WO2019046919A1 - Composition of growth-promoting prebiotic additives for animal feedstuffs and use thereof - Google Patents

Abstract

The present invention relates to a composition of growth-promoting prebiotic additives which can be used in animal feedstuffs, or as growth promoters, nutraceuticals, prebiotics or additives for controlling and preventing pathogenic bacteria in the intestinal tract of animals or farm animals. A composition of prebiotic additives was developed for animal feedstuffs, comprising 1,3 and 1,6 betaglucans and mannooligosaccharides (MOS) and fructooligosaccharides (FOS) and/or inulin. The composition of growth-promoting prebiotic additives can be used in animal feedstuff, particularly for the growth and fattening of chickens, for laying hens, for broiler breeder chickens, and in the initial and growth phase of swine.

Description

 COMPOSITION OF PREBIOTIC ADDITIVES GROWTH PROMOTERS

 FOR ANIMAL FEEDING AND ITS USE

APPLICATION FIELD

 The present invention relates to a composition for growth promoting prebiotic additives which can be used in animal feeds, or as growth promoters, or as nutraceuticals, or as prebiotics, or as additives for the control and prophylaxis of pathogenic bacteria in the intestinal tract of animals , or livestock.

STATE OF THE TECHNIQUE

 [002] Microorganisms in nature are constantly competing for resources (nutrients, space), and for that they use various resources. The term antibiosis refers to a natural process of selection through which one living being destroys another to ensure its survival.

 The term growth promoting antibiotic (APC), although not conceptually correct, is still widely used commercially. The term derives from the positive effect of the inclusion of antibiotics as food additives, at dosages below that used in the treatment of diseases, on weight gain in animals. Antibiotics are substances produced by fungi, yeasts or bacteria that act against other microorganisms present in the medium, causing the inhibition of their growth.

[004] Antibiotics and chemotherapeutics were considered the traditional growth promoters for poultry and swine. A large number of substances have proven effective in improving animal productivity over the past decades, and their use as a feed additive has still been widespread.

 [005] Antibiotics are the oldest and most successful substances used for the manipulation of the intestinal microbiota. When used in sub-dosages, they eliminate only the most sensitive individuals from some unwanted bacterial species of the intestinal microbiota. With the elimination of these individuals a greater efficiency in the feed conversion is achieved. That is, greater production of animal weight with the same amount of food. For this reason, they are known as growth promoting antibiotics, or APCs. The disadvantage of this application is that the use in sub-dosages promotes microbial resistance to the active principle, selecting only resistant microorganisms, which can be a very serious public health aggravating factor. This is because

REPLACEMENT SHEETS (RULE 26) resistance characteristic given by the genes of these microorganisms can be transmitted from one microorganism to another, reaching even the population of the human microbiota through the consumption of contaminated food.

 However, despite its effectiveness in animal production, its use as a PCA is criticized in a number of countries, especially since the 1990s. The main issue concerns possible residues of these antimicrobials in food of animal origin, which may have an allergenic and / or carcinogenic effect, and the risk of selection and development of strains of antibiotic resistant microorganisms that may affect human health, resulting in diseases that are difficult to control.

 Since 1998, the European Union (EU) has banned the use of some antimicrobials as pro-nutrients and the world trend is to develop new products capable of producing the same or even better effect than traditional antibiotics and which have innocuous effects or more natural properties, both for animals and for humans consuming animal products.

 Non-digestible food ingredients are known as prebiotics for both humans and animals that have the ability to induce the growth or activity of beneficial microorganisms such as the bifid bacteria of the intestinal tract (eg bacteria and fungi), particularly of the colon , improving the health of its host. Prebiotics act as food for probiotic bacteria. The use of prebiotics as a food ingredient may have a superior beneficial effect to the growth promoting antibiotics, since they do not leave residues in products of animal origin and do not induce the development of drug resistance, since they are essentially natural products.

 [009] Some of the general characteristics of prebiotics are: they are not metabolized or absorbed by the upper digestive tract, serve as a substrate for beneficial intestinal bacteria, stimulating them to grow or becoming metabolically active, alter the intestinal microflora in a health-friendly way of the host and induce the beneficial systemic or intestinal effects of the host.

 [010] Prebiotics act by feeding and stimulating the growth of various beneficial intestinal bacteria, whose metabolites also act to lower pH by increasing the amount of organic acids. On the other hand, they act by blocking

REPLACEMENT SHEETS (RULE 26) (mainly D-Mannose), immobilizing and reducing the ability of some pathogenic bacteria to attach to the intestinal mucosa and thus stimulating the immune system, reducing lesions in the walls of the intestinal mucosa and avoiding the indirect translocation of pathogens, which determine infections after reaching the bloodstream.

[011] Oligosaccharides such as fructooligosaccharides (FOS) are polysaccharides which have demonstrated excellent prebiotic effects by selectively feeding some species of Lactobacillus and Bifidobacterium, thereby reducing the amount of other bacteria such as Bacteroides, Clostridium and coliforms. Other prebiotics oligosaccharides are MOS (mananooligosaccharides), GOS (galactooligosaccharides), etc.

[012] Arabinosis, galactose, mannose and mainly lactose, are carbohydrates used as prebiotics.

