FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for affecting and modulating the gut microbiome of subjects.

BACKGROUND OF THE INVENTION

Pork is the world’s most consumed meat from terrestrial animals, see currently available link: www.fao.oig/ag/againfo/themes/en/meat/backgroimd.html. In modem swine breeding conditions, weaning is the most critical period in the course of the pig’s life due to sudden dietary, social, and environmental changes. Weaning is a sudden, stressful, short, and complex event, which profoundly impacts piglet health and leads to decreased performance and sometimes even to mortality.

The multiple stressors encountered at piglet weaning induce inter alia, unbalanced gut microbiota. Gut microbiota influences host physiology in multiple ways, such as digestion and fomentation of carbohydrates, production of vitamins, maintenance of normal functions of the intestinal villi, regulation of the immune responses, and protection from pathogenic bacteria. Among the physiological and gastro-intestinal (GI) factors impacted by the weaning transition, gut microbiota disruption is likely to be recognized as one of the keys leading to post-weaning diarrhea. The pig gut microbiota is a very complex ecosystem showing dynamic composition and diversity which shifts over time and along the entire GI tract. Colonization is initiated at birth and is shaped by consumption of the sow’s milk, which provides nutritional advantages to the population of lactic acid bacteria, building a milk-oriented microbiome.

At weaning, piglets are suddenly fed with a solid diet containing cereals, with a structure and composition radically-different from that of maternal milk. Most of the studies conducted during the weaning transition have reported a decrease in bacteria of the Lactobacillus group and a loss of microbial diversity. Such disturbances of the gut microbial ecosystem and loss of diversity at early stages of life can dramatically increase the risk of GI diseases. In particular, as Lactobacillus spp. are major players in disease prevention, their abrupt decrease during weaning transition can contribute to an increase in the risk of disease.

In recent years, the relation between gut microbiota and the host’ s health has been extensively addressed and investigated. It is well known that the amounts of bacteria in and on the host’s body (skin, intestines, genitalia etc.) outnumber his/her own cells by a ratio of 10: 1. These bacteria naturally occupy many areas in the body, preventing colonization by harmful pathogens. Moreover, these beneficial bacteria greatly contribute to food digestion and hence, they can influence a variety of factors such as: inflammation, immune system, physiology, general health and especially factors related to intestinal protection and pathologies. Therefore, it is of extreme importance to control and increase the numbers of these beneficial enteric microbe populations, in order to ameliorate digestion and minimize health-related conditions and use of antibiotics.

To date, increasing the amounts of beneficial gut bacterial populations can be achieved by supplementing the diet with either probiotics (live bacteria) or prebiotics, which are nutritional products that stimulate or support the growth of these bacteria and defined by the WHO as non- viable food component that confer health benefits) on the host associated with modulation of the microbiota. Even though a significant body of studies has demonstrated the favourable effects of consuming probiotics and prebiotics (see“Using probiotics in clinical practice: Where are we now? A review of existing meta-analyses”, Rondanelli, M. et al, Gut Microbes, 8(6): 521-543, 2017, and“Probiotics, prebiotics and synbiotics- a review”, Pandey. R.K., et al, J Food Sci Technol, 52(12):7577-7587, 2015), prebiotic supplements seem to be advantageous as they are not as sensitive as bacteria to temperature, stomach acidity, or time and they are less likely to trigger allergic reactions, adverse effects or to pose health risks for immune-compromised and susceptible patients, such as bacteremia (see www.healthline.com/nutrition/probiotics-side-effects#section 3 and “Genomic and epidemiological evidence of bacterial transmission from probiotic capsule to blood in ICU patients”, Yelin. I., et al, Nat Med 25, 1728-1732, 2019).

US patent 20190261651 to Yessinergy Holding discloses a composition for additives which can be employed in animal feeds, for the control, modulation and prophylaxis of pathogenic bacteria in the intestinal tract of animals or farm animals. The composition comprises sugars (Fructooligosaccharides, Galacto-oligosaccharides, Mannan- oligosaccharides and 1,3 and l,6 Beta-glucans).

US patent 20180028490 to Jaguar Health discloses methods of treating neonatal and young non-human animals suffering from diarrhea by administering to an animal in need a proanthocyanidin polymer composition isolated from a Croton spp. or a Calophyllum spp.

