Patent Application
20230069477
The present invention relates to a composition for growing the population of microorganisms in a gut of monogastric animals and methods for growing the same. The present invention also relates to a method for enhancing an average daily gain of monogastric animals.
It is known that healthy, disease-free animals grow faster or are more able to convert their feed efficiently into body tissue than sick or immune-challenged animals. Obviously, faster growth or a more efficient conversion of feed into desirable body tissue in an animal is both economically and ecologically important, especially in animals raised for food. For this, and other reasons, it is desirable to prevent animals from contacting diseases.
One approach to keeping animals healthy is to give the animals antibiotics. However, that approach is not desirable for animals raised for food because there can be antibiotic residues in the food. Furthermore, using antibiotics increases the risk to select antibioresistant bacteria, which is a human health concern of crucial importance.
In another approach, live yeast supplementation to ruminants has been shown to enhancing fiber digestion and improve the growth thereof. A number of in vivo experiments have demonstrated in ruminants the effect of live yeast in enhancing fiber digestion (Wohlt et al., 1988; Guedes et al., 2008; Marden et al., 2008), and have concluded that one of the main mechanisms by which this is achieved is by increasing the growth and activities of fibrolytic bacteria community, including Fibrobacteres succinogenes (Chaucheyras-Durand and Fonty, 2002; Mosoniet al., 2007; Wallace and Newbold, 2007). Fibrobacteres is a bacterial phylum first described in ruminants. Only the genus Fibrobacter has been described for this phylum, and this genus presently contains only formally cultured and described species, Fibrobacter intestinalis and Fibrobacter succinogenes. Fibrobacteres is known to possess a unique array of hemicellulose-degrading enzymes and is an efficient and prolific degrader of cellulose as its sole energy source (Suen et al., 2011).
The knowledge about Fibrobacteres in monogastrics, and more particurlarly in swine is however less advanced. Culture-dependent approaches using selective medium and culture-independent techniques targeting 16S rDNA identified a potential core population inhabiting the swine gut population, including the genera Fibrobacter. Fibrobacter intestinalis and Fibrobacter succinogenes have been both identified in the gut of swine. Very little is known about interactions between probiotics and Fibrobacteres in monogastric animals. The only published result is a study about supplementation of post-weaned piglets for 28 days post-weaning with a probiotic bacteria leads to a decrease of Fibrobacteres population (Li et al., 2016). Contrary to what it is known and observed in ruminants, the effects of yeast on the activity and the growth of fibrolytic community, and more precisely of Fibrobacteres, in monogastric animals are not documented.
Another low studied phylum is the phylum of Actinobacteria, a phylum considered as minor in terms of relative abundance, but for which importance of the functionality is more and more claimed. The family of Bifidobacteriaceae, an important fibrolytic family is part of the Actinobacteria. Similarly, the family of Coriobacteraceae belongs to the phylum of Actinobacteria. Several bacteria that are part of this family are involved in the biliary acids metabolism, an interesting target to promote feed efficiency and growth performance of animals.
Fiber degradation is important for monogastric animal, such as, for example swine, as among other benefits it produces short-chain fatty acids (SCFA), an energy source for the colonocytes.
In the same manner, conversion of primary biliary acids into secondary biliary acids to improve growth performance (Ipharaguerre et al., 2018) is also growing in importance. Modulating gut microbiota to promote balance between primary and secondary biliary acids is thus interesting to optimise feed utilization and growth performance of monogastric animals.
The phyla Fibrobacteres and Actinobacteria are minor bacterial phyla in terms of relative abundance, however, they can have a really important functional role due to the fact that they exert fibrolytic effects and that the phylum of Actinobacteria contain bacteria able to convert bile acids (for example Collinsella sp. and Olsenella sp.). In the gut, Coriobacteriaceae carry out functions of importance such as the conversion of bile salts and steroids as well as the activation of dietary polyphenols (Clavel et al., 2014). Coriobacteriaceae is a family within the order Coriobacteriales (phylum Actinobacteria).
