotrophic fungi in indoor environments (Haleem Khan and Mohan Karuppayil, 2012) There was a single strain of A niger isolated on the first day and most likely by chance Traditional Vietnamese alcoholic fermentation substantially differs from the fermentation of Japanese sake (Kitamoto, 2015) and Chinese maotai (Chen et al., 2014), where Aspergillus spp are the predominant This specificity might be due to high moisture content of the substrates used in Vietnamese alcoholic fermentation We found that the initial moisture contents of dough for starter preparation and cooked rice for alcoholic fermentation were in the range from 50% to 55% The condition would facilitate the growth of mucoraceous fungi which are well adapted to the substrates with high moisture contents (Richardson, 2009) and are capable of growing in microaerophilic and anaerobic environments (Hesseltine et al., 1985) With species identified, highest fungal diversity was observed at the beginning of solid state fermentation and the diversity reduced as the fermentation progressed On the second day, only three species, namely R microsporus, R arrhizus, and M indicus were detected Strains of R microsporus and R arrhizus are well known as strong amylase producers (Peixoto et al., 2003; Dung et al., 2006) M indicus is a dimorphic fungus that exhibits both filamentous and yeast growth M indicus is an efficient ethanol producer and can accumulate up to 5% of ethanol when growing in glucose containing medium (Karimi and Zamani, 2013) M indicus was the only fungus detected in the fermentation broth till day of days of fermentation Strains of M indicus obtained in this study showed very low or no amylase activity when growing on rice substrate Although occupied second position in terms of the number of strains obtained in this study, R arrhizus is the best known fungus in banh men R arrhizus (previously known as Amylomyces rouxii) was first described by Calmette in his comprehensive study on banh men dated back to 1892 (Calmette, 1892) Calmette considered the fungus as the major agent causing liquefaction of rice starch and developed a technology for alcohol production based on A rouxii and Saccharomyces strains (Calmette, 1895) The technology was utilized by the alcohol monopoly, Table Microorganisms isolated on different days during fermentation of rice using banh men starter Microbial species Fungi Rhizopus microsporus (=R oligosporus) Rhizopus arrhizus (=Amylomyces rouxii, =R oryzae) Mucor indicus Mucor circinelloides Cunninghamella elegans Aspergillus niger Unidentified Yeast-like fungi Saccharomycopsis fibuligera Yeast Saccharomyces cerevisiae Clavispora lusitaniae Wickerhamomyces anomalus (=Pichia anomala) Lindnera (Pichia) fabianii Pichia kudriavzevii (=Issatchenkia orientalis) Candida rugosa Candida tropicalis Bacteria Stenotrophomonas maltophilia Lactobacillus brevis Lactobacillus helveticus Acinetobacter baumannii Staphylococcus hominis Bacillus megaterium Enterobacter asburiae Pediococcus pentosaceus Number of isolates 41 17 3 1 16 16 29 19 2 2 1 44 13 10 1 Day of isolation + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 18 V.N Thanh et al / International Journal of Food Microbiology 232 (2016) 15–21 Fig Typical fingerprints generated by micro-satellite PCR of fungi associated with traditional alcoholic fermentation The lanes were manually rearranged Number in boxes indicated strains identified by DNA sequencing M - kb DNA ladder (Fermentas) the Société Des Distilleries de L'indochine for a large scale industrial production in colonial Vietnam from 1897 to 1945 (Peters, 2012) A rouxii was also utilized for upgrading of banh men for production of rice wine from purple glutinous rice (Dung et al., 2006) The name A rouxiii represents a special phenotype of R arrhizus that widely occurs in Asian amylolytic starters It is characterized by rapid growth, intensive formation of large