Asexual reproduction of yeasts. (a) Budding, typically observed in Saccharomyces cerevisiae, Candida, Kloeckera, Brettanomyces and (b) fission, typically observed in Schizosaccharomyces pombe.

strain to be utilized in the production of fermented beverages.

Frontiers and New Trends in the Science of Fermented Food and Beverages

2. Yeasts

Figure 1.

108

Non-Saccharomyces yeasts are a group of microorganisms genetically diverse with specific metabolic characteristics and high potential for using in fermentation processes. In the past, many of them have been considered contaminants due to the production of compounds that alters the sensory quality of wines [4, 5]. With the purpose of eliminating them and avoiding their fermentative activity, for instance, in wine processing, disinfection of fermentation tanks and containers with sulfite is commonly performed. However, over time, the importance of non-Saccharomyces yeasts in spontaneous fermentation has been demonstrated since they contribute positively to the definition of the sensory quality of wines. These yeasts predominate at the initial stage of the spontaneous fermentation [6–8] until certain concentration of ethanol is reached (usually between 4 and 5% v/v), which are then inhibited due to the effect of the ethanol and the depletion of dissolved oxygen [9, 10]. At the end of the process, Saccharomyces yeasts, the most resistant to ethanol, predominate and complete the fermentation. It has been reported that some non-Saccharomyces yeasts are able to survive toward the end of the spontaneous fermentation and exert their metabolic activity, thus contributing positively to the sensory quality of wines. Based on this evidence, in recent years, many researchers have focused their studies in understanding the nature and fermentative activity of the non-Saccharomyces yeasts [8, 11–21]. The findings demonstrated the enormous potential of these yeasts for use in the fermentation of traditional and nontraditional beverages. Despite the fact that most non-Saccharomyces yeasts show some technological disadvantages compared to Saccharomyces cerevisiae such as lower fermentative power and production of ethanol, non-Saccharomyces yeasts possess characteristics that in S. cerevisiae are absent, for instance, production of high levels of aromatic compounds such as esters, higher alcohols and fatty acids [22, 23]. In addition, it has been reported that the fermentative activity of these yeasts is manifested in the presence of small amounts of oxygen which leads to an increase in cell biomass and the decrease in ethanol yield, a strategy that can be used to reduce the ethanol content of wines produced in coculture with S cerevisiae [24–26]. With the aim of exploiting the positive characteristics of non-Saccharomyces yeasts and reducing their negative impact, fermentations with mixed and sequential cultures with S. cerevisiae can be performed to produce fermented beverages with different sensory profiles [27–29]. The most important fact is related to the potential for producing a broad variety of compounds of sensory importance necessary to improve the organoleptic quality of wines and beers. The findings reported so far in literature have led to rethink the role of these yeasts in

fermentative processes and to evaluate their use in the development of new products. Among the most studied non-Saccharomyces yeasts that reached special importance for researchers include Candida, Kloeckera, Hanseniaspora, Brettanomyces, Pichia, Lanchacea and Kluyveromyces, among others.

generation of energy is greater since glucose undergoes a complete oxidation, and as a result 36 net moles of ATP per mole of glucose are generated. The low-energy yield obtained by yeasts under anaerobic conditions forces the cell to increase the flow of glucose consumption in order to obtain a higher amount of energy in the form of ATP. As consequence, the ethanol accumulates in the fermentation medium and exerts its inhibitory effect, thus stopping the fermentative activity of the yeasts [31]. The low amount of energy generated under anaerobic conditions is used by the yeast cells in requirements for maintenance and growth. Glucose is easily transported and metabolized inside the cell; however, disaccharides such as sucrose, maltose or lactose must be first hydrolyzed to their simple forms (hexoses) which are then catabolized in the glycolysis pathway. Sucrose is hydrolyzed to fructose and glucose, maltose to two glucose units and lactose to glucose and galactose. The disaccharides are preferably hydrolyzed in the periplasmic space before entering the cytosol. Under anaerobic conditions besides ethanol, glycerol is also produced, thus contributing to restore the redox balance inside the cell. The production of glycerol increases in fermentations with musts of high specific gravity as a response to the osmotic stress [32]. It has been found that yeasts unable of metabolizing dihydroxyacetone (Figure 2) are not capable of producing glycerol, and as a consequence dihydroxyacetone accumulates and inhibits the fermentation. Moreover, glucose apart of being metabolized via glycolysis, it is also broken by complementary pathways that are not necessarily related to the generation of energy. The hexose monophosphate pathway (HMP) also known as the pentose phosphate cycle usually accompanies the glycolytic pathway [33]. In addition, yeasts during fermentation produce small amounts of acetic acid either from acetaldehyde or acetyl-CoA (Figure 2). Acetic acid is the main organic acid produced by yeasts during the fermentation of glucose, and it is responsible for the acidification and the decrease of pH of the medium. Ethanol is the most important fermentation by-product, and from the technological point of view, the production capacity of yeasts is an important parameter that determines their usability in fermentative processes. Gay Lussac defined a stoichiometric theoretical relationship to explain

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

the production of ethanol by Saccharomyces cerevisiae yeasts which is:

higher alcohols, esters, aldehydes and organic acids, among others.

! 2 C2H5OH ethanol

According to this relationship, from 180.0 grams of glucose, 92.0 grams of ethanol and 88.0 grams of carbon dioxide are produced, which results in a theoretical yield of 0.511 g ethanol/g glucose. However, in practice, besides ethanol and CO2, the production of biomass, glycerol and other minority compounds also hap-

C6H12O6 þ nitrogen ! C2H5OH þ CO2 þ glycerol þ biomass þ minority compounds

At industrial scale, a yield of 0.45 g ethanol/g glucose is acceptable [34]. In the case of fermentations with non-Saccharomyces yeasts, lower yields are commonly observed. Regarding to glycerol, in the case of S. cerevisiae, its production represents approximately 3% of the utilized sugar. Minor compounds are represented by

Oxygen is an important element during the complete oxidation of glucose since it serves as final acceptor of electrons under aerobic conditions. It is also essential for other metabolic processes such as the synthesis of structural components of the

þ 2 CO2 carbon dioxide (1)

(2)

1 C6H12O6 glucose

pens, that is:

111

3.1.1 Importance of oxygen

### 3.1 Fermentative metabolism of sugars

Either non-Saccharomyces or Saccharomyces yeasts share common pathways for the central metabolism of carbon; thus, both groups metabolize glucose through glycolysis. However, the mechanisms involved in the regulation of respirefermentative metabolism can differ significantly among them [30]. The glycolysis operates indistinctly under aerobic and anaerobic conditions, and through it, the glucose is metabolized to pyruvate by means of a series of biochemical reactions (Figure 2). Under anaerobic or oxygen-limited conditions, pyruvate is converted to acetaldehyde and then to ethanol, and as a result, two net moles of ATP are generated. Under fully aerobic conditions and in the absence of any repression effect, the

#### Figure 2.

Fermentative metabolism of glucose by yeasts: Glycolysis (black lines) and ethanol and glycerol production (blue lines). Enzymes: 1, hexokinase; 2, phosphoglucose isomerase; 3, phosphofructokinase; 4, fructose 1,6 bisphosphate aldolase; 5, triosephosphate isomerase; 6, glyceraldehyde 3-phosphate dehydrogenase; 7, phosphoglycerate kinase; 8, phosphoglycerate mutase; 9, enolase; 10, pyruvate kinase; 11, pyruvate decarboxylase; 12, alcohol dehydrogenase; 13, aldehyde dehydrogenase; 14, acetyl-CoA hydrolase; 15, acetyl-CoA synthetase; 16, pyruvate dehydrogenase; 17, glycerol 3-P dehydrogenase; 18, glycerol 3-phosphatase.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

generation of energy is greater since glucose undergoes a complete oxidation, and as a result 36 net moles of ATP per mole of glucose are generated. The low-energy yield obtained by yeasts under anaerobic conditions forces the cell to increase the flow of glucose consumption in order to obtain a higher amount of energy in the form of ATP. As consequence, the ethanol accumulates in the fermentation medium and exerts its inhibitory effect, thus stopping the fermentative activity of the yeasts [31]. The low amount of energy generated under anaerobic conditions is used by the yeast cells in requirements for maintenance and growth. Glucose is easily transported and metabolized inside the cell; however, disaccharides such as sucrose, maltose or lactose must be first hydrolyzed to their simple forms (hexoses) which are then catabolized in the glycolysis pathway. Sucrose is hydrolyzed to fructose and glucose, maltose to two glucose units and lactose to glucose and galactose. The disaccharides are preferably hydrolyzed in the periplasmic space before entering the cytosol. Under anaerobic conditions besides ethanol, glycerol is also produced, thus contributing to restore the redox balance inside the cell. The production of glycerol increases in fermentations with musts of high specific gravity as a response to the osmotic stress [32]. It has been found that yeasts unable of metabolizing dihydroxyacetone (Figure 2) are not capable of producing glycerol, and as a consequence dihydroxyacetone accumulates and inhibits the fermentation. Moreover, glucose apart of being metabolized via glycolysis, it is also broken by complementary pathways that are not necessarily related to the generation of energy. The hexose monophosphate pathway (HMP) also known as the pentose phosphate cycle usually accompanies the glycolytic pathway [33]. In addition, yeasts during fermentation produce small amounts of acetic acid either from acetaldehyde or acetyl-CoA (Figure 2). Acetic acid is the main organic acid produced by yeasts during the fermentation of glucose, and it is responsible for the acidification and the decrease of pH of the medium. Ethanol is the most important fermentation by-product, and from the technological point of view, the production capacity of yeasts is an important parameter that determines their usability in fermentative processes. Gay Lussac defined a stoichiometric theoretical relationship to explain the production of ethanol by Saccharomyces cerevisiae yeasts which is:

$$\underset{\text{glucose}}{1\,\text{C}\_6\text{H}\_{12}\text{O}\_6} \to 2\,\text{C}\_2\text{H}\_5\text{OH} + 2\,\text{CO}\_2\tag{1}$$

According to this relationship, from 180.0 grams of glucose, 92.0 grams of ethanol and 88.0 grams of carbon dioxide are produced, which results in a theoretical yield of 0.511 g ethanol/g glucose. However, in practice, besides ethanol and CO2, the production of biomass, glycerol and other minority compounds also happens, that is:

$$\text{C}\_6\text{H}\_{12}\text{O}\_6 + \text{nitrogen} \rightarrow \text{C}\_2\text{H}\_5\text{OH} + \text{CO}\_2 + \text{glycine} + \text{biomass} + \text{minority compounds} \tag{2}$$

At industrial scale, a yield of 0.45 g ethanol/g glucose is acceptable [34]. In the case of fermentations with non-Saccharomyces yeasts, lower yields are commonly observed. Regarding to glycerol, in the case of S. cerevisiae, its production represents approximately 3% of the utilized sugar. Minor compounds are represented by higher alcohols, esters, aldehydes and organic acids, among others.

