**3.2** *Saccharomyces* **yeasts**

For *Saccharomyces* isolate characterization, a PCR-RFLP analysis was done, and the results showed that 95% of the isolates belonged to *Saccharomyces cerevisiae*, while only 3% and 2%, respectively, were identified as *S. paradoxus* and S. *bayanus.*

However, to discriminate isolate samples within the *Saccharomyces* sensu stricto group, a mitochondrial DNA restriction analysis [13] was carried out by digestion with the restriction endonuclease enzyme *Hinf* I. Restriction fragments were separated by electrophoresis on agarose gel with GelGreen™ (Biotium), and the results were visualized using a GeneFlash documentation system.

The *Saccharomyces* isolates were clustered in 105 different mtDNA patterns (**Table 2**), reflecting a variability of nearly 73% which is very high if it is compared to their variety in cellars [24–26].

Genetic patterns which involved at least 20% of the isolates were named as the "majority profile". At plants A and C, two majority profiles were characterized; at B and D, there was only one; and none was found at plant E. In addition, sweet and fermented piquettes were the substrata from which the most profiles were identified. Although patterns tended to be typical of each plant, the majority profiles accounted for 57% of the isolates at plant B and 33% and 30% at plants C and A, respectively.

**Plants Sample Isolates Strains Variability Majority profile** Fresh piquette 28 16 57 — A Fermented piquette 11 5 45 27% Lees 10 5 50 30% Fresh piquette 9 8 89 — B Fermented piquette 13 12 92 — Lees 7 4 57 57% Fresh piquette 27 22 81 22% C Fermented piquette — — — — Lees 9 7 78 33% Fresh piquette — — — — D Fermented piquette 8 7 88 27% Lees — — — — Fresh piquette — — — — E Fermented piquette 22 19 86 — Lees — — — —

Fermented piquettes presented the greatest degree of *Saccharomyces* variability, although several strains coexisted in both lees and sweet piquettes.

#### **Table 2.**

*Distribution of Saccharomyces isolates and strains in sweet and fermented piquettes and lees at the ethanol plants studied.*

**173**

fermentation tests.

electrical method [29].

**rate at different temperatures**

characteristic in a fermentation process.

*Yeast from Distillery Plants: A New Approach DOI: http://dx.doi.org/10.5772/intechopen.86291*

system was established [27, 28].

*P. galeiformis* and *C. ethanolica*.

nol from agricultural and forest by-products.

**assimilation of carbon compounds**

**4. Biotechnological properties of non-***Saccharomyces***: fermentation and** 

with new fermentation profiles for potential applications in various fields. The carbon compounds assayed were D-glucose, D-galactose, L-arabinose, L-rhamnose, melibiose, lactose, raffinose, xylose, maltose, mannose, saccharose and cellobiose. The tests were carried out on a 96-well microtiter plate. Each well was filled with sugar solution, bromocresol green and cell suspensions (exhausting the endogenous carbon compound reserves). Finally, the wells were sealed with sterile vaseline, and the plates were incubated at 28°C/5 days. Depending on the time of the change and the intensity of colouration (from blue to yellow or yellow green), a classification

Fermentation of carbon compounds is particularly useful for identifying isolates

The majority of the isolates (*Torulaspora*, *Lachancea* and *Saccharomycodes* species and *C. lactis-condensi*) fermented D-glucose either in the first 12 h or on the 5th day.