EP patent 2672980 to LAVIVO discloses synbiotic compositions comprising probiotic bacterial strains and prebiotic substances (di saccharides, oligosaccharides, and/or polysaccharides) that, when combined exhibit synergistic behavior. The synergetic compositions stimulate the indigenous microflora to restore and reconstitute in vivo gut like conditions after antibiotic associated diarrhea and/or other gut infections caused by gastrointestinal pathogens, and relapses thereof, as well as the prevention of these disorders.

In view of all the above, there is still an unmet need in finding new and effective antibiotic free treatment modalities to restore gut microbial balance in subjects.

SUMMARY OF THE INVENTION

It is thus one object of the present invention to disclose an isotonic antibiotic-free composition for increasing beneficial gut bacterial populations in a subject, as compared to untreated control subject, the composition is characterized by at least three members of a group consisting of steviol glycoside, citric acid monohydrate, monosodium glutamate, glycine, mixtures and derivatives thereof.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, characterized by no residual chlorine taste.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above wherein the subject is human.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the subject is a human patient with a condition selected from a group consisting of: inflammatory bowel disease, Crohn's disease, ulcerative diseases, irritable bowel syndrome, intestinal infections, colitis, chronic pouchitis and any combination thereof.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the subject is selected from a group consisting of cattle, pigs, sheep, goats, horses, mules, asses, buffalo and camels, chickens, turkeys, ducks, geese, guinea fowl, squabs, dogs, and cats and litters thereof.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the composition is useful for maintaining intestinal microbial homeostasis.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the composition is useful for preventing, improving or otherwise treating intestinal dysbiosis.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the composition is useful for preventing, improving or otherwise treating intestinal health. It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the beneficial gut bacterial populations are selected from a group consisting of Lactobacillus, Bacteroides, Veilonella, Oscillospira and Ruminococcus.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the composition is configured to increase concentrations of volatile fatty acids in the intestines of a subject.

It is another object of the present invention to disclose an isotonic antibiotic-free composition as defined above, wherein the composition is in the form selected from the group consisting of a tablet, a capsule, a pill, lyophilized, powder, emulsion, powder, cream, ointment, enema, food additive, food ingredient, beverage, drink, paste, lotion, gel, liquid, a solution, a patch, suspension, granulated powder, liquid, syrup, cream, foam, capsule, suppository, enema, infusion, enteric-coated pill, enteric-coated capsule, mouth wash, toothpaste and any combination thereof.

It is another object of the present invention to disclose isotonic antibiotic-free composition as defined above, wherein the composition is configured to be administrable in a manner selected from a group consisting of fast release, slow release, sustained release, controlled release and any combination thereof.

It is another object of the present invention to disclose a method for increasing beneficial gut bacterial populations in a subject as compared to untreated control subject, comprising step of administrating the subj ect with an isotonic antibiotic-free composition, characterized by at least three members of a group consisting of steviol glycoside, citric acid monohydrate, monosodium glutamate, glycine, mixtures and derivatives thereof.

It is another object of the present invention to disclose the method as defined above, wherein the method further comprising a step of providing the composition with no residual chlorine taste.

It is another object of the present invention to disclose the method as defined above, wherein the method comprises administration useful for a human.

It is another object of the present invention to disclose the method as defined above, wherein the step of administration is useful for a human patient with a condition selected from a group consisting of: inflammatory bowel disease, Crohn's disease, ulcerative diseases, irritable bowel syndrome, intestinal infections, colitis, chronic pouchitis and any combination thereof.

It is another object of the present invention to disclose the method as defined above, wherein the subject is an animal selected from a group consisting of cattle, pigs, sheep, goats, horses, mules, asses, buffalo, camels, chickens, turkeys, ducks, geese, guinea fowl, squabs, dogs, cats and litters thereof.

It is another object of the present invention to disclose the method as defined above, wherein the method is useful for maintaining intestinal microbial homeostasis.

It is another object of the present invention to disclose the method as defined above, wherein the method is useful for preventing intestinal dysbiosis.

It is another object of the present invention to disclose the method as defined above, wherein the method is useful for improving intestinal health.

It is another object of the present invention to disclose the method as defined above, wherein the beneficial gut bacterial populations are selected from a group consisting of Lactobacillus, Bacteroides, Veilonella, Oscillospira and Ruminococcus.