#### 3.1.1 Importance of oxygen

Oxygen is an important element during the complete oxidation of glucose since it serves as final acceptor of electrons under aerobic conditions. It is also essential for other metabolic processes such as the synthesis of structural components of the

fermentative processes and to evaluate their use in the development of new products. Among the most studied non-Saccharomyces yeasts that reached special importance for researchers include Candida, Kloeckera, Hanseniaspora, Brettanomyces, Pichia,

Frontiers and New Trends in the Science of Fermented Food and Beverages

Either non-Saccharomyces or Saccharomyces yeasts share common pathways for the central metabolism of carbon; thus, both groups metabolize glucose through glycolysis. However, the mechanisms involved in the regulation of respirefermentative metabolism can differ significantly among them [30]. The glycolysis operates indistinctly under aerobic and anaerobic conditions, and through it, the glucose is metabolized to pyruvate by means of a series of biochemical reactions (Figure 2). Under anaerobic or oxygen-limited conditions, pyruvate is converted to acetaldehyde and then to ethanol, and as a result, two net moles of ATP are generated. Under fully aerobic conditions and in the absence of any repression effect, the

Fermentative metabolism of glucose by yeasts: Glycolysis (black lines) and ethanol and glycerol production (blue lines). Enzymes: 1, hexokinase; 2, phosphoglucose isomerase; 3, phosphofructokinase; 4, fructose 1,6 bisphosphate aldolase; 5, triosephosphate isomerase; 6, glyceraldehyde 3-phosphate dehydrogenase; 7, phosphoglycerate kinase; 8, phosphoglycerate mutase; 9, enolase; 10, pyruvate kinase; 11, pyruvate decarboxylase; 12, alcohol dehydrogenase; 13, aldehyde dehydrogenase; 14, acetyl-CoA hydrolase; 15, acetyl-CoA synthetase; 16, pyruvate dehydrogenase; 17, glycerol 3-P dehydrogenase; 18, glycerol 3-phosphatase.

Lanchacea and Kluyveromyces, among others.

3.1 Fermentative metabolism of sugars

Figure 2.

110

cytoplasmic membrane of yeasts. During alcoholic fermentation, as ethanol accumulates, it exerts a detrimental effect on the integrity and stability of the cytoplasmic membrane [31]. Under this condition, the supply of small amounts of oxygen to the medium through aeration promotes the synthesis of unsaturated fatty acids and sterols (mainly ergosterol) which are important components of the yeast cell membrane. Thus, the produced compounds can be used to replace the damaged fraction caused by the effect of ethanol that acts as a solvent [35, 36]. The replacement of unsaturated fatty acids and sterols is important to maintain the cell viability and allow the yeasts to complete successfully the fermentation. From the technological point of view, the supply of small amounts of oxygen is recommended in fermentations with musts of high specific gravity in order to avoid some drawbacks such as sluggish fermentation. It is also necessary for promoting the fermentative metabolism of non-Saccharomyces yeasts which are unable to ferment under fully anaerobic conditions [37]. The optimization of the aeration rate is very important to ensure the predominance of the fermentative metabolism and to reach the highest ethanol yield. In Crabtree-negative yeasts, as the concentration of oxygen in the medium increases above a certain value, the metabolism may become predominantly oxidative; thus, the ethanol yield decreases and the production of biomass increases. The highest ethanol yield is possible to achieve, adjusting properly the aeration rate of the fermentation medium. Aeration also affects the production of glycerol by yeasts; thus, as the concentration of oxygen increases, the production of glycerol decreases. From the technological point of view, aeration of the fermentation medium is an interesting tool to control the metabolic activity of non-Saccharomyces yeasts during fermentation, for instance, wines and beers [38, 39]. In addition, aeration can be also used in winemaking to improve the quality of wines since it provokes the transformation of phenols, which reduces the astringency.

## 3.2 Production of higher alcohols

During alcoholic fermentation, either non-Saccharomyces or Saccharomyces yeasts produce diverse volatile compounds of sensory importance such as higher alcohols, aldehydes, fatty acids and esters in different concentrations depending on the species of yeasts and the fermentation conditions. The harmonic balance of the compounds determines the sensory quality of the fermented beverage. Higher alcohols are a group of compounds that mostly confer unpleasant organoleptic character when present at high concentrations [40, 41]. In adequate concentrations, they contribute positively in defining the organoleptic quality of alcoholic beverage such as wines, beers and ciders. They are produced in the cytosol and then exported outside the yeast cell where it accumulates. Higher alcohols result from the decarboxylation of ketoacids that leads to the formation of the respective aldehydes, which are then reduced to form the corresponding higher alcohols (Figure 3). Ketoacids can be originated either from the metabolism of glucose or the catabolism of amino acids [42, 43], which are taken by the yeast cell from the fermentation medium. The synthesis of higher alcohols involves the participation of at least three enzymes: a transaminase, a carboxylase and an alcohol dehydrogenase. Factors that increase the metabolism of sugar and amino acids promote the synthesis of higher alcohols. The factors include temperature of fermentation, amino acid concentration and composition of the fermentation medium.

action of specific enzymes that catalyze the reaction between an alcohol and a volatile fatty acid (Figure 4). The synthesis of esters by yeast initially involves the activation of fatty acids to acyl coenzyme A mediated by energy and the subsequent condensation of the active compound with an alcohol present in the medium to form the corresponding ester [44]. From the sensory point of view, acetate esters are the most important compounds present in fermented beverages, which include ethyl acetate, butyl acetate, propyl acetate, phenyl ethyl acetate and amyl acetate, among others. The esters produced by S. cerevisiae involve the activity of at least three acetyltransferases (AAT, EC <IP_ADDRESS>): an alcohol acetyltransferase, an ethanol acetyltransferase and an isoamyl alcohol acetyltransferase [45, 46]. Other enzymes such as ester synthase were also reported to participate in the synthesis of esters.

Production of higher alcohols by yeast. Ehrlich's pathway and glucose catabolism.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

Figure 3.

Figure 4.

113

Mechanisms for the production of esters by yeasts.

#### 3.3 Production of esters

Esters are a group of compounds that mostly impart positive sensory characteristics to fermented beverages such as wines, beer and ciders. They are formed by the Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

Figure 4. Mechanisms for the production of esters by yeasts.

action of specific enzymes that catalyze the reaction between an alcohol and a volatile fatty acid (Figure 4). The synthesis of esters by yeast initially involves the activation of fatty acids to acyl coenzyme A mediated by energy and the subsequent condensation of the active compound with an alcohol present in the medium to form the corresponding ester [44]. From the sensory point of view, acetate esters are the most important compounds present in fermented beverages, which include ethyl acetate, butyl acetate, propyl acetate, phenyl ethyl acetate and amyl acetate, among others. The esters produced by S. cerevisiae involve the activity of at least three acetyltransferases (AAT, EC <IP_ADDRESS>): an alcohol acetyltransferase, an ethanol acetyltransferase and an isoamyl alcohol acetyltransferase [45, 46]. Other enzymes such as ester synthase were also reported to participate in the synthesis of esters.

cytoplasmic membrane of yeasts. During alcoholic fermentation, as ethanol accumulates, it exerts a detrimental effect on the integrity and stability of the cytoplasmic membrane [31]. Under this condition, the supply of small amounts of oxygen to the medium through aeration promotes the synthesis of unsaturated fatty acids and sterols (mainly ergosterol) which are important components of the yeast cell membrane. Thus, the produced compounds can be used to replace the damaged fraction caused by the effect of ethanol that acts as a solvent [35, 36]. The replacement of unsaturated fatty acids and sterols is important to maintain the cell viability and allow the yeasts to complete successfully the fermentation. From the technological point of view, the supply of small amounts of oxygen is recommended in fermentations with musts of high specific gravity in order to avoid some drawbacks such as sluggish fermentation. It is also necessary for promoting the fermentative metabolism of non-Saccharomyces yeasts which are unable to ferment under fully anaerobic conditions [37]. The optimization of the aeration rate is very important to ensure the predominance of the fermentative metabolism and to reach the highest ethanol yield. In Crabtree-negative yeasts, as the concentration of oxygen in the medium increases above a certain value, the metabolism may become predominantly oxidative; thus, the ethanol yield decreases and the production of biomass increases. The highest ethanol yield is possible to achieve, adjusting properly the aeration rate of the fermentation medium. Aeration also affects the production of glycerol by yeasts; thus, as the concentration of oxygen increases, the production of glycerol decreases. From the technological point of view, aeration of the fermentation medium is an interesting tool to control the metabolic activity of non-Saccharomyces yeasts during fermentation, for instance, wines and beers [38, 39]. In addition, aeration can be also used in winemaking to improve the quality of wines since it

Frontiers and New Trends in the Science of Fermented Food and Beverages

provokes the transformation of phenols, which reduces the astringency.

amino acid concentration and composition of the fermentation medium.

