GOFOS™ – The Benefits of sc-FOS vs. MOS in Pet Foods – Dr Fernando Schved article

The Benefits of sc-FOS vs. MOS in Pet Foods

sc-FOS (GOFOS™) vs. MOS in pet foods – Galam shares its findings from a dog-feces model experiment

By: Dr. Fernando Schved, VP R&D & CSO, Galam Ltd

Pet food professionals may encounter confusion when considering the use of either sc-FOS (short-chain fructo-oligosaccharides) or MOS (mannan-oligosaccharides) as potential beneficial ingredients to be included in their pet food formulations. Based on their resistibility in the upper digestion track of the animal, these non-digestible oligosaccharides (NDO) reach the colon intact. In the colon sc-FOS and MOS exert their beneficial physiological effects mainly via their effect on the naturally occurring microbiome housing the colon. While these NDO may serve as direct energy source they differ quite a lot in their mode of action as reflected by their effect on the pet digestive and overall health. In fact, one may favor the use of both in combination to create a complementary bioactivity effect i.e., they do not necessarily need to compete each with the other. In this short documentation we will share findings obtained from a comparative dog feces model, and elaborate on some of the differences between sc-FOS and MOS.

What are sc-FOS? and what are their main attributes?

     Short-chain fructo-oligosaccharides (sc-FOS) are amongst the most researched non-digestible soluble prebiotic dietary fibers consumed by humans and animal ^{1-6, 8-10, 12, 13}.

Sugar-based sc-FOS is composed of oligofructose molecules of the GFn-type i.e., oligofructose molecules composed of 2,3 or 4 fructosyl monomeric units (F) bonded to each other having a glucosyl terminal unit (G). Commercial enzymatically synthesized sc-FOS are characterized by a typical ratio of oligofructose molecules, i.e. Kestose (GF2) about 37%, Nystose (GF3) about 53% and Fructosyl-Nystose (GF4) at less than 10% by d.s. ISSAP’s definition of prebiotics is: “a substrate that is selectively utilized by host microorganisms conferring a health benefit”⁷. Based upon an history of more than 30 years of peer-review scientific publications sc-FOS meets the above international well accepted definition. A myriad of beneficial attributes has been documented for sc-FOS prebiotic beneficial activity including strong bifidogenic effect, acidification of colon by inducing short-chain fatty acid (SCFA) bacterial production, ability to lower counts of potential pathogens, immunomodulation, increased absorption of Ca⁺² and Mg⁺², and anti-tumor activity. sc-FOS prebiotic activity is strongly corroborated by clinical nutrition data, and therefore is also allowed as an ingredient used in the most sensitive application, namely infant formula demonstrating the scientific consensus around it (12).

What is MOS? and what are its main attributes?

To start, it is necessary to point out that in contrast to sc-FOS there are no real pure MOS products in the market. MOS-containing products are derived from yeast cell walls preparations. These products contain highly variable concentrations of MOS along with many other compounds. Thus, by referring to MOS one refers to a purified mixture of yeast cell-wall derived products. This may partially answer the variable responses observed when such products are fed to animals. Nevertheless, the main physiological effects of MOS consists of; [1] exclusion of pathogens from their sites of colonization in the gut by possibly binding to attachment sites on intestinal mucosa, thus limiting the space for pathogens to attach and proliferate, [2] MOS is believed to attach to pathogenic bacteria, therefore blocking them from attaching to the mucosa epithelium. In this regard, it is important to note that not all pathogens require attachment to the gut epithelium, thus MOS may be effective only on specific strains, [3] direct or indirect action with the mucosa and specifically with its immune system. Therefore, the end-result of MOS addition is expected to materialize via a potential reduced pathogen risk.

sc-FOS vs MOS – Similarities and differences

Although in some aspects (mainly in their role in immunomodulation) sc-FOS and MOS share some similarities, sc-FOS exerts a more complete prebiotic characteristic, and has different mechanisms of action, which may lead to different physiological effects in the target organism. sc-FOS is a prebiotic soluble fiber and serves as a preferred substrate for beneficial gut and colonic bacteria such as Bifidobacterium species and other species leading to rapid growth, and inhibition of colonization by potential pathogenic microbes. MOS’s mode of action is different i.e., it is mainly expressed via high affinity for specific molecular binding sites on certain pathogenic bacteria preventing their attachment to the intestinal epithelium. As a result, the potential pathogens flow out of the intestine allowing beneficial microorganisms a higher opportunity to attach and colonize. In contract to MOS, sc-FOS is uniquely preferred for fermentation by beneficial microbiota species such as Bifidobacterial and Lactobacillus. The mechanisms for its action are based on evolutional adaptation and is expressed in the form of enzymes assisting both in preferred translocation of sc-FOS into the microbial cells and/or rapid utilization of sc-FOS. MOS contribution is focused mainly on immuno-stimulation, which is only a part of the various components of the prebiotic activity attributed to sc-FOS. MOS is characterized by a weaker effect vs FOS when compared for SCFA production (17). For example, in dogs, MOS supplementation resulted in higher concentrations of ammonia when compared to FOS. This is an indication that MOS is capable to be fermented via putrefactive (proteolytic) fermentation when compared to sc-FOS (16).