There is therefore a need for maintaining or growing the population of microorganisms responsible of fiber digestion, conversion of bile acids, or both in a gut of monogastric animals and/or a litter thereof to enhance fiber digestion of the monogastric animals. There is also an increased need for enhancing average daily gain of monogastric animals.
The present disclosure provides a composition comprising at least one biologically pure culture of Saccharomyces cerevisiae strain in amount effective for enhancing an average daily gain of monogastric animals, and a suitable carrier.
The present disclosure also provides composition comprising at least one biologically pure culture of Saccharomyces cerevisiae strain in amount effective for maintaining or growing a population of microorganisms in a gut of monogastric animals and/or a litter thereof, and a suitable carrier. In an aspect of the present disclosure, the population of microorganisms is responsible of fiber digestion, of conversion of primary biliary acids into secondary biliary acids or both.
The present invention further provides a feed or food additive comprising the composition as defined in the embodiments of the present disclosure.
The present disclosure also provide a use of the composition as defined in the embodiments of the present disclosure, for enhancing an average daily gain of monogastric animals.
The present disclosure also provide a method of maintaining or growing the population of microorganisms in a gut of monogastric animals and/or a litter thereof, the method comprising feeding to the monogastric animals an effective amount of the composition as defined in the embodiments of the present disclosure.
The present disclosure also provide a method for enhancing an average daily gain of monogastric animals, the method comprising feeding to the monogastric animals an effective amount of the composition as defined in the embodiments of the present disclosure.
Most nutrients from the feed are chemically digested and absorbed by the small intestine. This is the case, in general, for protein, lipids, and digestible carbohydrates. However, a big portion of the non-digestible carbohydrates will reach the hindgut where they will be partially consumed by the local microbial communities, the microbiota. Short Chain Fatty Acids (SCFAs) are formed as a product of this microbial fermentation, and can be absorbed locally and used as a source of energy for the host. The fiber fraction of the feed is very heterogeneous and may include:
Soluble components, which are easily fermented such as, for example, fructans, gums and/or pectins;
Partially degradable structural components such as cellulose and/or hemicellulose;
Cell wall protecting substances, which are practically indigestible, like chitin and/or lignin.
The inclusion of fiber in swine diets stimulates the speed of the digestive transit in relation with its content in Neutral Detergent Fiber (NDF) and benefits animal welfare, reducing constipation incidence, stereotypic behaviors and stress (Gerrits and Verstegen, 2006).
The proportion of fermentable fiber is positively associated with the content of soluble fiber and negatively associated with the level of lignin. It is related with changes in the intestinal environment (pH, ammonia concentration, production of SCFAs). Both types of fiber, soluble low lignified (e.g., sugar beet pulp) and insoluble lignified (e.g., oats bran) affect swine intestinal health through different mechanisms: a) production of SCFA from hindgut fermentation, and b) improvement of intestinal motility and functionality (Nutritional requirements for swine, FEDNA, 2013)
Sows are well adapted to digest fiber. They are equipped with a more voluminous large intestine than piglets or fattening pigs. The digesta remains in the large intestine for 70-85% of the total digestion time, allowing it to be in contact with the hindgut microbiota. This particularity confers sows a much higher cellulolytic activity than young pigs for example. In fact, many of the bacteria able to digest fiber that are located in the rumen of a cow can also be identified in the colon of sows. It is important to note that cellulolytic bacteria need an anaerobic environment to proliferate and be metabolically active. However, this is not always the case due to the huge vascularization irrigating the intestinal mucosa that brings in oxygen and can have a negative impact on the microbial profile. The increase in fibrolytic bacteria population in the sows could be of interest also for the piglets, as it is well-demonstrated that the microbiota of the sows strongly influences the early colonization in the piglet.
Biliary acids emerge as a promising target for developing efficacious alternatives to the use of antibiotic as growth promoter. Indeed, it has been demonstrated that the use of antibiotics and zinc oxide at doses commonly used for stimulating growth or preventing post-weaning enteritis in pigs converge in promoting microbial production of bile acids (BA) in the intestine. This leads to tissue-specific modifications in the proportion of BA, thereby amplifying BA signaling in intestine, liver, and white adipose tissue. Activation of BA-regulated pathways ultimately reinforces the intestinal protection against bacterial infection and pathological secretion of fluids and electrolytes, attenuates inflammation in colon, alters protein and lipid metabolism in liver. Conceivably, these alterations could spare nutrients for growth and improve the metabolic efficiency of treated monogastric animals. Thus, promoting bacterial population able to produce these bile acids is of interest for promoting growth of animals.
