Antonie van Leeuwenhoek (2011) 100:497506 DOI 10.

1007/s10482-011-9605-y

ORIGINAL PAPER

Yeast communities associated with artisanal mezcal fermentations from Agave salmiana
A. Verdugo Valdez L. Segura Garcia M. Kirchmayr lez Esquinca rez Rodr guez A. Gonza P. Ram R. Coria A. Gschaedler Mathis

Received: 29 January 2011 / Accepted: 3 June 2011 / Published online: 17 June 2011 Springer Science+Business Media B.V. 2011

Abstract The aims of this work were to characterize the fermentation process of mezcal from San Luis xico and identify the yeasts present in the Potosi, Me fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/ D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the rst fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were
lez Esquinca A. Verdugo Valdez A. Gonza gicas, Universidad de Ciencias Facultad de Ciencias Biolo y Artes de Chiapas, Libramiento Norte 1150, colonia rrez, CHIS, Me xico Lajas Maciel, Tuxtla Gutie L. Segura Garcia M. Kirchmayr rez Rodr guez A. Gschaedler Mathis (&) P. Ram n y Asistencia en Tecnolog a y Centro de Investigacio o del Estado de Jalisco A.C, Av. Normalistas # 800, Disen colonia Colinas de la Normal, 44270 Guadalajara, xico JAL, Me e-mail: agschaedler@ciatej.net.mx; gschaedlera@hotmail.com R. Coria a Celular, Circuito Exterior S/N Instituto de Fisiolog noma Ciudad Universitaria, Universidad Nacional Auto xico, Coyoaca n 04510, DF, Me xico de Me

identied, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identied yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identications were conrmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, conrming the results obtained with classic techniques. Keywords Mezcal Yeast diversity Fermentation process RFLP DGGE

Introduction Mezcal is a traditional Mexican distilled beverage produced by fermenting the juices of cooked agave a in Spanish). Mezcal is produced plant core (pin from various species of Agave in a denomination of origin region, which includes the states of Durango, Guerrero, Oaxaca, San Luis Potosi, Zacatecas and some districts of Guanajuato and Tamaulipas (Mexican Ministry of Commerce and Industry a Mendoza 1998; Lappe-Oliveiras et al. 1994; Garc 2008). Agave salmiana is used mainly in Mexicos

123

498

Antonie van Leeuwenhoek (2011) 100:497506

n-Rodr guez et al. 2006, Altiplano region (De Leo 2008). The general steps in the production process are: harvesting the raw material, cooking, milling, fermentation, distillation, and in some cases, maturation. In San Luis Potosi cooking is done in rustic ovens where the heat is provided by steam injection, in this step the fructans contained in the agave are hydrolyzed into simple sugars, mainly fructose. The juice of cooked agave is obtained in a rudimentary mill (named tahona), which has a circular stone of about 1.5 m in diameter rotating in a circular pit over the cooked agave. During the milling process, water is added, and the resulting juice is fermented through a spontaneous process and then distilled. The taste and the aroma of the mezcal are provided by the composition of a complex mixture of compounds produced during the process (cooking, fermentation and distillation), and other elements that come n-Rodr guez directly from the agave plant (De Leo et al. 2006, 2008). Many studies have demonstrated that several species of non-Saccharomyces yeasts are predominant at the beginning of spontaneous wine fermentations (Fleet 2003), and these contribute signicantly to sensory characteristics of the beverage (Romano et al. 2003). In spite of the signicance of the process and the sensorial characteristics of the end product, few works have addressed the identication and characterization of the yeasts involved in the fermentation process of the different agave spirits. The rst study to address this goal was conducted on traditional tequila fermentation by Lachance (1995). He identied, using classic microbiological techniques, the yeast communities and showed the great diversity in these processes. Samples from the early fermentation process contained a rich mixture of yeast species. However, as fermentation progressed, the number of species present tended to diminish, and nally only one biotype of Saccharomyces cerevisiae became dominant. Another study carried out in mezcal fermentation from Agave salmiana (Escalante-Minakata et al. 2008), revealed lower biodiversity of yeasts through molecular identication methods. In the last decade, it has been shown that neither classic microbiological methods nor culture depended molecular methods accurately detect complex microbial diversities in artisanal fermentations (Ben Omar and Ampe 2000; Tu et al. 2010). The most widely used

culture-independent method for the study of microbial communities is analysis by PCR-DGGE (denaturing gradient gel electrophoresis). This method has been used to study microbial communities, for example, in wine (Cocolin et al. 2001; Renouf et al. 2007), in sourdough (Meroth et al. 2003; De Vuyst et al. 2009; Moroni et al. 2011), in cocoa bean (Nielsen et al. 2005; Lefeber et al. 2011), in sausages (Rantsiou and Cocolin 2006), in soybean paste (Kim et al. 2009) and in several other fermented products. The goal of this work is to characterize the fermentation process of mezcal of San Luis Potosi and to identify the yeasts present during the fermentation using molecular methods, by RFLP analysis of the 5.8S-ITS region (Esteve-Zarzoso et al. 1999), and sequencing of the D1/D2 domain (Kurtzman and Robnett 1998). Additionally, and for the rst time in an agave distilled spirit, a culture-independent method (PCR-DGGE) was used to study the succession of the different species of the yeast during the fermentation process.