Esters are a group of compounds that mostly impart positive sensory characteristics to fermented beverages such as wines, beer and ciders. They are formed by the

During alcoholic fermentation, either non-Saccharomyces or Saccharomyces yeasts produce diverse volatile compounds of sensory importance such as higher alcohols, aldehydes, fatty acids and esters in different concentrations depending on the species of yeasts and the fermentation conditions. The harmonic balance of the compounds determines the sensory quality of the fermented beverage. Higher alcohols are a group of compounds that mostly confer unpleasant organoleptic character when present at high concentrations [40, 41]. In adequate concentrations, they contribute positively in defining the organoleptic quality of alcoholic beverage such as wines, beers and ciders. They are produced in the cytosol and then exported outside the yeast cell where it accumulates. Higher alcohols result from the decarboxylation of ketoacids that leads to the formation of the respective aldehydes, which are then reduced to form the corresponding higher alcohols (Figure 3). Ketoacids can be originated either from the metabolism of glucose or the catabolism of amino acids [42, 43], which are taken by the yeast cell from the fermentation medium. The synthesis of higher alcohols involves the participation of at least three enzymes: a transaminase, a carboxylase and an alcohol dehydrogenase. Factors that increase the metabolism of sugar and amino acids promote the synthesis of higher alcohols. The factors include temperature of fermentation,

3.2 Production of higher alcohols

3.3 Production of esters

112

However, the relevance attributed to the activity of this enzyme is quite limited. Ethyl acetate is the most abundant ester present in wines and largely responsible for the sensory character. Studies carried out with non-Saccharomyces yeasts related to the ability of producing esters allowed to select species of Hanseniaspora and Pichia able to promote esterification of various alcohols such as ethanol, isoamyl alcohol and 2-phenyl ethanol to produce the corresponding esters [47].

and K. corticis were isolated from a variety of niches including the spontaneous fermentation of grape must and ciders [6, 8, 49, 59]. Most representatives present a lemon shape (apiculate yeasts) and asexual reproduction with bipolar budding. It was reported that these yeasts participate positively in the early stage of the spontaneous fermentation of wine [59, 60], strains of Kloeckera apiculata being the most dominant [19, 49, 51, 52]. During spontaneous fermentation, as the ethanol concentration increases, the fermentative activity of these yeasts slows down and stops toward the end of fermentation by the effect of the ethanol [61]. These yeasts are characterized by producing amounts of ethanol around 4–5% v/v, values typically found in commercial beers. It was reported that the control of aeration during fermentation has effect on the production of ethanol and compounds of sensory importance such as esters, higher alcohols and organic acids [14]. Based on the information available in literature, these yeasts are promissory for being used in brewing; however, before defining a strategy of exploitation, it is necessary to carry out more in-depth studies on the effect of temperature, wort composition and inoculation rate in the fermentative activity of these yeasts. In addition, it is also necessary to carry out studies on the behavior of these yeasts in fermentations with mixed and sequential cultures with Saccharomyces cerevisiae and the production of compounds of sensory importance. Studies carried out with pure cultures of Kloeckera corticis showed that these yeasts are capable of producing acetic acid, acetaldehyde, ethyl acetate and acetoin at high concentrations [62]. In addition, it has been reported that strains of Kloeckera apiculata are capable of producing higher concentrations of ethyl and isoamyl acetate than other non-Saccharomyces yeasts [14, 63]. From the technological point of view, techniques of cell immobilization can be an additional strategy to improve the fermentative behavior and the production of compounds of sensory importance. The ability of these yeasts to produce a variety of aromatic compounds with positive impact on the sensory quality makes

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

them attractive and potentially exploitable in fermentation processes.

Few studies have been conducted regarding the potential use of yeasts belonging to the genus Hanseniaspora (apiculate yeasts) in the production of fermented beverages. The studied yeasts were isolated from the spontaneous fermentation of grape musts [6, 8, 59] and include species of Hanseniaspora uvarum, H. osmophila and H. guilliermondii, among others. It has been shown that these yeasts play an important role during the early stage of spontaneous fermentation of wine and strains of Hanseniaspora uvarum (also called Kloeckera apiculata) are dominant [19, 51, 52]. They are characterized by tolerating and producing low amounts of ethanol that do not exceed the values of 5.0% v/v [61]. This limitation explains why these yeasts do not participate actively toward the end of spontaneous fermentation of wines where the ethanol content reaches values even higher than 10%v/v. However, the fermentative capability of these yeasts is enough to produce beers of standard ethanol content similarly to those found in the market (4.5–5%v/v). In addition, they are able to ferment a wide range of sugars including maltose, which is an important feature needed for the production of beers. Regarding the production of compounds of sensory importance, studies have reported that strains of Hanseniaspora osmophila are characterized by producing high concentrations of acetic acid, acetaldehyde and ethyl acetate [62]. Additionally, it was also found that strains of Hanseniaspora uvarum are able to produce a variety of esters that confer fruitiness to fermented beverages [11, 62, 64]. However, other studies reported that mixed cultures of H. uvarum with S. cerevisiae produce higher amounts of higher alcohols than monocultures with S. cerevisiae

4.3 Hanseniaspora yeasts

115

## 4. Most important non-Saccharomyces yeasts

#### 4.1 Candida yeasts

In the last years, the fermentative behavior of some Candida yeasts has been studied with respect to the production of wines and beers. The most studied species include Candida stellata, C. zemplinina and C. pulcherrima, among others [16, 20, 21, 48–50]. Representatives of Candida yeasts have been isolated from the early stages of spontaneous fermentation of different types of wines [8, 19, 51, 52]. The isolated species were characterized by being round in shape and smaller than S. cerevisiae. These yeasts are able to sediment toward the end of fermentation in a similar manner as S. cerevisiae [20]. Currently, the most important characteristics reported include the production of considerable amounts of ethanol and glycerol and a balanced production of volatile compounds of sensory importance, for instance, esters, fatty acids, aldehydes and higher alcohols. The production of ethanol is an important feature to define the use of yeasts in the production of fermented beverages with high ethanol contents such as wines. It has been reported that C. zemplinina strains are capable of producing ethanol up to 11.0% v/v [53], amount normally reached during the fermentation of sweet and semidry wines with S. cerevisiae. In addition, it has been demonstrated that Candida yeasts are capable of producing high amounts (up to 25.0 g/L) of glycerol [53–56], compound that contributes positively to the sensory quality of wines, beers and other beverages. The fermentative behavior of these yeasts was also evaluated as mixed cultures with S. cerevisiae [57]. The results were promising and interesting for being scaled-up to pilot fermentations. For instance, fermentation experiments of mixed cultures of C. stellata with S. cerevisiae produced higher levels of esters and fatty acids than monocultures of S. cerevisiae [19, 57]. Fermentations with mixed and even sequential cultures of yeasts are an interesting field of research to evaluate the potential use of non-Saccharomyces yeasts to produce sensory differentiated beverages. In addition, individual fermentations with C. stellata and C. zemplinina strains using immobilized systems have been also performed [53, 58]. The results showed the improvement of some technological properties such as the fermentation rate, ethanol production and the reusability of the strains in successive fermentations. Currently, studies to evaluate the usability of C. zemplinina strains in beer fermentation have been carried out using malt wort of 14 and 20°P, typically used in beer fermentation processes [21, 22]. The yeast strains showed a suitable fermentative behavior for the production of lager and ale beers. One interesting feature is that Candida zemplinina is unable to ferment maltose, the main fermentable sugar of the malt wort. This characteristic is of special importance since it would enable the production of beers with low ethanol content and particular sensory profiles.

#### 4.2 Kloeckera yeasts

Yeasts species belonging to this genus have recently become of interest for the production of fermented beverages. Species such as Kloeckera apiculata, K. javanica Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

and K. corticis were isolated from a variety of niches including the spontaneous fermentation of grape must and ciders [6, 8, 49, 59]. Most representatives present a lemon shape (apiculate yeasts) and asexual reproduction with bipolar budding. It was reported that these yeasts participate positively in the early stage of the spontaneous fermentation of wine [59, 60], strains of Kloeckera apiculata being the most dominant [19, 49, 51, 52]. During spontaneous fermentation, as the ethanol concentration increases, the fermentative activity of these yeasts slows down and stops toward the end of fermentation by the effect of the ethanol [61]. These yeasts are characterized by producing amounts of ethanol around 4–5% v/v, values typically found in commercial beers. It was reported that the control of aeration during fermentation has effect on the production of ethanol and compounds of sensory importance such as esters, higher alcohols and organic acids [14]. Based on the information available in literature, these yeasts are promissory for being used in brewing; however, before defining a strategy of exploitation, it is necessary to carry out more in-depth studies on the effect of temperature, wort composition and inoculation rate in the fermentative activity of these yeasts. In addition, it is also necessary to carry out studies on the behavior of these yeasts in fermentations with mixed and sequential cultures with Saccharomyces cerevisiae and the production of compounds of sensory importance. Studies carried out with pure cultures of Kloeckera corticis showed that these yeasts are capable of producing acetic acid, acetaldehyde, ethyl acetate and acetoin at high concentrations [62]. In addition, it has been reported that strains of Kloeckera apiculata are capable of producing higher concentrations of ethyl and isoamyl acetate than other non-Saccharomyces yeasts [14, 63]. From the technological point of view, techniques of cell immobilization can be an additional strategy to improve the fermentative behavior and the production of compounds of sensory importance. The ability of these yeasts to produce a variety of aromatic compounds with positive impact on the sensory quality makes them attractive and potentially exploitable in fermentation processes.

#### 4.3 Hanseniaspora yeasts

However, the relevance attributed to the activity of this enzyme is quite limited. Ethyl acetate is the most abundant ester present in wines and largely responsible for the sensory character. Studies carried out with non-Saccharomyces yeasts related to the ability of producing esters allowed to select species of Hanseniaspora and Pichia able to promote esterification of various alcohols such as ethanol, isoamyl alcohol

In the last years, the fermentative behavior of some Candida yeasts has been studied with respect to the production of wines and beers. The most studied species include Candida stellata, C. zemplinina and C. pulcherrima, among others [16, 20, 21, 48–50]. Representatives of Candida yeasts have been isolated from the early stages of spontaneous fermentation of different types of wines [8, 19, 51, 52]. The isolated species were characterized by being round in shape and smaller than S. cerevisiae. These yeasts are able to sediment toward the end of fermentation in a similar manner as S. cerevisiae [20]. Currently, the most important characteristics reported include the production of considerable amounts of ethanol and glycerol and a balanced production of volatile compounds of sensory importance, for instance, esters, fatty acids, aldehydes and higher alcohols. The production of etha-

nol is an important feature to define the use of yeasts in the production of

fermented beverages with high ethanol contents such as wines. It has been reported that C. zemplinina strains are capable of producing ethanol up to 11.0% v/v [53], amount normally reached during the fermentation of sweet and semidry wines with S. cerevisiae. In addition, it has been demonstrated that Candida yeasts are capable of producing high amounts (up to 25.0 g/L) of glycerol [53–56], compound that contributes positively to the sensory quality of wines, beers and other beverages. The fermentative behavior of these yeasts was also evaluated as mixed cultures with S. cerevisiae [57]. The results were promising and interesting for being scaled-up to pilot fermentations. For instance, fermentation experiments of mixed cultures of C. stellata with S. cerevisiae produced higher levels of esters and fatty acids than monocultures of S. cerevisiae [19, 57]. Fermentations with mixed and even sequential cultures of yeasts are an interesting field of research to evaluate the potential use of non-Saccharomyces yeasts to produce sensory differentiated beverages. In addition, individual fermentations with C. stellata and C. zemplinina strains using immobilized systems have been also performed [53, 58]. The results showed the improvement of some technological properties such as the fermentation rate, ethanol production and the reusability of the strains in successive fermentations. Currently, studies to evaluate the usability of C. zemplinina strains in beer fermentation have been carried out using malt wort of 14 and 20°P, typically used in beer fermentation processes [21, 22]. The yeast strains showed a suitable fermentative behavior for the production of lager and ale beers. One interesting feature is that Candida zemplinina is unable to ferment maltose, the main fermentable sugar of the malt wort. This characteristic is of special importance since it would enable the production of beers with low ethanol content and particular sensory profiles.