The present disclosure follows from the unexpected finding that feeding monogastric animals with a Saccharomyces cerevisiae strain, or a composition comprising thereof impacts on feed efficiency of monogastric animals and on an average daily weight gain of monogastric animals by enhancing fiber digestibility of monogastric animals by influencing lower gut fermentation; and also by stimulating a bacterial population related to bile acids metabolism.
Without being bound to a particular theory, it is believed that part of the mode of action of Saccharomyces cerevisiae in the gut of monogastric animals has to do with the fast consumption of oxygen carried out by the yeast in both caecum and colon. This creates better anaerobic conditions where the anaerobic bacteria, comprising cellulolytic bacteria and bacteria involved in the bile acids metabolism can proliferate. As a result, more energy in the form of SCFA is released from the same diet and in a shorter time; while the change in the bile acids metabolism leads to better growth efficiency.
The present disclosure provides a composition comprising at least one biologically pure culture of Saccharomyces cerevisiae strain in amount effective for enhancing average daily gain of monogastric animals, and a suitable carrier.
The present disclosure further provides a composition comprising at least one biologically pure culture of Saccharomyces cerevisiae strain in amount effective for maintaining or growing a population of microorganisms in a gut of monogastric animals and/or a litter thereof, and a suitable carrier. In an embodiment, the population of microorganisms is either responsible of fiber digestion, of conversion of primary biliary acids into secondary biliary acids or both.
In an embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is a probiotic strain. In a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is a Saccharomyces cerevisiae var. boulardii strain. In yet a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is a Saccharomyces cerevisiae var. boulardii strain deposited under accession number 1-1079 at the CNCM (i.e. at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, FRANCE).
The expression “in amount effective” when used herein will be understood to refer to an amount of at least one biologically pure culture of Saccharomyces cerevisiae strain which is at least sufficient to maintain or grow the population of microorganisms in a gut of monogastric animals and/or a litter thereof. The expression also refer to an amount of at least one biologically pure culture of Saccharomyces cerevisiae strain which is at least sufficient to enhance an average daily gain of monogastric animals.
In an embodiment, the amount of at least one biologically pure culture of Saccharomyces cerevisiae strain which is at least sufficient to maintain or grow the population of microorganisms in a gut of monogastric animals and/or a litter thereof; or to enhance an average daily gain of offspring from monogastric animals is an amount of at least 1×107 CFU per kilogram of the composition, at least 1×108 per kilogram of the composition or 1×109 CFU per kilogram of the composition. In a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is in an amount ranging from 1×107 CFU to 1×1012 CFU per kilogram of the composition. In yet a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is in an amount ranging from 1×108 CFU to 1×1012 CFU per kilogram of the composition. In still a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is in an amount ranging from 1×109 CFU to 1×1012 CFU per kilogram of the composition.
In an embodiment, the suitable carrier is feed or food, more specifically any orally ingestible animal feed or food suitable for monogastric animals. The skilled person in the art will appreciate that feed or food may vary from one monogastric animals to another. The feed and the food including but not limited to a soup, pellets or a meal-based diet for swine and poultry, or kibbles, wet food and treats for dogs and cats. In a further embodiment, at least one biologically pure culture of Saccharomyces cerevisiae strain can be admixed with the basal diet of the monogastric animals. In an alternate embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain can be top-dressed over feed or food. In a further alternate embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain can be top-dressed over the basal diet of the monogastric animals.
In one embodiment, the monogastric animals are swine, dogs, cats, horses, rabbits or poultry. In a further embodiment the swine include sows, growing and fattening pigs and piglets. In yet a further embodiment the poultry include hens and chicks. In a furthermore embodiment, the monogastric animals are offspring thereof.