chlamydospores, and the lack of black pigmented sporangia (Hesseltine, 1983) A rouxiii itself is a heterogeneous group A rouxiii strains isolated from banh men produce malic acid but not lactic acid while strains from look-pang and ragi-tape produce majorly lactic acid (Kito et al., 2009) The separation of the two groups is supported by RFLP and DNA sequencing data Two varieties, R arrhizus var delemar for lactic acid negative strains from banh men and R arrhizus var arrhizus for lactic acid producing strains from look-pang and ragi-tape, have been proposed (Kito et al., 2009; Dolatabadi et al., 2014a) In this study, we have found two genetic groups of R arrhizus (see Fig 1) Similar to the case of R arrhizus, the domesticated form of R microsporus having reduced sporulation activity was treated as a separate species R oligosporus or a variety (R microsporus var oligosporus) However, this variety along with others (azygosporus, chinensis, oligosporus, microsporus, rhizopodiformis and tuberosus) are not supported genetically by matting compatibility and sequencing data of ITS, ACT, 1-α (TEF) genes (Dolatabadi et al., 2014b) Strains of R microsporus can produce rhizoxins, a potent antimitotic macrocyclic polyketide toxins It was revealed that the fungus cannot produce toxin by itself and the toxin is produced by bacteria of the genus Burkholderia that live inside the fungal cells (Partida-Martinez et al., 2007) Alarming was the fact that the bacteria have been found in R microsporus CBS 111563, a strain originally isolated from banh men in Vietnam (Lackner et al., 2009) When the strain was tested for sufu and tempe production, considerable amounts of rhizoxins could be detected in the final products (Rohm et al., 2010) It is not clear about the extent of toxigenic phenotype amongst R microsporus populations associated with traditional Vietnamese alcoholic fermentation Beside amylolytic mucoraceous fungi, Sm fibuligera, an active amylase producer was also found in high density (up to 8.2 log CFU/g) during traditional Vietnamese alcoholic fermentation (see Table and Fig 2) Sm fibuligera produces extracellular α-amylase, glucoamylase and raw starch digesting glucoamylase (Chi et al., 2009) In culture medium, Sm fibuligera produces extracellular C14-C18 2-D-hydroxy fatty acids in the form of needle shaped crystals (Kurtzman et al., 1973) Sm fibuligera strains obtained in this study also exhibited similar phenotype There is no information on the possible effect of these compounds on microbial community and organoleptic quality of the fermentation products, although it seems quite obvious Kurtzman et al (1973) informed that the compounds had little or no antibacterial activity However, in the study, the target microorganisms were restricted to common enteric pathogens (Kurtzman et al., 1973) Amongst the fermentative yeasts isolated during rice fermentation, S cerevisiae was the most prevalent The yeast occurred in all samples from beginning to the end of fermentation On the last day of fermentation, beside bacteria, S cerevisiae existed practically as a pure culture (see Table and Fig 2) The presence of S cerevisiae populations in Asian amylolytic fermentation is not a result of spontaneous introduction from environment but a consequence of long term domestication Recent phylogenetic analysis based on multi-gene sequencing data Fig Microbiological (A) and biochemical (B) parameters during the course of rice fermentation using banh men starter Error bar represents differences between thee independent fermentations V.N Thanh et al / International Journal of Food Microbiology 232 (2016) 15–21 Fig Mobility in starch containing gel of amylases extracted from pure cultures of mucoraceous fungi and Sm fibuligera grown on rice indicated that S cerevisiae strains obtained from Japanese sake, Chinese rice wine and Indonesian ragi represent a well-supported clade and distant