Yeasts species belonging to this genus have recently become of interest for the production of fermented beverages. Species such as Kloeckera apiculata, K. javanica

and 2-phenyl ethanol to produce the corresponding esters [47].

Frontiers and New Trends in the Science of Fermented Food and Beverages

4. Most important non-Saccharomyces yeasts

4.1 Candida yeasts

4.2 Kloeckera yeasts

114

Few studies have been conducted regarding the potential use of yeasts belonging to the genus Hanseniaspora (apiculate yeasts) in the production of fermented beverages. The studied yeasts were isolated from the spontaneous fermentation of grape musts [6, 8, 59] and include species of Hanseniaspora uvarum, H. osmophila and H. guilliermondii, among others. It has been shown that these yeasts play an important role during the early stage of spontaneous fermentation of wine and strains of Hanseniaspora uvarum (also called Kloeckera apiculata) are dominant [19, 51, 52]. They are characterized by tolerating and producing low amounts of ethanol that do not exceed the values of 5.0% v/v [61]. This limitation explains why these yeasts do not participate actively toward the end of spontaneous fermentation of wines where the ethanol content reaches values even higher than 10%v/v. However, the fermentative capability of these yeasts is enough to produce beers of standard ethanol content similarly to those found in the market (4.5–5%v/v). In addition, they are able to ferment a wide range of sugars including maltose, which is an important feature needed for the production of beers. Regarding the production of compounds of sensory importance, studies have reported that strains of Hanseniaspora osmophila are characterized by producing high concentrations of acetic acid, acetaldehyde and ethyl acetate [62]. Additionally, it was also found that strains of Hanseniaspora uvarum are able to produce a variety of esters that confer fruitiness to fermented beverages [11, 62, 64]. However, other studies reported that mixed cultures of H. uvarum with S. cerevisiae produce higher amounts of higher alcohols than monocultures with S. cerevisiae

[4, 19]. Regarding fermentation parameters, the control of aeration and temperature exerts an important effect on the dynamics and activity of Hanseniaspora yeasts. Both parameters are important to control the production of compounds of sensory importance, which influence the quality of fermented beverages [11, 65]. However, in view of the scarce information on the fermentative behavior of Hanseniaspora yeasts, particularly referring to the production of fermented beverages, additional studies are needed to perform in order to find the adequate conditions for their usage, for instance, in the production of beers with new sensory profiles.

5. Production of special wines

DOI: http://dx.doi.org/10.5772/intechopen.81868

sensory qualities.

117

6. Production of craft beers

It is of common agreement that non-Saccharomyces yeasts contribute beneficially to the sensory quality of spontaneously fermented wines, an evidence that served as a starting point to pay attention to particular yeast species that could be exploited in fermentations of commercial and noncommercial fermented beverages. Non-Saccharomyces species are characterized by producing a greater

diversity of compounds of sensory importance than S. cerevisiae yeasts. Although these yeasts show a low fermentation power, some species possess important

Hanseniaspora yeasts produce a variety of compounds of sensory impact, particularly esters at concentrations even higher than S. cerevisiae. On the other hand, Candida zemplinina, a fructofilic yeast, has been shown to produce glycerol in higher concentrations than S. cerevisiae. It is also capable of producing ethanol in concentrations high enough to produce different types of wines. In view of the complementary characteristics of both groups of yeasts (Saccharomyces and non-Saccharomyces), the use of non-Saccharomyces yeasts can be proposed in fermentations with mixed or sequential cultures with S. cerevisiae as an important strategy to improve sensory complexity and mouthfeel of wines [19, 73]. The fermentative versatility of non-Saccharomyces yeasts would enable the production of special wines with different and innovative sensory characteristics. In addition, among the techniques that can be implemented for enabling their practical exploitation include the selection of new strains, the development of fermentation strategies (mixed or sequential cultures with two or more yeast strains), the ratio of both strains in the inoculum (non-Saccharomyces/Saccharomyces cerevisiae) and the inoculation rate at the beginning of fermentation [57, 76]. Finally, some technological characteristics of non-Saccharomyces yeasts can be also modified by using cultivation techniques in bioreactors with the aim of improving, for instance, the fermentation rate. The possibility of commercializing as starter cultures is an attractive opportunity for the production of different types of wines with special

In the last 10 years, the market of craft beers has increased in the USA, Latin America and some countries of Europe [77, 78]. This phenomenon is related to the expectation of consumers for discovering in these beers sensory characteristics different from those routinely found in commercial beers [74]. Current consumers are curious and interested in sensing new flavors and aromas that can satisfy their preferences. As consequence, new market segments have emerged in response to the broad possibility of offering new types of beers produced using different methods and techniques of fermentation. The production of craft beer is generally

processing methods. Craft beers are not usually filtered; due to this, their shelf life is relatively short, and therefore, their consumption must be within few days after bottling. There are a variety of innovative alternatives to produce different types of craft beers which include the use of new types of adjuncts either amylaceous (cereal grains) or non-amylaceous (fruit pulps or juices) and selected strains of non-Saccharomyces yeasts which have an enormous exploitation potential. Although most non-Saccharomyces yeasts produce low concentrations of ethanol, the fermentative capacity of some representatives of Kloeckera and Hanseniaspora yeasts is adequate to produce beers with an ethanol content typically found in the market

carried out in small-scale breweries and involves the use of non-technified

fermentative features, for instance, representatives of Kloeckera and

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

### 4.4 Brettanomyces yeasts

Yeasts of this genus do not have a good reputation in fermentation processes such as in winemaking. For instance, representatives of Brettanomyces bruxellensis are considered detrimental due to the production of compounds such as 4 ethylguaiacol, 4-ethylphenol and 4-ethylcatechol which impart unpleasant sensory character to wines known as "Bretty" [5, 66]. These compounds result from the activity of a decarboxylase that acts on hydroxycinnamic acids followed by a reduction reaction [67]. The hydroxycinnamic acids are phenolic compounds naturally present in the skin and seeds of grapes. The common representatives of this genus were isolated from the spontaneous fermentation of wine, beer, cider and even kombucha [68–70]. It was also isolated from equipment and utensils utilized in fermentation processes, which are difficult to sanitize. The commonly isolated species include Brettanomyces bruxellensis, B. lambicus, B. intermedius and B. anomalus, among others [68, 69]. Particularly, strains of B. bruxellensis are able to ferment only in the presence of oxygen (positive Crabtree effect), a broad spectrum of sugars and even maltooligosaccharides which are not fermentable by S. cerevisiae [71]. Under anaerobic conditions, these yeasts are unable to ferment and produce ethanol; thus, at low concentration of sugar in the medium, the fermentation of glucose to ethanol is blocked. On the contrary, the fermentation is stimulated in the presence of oxygen, an effect known as Custer or negative Pasteur [72]. Apart from producing ethanol in the presence of oxygen, Brettanomyces bruxellensis also produces high concentrations of acetic acid, which acidifies and lowers the pH of the medium. However, yeasts of this genus are not entirely undesirable; some representatives participate, for instance, during the fermentation of certain beers known as "Lambic" and "Gueuze" consumed commonly in Belgium and "Coolship Ales" in North America. The fermentation of "Lambic" beer is a spontaneous process which goes through a complex succession of microorganisms where Brettanomyces bruxellensis participates during the final stage acidifying the product [73]. The participation of these yeasts gives the beer its characteristic acidity and dryness and additionally is responsible for the production of compounds such as ethyl phenol, ethyl acetate, ethyl caprylate, ethyl decanoate and ethyl lactate, which synergistically confer their typical aroma character [18, 74]. It has been shown that esters soften the sour taste and add fruity notes to this kind of beers [75]. Based on these findings, it was demonstrated that these yeasts and particularly B. bruxellensis contribute positively to defining the floral and fruity character of "Lambic" beers [18]. Beyond the contribution of Brettanomyces yeasts in spontaneous fermentation processes, in recent years, their use in controlled fermentations has been investigated, both in pure and in coculture with S. cerevisiae [15, 17]. Interesting findings were reported, indicating that the control of aeration during fermentation is a critical point to guide the fermentative metabolism toward the production of important volatile compounds that may contribute to the organoleptic character of fermented beverages.

## 5. Production of special wines

[4, 19]. Regarding fermentation parameters, the control of aeration and temperature exerts an important effect on the dynamics and activity of Hanseniaspora yeasts. Both parameters are important to control the production of compounds of sensory importance, which influence the quality of fermented beverages [11, 65]. However, in view of the scarce information on the fermentative behavior of Hanseniaspora yeasts, particularly referring to the production of fermented beverages, additional studies are needed to perform in order to find the adequate conditions for their usage, for instance, in the production of beers with new

Frontiers and New Trends in the Science of Fermented Food and Beverages

Yeasts of this genus do not have a good reputation in fermentation processes such as in winemaking. For instance, representatives of Brettanomyces bruxellensis are considered detrimental due to the production of compounds such as 4-

ethylguaiacol, 4-ethylphenol and 4-ethylcatechol which impart unpleasant sensory character to wines known as "Bretty" [5, 66]. These compounds result from the activity of a decarboxylase that acts on hydroxycinnamic acids followed by a reduction reaction [67]. The hydroxycinnamic acids are phenolic compounds naturally present in the skin and seeds of grapes. The common representatives of this genus were isolated from the spontaneous fermentation of wine, beer, cider and even kombucha [68–70]. It was also isolated from equipment and utensils utilized in fermentation processes, which are difficult to sanitize. The commonly isolated

species include Brettanomyces bruxellensis, B. lambicus, B. intermedius and

B. anomalus, among others [68, 69]. Particularly, strains of B. bruxellensis are able to ferment only in the presence of oxygen (positive Crabtree effect), a broad spectrum of sugars and even maltooligosaccharides which are not fermentable by S. cerevisiae [71]. Under anaerobic conditions, these yeasts are unable to ferment and produce ethanol; thus, at low concentration of sugar in the medium, the fermentation of glucose to ethanol is blocked. On the contrary, the fermentation is stimulated in the presence of oxygen, an effect known as Custer or negative Pasteur [72]. Apart from producing ethanol in the presence of oxygen, Brettanomyces bruxellensis also produces high concentrations of acetic acid, which acidifies and lowers the pH of the medium. However, yeasts of this genus are not entirely undesirable; some representatives participate, for instance, during the fermentation of certain beers known as "Lambic" and "Gueuze" consumed commonly in Belgium and "Coolship Ales" in North America. The fermentation of "Lambic" beer is a spontaneous process which goes through a complex succession of microorganisms where Brettanomyces bruxellensis participates during the final stage acidifying the product [73]. The participation of these yeasts gives the beer its characteristic acidity and dryness and additionally is responsible for the production of compounds such as ethyl phenol, ethyl acetate, ethyl caprylate, ethyl decanoate and ethyl lactate, which synergistically confer their typical aroma character [18, 74]. It has been shown that esters soften the sour taste and add fruity notes to this kind of beers [75]. Based on these findings, it was demonstrated that these yeasts and particularly B. bruxellensis contribute positively to defining the floral and fruity character of "Lambic" beers [18]. Beyond the contribution of Brettanomyces yeasts in spontaneous fermentation processes, in recent years, their use in controlled fermentations has been investigated, both in pure and in coculture with S. cerevisiae [15, 17]. Interesting findings were reported, indicating that the control of aeration during fermentation is a critical point to guide the fermentative metabolism toward the production of important volatile compounds that may contribute to the organoleptic character of

sensory profiles.

