Yeast derivatives (inactivated whole yeasts or yeast cell walls) are well known for their benefits in animal and human nutrition. They are particularly used to help balance the intestinal microflora and help stimulate the host natural defenses.

Most yeast derivatives on the market today are by-products of

the fermentation industry, such as biofuel production. They are usually characterized according to their biochemical composition: level of mannan-oligosaccharides (MOS), yeast β-glucans or protein contents. If such an approach is interesting to evaluate product purity, it does not totally reflect their functionality.

As experts in yeast fermentation, Lallemand teams have conducted a collaborative research and development program in partnership with renowned research institutes aiming toward a better understanding of yeast fractions. In particular, the relationship between composition and function, with a view to design a new generation of specific yeast fractions with optimal characteristics in terms of pathogen binding and modulation of the immune system.

Looking at yeast fractions at the molecular-level

Cutting-edge techniques such as atomic-force microscopy (AFM) and single-molecule force spectroscopy (SMFS) were used to study yeast fractions under a totally new light. These techniques represent powerful tools to investigate the forces and motions associated with single molecules.

The principle is to use a force sensor (a micrometer-sized bead or a cantilever) to measure the force or tension associated with a biopolymer immobilized on a surface (in our case, the yeast outer cell wall polysaccharides). Hence, the sensor is able to “read” through the surface of the

sample (yeast fraction in our case) and draw the topography of the binding forces (Figure 1, middle images).

In the case of yeast, the force studied was the binding force between yeast surface polysaccharides and Type 1 fimbriae. Type 1 fimbriae are adhesion organelles (pili) expressed by many gram-negative bacteria (e.g. Coliform, Salmonella, Campylobacter) and are responsible for the adhesion of these bacteria to the intestinal epithelial cells.

Not all yeasts are equalFor the first time, we were able to “visualize” very precisely the

yeast surface topography in terms of pathogen binding potential.

DESIGNING YEAST DERIVATIVE 2.0Thanks to innovative yeast screening strategy, a new generation of formulated yeast derivatives

could be developed that shows promising results in helping livestock animals face stresses, such as pathogen pressure, weaning or feed transition in a context of lower antibiotic usage.

Figure 1: Different yeast strains exhibit different adhesive capacity. Three steps of AFM analysis. Left: Microscopic topography of the yeast cell. Middle: representation of adhesion forces along the yeast cell (Unit=pNewton). Right: analysis of binding capacity. Strain 2 shows higher adhesion properties

by Bruno Bertaud, Yeast Derivatives Product Manager Lallemand Animal Nutrition and Sylvie Roquefeuil, Communications and PR Manager, Lallemand Animal Nutrition

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It was shown that binding molecules were arranged differently depending on the yeast sample. For certain yeast strains, they are arranged as “sticky patches”, while in others, they are scattered along the surface. In terms of functionality, the sticky patches show higher adhesive properties.

This finding clearly shows that, while biochemical parameters are important, they are not sufficient to account for the functionality of a given yeast fraction. The distribution of these molecules along the cell wall is also very important. These studies demonstrate that all yeast strains do not share the same biophysical structure, or topography. Indeed, ”Sticky patches “ on a yeast cell wall surface can be compared to scratch tissue sticking to pathogens.

Using SMFS it was possible to measure the length of the

polysaccharide chains: a characteristic linked to the binding capacity (the longer the polysaccharide chain, the better the binding potential). These findings also unveiled differences between yeast strains (see Figure 1 as example). Yeast cell wall properties are linked to the strain, each strain exhibiting specific structure and functionality in terms of binding properties.

It was also shown that, for a given strain, binding properties could differ according to the production and inactivation process involved. Hence, for every selected strain, it is essential to determine the optimal production conditions: fermentation, but also the treatment of the live yeast to obtain the yeast fractions (inactivation technique).

This cannot be controlled when yeast cell walls are the byproduct of fermentation processes, since in this case the industrial processes are designed according to the primary production. When producing custom yeast cells, however, the production process for each strain can be adapted to reach the desired characteristics of the yeast derivative.

Specific formulationBased on this knowledge, the single-molecule techniques

were applied to screen different yeast strains and select the best candidates for optimal pathogen binding potential. Different strains from two different yeast species (Saccharomyces cerevisiae and Cyberlindnera jadinii) were selected for their complementary properties (Figure 2). For each strain, optimal production process was then developed.