from natural populations (Wang et al., 2012; Li et al., 2014) Yeasts W anomalus (= P anomala), Pichia kudriavzevii (=Issatchenkia orientalis), C lusitaniae were isolated sporadically at different days of fermentation (Table 1) They have also been reported to occur in banh men (Lee and Fujio, 1999; Dung et al., 2007; Thanh et al., 2008) W anomalus is a regular component in several types of Asian alcoholic fermentation starters (Haard et al., 1999; Limtong et al., 2002) The yeast can accumulate up to 5% (w/v) ethanol in broth culture (Limtong et al., 2002) W anomalus strains are known to produce killer toxin, a glycoprotein that inhibits the growth of other yeasts (Schneider et al., 2012) W anomalus produces a spectrum of small volatile compounds, such as ethyl acetate, ethyl propanoate, phenyl ethanol and 2-phenylethyl acetate (Passoth et al., 2006) These volatile compounds might contribute to the aroma of rice liquor Bacteria of banh men received little attention and were often regarded as contaminant and unwanted (Haard et al., 1999) In this study, a rather high count (up to 8.8 log CFU/g) of bacteria was observed in the fermentation mash (see Fig 2) Following species were identified: Stenotrophomonas maltophilia, L brevis, L helveticus, Acinetobacter baumannii, Staphylococcus hominis, Bacillus megaterium, Enterobacter asburiae, and P pentosaceus (see Table 1) Fungal and yeast composition was consistent and all three fermentation batches shared the same dominating species (R microsporus, R arrhizus, M indicus, Sm fibuligera, and S cerevisiae) Meanwhile, the bacterial composition was inconsistent Sten maltophilia dominated in one fermentation batch but was not detected in the other two Similar finding was reported for the microbial composition of banh men (Thanh et al., 2008) Taken that bacteria present in high cell density but with unstable species composition, controlling of bacterial microbiota might help in improving quality consistency of traditional Vietnamese alcoholic fermentation product 19 bacteria and mucoraceous fungi After 24 h of fermentation, the bacterial count reached 8.7 log CFU/g, meanwhile the count for mucoraceous fungi reached 5.6 log CFU/g Beside lactic acid bacteria (L brevis, L helveticus, Staph hominis, Pd pentosaceus), other bacterial species detected (Sten maltophilia, A baumannii, and E asburiae) are also known to produce acid when growing on sugar substrates (Brenner et al., 1986; Juhnke and des Jardin, 1989; Constantiniu et al., 2004) R microsporus and R arrhizus, the major mucoraceous fungi during the onset of fermentation can produce fumaric, malic and lactic acids (Kitpreechavanich et al., 2008; Kito et al., 2009) It should be noted that quantification of mucoraceous fungi based on colony forming unit may be bias From our observation, in the fermentation mash, Rhizopus formed extensive filamentous growth with hyphae penetrating deep inside the rice grains This pattern of growth would be difficult to quantify The comparative contributions of bacteria and mucoraceous fungi in the acidification of fermentation mash, thus remained unclear Acidic condition would inhibit the growth of spoilage bacteria and facilitate the growth of yeast and fungi Rapid changes in microbial composition observed during fermentation correlated with ethanol accumulation (Fig 2) On the first day of solid state fermentation, non-Saccharomyces dominated amongst yeast populations Yeast growth peaked at 60 h, both in terms of cell density (8.9 log CFU/g) and diversity At that point, ethanol concentration was 2.2% (v/v) Soon after, the diversity rapidly decreased and S cerevisiae thrived to be the dominant At 78 h, ethanol concentration reached 4.9% (v/v) only two types of yeasts were detected At 120 h, when ethanol concentration was 7.6% (v/v), S cerevisiae existed practically as a pure culture Starting with fungal species, mainly the ones associated