Materials and methods Mezcal fermentations and sampling procedures Sampling was carried out at the distillery of Laguna Seca in San Luis Potosi (Mexico). After milling, the crushed cooked agave is transferred in a special vat (called a lavadero) where water is added and the agave is washed in order to extract the sugars. Then, the juice (without the bers) is transferred to fermentation tanks. A smaller tank is used to propagate a starter ferment in agave juice at 10Bx supplemented with (NH4)2PO4 (1 g/l). The yeasts used in this starter culture are the autochthonous yeasts of the factory, which were preserved at 4C from a previous fermentation. Fermentation is carried out in 7000 l of agave juice supplemented with (NH4)2PO4 (1 g/l). The sugar concentration varies, depending on the raw material. Multiple samples were taken from two separate fermentations (fermentation I and II): from the lavadero; the starter culture; from the initial juice before inoculation; and at various points along the fermentation process. After sampling, yeast cultures were performed in the distillery, and aliquots of the same samples were frozen immediately until they

123

Antonie van Leeuwenhoek (2011) 100:497506

499

were processed in the laboratory (analytical methods and PCR-DGGE). Yeast isolation For all fresh samples, decimal dilutions in saline physiological solution (9 g/l NaCl) were prepared and used to inoculate WL nutrient agar (Fluka) plates, supplemented with 0,01% chloramphenicol. The plates were incubated at 29C for 35 days for colony development. The various colony types were counted, and representative colonies of each type were isolated and subcultured in YPD (yeast extract 10 g/l; peptone 20 g/l; dextrose 20 g/l; agar 20 g/l) for subsequent identication. Analytical methods For the determination of major volatile compounds (ethanol, methanol, amyl alcohols, acetaldehyde, isobutanol, 1-propanol and ethyl acetate), the must samples were volatilized in a Hewlett Packard headspace sampler HP 7694E and analyzed in a Hewlett Packard 6890 series gas chromatograph equipped with a ame ionization detector (FID), and a 60 m 9 320 9 0.25 lm lm thickness HP-Innowax capillary column. The chromatographic conditions were 45C for 7 min, increased at 10160C, 20220C/min, and maintained at this temperature for 8 min. Helium was used as carrier gas at a ow rate of 1.8 ml/min, and the injector and detector temperature were at 250C. Reducing sugar concentration was determined by the dinitro-salicylic acid (DNS) method (Miller 1959) using fructose as the standard. Molecular identication Yeasts were directly collected from the colonies and suspended in a PCR reaction mixture. For amplication of the ITS-5.8S rRNA region, the primers ITS1 (50 -TCC GTA GGT GAA CCT GCG-30 ) and ITS4 (50 -TCC TCC GCT TAT TGA TAT GC-30 ) were used. PCR conditions described by Esteve-Zarzoso et al. (1999) were followed and the enzymatic digestions were carried out using restriction enzymes HhaI, HaeIII and HinfI. Restriction fragments were analyzed by electrophoresis in 3%(w/v) agarose gels (Invitrogen, Carlsbad, CA, USA). The migration was

conducted at 100 V for 1 h in TAE 1X buffer (SigmaAldrich). The gels were stained with ethidium bromide (SigmaAldrich, Steinheim, Germany), visualized under UV light using an GelDoc system (Bio-Rad, Hercules, CA, USA), the size of the fragments was estimated by comparison with TrackIt 100 bp DNA ladder (Invitrogen), and analyzed using the Quantity one software (Bio-Rad). Identity of the yeasts was conrmed by sequencing the variable domain D1/D2 of the large (26S) ribosome subunit (Kurtzman and Robnett 1998). The PCR products were sequenced by Macrogen (Rockville, MD, USA). The resultant sequences were aligned in GenBank using the BLAST program for identication. PCR-DGGE analysis For direct DNA extraction from musts samples, the MasterPure Yeast Purication Kit (Epicentre Biotecnologies, Madison, WI) was used. The D1 region of the 26S rRNA gene was amplied by PCR, using the primers NL1GC (50-GCG GGC CGC GCG ACC GCC GGG ACG CGC GAG CCG GCG GCG GGC CAT ATC AAT AAG CGG AGG AAA AG-30) (the GC clamp is underlined) and LS2 (50ATT CCC AAA CAA CTC GAC TC30), following the conditions described by Cocolin et al. (2001). In case of direct amplication from isolated yeasts, an initial step of denaturalization was added (25 min at 95C). Amplied products were analyzed on 3% ultrapure agarose (Invitrogen) gels containing 0.5 mg/ml ethidium bromide, visualized under UV light and analyzed with the Quantity one software (BioRad). The DcodeTM Universal Mutation Detection System (BioRad) was used for DGGE analysis. Electrophoreses were performed in a 1.0 mm polyacrylamide gel [8% (w/v) acrylamide:bisacrylamide 37.5:1] using a denaturant gradient increasing in the direction of the electrophoretic run. Amplicons produced by PCR were analyzed in a denaturant gradient from 45 to 75%. Electrophoretic runs were carried out at a constant temperature of 60C in 1X TAE at 100 V for 16 h. After electrophoresis, the gels were stained for 10 min in 1X TAE containing ethidium bromide 0.1 ll/ml, visualized under UV light, and analyzed with the Quantity One software (BioRad).