In an alternative embodiment, the compositions described above can further comprise at least one additional microorganism strain. The additional microorganism strain including but not limited to Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, Enterococcus faecium, Pediococcus acidilacti, Lactococcus lactis, Lactobacillus acidophilus, L. casei, L. plantarum, L. rhamnosus or a mixture thereof.
In another alternative embodiment, the compositions comprising at least one biologically pure culture of Saccharomyces cerevisiae strain do not comprise any Enterococcus strain. The compositions may not comprise any bacterial strain and/or may not comprise any additional yeast strain. The compositions may not comprise any additional microorganism strain. The composition may consist or consist essentially of one or more biologically pure culture of Saccharomyces cerevisiae strain, and a suitable carrier.
The compositions in accordance with the present description can be in any suitable form to be served to the monogastric animals. The skilled person in the art would know what form is preferable for each monogastric animal species. For the sake of exemplifying the various form available, the compositions in accordance with the present disclosure can be in the form of a gelatin capsules, a pressed tablets, a gel caps, an animal feed or supplements, animal food or liquid beverages.
The expression “population of microorganisms responsible of fiber digestion” when used herein will be understood to refer generally to microorganisms that are able to process complex plant polysaccharides thanks to their capacity to synthesize cellulolytic and hemicellulolytic enzymes (often referred to as Fibrolytic bacteria). Polysaccharides are present in plant cellular cell walls in a compact fiber form where they are mainly composed of cellulose and hemicellulose.
The expression “population of microorganisms responsible of conversion of primary biliary acids into secondary biliary acids” when used herein will be understood to refer generally to microorganisms that are able to metabolize primary biliary acids. Biliary acids are produced from cholesterol and other acid steroids by the liver of mammals.
In an embodiment, the population of microorganisms responsible of fiber digestion is from the Fibrobacteres phylum, from the Actinobacteria phylum, or both. In a further embodiment the population of microorganisms responsible of fiber digestion is from the Fibrobacter genus, Bifidobacteriaceae family or both. In still a further embodiment, the population of microorganisms responsible of fiber digestion is a Fibrobacter intestinalis, a Fibrobacter succinogenes, or both.
In an embodiment, the microorganisms responsible of conversion of primary biliary acids into secondary biliary acids are from Actinobacteria phylum. In a further embodiment, the microorganisms responsible of conversion of primary biliary acids into secondary biliary acids are from Coriobacteraceae family. In yet a further embodiment, the microorganisms responsible of conversion of primary biliary acids into secondary biliary acids are from Collinsella sp., Olsenella sp., or both.
In either cases, the population of microorganisms also allows for enhancing an average daily gain of monogastric animals.
The present disclosure further provides for a feed or a food additive comprising a composition as defined in all the previous embodiments
The present disclosure further provides for a use of the composition described in all the embodiments above to enhance fiber digestibility of monogastric animals. In an embodiments of the use, the fiber digestibility of monogastric animals fed with the composition as defined in the different embodiments above enhanced fiber digestibility of monogastric animals by at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% when compared to the fiber digestibility of a monogastric animal not fed with the composition.
In another embodiment, the composition described in the above embodiments is used for maintaining or growing the population of microorganisms in a gut of monogastric animals and/or a litter thereof. In a further embodiment of the use, the population of microorganisms in a gut of monogastric animals fed with the composition is enhanced by at least 10, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295 or 300% when compared to the microorganism population in a monogastric animal not fed with the composition. In a further embodiment of the use, the population of microorganisms is responsible of fiber digestion, of conversion of primary biliary acids into secondary biliary acid or both.
In yet a further embodiment, the composition described in all the embodiments above is used for enhancing an average daily gain of monogastric animals. In a further embodiment, the average daily gain of monogastric animals fed with the composition is enhanced of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% when compared to the average daily gain of monogastric animals not fed with the composition.