Dog feces model

Galam conducted a comparative trial employing a model of dog feces to evaluate the effect of several non-digestible oligosaccharides on fermentation patterns and resulting metabolites. The project was designed and executed during 2021 at IATA (Instituto de Agroquimica y Tecnologia de Alimentos, CSIC) in Madrid. The protocol employed consisted of 10 healthy dogs (6 males, 4 females) having a mean age of 6.9 years and a mean weight of 19.2 kg. Fecal samples were collected in sterile tubes and kept in an anaerobic device to ensure the viability of anaerobic bacteria. Shortly after the collection (less than 12 hours), samples were used to simulate the fermentation occurring in the colon through the activity of the fecal microbiota. Samples were homogenized and diluted. Samples were mixed to generate a unique pool representative of the dog colonic microbiota. Each tested prebiotic was added to samples taken from the pooled fecal suspension. In total, each prebiotic was tested in 6 independent experiments (replicates). In parallel, a “positive control” replacing the prebiotic by glucose was also tested, including six replicates as well. In addition, six replicates of pooled fecal suspension with no added carbon source (glucose or prebiotic) were evaluated as a “negative control”. All samples were incubated for 24 h under anaerobic conditions. The fermentation process was confirmed by acidification of the medium quantified by pH measurements. Samples were collected initially (t=0) and after the incubation leading to fermentation to analyze the composition of the microbiota at baseline and after the fermentation as well as the metabolites generated.

The metabolome analysis included quantification of SCFA (lactic, propionic, butyric, valeric acid, caproic acid, heptanoic acid, isobutyric acid, 2-methylbutyric acid, and isovaleric acid), ammonia and BCAA (valine, leucine, and isoleucine). Changes in pH of fermented medium was monitored as well. To assess the microbiota composition in the different experimental conditions the V3-V4 hypervariable regions of the 16S ribosomal ribonucleic acid (rRNA) gene were amplified from total DNA and sequenced. For the taxonomic identification of OTUs, the 16S reads were mapped onto reference databases.

Fermentation results in the production of such as organic acids like acetic, butyric, or propionic acids (secondary metabolites, all classified as short-chain fatty acids or SCFA) reflected in a pH drop.  Acidification of the medium after fermentation would indicate the actual metabolization of the prebiotics tested by the bacteria present in the collected feces. As expected, addition of glucose (sample “gluc”) to stool suspension caused a statistically significant drop (1.6 pH units) in pH (“positive control”). The pH measurements after 24-hour fermentation with both sc-FOS and MOS revealed a statistically significant decrease in pH of stool suspension for all prebiotics tested (0.4 pH units in the case of MOS (sample M) vs 2.0 pH units for sc-FOS sample G and K, figure 1). These results suggest MOS influenced bacterial fermentation with lower efficiency when compared to sc-FOS. The improved ability of sc-FOS to induce fermentation leading to increased SCFA production, alongside to a stronger acidification of the colon are two of the main manifestation patterns of sc-FOS improved prebiotic effect.

Yet, another aspect of the mechanistic difference between sc-FOS and MOS effects of fermentation of the microbial population in the dog-feces model was their distinctive effect of ammonia accumulation (fig. 2).  Ammonia is produced by bacterial degradation of proteins being potential substrates for bacterial growth in the intestine (i.e., proteolytic fermentation). The source for such proteins may origin both from the diet but also include host proteins and those from bacterial turnover and renewal. Proteolytic fermentation may result in accumulation of harmful metabolites such as ammonia. Ammonia derived from intestinal fermentation has been related in the pathogenesis of several diseases (14,18). Figure 2 shows the ammonia concentration after 24-hour fermentation with the prebiotic samples tested in the model. The results obtained clearly demonstrate that MOS (sample M) presents the most distinctive profile compared to the other prebiotics and controls, namely it strongly elevates the levels of ammonia after fermentation when compared to sc-FOS (labeled as samples G & K). Moreover, sc-FOS also indued a reduced ammonia accumulation when compared to the “positive control” of glucose (labeled as C+). The ability of sc-FOS to induce a lower level of ammonia accumulation has been also demonstrated when compared to MOS in experiments where dogs were orally fed with oligosaccharides added to their food (16). These results demonstrate the minimal effects sc-FOS has on proliferation of bacteria capable to ferment proteinaceous molecules to release ammonia when compared to MOS.

The effect which sc-FOS exerted on the microbiome in the dog-feces model was also demonstrated and summarized in the diagram shown in fig 3 below as a multidimensional scaling (MDS) ordination plot at genus level of the microbiome 16S dataset. On the MDS plot (fig. 3), points that are closer together are more alike than those further apart. MDS component 1 separates t0 and negative control (CN) from the rest of samples, indicating that the higher differences in microbiome depend on the addition of glucose or

prebiotics to the medium. MDS component 2 is separating fermentation effect as second source of variability. Worthy of note is the separation of MOS labelled as M) (microbial composition is different) compared to sc-FOS labels G & K) clearly indicating that these two types of oligosaccharides induce the growth of different bacterial populations in this dog-feces model.

Concluding, the dog-feces model clearly demonstrates that sc-FOS differs in its mechanistic mode of action compared to MOS. sc-FOS, being a pure strong prebiotic, acts   by specifically affecting the growth of bacterial populations (existing in the feces) allowing a stronger release of metabolites such as SCFA which reduced the pH of medium to a greater extent when compared to MOS. The above difference is certainly an added value trait when considering the addition of sc-FOS into food for dogs. Moreover, the reduced  ability of sc-FOS to induce growth of bacteria having fermentative characteristics resulting in ammonia accumulation (as a secondary metabolite)  is yet an additional contribution factor for its incorporation. This effect may manifest itself in reduced unwanted odors if incorporated into dog foods which is in addition to inducing lower risks associated with ammonia accumulation. Nevertheless, formulators may consider the addition of both sc-FOS and MOS in combination since their different modes of action may provide enhanced beneficial health results in practice.

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