None of the isolates fermented xylose, lactose, arabinose, melibiose and rham-

Only one *H. uvarum* isolate and one *H. vinae* isolate weakly fermented cellobiose, which is a sugar of great biotechnological interest in the production of bioetha-

The compounds used for the assimilation assay were mono- and disaccharides (D-glucose, maltose, lactose, L-rhamnose, xylose and cellobiose), polysaccharides (starch, carboxymethylcellulose and lignin) and alcohols (ethanol and methanol). The tests were carried out in agar plates containing the carbon source and YNB without amino acids (Difco™). The assimilation profile was noticed as (++) abun-

Assimilation of carbon compounds, glucose and maltose were the most commonly used and, to a lesser extent, xylose and methanol. Three species of *Candida*, *C. viswanathii, C. ethanolica* and *C. sake*, and one *P. galeiformis* isolate assimilated carboxymethyl cellulose, while three *Pichia* isolates used starch. The majority of *Torulaspora* isolates and a few isolates of *P. kudriavzevii*, *P. galeiformis* and *H. osmophila* assimilated xylose. All of the *H. osmophila, H. uvarum* and *S'codes ludwigii* isolates effectively assimilated cellobiose. Ethanol was assimilated by a few *P. galeiformis* and *P. anomala* isolates. Finally, only some *L. thermotolerans, P. kudriavzevii*, *C. sake* and *C. viswanathii* isolates assimilated methanol. Thus, differences between isolates of the same species were observed, as can be seen in the

**5. Biotechnological properties of** *Saccharomyces***: cell vitality and growth** 

Cell vitality and growth rates at different temperatures were carried out with the 105 strains. These properties were selected because they are considered a relevant

Cell vitality was evaluated as a measure of fermentative activity by an indirect

*C. lactis-condensi* fermented the majority of the sugars at a major or minor intensity. On the other hand, for galactose, raffinose and saccharose fermentation, variability was observed in species such as T. *delbrueckii*, *C. lactis-condensi*,

D-mannose and saccharose were fermented to a lesser extent.

dant growth, (+) normal growth and (−) absence of growth.

nose, and some only weakly fermented galactose, maltose and raffinose.

*Advances in Grape and Wine Biotechnology*

[4] in tequila and agave beverages.

to their variety in cellars [24–26].

**3.2** *Saccharomyces* **yeasts**

*bayanus.*

respectively.

Spanish industry is an interesting yeast niche. Additionally, some of these genera and species were also found by Amaya-Delgado et al. [5] and Lappe-Oliveras et al.

For *Saccharomyces* isolate characterization, a PCR-RFLP analysis was done, and the results showed that 95% of the isolates belonged to *Saccharomyces cerevisiae*, while only 3% and 2%, respectively, were identified as *S. paradoxus* and S.

However, to discriminate isolate samples within the *Saccharomyces* sensu stricto group, a mitochondrial DNA restriction analysis [13] was carried out by digestion with the restriction endonuclease enzyme *Hinf* I. Restriction fragments were separated by electrophoresis on agarose gel with GelGreen™ (Biotium), and the results

The *Saccharomyces* isolates were clustered in 105 different mtDNA patterns (**Table 2**), reflecting a variability of nearly 73% which is very high if it is compared

Genetic patterns which involved at least 20% of the isolates were named as the "majority profile". At plants A and C, two majority profiles were characterized; at B and D, there was only one; and none was found at plant E. In addition, sweet and fermented piquettes were the substrata from which the most profiles were identified. Although patterns tended to be typical of each plant, the majority profiles accounted for 57% of the isolates at plant B and 33% and 30% at plants C and A,

Fermented piquettes presented the greatest degree of *Saccharomyces* variability,

**Plants Sample Isolates Strains Variability Majority profile**

A Fermented piquette 11 5 45 27%

B Fermented piquette 13 12 92 —

C Fermented piquette — — — —

D Fermented piquette 8 7 88 27%

E Fermented piquette 22 19 86 —

*Distribution of Saccharomyces isolates and strains in sweet and fermented piquettes and lees at the ethanol* 

Fresh piquette 28 16 57 —

Lees 10 5 50 30% Fresh piquette 9 8 89 —

Lees 7 4 57 57% Fresh piquette 27 22 81 22%

Lees 9 7 78 33% Fresh piquette — — — —

Lees — — — — Fresh piquette — — — —

Lees — — — —

although several strains coexisted in both lees and sweet piquettes.

were visualized using a GeneFlash documentation system.

**172**

**Table 2.**

*plants studied.*