123

500

Antonie van Leeuwenhoek (2011) 100:497506

Results Fermentation kinetics and general yeast counts Figure 1 shows the time course of the two alcoholic fermentations sampled. Generally, fermentation times are short; the process is completed in 2448 h. The sugar and ethanol concentrations are higher in fermentation I than in fermentation II. However, the conversion yields of sugar to ethanol are still identical: 0.39 and 0.38 g/g for fermentation I and II, respectively. An important characteristic of this type of fermentation is the high temperature of the must, which is maintained between 33 and 35C. The yeast population in the inoculums reached 4 9 107 cells/ml. The maximum concentration during fermentation was reached after 10 h and decreased slightly until the end of the process. Figure 1 also shows the concentration of the major volatile

compounds found in the must. In general, fermentation I (A) generated greater concentrations of these compounds than fermentation II (B).The amyl alcohols are the predominant compounds, followed by acetaldehyde, isobutanol, 1-propanol and ethyl acetate. The concentration of higher alcohols (sum of concentrations of amyl alcohols, isobutanol and 1-propanol) was 54.8 and 27.5 mg/l in fermentation I and II respectively. Molecular identication and enumeration of yeasts One hundred ninety-two (192) different colonies were isolated from WL plates and identied by RFLP analysis of the ITS1-5.8S-ITS2 of rRNA gene. Twenty-six (26) different morphologies were distinguished, resulting in eight different restriction proles of RFLP. Table 1 shows the nucleotide fragment size

Fig. 1 Evolution of alcoholic fermentation and production of volatile compounds in Fermentation I (a) and II (b) in distillery , Me xico Laguna Seca in San Luis Potos

123

Antonie van Leeuwenhoek (2011) 100:497506

501

(pb) of the ITS-5.8S region amplied by PCR and digested with restriction endonucleases, which were compared to the data previously described (EsteveZarzoso et al. 1999). The identied yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identications were conrmed by sequencing the D1D2 region of 26S rRNA gene for each RFLP prole. Table 2 shows the species found in different areas of the distillery, the lavadero where the cooked and crushed agave is washed; in the agave juice before the inoculation; and in the tank of starter culture. S. cerevisiae, K. marxianus, S. exiguus and T. delbrueckii were detected at every site. C. ethanolica was only detected in the starter culture, and P. kluyveri and Z. bailii only in the agave juice before inoculation. During fermentation (Table 3), S. cerevisiae was the predominant yeast. Large differences in the populations of non-Saccharomyces were observed between the two fermentations. At the beginning, fermentation I exhibited high diversity, while in fermentation II less non-Saccharomyces species were detected. At the end of fermentation I

only K. marxianus and T. delbrueckii were detected whereas, in fermentation II, K. marxianus, P. kluyveri, Z. bailii, C. lusitaniae, T. delbrueckii, and S. exiguus were found together with a signicant population of Z. bailii. PCR-DGGE analysis The PCR-DGGE proles obtained from the extracted musts samples are shown in Fig. 2 and compared with proles from the different isolated strains. Bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii are clearly detected and conrmed the results obtained with the traditional techniques. A few bands were observed which didnt correspond to any of the isolated yeast. However, these bands were a result of either secondary structures of the isolated species or belonged to fungi genomes (Penicillium spp.).