4.4 Brettanomyces yeasts

fermented beverages.

116

It is of common agreement that non-Saccharomyces yeasts contribute beneficially to the sensory quality of spontaneously fermented wines, an evidence that served as a starting point to pay attention to particular yeast species that could be exploited in fermentations of commercial and noncommercial fermented beverages. Non-Saccharomyces species are characterized by producing a greater diversity of compounds of sensory importance than S. cerevisiae yeasts. Although these yeasts show a low fermentation power, some species possess important fermentative features, for instance, representatives of Kloeckera and Hanseniaspora yeasts produce a variety of compounds of sensory impact, particularly esters at concentrations even higher than S. cerevisiae. On the other hand, Candida zemplinina, a fructofilic yeast, has been shown to produce glycerol in higher concentrations than S. cerevisiae. It is also capable of producing ethanol in concentrations high enough to produce different types of wines. In view of the complementary characteristics of both groups of yeasts (Saccharomyces and non-Saccharomyces), the use of non-Saccharomyces yeasts can be proposed in fermentations with mixed or sequential cultures with S. cerevisiae as an important strategy to improve sensory complexity and mouthfeel of wines [19, 73]. The fermentative versatility of non-Saccharomyces yeasts would enable the production of special wines with different and innovative sensory characteristics. In addition, among the techniques that can be implemented for enabling their practical exploitation include the selection of new strains, the development of fermentation strategies (mixed or sequential cultures with two or more yeast strains), the ratio of both strains in the inoculum (non-Saccharomyces/Saccharomyces cerevisiae) and the inoculation rate at the beginning of fermentation [57, 76]. Finally, some technological characteristics of non-Saccharomyces yeasts can be also modified by using cultivation techniques in bioreactors with the aim of improving, for instance, the fermentation rate. The possibility of commercializing as starter cultures is an attractive opportunity for the production of different types of wines with special sensory qualities.

## 6. Production of craft beers

In the last 10 years, the market of craft beers has increased in the USA, Latin America and some countries of Europe [77, 78]. This phenomenon is related to the expectation of consumers for discovering in these beers sensory characteristics different from those routinely found in commercial beers [74]. Current consumers are curious and interested in sensing new flavors and aromas that can satisfy their preferences. As consequence, new market segments have emerged in response to the broad possibility of offering new types of beers produced using different methods and techniques of fermentation. The production of craft beer is generally carried out in small-scale breweries and involves the use of non-technified processing methods. Craft beers are not usually filtered; due to this, their shelf life is relatively short, and therefore, their consumption must be within few days after bottling. There are a variety of innovative alternatives to produce different types of craft beers which include the use of new types of adjuncts either amylaceous (cereal grains) or non-amylaceous (fruit pulps or juices) and selected strains of non-Saccharomyces yeasts which have an enormous exploitation potential. Although most non-Saccharomyces yeasts produce low concentrations of ethanol, the fermentative capacity of some representatives of Kloeckera and Hanseniaspora yeasts is adequate to produce beers with an ethanol content typically found in the market

(4.5–5%v/v). Among non-Saccharomyces yeasts considered important in beer fermentation, Brettanomyces lambicus is the most representative which is involved in the production of "Lambic" and "Gueuze" beers. Currently, some studies with Candida zemplinina strains were performed in fermentations with pure malt wort and with different adjuncts (grape or apple juice) at different temperatures and specific gravities. The findings were promissory and showed the capability of these yeasts to ferment at low temperatures (14°C) and in medium with high specific gravity (16°P), which demonstrates the possibility for being exploited in the production of craft beers. In addition, it was also proposed that these yeasts can be used for the production of beers with low ethanol content since they are not able to ferment maltose, the main and most abundant sugar present in the wort [20, 21]. Additionally, other non-Saccharomyces yeasts such as Dekkera anomala, Naumovozyma dairenensis and Debaryomyces spp. have been also reported with a high potential for being used in the fermentation of beers. In view of the different fermentative behavior of non-Saccharomyces yeasts and the variety of compounds of sensory importance that they can produce during fermentation, their use in controlled fermentations has aroused the interest of brewers for producing beers with distinctive sensory features [23, 79].

## 7. Conclusion

Non-Saccharomyces yeasts show a great potential to be used in the production of fermented beverages mainly wines and beers. These yeasts show a variety of fermentative patterns, and depending on the fermentation conditions, they produce a wide range of volatile compounds of sensory importance. For their practical application in a particular fermentative process, it is necessary knowing the parameters that directly influence on the fermentative activity and the production of desirable volatile compounds. Among the non-Saccharomyces yeasts that have attracted interest of researchers due to their fermentative qualities include strains of Candida stellata, C. zemplinina, Kloeckera apiculata and Hanseniaspora uvarum. Particularly, strains of Candida stellata and C. zemplinina have become very attractive for using in fermentations of different types of wines and beers. These yeasts are capable of producing significant concentrations of glycerol, an important compound that imparts a positive impact on the sensory quality of wines and beers. Candida yeasts, especially C. zemplinina, also produce high concentrations of ethanol, high enough to drive fermentation processes of wines. On the other hand, species of Kloeckera and Hanseniaspora yeasts are characterized by producing considerable amounts of acetate esters, valuable compounds that contribute positively to the sensory character of beers. Based on this, if a fermentation process that involves the use of non-Saccharomyces yeasts is going to be implemented, it is necessary to select the best representatives and then define the appropriate fermentation conditions for the production of fermented beverages with the desired sensory qualities.

Author details

119

Waldir Desiderio Estela Escalante

provided the original work is properly cited.

Laboratory of Bioprocessing and Technology of Fermentation, Faculty of Chemistry and Chemical Engineering, Universidad Nacional Mayor de San Marcos, Lima, Peru

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to<EMAIL_ADDRESS>
Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

DOI: http://dx.doi.org/10.5772/intechopen.81868

## Conflict of interest

The author certifies that he has no affiliation with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

## Author details

(4.5–5%v/v). Among non-Saccharomyces yeasts considered important in beer fermentation, Brettanomyces lambicus is the most representative which is involved in the production of "Lambic" and "Gueuze" beers. Currently, some studies with Candida zemplinina strains were performed in fermentations with pure malt wort and with different adjuncts (grape or apple juice) at different temperatures and specific gravities. The findings were promissory and showed the capability of these yeasts to ferment at low temperatures (14°C) and in medium with high specific gravity (16°P), which demonstrates the possibility for being exploited in the production of craft beers. In addition, it was also proposed that these yeasts can be used for the production of beers with low ethanol content since they are not able to ferment maltose, the main and most abundant sugar present in the wort [20, 21]. Additionally, other non-

Frontiers and New Trends in the Science of Fermented Food and Beverages

Saccharomyces yeasts such as Dekkera anomala, Naumovozyma dairenensis and Debaryomyces spp. have been also reported with a high potential for being used in the fermentation of beers. In view of the different fermentative behavior of non-Saccharomyces yeasts and the variety of compounds of sensory importance that they can produce during fermentation, their use in controlled fermentations has aroused the interest of brewers for producing beers with distinctive sensory features

Non-Saccharomyces yeasts show a great potential to be used in the production of fermented beverages mainly wines and beers. These yeasts show a variety of fermentative patterns, and depending on the fermentation conditions, they produce a wide range of volatile compounds of sensory importance. For their practical application in a particular fermentative process, it is necessary knowing the parameters that directly influence on the fermentative activity and the production of desirable volatile compounds. Among the non-Saccharomyces yeasts that have attracted interest of researchers due to their fermentative qualities include strains of Candida stellata, C. zemplinina, Kloeckera apiculata and Hanseniaspora uvarum. Particularly, strains of Candida stellata and C. zemplinina have become very attractive for using in fermentations of different types of wines and beers. These yeasts are capable of producing significant concentrations of glycerol, an important compound that imparts a positive impact on the sensory quality of wines and beers. Candida yeasts, especially C. zemplinina, also produce high concentrations of ethanol, high enough to drive fermentation processes of wines. On the other hand, species of Kloeckera and Hanseniaspora yeasts are characterized by producing considerable amounts of acetate esters, valuable compounds that contribute

positively to the sensory character of beers. Based on this, if a fermentation process that involves the use of non-Saccharomyces yeasts is going to be implemented, it is necessary to select the best representatives and then define the appropriate fermentation conditions for the production of fermented beverages with the desired

The author certifies that he has no affiliation with or involvement in any organization or entity with any financial interest or non-financial interest in the subject

[23, 79].

7. Conclusion

sensory qualities.

118

Conflict of interest

matter or materials discussed in this manuscript.