 [013] Oligosaccharides can be obtained in the natural form in seeds and roots of some vegetables such as Yacon, chicory, onion, garlic, artichokes, leeks, asparagus, barley, rye, wheat, soybeans, chickpeas and lupine, for example. There are obtained oligosaccharides extracted by cooking, enzymatic or alcoholic action, and those obtained synthetically through the direct polymerization of some yeast cell wall disaccharides or fermentation of polysaccharides.

 [014] Bacteria present in the intestinal microbiota fulfill a number of functions useful and beneficial to hosts, whether humans or animals, among them the ability to metabolize nondigestible saccharides, regulate lipid metabolism, and biosynthesize vitamins. However, this benefit goes much further, since the existence of a beneficial microbial population works by stimulating cell growth, suppressing the growth of harmful microorganisms, training the immune system to respond only to pathogens, and promoting defense against some diseases. Pathogens can cause undesirable pathologies, as well as being prone to allergies, caused by excessive reactions of the immune system, due to the prevalence of C. difficile and S. aureus, and the lower prevalence of beneficial microorganisms such as Bacteroides and Bifidobacteria.

[015] Salmonella is a genus of bacteria commonly called salmonella belonging to the family Enterobacteriaceae. They are gram-negative, rod-shaped bacteria, mostly mobile (with peritrichal flagella), non-sporulated, uncapped, have fimbriae, most of which do not ferment lactose. The salmonella

REPLACEMENT SHEETS (RULE 26) are an extremely heterogeneous genus, composed of three species, Salmonella subterranea, Salmonella bongori and Salmonella enterica, the latter having about 2,610 serotypes. The intestinal tract of man and animals is the main natural reservoir of this pathogen, with food of avian origin being important transmission routes.

[016] Escherichia coli is a Gram-negative, anaerobic, facultative bacillary bacterium that does not produce spores. They have fimbriae or adhesins that allow their fixation, preventing the entrainment through the urine or diarrhea. They are part of the human microbiota and are mostly non-pathogenic. However some strains are considered pathogenic to produce enterotoxins. They have lipopolysaccharide (LPS), like most Gram-negative bacteria, which disproportionately activate the immune system and excessive vasodilatation caused by cytokines produced, which can lead to septic shock and death in cases of septicemia.

 Among the various possible fronts for attacking undesirable bacteria, such as Salmonella and E. coli, are the prebiotics binding bacteria like MOS (Mannololigosaccharides), immune modulators such as Betaglucans and the prebiotics that promote the competitive exclusion, such as that caused by the bifidogenic effect of prebiotics such as FOS (Fruit-oligosaccharides) and GOS (Galactooligosaccharides), among others. The ideal would be to ally these 3 fronts of attack in a single product.

 Thus, the urgent replacement of animal feed antibiotics with prophylactic additives of pathogens in the human and animal microbiota indicates that one of the possibilities is the use of prebiotics in animal feed.

 [019] Prebiotics are defined as non-digestible food ingredients that benefit health as they stimulate the growth or activity of beneficial bacteria in our colon or intestinal tract.

 [020] Fructooligosaccharides (FOS) and galactooligosaccharides (GOS) are the most extensively studied prebiotics and have a proven bifidogenic effect.

[021] Fructooligosaccharides or FOS, are prebiotics that are also called oligofructose or oligofrutans, because they constitute the fructose oligosaccharides that can be used as sweeteners. They emerged in the 1980s as a response to consumers' tendency to employ low-calorie sweeteners. Fructooligosaccharides

REPLACEMENT SHEETS (RULE 26) (FOS) are obtained by processes of extraction and hydrolysis of the inulin molecule present in many plants and by the enzymatic reaction of transfructosylation of the sucrose molecule. Inulin is a fructose polymer, or a polyfructose, with polymerization degree of 10 to 60. Inulin can also be hydrolyzed into smaller molecules by chemical and enzymatic methods, generating mixtures of oligosaccharides with the general structure Glucose-Fructose or Gli- Fru _n (abbreviation GF _n ) and frum (Fm), with n ranging from 2 to 9 in varying from 2 to 10. This process occurs naturally in nature, so that these oligosaccharides can also be found in many vegetables such as artichoke, chicory and agave. The compositions of the commercial products may comprise questose (GF2), nystose (GF3), fructofuranosylnysthosis (GF3), inulobiosis (F2), inulotriose (F3) and inulotetraose (F4). The second type of FOS is obtained by transfructosylation by the enzymes β-fructofuranosidade (invertase) and fructosyltransferase, present in many microorganisms, as for example Aspergillus niger, Aspergillus sp., Aureobasidium sp. This process generates a mixture of the general formula GFn with n ranging from 1 to 5. All these isomers and oligomers are called FOS.

[022] Interest in the use of mannan-oligosaccharides (MOS) to improve gastrointestinal health began in the 1980s. Scientists at the time found that MOS mannose inhibited infections caused by Salmonella. Studies have shown that Salmonella can bind to mannose by projections of type 1 fimbriae, thus reducing the risks of pathogenic colonization in the intestinal tract. Different types of mannose-like sugars interact differently with fimbriae type 1. The present form on the walls of Saccharomyces cerevisiae cells (branched manns alpha-1,3 and alpha-1,6) are effective in the pathogenic connections. Upon binding to mannose, the bacteria are physically and definitively captured by the MOS-containing particles and loaded out of the digestive tract along with the faecal cake. The way of measuring the efficiency of each MOS source is by the amplitude and intensity of agglutination for the various species and serotypes of Salmonellas and E coli.