Figure 2: Example of yeast fractions pathogen binding properties (agglutination proportion measured 1 hour after incubation in vitro). The yeast fractions 1, 2 and 3 showing different levels of agglutination, were selected and formulated together.

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Once an optimal formulation had been designed based on in vitro studies, the model was further validated in vivo. For example, a study conducted in piglets confirmed a significant reduction of E. coli contamination in feces after a pathogen challenge (Schothorst Research Center, 2015).

Further mechanistic studies also indicated that the different yeast fractions exert a synergistic activity on the immune system. A patent has been filed concerning this immune effect.

Benefits in young livestockThe formula was then tested in animal production in

commercial farms and research centers stings. Trials performed in different species have shown that the formula (YANG, for Yeast Association New Generation) is effective in helping animals face stresses, such as pathogen pressure, weaning or feed transition in a context of lower antibiotic usage.

SwineTrials in post-weaning piglets have shown benefits, particularly

in the context of antimicrobial withdrawal in feed: improved feces aspect, absence of diarrhea despite challenging conditions (antibiotic and high dose zinc oxide withdrawal). Those benefits were reflected by a reduced feed conversion ratio in the starter phase in absence of zinc oxide (1.66 for the Control vs. 1.59 with YANG), and up to a 4.7% increase in Average Daily Gain during the whole transition period (commercial trial, Hungary, 2015).

RuminantsIn young ruminants, commercial trials in calves, goats and

lambs have also proven benefits on immunity and performance at the time of digestive challenge, such as weaning. For example, kid goats, which are very sensitive to digestive problems and the challenge of diseases at weaning, have shown higher body weight gain, especially in challenging conditions (diarrhea): average daily gain was improved by 9% in mid-challenging conditions and up to 11.9% in high challenging conditions (high diarrhea incidence) (commercial farm, France, 2015).

In calves, similar trends were shown: a Spanish trial performed in a commercial farm (2014) showed that the yeast derivative supplementation helped improve average daily gain by about 50 g/day during the pre-weaning adaptation phase and around weaning (Figure 3). This is mainly explained by lower morbidity levels shown during this stressful period.

The percentage of animals treated at least once with antibiotics is reduced by 39%, and animals treated for diarrhea reduced by 70%. In addition to better growth performance, investigators measured a decrease of a biomarker of inflammation (haptaglobin). Users repeatedly notice improved pen homogeneity in those young animals.

AquacultureIn recent years, the shrimp industry has paid a heavy toll

to early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND). Its causative agent was identified in 2013 as a specific pathogenic strain of the bacteria Vibrio parahaemolyticus. A trial was conducted in Vietnam in 2015 on juvenile white shrimps (Litopenaeus vannamei) subjected to a pathogen challenge.

Survival rate of the shrimps fed the yeast derivatives was 3.9-fold higher than in the control group (Figure 4). Such a result is very promising to limit use of antibiotics and lower the impact on the environment.

In fish, significant immune and performance benefits were also highlighted at the field level. In seabass for example (Plymouth University, UK, 2014), in conditions of digestive stress (incorporation of high level plant-based raw materials in the diet – 40% soybean meal), zootechnical performance of individuals fed the diet supplemented with the yeast derivatives product are significantly improved, such as weight gain (21.64 g/fish vs. 18.45 in control), daily growth rate (1.39% vs. 1.25% in control group), and feed conversion ratio (1.45 vs. 1.66 in control.

This can be linked to a noticeable positive effect on gut morphology: the density of microvilli of the intestinal wall is increased, and a significant difference has also been observed in the length of microvilli and gut perimeter. Hence nutrient absorption is optimized, which could be linked to improved feed utilization.

Moreover, in this trial, a strong correlation between the use of the yeast derivatives formulation in feed and the expression of genes involved in the immune response was shown.

ConclusionIn conclusion, the use of an original, single-molecule

microscopic-based approach to yeast strain selection and yeast derivatives development has led to new generation of yeast derivatives, with promising applications at a time when increased pressure is put on the feed industry to reduce the use of antibiotics in feeds.

Figure 4: Effect of yeast derivative formula in juvenile shrimp feed on survival rate following a Vibrio challenge (Vietnam, 2015).

Figure 3: Effect of the yeast derivative formula on the growth of Holstein male calves (Spain, 2014).

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