It is another object of the present invention to disclose the method as defined above, wherein the method comprising step of selecting the composition form a group consisting of a tablet, a capsule, a pill, food, food additive, drink, beverage, lyophilized, powder, emulsion, powder, cream, ointment, paste, lotion, gel, liquid, a solution, a patch, suspension, granulated powder, liquid, syrup, cream, foam, capsule, suppository, enema, infusion, enteric-coated pill, enteric- coated capsule, mouth wash, toothpaste and any combination thereof.

It is another object of the present invention to disclose the method as defined above, wherein the method comprising step of configuring the composition to be administrable in a manner selected from a group consisting of fast release, slow release, sustained release, controlled release and any combination thereof.

It is another object of the present invention to disclose a medical device useful for increasing beneficial gut bacterial populations in a subject, comprising a delivery mechanism configure to accommodate a flowing isotonic composition; and a container in a fluid connection with the delivery mechanism, wherein the composition comprises at least three members of a group consisting of steviol glycoside, citric acid monohydrate, monosodium glutamate, glycine, mixtures and derivatives thereof.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention, wherein

Fig. 1 depicts relative abundance of iMctobacillus in pigs receiving Tonisity Px between days 2 and 8 of life; and

Fig. 2 depicts relative abundance of Bacteroides in pigs receiving Tonisity Px between days 2 and 8 of life;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description is provided, alongside all chapters of the present invention, so as to enable any person skilled in the art to make use of the invention and sets forth the best modes contemplated by the inventor of carrying out this invention. Various modifications, however, are adapted to remain apparent to those skilled in the art, since the generic prindples of the present invention have been defined specifically to provide compositions and methods for affecting microbiome.

The term microbiome refers hereinafter in a non-limiting manner to a community of microorganisms, such as bacteria, fungi, and viruses that inhabit a particular environment and especially the collection of microorganisms living in or on the host’s body; or to the collective genomes of microorganisms inhabiting a particular environment and especially the mammalian and avian body. The microbiota includes inter alia bacteria, fungi, archaea and viruses.

The term beneficial gut bacterial populations refers hereinafter to bacteria inhabiting the intestines of a subject and providing health-related benefits for the host, such as physically occupying niches to avoid pathogen colonization, vitamin production, and dietary fibers fermentation resulting in improved digestion. The subject has any type of digestive system: Monogastric (simple); Avian; Auminants (polygastric) and Pseudo-ruminants.

The term volatile fatty acids (VFAs) refers hereinafter to saturated aliphatic organic acids among which acetic add, propionic add and butyric add are the most abundant VFAs in the colon. VFAs are produced by gut microbiota in the large bowel upon fermentation of unabsorbed or undigested carbohydrates and therefore, they serve as an indicator of beneficial gut bacteria activity.

The terms dysbiosis or dysbacteriosis refer hereinafter in a non-limiting manner to microbial imbalance, or maladaptation on or inside the body, such as impaired microbiota. For example, a part of the human microbiota, e.g., skin flora, gut flora, or vaginal flora, can become deranged with normally dominating species underrepresented and normally outcompeted or contained species increasing to fill the void. Dysbiosis is most commonly reported as a condition in the gastrointestinal tract.

The term steviol refers hereinafter in a non-limiting manner to steviol glycoside, and/or rebaudioside A extract.

The terms Tonisity Px™, Px™ or TPX interchangeably refer hereinafter in a non-limiting manner to the commercially available isotonic composition by Tonisity International Ltd, see currently available site www.tonisity.com. Typical Px composition is described in Table 1 bellow.

The Px solution additional comprises cereals as a protein source; enzyme co-factors; and a monosaccharide.

The current invention further discloses compositions and methods for affecting gut microbiota in subjects. Specifically, the current invention pertains to dietary manipulation of Px on porcine gut microbiota. Administration of Tonisity Px in the form of peri-weaning (pre- and post-weaning) application modulates the intestinal microbial profile in piglets, by increasing the abundance of beneficial bacterial populations, including inter alia Lactobacillus, Bacteroides, Veilonella, Oscillospira and Ruminococcus, as compared to the total number of sampled gut bacteria. Overall, Px improves gut health, assists with gut development, digestion and post-weaning feed intake while maintaining GI microbial homeostasis, preventing dysbiosis and modulating the host gut’s microbiota.

The compositions of the present invention comprise at least two members of a group consisting of inter alia, steviol glycoside, citric acid monohydrate, monosodium glutamate and glycine. Alternatively, this composition is characterized by a composition which inter alia comprises at least three members of a group consisting of steviol glycoside, citric acid monohydrate, monosodium glutamate and glycine. Additionally or alternatively, this composition is characterized by a composition comprising, inter alia, steviol glycoside, citric acid monohydrate, monosodium glutamate and glycine.