Discussion The fermentation process of Agave salmiana was analyzed, including a general characterization of the fermentation process; the generation of some volatile

Table 1 Yeast identied and sizes of ITS-5.8S region amplied by PCR (AP) and the fragments obtained after digestion with restriction endonucleases HhaI, HaeIII and HinfI Yeast identied AP (pb) Restriction fragments HhaI Saccharomyces cerevisiae Kluyveromyces marxianus Clavispora lusitaniae Saccharomyces exiguus Torulaspora delbrueckii Zygosaccharomyces bailii Pichia kluyveri Candida ethanolica 875 784 380 658 800 723 465 450 373347138 29819617190 16599 361297 325220153-100 3212849494 16810886 152101 HaeIII 321241180133 64485 379 496246 795 700 31695 301 HinfI 374127 25619011887 257217 354248137 410378 339226158 257207 250201

Table 2 Yeast identication from different sites and fermentations process from the factory Laguna Seca in the State of , Mexico San Luis Potos

Isolation site

Lavadero

Starter culture

Fermentation tank (before inoculation) S. cerevisiae S. exiguus K. marxianus T. delbrueckii P. kluyveri Z. bailii

Isolated yeast

S. cerevisiae K. marxianus S. exiguus T. delbrueckii

S. cerevisiae K. marxianus S. exiguus T. delbrueckii C. ethanolica

123

502 Table 3 Occurrence of yeast populations in fermentation tank at the beginning and at the end of the fermentation Species Fermentation ages Initial (%) Fermentation I S. cerevisiae S. exiguus K. marxianus T. delbrueckii P. kluyveri Z. bailii C. lusitaniae Fermentation II S. cerevisiae S. exiguus K. marxianus T. delbrueckii P. kluyveri Z. bailii C. lusitaniae 97.63 0.18 2.19 77.26 1.68 1.43 0.67 13.68 5.28 94.00 0.90 2.20 0.50 0.50 1.40 0.50 2.00 1.78 96.22 End (%)

Antonie van Leeuwenhoek (2011) 100:497506

compounds; and a detailed study of the yeast populations using culture-dependent and -independent methods. Fermentation process and generation of volatile compounds The Agave salmiana used for the elaboration of this mezcal is not cultivated but is collected in the Mexican Altiplano. The characteristics of the agave depend where it is collected. In particular, the concentration of

fructans, which are the stored sugar of the plant, may vary. In fact, the agave plants used for each fermentation arrived from different sites and had large difference in the initial sugar concentrations. Since it had less sugar, fermentation II produced less ethanol than fermentation I. A similar effect was observed with the volatile compounds: amyl alcohols, isobutanol and 1-propanol, since sugar concentration affects this process as well. In tequila, synthesis of isobutanol and amyl alcohols is increased when the C/N ratio is increased (Arrizon and Gschaedler 2007). In our case, initial nitrogen concentration was similar in the two fermentations (addition of (NH4)2PO4, 1 g/l) so the differences in the volatile compounds proles could be due to different sugar contents in the raw material. Although only a few compounds could be directly measured in the must, these reect the overall behavior of the volatile com n-Rodr guez et al. (2006) analyzed pounds. De Leo sixteen mezcal brands from San Luis Potosi and identied thirty-seven compounds; nine of them were classied as major compounds. Five of the compounds determined in this study belonged to this group, and had an impact on the organoleptic properties and the bouquet of the nal product. The rst conclusion of this work is that it is essential to have quality standards for the raw material, particularly in the sugar concentration, in order to generate a fermented must with similar concentrations of ethanol and volatile compounds. Yeast identication succession and generation of volatile compounds Like numerous previous studies (Zott et al. 2008; Tofalo et al. 2009; Csoma et al. 2010; Li et al. 2010;

Fig. 2 Migration prole of PCR-DGGE from fermentation I (a) and II (b). Line M corresponds to mixture of pure strains isolated on WL medium and identied by RFLP; lines 19 DGGE proles of the DNA amplicons obtained directly from musts corresponding to 7.5, 10, 15, 23, 25.5, 27, 30, 32 and

47 h in (a); lines 17 corresponding to 0, 3, 6, 9, 11, 13 and 24 h in (b). Abbreviations: C. sake (Cs), T. delbrueckii (Td), K. marxianus (Km), Z. bailii (Zb), S. cerevisiae (S.c.), R. mucilaginosa (Rm), S. exiguus (Se), C. ethanolica (Ce) and P. membranifaciens (Pm)

