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Cuban Journal of Agricultural Science

Print version ISSN 0864-0408On-line version ISSN 2079-3480

Cuban J. Agric. Sci. vol.54 no.4 Mayabeque Oct.-Dec. 2020  Epub Dec 01, 2020


Review Article

Development of yeast additives for feeding ruminants in Cuba

1Instituto de Ciencia Animal, Carretera Central km 47 1/2, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba

2Universidad Autónoma de Chihuahua, México


The information obtained in the Institute of Animal Science of Cuba about the effect of additives with viable yeasts for ruminants is presented. Institutions from Mexico, Colombia and Brazil helped to gather all this information. The document is structured as follows: 1) effect of the yeast Saccharomyces cerevisiae L25/7/13 on ruminal fermentation and milk production, 2) isolation, characterization and molecular identification of yeast strains from the ruminal ecosystem under the edaphoclimatic conditions of Cuba, 3) Effect of Levica-25 (Candida tropicalis) strain on ruminal microbial fermentation of animals that consume fibrous diets and 4) Evaluation of factors affecting the use of yeasts as microbial additives for ruminants (strain, culture medium, inclusion level and diet). This review is a valuable contribution, mainly intended for specialists in nutrition biochemistry, who project the use of viable yeasts as activating additives for ruminal fermentation.

Key words:  ruminal activators; Saccharomyces; Candida; Pichia

In animal feed, the use of yeasts as microbial additives activating ruminal fermentation contributes to improvement health and to a better use of food, allowing the increase of productive yields and, consequently, availability of milk and meat destined for population (Di Francia et al. 2008 and Bruno et al. 2009). Among the most described actions of yeasts are the stimulation of the number and activity of ruminal bacteria, mainly cellulolytic (Kumar et al. 2013 and Moya et al. 2017) and, therefore, fiber degradability (Elghandour et al. 2014). Increases of fungi (Mao et al. 2013), protozoa (Kumar et al. 2013) and acetogenic bacteria (Hristov et al. 2013) populations were also observed, as well as it is stated that they can reduce the concentration of lactic acid (Mohammed et al. 2017) and the production of short chain fatty acids (SCFA) (Thurne et al. 2009).

Since the last decade of the last century, available scientific references demonstrate the use of several additive products that use commercial yeast strains, mainly of S. cerevisiae species, which is defined as one of the most promising microorganisms (Newbold et al. 1998). However, other species have been studied recently, which have also demonstrated a stimulating effect on ruminal fermentation of different foods (Castillo et al. 2016 and Fernandes et al. 2019).

There are different hypotheses about the action mechanisms of yeasts to exert their effects on the digestive processes of ruminants (Salazar et al. 2016), but it was also confirmed that the response to the inclusion of yeasts can vary due to multiple factors such as diet, species or strain used, dose and even the animal (Fonty and Chaucheyras 2006). For all the above, it is considered necessary to carefully select the microbial additives to be used, according to the specific characteristics of each production system.

For the above reasons, in Cuba, the Institute of Animal Science (ICA, initials in Spanish) conducted research aimed at obtaining additives with yeasts that have a stimulating effect on ruminal fermentation of diets with high fiber content, as one of the ways described to manipulate ruminal microbial fermentation (Galindo and Marrero 2005). Thus, studies began with S. cerevisiae L25/7/13 strain, which is used in all distilleries for alcohol production in Cuba and as a protein source for animal feed (Solano et al. 2001 and Carro et al. 2006). Subsequently, researches with other types of yeast, isolated from the ruminal ecosystem, were widened under edaphoclimatic conditions of the country and their potentialities under in vitro and in vivo conditions were studied.

The objective of the following review was to present the main results of the characterization and evaluation of yeasts as additive candidates for ruminant feeding in Cuba.


Studies were conducted with S. cerevisiae L25/7/13 strain from the collection of the Instituto Cubano de Derivados de la Caña de Azúcar (ICIDCA) in order to study its possible activating action of ruminal fermentation. These were carried out in Cuba and Colombia and incubation techniques were used in fermentation tubes and gas production, respectively, with the use of star grass (Cynodon nlemfuensis) as substrate. The maximum growth time of this strain and the effect of inclusion (at a rate of 20% of total incubation volume) of its live cells and the product of its metabolism were determined in the microbial population and some ruminal fermentative indicators (Marrero et al. 2006 b and 2010).

