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World Journal of Microbiology & Biotechnology 20: 1924, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

19

Fermentation characteristics as criteria for selection of cachac a yeast


Evelyn Souza Oliveira1,*, Carlos A. Rosa2, Marcelo Antonio Morgano3 and Gil Eduardo Serra4 1 Departamento de Alimentos, Faculdade de Farmacia, Universidade Federal de Minas Gerais, CEP 30.180-112 Minas Gerais, Brasil 2 Departamento de Microbiologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Brasil 3 Centro de Qumica de Alimentos e Nutric ao Aplicada, Instituto de Tecnologia de Alimentos, Brasil 4 Departamento de Tecnologia de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Brasil *Author for correspondence: Address: Av. Olegario Maciel, 2360 Bairro Lourdes, Belo Horizonte, Minas Gerais, CEP 30.180-112, Brasil, Tel.: 31-3339-7634, Fax: 31-3339-7632, E-mail: evelyn@farmacia.ufmg.br
Received 17 February 2003; accepted 31 July 2003

Keywords: alcoholic fermentation, cachac a, Saccharomyces cerevisiae, yeast

Summary The fermentation characteristics of 24 strains of Saccharomyces cerevisiae and one strain of Candida apicola, C. famata, C. guilliermondii, Hanseniospora occidentalis, Pichia subpelicullosa and Schizosaccharomyces pombe were evaluated for the production of cachac a. They were isolated from small cachac a distilleries (27), industrial cachac a distilleries (2) and one sugarcane alcohol distillery. The yeasts showed signicant dierences in ethanol yield, substrate conversion, eciency, conversion factors of substrate into ethanol (Yp/s), cells (Yx/s), organic acids (Yac/s) and glycerol (Yg/s), and maximum specic growth rate (lmax). In general the S. cerevisiae strains showed better fermentation potential, with yields between 83 and 91% and lmax between 0.450 and 0.640 h)1, several of them being comparable with the high performance yeast used in the industrial production of ethanol, which was adopted as a reference. The non-Saccharomyces strains showed high eciency, very low ethanol yield and very high Yac/s and Yg/s values, except Pichia subpelliculosa, which behaved very similarly to the S. cerevisiae strains. Hierarchical Cluster Analysis and Principal Component Analysis showed the fermentation yield (or substrate conversion) as being the variable which contributed most to the separation of the strains into dierent groups. Introduction Cachac a is a typical Brazilian beverage with an increasing focus on quality and the international market, because of its special avor and aroma. It is obtained by distillation of the fermented must from sugarcane juice. The alcohol content of cachac a can reach 3854% (v/v) at 20 C. Brazilian production of cachac a is estimated at 1.3 109 1/year. The production of cachac a on a small scale (20,000 to 200,000 l/year) by rural producers is processed in batch copper pot stills, and the product is quite different from that obtained on a large scale using stainless steel continuous distillation columns. The State of Minas Gerais is in the forefront of the production of cachac a by rural producers, leading to a concept of a quality typical for this beverage. More than 8000 such distilleries exist in the State of Minas Gerais, producing about 130 106 l/ year during the months from May to December. The production of cachac a by rural distillers is characterized by the fermentation of the sugarcane juice by a mixed culture of yeasts, with predominance of Saccharomyces cerevisiae. Apiculate yeasts (mainly Kloeckera japonica) and species of Candida, Kluyveromyces and Pichia are also frequently isolated (Morais et al. 1997; Pataro et al. 2000). The yeast populations present in these fermentations are in constant change, because of the introduction of new strains from the sugarcane juice, and also because the process is not sterile (Pataro et al. 1998). With the progress in alcoholic fermentation technology and yeast taxonomy, it was possible to verify that the predominant strain is that which possesses the most suitable characteristics for adaptation to the different processing conditions, such as the sugar content of the mash, ethanol content of the wine, wine acidity, fermentation temperature, equipment or process traps and process conditions. When the processing conditions are altered, this certainly provides the possibility of establishing a new equilibrium between the yeast strains, with the rise of a new dominant strain. The simple inoculation of a selected strain, or a dominant strain isolated from a certain distillery, is not sucient to guarantee its permanence and improvement in the eciency of distilleries (Basso et al. 1993; Andrietta et al. 1997). The alteration and adjustment of process