123

Antonie van Leeuwenhoek (2011) 100:497506

503

Cordero-Bueso et al. 2011), PCRRFLP analysis was successfully used to identify yeast species. Sequencing was used to conrm the identities obtained, by comparing the RFLP patterns with similar published data. Escalante-Minakata et al. (2008) reported K. marxianus, P. fermentans and C. lusitaniae, in another Agave salmiana fermentation in the same region. However, the use of only one restriction enzyme, Hae III, and different solid mediums for yeast isolation, may explain the dissimilarities in diversity and species encountered. The use of WL medium in this study was very useful for the detection of yeast diversity (Pallmann et al. 2001; Cocolin et al. 2006; Urso et al. 2008; Li et al. 2010); based on not only morphological characteristics, but also on differences in color of the colonies. In addition to the latter, we used a medium supplemented with agave juice, and we found the same yeasts (data not shown). In tequila, another agave distilled spirit, Lachance (1995) found S. cerevisiae, Z. bailii, Candida milleri and Brettanomyces anomala, as dominant yeasts and B. bruxellensis, Hanseniaspora guilliermondii, H. vinae, P. membranaefaciens, T. delbrueckii and K. marxianus as secondary yeasts in the fermentation process. On the y drosophila, which is a vector for yeast, Lachance found P. kluyveri. However, it wasnt detected in the fermentation. So, ve of the eight yeasts detected in these mezcal fermentations were also found in the tequila fermentation. Regarding this study, tequila fermentations present more diversity of yeasts than the Agave salmiana fermentations. One reason could be the characteristics of the raw material. Agave salmiana contains a high level of saponins (Zamora et al. 2010), and these compounds are known to be inhibitors of yeasts growth (Miyakoshi et al. 2000). In another work, Cira et al. (2008) showed that the heterologous expression of Fusarium oxysporum tomatinase (which detoxies steroidal saponins) in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave must. Finally, another probable reason could be the geographical location of the distillery: the Mexican altiplano is an arid semi-desert region with a low population of insects, which are the possible vectors of yeasts in this kind of fermentation as demonstrated by Lachance (1995). In wine must, and recently in vineyards and wineries, various studies have been carried out in

order to characterize the current yeast populations, emphasizing non-Saccharomyces yeasts. In a study of yeasts from grape berries, Clavijo et al. (2010) found that 84% of the total yeast population was nonSaccharomyces species, and that Kluyveromyces thermotolerans, H. guilliermondii, H. uvarum and n Issatchenkia orientalis represented the 42.7%. Oco et al. (2010) studied the yeasts present in the facilities and cellars of four wineries from the D.O.Ca. Rioja Region. Pichia and Cryptococcus genera and the Pichia anomala species were found in all four wineries; T. delbrueckii and P. membranifaciens were detected in four wineries; and Aerobasidium pullulans, Kloeckera apiculata and Debaryomyces hansenii were isolated in two wineries. Zott et al. (2008) found 19 yeasts species in the wine elaboration process in France, which includes a cold maceration prior to fermentation. Hanseniaspora uvarum and Candida zemplinina were the predomi lez et al. nant non-Saccharomyces yeasts. Gonza (2006) in Spain found 27 species with a high number of Candida and Pichia. In Argentina, 11 species were isolated by Combina et al. (2005). In general, the diversity of species is higher in wine fermentations than in the studied mezcal process. Here, the raw material (agave) is rst cooked, which eliminates all microorganisms present in the raw material. In wine, in contrast, the principal source of non-Saccharomyces yeasts are the grapes which are only crushed, so any microorganisms that are present remain alive and inoculate the fresh wine must. Kluyveromyces spp, Zygosaccharomyces spp and Torulaspora spp, the principal non-Saccharomyces yeasts found in this study, have been also detected in wine fermentations; however, only as minor species. K. marxianus has been isolated from a great variety of habitats and has great potential in biotechnological applications, particularly in the production of enzymes (Fonseca et al. 2008). K. marxianus seems to be closely related to fermentations carried out with Agave as raw material. rez-Brito et al. (2007) reported K. marxianus in Pe plants and must of henequen (Agave fourcroydes); Lachance (1995) found it in the tequila fermentation; Lappe and Ulloa (1993) in pulque, which results in the spontaneous fermentation of the sap or aguamiel of different Agave species. The behavior of the different yeasts populations is quite different between the two fermentations. For fermentation II, the dominant yeast is S. cerevisiae,

123

504

Antonie van Leeuwenhoek (2011) 100:497506

the population of non-Saccharomyces is higher than in fermentation I: at the end of the process, ve different species are detected (Table 3). Its well known that non-Saccharomyces has low ethanol tolerance, so with lower ethanol content then in fermentation I, the diversity of non-Saccharomyces is still high at the end of fermentation II. These strains, particularly Z. bailii and C. lusitaniae are able to grow with ethanol concentration of 12 g/l whereas in fermentation I with 24 g/l of ethanol they didnt survive until the end of the fermentation. Zott et al. rez(2008), like other authors (Nissen et al. 2003; Pe Nevado et al. 2006), proposed that there is some negative interaction between the S. cerevisae and the non-Saccharomyces. In our case, the quantity of Saccharomyces is higher in fermentation I than in fermentation II so that could be another reason of a lower non-Saccharomyces population. However, it will be important to study more fermentations and the behavior of the isolated yeasts in controlled laboratory conditions in order to understand this specic point. However, these changes in the yeast population probably have a great impact on generation of volatile compounds, as demonstrated in wine fermentation (Romano et al. 2003). Few studies have dealt with the behavior of specic yeasts isolated from agave fermentations and their role in the generation of volatile compounds. Arrizon et al. (2006) demonstrated great differences between agave and grape yeasts, particularly in the production of volatile compounds in must prepared with agave and grape juice. Although the non-Saccharomyces species, e.g. K. marxianus, are well known to produce high amounts of volatile compounds, particularly esters (Fonseca et al. 2008), it is barely possible to associate the levels of volatiles with the succession of the global or specic yeast populations in the studied fermentation. Even though in this work the bacterial community wasnt studied, we detected considerable amounts of bacteria during the process which may be an another important factor in the generation of volatile compounds. Previous studies demonstrated the presence of lactic and acetic bacteria as well as Zymomonas mobilis in these kinds of fermentations (Escalante ez-Zapata et al. 2010). Minakata et al. 2008; Narva The real contribution of these microorganisms is still unknown and needs further research in order to elucidate its role.