As results, S. cerevisiae L25/7/13, in its culture medium, favored the development of cellulolytic bacteria and total populations (table 1) and fungi. However, no effect was found on protozoan population. This increase in microbial populations generated a positive response on in vitro fiber degradation rate, and, as a consequence, on accumulated gas production (figure 1). However, the inclusion of yeast pellets showed a positive effect with the use of DM fraction and not in the NDF. Yeast culture supernatant caused deleterious effects on all response variables.

Table 1 Effect of the inclusion of S. cerevisiae L25/7/13 on cellulolytic bacteria population (103 cfu mL-1) according to Marrero et al. (2006 b) 

Hours Treatments SE ± Sign
Control without yeast Culture medium Culture of S. cerevisiae Pellet Supernatant
0 0.28ab (2.12) 0.31abc (2.55) 0.27a (1.60) 0.41cde (7.42) 0.39bcd (4.90) 0.03***
4 0.36abcd (4.07) 0.40cd (6.37) 0.56f (29.12) 0.52ef (17.97) 0.46de (15.70)
8 0.32abc (2.85) 0.33abc (4.15) 0.58f (40.12) 0.57f (31.32) 0.40cd (6.02)
12 0.26a (2.45) 0.32abc (2.95) 0.57f (33.50) 0.51ef (17.32) 0.33abc (3.00)

Data transformed according to Ln X. Original means between parentheses

abcdefDifferent letters differ at p<0.05 (Duncan 1955) *** P<0.001

Figure 1 Effect of inclusion of S.cerevisiae L25/7/13 on accumulated gas production with DM of C. nlemfuensis as substrate modelled with Gompertz (P<0.05) according to Marrero et al. (2010)  

Previously described in vitro studies indicated the activating effect of ruminal fermentation of S. cerevisiae L25/7/13 yeast, associated with the actions of its live cells and the products of its metabolism. In this sense, Montes de Oca et al. (2016) described several action mechanisms for S. cerevisiae, such as consumption of the oxygen present in the rumen that favors the growth and activity of anaerobic microorganisms, pH regulation and limitation of acidosis risks through the control of bacteria populations that produce and consume lactate as well as the fact of providing nutrients such as peptides, vitamins, organic acids and cofactors required by ruminal microorganisms. Results were encouraging, considering that not all strains of S. cerevisiae are capable of stimulating digestion in the rumen as described by Newbold et al. (1998) in the 90s of last century. Therefore, it was verified whether the effects of yeast in the fermentation of fibrous substrates were reflected in the productivity of the animals.

An experiment was carried out in a productive unit of the Institute of Animal Science, under controlled conditions, with the aim of studying the effect of inclusion of S. cerevisiae L25/7/13 on milk production (García et al. 2020). Medium potential commercial Holstein cows were used, which were in the middle stage of lactation and consumed a high fiber diet. They were provided with 10g L-1 of yeast microbial preparation every day. The preparation increased (P <0.01) milk production by 0.7 liters and influenced directly on milk protein and total solids, which indicates a better use of final resources of digestion. These productive responses were in correspondence with the increases found in ruminal populations of yeast, bacteria and cellulolytic fungi.

Studies conducted with S. cerevisiae L25/7/13 strain were very promising for obtaining microbial additives for ruminants. However, many questions remained to be answered. In this sense, it is known that yeasts do not belong to the ruminal ecosystem and their representation in there is mainly due to their presence in the food consumed by animals, which is why they are classified as allochthonous. This knowledge was evident with S. cerevisiae L25/7/13 strain, which was able to remain in the rumen for a certain period of time and stimulate fermentative processes that occur in it. However, it is known (Zoumpopoulou et al. 2018) that adaptation to a certain ecosystem is an important characteristic in the selection of probiotic candidates and the efficacy of selected microorganisms may depend on the original host.

Considering the above concepts, a new field of research was opened in ICA for obtaining additives with yeasts for ruminants. In this sense, it was logical to think that when foods rich in yeasts are offered to an animal for a certain time, there could be some species resistant to the stressful conditions offered by the rumen and, therefore, their action within it could be more effective.