The present disclosure further provides for a method of maintaining or growing the population of microorganisms in a gut of monogastric animals and/or a litter thereof, the method comprises feeding to the monogastric animals an effective amount of the composition as defined in the embodiments above. In an embodiment, the population of microorganisms allows for or is responsible of fiber digestion, of conversion of primary biliary acids into secondary biliary acids or both In another embodiment of the method, the growth of the population of microorganisms in the gut of monogastric animals is enhanced by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295 or 300% when compared to the microorganism population in a monogastric animal not fed with the composition. In another embodiment of the method, the composition can be admixed with the basal diet of the animal.
In a further embodiment of the method, the amount of at least one biologically pure culture of Saccharomyces cerevisiae strain is an amount of at least 1×107 CFU per kilogram of the composition, at least 1×108 per kilogram of the composition or 1×109 CFU per kilogram of the composition. In a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is in an amount ranging from 1×107 CFU to 1×1012 CFU per kilogram of the composition. In yet a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is in an amount ranging from 1×108 CFU to 1×1012 CFU per kilogram of the composition. In still a further embodiment, the at least one biologically pure culture of Saccharomyces cerevisiae strain is in an amount ranging from 1×109 CFU to 1×1012 CFU per kilogram of the composition.
In an embodiment of the method, the step of feeding to the monogastric animals an effective amount of the composition is done on a daily basis. In yet a further embodiment of the method, the step of feeding to the monogastric animals an effective amount of the composition is done on a daily basis for a period of at least 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days.
In an embodiment of the method described above, the monogastric animals is in gestation, in lactation, weaning or growing.
In an embodiment of the method, the step of feeding to the monogastric animals an effective amount of the composition is done at least 5 days before whelping. It was found that feeding monogastric animal with the composition in accordance with the present description was also beneficial for the gut of offspring. In a further embodiment, the growth of the population of microorganisms in a gut of offspring from monogastric animals fed with the composition is enhanced when compared to the population of microorganisms responsible of fiber digestion in a gut of offspring from monogastric animals not fed with the composition. More specifically, the growth of the population of microorganisms responsible of fiber digestion in the gut of the offspring from monogastric animals fed with the composition is enhanced by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295 or 300% when compared to the microorganism population in the offspring from monogastric animals not fed with the composition.
In an alternate embodiment of the method described in the embodiment above, the step of feeding the composition to the monogastric animals can be done in combination with at least one antibiotic and/or Zinc Oxide. The antibiotics are those usually used by in agriculture for growing monogastric animals. The antibiotic used can be, for example, but not limited to is Amoxicillin, Doxycycline, Lyncomycin, colistin, Tiamulin, or any combinations thereof. Other suitable antibiotics can also be used in combination with the composition in accordance with the present disclosure in the method described above.
The present disclosure further provides for a method for enhancing an average daily gain of offspring from monogastric animals, the method comprising feeding to the monogastric animals an effective amount of the composition as described in all the embodiments above. In an embodiment of the method, the average daily gain of offspring from monogastric animals fed with an effective amount of composition in accordance with the present description is increased within a at least 21 days period compared to the average daily gain of offspring from monogastric animals not fed with an effective amount of the composition. In another embodiment of the method, the average daily gain of offspring from monogastric animals fed with the composition is enhanced of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% when compared to the average daily gain of offspring from monogastric animals not fed with the composition.
In any of the uses and methods described above, the composition comprising at least one biologically pure culture of Saccharomyces cerevisiae strain may not comprise any Enterococcus strain. The composition may not comprise any bacterial strain and/or may not comprise any additional yeast strain. The composition may not comprise any additional microorganism strain. The methods and uses may not comprise administration of any Enterococcus strain. The methods and uses may not comprise administration of any additional bacterial strain and/or any additional yeast strain. The methods and uses may not comprise administration of any additional microorganism strain,
The uses described above may be non-therapeutic. The methods described above may be non-therapeutic. Also described herein is a composition as described above for use in a method for enhancing an average daily gain of monogastric animals, or for use in a method for maintaining or growing the population of micro-organisms in a gut of monogastric animals and/or a litter thereof. The method may be a method for treatment of the animal body by therapy.