 [023] The agglutinating effect of bacteria harboring type 1 fimbriae, promoted by MOS, can be considered a physical attack against these bacteria. In practice it is possible to understand the action of MOS as a physical trap to capture these bacteria in the digestive tract. Thus, they are prevented from attaching to the walls of the intestine and

REPLACEMENT SHEETS (RULE 26) enteritis, whose scarring calluses reduce the nutrient absorption capacity of the intestine.

 [024] MOS is particularly important in the case of animals since the health of the intestine allows a better absorption of the feed components. For many decades antibiotics have been added to the composition of animal feeds at non-therapeutic levels, prophylactically, to prevent disease, improve the feed conversion ratio of feeds and accelerate growth, thus increasing the profitability of animal producers. Today there is a worldwide trend that wants to prevent this practice because it greatly increases the number of bacteria resistant to antibiotics, which are transmitted and can cause serious risks for humans. This fact has fueled interest in the development of functional foods, with MOS being among the main prebiotics with the capacity to substitute antibiotics in animal feeds. Studies have shown how NIMs impair bacterial fixation in the intestinal wall of chickens, pigs and laying hens, and the reduction in the prevalence and concentration of several types of Salmonella (causing zoonosis in animals whose symptoms are diarrhea) is reported. Escherichia coli (E. coli), Clostridium, among others. Researchers also found that MOS provoke increased mucus production by protecting intestinal microvilli in animals.

 [025] β-Glucans comprise a group of β-D-glucose polysaccharides that occur naturally in the walls of cereal, yeast, bacterial and fungal cells, exhibiting different physicochemical properties that depend on their source. Typically, β-glucans have a linear structure with 1,3 β-glycosidic bonds, varying in their molecular weights, solubility, viscosity, branching, gelling properties, thus causing various effects on animal physiology.

[026] Betaglucans and mannans are part of the cell wall structure of yeast and fungi. For the industrial extraction of these and production of prebiotics the most common and desirable are those extracted from yeasts of the genera Saccharomyces and Candida.

 [027] The immunomodulatory effect of beta-glucans can be considered a physiological attack of the organism against these harmful bacteria. It manifests itself in the form of faster healing of the enteritis produced in the walls of the intestine, by these

REPLACEMENT SHEETS (RULE 26) bacteria, allowing a rapid coating of these walls by the protective mucus.

 [028] The use of mammalian intestinal tract immunomodulatory oligosaccharides is already known to man in the art.

 BR 0403979 relates to a nutritional or pharmaceutical composition for inhibiting the adhesion of pathogens to mammalian cells, or to preventing, reducing or inhibiting the invasion and infection of mammalian cells by pathogen, wherein the mammalian cells (GOS), curdlan (beta 1, glycosylcholines), glycosylcholines (GOS), glycogenoligosaccharides (GOS), glyceroligosaccharides, 3-glucan), sialyl-oligosaccharides, isomalto-oligosaccharides, oligogalacturonide, partially hydrolyzed agar, gentiololigosaccharides, arabino-oligosaccharides, pectin, lactose, lactulose, lactosucrose and long chain isomaltooligosaccharides.

BR 0813484 relates to a composition comprising: (A) an acidifying agent which may be selected from the group consisting of 2-hydroxy-4-methylthiobutanoic acid, formic acid, butyric acid, fumaric acid, lactic acid, benzoic acid , phosphoric acid, propionic acid, sorbic acid, and citric acid; (B) an immuno-stimulating agent which may be selected from the group consisting of a yeast derived product, a bacterial derived product and combinations thereof; and (C) an antioxidant which may be selected from the group consisting of 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, tertiary hydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, gallic acid or acid derivative gallic, lecithin, ascorbic acid and tocopherol.

BR 0611535 relates to a method for processing microorganism cells comprising: (A) autolysing the cells of the microorganism to release the cell walls of the microorganisms; (B) incubating the cell walls of the microorganism with an exogenous protease; (C) separating the cell walls of the microorganism into a glucan-enriched component and a mannan-enriched component; and (D) ultrafiltrating the mannan-enriched component of step (c) to form a filtrate and retentate, the mannan rich fraction being employed in animal feeds.

 [032] US 2012202770 relates to veterinary product or to animal feed.

REPLACEMENT SHEETS (RULE 26) animals for the treatment of diarrhea or intestinal disease associated with dietary Fabaceae protein in an animal, wherein said animal consumes the Fabaceae protein and wherein said treatment comprises the consumption of glucan or mannan.

US 2011250235 relates to a method of obtaining an immunomodulatory composition comprising soluble yeast mannan, wherein the saccharide fraction comprises betal-6 glucan in 0.001 to 10% by dry weight percent and optionally said composition comprises mannan at 5 to 25% dry weight percent and the amount of β-glucan or β-glucans is less than 5%, dry weight percent.

 [034] Many products of the prior art are presented as compositions which refer to the use of various prebiotics in an alternative manner. For example, a particular composition may contain Betaglucans or MOS or FOS, and not cumulatively, for example, a composition containing Betaglucans and MOS and FOS as essential elements.