20 procedures is certainly an important preliminary step for altering the yeast ora. It is important to note that dominant yeasts per se do not exist. Yeasts are dominant under certain conditions. The selection of the most ecient mixed strains, producing the highest yield of ethanol, while taking the beverage quality into consideration, must be sought in the adoption of an adequate process that operates under the most stable possible conditions. The yeast communities present in the fermentation of cachac a by rural distillers, present many species and strains, showing the occurrence of yeasts with wide variations in their fermentation parameters and in the production of secondary compounds. What is of real interest is the occurrence of a microbiota, and of yeasts in particular, which lead to high fermentation yield and the best beverage quality. Fermentation parameters such as ethanol yield, productivity and maximum specic velocity of cell growth are considered to be essential for good performance in a fermentation process. In Brazil, these and other parameters are frequently used to characterize yeasts for industrial alcohol production (Oliveira 1988; Andrietta et al. 1999). However in the production of distilled cachac a this information is mostly lacking. The objective of this study is the evaluation of the fermentative behaviour of yeasts isolated from small cachac a distilleries of the State of Minas Gerais in Brazil. The knowledge of the fermentation parameters of these yeasts, as well as the variation within the strains, permits the selection of yeast strains for small-scale fermentation. The application of multivariate statistical methods allows the distribution of the isolated yeasts into differentiated groups, and the determination of the most important parameters for their grouping. It also aims to detect the variability of yeast fermentation parameters.

E.S. Oliveira et al.


Table 1. Origin and identication of yeast strains. Distilleries Year 1996 Brumado Velho (MG) Sc1 UFMG-A957 Sc2 UFMG-A968 1997 Sc3 UFMG-A1605 Sc4 UFMG-A1667 Sc5 UFMG-A1656 Sc6 UFMG-A1676 Sc7 UFMG-A1683 Ps UFMG-A960 Ca UFMG-A972 Sc13 UFMG-A1492 Sc14 UFMG-A1497 Sc15 UFMG-A1607 Sc16 UFMG-A1626 Sc17 UFMG-A1632 Sc18 UFMG-A1641 Cg UFMG-A882 Ho UFMG-A893 Cf UFMG-A1029 Sp UFMG-A1113 Sc19 UNICAMP-V1 Sc20 UNICAMP-V2 Sc21 UNICAMP-V3 Sc22 UNICAMP-R1 Sc23 UNICAMP-R2 Sc24 UNICAMP-CL14/97

Germana (MG)

Sc8 UFMG-A905 Sc9 UFMG-A1119 Sc10 UFMG-A1127 Sc11 UFMG-A1278 Sc12 UFMG-A1265

Vale Verde (MG)

Rosa (SP) Clealco

Sc: S. cerevisiae, Ca: Candida apicola, Cf: C. famata, Cg: C. guilliermondii, Ho: Hanseniaspora occidentalis (anamorph: Kloeckera javanica), Ps: Pichia subpelliculosa, Sp: Schizosaccharomyces pombe.

Materials and methods Thirty yeast strains were evaluated, of which 24 were Saccharomyces cerevisiae and six belonging to such other species as Candida apicola, C. famata, C. guillermondii, Hanseniospora occidentalis (teleomorph of Kloeckera javanica), Pichia subpelliculosa and Schizosaccharomyces pombe (Table 1). These strains were isolated from three small scale cachac a distilleries in the State of Minas Gerais, with the exception of strain Sc22 and 23 which came from an industrial cachac a distillery, and Sc24 from an industrial alcohol distillery, the latter being used as a reference strain since it presents characteristics considered desirable for alcohol fermentation (Andrietta et al. 1997). All the strains were identied by standard methodology with morphological and physiological standards and biochemical tests (Kurtzman & Fell 1998). The strains were isolated from samples collected from the distilleries at different times of the year. The S.