The inclusion of a fermented product that contained populations between 106-108 cfu mL-1 of live yeasts, in the diet of cows, allowed to increase the populations of these microorganisms in the rumen. In this way, a total of 24 strains were isolated, which demonstrated a permanence in the organ for up to 12 hours after the product was ingested by the animal. These strains were characterized by biochemical tests and it was determined that none belonged to Saccharomyces genus. These studies made it possible to propose a new methodology for isolating and characterizing yeast strains present in the ruminal ecosystem (Marrero et al. 2005) that was applied in joint research projects with the Autonomous University of Chihuahua, Mexico (Castillo et al. 2016).

The result of the characterization was verified with karyotyping technique and it was confirmed that the electrophoretic profile of 20 of the isolated strains was different from that of S. cerevisiae. Later, 14 of the strains isolated from the ruminal ecosystem were identified by using polymerase chain reaction (PCR). For this, primers that amplified for D1/D2 region of the larger 26S rDNA subunit were used. Out of the 14 strains under study, seven belong to Pichia genus with a similarity percentage between 88 and 98% and it was also confirmed that six of them belong to Pichia guillermondii species with more than 90% of maximum identity except of strain 17 that had 88%. Five other strains were identified as Issatchenkia orientalis, strain 18 as Rodotorula mucilaginosa, and strain 25 as Candida tropicalis. These strains were named, for the purposes of the Laboratory of the Institute of Animal Science, as Levica plus the isolate number (Marrero et al. 2013).

In another experiment, gas production was studied, in which C. nlemfuensis was used as substrate with the inclusion of 20 mg of DM mL-1 of isolated yeasts. It showed, through a grouping of the values by cluster (table 2), that Levica-25 strain produced the greatest stimulating effect of ruminal fermentation.

Table 2 Accumulated gas production at 24 hours of fermentation in yeast groups isolated from ruminal ecosystem 

Groups Accumulated gas production (mL) SE ( Sign
Group 1 (13 strains) 55.82d 0.85*
Group 2 (3 strains) 38.01b
Group 3 (7 strains) 47.53c
Group 4 (strain 25) 70.83e
Group 5 (control without yeast and S. cerevisiae) 28.90a

abcdeDifferent letters differ p<0.05 (Duncan 1955)

All identified strains were preserved in a specific culture medium for freezing conditions that allows better conservation of the collection for future studies. Viability of these strains was evaluated after six months of cryopreservation at -80 oC, in a malt extract medium with glycerol and the effectiveness of the method was confirmed (Sosa et al. 2017). Yeast collection was registered in GenBank and it is part of the ICA collection in the World Data Center for Microorganisms (WDCM) with registration number 980.

Taking into account the results with Levica-25, another study was carried out that included new biochemical tests and the phylogenetic study due to the associated pathogenicity of Candida tropicalis species. Thus, the sequence obtained from strain 25 was compared with the sequence of C. tropicalis reported in GenBank (HM627137.1) and the phylogenetic tree based on D1/D2 region of the 26S rDNA gene was studied. The analysis showed that Levica-25 strain is a new strain, very close to Candida tropicalis that could present different characteristics regarding pathogenicity referred for this species (Marrero et al. 2011).

Results described in the previous sections were encouraging considering that there were not abundant studies in which strains other than S. cerevisiae were used as additives for ruminants, so studies were continued with Levica-25 strain and S. cerevisiae L25/7/13 was used as a pattern. However, it was known that species and yeast strains, and the inclusion dose, as well as the diet used, have an important influence on physiological response and, therefore, on animal productivity (Enjalbert et al. 1999). For this reason, in parallel, comparative studies of all isolated strains were carried out with the use of different diets and inclusion doses.


A Latin square design was used to study the effect of biological preparations with viable yeasts in the ruminal microbial population and fermentative indicators in cows consuming fibrous diets (Marrero et al. 2006a). For this purpose, preparations of S. cerevisiae L25/7/13 and Levica-25 yeast, alone and combined, in a dose of 10 g/animal/d were included in diets. The inclusion of preparations caused a stimulation of the ruminal microbial population and, especially, of the cellulolytic one. The most marked effect was caused by Levica-25. However, no effects of yeasts were found on the populations of proteolytic bacteria and protozoa in the rumen, nor on pH values and SCFA concentrations.

