Advance and Applications of Active Dry Yeast and Yeast Culture Ruminant Animals

Abstract: Feed-grade active dry yeast and yeast cultures possess the characteristics of improving the production performance of ruminants, enhancing the body's immunity, and being safe without residues. They have been widely applied in production. This paper elaborates on the four aspects of active dry yeast and yeast cultures, namely strain selection, production characteristics, mechanism of action, and application effects, with the aim of providing a reference for the research and development of yeast products in ruminants.

Key words: Feed-grade active dry yeast, Yeast culture, Ruminants, Strain screening, Application effect.

Active dry yeast for feed (ADY) is a yeast cell product obtained by liquid fermentation and ventilation cultivation of active yeast strains, followed by dehydration and drying. Its content is no less than 1.5×1010 CFU/g, and the addition amount in the feed is usually 1-40g/(head·day). Yeast cultures (YC) are products obtained by concentrating and drying after liquid and solid fermentation of active yeast. They are rich in yeast cell walls (containing immune-active polysaccharides, β-glucans, and mannan oligosaccharides), cell solubles (containing small peptides, amino acids, chelated minerals, vitamins, various enzymes, and nucleic acids), extracellular metabolites (containing small peptides, organic acids such as malic acid, fructo-oligosaccharides and mannan-oligosaccharides, etc.) and various nutrients contained in the fermentation medium. Their composition mainly depends on the fermentation strain, the ratio of the culture medium, and the fermentation process conditions. The addition amount in the feed is usually 50-300g/(head·day). Some yeast cultures in some studies refer to solid culture media or liquid yeast cultures with a high number of viable cells. These products have the characteristics of both live yeast and yeast cultures. Yeast-related feed products are diverse, including active dry yeast for feed, yeast cultures, yeast cell walls, yeast hydrolysates, yeast nucleic acids, yeast single-cell proteins, yeast fermented feed, enriched trace element yeast, etc. This article reviews the mechanism of action, application research, and strain selection of active dry yeast for feed and yeast cultures in ruminants, providing a reference for research and application in ruminant feed nutrition.

1.Selection of yeast strains

The variety of yeast species in nature is abundant. Currently, there are approximately 60 different yeast genera, totaling about 500 species, and they are widely distributed in animal feed. Most of these species are neither harmful nor beneficial to humans and animals. Among them, Saccharomyces cerevisiae and Candida utilis are common beneficial bacteria, while Candida species and Cryptococcus species are harmful bacteria. The selection of yeast strains is obviously crucial. This is not only a consideration of the use effect, but also involves the issue of biological safety. The selected strains must be included in the feed additive directory, such as Saccharomyces cerevisiae and Candida utilis. In the production of live yeast and yeast cultures, Saccharomyces cerevisiae is mainly used as the production strain, which has the characteristics of high protein content and large cell volume. While Candida utilis is characterized by its ability to utilize not only hexoses but also a wide range of pentoses, glucuronic acid, etc., and it also has strong disease resistance and a short growth cycle. During the fermentation process, it produces a large number of false hyphae, with a lower colony count than Saccharomyces cerevisiae, and is mainly used for the production of cell protein. Currently, there are relatively few related studies on its application in ruminants, and there are no comparative data on its impact on ruminants compared with Saccharomyces cerevisiae. Bradia yeast is one of the most studied probiotics. It is a variant of Saccharomyces cerevisiae and has the functions of improving intestinal health and preventing and treating diarrhea. It has been widely applied in the field of animal nutrition. Compared with other yeasts, the optimal growth temperature of Bradia yeast is 37℃, and it can tolerate an environment with a pH of 1.5. Studies have shown that Bradia yeast can effectively grow in the intestine and competitively inhibit the proliferation of harmful bacteria, thereby preventing and treating diarrhea. In terms of intestinal immune regulation function, Bradia yeast is superior to other probiotics, such as animal bifidobacterium and Lactobacillus casei.