Even though the examples depicted in the current invention relate to pigs, administration of the current composition to other mammals, especially humans, can improve GI conditions and pathologies by increasing the amounts of beneficial gut bacteria populations. According to several publications, the potential extrapolation of data obtained from porcine gut microbiota research to human is feasible and reasonable. Even though no animal model could perfectly and completely mimic human beings, several studies have demonstrated that pigs are the closest to resemble human digestion physiology and GI conditions and therefore are considered a superior model over other non-primate animal models. As humans, pigs are omnivorous and they share similar nutritional requirements and other attributes. In addition, the two mammals are very similar in terms of metabolic processes, digesta transit times, compositions of enteric microbiota and villus structure and epithelial cell types. As far as intestinal length, pigs have undoubtedly longer organs, but when bodyweight is factored in and accounted for, the end result is a value of around 0.1 meters of intestine per kilogram bodyweight for humans and pigs alike (see“The Pig as an Experimental Model for Elucidating the Mechanisms Governing Dietary Influence on Mineral Absorption”, PATTERSON, J.K. et al, Exp Biol Med 233:651-664, 2008 and“Swine as Models in Biomedical Research and Toxicology Testing”, Swindle M. M. et al, Vet Pathol, online publication, DOI: 10.1177/0300985811402846, 2011). Given the above described similarities, it is likely that dietary and/or nutritional prebiotic products that might support beneficial enteric microbiota in pigs, could have the same effect in human subjects. The present disclosure relates to compositions and methods for affecting and modulating the gut microbiome of subjects. The beneficial effect of the composition administration can be observed in all types of digestive systems of the subjects: mono-gastric (simple), avian, ruminants (poly-gastric) and pseudo-ruminants.

The term 'about* refers hereinafter to a value being 25% lower or greater than the defined measure.

EXAMPLE 1

Objective Since intestinal microbiota in piglets has been shown to be an important factor affecting life-long production and health of pigs, the effects of Tonisity Px on intestinal microbiota were studied. The aim of this study was to assess whether the gut-modulating effects of Tonisity Px were linked with changes in intestinal beneficial bacterial populations, and to determine whether effects observed on intestinal microbiota are stable over time.

Materials and methods The tested material comprised of an isotonic protein solution (namely Tonisity Px™), which provides easily-absorbable nutrients, e.g., glucose, amino acids, peptides and electrolytes that can be utilized and metabolized directly by the enterocytes.

15 gilts (Yorkshire x Landrace x Duroc) and their litters (161 piglets) were enrolled in the study which was performed in a dedicated high-health research facility. Litters were allocated to one of two study groups, namely a control group, and a second group that was supplemented with TPX. Control litters received no supplementation during the pre-weaning phase, and were given creep feed from weaning up until two days after weaning. From days 2-8 of age, TPX litters were supplemented with 500 mL of 3% Tonisity Px solution once daily in an open pan. TPX litters also received the 3% solution 3 and 2 days before weaning followed by a gruel mixture of creep feed and the 3% protein solution from 1 day before weaning up until 2 days after weaning. Both groups had ad lib access to normal feed after weaning. Weaning was at about 21 days. A total of 56 piglets was randomly selected from each litter for euthanasia at 9 and 17 days of age and a further 20 piglets were randomly selected from each treatment group at 30 days of age.

Two pigs/litter were randomly selected from both the control and TPX groups at days 9 (n= 13 vs 12), 17 (n= 13 vs 14) and 30 (n= 10 vs 8) of the trial (see Table 2). Fecal samples were collected for 16S rRNA gene sequencing. 16S rRNA gene sequencing was performed using the MiSeq (Ulumina) platform after PCR amplification of the 16S v4 region. Data were normalized to the lowest number of reads per pig (3, 157), as the rarefaction curves revealed a plateau after this point.

Table 2. Number of pigs per treatment analyzed for each age per treatment group

Statistical analysis Data were analyzed using a generalized mixed model within the GLIMMIX procedure in SAS 9.4 using a binomial distribution. Treatment, age and their interaction were included as main effects in the model. Body weight at euthanasia was included as a covariate and the litter origin was included as a random effect in the model. Relative abundance of microbiota was calculated as the number of reads for individual taxa divided by the total number of reads per sample. Data are presented as means and standard errors of the mean (SEM). Values of P < 0.05 were considered statistically significant, while 0.05 < P < 0.10 were considered a near-significant trend. Since data were non-normally distributed, they were normalized by logit transformation within the model to facilitate analysis and then transformed back to the original data scale.