In all the embodiments described above, the monogastric animals may be swine, dogs, cats, horses, rabbits, poultry or offspring thereof. The swine may be sows, growing and fattening pigs, and piglets. The monogastric animals can be in gestation, in lactation, weaning or growing
Fecal samples were collected during a 50 day trial conducted on 288 piglets after weaning. Piglets were weaned at 21 day of age and followed a 3-phase feeding program. Basal diet was supplemented with antibiotics and ZnO in the first two phases (D0-D11 and D12-D33) and only with antibiotics in the third phase (D34-D50). Piglets were allocated to three groups: basal diet, basal diet supplemented with 2×109 CFU/kg Saccharomyces cerevisiae var. boulardii CNCM I-1079, diet without medication after D11 and with 2×109 CFU/kg Saccharomyces cerevisiae var. boulardii CNCM I-1079. Fecal samples were collected from 24 piglets per treatment group at D10, D34 and D50. Zootechnical performances were also measured: body weight (BW) was individually registered at the beginning and at the end of the trial and at each diet change. The feed intake (FI) was measured per pen at each change of diet.
Microbial DNA Extraction and 16S rRNA Gene Sequencing
Fecal samples collected were processed using a DNA extraction kit from Qiagen. Microbial DNA was extracted from 40-60 mg of feces using ZR-96 Soil Microbe DNA Kit™ (Zymo Research, Freiburg, Germany) according to the manufacturer's instruction. A 15 min bead beating step at 30 Hz was applied using a Retsch MM400 Mixer Mill. The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified using the following primers:
High-throughput sequencing was performed on a MiSeq sequencer using the Reagent Kit v3, according to the manufacturer's instruction (Illumina Inc., San Diego, Calif.).
Bioinformatics analyses were performed using GenoToul bioinformatics facility (Toulouse, France). Generated paired-end 250 bp sequences were assembled using Flash software (10 bp minimum overlap, 10% maximum mismatch). Assembled sequences were processed using FROGS pipeline (Escudié et al., 2018). Briefly, sequences were clustered in Operational Taxonomic Units (OTUs) using SWARM algorithm (Mahé et al., 2014). Chimeric sequences were then detected by samples using UCHIME algorithm and removed from all samples (Edgar et al., 2011). A rarefaction step was applied to each sequencing dataset to avoid bias due to differences in sequencing depth. A filtering step was applied to remove all singletons (i.e. OTU represented by only one read). The generated OTU count table was normalized by total sum scaling. Taxonomic annotation of the OTUs was performed using the SILVA SSU database and BLAST+ and RDP algorithms. BLAST hits with identity and coverage alignments higher than 99% were kept for annotation. Otherwise, species were annotated as unknown and RDP classifier results were used for higher rank. Bootstrap thresholds were set to 0.9 and 0.8 respectively for annotation at the genus rank and higher ranks. Phylum, family and genus relative abundance tables were generated.
Zootechnical performances. Average daily gain (ADG), feed intake (FI), and feed conversion ratio (FCR) were performed by a MIXED procedure of SAS (The SAS Stat. v.9.3) for repeated measurements on pen basis. The statistical model accounted for the main effects of treatment, sex, and time, also considering the interaction between treatment and time, treatment and sex, and treatment×time×sex. The experimental unit was the pen. Significance level was fixed for A,B P≤0.01 and a,b P≤0.05, while 0.05<P≤0.1 was considered as a trend.
Microbiota analysis. Microbiota statistical analyses were carried out using R software and RStudio software. A Kruskal-Wallis Rank Sum Test followed by Pairwise Test for Multiple Comparisons of Mean Rank Sums (Conover-Test, PMCMR R package) were used to analyze compositional data. Differential analyses were performed only on taxa represented by more than 0.005% of the total sequences. The Benjamini-Hochberg procedure was used to adjust generated P values. Contingency data were analyzed using a Fisher's exact test.