cerevisiae strains presented viable cell counts in the range from 107 to 109 cells/ml of wine, while those of the other genera were between 105 to 106 cells/ml. The assays for the determination of the fermentation parameters were conducted in triplicate, in 250 ml asks containing 100 ml sterile culture medium (glucose 150 g/l; monopotassium phosphate 5.0 g/l; ammonium chloride 1.5 g/l; magnesium sulphate heptahydrate 1.0 g/l; potassium chloride 1.0 g/l; yeast extract 6.0 g/l, pH 6.0). The asks were closed with rubber stoppers with a central hole, through which an L-shaped glass tube (with a cotton wool lter) was passed, for the escape of CO2. The inoculum was prepared by resuspending in distilled water, the yeast slant cultures maintained in GYMP agar (2% glucose, 0.5% yeast extract, 1% malt extract, 0.2% NaH2PO4 and 2% agar). The asks containing the sterile medium were inoculated with a volume equivalent to 10% v/v of the suspension of cells (inoculum), and incubated in a shaker at 32 1 C and 150 rev/min for 24 h. A sample from each ask was then centrifuged and total reducing sugars, glycerol and total acidity were analysed in the supernatant. For ethanol determination, samples from the fermented media were distilled by steam stripping in a micro-distillation apparatus. The initial total reducing sugar content, the dry cell mass in the inoculum and the dry cell mass in the wine were also

Selection of cachac a yeast determined. The value for lmax was determined in shaker asks containing 40 g/l glucose. The cell growth was evaluated by reading the optical density at 600 nm and calculating the angular coecient corresponding to the exponential growth phase (Pirt 1985). The following parameters were determined, submitting the fermenting asks to a mass balance: fermentation yield; substrate conversion; eciency; conversion factor of substrate into ethanol (Yp/s), into cells (Yx/s), into acetic acid (Yac=s ) and into glycerol (Yg/s). The ethanol concentration was determined spectrophotometrically (Zimmermann 1963; Salik & Povh 1993), based on the oxidation of ethanol to acetic acid using potassium dichromate and sulphuric acid; these reagents turn the solution into a greenish colour directly proportional to the ethanol concentration, and at 600 nm wavelength the Cr3+ can be read without interferences. Glucose was determined by the glucose oxidaseperoxidase method (Henry et al. 1974), glycerol using the Labtest Triglyceride Kit (MacGowan et al. 1983), total acidity by titration and cell mass by oven drying at 55 C. The several fermentation parameters were distributed into a maximum of ve levels, which were settled considering the wide range of variation of some parameters. The value of the reference yeast strain Sc24 was a basis for comparison and distribution of the other values in the levels. This strain is one of best performance in Brazilian ethanol distilleries (Andrietta et al. 1997), with high yield, conversion, eciency, Yp/s, Yx/s and lmax, but low Yac/s and Yg/s. The intervals were settled for each parameter considering the individual values and the distribution within the limits between the lower and higher value. The levels were adopted in order to show the results in a model that provides both the average results and behaviour of yeasts in relation one to the others. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) (PIROUETTE computer program) were used to analyse the fermentation parameters. The analysis of variance (ANOVA) was also carried out and the Tukey test applied, using SAS Statistics Program (SAS Institute 1993).
1.0 Sp Ho
Group 1

21
0.8 0.6 0.4 0.2 0.0

Sc15 Sc21 Sc17 Sc16 Ps Sc3 Sc4 Sc2 Sc1 Sc24 Sc22

Group 2

Sc23 Sc13 Sc20 Sc7 Sc6 Sc5 Sc19 Sc8 Sc18

Group 3

Sc14 Sc12 Sc10 Sc9

Group 4

Ca Sc11 Cf Ca

Figure 1. Dendrogram of yeast groups showing the similarity fermentation parameters for the yeast strains.