The growth characteristics and metabolic products of yeast are among the main factors determining whether a product can have efficacy. The main nutrients within yeast cells include amino acids, peptides, carbohydrates, nucleic acids, enzymes, and cofactors. There have been numerous related studies in China on the screening of feed yeast strains, including in vitro fermentation experiments, lactate utilization ability, cellulose production ability, production of nutritional active substances (β-glucans, mannan polysaccharides, organic acids, amino acids, and peptides), high glycerol production, rumen endogenous yeast strain screening, and low gas production. Although it is known that yeast strains cannot effectively colonize in the rumen, some strains can maintain metabolic activity in rumen fluid for up to 48 hours. The optimal pH environment for yeast is 4.5 to 7.0, and the most suitable growth temperature is 28 to 30℃. However, most yeast strains can still grow in an environment with a pH of 3.0, and some yeast strains can tolerate a pH of 1.5. Yeast can also grow normally at 37℃. Although Saccharomyces cerevisiae can survive in a microoxic environment, oxygen is very important for maintaining cell activity. Yeast not only needs oxygen as the terminal electron acceptor for respiration but also requires oxygen as a necessary growth factor for the biosynthesis of cell membrane fatty acids and sterols. Therefore, in anaerobic conditions, yeast proliferation slows down, and its metabolic products also change.

2. Differences between active dry yeast and yeast culture in production

The main evaluation index for active dry yeast is the number of viable bacteria. How to increase the quantity and activity of yeast has always been the focus of product development research. Some scholars have studied the optimization of high-density fermentation and drying processes for feed yeast, resulting in a product with an active cell count of over 200 billion per gram. Sullivan et al.'s research found that the number of viable bacteria in active dry yeast decreases by 90% each month when stored at 40°C; the live yeast added to vitamin premixes or grains maintains its activity without significant reduction when stored at 22°C for 2 weeks, indicating that the use of active dry yeast has certain requirements for the temperature environment. Moist heat conditions are one of the most effective methods for killing microorganisms. The feed granulation process significantly reduces the activity of yeast. Currently, there are also methods to produce special live yeast that is resistant to high temperatures, which can effectively meet the needs of the feed industry for granulation. However, some research has found that although the number of live yeast in the feed decreases from 106 CFU/g to 103 CFU/g after granulation, with a yeast survival rate of less than 1%, it can still exert a good growth-promoting effect.

The production process of yeast culture mainly includes liquid culture, aerobic solid fermentation and anaerobic solid fermentation. Yeast cells rapidly proliferate under aerobic conditions, generating a large amount of cell proteins, yeast polysaccharides, vitamins and other beneficial components. In the anaerobic environment, the metabolic products may be similar to those of live yeast in the anaerobic environment of the rumen and intestinal tract, and thus can also produce similar application effects as active dry yeast. He Jiayun found that the yeast culture product without anaerobic solid fermentation had a reduced effect in controlling pig diarrhea. The high-temperature processing of the feed has no significant effect on the activity of yeast metabolites, so the granulation process does not affect the effect of the yeast culture product.

Some people hold the view that most of the nutrients of yeast are contained within the yeast cells, and the cell wall hinders the digestion and absorption of nutrients by the body. Therefore, the yeast cells must undergo autolysis and lysis. The differences in amino acid and vitamin digestibility and bioavailability between live yeast and dead yeast are still unclear. During the drying process of the feed, the Maillard reaction caused by high temperature will reduce the utilization rate of amino acids and accelerate lipid oxidation, thereby reducing the biological activity of fat-soluble vitamins. Considering that the production of dead yeast products requires a higher drying temperature, the digestibility and bioavailability of live yeast cells may be greater. The application effect of yeast culture is affected by factors such as strain and fermentation process, and the effects of different products vary greatly. For yeast cultures produced by different production processes, the appropriate addition amount, application effect under specific feeding conditions, and its impact on the gastrointestinal microbial flora still need further in-depth research. The solid-state fermentation substrates of yeast culture mainly include corn, soybean meal, bran, miscellaneous meal, distillery residue, molasses, etc. The proportion of each component, pretreatment process, and fermentation process can all have certain effects on the fermentation process. How to increase the content of the main active components has always been the focus of research. The research on the production process of yeast culture mainly focuses on fermentation conditions and yeast autolysis conditions. The solid-state fermentation process is more difficult to control than the liquid-state fermentation process, and it is prone to phenomena such as uneven fermentation, insufficient heat dissipation, uneven moisture, and microbial contamination. How to reasonably and effectively control the fermentation process to ensure the quality and safety of fermentation is always a focus of attention in production.