PCR-DGGE Recently, numerous authors have employed a combination of culture-dependent and culture-independent methods, in order to study the behavior of the microbiota that participates in the elaboration of fermented products (Cocolin et al. 2002; Prakitchaiwattana et al. 2004; Nielsen et al. 2005; Rantsiou et al. 2005; Cocolin et al. 2006; Rantsiou and Cocolin 2006; Obodai and Dodd 2006; Dolci et al. 2008; Oelofse et al. ` et al. 2010; Lacerda 2009; Kim et al. 2009; Andorra Ramos et al. 2010) and to understand the ecological relationship between the microorganisms and the inuence of this diversity on the characteristics of the end product. As in wine, PCR-DGGE has been shown to be a reliable method for direct qualitative assessment of the yeast populations present in mezcal fermentations. According to Cocolin et al. (2001), PCRDGGE avoids the problems often associated with microbial enrichments. Moreover, it can be performed in a reasonably rapid fashion (one day) and with minimal sample volume. In this study, the PCR-DGGE detected a microbial consortium composed of S. cerevisiae, T. delbrueckii and K. marxianus throughout the fermentation process. In complex mixed yeast populations, this method detected species present at 10100 fold less than other species, but not when the ratio exceeded 100 fold (Prakitchaiwattana et al. 2004). When yeast populations fell below 104 CFU/ml, the corresponding DGGE bands faded or disappeared. This threshold is likely the result of a larger quantity of Saccharomyces DNA in these samples competing with the smaller amounts of template from the nonSaccharomyces yeasts for amplication of the rDNA (Mills et al. 2002). This can explain the fact that in the case of the minority yeasts, S. exiguus, P. kluyvery and Z. bailii, the detected bands were very weak.
Acknowledgments This study was developed within the PhD gicas) from Universidad research program (Ciencias Biolo noma de Me xico, and supported by the SEPNacional Auto CONACYT # 24556 project. The authors thank Consejo a (CONACyT) for economic Nacional de Ciencia y Tecnolog support (grant for the PhD to Verdugo Valdez A.) and the distillery Real de Magueyes for their interest and help.

References
` I, Landi S, Mas A, Esteve-Zarzoso B, Guillamo n J Andorra (2010) Effect of fermentation temperature on microbial

123

Antonie van Leeuwenhoek (2011) 100:497506 population evolution using culture-independent and dependent techniques. Food Res Int 43:773779 Arrizon J, Gschaedler A (2007) Effects of the addition of different nitrogen sources in the tequila fermentation process at high sugar concentration. J Appl Microbiol 102:11231131 Arrizon JP, Fiore C, Acosta G, Romano P, Gschaedler A (2006) Fermentation behaviour and volatile production by agave and grape must yeasts in high sugar and grape must fermentations. Antonie van Leeuwenhoek 8:181189 Ben Omar N, Ampe F (2000) Microbial community dynamics during production of the fermented maize dough pozol. Appl Environ Microbiol 66:36643673 lez GA, Torres JC, Pelayo C, Gutie rrez M, Cira LA, Gonza rez J (2008) Heterologous expression of Fusarium Ram oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must. Antonie van Leeuwenhoek 93:259266 n IL, Paneque P (2010) Diversity of SacClavijo A, Caldero charomyces and non-Saccharomyces yeasts in three red a de Ronda (Spain) grape varieties cultured in the Serran n. Int J Food Microbiol 143:241245 vine-growing regio Cocolin L, Heisey A, Mills DA (2001) Direct identication of the indigenous yeast in commercial wine fermentations. Am J Enol Viticul 52:4953 Cocolin L, Manzano A, Rebecca S, Comi G (2002) Monitoring of yeast population changes during a continuous wine fermentation by molecular methods. Am J Enol Viticul 53:2427 Cocolin L, Urso R, Rantsiou K, Cantoni C, Comi G (2006) Dynamics and characterization of yeasts during natural fermentation of Italian sausages. FEMS Yeasts Res 6:692701 a A, Mercado L, Catania C, Ganga A, Martinez Combina M, El C (2005) Dynamics of indigenous yeast population during spontaneous fermentation of wines form Mendoza, Argentina. Int J Food Microbiol 99:237243 Cordero-Bueso G, Arroyo T, Serrano A, Tello J, Aporta I, lez MA, Valero E (2011) Inuence of the farming Ve system and vine variety on yeast communities associated with grape berries. Int J Food Microbiol 99:237243 Csoma H, Zakany N, Capece A, Romano P, Sipiczki M (2010) Biological diversity of Saccharomyces yeasts of spontaneously fermenting wines in four wine regions: Comparative genotypic and phenotypic analysis. Int J Food Microbiol 140:239248 n-Rodr guez A, Gonza lez-Herna ndez L, Barba de la De Leo pez MG (2006) Rosa AP, Escalante-Minakata P, Lo Characterization of volatile compounds of mezcal, an ethnic alcoholic beverage obtained from Agave salmiana. J Agri Food Chem 54:13371341 n-Rodr guez A, Escalante-Minakata P, Barba de la Rosa De Leo AP, Blaschek HP (2008) Optimization of fermentation conditions for the production of the mezcal from Agave salmiana using response surface methodology. Eng Process 47:7682 De Vuyst L, Vrancken G, Ravyts F, Rimaux T, Weckx S (2009) Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota. Food Microbiol 26:666675

