vNU journal of science, Natural L sciences and rechnologv 23, No 1s (2002) 1g1-1g6 Microorganisms in yeast cakes controlling the quality and taste of traditional fermentation products -r- ::rl" \ Tran Thi Le Quyent, Bui Thi Viet Hal,*, Dao Thi Luong2, Dinh Th.ry Hang2, Duong Van Hop2 ;r tFaculty 2lnstitute of Biotogt, college of science, vNU, 334 Nguyen Trai, Hanoi, I/ietlant of Microbiolog,t and Biotechnologt, WU, 144 Xuan Thuy, Honoi, viefirum Received 15 August 2007 Abstract Alcohol fermentation was carried out with sticky rice using 15 most popular sorts of yeast cakes in Vietnam to produce different kinds of alcoholic beverages The products were obtained by traditional distillation method and compared with each other for flavour and taste according to the standard qualification scale for alcoholic products It revealed that Ruou Can yeast cake which has been widely used for hundreds years by local commirnities yielded the best product and therefore was selected for further studies From this yeast cake, two yeast shains were first time isolated and identifie d as Saccharomyces cerevisiae and, Sqccharomycopsis fibuliger based on morphology and 263 rDNA Dl/D2 sequencing analyses A sort yeast cake remade by using these two yeast strains yielded alcoholic product as good as the product obtained from using Ruou Can yeast cake hec x- PCR/DGGE analysis of 165 rDNA (for the Prokaryotes) and l8S rDNA (for the Eukaryotes) showed differences in structures of microbial communities in the studied yeast cakes However, in all cases, Saccharomyces sp., Saccharomycopsis sp., and Lactobacillus sp accounted for the most abundant populations Interestingly, the both best sorts ofyeast cakes, i.e the Ruou Can yeast cake and the remade yeast cake, contain mainly these groups of microorganisms l\E Keywords : yeast cake, traditional fermentation, alcohol fermentation Irii 11,w h4 roc Introduction on the sorts of "yeast cake", water source and the nature of starch source [1] Among the above mentioned factors, yeast cake with high stability is the most important for the success of fermentation process and the taste of product, In this study we investigate the relationship between the microbial community in yeast cakes and the quality of alcoholic drinks The obtained results would be applied in improvement of the production process of a good sort of yeast cake with stable microbial hsp Irl Yeast cake is a traditional microbial product which has been used widely in Vietnam as starter in fermentation to produce alcoholic drinks It is rich in microorganisms, especially those involve in the transformation of starch into fermentable sugars, then into ethyl alcohol Quality of alcoholic drinks pnmarily depends to.r.rp*ang author Tel : 84-4-85 E-mail: habtv@mu.edu.vn 88 856 181 182 T.T.L Quyen et al / content Wll lournal of Science, Natural for long term production of highly qualifi ed alcoholic drink Materials and methods Yeast cakes 15 yeast cake samples were collected from different provinces which are well known for traditional alcoholic dnnk products in Northern Vietnam (data not shown) Fermentation '\east cakes" were ground to dust, mixed with cooked sticlry rice and incubated for 5-8 days at room temperature Alcoholic products were collected by distillation and examined for quality according to the national standard TCW3217-79 Sequencing ond phylogenetic analysis Cells rvere gro\rm in the logarithmic $owth phase Isolation and purification of nuclear DNA were done according to Takashima and Nakase (2000)121 The sequence of the DllDz regron of 265 rDNA, were determined after ampliffing the DNA using PCR The strains were sequenced directly t3] Generated sequences were aligned with related species by using the CLUSTAL W ver 1.83 sorftware program [4] Reference sequences used for the phylogenetic study were obtained from the Sciences and Technology 23, No 15 (2007) 181-1.g6 the primers EuklA (5'- CTG GTT GAT CCT GCC AG-3') and Euk5l6r (5'- ACC AGA CTT GCC CTC C-3') In order to stabilize the melting behavior