3. The regulatory mechanisms of active dry yeast and yeast cultures on ruminants

3.1 Mechanisms of Regulation of Rumen Fermentation Function

In the rumen of the lamb, the quantity of live yeast remained close to the initial addition level within approximately 30 hours. It could be detected in the feces 8 hours after feeding, and could no longer be detected 102 hours after intake. Live yeast cannot establish a colonization in the rumen. 17% to 23% of the yeast can still survive after passing through the digestive process of the gastrointestinal tract and may play a role in the intestine. One of the main ways that live yeast cells exert their effects is by reducing the oxygen concentration in the rumen through respiration, thereby maintaining the anaerobic environment of the rumen and promoting the growth of anaerobic bacteria. Anaerobic fungi in the rumen, which play a dominant role in the degradation of fiber substances, can still survive after the digestive process through the gastrointestinal tract. Feeding live yeast to newborn calves can reduce the redox potential (oxygen content) in rumen fluid, increase the colonization speed of microorganisms, and significantly improve the rumen fermentation function. However, Oeztuerk's external culture experiment of rumen fluid found that the stimulation effect of Brade yeast after high-pressure sterilization on rumen microorganisms was not significantly different, indicating that the nutritional components of the yeast cells themselves have certain efficacy. It was found that under high-concentrate substrate conditions, yeast cells compete with lactic acid-producing bacteria (Bovis Streptococcus and lactic acid bacteria) for the utilization of monosaccharides in the rumen and stimulate the growth of lactic acid-utilizing bacteria (such as Escherichia giganteus). The latter can convert lactic acid into butyric acid and propionic acid, thereby reducing the accumulation of lactic acid in the rumen and increasing the pH. Thrune's research found that the total volatile fatty acid content in the rumen of the group with added live yeast showed a downward trend, and the average pH (6.53 ± 0.07) was significantly higher than that of the control group (6.32 ± 0.07), with the maximum and minimum values of rumen pH also significantly higher than those of the control group; under subacute rumen acidosis conditions, the low pH of the rumen fluid after adding yeast was significantly lower than that of the control group. Adding active dry yeast can increase the activity of enzymes related to fiber degradation in the rumen of Qinchuan cattle, including β-glucosidase, carboxymethyl cellulase, and xylanase, and improve the digestibility of acid-insoluble fiber (ADF) and neutral detergent fiber (NDF).

The regulatory mechanism of yeast culture and live yeast on rumen fermentation function is similar. The nutrients contained in them can stimulate the growth of rumen microorganisms, maintain an anaerobic environment, and improve the efficiency of rumen fermentation. Studies have found that yeast culture can enhance rumen fermentation function by improving the growth activity of fiber-degrading bacteria, increase the synthesis of microbial protein (MCP), stimulate the growth of lactic acid-utilizing bacteria, reduce lactic acid accumulation, especially in cases of low feed quality. Experiments have shown that adding yeast culture to the diet can increase the relative abundance of Prevotella brevis, R. flavefaciens, ciliates, and Bifidobacterium spp. In the diets of lactating dairy cows, adding rich glycerol yeast culture can promote gluconeogenesis by up-regulating the activity of phosphoenolpyruvate carboxykinase (PEPCK), the mRNA and protein expression levels of PEPCK-C, and the activity and mRNA expression levels of glucose kinase (GK), thereby improving the blood glucose level of dairy cows and improving the energy balance of dairy cows. It has been reported that adding yeast culture to the ruminant diet can change the hydrogenation metabolic pathway of rumen microorganisms. The linoleic acid, linolenic acid, and oleic acid from the feed source, as well as beneficial biologically hydrogenated intermediate products, will escape complete biohydration in the rumen and be absorbed and utilized by the mammary tissue and muscle tissue, thereby improving the quality of milk and meat. It has been found that yeast culture and yeast cell walls can induce or promote the expression of epithelial cell defensins in ruminant epithelial cells, forming protection for the gastrointestinal mucosa, inhibiting and killing harmful bacteria and regulating the quantity and type of beneficial bacteria to maintain the microecological balance of the gastrointestinal tract. In addition, some researchers have found that yeast culture has the tendency to reduce the ammonia nitrogen concentration in the rumen, reduce fecal ammonia and methane emissions, increase the synthesis yield of microbial protein, and provide more amino acids to the hindgut.