with banh men, fungal growth reached maximum after days and only species (R microsporus, R arrhizus, and M indicus) thrived Upon addition of water, fermentation turned into alcohol production phase, species of Rhizopus died out but M indicus could still grow until the near end of fermentation (144 h, 8.9% ethanol) Similarly, Sm fibuligera achieved maximum density during solid state fermentation and died out after 96 h (6.9% ethanol) Bacteria reached maximal density and diversity after 24 h of fermentation, roughly a day earlier than yeast and fungi Although reduced in diversity, bacterial populations maintained high cell density until the end of fermentation (see Fig 2, Table 1) During the first 24 h of solid state fermentation, amylase activity and concentration of free sugar were low (see Fig 2) At this stage, microbial growth perhaps was supported by the ready available nutrients present in cooked rice During the next 24 h, rapid increase in amylase activity was observed The rate of starch degradation surpassed sugar consumption and led to the accumulation of free sugars which peaked at 7.5% at the end of solid state fermentation At this moment, ethanol fermentation still did not start although yeast density reached maximum Upon 3.2 Microbial succession during traditional alcoholic fermentation Microbial succession and changes in biochemical parameters during the course of traditional alcoholic fermentation is presented in Fig During the first 24 h, the pH of the fermentation mash rapidly dropped from 6.5 to 3.5 and this coincided with the growth of acid producing Fig Zymography of amylases extracted from pure culture of R arrhizus BMF4, Sm fibuligera BMQ908, banh men starter, and fermentation mash samples collected at different time intervals 20 V.N Thanh et al / International Journal of Food Microbiology 232 (2016) 15–21 Fig Influence of moisture content on the production of amylases by pure and mixed cultures of Sm fibuligera BMQ908 and R microsporus BMF40 grown on rice substrate Amylase was extracted after 48 h of fermentation Lanes to are substrates with increasing moisture contents: 1–23%; 2–29%; 3–34%; 4–38%; 5–48%; 6–55%; 7–60% addition of water, ethanol fermentation took place, the concentration of free sugar decreased rapidly although amylase activity was still on the rise for another 12 h During days of ethanol fermentation, yeast remained in high density but shifted from multispecies to practically pure culture of S cerevisiae Amylase activity decreased after 72 h of fermentation and stabilized at low level At the end of fermentation, ethanol concentration reached an average value of 9.3% (v/v) and the mash exhausted in free sugar 3.3 Zymography and the origin of amylases in fermentation mash The correlation of amylase activity in the fermentation mash with the growth of mucoraceous fungi and Sm fibuligera (see Fig 2) has prompted the question concerning the contribution of each group in starch degradation There are compelling opinions regarding the role of Sm fibuligera and Rhizopus spp in saccharification of starch (Uchimura et al., 1990; Limtong et al., 2002; Dung et al., 2006) We decided to trace the origin of amylase in fermentation mash by comparing the electrophoretic mobility of the amylase extracted from fermentation mash with the ones produced by pure cultures R microsporus, R arrhizus, M indicus, M circinelloides, Cn elegans, and Sm fibuligera strains were grown in cooked rice Except M indicus, all tested strains demonstrated amylase activity at different extents Amylases produced by mucoraceous fungi had low electrophoretic mobility and could be differentiated from the ones produced by Sm fibuligera (see Fig 3) When comparing the amylase extracted from the starter (banh men) with amylases produced by pure cultures, it become clear that banh men contained both amylases from mucoraceous fungi and Sm fibuligera (Fig 4) It was