of the amplified fragments in the gradient denaturing gel, a GC-clamp (CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGGG) was added at 5'-end of the primers GM5F (for prokaryote) and Euk516r (for eukaryotes) PCR were performed in 50 r,1 volume, containing pl of template DNA, pl buffer x 10, rr1 BSA (3mg/m1), lpl dNTPs 25mM, lpl primer (20p1), 0.6y1 Taq DNA polymerase (2.5u/pl) The PCR program for eukaryotic primer set included an initial denaturation at 94 oC for 130 s, followed by 35 cycles of denaturation at 94 oC for 30 s, annealing at 56 oC for 45s, and extensionatT2 oC for 130 s The PCR program for prokaryotic primer set included an initial denaturation at 94 oC for I and 20 touchdown cycles of denaturation at 94 "C for min, annealing at 65 'C (with the temperature decreasing 0.5 oC each cycle) for min, and extension at 72 oC for min, followed by 20 cycles of 94 oC for min, 55 oC for min, and 72 oC for During the last cycle of both programs, the length of the extension step was increased to l0 database The phylogenetic kee was constructed DGGE was run in DCode system (Bio-Rad) from the evolutionary distance data according to Kimura using the neighbor-joining method ls l Denaturing gradient gel electrophoresis (DGGE) Total DNA from "yeast cakes" was as described by Muyzer et al l3l A 6% polyacrylamide gel with a gradient of DNAdenaturant agent was cast by mixing solution of Uoh and 80% denaturant agent (100% :7 M urea and 40o/o deionized formamide) Linear gradients of denaturants (20-70% for extracted by using method described by Zhou et al [6] with some modifications Fragments of the 163 rDNA (550 bp) for DGGE analysis were obtained with the bacterial-specific primer pair GM5F (5'- CCT ACG GGA GGC AGC AG-3) and 907R (5'- CCG TCA ATT CCT TTR AGT TT-3') Fragments of the 18S rDNA (560 bp) for DGGE analysis were obtained with prokaryotic DNA, 30-60% for eukaryotic DNA) rvere used PCR products were loaded on the gels rvith the volume of 50 pl for each sample and the electrophoresis was run at 200 V for 3.5 h at 60 oC in IxTAE buffer Aftenvard, the gels were stained with ethidium bromide (5 mg/ml) for 30 min, rinsed with wi Pr ml 40 pn usl cal pr( eul per seq BL ph: 3.I yei of pro' yei alco othe Acc 321',, yea "y"u micr ,] isola 84.l whor reprc 84.2 cells budd resen Saccl reSper rDN.a 84.1 T.T.L Quyen et al ; / WLI lournal of Science, Natural and visualized under IfV Prominent bands were excised from the gels, mounted in sterilized Mili-Q water overnight at 4'C The DNA was reamplified (using the same primer sets without GC-clamp) and purified using QIAgen kit Sequencing reactions u'ere carried out with the primers GM5F for prokaryotic DGGE bands, EuklA for eukaryotic DGGE bands and sequencing rvas performed in an automatic ABI PRISN{ 3100 sequencer Sequences were submitted to the BLAST Search of Gene Bank to determine the phylogenetic affiliation [7] water for rI : J : J't iE- Sciences and Technology 23, No 15 (2007) 181-186 183 Saccharomyces cerevisiae (l00Yo homology) and Saccharomycopsis fibuliger (100% homology), respectively (Fig 1) [9] In order to verify the role of the isolated yeast strains in the "yeast cake" used for alcoholic fermentation, we used them as the only source of microorganisms for making a new sort of yeast cake, called remade yeast cake (BM16) The alcoholic drink obtained with the remade "yeast cake" showed high quality and pleasant taste, similarly to the drink produced with Ruou Can "yeast cake" The obtained results indicated that the isolated yeast strains represented the key organisms, responsible for the fermentation process Results and discussion y{ of 55 "1c 'C ; h ^ r,]t It is known that "yeast cakes" contain highly Fermentation 15 most popular sorts of "yeast cakes" presently used for the production of alcoholic drinks were collected from provinces in Northem Vietnam By using these "yeast cakes" for fermentation, we obtained 15 alcoholic products which differed from each other in quality, alcohol content and taste According to the