 Many products of the prior art are presented as compositions which represent product with consequent characterizations of the type of process of obtaining the prebiotic, with type of raw material (sugar cane, or corn, or agave, or rice), type of fermentation and enzymes employed, different, and also process conditions such as temperature, fermentation time, additives employed, etc., also different from each other.

[036] Generic prebiotic terms, such as FOS, MOS, GOS, etc., hide complexities in their compositions, and are rarely pure. And even pure, several polymers carry the generic name of the group to which they belong, as is the case of lactulose and of the lactose itself, which are called by the man of the technique as being GOS. Apart from this, the man of the technique calls FOS a product as if it were a simple compound and not a complex mixture of polymers, and still of different origins, and make comparisons of its performances against the microorganisms in general, not considering that the MOS by For example, it has better action on bacteria with type 1 fimbriae, beta-glucans serve to stimulate phagocytosis, and FOS and GOS have bifidogenic effects. The effects of FOS, MOS, GOS and betaglucans can not be compared linearly, neither individually nor with each other.

 [037] Other than that, the FOS often understands in its MOS formulation, due to the

REPLACEMENT SHEETS (RULE 26) (mannan-oligosaccharides from yeast cell walls used to obtain FOS).

 [038] It is important to also take into account that prebiotics with different modes of action on undesirable bacteria may present synergy when combined in a single product. Thus, neither beta-glucans nor MOS and FOS alone provide improved feed conversion of domestic animals higher than growth-promoting antibiotics (APCs). However, when suitably combined in certain proportions and administered in correct dosages, they may provide feed conversion improvements greater than that of APCs. This is what the mentioned synergy is about.

 [039] In addition, by attacking undesirable bacteria in the digestive process on several fronts, or by various modes of action, the possibility of selecting resistant bacteria becomes negligible.

 The most limiting factor for industry is the price of the prebiotic to be used, which makes its use as a substitute for antibiotics and organic acids, which are much cheaper, and have good efficiency, but provoke the development of resistance by part of the bacteria. In addition to side effects for animals, such as chronic headaches and heartburn, and humans as antibiotic resistant bacteria. OBJECTS OF THE INVENTION

 [041] A composition for use as an animal feed growth promoting additive having the following properties or advantages has been developed: A - It comprises in its formulation specific amounts of prebiotics: with different modes of action, so as to be capable of acting on several fronts in the broad spectrum of bacteria of the animal microbiota and, had comparable or superior efficiency to that of the growth promoting antibiotics;

 B - Represented a product that modulates the intestinal microbiota: it contains a certain diversity of prebiotic types capable of having synergy between them, which have a strong growth promoting effect and a reduction in the population of undesirable bacteria, especially the various species and serotypes of Salmonella, Clostridium and E. co //, acting on 5 simultaneous fronts:

 * stimulating the immune system;

REPLACEMENT SHEETS (RULE 26) * provoking the agglutination of bacteria with fimbriae type 1;

 * promoting their competitive exclusion from undesirable bacteria;

 * increasing the production of short chain organic acids in the cecum;

* increasing the production of protective mucus and the villus height of the wall of the intestine.

 C - Be efficient in the long term: since the current strategies of antibiotics and their substitutes are based on the use of a single bactericidal substance, which is not efficient in the long term, since there is selection of individuals resistant to these substances, in the organisms that make up the intestinal microbiota.

 D - Had a low final cost, for use in animal nutrition: the most important is that the composition used prebiotics and proportions that would allow its final cost, for large-scale employment in the animal feed industry.

 E - Had the capacity to reduce Samonella: and to promote a higher rate of growth of animals or creations.

SUMMARY OF THE INVENTION

 The present invention relates to a composition for additives suitable for use in animal feeds, such as growth promoters, or as nutraceuticals, or as prebiotics, or as additives for the control and prophylaxis of pathogenic bacteria in the intestinal tract of animals or farmed animals , which comprises 1,3 and 1,6 beta glucans and mannanoligosaccharides (MOS) and fructooligosaccharides (FOS) and / or inulin.

The composition of growth promoting prebiotic additives according to the invention may be employed in animal feeds, particularly for broiler growth and fattening, for heavy and laying matrices and in the initial and growth stages of pigs.

 DETAILED DESCRIPTION OF THE INVENTION

 [044] Prebiotics act by feeding and stimulating the growth of several beneficial intestinal bacteria (mainly Lactobacillus and Bifidobacteria), whose metabolites also act to reduce the pH of the intestinal endothelium by increasing the amount of organic acids. As the faecal cake has alkaline pH, the greater the production of organic acids (lactic and butyric) near the intestinal wall, where most of the Lactobacillus and Bifidobacteria live, the greater the repellency against the

REPLACEMENT SHEETS (RULE 26) the undesirable bacteria in the faecal cake. On the other hand, they act by blocking adhesion sites (mainly D-Mannose), immobilizing and reducing the fixing capacity of some pathogenic bacteria in the intestinal mucosa, reducing lesions in the walls of the intestinal mucosa and avoiding the indirect translocation of pathogens, which infections after reaching the bloodstream. Finally, by continuously accessing the lymphatic system to the intestinal endothelium, microfragmented beta-glucans mimic constant bacterial attacks, which end up exerting phagocytosis and multiplying the macrophages, so that undesirable bacteria that eventually reach the intestinal endothelium are rapidly phagocytosed , preventing the formation of enteritis. Or, at least, allowing a quick healing of these.