Results and discussion The similarity among the yeast strains in terms of the fermentation parameters yield, conversion, efciency, Yx/s and lmax can be seen in the dendrogram of Figure 1. The scale (0 to 1) represents the index of similarity. The yeasts were distributed into ve groups with a similarity of 0.83. Groups 2 and 3 contained the majority of the S. cerevisiae strains (63.3%) and P. subpelliculosa. Group 1 consisted of S. cerevisiae together with Schizosaccharomyces pombe and Hanseniaspora occidentalis. Groups 4 and 5 were comprised of the Candida strains, with the exception of one Saccharomyces (Sc11) strain and showed no similarity with the other groups, indicating that these yeasts are considerably dierent. The

statistical analysis carried out with the inclusion of the factors Yac/s and Yg/s did not alter the results. The majority of the S. cerevisiae strains came from Brumado Velho (7 strains) and Germana (11 strains) distilleries. The Brumado Velho strains showed less variability in the fermentation parameter values and all belonged to Group 2, as did the Sc22 and 23 strains, which came from an industrial cachac a distillery. These strains showed characteristics similar to those of the reference strain (Sc24), which is a strain used for the industrial production of alcohol, since it exhibits good fermentation characteristics. These results demonstrate that in the Brumado Velho Distillery, strain selection occurred according to higher ethanol yield, possibly because the process was conducted under stable operational conditions. The Germana strains were distributed among the ve groups, thus presenting greater variability in their fermentation characteristics, including strains with both very high yields (Sc8) and very low yields (Sc11). In a similar way, Andrietta et al. (1999) observed that yeasts isolated from two alcohol distilleries showed completely dierent behaviour; those from one distillery showing fairly uniform behaviour of the fermentation parameters and the other

Group 5

22
8
group 5
Cf

E.S. Oliveira et al.


Sc8

efficiency

group 3
Sc18

Sc19

4
Cg

0.6
Sc5 Sc7 Sc15 Sc12 Sc14 Sc21

PC2

group 1

Sc10

PC2

Sc22 Sc6 Sc24 Sc13 Sc23 Sc20

yield

Ho

Sc9 Sc17

Ps Sc3 Sc4 Sc2 Sc1

0.2
Y x/s max

-4

group 4

Ca

Sp

Sc16

group 2

-0.2
-8
Sc11

substrate conversion

-80

-60

-40 PC1

-20

20

0.0

0.2 PC1

0.4

0.6

0.8

Figure 2. Graph of the PC1 vs. PC2 scores for the yeast strains (PC principal component).

Figure 3. Graph of the PC1 vs. PC2 loadings for the fermentation parameters.

showing great variability. This result also indicates that the process conditions inuence the selection of the yeast strain and that unstable processing conditions lead to a more heterogeneous yeast population with respect to fermentation characteristics. This heterogeneity results in the production of a less standardized cachac a quality throughout the season, and oscillating yields. The separation between the yeast strains was described in the rst principal component, as can be seen in Figure 2 (PC1 vs. PC2), which described 98% of the total data variance. When Figures 2 and 3 are compared with each other, the separation between the strains was almost totally due to ethanol yield (or substrate conversion, since they are dependent variables, as can be seen from the correlation coefcient r 0:99, p 0.001). Analysis of the scores and loadings for the rst two PC, in Figures 2 and 3, respectively, shows that the strains from Groups 2 and 3 present positive scores for PC1 (yield), while the other groups show negative scores. Groups 2 and 3 have positive scores since they present yield values considered as medium, high and very high (Table 2), signifying groups where yield is a maximized attribute; the remaining groups presented values classied as low and very low. The statistical analysis of the principal components (PCA) showed that the yield was the parameter that determined the separation of the yeasts into ve groups (Figure 1). The second principal component (PC2) only described 2% of the variance, and fermentation efciency was the most signicant parameter, being responsible for the separation of the strains in Group 5. The results for the fermentation parameters can be found in Table 2. They are classied in levels from low to very high and the yeast strains grouped accordingly. These levels were dened considering the amplitude, the statistical analysis of the results, and the classication proposed by Andrietta et al. (1999), who grouped the yeasts from industrial production of alcohol into low and high levels according to their fermentation parameters. The ranges observed were: substrate conversion, <90.0 and >98.5; conversion factor for substrate into