Six pigs from the same litter in the TPX group received antibiotics during the trial and were therefore removed from the analysis, as antibiotics are known to severely alter the intestinal microbial population and could skew the data.

initially, a phylum level analysis was performed for the 9 main phyla that accounted for >90% of the sequences from this study. The analysis also showed that a core of 23 genera with a relative abundance of >0.5% comprised the majority of reads analyzed over days 9, 17 and 30. These amounted to 91.2%, 87.3%, 86.0% of the total abundance of genera uncovered in these samples at days 9, 17 and 30, respectively and represent the main bacterial groups that are present in all pigs. A genus-level analysis was then performed to reveal any changes within bacteria that have established roles in the intestine. Results and discussion Both treatment groups had a similar richness and diversity of bacterial population. Age had a significant effect on all bacterial populations studied and therefore data were stratified by age to investigate treatment differences at each time point. Microbiome at Day 30 of age (which was post-weaning) showed distinct shifts in population compared to days 9 and 17 of age (which was pre-weaning) population. This was most likely due to introduction of solid feed at time of weaning; and show to severely affect the microbial balance around weaning transition.

Phylum-level analysis was correlated to what was found at the genus level analysis and therefore the results presented here are at the genus level. The results are presented based on the potential role of the bacteria in the gut, specifically regarding beneficial bacteria, such as Lactobacillus.

Tonishy Px effects on beneficial bacteria Lactobacillus - Toni si ty Px administered at days 2-8 of life resulted in a highly significant 3.5-fold increase in Lactobacillus at day 9 of life compared to control (4.0% vs 1.2%, respectively; P = 0.0001), amounting to a 233% increase (see Figure 1). lactobacilh are considered an indicator for beneficial gut bacteria and are linked in the art to improved gut maturation and digestion. The presence of lactobacilh in the gut has been shown to provide the host with advantages such as fighting off potential pathogens and modulating the immune response. Furthermore, lactobacilh have been also shown in the art to improve pig growth when administered as probiotics. In the current trial, the availability of the Tonisity Px as a rapidly-digestible ingredient in the intestine could have stimulated the lactobacilh to proliferate, since these bacteria are specialized in using simple carbohydrates.

Bacteroides - Bacteroides are another genus of bacteria considered to be beneficial in the intestine and were significantly more abundant at day 17 in the TPX group compared to the control group (8.5% vs 4.3%; P < 0.05). An increase in Bacteroides observed at day 17 was most likely due to their reciprocal relationship with Lactobacillus. While both Lactobacillus and Bacteroides are known for their beneficial effects on the host, their abundance has been shown to be negatively correlated, likely due to competition for specific nutrients. Therefore, at day 17, the Bacteroides may have occupied the niche that had been occupied by Lactobacillus at day 9. Nevertheless, higher population of Bacteroides in the intestine is considered beneficial, since they have been shown to improve gut function, and to modulate the host immune system.

A numerical reduction in Bacteroides in the TPX group compared to the control group (9.6% vs 14.8%; P > 0.10) was observed at day 9, possibly due to the above-mentioned reciprocal relationship with Lactobacillus ; although it did not reach statistical significance (see Figure 2). Since no detrimental effects are associated with lower intestinal Bacteroides, the numerical decrease at day 9 is not believed to negatively affect the host, especially as this occurred in conjunction with an increase in the abundance of the beneficial Lactobacillus.

Ruminococcus, Oscillospira, and Veillonella are all less well-known species of beneficial gut bacteria. All of them were significantly more abundant in the TPX group at day 9. Ruminococcus (P = 0.08) and Veillonella (P < 0.01) were both increased at day 30. These changes indicate that the effect of Tonisity Px persists post-weaning, in relation to improving fiber fermentation ( Ruminococcus ) and immunomodulation (Veillonella).