Waldir Desiderio Estela Escalante Laboratory of Bioprocessing and Technology of Fermentation, Faculty of Chemistry and Chemical Engineering, Universidad Nacional Mayor de San Marcos, Lima, Peru

\*Address all correspondence to<EMAIL_ADDRESS>
© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## References

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[2] Clavijo A, Calderón IL, Paneque P. Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region. International Journal of Food Microbiology. 2010;143:241-245

[3] Barnett JA, Payne RW, Yarrow D. Yeasts: Characteristics and Identification. 2nd ed. Cambridge, UK: Cambridge University Press; 1990

[4] Ciani M, Maccarelli F. Oenological properties of non-Saccharomyces yeasts associated with wine-making. World Journal of Microbiology and Biotechnology. 1998;14:199-203

[5] Loureiro V, Malfeito-Ferreira M. Spoilage activities of Dekkera/ Brettanomyces spp. In: Blackburn C, editor. Food Spoilage Microorganisms. Cambridge: Woodhead Publishers; 2006. pp. 354-398

[6] Heard GM, Fleet GH. Growth of natural yeast flora during the fermentation of inoculated wines. Applied and Environmental Microbiology. 1985;50:727-728

[7] Fleet GH. Yeast interaction and wine flavour. International Journal of Food Microbiology. 2003;86:11-22

[8] Grangeteau C, Gerhards D, Terrat S, Dequiedt S, Alexandre H, Guilloux-Benatier M, et al. FT-IR spectroscopy: A powerful tool for studying the inter- and intraspecific biodiversity of cultivable non-Saccharomyces yeasts isolated from

grape must. Journal of Microbiological Methods. 2016;121:50-58

[15] Estela-Escalante WD, Rychtera M, Melzoch K, et al. Efecto de la aireación en la producción de compuestos volátiles por cultivo mixto de Brettanomyces intermedius y Saccharomyces cerevisiae durante la fermentación de sidra. TIP Revista Especializada en Ciencias Químico-

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[24] Quiros M, Rojas V, Gonzalez R,

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[9] Pardo I, Garcia MJ, Zuñiga M, Uruburu E. Dynamics of microbial populations during fermentation of wines from the Utiel-Requena region of Spain. Applied and Environmental Microbiology. 1989;55:539-541

[10] Tamang JP, Fleet GH. Yeasts diversity in fermented foods and beverages. In: Satyanarayana T, Kunze G, editors. Yeast Biotechnology. Netherlands: Diversity and Applications. Springer; 2009. pp. 169-198

[11] Estela Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de Hanseniaspora uvarum y su importancia en la producción de bebidas fermentadas. Revista de la Sociedad Venezolana de Microbiología. 2011;31:57-63

[12] Estela Escalante W, Rychtera M, Melzoch K, Quillama Polo E, Hatta Sakoda B. Study of the fermentative activity of Hansenula anomala and production of chemical compounds of sensory importance. Revista Peruana de Biología. 2011;18(3):325-334

[13] Estela-Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de Saccharomycodes ludwigii y evaluación de la síntesis de compuestos de importancia sensorial durante la fermentación de jugo de manzana. TIP Revista Especializada en Ciencias Químico-Biológicas. 2011;14(1):12-23

[14] Estela-Escalante WD, Rychtera M, Melzoch K, Guerrero-Ochoa MR. Influence of aeration in the fermentative activity of Kloeckera apiculata during fermentation of apple juice. Acta Biológica Colombiana. 2012; 17(2):309-321

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

[15] Estela-Escalante WD, Rychtera M, Melzoch K, et al. Efecto de la aireación en la producción de compuestos volátiles por cultivo mixto de Brettanomyces intermedius y Saccharomyces cerevisiae durante la fermentación de sidra. TIP Revista Especializada en Ciencias Químico-Biológicas. 2014;17(1):5-14

References

2007;24:403-412

[1] Mercado L, Dalcero A, Masuelli R, Combina M. Diversity of Saccharomyces strains on grapes and winery surfaces: Analysis of their contribution of

Frontiers and New Trends in the Science of Fermented Food and Beverages

grape must. Journal of Microbiological

[9] Pardo I, Garcia MJ, Zuñiga M, Uruburu E. Dynamics of microbial populations during fermentation of wines from the Utiel-Requena region of Spain. Applied and Environmental Microbiology. 1989;55:539-541

[10] Tamang JP, Fleet GH. Yeasts diversity in fermented foods and beverages. In: Satyanarayana T, Kunze G, editors. Yeast Biotechnology.

Springer; 2009. pp. 169-198

uvarum y su importancia en la producción de bebidas fermentadas. Revista de la Sociedad Venezolana de

Microbiología. 2011;31:57-63

Biología. 2011;18(3):325-334

Netherlands: Diversity and Applications.

[11] Estela Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de Hanseniaspora

[12] Estela Escalante W, Rychtera M, Melzoch K, Quillama Polo E, Hatta Sakoda B. Study of the fermentative activity of Hansenula anomala and production of chemical compounds of sensory importance. Revista Peruana de

[13] Estela-Escalante W, Rychtera M, Melzoch K, Hatta-Sakoda B, et al. Actividad fermentativa de

Saccharomycodes ludwigii y evaluación de la síntesis de compuestos de importancia sensorial durante la fermentación de jugo de manzana. TIP Revista Especializada en Ciencias Químico-Biológicas. 2011;14(1):12-23

[14] Estela-Escalante WD, Rychtera M, Melzoch K, Guerrero-Ochoa MR. Influence of aeration in the fermentative activity of Kloeckera apiculata during fermentation of apple juice. Acta Biológica Colombiana. 2012;

17(2):309-321

Methods. 2016;121:50-58

fermentative flora of Malbec wine from Mendoza (Argentina) during two consecutive years. Food Microbiology.

[2] Clavijo A, Calderón IL, Paneque P. Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region. International Journal of Food Microbiology. 2010;143:241-245

[3] Barnett JA, Payne RW, Yarrow D.

Identification. 2nd ed. Cambridge, UK: Cambridge University Press; 1990

[4] Ciani M, Maccarelli F. Oenological properties of non-Saccharomyces yeasts associated with wine-making. World

[5] Loureiro V, Malfeito-Ferreira M. Spoilage activities of Dekkera/ Brettanomyces spp. In: Blackburn C, editor. Food Spoilage Microorganisms. Cambridge: Woodhead Publishers;

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[21] Estela-Escalante WD, Moscosa-Santillán M, González-Ramírez JE, Rosales-Mendoza S. Evaluation of the potential production of ethanol by Candida zemplinina yeast with regard to beer fermentation. Journal of the American Society of Brewing Chemists. 2017;75(2):130-135

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[23] Cordero-Bueso G, Esteve-Zarzoso B, Cabellos J, Gil-Díaz M, Arroyo T. Biotechnological potential of non-Saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv. L.). European Food Research and Technology. 2013;236(1): 193-207. DOI: 10.1007/s00217-012- 1874-9

[24] Quiros M, Rojas V, Gonzalez R, Morales P. Selection of non-Saccharomyces yeast strains for reducing alcohol levels in wine by sugar respiration. International Journal of Food Microbiology. 2014;181:85-91

[25] Contreras AC, Hidalgo PA, Henschke PJ, Chambers C, Curtin C, Varela C. Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine. Applied and Environmental Microbiology. 2015;80: 1670-1678

[26] Ciani M, Capece A, Comitini F, Canonico L, Siesto G, Romano P. Yeast interactions in inoculated wine fermentation. Frontiers in Microbiology. 2016;7(555)

[27] Gobbi M, Comitini F, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine. Food Microbiology. 2013;33:271-281. DOI: 10.1016/j.fm.2012.10.004

[28] Loira I, Vejarano R, Banuelos MA, Morata A, Tesfaye W, Uthurry C, et al. Influence of sequential fermentation with Torulaspora delbrueckii and Saccharomyces cerevisiae on wine quality. LWT- Food Science and Technology. 2014;59:915-922

[29] Canonico L, Agarbati A, Comitini F, Ciani M. Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content. Food Microbiology. 2016;56:45-51. DOI: 10.1016/j.fm. 2015.12.005

[30] Flores CL, Rodríguez C, Petit T, Gancedo C. Carbohydrate and energyyielding metabolism in non-conventional yeasts. FEMS Microbiology Reviews. 2000;24:507-529

[31] Puligundla P, Smogrovicova D, Obulam VSR, Ko S. Very high gravity (VHG) ethanolic brewing and fermentation: A research update. Journal of Industrial Microbiology & Biotechnology. 2011;38:1133-1144. DOI: 10.1007/s10295-011-0999-3

[32] Prior BA, Hohmann S. Glycerol production and osmoregulation. In: Zimmermann FK, Entian KD, editors. Yeast sugar metabolism. Lancaster: Technomic Publishing; 1997. pp. 313-337

[33] Steel CC, Grbin PR, Nichol AW. The pentose phosphate pathway in the yeasts Saccharomyces cerevisiae and Kloeckera apiculata, an exercise in comparative metabolism for food and wine science students. Biochemistry and Molecular Biology Education. 2001;29: 245-249

[34] Boudarel MJ. Contribution ál étude de la fermentation alcoolique á partir de jus de Betteraves avec. Saccharomyces cerevisiae [Thèse de Doctorat]. Université de Dijon, Francia; 1984

[35] Buttke TM, Jones SD, Bloch K. Effect of sterol side chains on growth and membrane fatty acid composition of Saccharomyces cerevisiae. Journal of Bacteriology. 1980;144(1):124-130

[44] Nordstrom K. Possible control of volatile ester formation in brewing. In: Proc. Eur. Brew. Conv. 10th, Stockholm.

DOI: http://dx.doi.org/10.5772/intechopen.81868

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[52] Beltran G, Torija MJ, Novo M, Ferrer N, Poblet M, Guillamon JM, et al. Analysis of yeast populations during alcoholic fermentation: A six year follow-up study. Systematic and Applied Microbiology. 2002;25(2):

[53] Englezos V, Rantsiou K, Torchio F, Rolle L, Gerbi V, Cocolin L. Exploitation

Starmerella bacillaris (synonym Candida zemplinina) in wine fermentation: Physiological and molecular

characterizations. International Journal of Food Microbiology. 2015;199:33-40

[54] Zara G, Mannazzu I, Del Caro A, Budroni M, Pinna MB, Murru M, et al. Wine quality improvement through the combined utilisation of yeast hulls and Candida zemplinina/Saccharomyces cerevisiae mixed starter cultures. Australian Journal of Grape and Wine

[55] Mestre MV, Maturano YP, Combina M, Mercado LA, Toro ME, Vazquez F. Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: A strategy to obtain wines with reduced ethanol content. FEMS Yeast Research. 2017;17(2):1-10.

[56] Rantsiou K, Englezos V, Torchio F, Risse PA, Cravero F, Gerbi F, et al. Modeling of the fermentation behavior of Starmerella bacillaris. American Journal of Enology and Viticulture.

[57] Englezos V, Torchio F, Cravero F, Marengo F, Giacosa S, Gerbi V, et al. Aroma profile and composition of Barbera wines obtained by mixed fermentations of Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae. LWT- Food Science and Technology. 2016;73:567-575

[58] Balli D, Flari V, Sakellaraki E, Schoina V, Iconomopoulou M,

Research. 2014;20:199-207

DOI: 10.1093/femsyr/fox010

2017;68:378-385

of the non-Saccharomyces yeast

287-293

[45] Yoshioka K, Hashimoto N. Ester formation by alcohol acetyltransferase from brewers' yeast. Agricultural and Biological Chemistry. 1981;45(10):

[46] Lilly M, Lambrechts MG, Pretorius IS. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Applied and Environmental Microbiology. 2000;

[47] Rojas V, Gil JV, Manzanares P, Gavara R, Piñaga F, Flors A.