ethanol (Yp/s), <0.42 and >0.45; conversion factor for substrate into cells (Yx/s), <0.041 and >0.044; maximum specic velocity for growth (lmax), <0.45 and >0.55. The majority of the strains (63%) were included in the range from 70.1 to 87.0% (low to medium levels) for ethanol yield (Table 2), and only 13% presented yields between 87.1 and 91.0% (high to very high levels). The Sc8 and Sc19 strains were the two that presented the highest yields (90.5 and 89.3% respectively), greater than that of the reference strain Sc24 (88.4%). The non-Saccharomyces yeasts together with strains Sc11 and Sc15 presented lower yields (24.070.0%). One exception was the P. subpelliculosa strain, which presented an average yield of 84.5%. The fermentation yield is an important parameter in production economics and, in the case of distilled liquors, it can also directly affect the quality. Fermented mash with a standardized alcohol content will certainly give rise to equally well-standardized distillates. The data in Table 2 also shows that the conversion factor of substrate into ethanol )Yp/s (ethanol/g glucose consumed) or % eciency of the yeast, was in the medium range for the majority (70%) of the strains. Almost all of the S. cerevisiae strains (91.7%) showed a medium level for Yac/s, while the strains belonging to other genera were in the high or very high ranges. With respect to Yg/s, 91.7% of the S. cerevisiae strains were in the low or medium ranges, while those of the other genera were in the high or very high ranges, with the exception of P. subpelliculosa, which presented a low value for Yg/s. A negative correlation was observed between the ethanol yield and the factors Yg/s and Yac/s for the yeast strains, with correlation coecients of r )0.93 (P 0.001) and r )0.59 (P 0.001), respectively, signifying that there will always be a reduction in the yield when a large amount of sugar is diverted to the production of glycerol and organic acids. On the other hand, Saccharomyces strains showed a lower production of glycerol and organic acids. A positive correlation was also observed between Yg/s and Yac/s, with r +0.67 (P 0.001), indicating that both products were synthesized proportionally by the yeasts during fermentation.

Selection of cachac a yeast


Table 2. Average ranges of variation for the fermentation parameters levels of the yeast strains. Parameters Levels Very low Yield (%) 24.070.0 Sc 11,15, Ca, Cf, Cg, Ho, Sp (7) 25.060.0 Sc 11, Ca, Cf, Cg (4) Low 70.183.0 Sc 1,9,10,12,14, 16,17,18,21 (9) 60.190.0 Sc 1,2,10,12,15, 16,Ho, Sp (8) Medium 83.187.0 Sc 2,3,4,5,6,13, 20,22,23, Ps (10) 90.097.0 Sc 9,14,18,22 (4) 82.088.0 Sc 1,2,3,4,6,7,9,11, 12,13,14,16,17,20,21, 22, 23, Sp, Ca, Ho, Ps (21) 0.4200.450 Sc1,2,3,4,6,7,9,11,12, 13,14,16,17,20,21,22, 23, Ca, Sp, Ps, Ho (21) 0.0390.040 Sc11,Sp 0.0410.043 Sc 5,9,10,14 High 87.189.0 Sc 7,24 Very high 89.191.0 Sc 8,19

23

(2) 97.199.0 Sc 5,8,13,23 (4) 88.195.0 Sc 5,8,10, 15,18,19,24

(2) 99.1100.0 Sc 1,2,3,4,6,7, 19,20,24, Ps (10) 95.199.5 Cf, Cg

Conversion (%)

Eciency (%)

(7) 0.4510.490 Sc 5,8,10,15, 18,19,24 (7) 0.0440.060 Sc 1,2,3,4,6,7,8, 12,13,15,16,17, 18,19,20,22,23, 24,Cf, Ps (20) 0.01310.0200 Sc3,13,Ca,Cg, Ho,Ps (6) 0.0510.080 Sc 11,15, Ho (3) 0.5510.600 Sc3,4,7,8,14,20, Ca, Cf (8)