Zootechnical performances. Overall, there were significant differences (P<0.01) found in average daily gain (ADG), piglets fed T1 (yeast+antibiotic) being the ones growing faster than piglets fed Ctl (Ctl+antibiotic) and T2 (yeast alone) (
Microbiota analysis. The relative abundance of the phyla Fibrobacteres increased with time (P<0.001) whatever the treatment was, probably due to the increase of fiber quantity reaching the colon. The supplementation with yeast resulted in an increase of Fibrobacteres mean abundance (expressed as %) and in Fibrobacteres detection frequency (in %, P<0.05;
The relative abundance of the phylum Actinobacteria and of the family of Coriobacteriaceae was also found to be increased in feces from piglets fed with Saccharomyces cerevisiae var. boulardii CNCM I-1079 on day 10 and on day 50 (
Fecal samples were collected during a trial conducted on sows (from 4 weeks before farrowing until weaning) and associated post-weaning piglets. Sows were fed a diet supplemented or not with 109 CFU/kg Saccharomyces cerevisiae var. boulardii CNCM I-1079 (Ctl or LSB diet respectively). Piglets were weaned at 21 day of age and followed a 2-phase feeding program. Piglets born from control or Saccharomyces cerevisiae var. boulardii CNCM I-1079-treated sows were subsequently allocated to three groups: basal diet (Ctl), basal diet supplemented with 2500 ppm ZnO, 420 ppm of antibiotics (Antibiotic group; AB), basal diet supplemented with 2×109 CFU/kg Saccharomyces cerevisiae var. boulardii CNCM I-1079 (SB). Fecal samples were collected from 13 and 22 sows per treatment, respectively for primiparous and parity 2 sows. The same sows were sampled 2 days after move to farrowing house, and one day after farrowing. Fecal samples were collected from 10 piglets per treatment group, so 60 piglets in total. Piglets were sampled on weaning day, at day 6 and day 20 post-weaning.
Microbial DNA Extraction and 165 rRNA Gene Sequencing
Fecal samples collected were processed as followed: microbial DNA was extracted from 40-60 mg of feces using ZR-96 Soil Microbe DNA Kit™ (Zymo Research, Freiburg, Germany) according to the manufacturer's instruction. A 15 min bead beating step at 30 Hz was applied using a Retsch MM400 Mixer Mill. The V4 and V5 hypervariable regions of the 16S rRNA gene were amplified using the following primers:
High-throughput sequencing was performed on a MiSeq sequencer using the Reagent Kit v3, according to the manufacturer's instruction (Illumina Inc., San Diego, Calif.).
Bioinformatics analyses were performed using GenoToul bioinformatics facility (Toulouse, France). Generated paired-end 250 bp sequences were assembled using Flash software (10 bp minimum overlap, 10% maximum mismatch). Assembled sequences were processed using FROGS pipeline (Escudié et al., 2018). Briefly, sequences were clustered in Operational Taxonomic Units (OTUs) using SWARM algorithm (Mahé et al., 2014). Chimeric sequences were then detected by samples using UCHIME algorithm and removed from all samples (Edgar et al., 2011). A rarefaction step was applied to each sequencing dataset to avoid bias due to differences in sequencing depth. A filtering step was applied to remove all singletons (i.e. OTU represented by only one read). The generated OTU count table was normalized by total sum scaling. Taxonomic annotation of the OTUs was performed using the SILVA SSU database and BLAST+ and RDP algorithms. BLAST hits with identity and coverage alignments higher than 99% were kept for annotation. Otherwise, species were annotated as unknown and RDP classifier results were used for higher rank. Bootstrap thresholds were set to 0.9 and 0.8 respectively for annotation at the genus rank and higher ranks. Phylum, family and genus relative abundance tables were generated.
Zootechnical performances. Average daily gain (ADG), feed intake (FI), and feed conversion ratio (FCR) were performed by a MIXED procedure of SAS (The SAS Stat. v.9.3) for repeated measurements. The statistical model accounted for the main effects of treatment of the sow, treatment of the piglet, and time, also considering the interactions. Significance level was fixed for A,B P≤0.01 and a,b P≤0.05, while 0.05<P≤0.1 was considered as a trend.
Microbiota statistical analyses. They were carried out using R software and RStudio software. A Kruskal-Wallis Rank Sum Test followed by Pairwise Test for Multiple Comparisons of Mean Rank Sums (Conover-Test, PMCMR R package) were used to analyze compositional data. Differential analyses were performed only on taxa represented by more than 0.005% of the total sequences. The Benjamini-Hochberg procedure was used to adjust generated P values. Contingency data were analyzed using a Fisher's exact test.