In a second study, in vitro gas production technique was used to evaluate the inclusion of the same strains at a rate of 20% of total incubation volume, in the fermentation process of C. nlemfuensis with the use of water buffalo rumen liquid (Galindo et al. 2010). The inclusion of microbial preparation with Levica-25 increased in vitro gas production and S. cerevisiae L25/7/13 produced intermediate gas values between the control and Levica-25. Gas production was modified with respect to fermentation time and the highest values were found at 24 h of fermentation. On the other hand, S. cerevisiae increased the population of cellulolytic bacteria by 1.75 times and Levica-25 multiplied the total viable population by 2.25. In addition, both yeasts reduced the populations of methanogens after four hours of fermentation. This was reflected in the ruminal methane concentrations (table 3).

Table 3 Effect of S. cerevisiae L25/7/13 and Levica-25 on in vitro methane production (μL) at 24 h of fermentation, according to Galindo et al. (2010)  

Control S. cerevisiae Levica-25 SE (±) Sig
78.97 c 45.21 b 21.52 a 6.74**
Time, h
4 8 12 24 SE (±) Sig
13.12a 56.54b 59.61b 64.99b 9.32*

a,bMeans with different letters in the same line differ at P < 0.05 (Duncan 1955)

* P < 0.05 **P < 0.01

In other experiments developed at the Facultad de Zootecnia of the Universidad Autónoma de Chihuahua (UACH), Mexico, the effect of yeast inclusion of Candida genus on in vitro ruminal fermentation of different fibrous substrates was studied: oat straw (Avena sativa) and alfalfa (Medicago sativa). In both, the previously described strain Levica-25 (Candida tropicalis) was compared to the so-called Levazoot 15 (Candida norvegensis), also of ruminal origin and belonging to the collection of the Mexican institution (Castillo et al. 2016).

It was possible to verify (figure 2) that both strains stimulated (P <0.0001) ruminal fermentation of the substrates under study. However, Levazoot 15 strain stimulated the fermentation of alfalfa DM by 21.43% above Levica -25 (Marrero et al. 2015). The foregoing corroborated the influence of the strain and diet on the action of yeasts within the rumen and the importance of selecting the correct strain under each production condition. These results laid the foundations for studies of other factors that could influence on the action of yeasts in the rumen.

Figure 2 Effect of the inclusion of Levica-25 and Levazoot 15 strains on accumulated gas production of DM of oat straw (left) and alfalfa (right) P<0.0001, according to Marrero et al. (2015)  


For the study of factors that affect the use of yeasts as microbial additives for ruminants, gas production technique was used and several experiments were carried out. Marrero et al. (2014), in a first study, evaluated the dose of 5 mg of DM mL-1 equivalent to 10 g of DM/animal of yeast cultures, as it is the one that is mostly reported in references. Strains isolated from the ruminal ecosystem were compared with the reference strain S. cerevisiae L25/7/13 and a S. cerevisiae strain isolated from LEVUCELL ®SC product was also included. In addition, a control treatment without yeast and a control with the culture medium without inoculation were included. It was found that all treatments had superior performance to control except in which strain 15 was included (figure 3, table 4). Although Levica-25 strain stimulated gas production, the best-performing treatment was the one that included Levica-27 strain, which belongs to Pichia guillermondii species. Hence, in a second study, the inclusion of two levels (5 and 10 mg of DM mL-1) of it in the ruminal fermentation of corn stubble was evaluated and the best results were obtained when the highest dose of yeast was included.

Figure 3 Accumulated gas production (mL) for 24 hours of in vitro fermentation of C. nlemfuensis. Inclusion dose: 5 mg of DM.mL-1, according to Marrero et al. (2014)  

Table 4 Curve comparison by CIAC test and group formation by Tukey test, according to Marrero et al. (2014)  

C11a C13b C15c C17d C22d C28be C18Bf C18Rf
C23beg C24abe C29ae C33fg C27h CLLScca C25bfg Sc ICIDCAbeg

Different letters represent significant differences at P < 0.05

Recently, a study was carried out under similar in vitro conditions in order to corroborate the effect of diet on the inclusion of different species of yeasts in ruminal fermentation. For this, in addition to C. nlemfuensis, a corn-soy bean supplement was added (Marrero et al. 2020). Gas production technique was used and the evaluated strains are part of the collection of the Institute of Animal Science that previously showed effectiveness as activators of ruminal fermentation. The same dose of the previous study, equivalent to 10 g of DM in an adult bovine, was added and the presence of live cells in them was in the order of 106cel mL-1. The stimulatory effect of yeast addition on gas production was observed with increases that varied from 6% (strain 23) to 37% (strain 28) (figure 4). In this case, although P. guillermondii Levica-27 strain caused increases in gas production, strain 28, which belongs to the same species, achieved better results.