(2) 0.4910.510 Cf, Cg

Yp/s (g/g)

(2) 0.0610.072 Sc21,Ca, Cg, Ho

Yx/s (g/g)

(2) Yac/s (g/g)

(4) 0.00800.0130 Sc1,2,4,5,6,7,8,9,10, 11,12,14,15,16,17,18, 19,20,21,22,23,24 (22)

(4) 0.02010.0240 Cf, Sp

(2) 0.0810.120 Cg, Cf, Ca, Sp (4) 0.6010.670 Sc2,5,6,12,19, 23, 24, Ps (8)

Yg/s (g/g)

0.0290.040 Sc 1,2,3,4,5,6,7, 8,9,22,23,24, Ps (13) 0.3800.450 Cg, Sp (2)

0.0410.050 Sc 10,12,13,14,16, 17,18,19,20,21 (10) 0.4510.550 Sc1,9,10,11,13,15,16, 17,18,21,22, Ho (12)

lmax (h)1)

The numbers between brackets correspond to the number of strains in each range. Sc: S. cerevisiae, Ca: Candida apicola, Cf: C. famata, Cg: C. guilliermondii, Ho: Hanseniospora occidentalis, Ps: Pichia subpelliculosa, Sp: Schizosaccharomyces pombe, Sc24: reference yeast.

Nearly 40% of the yeast strains presented lmax values in the medium range, and 53.3% in the high to very high ranges; the strains C. guilliermondii and S. pombe presented the lowest values for lmax (0.414 and 0.382 h)1, respectively). Subden (1990) also referred to S. pombe as a yeast with a low growth rate. The positive correlation between yield and lmax for the strains, with r +0.50 (P  0:001), showed that the selection of yeasts with higher yields has a tendency for also selecting those with higher growth rates, and, therefore, with a greater potential for dominating fermentation. The results for maximum growth rate showed that almost all the strains isolated possessed a lmax value

greater than median, indicating the importance of this parameter in dominant strains or those appearing with greater frequency in fermentations. The lmax value provides an indication of the capacity of a given strain to dominate the process. The higher the lmax, the greater the possibility of a strain remaining in the process throughout the season. This will not occur if any operational condition does not favour the continuation of the yeast in the process (yeast occulation, oscillating sugar content of mash, etc.). The high correlation between fermentation yield and the amount of ethanol in the fermented mash, with r +0.99 (P 0.001), indicates that the selection of yeasts with greater yields and more rapid fermentation

24 occurs, i.e., the maintenance of a high ethanol level in the wine assures the selection of more productive strains. The strain Pichia subpelliculosa (Ps) exhibits fermentation characteristics similar to those of Saccharomyces cerevisiae, with a notably greater conversion into organic acids (Yac/s) and higher specic growth rate (0.661 h)1). The potential of this strain to dominate the fermentation process can be supported by the results reported by Pataro et al. (2000), who observed the presence of P. anomala at a concentration of 4 109 cells/ml in a small-scale production of cachac a. Conclusions The strains of S. cerevisiae and of other species isolated from small-scale cachac a distilleries exhibited well-differentiated fermentation parameters. The statistical methods used demonstrated that the fermentation yield (or substrate conversion) was the most important attribute for grouping the strains. The fermentation yield, ethanol content in the wine and the maximum specic rate for cell growth were identied experimentally as important parameters in the selection of strains that could dominate the fermentation, resulting in leading to greater yields. Several strains of S. cerevisiae presented values for the fermentation parameters similar to those of an industrial strain used for the production of alcohol. The nonSaccharomyces yeast strains presented parameters showing their low potential for alcohol production and for competing with the Saccharomyces (with the exception of Pichia subpelliculosa), as well as the fact that they are greater producers of organic acids and glycerol. Their low population and low fermentative activity in an alcoholic fermentation, in competition with Saccharomyces strains, could minimize their importance.

E.S. Oliveira et al.


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