505 Dolci P, Alessandria V, Rantsiou K, Rolle L, Zeppa G, Cocolin L (2008) Microbial dynamics of Castelmagno PDO, a traditional Italian cheese, with a focus on lactic acid bacteria ecology. Int J Food Microbiol 122:302311 Escalante-Minakata P, Blaschek HP, Barba de la Rosa AP, n-Rodr guez A (2008) Identication of yeast Santos L, Leo and bacteria involved in the mezcal fermentation of Agave salmiana. Lett Appl Microbiol 46:626630 Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A (1999) lisis of the 5.8S Identication of yeasts by RFLP ana rRNA gene and the two ribosomal internal transcribed spacers. Int J System Bacteriol 49:329337 Fleet G (2003) Yeast interactions and wine avour. Int J Food Microbiol 86:1122 Fonseca GG, Heinzle E, Wittmann C, Gombert AK (2008) The yeast Kluyveromyces marxianus and its biotechnological potential. Appl Microbiol Biotechnol 79:339354 a Mendoza A (1998) Con sabor a maguey: Gu a de la Garc n nacional de Agavaceas y Nolina n ceas del jard coleccio nico. Instituto de Biolog a-Universidad Nacional bota noma de Me xico, Me xico Auto lez SS, Barrio E, Querol A (2006) Molecular identiGonza cation and characterization of wine yeasts isolated from Tenerife (Canary Island, Spain). J Appl Microbiol 102: 10181025 Kim TW, Lee JH, Kim SE, Park MH, Chang HC, Kim HY (2009) Analysis of microbial communities in doenjang, a Korean fermented soybean paste, using nested PCRdenaturing gradient gel electrophoresis. Int J Food Microbiol 131:265271 Kurtzman CP, Robnett CJ (1998) Identication and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial subunit. Antonie van Leeuwenhoek 73:331371 Lacerda Ramos C, Gonzaga de Almeida E, De Melo Pereira GV, Gomes Cardoso P, Souza Dias E, Freitas Schwan R (2010) Determination of dynamic characteristics of microbiota in a fermented beverage produced by Brazilian Amerindians using culture-dependent and culture-independent methods. Int J Food Microbiol 140:225231 Lachance MA (1995) Yeast communities in a natural tequila fermentation. Antonie van Leeuwenhoek 68:151160 Lappe P, Ulloa M (1993) Microbiologia del Pulque. In: Wacher genas de MC, Lappe P (eds) Alimentos fermentados ind xico. Universidad Nacional Auto noma de Me xico, Me xico, pp 7580 Me o J, Lappe-Oliveiras P, Moreno-Terrazas R, Arrizon-Gavin a-Mendoza A, Gschaedler-Mathis Herrera-Suarez T, Garc A (2008) Yeasts associated with the production of Mexican alcoholic non-distilled and distilled Agave beverages. FEMS Yeast Res 8:10371052 Lefeber T, Gobert W, Vrancken G, Camu N, De Vuyst L (2011) Dynamics and species diversity of communities of lactic acid bacteria and acetic acid bacteria during spontaneous cocoa bean fermentation in vessels. Food Microbiol 28:457464 Li S, Cheng Ch, Li Z, Chen J, Yan B, Han B, Reeves M (2010) Yeast species associated with wine grapes in China. Int J Food Microbiol 138:8590 Meroth CB, Hammes WP, Hertel C (2003) Identication and population dynamics of yeasts in sourdough fermentation