Tonisity Px effects on bacteria with variable roles Streptococcus is a bacterial genus which contains species with variable roles. Their ability to cause pathology or act as beneficial bacteria is strain-specific. While some Streptococci are known pathogens, intestinal streptococci have been used as probiotics in human and pig nutrition and are one of the most consumed probiotics worldwide. A higher population of Streptococcus was found in the TPX pigs at day 30 (1.5 vs 0.2%; P < 0.01) and a tendency for higher Streptococcus was observed at day 9 (0.5% vs 0.4%; P = 0.08). The streptococci have also been shown to have beneficial immune-stimulating effects and are normal inhabitants of the digestive tract of pigs. Resident intestinal streptococci do not seem to be associated with pathology, as it is known that the streptococci from the respiratory tract and tonsils are the strains most likely to cause pathologies. Since the microbiota sequencing only classified the bacteria to genus level, there was no information as to the identity at a species level. However, differences observed in this study did not translate to any pathology present in these pigs, either respiratory or intestinal.

Conclusion Tonisity Px has been shown to promote a more beneficial microbial profile in the intestine of the treated pigs.

The results from the current study indicate that administration of Tonisity Px between days 2 and 8 of life and in the peri-weaning phase improved the intestinal microbial profile in piglets, by increasing the abundance of the following beneficial gut bacterial populations: Lactobacillus, Bacteroides, Veilonella, Oscillospira and Ruminococcus.

These findings show that the isotonic protein drink“Tonisity Px” has the ability to influence some of the major intestinal beneficial genera in the pig intestine. The protection provided by the Lactobacillus and Bacteroides at days 9 and 17 of life, respectively, is likely to provide a better gut environment for weaning, which is a maj or stressor in a pig’ s life. While the changes observed at days 9 and 17 of life (pre-weaning phase) could be linked to nutritional effects of the Tonisity Px and occur in well-known beneficial populations, the post-weaning changes, at a time of great restructuring for the gut microbiota are also worth noting. Increase and recovery of fiber-fermenting populations, along with an increase in the Veillonella, a potential immune modulator, is provided useful for long-term health of the intestine.

Summary

Administration of Tonisity Px had a probiotic effect. Increases were observed in bacteria considered to support GI health. Table 3 summarizes the different genera affected by the administration of Tonisity Px as assayed by 16s RNA sequencing.

The changes observed in beneficial genera from the current study are generally linked to better overall intestinal health and immune modulation, reduced inflammation and decreased intestinal pathology.

Such changes provide long lasting benefits, as it is known that a window of opportunity to modify gut physiology and immunology exists in the early stages of life. Development of a healthy microbiome in early life can modulate the immune system and provide lifelong benefits.

Table 3. Overview of the changes in gut microorganisms in response to Tonisity Px administration*

* Representative species are listed in parentheses, but were not measured individually

EXAMPLE 2

An experiment was conducted in a commercial farm in Minas Gerais, Brazil . The experimental protocol was submitted to the Ethics Committee of the Federal University of Lavras. The study was divided into two stages: Farrowing and Nurseiy.

The experiment included 120 litters of the same commercial origin with high genetic potential based on the age and farrow numbers of sows and the initial weight of her litter. The litters were identified by sow cards in the pens and the piglets were individually identified by numbered ear tags and with different colors so that after the treatments were established the piglets were no longer transferred between pens and treatments.

Experimental treatments The experimental design was by randomized blocks, with 4 treatments with 30 replicates (litters). The block was performed according to the order of delivery and the experimental unit was the litter. The trial included the following groups:

T1 - Control, without the addition of any additive;

T2 - Tonisity Px from 2 to 8 days of life (500 ml of the 3% solution per litter);

T3 - Tonisity Px 3 and 2 days before weaning (500ml of the 3% solution per litter) and day 1 before weaning and 3 days after weaning (gruel with 3% solution);

T4 - Tonisity Px from 2 to 8 days of age and at day 3 and 2 before weaning (500ml of the 3% solution per litter) and day 1 before weaning and 3 days after weaning (gruel with 3% solution);

Feed management During the phase of farrowing, in addition to the mother’s milk, the animals had access to the Tonisity Px according to the specific treatment. In addition, all litters were given water ad libitum with the addition of organic acids in different bowls of the product. From the 4 ^th day of life, they received equal doses of creep feeding.

In the nursery phase, all groups received the same feed plus gruel. Groups 1 and 2 received gruel made up with water, while groups 3 and 4 received gruel made up with Tonisity Px. The same feed used in the creep feeding was used to feed the animals in the nursery' phase.

Experimental design and statistical description The experimental design was in randomized blocks, with four treatments and thirty repetitions. The experimental unit w¾s the piglet. In the nursery phase, nine replicates (litters) were selected per treatment group, using the same number of piglets per litter and the same installation shed. The data were transformed through PROC RANK to achieve normality.