 [045] Oligosaccharides such as fructooligosaccharides (FOS) are polysaccharides which have demonstrated excellent prebiotic effects by selectively feeding some species of Lactobacillus and Bifidobacterium, thereby reducing the amount of other bacteria such as Bacteroides, Clostridium and coliforms. Other prebiotics oligosaccharides are MOS (mananooligosaccharides), GOS (galactooligosaccharides), etc.

The immunomodulatory and growth promoter composition and control of the undesirable bacterial populations of the intestinal microbiota in animals (including the various species and serotypes of Salmonella and E. coli) has a growth promoting effect analogous or superior to that of promoter antibiotics used for this purpose (APCs).

[047] The prebiotics with bifidogenic effect, which make up the invention, mostly represented by FOS and MOS, have the purpose of promoting a strong growth of the populations of Lactobacillus and Bifidobacteria, existing in the intestinal cecum. The strong increase in the populations of these beneficial bacteria has the objective of performing the competitive exclusion of undesirable bacteria, acidifying the faecal cake in the region of its contact with the intestinal walls and increasing the production of protective mucus, as well as the quantity and the size of the villi intestinal diseases. All these benefits are aimed at improving feed conversion and assisting in the control of undesirable bacteria, especially Salmonella, Clostridium and E coli.

 [048] The composition according to the invention comprises 1,3 and 1,6 beta glucans, mannanoligosaccharides (MOS), fructooligosaccharides (FOS) and / or inulin.

REPLACEMENT SHEETS (RULE 26) [049] Mannanoligosaccharides (MOS) are carbohydrates composed of 2 to 10 mannose residues, which can be obtained from galactomannans (GM). GMs, on the other hand, are polysaccharides composed of D-mannopyranose residues on β (1 - 4) bonds.

Mannanoligosaccharides (MOS) are oligosaccharides whose source is yeast cell wall (dry yeast fermentation extract of the Saccharomyces or Candida genera). MOS contains fragments of the cell wall, as it is present on the surface of yeast cells and their walls where they represent 25 to 50% of their dry weight. The cells are broken down by autolysis, enzymatic hydrolysis or mechanical fragmentation, and the resulting mass is centrifuged to isolate the cell wall components, which are subsequently washed and dried by spray drying. The yeast wall has 80 to 85% polysaccharides, mainly glucans and mannans.

[051] MOS are oligosaccharides that have mannose in their composition. Unlike FOS, it is not fermented by most intestinal bacteria. The presence of mannose provides a binding site for the pathogenic bacteria, which bind to the MOS and are carried by the peristaltic movements into the external environment, making colonization of the intestinal tract difficult.

 [052] They have the ability to modulate the immune system and intestinal microflora by binding to a wide variety of mycotoxins and preserving the integrity of the surface of intestinal absorption. Pathogenic bacteria colonize the gastrointestinal tract by attaching themselves to the surface of epithelial cells, and by binding MOS particles they prevent this colonization, blocking the adhesion of pathogenic bacteria by occupying the receptors of the intestinal epithelial cells.

 [053] MOS absorbs gram-negative bacteria containing type I fimbriae, preventing adhesion to intestinal villi and competition at the intestinal epithelial attachment sites, as well as modulating the host immune system. Approximately 70% of E. coli and 53% of Salmonella sp. have type I fimbriae and one of the major concerns in the poultry industry is the threat of pathogenic bacteria associated with poultry products. Campylobacter, Clostridium, Escherichia coli and Samonella are the major pathogens associated with birds that cause disease in humans.

[054] The mechanism of action of the manno-oligosaccharides (MOS) is the agglutination of bacteria having type 1 fimbriae, which are undesirable, such as those of the genera

REPLACEMENT SHEETS (RULE 26) Salmonella and E. coli, because they adhere the intestinal walls, causing enteritis that prevent an improvement of the feed conversion. A secondary mechanism of action of MOS is the inhibition of the presence of certain fungi, which are equally undesirable for improved feed conversion.

 [055] The MOS or mannanoligosaccharides of the composition of this invention are responsible for the agglutination of bacteria having type 1 fimbriae and must come from yeast cell walls of the genera Saccharomyces or Candida. Preferably, from Saccharomyces cerevisiae from the production of ethanol from sugarcane. For purposes of this invention, MOS is preferably to be extracted from the hydrolysis promoted by the action of commercial proteases added on yeasts of Saccharomyces cerevisae. Although, not preferentially, other types of MOS originating from other species of yeast and obtained by other extraction processes, may also be used in the composition of this invention. Including, the MOS contained in whole dry yeasts and non-hydrolysed cell walls of yeasts in general, produced in different musts, whether in primary or secondary fermentations.

[056] The prebiotic additive composition comprises 11% to 3% by weight of MOS or mananooligosaccharides. Preferably, the composition should contain 7% MOS.

[057] Beta-glucans (β-glucans) are polysaccharides of D-glucose monomers linked by β-type glycosidic linkages. Beta-glucans vary in molecular mass, solubility, viscosity and three-dimensional configuration. In nature they occur as cellulose in plants, or in bran cereal grains, yeast cell walls, in certain fungi, mushrooms and bacteria.

 [058] The most active forms of beta-glucans are those comprising D-glucose units attached to one another in position (1,3) with D-glucose side chains attached at the (1,6) position, which are called of beta-glucans 1,3 / 1,6 glucan. The frequency, location, and length of the side chain rather than the backbone that determines its activity along the immune system is believed.