123

506 processes by PCR-denaturing gradient gel electrophoresis. Appl Environ Microbiol 69:74537461 Mexican Ministry of Commerce and Industry (1994) Regulations: NOM-070-SCFI-1994. Alcoholic drinks-Mezcal n: Me xico Specications. Diario Ocial de la Federacio Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426428 Mills DA, Johannsen EA, Cocolin L (2002) Yeast diversity and persistence in botrytis affected wine fermentations. Appl Environ Microbiol 68:48844893 Miyakoshi M, Masuda H, Mizutani K, Tanaka O, Ikeda T, Ohtani K, Kasai R, Yamasaki K (2000) Antiyeast steroidal saponins from Yucca shidigera (Mohave Yucca), a new anti-food-deteriorating agent. J Nat Prod 63:332338 Moroni AV, Arendt EK, Dal Bello F (2011) Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs. Food Microbiol 28:497502 ez-Zapata JA, Rojas Herrera RA, Rodr guez-Luna IC, Narva Larralde-Corona CP (2010) Culture-independent analysis of lactic acid bacteria diversity associated with mezcal fermentation. Curr Microbiol 61:444450 Nielsen DS, Hnholt S, Tano-Debrah K, Jespersen L (2005) Yeast populations associated with Ghanaian cocoa fermentations analyzed using denaturing gradient gel electrophoresis (DGGE). Yeast 22:271284 Nissen P, Nielsen D, Arneborg N (2003) Viable Saccharomyces cerevisiae cells at high concentration cause early growth arrest of non-Saccharomyces yeast in mixed cultures by cell a cell contact-mediated mechanism. Yeast 20:331341 Obodai M, Dodd CER (2006) Characterization of dominant microbiota of a Ghanaian fermented milk product, nyarmie, by culture- and non culture-based methods. J Appl Microbiol 100:13551363 n E, Gutie rrez AR, Garijo P, Lo pez R, Santamar a P (2010) Oco Presence of non-Saccharomyces yeasts in cellar equipment and grape juice during harvest time. Food Micribiol 27:10231027 Oelofse A, Lonvaud-Funel A, Du Toit M (2009) Molecular identication of Brettanomyces bruxellensis strain isolated from red wines and volatile phenol production. Food Microbiol 30:19 Pallmann CL, Brown JA, Olineka TL, Cocolin L, Mills DA, Bisson LF (2001) Use of WL medium to prole native ora fermentations. Am J Enol Viticult 53:198203 rez-Brito D, Tapia-Tussell R, Quijano-Ramayo A, Larque Pe Saavedra A, Lappe P (2007) Molecular characterization of

Antonie van Leeuwenhoek (2011) 100:497506 Kluyveromyces marxianus strains isolated from Agave fourcroydes (Lem.) in Yucatan, Mexico. Mol Biotechnol 37:181186 rez-Nevado F, Albergaria H, Hogg T, Girio F (2006) CelPe lular death of two non-Saccharomyces wine-related yeasts during mixed fermentations with Saccharomyces cerevisiae. Int J Food Microbiol 108:336345 Prakitchaiwattana CJ, Fleet G, Heard GM (2004) Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes. FEMS Yeast Res 4:865877 Rantsiou K, Cocolin L (2006) New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods: a review. Int J Food Microbiol 108:255267 Rantsiou K, Urso R, Iacumin L, Cantoni C, Cattaneo P, Comi G, Cocolin L (2005) Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages. Appl Environ Microbiol 71:19771986 Renouf V, Claisse O, Lonvaud-Funel A (2007) Inventory and monitoring of wine microbial consortia. Appl Microbiol Biotechnol 75:149164 Romano P, Fiore C, Parragio M, Caruso M, Capece A (2003) Function of yeast species and strains in wine avour. Int J Food Microbiol 86:169180 pez C, Di Fabio F, Schirone M, Felis GE, Tofalo R, Chaves-Lo Torriani S, Paparella A, Suzzi G (2009) Molecular identication and osmotolerant prole of wine yeasts that ferment a high sugar grape must. Int J Food Microbiol 130:179187 Tu RJ, Wu HY, Lock YS, Chen MJ (2010) Evaluation of microbial dynamics during the ripening of a traditional Taiwanese naturally fermented ham. Food Microbiol 27:460467 Urso R, Rantsiou K, Dolci P, Rolle L, Comi G, Cocolin L (2008) Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods. FEMS Yeast Res 8:10531062 rez F,JR, Aguirre R,D, Ortiz P,CI, God nez Zamora P,C,BI, Jua lvarez F (2010) Variacio n de la concentracio n de H,G, A n del maguey cares y saponinas durante la coccio azu mezcalero potosino. e-Gnosis [online] 8:111 Zott K, Miot-Sertier C, Claisse O, Masneuf-Pomarede I, Lonvaud-Funel A (2008) Dynamics and diversity of nonSaccharomyces yeasts during the early stages in winemaking. Int J Food Microbiol 125:197203

123