3.2 The mechanism of action on stress resistance and immune regulation in ruminants

Many diseases in animal bodies are caused by oxidative stress. Yeast cells and their metabolites contain a large amount of substances with antioxidant activity. Studies have found that the content of glutathione in yeast autolysis products is relatively high, and they also have high total antioxidant capacity, DPPH free radical scavenging ability, nitric oxide scavenging ability, and hydroxyl radical scavenging ability. They can be used as new natural antioxidants and immune stimulants in functional foods or drugs. Yeast polysaccharides are one of the main active components of yeast, and they can improve the oxidative-reductive state of lymphocytes stimulated by polyhydroxyalkanoates (PHA), reduce oxidative damage caused by heat stress, and thereby regulate the expression of apoptosis-related genes and enhance the adaptability of lymphocytes to heat stress conditions.

Under heat stress conditions, adding live yeast to the diet of lactating cows can increase the yield of energy-adjusted milk (ECM), while the dry matter intake (DMI) remains unaffected. The efficiency of converting dry matter to ECM is increased by 7.4%. Adding live yeast to the cow's diet helps to enhance the expression of epithelial barrier genes and anti-inflammatory-related genes in the rumen and colon, and increases the expression of the antimicrobial peptide gene defensin β1 (DEFB1).

The yeast culture can down-regulate the mRNA expression level of heat shock protein 70 (HSP70) in the peripheral blood lymphocytes of heat-stressed dairy cows, reduce the expression level of apoptosis-related gene Bax-α mRNA, inhibit cell apoptosis; stimulate ConAn-mediated T cell transformation, increase the levels of interleukin-2 (IL-2) and immunoglobulin G (IgG) in the lymphocytes of dairy cows, and enhance the cellular and humoral immunity of heat-stressed dairy cows. Vailati-Riboni et al. found through transcriptomics research that adding yeast culture to the dairy cow diet can increase the expression of cell immune-related genes, epithelial tissue protection-related genes and anti-inflammatory activity-related genes, and effectively enhance the antibacterial ability of mammary cells.

3.3 Impact on Methane Production in Ruminants and Mechanism of Action

During the fermentation process in the gastrointestinal tract of ruminants, a large amount of methane is produced (ruminant rumen fermentation accounts for 87% - 90%, while intestinal fermentation accounts for 10% - 13%). The average daily methane emission of adult beef cattle can reach as high as 161 - 323 grams, causing energy losses of 2% - 12% of the feed intake. Studies have shown that the methane production of ruminants is influenced by various factors such as feed types, antibiotics (such as monensin), and environmental temperature. There is still much controversy regarding the impact of yeast products on ruminant methane production. In vitro studies have shown that when acetic acid-producing bacteria and methane-synthesizing bacteria are co-cultured, hydrogen is mainly used for methane synthesis. After adding live yeast, the utilization rate of hydrogen by acetic acid-producing bacteria significantly increases, and the methane production decreases. In in vitro studies, when different sources of live yeast preparations were added to high-concentrate diets, the methane concentration in each experimental group was lower than that of the control group, but the difference was not significant. Xu Binbin investigated the effect of different doses of active brewer's yeast on methane emissions from beef cattle's intestines. The results showed that adding 6g/(head·day) could significantly reduce methane emissions from beef cattle. In the study by Oh et al., the yeast-based microecological product had no significant effect on methane production in lactating dairy cows. A Meta-analysis discussed the impact of yeast products on methane production in dairy cows and beef cattle, including 6 studies. The analysis results indicated that yeast products as feed additives had no significant effect on reducing methane production in dairy cows and beef cattle. There are significant differences in the experiments on the impact of different yeasts on methane production and methane/DMI, which may be due to the influence of yeast strains, feed composition, and environmental factors. The mechanism of the impact of yeast products on ruminant rumen methane production and its effects are not yet clear, and further in-depth research is needed.