national standard TCW j217-79, the drink produced by Ruou Can "yeast cake" showed best quality and taste, the "yeast cake" was therefore chosen for B,Ci 5',1,r L\- rof !fr E@ fqr crttf Icm ash 3m f,Er [rr[M r.ffi microbiological studies Two yeast strains, B4.1 and B4.2, u'ere isolated from the Ruou Can "yeast cake" Strain B4.1 has round, smooth, white-cream clonies whose cells are spheroidal or oval and reproduce by budding On the other hand, strain P.4.2 has round, rough, white colonies, whose cells are of branched form and reproduce by budding Thus, morphologically these strains resemble the representatives of genera Saccharomyces and Saccharomycopcis, respectively [8] Phylogenetic analyses of 265 rDNA DllDz sequencing indicated that strains B4.1 and B4.2 could be affiliated to species diverse communities of microorganisms, including yeasts, molds and bacteria that directly or indirectly involve in the fermentation process and determine the quality of alcoholic products [10] To investigate the communities of microorganisms existing in different sorts of "yeast cakes" and look inside into the role of microorganisms in fermentation we carried out analyses of PCR major groups of amplified fragments of 165 rDNA and 18S rDNA from the total DNA pool of "yeast cakes' using DGGE method PCR fragments of prokaryotic 165 rDNA (550 bp) and eukarl'otic 18S rDNA (560 bp) arnplifred from DNA pool of eight different "1'east cakes" x-ere separated on denaturing -eradient pol-varylamide gels (Fig and 3) -{s references, PCR fragments arrplified *-ith ttrre same primer set from the 1'east isotrates B\I4.1 and BM4.2 were also included" " 1'east cake" (BM4), and the remade ")'east cake" @M16) showed similar DCrGE pattern in eukaryotic rDNA analyses (Fig 2, lanes and land 9) and consisted two DCrGE bands (Fig 2, E3 and E4) that were identical to the bands produced by pure cultures Rou Can l.84 T.T.L Quyen et al / WU lournal of Science, Natural Saccharomyces cerevisiae BM4.1 and Saccharomycopsis filuliger 8M4.2 respectively (Fig.l, lane 10, 11) On the other hand, other "yeast cake" samples contained besides these two bands some other bands (Fig.1,lane 1,2,48), representing yeast populations other than Saccharomyceis and Saccharomycopsis such as Lepidoglyphus (Fig.1, band E5) Sciences and Technology 23, No 15 (2007) 787-186 have influences on the flavour and taste of the final products [4] Further investigation on the role of bacteria such as Lactobacillus sp 1: production of specific flavour or taste o: alcoholic drinks are in process Srdrm]66i86 Since Sacdrffi.r6ceDuisiae Saccharomyces and Saccharomycopsis yeasts are responsible for two steps in fermentation process, the starch hydrolysis and alcoholic fermentation on sugar, the presence of these populations in Ruoucan and the remade "yeast cakes" could explain for the high quality of the products At the same time, it seemed that the existence of yeast populations other , C[ Sacdr@]@shtuiilzwi SecdrwlesDaloc Sacdrffi)espasbrin Saodr@I,}osnagmiCli Seodrmlsshmdiffi Srcdrmr€stsinds Secdrardrrlcas Saodarurucop.ds SacdrarnlopdsmabrgE Strdlamt@pdsltDd&Dr Weissella and Lactobacillus groups, of bacteria are known for the production of acids or respectively These groups antimrcrobial substances and therefore might inhibit souring microorganisms, and at the same time support growth of yeast populations In addition, these groups of bacteria could also J.( @r&_wi Dobaympswrib Cardida&ttu it is presented for the first time that "yeast cakes" with less diverse yeast Although not being involved directly in the process, prokaryotic fermentation microorganisms such as many kinds of bacteria in "yeast cakes" could be responsible for the taste of the drink products, Different "yeast cake" samples showed different DGGE pattern in prokaryotic rDNA analysis (Fig.3), even the two similar samples Ruou Can and the remade "yeast cakes" The most prominent bands P1, P2 and P3 showed affiliation to Acetobacter, crebogdrsb Saodrmlopsisviri this results, Saccharomyces and