 [059] Insoluble beta-glucans (1.3 / 1.6) have higher biological activity than soluble beta-glucans (1.3 / 1.4).

 [060] One of the most common sources of beta-glucans is from yeast walls such as Saccharomyces cerevisae.

REPLACEMENT SHEETS (RULE 26) [061] The mechanism of action of the 1,3 and 1,6 Betaglucans is the modulation of the immune system in general and especially the one connected to the digestive system, stimulating the multiplication of the macrophages and their phagocytosis activity of the unwanted bacteria that adhere to the intestinal walls or that eventually invade the lymphatic system. Both are undesirable to an improved feed conversion.

 [062] The prebiotic additive composition comprises 20% to 7% by weight of beta glucans, particularly 1,3 and 1,6 beta glucans. Preferably, the composition should contain 14% of 1,3 and 1,6 Betaglucans.

 Betaglucans of the composition of this invention are responsible for stimulation of the immune system and must be derived from yeast cell wall extraction from the Saccharomyces or Candida genera, preferably from Saccharomyces cerevisiae, from the sugar cane ethanol production. For the purposes of this invention, the preferred process of extraction and fractionation of Betaglucans is based on the use of commercial proteases and betaglucanases added on the yeasts of Saccharomyces cerevisae, derived from the production of ethanol from sugar cane. Although not preferably 1.3 and 1.6 Betaglucans from primary or secondary fermentations of other types of must, from other species and extracted by other processes, including non-enzymatic processes, may also be used in the composition of this invention.

[064] Natural fructooligosaccharides (FOS) are extracted from Blue Agave as well as from fruits and vegetables such as bananas, onions, chicory, garlic, asparagus, etc.

[065] FOS are sugar polymers rich in fructose. With a similar structure, but of different size, there is inulin, a polymer found in vegetables. The FOS are fermented mainly by Bifidobacterium and Lactobacillus, in such a way that they stimulate the growth of populations beneficial to the intestine. These bacteria mainly produce lactic and butyric acid, which is reflected in the reduction of the pathogenic population by the acidification of the environment near the intestinal endothelium.

 [066] Commercially available fructooligosaccharides are obtained by degradation of inulin or polyfructose, a polymer of β-fructose residues linked by β (1 - 2) linkages with a β-linked linkage of D (1 - 2). The degree of polymerization of inulin ranges from 10 to 60. Inulin can be enzymatically or chemically degraded in a mixture of oligosaccharides having a general structure Glu-Frun (abbreviation GFn) and Frum

REPLACEMENT SHEETS (RULE 26) (Fm), with neither ranging from 1 to 7. As this process also occurs in nature we can find these oligosaccharides in plants as well. The main components of the commercial products are kestose (GF2), nystose (GF3), fructosylnysteosis (GF4), bifurcose (GF3), inulobiosis (F2), inulotriose (F3) and inulotetraose (F4).

 [067] Another route of FOS synthesis addresses the transfructosylation by the action of β-fructosidase from Asperguillus niger or Aureobasidium sp. on sucrose molecules. The resulting product has the general formula GFn, where n can vary from 1 to 5. Unlike the process of obtaining FOS by inulin, in this type of process not only β-type bonds (1 - 2) can occur, but other types also in smaller numbers. Due to the presence of the glycosidic bonds, the fructooligosaccharides resist the hydrolysis by the action of the saliva and digestive enzymes of the intestine. In the colon they are fermented by anaerobic bacteria, for this reason they represent food with low calorific content, however they contribute to the fiber fraction of the diet. Fructooligosaccharides are more soluble than inulins.

 [068] This invention employs FOS comprising in particular kestose (GF2), nystose (GF3), fructosylnysteosis (GF4).

 [069] FOS molecules that are not digestible in the small intestine are fermented in the large intestine by the anaerobic bacteria that make up the intestinal microbiota, producing large amounts of short-chain fatty acids or volatile fatty acids, among them: acetic acid, propionic acid and butyric acid, in addition to CO2, ammonia and H2 and, as a result, the pH, together with the intestinal endothelium of the large intestine, chasing undesirable bacteria and increasing the absorption of minerals, mainly calcium. Bifidobacteria and Lactobacillus are resistant to the acid medium, while harmful bacteria like Clostridium, E. coli, Salmonella, among others, are sensitive to this medium.

The FOS or Fructooligosaccharides of the composition of this invention should preferably be of the type containing only short chain polymers which have a higher bifidogenic effect, i.e. comprise GF2, GF3 and GF4. Preferably, these are derived from the fermentation and biotransformation of sugarcane sucrose by enzymes produced by yeasts of the genus Aureobasidium. Although not preferably other types of FOS, extracted from plants or products from the enzymatic transformation of other sugars by other species of yeast or bacteria

REPLACEMENT SHEETS (RULE 26) may also be used as the FOS component of the composition of this invention.

[071] The prebiotic additive composition comprises 12% to 45% by weight of FOS or fructooligosaccharides. Preferably, the composition should contain 35% FOS.

[072] Inulin according to the invention may be represented by any fructan.