All data were analysed using SAS 9.4 (SAS, Cary, NC, USA). Tests were two-tailed and carried out with a risk of alpha == 5%. Results were considered significant at P < 0.05 and considered a near-significant trend at 0.05 < P < 0.10.

Microbiological and morphometric information Only animals that were selected for slaughter were evaluated. Microbiological variables were submitted to analysis of variance, using treatments as variables. When there was a significant difference by the F test (p <0.05), the Tukey-Kramer test was used to compare the means.

Experimental procedures The animals remained in farrowing pens in the presence of the mother and were submitted to the standard procedures of the farm, where the navel cure, anticoccidial application, tail cutting, castration performed at seven days of age and animals that presented diseases were treated with antibiotics specific for the disease. Penicillin was used for the treatment of arthritis and, for diarrhoea, fosfomycin or tylosin was used. All the animals that underwent some therapeutic treatment were listed in a specific file.

After the second day of life the administration of the supplement was started. From there, the piglets were not transferred between the stalls until the day of weaning (24 days of life). The supplement was diluted in water and supplied in feed troughs. 500 ml of the product (3% solution) was supplied per litter from 2 to 8 days of age and weaning. The solutions were prepared daily.

From the 9th day of life until day 3 before weaning, the organic acid and creep feeding (same amount for each litter) were supplied according to the standard farm procedure.

In order to determine die performance of the animals, weighing was carried out at the second day of life of the animals after standardization, another weighing on the eighth day and the day before weaning. The animals were selected for the nursery phase according to the housing shed and the number of animals per litter, and 106 animals w ^r ere selected per treatment.

For the nursery phase the selected animals were transferred to a new facility where each treatment group was placed in a separate pen. These animals received the gruel in the morning in an accessory bowl.

There were two weighings on days 7 and 14 of nursery to determine the performance of the animals and evaluation of the residual effect of the treatments. On day 8, the animals were slaughtered, and 16 animals were selected per treatment, using the same weight average for the treatments. After slaughter, a jejunum sample (2.0 cm) was collected for evaluation of mucosal morphology. The samples were previously washed with saline solution, fixed in 10% formaldehyde for 24 hours, and transferred to 70 ° alcohol solution until the slides were manufactured. The histological analysis was performed in segments embedded in paraffin, sectioned at 4pm and stained with hematoxylin and eosin. The slides were photographed through the trinocular microscope (CX31, Olympus Optical do Brasil Ltda., Sao Paulo, SP) and digital image capture camera (SC30, Olympus Optical do Brasil Ltda., S&o Paulo, SP). The villi height and crypt depth were measured using the ImajeJ program using ten villi and well- directed crypts. The villi/crypt ratio was calculated, and the analyses were performed by a single person. Histology analyses were performed at the Laboratory of Histology and Immunohistochemi stry of the Department of Zootechnics of the Federal University of Lavras. For the microbiological analysis samples of cecal content were collected shortly after slaughter. The analyses of the populations were carried out by culture method in a selective medium specific for iMctobacillus spp. The colony counts (CFU/g) were submitted to logarithmic transformation (loglO) before statistical analysis. The analyses were carried out in the Laboratory of Microbiology of the Department of Animal Science of the Federal University of Lavras.

The analysis of volatile fatty acids (VFAs; acetic, propionic and butyric acids) was performed on cecal contents collected after slaughter. To a 2 g sample of the contents was added 4 mL of formic acid (17%) to extract and conserve the fatty acids present. The samples remained for two days at a temperature of 4 ° C, centrifuged at 3300 rpm for 10 minutes and the supernatant stored at-20°C to perform the analysis by gas chromatography. Gas chromatography analyses were performed at the Laboratory of Chromatography of the Department of Nutrition and Animal Production of the Faculty of Veterinary Medicine and Animal Science of the University of S5o Paulo, Pirassununga/SP. The small intestines of slaughtered animals were dissected and measured for analysis of their lengths.

Results A significant effect (p <0.05) for daytime weight at day 7 and day 14 was observed, where group T4 presented higher w'eight when compared to the control group (treatment 1). For weight gain on the fourteenth day of nursery, group T4 also presented a significant result when compared to the control group. For the variable‘antibiotics applications’ group T3 presented a significant value higher than the other treatments. For the other variables, no significant effects were observed.