Zootechnical performance. A supplementation of sows with Saccharomyces cerevisiae var. boulardii CNCM I-1079 resulted in a significant increase in body weight of the piglets at weaning (Tableau 1). Furthermore, an effect of the supplementation of the sows is showed on growth performance on post-weaning performance, up to 35 days of age (Table 2).
These results demonstrates that when the sows were fed with Saccharomyces cerevisiae var. boulardii CNCM I-1079, the growth of their piglets was stronger than the growth obtained in the piglets which mothers were not fed with Saccharomyces cerevisiae var. boulardii CNCM I-1079.
boulardii CNCM I-1079 (LSB) of the sow and the post-weaning
Microbiota analysis. In sows, we evidenced an effect of Saccharomyces cerevisiae var. boulardii CNCM I-1079 supplementation on the microbiota alpha-diversity. A higher homogeneity of the samples was observed within Saccharomyces cerevisiae var. boulardii CNCM I-1079 group.
Saccharomyces cerevisiae var. boulardii CNCM I-1079 supplementation in sows was associated with a significant higher relative abundance of the phylum of Fibrobacteres before farrowing (
In addition, we observed a higher frequency of Fibrobacter intestinalis in piglets whose mothers were fed with Saccharomyces cerevisiae var. boulardii CNCM I-1079 supplementation (
A positive correlation between Fibrobacteres relative abundance in sows before farrowing and piglets performance (average weaning weight) was also seen, as shown in
All these results taken together suggest that the supplementation with Saccharomyces cerevisiae var. boulardii CNCM I-1079 led to an increase in the population of Fibrobacteres in the colon of the sows, but also in the colon of the piglets which sows were fed with Saccharomyces cerevisiae var. boulardii CNCM I-1079. In the first example, in addition to the effect on Fibrobacteres, we observed an effect on Actinobacteria phylum, and especially on Collinsella and Olsenella, 2 genera described to be able to metabolize primary bile acids. In the 2 provided examples, the piglets, at the same time, exhibited greater growth.
While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
Further embodiments of the invention:
1. A composition comprising at least one biologically pure culture of Saccharomyces cerevisiae strain in amount effective for enhancing an average daily gain of monogastric animals, and a suitable carrier.
2. A composition comprising at least one biologically pure culture of Saccharomyces cerevisiae strain in amount effective for maintaining or growing a population of microorganisms in a gut of monogastric animals and/or a litter thereof, and a suitable carrier.
3. The composition of embodiment 2, wherein:
4. The composition of embodiment 2 or 3, wherein the population of microorganisms allows for enhancing an average daily gain of monogastric animals.
5. The composition of any one of embodiment 1 to 4, wherein:
6. A feed or food additive comprising the composition as defined in any one of embodiments 1 to 5.
7. Use of the composition as defined in any one of embodiments 1, 4 and 5, for enhancing an average daily gain of monogastric animals.
8. The use of embodiment 7, wherein the average daily gain of monogastric animals fed with the composition is enhanced of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% when compared to the average daily gain of monogastric animals not fed with the composition.
9. Use of the composition as defined in any one of embodiments 2 to 5, for maintaining or growing the population of microorganisms in a gut of monogastric animals and/or a litter thereof.
10. The use of embodiment 9, wherein:
11. A method of maintaining or growing the population of microorganisms in a gut of monogastric animals and/or a litter thereof, the method comprising feeding to the monogastric animals an effective amount of the composition according to any one of embodiments 2 to 5.
12. The method of embodiment 11, wherein:
13. A method for enhancing an average daily gain of monogastric animals, the method comprising feeding to the monogastric animals an effective amount of the composition according to any one of embodiments 1, 4 and 5.
14. The method of embodiment 13, wherein:
| Number | Date | Country | Kind |
|---|---|---|---|
| 19305767.6 | Jun 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/083172 | 11/29/2019 | WO |