[48] Soden A, Francis IL, Oakey H, Henschke PA. Effects of co-

Research. 2000;6:21-30

2002;38:319-324

2001;79(3):345-352

123

fermentation with Candida stellata and Saccharomyces cerevisiae on the aroma and composition of chardonnay wine. Australian Journal of Grape and Wine

[49] Zohre DE, Erten H. The influence of

Kloeckera apiculata and Candida pulcherrima yeasts on wine

fermentation. Process Biochemistry.

[50] Tofalo R, Schirone M, Torriani S, Rantsiou K, Cocolin L, Perpetuini G, et al. Diversity of Candida zemplinina strains from grapes and Italian wines. Food Microbiology. 2012;29:18-26

[51] Torija MJ, Rozes N, Poblet M, Guillamon JM, Mas A. Yeast population dynamics in spontaneous fermentations comparison between two different wine-producing areas over a period of three years. Antonie Van Leeuwenhoek.

Measurement of alcohol acetyltransferase and ester hydrolase activities in yeast extracts. Enzyme and Microbial Technology. 2002;30(2):224-230. DOI: 10.1016/S0141-0229(01)00483-5

1965. pp. 195-208

2183-2190

66(2):744-753

[36] Gibson BR. 125th anniversary review: Improvement of higher gravity brewery fermentation via wort enrichment and supplementation. Journal of the Institute of Brewing. 2011;117(3):268-284. DOI: 10.1002/ j.2050-0416.2011.tb00472.x

[37] Visser W, Scheffers WA, Batenburg-Van Der Vegte WH, Van Dijken JP. Oxygen requirements of yeasts. Applied and Environmental Microbiology. 1990;56(12):3785-3792

[38] González R, Quiros M, Morales P. Yeast respiration of sugars by non-Saccharomyces yeast species: A promising and barely explored approach to lowering alcohol content of wines. Trends in Food Science and Technology. 2013;29(1):55-61. DOI: 10.1016/j.tifs. 2012.06.015

[39] Rodrigues AJ, Raimbourg T, Gonzalez R, Morales P. Environmental factors influencing the efficacy of different yeast strains for alcohol level reduction in wine by respiration. LWT-Food Science and Technology. 2016;65: 1038-1043

[40] Romano P, Suzzi G, Comi G, Zironi R. Higher alcohol and acetic acid production by apiculate wine yeasts. The Journal of Applied Bacteriology. 1992;73:126-130

[41] Vidrih R, Hribar J. Synthesis of higher alcohols during cider processing. Food Chemistry. 1999;67:287-294

[42] Ehrlich F. Uber eine Methode zur spaltung racemischer aminosauren mittels Hefe. Biochemische Zeitschrift. 1906;1:8

[43] Webb AD, Ingraha JL. Fusel oil. Advances in Applied Microbiology. 1963;5:317-353

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

[44] Nordstrom K. Possible control of volatile ester formation in brewing. In: Proc. Eur. Brew. Conv. 10th, Stockholm. 1965. pp. 195-208

Influence of sequential fermentation with Torulaspora delbrueckii and

2014;59:915-922

2015.12.005

2000;24:507-529

pp. 313-337

245-249

122

Saccharomyces cerevisiae on wine quality. LWT- Food Science and Technology.

Frontiers and New Trends in the Science of Fermented Food and Beverages

Saccharomyces cerevisiae. Journal of Bacteriology. 1980;144(1):124-130

[36] Gibson BR. 125th anniversary review: Improvement of higher gravity

brewery fermentation via wort enrichment and supplementation. Journal of the Institute of Brewing. 2011;117(3):268-284. DOI: 10.1002/

j.2050-0416.2011.tb00472.x

[37] Visser W, Scheffers WA, Batenburg-Van Der Vegte WH,

2012.06.015

1038-1043

1992;73:126-130

1906;1:8

1963;5:317-353

Van Dijken JP. Oxygen requirements of yeasts. Applied and Environmental Microbiology. 1990;56(12):3785-3792

[38] González R, Quiros M, Morales P. Yeast respiration of sugars by non-Saccharomyces yeast species: A

promising and barely explored approach to lowering alcohol content of wines. Trends in Food Science and Technology. 2013;29(1):55-61. DOI: 10.1016/j.tifs.

[40] Romano P, Suzzi G, Comi G, Zironi R. Higher alcohol and acetic acid production by apiculate wine yeasts. The Journal of Applied Bacteriology.

[41] Vidrih R, Hribar J. Synthesis of higher alcohols during cider processing. Food Chemistry. 1999;67:287-294

[42] Ehrlich F. Uber eine Methode zur spaltung racemischer aminosauren mittels Hefe. Biochemische Zeitschrift.

[43] Webb AD, Ingraha JL. Fusel oil. Advances in Applied Microbiology.

[39] Rodrigues AJ, Raimbourg T, Gonzalez R, Morales P. Environmental factors influencing the efficacy of different yeast strains for alcohol level reduction in wine by respiration. LWT-Food Science and Technology. 2016;65:

[29] Canonico L, Agarbati A, Comitini F, Ciani M. Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content. Food Microbiology. 2016;56:45-51. DOI: 10.1016/j.fm.

[30] Flores CL, Rodríguez C, Petit T, Gancedo C. Carbohydrate and energyyielding metabolism in non-conventional yeasts. FEMS Microbiology Reviews.

[31] Puligundla P, Smogrovicova D, Obulam VSR, Ko S. Very high gravity

[32] Prior BA, Hohmann S. Glycerol production and osmoregulation. In: Zimmermann FK, Entian KD, editors. Yeast sugar metabolism. Lancaster: Technomic Publishing; 1997.

[33] Steel CC, Grbin PR, Nichol AW. The pentose phosphate pathway in the yeasts Saccharomyces cerevisiae and Kloeckera apiculata, an exercise in comparative metabolism for food and wine science students. Biochemistry and Molecular Biology Education. 2001;29:

[34] Boudarel MJ. Contribution ál étude de la fermentation alcoolique á partir de jus de Betteraves avec. Saccharomyces cerevisiae [Thèse de Doctorat]. Université de Dijon, Francia; 1984

[35] Buttke TM, Jones SD, Bloch K. Effect of sterol side chains on growth and membrane fatty acid composition of

(VHG) ethanolic brewing and fermentation: A research update. Journal of Industrial Microbiology & Biotechnology. 2011;38:1133-1144. DOI:

10.1007/s10295-011-0999-3

[45] Yoshioka K, Hashimoto N. Ester formation by alcohol acetyltransferase from brewers' yeast. Agricultural and Biological Chemistry. 1981;45(10): 2183-2190

[46] Lilly M, Lambrechts MG, Pretorius IS. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Applied and Environmental Microbiology. 2000; 66(2):744-753

[47] Rojas V, Gil JV, Manzanares P, Gavara R, Piñaga F, Flors A. Measurement of alcohol acetyltransferase and ester hydrolase activities in yeast extracts. Enzyme and Microbial Technology. 2002;30(2):224-230. DOI: 10.1016/S0141-0229(01)00483-5

[48] Soden A, Francis IL, Oakey H, Henschke PA. Effects of cofermentation with Candida stellata and Saccharomyces cerevisiae on the aroma and composition of chardonnay wine. Australian Journal of Grape and Wine Research. 2000;6:21-30

[49] Zohre DE, Erten H. The influence of Kloeckera apiculata and Candida pulcherrima yeasts on wine fermentation. Process Biochemistry. 2002;38:319-324

[50] Tofalo R, Schirone M, Torriani S, Rantsiou K, Cocolin L, Perpetuini G, et al. Diversity of Candida zemplinina strains from grapes and Italian wines. Food Microbiology. 2012;29:18-26

[51] Torija MJ, Rozes N, Poblet M, Guillamon JM, Mas A. Yeast population dynamics in spontaneous fermentations comparison between two different wine-producing areas over a period of three years. Antonie Van Leeuwenhoek. 2001;79(3):345-352

[52] Beltran G, Torija MJ, Novo M, Ferrer N, Poblet M, Guillamon JM, et al. Analysis of yeast populations during alcoholic fermentation: A six year follow-up study. Systematic and Applied Microbiology. 2002;25(2): 287-293

[53] Englezos V, Rantsiou K, Torchio F, Rolle L, Gerbi V, Cocolin L. Exploitation of the non-Saccharomyces yeast Starmerella bacillaris (synonym Candida zemplinina) in wine fermentation: Physiological and molecular characterizations. International Journal of Food Microbiology. 2015;199:33-40

[54] Zara G, Mannazzu I, Del Caro A, Budroni M, Pinna MB, Murru M, et al. Wine quality improvement through the combined utilisation of yeast hulls and Candida zemplinina/Saccharomyces cerevisiae mixed starter cultures. Australian Journal of Grape and Wine Research. 2014;20:199-207

[55] Mestre MV, Maturano YP, Combina M, Mercado LA, Toro ME, Vazquez F. Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: A strategy to obtain wines with reduced ethanol content. FEMS Yeast Research. 2017;17(2):1-10. DOI: 10.1093/femsyr/fox010

[56] Rantsiou K, Englezos V, Torchio F, Risse PA, Cravero F, Gerbi F, et al. Modeling of the fermentation behavior of Starmerella bacillaris. American Journal of Enology and Viticulture. 2017;68:378-385

[57] Englezos V, Torchio F, Cravero F, Marengo F, Giacosa S, Gerbi V, et al. Aroma profile and composition of Barbera wines obtained by mixed fermentations of Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae. LWT- Food Science and Technology. 2016;73:567-575

[58] Balli D, Flari V, Sakellaraki E, Schoina V, Iconomopoulou M,

Bekatorou A, et al. Effect of yeast cell immobilization and temperature on glycerol content in alcoholic fermentation with respect to wine making. Process Biochemistry. 2003;39: 499-506

[59] Suarez-Valles B, Pando Bedrinana R, Ferandez Tasco N, Querol Simon A, Rodriguez Madrera R. Yeast species associated with the spontaneous fermentation of cider. Food Microbiology. 2007;24:25-31

[60] Zott K, Miot-Sertier C, Claisse O, Lonvaud-Funel A, Masneufpomarede I. Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking. International Journal of Food Microbiology. 2008;125: 197-203

[61] Di Maro E, Ercolini D, Coppola S. Yeast dynamics during spontaneous wine fermentation of the Catalanesca grape. International Journal of Food Microbiology. 2007;117(2):201-210. DOI: 10.1016/j.ijfoodmicro.2007.04.007

[62] Granchi L, Ganucci D, Messini A, Vincenzini M. Oenological properties of Hanseniaspora osmophila and Kloeckera corticis from wines produced by spontaneous fermentations of normal and dried grapes. FEMS Yeast Research. 2002;(2):403-407