[073] The combination of these components shall obey the following percentage ratio by mass in relation to the composition mass indicated in Table 1 below:

 Table 1

 [074] Dosages of the components of the composition to be used in g / t feed are shown in Table 2 below:

 Table 2

 The composition may further contain acidifying agents (short chain organic acids), minerals (in inorganic form or linked to organic molecules), probiotics, tannins, essential oils, etc.

 [076] Still with the same immuno modulating and controlling function of the population of undesirable bacteria of the intestinal microbiota, the composition according to the invention may contain mineral micronutrients, in inorganic forms or associated with organic molecules, preferably Zinc, Manganese, Magnesium and Copper chelated and organic selenium, whose action is immunomodulatory.

 [077] The present invention is especially intended for creations where control of undesirable bacteria is more important, as is the case of Salmonellas in the fattening stage of chickens and Clostridium, E coli and Salmonella in swine (diarrhea-causing agents), and Salmonella galinarum in laying hens. Namely, the composition according to the invention is intended primarily for the control of undesirable bacteria, although it is also a good growth promoter. The most indicated creations are growth and fattening of

REPLACEMENT SHEETS (RULE 26) chickens, laying hens, heavy chicks and early stages and growth of pigs.

[078] The examples below are for illustrative purposes only and should not be construed as limiting the invention.

Example 1:

 [079] A composition according to the invention was developed which utilized the raw materials according to table 3 below:

 Table 3

 [150] 150 piglets of PIC genetics were tested in the 41 days of the initial phase for their productive performance (feed conversion), with rations containing several growth promoting additives. Five treatments were done containing 6 piglets each, with 5 replicates.

 • Treatment 1 = Basal diet with halquinol addition as growth promoter;

• Treatment 2 = Basal diet with addition of GLUCAN MOS, (2kg / t of feed);

 • Treatment 3 = Basal diet with addition of GLUCAN MOS, (3kg / t of feed);

 • Treatment 4 = Basal diet with addition of GLUCAN MOS (67%) + FOS (33%), (2kg / t of ration);

 • Treatment 5 = Basal diet with addition of GLUCAN MOS (67%) + FOS (33%), (3 kg / t of feed).

 [081] Results: the results of the treatments are shown in Tables 4, 5, 6 and 7, below.

 [082] Table 4 below indicates the average daily feed intake (CDR), daily gain of weight (GW) and feed conversion rate (CA) of piglets during the first 15 days of experiment (Pre-Initial Phase I):

REPLACEMENT SHEETS (RULE 26) Table 4

 [083] Table 5 below indicates the average daily feed intake (CDR), daily gain of weight (GW) and feed conversion (CA) of piglets during the first 28 days of experimentation (Pre-Initial and Pre-Initial Phases II):

 Table 5

 [084] Table 6 below indicates the average daily feed intake (CDR), daily gain of weight (GW) and feed conversion (CA) of piglets during the total experimental period (41 days);

 Table 6

 [085] Table 7 below indicates the average daily feed intake (CDR), daily gain of weight (GW) and feed conversion (CA) of piglets during the total experimental period (41 days);

 Table 7

 [086] Table 8 below indicates the economic results of the treatments, showing daily costs of the additives (ADC), the daily benefits (BD - CDR and GDP) and the benefit / cost ratio provided by treatments, piglets during the period

REPLACEMENT SHEETS (RULE 26) total experimental (41 days):

 Table 8

 The above tables show that the composition according to this invention was able to substitute the antibiotic as growth promoter (APC), presenting better feed conversion rates in all phases tested. The use of only beta-glucans as growth promoters was not sufficient to replace APC.

[088] The economic result of the treatments, when evaluated through the benefit / cost ratio of each additive, shows that the composition according to the invention was very competitive with APC. Both for inclusion rate of 2kg and for 3kg / ton of feed.

Examples 2:

 [089] 3 prebiotic compositions were made according to the invention to be tested as growth promoting and auxiliary additives in the control of Salmonella in chickens inoculated with Salmonella enteritidis. The compositions used the raw materials listed below in Table 9 below:

 Table 9

 The compositions of Examples 2, 3 and 4 presented mass contents of the components on the final composition according to the invention as shown in Table 10 below:

REPLACEMENT SHEETS (RULE 26) 1.3 and 1.6

 Composition MOS FOS GOS

 Betaglucans

 Example 2 16% 8% 24% 0%

 Example 3 16% 8% 24% 0%

 Example 4 13% 7% 18% 6%

 Table 10

 [091] The compositions of examples 2, 3 and 4 were used as growth promoting additives for growing chicken feeds, that is, for the first 28 days of life, to evaluate the productive performance and control of Salmonella enteritidis. A total of 300 broilers of the commercial Ross lineage were divided into 5 treatments with six replicates of 10 animals each. On the fourth day all birds were inoculated with Salmonella enteritidis. Weight control, feed intake and suabe propé test were performed weekly to evaluate the presence of Salmonella in aviaries' bed.

[092] The treatments were as follows:

 Tl: Basal diet + Antibiotic Fosbac 350 - 30mg / t of ration

 T2: Basal diet + GlucanMOS - 2 kg / t of ration

 T3: Basal Diet + Composition of Example 3 - 2 kg / t of feed

 T4: Basal Diet + Composition of Example 4 - 3 kg / t of ration

 T5: Basal Diet + Composition of Example 5 - 2 kg / t of feed

[093] The productivity results obtained are shown in Table 11 below:

 Table 11

 [094] Where PF is the mean final weight of the animals of each treatment, CM is the average feed intake of the animals from each treatment and the feed conversion ratio of each treatment.