surprising that although Sm fibuligera dominated amongst amylase producers (0.8 to 1.9 log CFU higher) in the fermentation mash (see Fig 2), amylase of Sm fibuligera was not detected throughout the fermentation process (see Fig 4) Instead, amylase with mobility similar to that of Rhizopus was the main component Light bands presumably of Cn elegans were also observed In order to verify the consistency of the phenomenon, fermentation test was repeated using additional starters Also, fermentation samples from a traditional alcohol producer (Van village, Bac Ninh province) were collected and analyzed The results were similar, i.e amylase of Sm fibuligera was detected in the starters but not in fermentation mashes (data not shown) Thus, it could be concluded that, although present in high density during solid state fermentation, M indicus and Sm fibuligera play little or no role in starch degradation and Rhizopus is the main amylase producer Due to similar electrophoretic mobility, it was not possible to discriminate between amylases produced by R microsporus and R arrhizus 3.4 Substrate moisture content as regulating factor for amylase production in co-culture The fact that amylase of Sm fibuligera presents in the starter but not in the fermentation mash has prompted the question concerning the regulation of amylase production in Sm fibuligera Since banh men is relatively drier comparing with the fermentation mash, it was hypothesized that moisture content could play a regulating role Sm fibuligera BMQ908 and R microsporus BMF40 were grown in pure and mixed cultures on cooked rice with different moisture contents (see Fig 5) and amylases were extracted It was shown that Sm fibuligera BMQ908 and R microsporus BMF40 produced amylase at all tested moisture contents but most optimum at 29–38% (lanes 2, 3, 4) In mixed culture, while R microsporus BMF40 maintained the same amylase production pattern, Sm fibuligera BMQ908 ceased to produce amylase when the moisture content was equal or above 34% Taken that cooked rice used in traditional Vietnamese alcoholic fermentation has the moisture content in the range from 50% to 55%, the absence of Sm fibuligera's amylase in fermentation mash is thus understandable It is most likely that free sugar released by the action of Rhizopus amylase repressed amylase production in Sm fibuligera, and such interaction is facilitated in the substrates with high moisture contents Thus, despite the presence of a rather complex microbiota in the starter, traditional Vietnamese alcoholic fermentation is a stable process that involves a limited number of functional species The role of satellite microorganisms, however should not be overlooked since they may contribute to the organoleptic quality of final product Surprising behavior of Sm fibuligera has highlighted the need for understanding the system in interaction Since banh men is similar to Chinese yeast ball, murcha (India, Nepal), bubod (Philippines), loog-pang (Thailand), ragi (Indonesia, Malaysia) in terms of microbiological composition and method of application (Hesseltine et al., 1988), what described here for traditional Vietnamese alcoholic fermentation might also be relevant to other processes Acknowledgements We thank Truong A Tai and Tran Thi Thom for valuable assistance This study was supported by Grant from The National Foundation for Science and Technology Development (NAFOSTED) #106.03.146.09 V.N Thanh et al / International Journal of Food Microbiology 232 (2016) 15–21 Appendix A Supplementary data Supplementary data to this article can be found online at http://dx doi.org/10.1016/j.ijfoodmicro.2016.05.024 References Aidoo, K.E., Nout, M.J., Sarkar, P.K., 2006 Occurrence and function of yeasts in Asian indigenous fermented foods FEMS Yeast Res 6, 30–39 Brenner, D.J., McWhorter, A.C., Kai, A., Steigerwalt, A.G., Farmer, J.J., 1986 Enterobacter asburiae sp nov., a new