Saccharomycopsis groups, would be the key for a successful fermentation and produce alcoholic drinks of high quality L B] Secdlrylopsislamit than only Jakdrrrafl ds F \\' Seodrmlopsis&manbrs Saccharomyces and Saccharomycopsis was the reason for souring problems in products obtained with other sorts of "yeast cakes" With communities, ideally contain F-s'l-{ Sacdrm]@sporEam Nol Pichbhntui Phylogenetic tree based on 265 rDNA DliDl sequences showing the affiliation of yeasts isolates from Ruou Can yeast cake with other relative Fig cho ofi pler specles Ruc Sac Sac, mor "Ye pro( Ruo Fig DGGE pattem of PCR fragments amplified with 18S rDNA eukaryotic primers Lane 1-8M1, Lane 2-8M2, Lane 3-BM4, Lane 4-BM7,Late 5BM9, Lane 6-8M10, Lane 7-BM1l, Lane 8-BM15 Lane 9-BM16, Lane 10-B4.1, Lane ll-P.4.2 rDN Sacc Lact popL qual T.T.L Quyen et al I WU lournal of Science, Natural Sciences and Technology 23, No 75 (2007) 1.81-1.86 185 References f the the [] p in eof Nguyen Thi Hien et al., Technologt for production of L-glutamic acid and traditional products, Science and Technique Publishing House Ha Noi,2006 (in Vietnamese) [2] M Takashima, T Nakase, Four new spesies of q genus Sporobolomyces isolated from leaves in Thailand, Mycoscience 41 (2006) 65 [3] C P Kurtzman, C.J Robnett, Identification of clinically important ascomycetous yeasts Fig DGGE pattern of PCR fragments amplified based on nucleotide divergence in the 5'end of the large- subunit (265) ribosomal DNA gene J.Clin Microbiol.35 (1997) 1216 with l6s rDNA prokaryotic primers Lane l-BMl, Lane2-8M2, Lane 3-BM4, Lane 4-BM7,Lane 5BM9, Lane 6-BM10, Lane 7-BM11, Lane 8-BM15' Lane 9-BMl [4] Conclusions l) Among D1,D2 ,olates i'r'e 15 most popular "yeast cakes" in Northern Vietnam, Ruoucan "yeast cake" was choosen as the best "yeast cake" for production of alcoholic drink with high quality and soft, [5] N Sa:tou, M Nei, The neighbor-joining method: a nel method for reconstructing phylogenetic trees, MoL from Ruoucan "yeast cake" and were identified environmental samples, Appl Environ Microbio, 62 (1996) 316 Sanchez, Isabel Ferrera, Nuria Vigues, Tirso Garcia de Oteyza, Joan O Grimalt, Jordi Mas., Presence of opportunistic oil-degrading microorganisms operating at the initial steps of [7] Olga as cerevisioe and Saccharomyces Saccharomycopsis filuliger base on morphology and 265 rDNA Dl/Dz sequencing "Yeast cake" remaded with the isolated yeasts produced product as good as the original plified -8M1, ane 5-BM15 A.2 Biol Evol.4 (1987) 406 [6] Zhoul et al., Isolation of genomic DNA from pleasant taste 2) Two yeast strains were isolated J D Thompson, T J Gibson, F Plewniak, F Jeanmougin, D G Higgins, The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tool, Nucleic Acids Res.25 (1997) 4876 oil extraction Mitobiologt [8] and handling, International (2006) l19 Anna Kockova-Kratochvilova, Yeast and Yeash Ruoucan "yeast cake" like Organism,s, Alfa, Publishers of Technical and Economical Literature Bratislava, 3) DGGE analyses of 165 rDNA and 18S rDNA of different yeast cakes showed that Vu Saccharomyces, Saccharomycopsis and Lactobacillus were the major microbial populations in the "yeast cakes", controlling the quality and taste of the final drink products Czechoslovakia, 1990 tel Nguyen Thanh, Yeat Biodiversity and Evolution, Vol (2006), Part B ll0l Muyezer et ?1., Denaturing Gradient Gel Electrophoresis (DGGE), Mol Miuob Ecol Manual 3.4.4 (1996) l ... divergence in the 5'end of the large- subunit (26 5) ribosomal DNA gene J.Clin Microbiol.35 (1997) 121 6 with l6s rDNA prokaryotic primers Lane l-BMl, Lane2-8M2, Lane 3-BM4, Lane 4-BM7,Lane 5BM9, Lane... Reference sequences used for the phylogenetic study were obtained from the Sciences and Technology 23 , No 15 (20 07) 181-1.g6 the primers EuklA (5'- CTG GTT GAT CCT GCC AG-3') and Euk5l6r (5'- ACC AGA... containing pl of template DNA, pl buffer x 10, rr1 BSA (3mg/m1), lpl dNTPs 25 mM, lpl primer (20 p1), 0.6y1 Taq DNA polymerase (2. 5u/pl) The PCR program for eukaryotic primer set included an initial