[63] Plata C, Millan C, Mauricio JC, Ortega JM. Formation of ethyl acetate and isoamyl acetate by various species of wine yeasts. Food Microbiology. 2003;20:217-224. DOI: 10.1016/ S0740-0020(02)00101-6

[64] Caridi A, Tini V. Caratteristiche enologiche di Hanseniaspora guilliermondii. Vini d'Italia. 1991;23: 51-57

[65] Maturano YP, Mestre MV, Esteve-Zarzoso B, Nally MC, Lerena MC, Toro ME, et al. Yeast population dynamics during prefermentative cold soak of

cabernet sauvignon and Malbec wines. International Journal of Food Microbiology. 2015;199:23-32. DOI: 10.1016/j.ijfoodmicro.2015.01.005

traditional spontaneously fermented lambic beer. PLoS One. 2014;9(4):1-13. DOI: 10.1371/journal.pone.0095384

DOI: http://dx.doi.org/10.5772/intechopen.81868

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages

[74] Vanderhaegen B, Neven H, Coghe S, Verstrepen KJ, Derdelinckx G, Verachtert H. Bioflavoring and beer refermentation. Applied Microbiology and Biotechnology. 2003;62:140-150

[75] Verstrepen KJ, Derdelinckx G, Dufour JP, Winderickx J, Thevelein JM, Pretorius IS, et al. Flavor-active esters: Adding fruitiness to beer. Journal of Bioscience and Bioengineering. 2003;96: 110-118. DOI: 10.1263/jbb.96.110

[76] Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiology. 2011;28:

[77] Kell J. What You Didn't Know About The Boom In Craft Beer. 2016. Available from: http://fortune.com/ 2016/03/22/craft-beer-sales-rise-2015/.

[78] Brewers-Association. National beer sales and production data. The New Brewer. 2017. Available from: https://

statistics/national-beer-sales production

[79] Johnson E. Biotechnology of non-Saccharomyces yeasts-the ascomycetes.

Biotechnology. 2013;97(2):503-517. DOI: 10.1007/s00253-012-4497-y

[Accessed: 2018-07-20]

www.brewersassociation.org/

data/. [Accessed: 2018-07-18]

Applied Microbiology and

125

873-882

[66] Romano A, Perello MC, Lonvaud-Funel A, Sicard G, de Revel G. Sensory and analytical re-evaluation of "Brett character". Food Chemistry. 2009;114: 15-19

[67] Suarez R, Suárez-Lepe JA, Morata A, Calderón F. The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: A review. Food Chemistry. 2007;102:10-21

[68] Martens H, Iserentant D, Verachtert H. Microbiological aspects of a mixed yeast-bacterial fermentation in the production of a special Belgian acidic ale. Journal of the Institute of Brewing. 1997;103:85-91

[69] Morrissey WF, Davenport B, Querol A, Dobson ADW. The role of indigenous yeasts in traditional Irish cider fermentations. Journal of Applied Microbiology. 2004;97:647-655

[70] Teoh AL, Heard G, Cox J. Yeast ecology of Kombucha fermentation. International Journal of Food Microbiology. 2004;95:119-126

[71] Verachtert H, Derdelinckx G. Belgian acidic beers: Daily reminiscences of the past. Cerevisia. 2014;38(4):121-128. DOI: 10.1016/j. cervis.2014.04.002

[72] Wijsman MR, van Dijken JP, van Kleeff BH, Scheffers WA. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie Van Leeuwenhoek. 1984;50: 183-192

[73] Spitaels F, Wieme AD, Janssens M, Aerts M, Daniel HM, Van Landschoot A, et al. The microbial diversity of

Perspectives and Uses of Non-Saccharomyces Yeasts in Fermented Beverages DOI: http://dx.doi.org/10.5772/intechopen.81868

traditional spontaneously fermented lambic beer. PLoS One. 2014;9(4):1-13. DOI: 10.1371/journal.pone.0095384

Bekatorou A, et al. Effect of yeast cell immobilization and temperature on

Frontiers and New Trends in the Science of Fermented Food and Beverages

cabernet sauvignon and Malbec wines.

[66] Romano A, Perello MC, Lonvaud-Funel A, Sicard G, de Revel G. Sensory and analytical re-evaluation of "Brett character". Food Chemistry. 2009;114:

[67] Suarez R, Suárez-Lepe JA, Morata A, Calderón F. The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: A review. Food Chemistry. 2007;102:10-21

[68] Martens H, Iserentant D, Verachtert H. Microbiological aspects of a mixed yeast-bacterial fermentation in the production of a special Belgian acidic ale. Journal of the Institute of Brewing.

[69] Morrissey WF, Davenport B, Querol A, Dobson ADW. The role of indigenous

yeasts in traditional Irish cider fermentations. Journal of Applied Microbiology. 2004;97:647-655

[70] Teoh AL, Heard G, Cox J. Yeast ecology of Kombucha fermentation. International Journal of Food Microbiology. 2004;95:119-126

[71] Verachtert H, Derdelinckx G. Belgian acidic beers: Daily

cervis.2014.04.002

183-192

reminiscences of the past. Cerevisia. 2014;38(4):121-128. DOI: 10.1016/j.

[72] Wijsman MR, van Dijken JP, van Kleeff BH, Scheffers WA. Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect). Antonie Van Leeuwenhoek. 1984;50:

[73] Spitaels F, Wieme AD, Janssens M, Aerts M, Daniel HM, Van Landschoot A,

et al. The microbial diversity of

International Journal of Food Microbiology. 2015;199:23-32. DOI: 10.1016/j.ijfoodmicro.2015.01.005

15-19

1997;103:85-91

[59] Suarez-Valles B, Pando Bedrinana R, Ferandez Tasco N, Querol Simon A, Rodriguez Madrera R. Yeast species associated with the spontaneous fermentation of cider. Food Microbiology. 2007;24:25-31

[60] Zott K, Miot-Sertier C, Claisse O, Lonvaud-Funel A, Masneufpomarede I.

[61] Di Maro E, Ercolini D, Coppola S. Yeast dynamics during spontaneous wine fermentation of the Catalanesca grape. International Journal of Food Microbiology. 2007;117(2):201-210. DOI: 10.1016/j.ijfoodmicro.2007.04.007

[62] Granchi L, Ganucci D, Messini A, Vincenzini M. Oenological properties of Hanseniaspora osmophila and Kloeckera corticis from wines produced by spontaneous fermentations of normal and dried grapes. FEMS Yeast Research.

[63] Plata C, Millan C, Mauricio JC, Ortega JM. Formation of ethyl acetate and isoamyl acetate by various species of wine yeasts. Food Microbiology. 2003;20:217-224. DOI: 10.1016/ S0740-0020(02)00101-6

[64] Caridi A, Tini V. Caratteristiche

guilliermondii. Vini d'Italia. 1991;23:

[65] Maturano YP, Mestre MV, Esteve-Zarzoso B, Nally MC, Lerena MC, Toro ME, et al. Yeast population dynamics during prefermentative cold soak of

enologiche di Hanseniaspora

51-57

124

Dynamics and diversity of non-Saccharomyces yeasts during the early stages in winemaking. International Journal of Food Microbiology. 2008;125:

glycerol content in alcoholic fermentation with respect to wine making. Process Biochemistry. 2003;39:

499-506

197-203

2002;(2):403-407

[74] Vanderhaegen B, Neven H, Coghe S, Verstrepen KJ, Derdelinckx G, Verachtert H. Bioflavoring and beer refermentation. Applied Microbiology and Biotechnology. 2003;62:140-150

[75] Verstrepen KJ, Derdelinckx G, Dufour JP, Winderickx J, Thevelein JM, Pretorius IS, et al. Flavor-active esters: Adding fruitiness to beer. Journal of Bioscience and Bioengineering. 2003;96: 110-118. DOI: 10.1263/jbb.96.110

[76] Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, et al. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiology. 2011;28: 873-882

[77] Kell J. What You Didn't Know About The Boom In Craft Beer. 2016. Available from: http://fortune.com/ 2016/03/22/craft-beer-sales-rise-2015/. [Accessed: 2018-07-20]

[78] Brewers-Association. National beer sales and production data. The New Brewer. 2017. Available from: https:// www.brewersassociation.org/ statistics/national-beer-sales production data/. [Accessed: 2018-07-18]

[79] Johnson E. Biotechnology of non-Saccharomyces yeasts-the ascomycetes. Applied Microbiology and Biotechnology. 2013;97(2):503-517. DOI: 10.1007/s00253-012-4497-y

**127**

**Chapter 8**

**Abstract**

*Torulaspora delbrueckii*

product differ greatly between recipes.

**1. Introduction**

*Torulaspora delbrueckii*: Towards

Innovating in the Legendary

*Antonio Garcia-Triana and Rosa Lidia Solís-Oviedo*

Baking and brewing are among the oldest bioprocesses refined by human societ-

**Keywords:** alcoholic beverages, beer production, baking industry, brewing industry,

Bread and beer are among the oldest foods in the human history. The consumption of both of these fermented products has been rooted as a basic human food, and at the present time, these are still among the most consumed foods around the world. The ubiquity of their production allowed a diversification and development of refined, artisan techniques, which currently comprises innumerable recipes [1, 2]. All the recipes include essentially the same basic ingredients such as cereals, yeast, and water. However, the organoleptic properties (aroma, flavours, etc.) of the final

Since early times, both cereals and water were identified as fundamental ingredients for the preparation of beer or bread. Despite the fact that these ingredients have been recognised as essentials for centuries, the experimental approaches developed by Pasteur during the mid-nineteenth century revealed the existence of a third element much more essential to the fermentation process: yeast. The fermentation performed by yeast is undoubtedly the oldest and the largest biotechnology

ies. Both fermentative processes have successfully used domesticated strains of *Saccharomyces cerevisiae* in their process as the biocatalyst throughout their evolution. However, the dominance of *S. cerevisiae* has limited the capability for diversification of many organoleptic properties of the final product, such as aroma and flavours. The use of non-*Saccharomyces* yeasts can be an enormous source of opportunities for innovation in both fermentative processes. *Torulaspora delbrueckii* is a ubiquitous yeast species, and numerous strains have been isolated from many different bioprocesses. The strains of *T. delbrueckii*, once considered microbial contamination, have recently shown several advantages over *S. cerevisiae* strains, including higher ethanol tolerance; better capabilities to consume wort sugars; higher resistance to hop/pH/osmotic stress; and freeze-thaw resistance, among others. This chapter aims to present a comprehensive review of frontier research on *T. delbrueckii* regarding its

*Ángel De La Cruz Pech-Canul, David Ortega,*

potential and prospects for the baking and brewing industries.

Baking and Brewing Industries

## **Chapter 8**