3.4 Impact on Feed Mycotoxins

Mycotoxins are an important class of natural toxins in feed, posing significant risks to animal health and food safety. Regarding the effect of yeast on mycotoxins, Pfliegler et al. and Shetty et al. have provided detailed reviews. Studies have shown that the main active components of yeast cell walls (β-glucans and mannan oligosaccharides) have the effect of improving immune function. They can adsorb the main mycotoxins (Aflatoxin B1, ochratoxin A, T-2 toxin, and zearalenone) in feed in the animal digestive tract, effectively alleviating the adverse effects of toxins on animal health and production. It has been found that adding yeast cell walls or their main active component β-glucans to the diet of dairy goats can effectively adsorb Aflatoxin B1 (AFB1), reduce the content of Aflatoxin M1 (AFM1) in goat milk, and have a positive effect on food safety.

4. Application of Active Dry Yeast and Yeast Culture in Ruminants

4.1 Application in Young Ruminants

Both live yeast and yeast cultures can accelerate the colonization of microorganisms in the digestive system of newborn calves, which is of great significance for their subsequent rumen function, immune system development, and intestinal health [58]. However, the specific effects are related to the age of the calves, the composition of the diet, the management level of feeding, and the yeast strains, etc. [59]. Studies have shown that Saccharomyces cerevisiae can increase the dry matter intake (DMI), feed conversion rate, and growth performance of calves, and also effectively promote rumen development. The effect is most obvious before weaning, which can effectively reduce the diarrhea score, shorten the number of diarrhea days, thereby reducing the frequency of antibiotic treatment during the calf stage and improving growth performance.

Galvão et al. found that supplementing live yeast to calves with low maternal antibody levels can enhance the immunity of the calves and improve their growth performance. Feeding live yeast to calves from 0 to 60 days of age has a certain impact on the uniformity and diversity of the rumen microbiota (excluding archaea), and these effects can last for 32 months. However, the impact on production performance is not significant. Supplementing live yeast to calves from birth helps increase the production of endogenous immunoglobulin A (IgA) in the intestines, thereby reducing the risk of pathogen invasion. In a hot stress environment, adding live Brade yeast to milk can lower the rectal temperature, heart rate, and diarrhea rate of calves.

Feeding yeast cultures can significantly increase the daily weight gain of calves before and after weaning, raise the pH of the rumen fluid of calves, and alleviate weaning stress. Mitchell's research indicates that feeding yeast cultures to weaned calves under different roughage conditions (alfalfa grass, oat grass, and corn silage) can improve the composition of rumen bacteria, but has no significant effect on rumen fluid pH, feed intake, growth performance, or nutrient digestibility. Calves during the postpartum period fed yeast cultures can increase the level of immunoglobulins in colostrum, thereby enhancing the passive immunity level of calves. There are certain differences in the effects of different strains of yeast cultures on animal production performance. Tripathi et al. verified 4 yeast cultures, and the results showed that 3 of the yeast cultures could improve the production performance of lambs, while 1 yeast culture could reduce the production performance. Young ruminants and adult ruminants have significant differences in the digestive system, feed composition, and disease management. To meet the growth characteristics and multiple needs of young ruminants, further research and development of yeast products are needed to enhance the professionalism and targeting of related products.

4.2 Application on Cows

Desnoyer et al. conducted a quantitative Meta-analysis of 110 research papers, 157 experiments and 376 treatments, and found that adding yeast products (including live yeast and yeast cultures) to the ruminant diet increased the average milk yield by 0.8 kg/d. The higher the proportion of concentrated feed and neutral detergent fiber in the diet, the greater the improvement in organic matter digestibility. Poppy et al. also conducted a similar Meta-analysis, and their conclusion was that commercial brewing yeast products increased milk yield by 1.18 kg/d (fat-corrected milk 1.61 kg/d), increased milk fat yield by 0.06 kg/d, increased milk protein yield by 0.03 kg/d, increased DMI in the early lactation period by 0.62 kg/d, and increased DMI in the late lactation period by 0.78 kg/d. From the feeding behavior perspective, after adding live yeast to the diet of dairy cows, the feeding interval was shortened (20.0 min vs. 25.8 min), the number of daily feedings increased (9.0 times/d vs. 7.8 times/d), the rumination time increased (570.3 min/d vs. 544.9 min/d), and the milk fat content increased (3.71% vs. 3.55%). Under high starch and low starch diets, yeast cultures could increase milk yield; under high concentrate diets, yeast cultures could reduce rumen fluid pH and rumen lactic acid concentration, thereby reducing the risk of subacute ruminal acidosis. Rich glycerol yeast preparations could significantly increase plasma calcium and blood glucose levels, significantly reduce plasma β-hydroxybutyric acid and non-esterified fatty acids (NEFA) levels, and significantly reduce the incidence of clinical and subclinical ketosis. Overall, live yeast or yeast cultures had a significant effect on milk yield, but there was some uncertainty in the effects on the content of main milk components such as milk protein, which may be related to factors such as lactation stage, lactating cow production potential, and many other influencing factors.