# 70 % of corn, 19 % of soy bean, 2 % of salt and 2 % of minerals

Figure 4 Gas production in 24h of a mixture of 70% of C. nlemfuensis y 30% of a supplement incubated alone (control) or with different yeast strains, according to Marrero et al. 2020  

Finally, the culture medium was considered as a factor that could influence the activating effect of microbial additives. There are specifically formulated media for yeast growth, such as malt extract broth (MEB) and yeast-peptone-glucose (YPG) extract. Previous studies with P. guilliermondii (Levica-27) demonstrated with indirect methods of growth determination (biomass and optical density) that there are differences between both culture media at 16 h. It was found that the highest biomass concentrations were obtained for YPG medium, with values of 3.88 mg mL-1, while it was 2.33 mg mL-1 in MEB. An increase in pH was also observed when the strain was cultivated in MEB medium (Sosa et al. 2015). These results indicated that the culture medium influences on the nature of metabolites produced by the strain, so it is possible that it also affects the ruminal fermentative process, when it is included as an additive in animal feed.

With this background, another study was carried out to evaluate the effect of the cultivation of P. guilliermondii, in different media, on the in vitro fermentation of C. nlemfuensis (Marrero et al. 2016). Gas production technique was used and five treatments were evaluated, including P. guilliermondii strain (Levica-27), grown in the malt extract broth (MEB) and yeast-peptone-glucose (YPG) extract media. The supernatant of each medium was used after centrifugation and resuspension of the pellet of cells in a buffered medium. A control without yeast was also included. Results showed that Levica-27, grown in YPG medium, stimulated the gas production of C. nlemfuensis in a greater proportion (P <0.001) than when it was cultivated in MEB medium. The influence of metabolites produced by yeasts and their stimulating effect on a certain culture medium was confirmed and the importance of selecting the appropriate strains and culture medium for their use as an additive in ruminant diets, according to the food to be used, was confirmed.

It is known that the design of the culture medium is one of the most important tasks in biological technology. According to Winkler (1988), in the total cost of biotechnological products, raw materials can represent between 30 and 80%. Furthermore, the composition of the culture medium has to satisfy all nutritional requirements of the microorganism. Therefore, studies were conducted with the objective of evaluating different culture media that would allow selecting the most appropriate and economical one for obtaining Levica-27 on a larger scale.

With these studies, a more economical culture medium was obtained with national sources with which a higher cell concentration and maximum growth rate were achieved with respect to the commercial medium and, consequently, a lower biomass duplication time (González et al. 2018). This constituted the prelude for additive production and its subsequent scaling and evaluation in animals under production conditions.

In sum, field results of obtaining additives with yeasts for their use in ruminants in Cuba demonstrate its complexity and the influence of multiple factors to be taken into account to achieve good productive results. They also confirm that it is necessary to obtain products that respond to specific production situations, based on the use of medium and low quality fibrous diets as a characteristic of Cuba and tropical countries.


Studies conducted in Cuba with yeasts demonstrate the potential of these microorganisms as activators of ruminal fermentation and their contribution to the use of fibrous diets and milk production.

The methodology for isolation and characterization of yeasts from the ruminal environment and for additive evaluation is available, useful by the Cuban and international scientific community.

There is a collection of autochthonous yeast strains, isolated from the ruminal ecosystem, with potential for use as additive for ruminants that consume fibrous diets and their records are included World Data Center for Microorganisms (WDCM) and Gen Bank

The determining effect of diet, dose, species, strain and culture medium of yeasts in ruminal fermentation is confirmed, which states the importance of selecting the appropriate strains to be used as additive in ruminant diets, in accordance with the food to be use.

It is necessary to accelerate studies to achieve the production technologies for a yeast-based additive and increase animal production with its use.


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Received: September 23, 2020; Accepted: November 11, 2020


Los autores declaran no presentar conflicto de intereses

Los autores declaran presentar contribución igualitaria en la concepción de la investigación, obtención y procesamiento de los datos y redacción del documento

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