The performance results of the animals during the experimental period are shown in Table 4 and Table 5. Table 4. Effect of experimental diets on initial and final weight, mortality, viability, weaning weight, maternal weight gain (GPD), initial and final coefficient of variation (CV), day weight and weight gain at 7 and 14 days of nursery and medication applications in piglets.

<0.05.

SEM- Standard Error of the Mean. in the analyses of the variables described in table 5, a significant effect (p <0.005) was observed for villus length, where groups T2, T3 and T4 had higher villi compared to group Tl. Similarly, villus height/crypt depth ratio was higher in groups T3 and T4 compared with the control.

Table 5. Effect of experimental diets on intestinal morphometry (villus, crypt, villi/crypt ratio), small intestine length, beneficial intestinal microbiota (Lactobacillus) and volatile fatty adds (acetic, propionic and butyric).

To assess whether the day 2-8 Tonisity Px administration stimulates lactobadlli, the current dataset was grouped based on the pigs’ pre- and peri-weaning treatments (Pre-weaning T1+T3 = Control; T2+T4=TPX 2-8; Peri-weaning Tl+T2=Control; T3+T4=TPX). This grouping increased the power of the analysis and revealed a significant 0.39 log increase in Lactobacillus in TPX pigs (P=0.05; Table 6). It also became apparent that administration of Tonisity Px peri- weaning increased the production of volatile fatty acids (P=0.05; Table 6).

Table 6. Effects of experimental diets on caecal bacterial populations and production of volatile fatty acids

Volatile fatty acids (VFAs) are saturated aliphatic organic acids that consist of one to six carbons of which acetic acid (C2), propionic (C3), and butyric acid (C4) are the most abundant, representing 90-95% of the VFAs in the colon. VFAs are produced by gut microbiota in the large bowel upon fermentation of unabsorbed or undigested carbohydrates. The Bacteroidetes phylum mainly produces acetate and propionate, whereas other genera produce butyrate as their primary metabolic end product.

According to various publications, VFAs have multiple benefits for the host, as they might be associated with the prevention and treatment of the metabolic syndrome, bowel disorders, and certain types of cancer. In clinical studies VFAs administration positively influenced the treatment of ulcerative colitis, Crohn’s disease, and antibiotic-associated diarrhea. Furthermore, VFAs have been demonstrated to contribute to shaping the gut environment, influence the physiology of the colon, they can be used as energy sources by host cells and the intestinal microbiota, induce the expansion of colonic T-reg cells and they also participate in different host-signaling mechanisms (see“Intestinal Short Chain Fatty Acids and their Link with Diet and Human Health”, Rios-Covia, D. et al. Front. Microbiol., 7,185, 2016 and“The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism”, Den Besten, G. et al, Journal of Lipid Research, 54. 2325-2340, 2013, and“The Gut Microbiota in the Pathogenesis and Therapeutics of Inflammatory Bowel Disease.” Zuo, T., Front Microbiol, 2018).

In the current study, administration of Tonisity Px from days 2-8 and peri-weaning resulted in improved pig performance. These effects were associated with, an enrichment in Lactobacilli, higher VFA production and higher villi in the intestine of TPX pigs. EXAMPLE 3

Increasing the beneficial gut bacterial populations by consumption of probiotic or prebiotic supplements has been shown to improve various bowel conditions and pathologies in mammalian subjects, such as humans and mice. For instance, taking a probiotic preparation of eight live freeze-dried bacterial species was demonstrated to reduce active inflammation and sustain remission in 1BD patients (see “The Gut Microbiota in the Pathogenesis and Therapeutics of Inflammatory Bowel Disease”. Zuo, T., Front Microbiol, 2018”) and consumption of iMctobacilh was shown to induce remission of Crohn's disease in children and to prolong the relapse-free time in ulcerative diseases patients (see“Probiotics and prebiotics in inflammaioiy bowel disease: microflora 'on die scope”. Damaskos, D. Clin Pharmacol, 66(2):339, 2008). Prebiotic supplements have been shown to positively effect intestinal pathophysiology as well. Materials and compounds, such as lactulose, genninated barley, dietary fibres, inulin, fructo-oligosaccharides and oligosaccharides from goat’s milk have all been proven to be involved in attenuating bowel inflammations or maintaining remission of Gl diseases (see“Prebiotics in chronic intestinal inflammation”, Looijer-van Langen, M.A., Inflamm Bowel Dis, 15(3):454-62, 2009).