4.3 Application on Beef Cattle and Sheep

Ding et al. found that Saccharomyces cerevisiae significantly affects the degradation rates of dry matter and NDF in beef cattle feed, increasing the number of rumen total bacteria, fungi, protozoa, and lactic acid-utilizing bacteria, and reducing the number of starch-degrading bacteria and lactic acid-producing bacteria. Cao Wenxin's research discovered that adding inactivated yeast culture to the diet has a tendency to increase the daily weight gain of lambs, significantly reduce the feed-to-weight ratio, and improve the apparent digestibility of dry matter, crude protein, and total energy, as well as the concentrations of rumen propionic acid and total volatile fatty acids. Feeding active dry yeast can significantly improve the rumen fermentation-related indicators of beef cattle, increase the effective degradation rates of alfalfa hay dry matter, NDF, crude protein, and the dry matter of ensiled corn and rapeseed meal crude protein. The results of a Meta-analysis by Wagner et al. indicate that feeding yeast cultures to beef cattle helps increase the average daily feed intake (6.5%), DMI (1%), and feed-to-weight ratio (2.6%), and the effect is more significant with longer feeding time of yeast cultures. These differences may be partly attributed to different basal diets, especially the level of concentrate in the diet. Previous studies have shown that the probiotic effects of live yeast and yeast cultures are related to their stabilizing effect on rumen pH, and rumen pH is affected by the level of concentrate in the diet. Therefore, the effects of live yeast on nutrient degradation characteristics and rumen microbial community composition may be related to the ratio of concentrate to roughage in the diet. Additionally, live yeast and yeast cultures have a tendency to improve the carcass quality grade of beef cattle.

5. Summary

Feed-grade active dry yeast and yeast cultures have been widely applied in the field of ruminant nutrition. The selection of yeast strains has many directions. Whether it is the metabolic products of the yeast or its own nutritional components, they all have certain promoting effects on the production performance of ruminants. The research directions of active dry yeast and yeast cultures in production processes are different. The research on active dry yeast mainly focuses on increasing the number of viable yeast cells during production, storage and processing; while the production research of yeast cultures mainly focuses on optimizing the production process and improving the active products. When using active dry yeast, it is necessary to avoid excessively high environmental temperatures. When granulating, it is advisable to choose live yeast products that can withstand high-temperature processes. Active dry yeast, similar to other probiotics, mainly functions through the metabolic products of the yeast body in the gastrointestinal tract and the nutrients of the yeast body itself; yeast cultures mainly function through the yeast cells, metabolic products of the yeast cells and a large number of active nutritional substances in the denatured culture medium. With the development of analytical techniques, techniques such as gas chromatography-ion mobility (GC-IMS) for analyzing the composition and content of volatile organic compounds during fermentation, LC-MS non-targeted metabolomics for analyzing differential metabolites, whole-genome sequencing and functional analysis of fermentation strains have been widely applied in the field of microbial fermentation. In the future, they will undoubtedly be the research and development directions for yeast strain selection and fermentation process optimization. A large number of studies have shown that the application effects of the two yeast products have certain similarities. In young ruminants, they can promote rumen development, reduce diarrhea rates, and improve growth performance; in adult ruminants, they can effectively maintain the anaerobic environment of the rumen, regulate the composition of rumen microorganisms, stabilize rumen fluid pH, and improve the animal's stress resistance. At the same time, with the development of multi-omics technologies (metagenomics, metatranscriptomics, metaproteomics and metabolomics), the research on rumen microorganisms and their metabolic functions of ruminants has entered a new era [81]. The research on the mechanism of action of active dry yeast and yeast cultures on ruminants will also be more in-depth.