species found in clinical specimens, and reassignment of Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb nov and Enterobacter nimipressuralis comb nov J Clin Microbiol 23, 1114–1120 Calmette, A., 1892 La levure chinoise, ferment de l'amidon Ann Inst Pasteur 6, 604–620 Calmette, A., 1895 La fabrication des alcools de riz en Extrême-Orient Arch Méd Nav Colon 64 264-276 & 342-368 Chen, B., Wu, Q., Xu, Y., 2014 Filamentous fungal diversity and community structure associated with the solid state fermentation of Chinese Maotai-flavor liquor Int J Food Microbiol 179, 80–84 Chi, Z., Chi, Z., Liu, G., Wang, F., Ju, L., Zhang, T., 2009 Saccharomycopsis fibuligera and its applications in biotechnology Biotechnol Adv 27, 423–431 Constantiniu, S., Romaniuc, A., Iancu, L.S., Filimon, R., Taraşi, I., 2004 Cultural and biochemical characteristics of Acinetobacter spp strains isolated from hospital units J Prev Med 12, 35–42 Da, P.X., 2009 Study about raw materials and consumption areas of traditional wine in Vietnam Vietnam J Pract Med 650, 58–60 Dolatabadi, S., de Hoog, G.S., Meis, J.F., Walther, G., 2014a Species boundaries and nomenclature of Rhizopus arrhizus (syn R oryzae) Mycoses 57, 108–127 Dolatabadi, S., Walther, G., Gerrits van den Ende, A.H.G., de Hoog, G.S., 2014b Diversity and delimitation of Rhizopus microsporus Fungal Divers 64, 145–163 Dung, N.T.P., Rombouts, F.M., Nout, M.J.R., 2005 Development of defined mixed-culture fungal fermentation starter granulate for controlled production of rice wine Innov Food Sci Emerg Technol 6, 429–441 Dung, N.T.P., Rombouts, F.M., Nout, M.J.R., 2006 Functionality of selected strains of molds and yeasts from Vietnamese rice wine starters Food Microbiol 23, 331–340 Dung, N.T.P., Rombouts, F.M., Nout, M.J.R., 2007 Characteristics of some traditional Vietnamese starch-based rice wine fermentation starters (men) LWT Food Sci Technol 40, 130–135 Esteve-Zarzoso, B., Belloch, C., Uruburu, F., Querol, A., 1999 Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers Int J Syst Bacteriol 49, 329–337 Haard, N.F., Odunfa, S.A., Lee, C.H., Quintero-Ramírez, R., Lorence-Quinones, A., WacherRadarte, C., 1999 Fermented cereals a global perspective FAO Agricultural Services Bulletin No 138 Haleem Khan, A.A., Mohan Karuppayil, S., 2012 Fungal pollution of indoor environments and its management Saudi J Biol Sci 19, 405–426 Hesseltine, C.W., 1983 Microbiology of oriental fermented foods Annu Rev Microbiol 37, 575–601 Hesseltine, C.W., Featherston, C.L., Lombard, G.L., Dowell, V.R.J., 1985 Anaerobic growth of molds isolated from fermentation starters used for foods in Asian countries Mycologia 77, 390–400 Hesseltine, C.W., Rogers, R., Winarno, F.G., 1988 Microbiological studies on amylolytic oriental fermentation starters Mycopathologia 101, 141–155 Juhnke, M.E., des Jardin, E., 1989 Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere environments Appl Environ Microbiol 55, 747–750 Karimi, K., Zamani, A., 2013 Mucor indicus: biology and industrial application perspectives: a review Biotechnol Adv 31, 466–481 Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H., Yi, H., Won, S., Chun, J., 2012 Introducing EzTaxon: a prokaryotic 16S rRNA gene sequence 21 database with phylotypes that represent uncultured species Int J Syst Evol Microbiol 62, 716–721 Kitamoto, K., 2015 Cell biology of the Koji mold Aspergillus oryzae Biosci Biotechnol Biochem 79, 863–869 Kito, H., Abe, A., Sujaya, I.N., Oda, Y., Asano, K., Sone, T., 2009 Molecular characterization of the relationships among Amylomyces rouxii, Rhizopus oryzae, and Rhizopus delemar Biosci Biotechnol Biochem 73, 861–864 Kitpreechavanich, V., Maneeboon, T., Kayano, Y., Sakai, K., 2008 Comparative characterization of L-lactic acid-producing thermotolerant Rhizopus fungi J Biosci Bioeng 106, 541–546 Kurtzman, C.P., Robnett, C.J., 1998 Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences Antonie Van Leeuwenhoek 73, 331–371 Kurtzman, C.P., Vesonder, R.F., Smiley, M.J., 1973 Formation of extracellular C14-C18 2-Dhydroxy fatty acids by species of Saccharomycopsis Appl Microbiol 26, 650–652 Lackner, G., Möbius, N., Scherlach, K., Partida-Martinez, L.P., Winkler, R., Schmitt, I., Hertweck, C., 2009 Global distribution and evolution of a toxinogenic BurkholderiaRhizopus symbiosis Appl Environ Microbiol 75, 2982–2986 Lane, D.J., 1991 16S/23S rRNA sequencing In: Stackebrandt, E., Goodfellow, M (Eds.), Nucleic Acids Techniques in Bacterial Systematics Wiley, Chichester, pp 115–175 Lee, A.C., Fujio, Y., 1999 Microflora of banh men, a fermentation starter from Vietnam World J Microbiol Biotechnol 15, 51–55 Li, Y., Zhang, W., Zheng, D., Zhou, Z., Yu, W., Zhang, L., Feng, L., Liang, X., Guan, W., Zhou, J., Chen, J., Lin, Z., 2014 Genomic evolution of Saccharomyces cerevisiae under Chinese rice wine fermentation Genome Biol Evol 6, 2516–2526 Limtong, S., Sintara, S., Suwannarit, P., Lotong, N., 2002 Yeast diversity in Thai traditional alcoholic starter Kasetsart J (Nat Sci.) 36, 149–158 Nelson, M., 1944 A photometric adaptation of the Somogyi method for the determination of glucose J Biol Chem 153, 375–380 Partida-Martinez, L.P., de Looss, C.F., Ishida, K., Ishida, M., Roth, M., Buder, K., Hertweck, C., 2007 Rhizonin, the first mycotoxin isolated from the zygomycota, is not a fungal metabolite but is produced by bacterial endosymbionts Appl Environ Microbiol 73, 793–797 Passoth, V., Fredlund, E., Druvefors, U.A., Schnürer, J., 2006 Biotechnology, physiology and genetics of the yeast Pichia anomala FEMS Yeast Res 6, 3–13 Peixoto, S.C., Jorge, J.A., Terenzi, H.F., Polizeli Mde, L., 2003 Rhizopus microsporus var rhizopodiformis: a thermotolerant fungus with potential for production of thermostable amylases Int Microbiol 6, 269–273 Peters, E.J., 2012 Appetites and Aspirations in Vietnam: Food and Drink in the Long Nineteenth Century AltaMira Press Richardson, M., 2009 The ecology of the Zygomycetes and its impact on environmental exposure Clin Microbiol Infect 15, 2–9 Rohm, B., Scherlach, K., Möbius, N., Partida-Martinez, L.P., Hertweck, C., 2010 Toxin production by bacterial endosymbionts of a Rhizopus microsporus strain used for tempe/ sufu processing Int J Food Microbiol 136, 368–371 Sambrook, J., Fritsch, E.F., Maniatis, T., 1989 Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Schneider, J., Rupp, O., Trost, E., Jaenicke, S., Passoth, V., Goesmann, A., Tauch, A., Brinkrolf, K., 2012 Genome sequence of Wickerhamomyces anomalus DSM 6766 reveals genetic basis of biotechnologically important antimicrobial activities FEMS Yeast Res 12, 382–386 Somogyi, M., 1952 Note on sugar determination J Biol Chem 195, 19–23 Thanh, V.N., Mai, L.T., Tuan, D.A., 2008 Microbial diversity of traditional Vietnamese alcoholic fermentation starters (banh men) as determined by PCR-mediated DGGE Int J Food Microbiol 128, 268–273 Uchimura, T., Kojima, Y., Kozaki, M., 1990 Studies on the main saccharifying microorganism in the Chinese starter of Bhutan, “chang poo” J Brew Soc Jpn 85, 881–887 Wang, Q.M., Liu, W.Q., Liti, G., Wang, S.A., Bai, F.Y., 2012 Surprisingly diverged populations of Saccharomyces cerevisiae in natural environments remote from human activity Mol Ecol 21, 5404–5417 ... Materials and methods 2.1 Rice fermentation Rice fermentation was carried out in the laboratory using traditional procedures Three fermentation experiments were conducted separately using one of... during traditional alcoholic fermentation Microbial succession and changes in biochemical parameters during the course of traditional alcoholic fermentation is presented in Fig During the first 24... in fermentation mashes (data not shown) Thus, it could be concluded that, although present in high density during solid state fermentation, M indicus and Sm fibuligera play little or no role in