 The results in Table 11 above show that the compositions of Examples 2, 3 and 4 were superior in feed conversion (CA), when compared to the performance of the antibiotic treatment, which was expected, since

REPLACEMENT SHEETS (RULE 26) treated as a therapeutic antibiotic. Among the compositions tested, those of Example 2 and 3 were the ones that presented better feed conversion. However, as shown in Table 12 below, the best Benefit / Cost (BC) ratios were obtained with GlucanMOS and the composition of Example 4. That is, when it comes to feed conversion, the best technical solution is not always the best economic development.

 Table 12

 [096] Where CMA is the average cost of the additive, per animal, during the experiment period (28 days). BM is the average benefit provided by each additive (variation of feed intake and daily weight gain) during the 28 days of the experiment. And the B / C is the benefit / cost ratio provided by each additive when analyzing only the CA.

 Table 13

 [097] The results from Table 13 above (indicating results of re-isolation of Salmonella in bed, Suabe propé Positive tests), show the rate of reduction of the presence of Salmonella in avian bed of each treatment. The above results show that the treatment that used the composition of Example 3, as a growth promoting additive, was the one that showed the highest efficiency in eliminating the presence of Salmonella. This may be an indication that compositions with higher levels of FOS are the most indicated, when control of Salmonella is necessary, in addition to promoting growth.

 [098] In this respect, the composition of Example 3 was economically more efficient

REPLACEMENT SHEETS (RULE 26) than the therapeutic antibiotic, since it eliminated Salmonella from the aviary bed in less than two weeks. The composition of Example 2, which is the same as that of Example 3, but at a lower dosage (2kg / t of feed), took 3 weeks to control Salmonella from avian litter, which is indicative that the dosage of 720g of FOS / t of ration of Example 3 is most efficient for the purposes of this invention.

REPLACEMENT SHEETS (RULE 26)

Claims

Claims  1. COMPOSITION OF PREBIOTIC ADDITIVES GROWTH PROMOTERS FOR ANIMAL FEEDING characterized by comprising 1,3 and 1,6 betaglucans and mannanoligosaccharides (MOS) and fructooligosaccharides (FOS) and / or inulin.  A composition according to claim 1, characterized in that the MOS can be derived from whole dry yeasts and non-hydrolyzed cell walls of yeasts in general produced in different musts, whether in primary or secondary fermentations.  A composition according to claim 1 or 2, characterized in that the MOSs originate from yeast cell walls of the genera Saccharomyces or Candida.  A composition according to claim 3, characterized in that Saccharomyces cerevisiae is derived from the production of ethanol from sugar cane.  The composition according to claim 1, characterized in that the betaglucans are derived from yeast cell wall extraction from the Saccharomyces or Candida genera.  A composition according to claim 5, characterized in that beta-glucans are derived from Saccharomyces cerevisiae from the production of ethanol from sugar cane.  A composition according to claim 1, characterized in that the 1,3 and 1,6 Betaglucans originate from primary or secondary fermentations of other types of must, from other species and extracted by processes other than sugar cane , including non-enzymatic.  The composition according to claim 1, characterized in that the FOS comprise short chain polymers.  A composition according to claim 8, characterized in that the FOS comprise kestose (GF2), nystose (GF3) and fructosylnysteine (GF4).  A composition according to claim 1, characterized in that the inulin can be represented by any fructan.  A composition according to claim 1, characterized in that it comprises by weight in relation to the total mass of the mixture from about 7% to about 20% by weight. REPLACEMENT SHEETS (RULE 26) % of 1,3 and 1,6 beta glucans.  A composition according to claim 1, characterized in that it comprises by weight in relation to the total mass of the mixture about 3% to about 11% of mannanoligosaccharides (MOS).  A composition as claimed in claim 1, characterized in that it comprises by weight in relation to the total mass of the mixture about 12% to about 45% of fructooligosaccharides (FOS) and / or inulin.  A composition according to claim 1, characterized in that it contains acidifying agents (organic acids of short chain), minerals (in inorganic form or linked to organic molecules), probiotics, tannins, essential oils, etc.  A composition according to claim 1, characterized in that it contains mineral micronutrients, in inorganic forms or associated with organic molecules, preferably Chelated Zinc, Manganese, Magnesium and Copper and Organic Selenium, the action of which is immunomodulatory.  A composition according to claim 1, characterized in that it comprises 140 g / t to 800 g / t of 1,3- and 1,6-beta-glucans, 60 g / t to 440 g / t of mannanoligosaccharide (MOS) feed and 240 g / t at 1800 g / t FOS feed and / or inulin.  Use of the composition of growth promoting preservative additives for animal feeds as described in the previous claims, characterized in that it is intended for broiler growth and fattening diets.  USE OF THE COMPOSITION OF PREBIOTIC GROWTH PROMOTING ADDITIVES FOR ANIMAL FEEDING, as described in the previous claims, characterized in that it is intended for feeds of laying hens and heavy matings of broiler chicks.  Use of the composition of growth promoting prebiotic growth additives as described in the preceding claims, characterized in that it is intended for early stage and swine growth rations. REPLACEMENT SHEETS (RULE 26)