BR102012030910A2 - Soybean meal extract as a bacterial inhibitor and process for producing same - Google Patents

Abstract

SOYBEAN EXTRACT AS BACTERIAL INHIBITOR AND PROCESS FOR THE PRODUCTION OF THE SAME. The present invention deals with an "EXTRACT OF SOY MALT AS BACTERIAL INHIBITOR AND PROCESS FOR THE PRODUCTION OF THE SAME". The invention comprises processes for the production of extracts containing high levels of proteins and carbohydrates, capable of adsorbing enterobacteria, preventing gastrointestinal infections in animals. Specifically, it deals with methods and compositions to prevent infections caused by gram-negative bacteria. It is proposed to produce these extracts from soybean meal and subsequent extraction and purification processes.

Description

Extract of soybean meal as a bacterial inhibitor and process for the production of the same Field of Invention

The present invention relates to a soybean meal extract containing 5 high protein and carbohydrate contents capable of adsorbing enterobacteria, preventing gastrointestinal infections in animals. Specifically, it deals with methods and compositions for preventing infections caused by gram-negative bacteria. More specifically, it deals with the manufacturing process of said material used in said methods and compositions. 10 Foundations of the Invention and State of the Art

Soybean meal

Brazil is the second largest soybean producer worldwide with a production of 74 million tons in the 2011 crop (USDA, 2011; EMBRAPA, 2011a; IBGE, 2012). The largest producing states are Mato Grosso and Paraná with productions in 2010 of 20.4 and 15.4 million tons, respectively (EMBRAPA, 2011b).

Soy is a highly versatile grain originating products and by-products used by the agribusiness, chemical and food industry. Its best known application is to obtain oil, which is a mechanical and / or chemical process that generates large quantities of a byproduct called soybean meal.

Soybean meal is a relatively inexpensive product with great potential for use as a supplement, nitrogen source, protein, polysaccharide or substrate in biotechnological processes. Bran represents about 70% of soybean seed. Its composition may vary from approximately 25 to 50% protein, 40% carbohydrate and 10% ash and other components (BAINY et al., 2008).

The carbohydrate fraction includes simple sugars (monosaccharides and disaccharides), oligosaccharides and polysaccharides (cellulose, hemicelluloses, pectin and starch) (LIU, 1999). Its high protein content makes soybean meal the main raw material in the manufacture of animal feed. Almost 70% of the protein meal in animal feed comes from soybeans (EMBRAPA, 2011c). Although much of this waste is destined for animal feed, several researches and processes have been developed in order to obtain refined products with a higher added value (STRÕHER, 2010).

Soy carbohydrates are the second largest component after

proteins indicating an important economic value for the soy industry (BAINY et al., 2008). According to Aspinall (1988), soy polysaccharides can be separated into a non-cellulosic main fraction and a smaller cellulosic fraction, the non-cellulosic fraction consisting of 10 acid polysaccharides and arabinogalactans. According to Aspinall et al. (1967a), the main acid sugar present in soy is galacturonic acid. These acid polysaccharides then have D-galacturonic acid and L-rhamnose in the backbone with side chains consisting mainly of galactose and arabinose units (HUISMAN; SCHOLS; VORAGEN, 1998; FURUTA et al, 1998).

Bainy et al. (2008) found 45% carbohydrate content for bran

soybeans, being galactose, arabinose and uronic acids the most common monosaccharides. Glucose was found in low amounts, which is in agreement with the work of Huisman, Schols and Voragen (1998). Furuta et al. (1998), in addition to galactose, arabinose and uronic acids detected the presence of mannose in their fractions, which is indicative of incomplete removal of soybean hulls, since they have high levels of galactomannans (WHISTLER; SAARNIO, 1957 ).

Among the polysaccharides obtained from soybean, arabinogalactans have been studied intensively (ASPINALL et al., 1967a; ASPINALL et al., 25 1967b; MORITA, 1965; LABAVITCH; FREEMAN; ALBERSHEIM, 1976) and shown to contain pD-galactopyranose major chains. - (1-4) -linked, where some units carry at C-3 a (1-4.5) -linked L-arabinofuranose side chain. However, a large variation is present in the degree of branching and the distribution of substituents in the chain (HUISMAN; SCHOLS; 30 VORAGEN, 1998). Aspinall and Cottrell (1971) isolated a highly branched arabinan containing (1-3) and (1-5) -linked arabinofuranose. Liao et al. (2001) found soy polysaccharides with α-D-glucan characteristics. Its central chain was formed by α-Dglucopyranose- (1-6) -linked, with galactose and arabinose units (1-> 3) or (1-> 4) -linked on glucose units.

5 Fransen et al. (2000) isolated soybean meal arabinogalactans with

side chains replaced at 0-4 by rhamnose units. Its structure is formed by a highly branched p- (1-> 4) -linked galactan backbone with arabinan chains.

Nakamura et al. (2001) isolated from delipidified and deproteinized soybean cotyledons a polysaccharide named by the authors of Soluble Soy Polysaccharide (SSPS). The detailed structure of SSPS was determined by enzymatic degradation. The molecules are acid polysaccharides containing 18% galacturonic acid (NAKAMURA et al., 2002b). The main chain consists of galacturonan (NG) and ramnogalacturonan (RG). (1-4) Galactan side chains are branched with fucose and arabinose residues; and the (1-3) and (1-5) -α-arabinan side chains are attached to the C-4 of the rhamnose residues in the RG regions. Some galacturonic acids are modified at C-3 by xylosyl oligosaccharides, which are composed of linked β- (1-4) - and β- (1-> 2) ’20 xylose (NAKAMURA et al., 2002a).

Yamaguchi, Otab and Hatanaka (1996) have isolated okara (byproduct resulting from soymilk extraction) pectic polysaccharides comprising regions of galacturonan and ramnogalacturonan with side chains composed mainly of arabinans and galactans. The 25 galacturanane regions are distributed at the reducing and non-reducing ends of the polymer.

Huisman et al. (2000), through alkaline extraction, extracted polysaccharide fractions of soybean meal containing xyloglucans. The xyloglucans found are fucosylated and are composed of XXXG1 units similar to 30 other xyloglucans originating from other plants. Whistler and Saarnio (1957) isolated galactomannans from soybean hulls. Analyzes of these extracted polysaccharides indicated a (1-4) -Dmanp chain with D-galactopyranosyl units attached to some (1-6) -linked Dmanose units. Aspinall1 Hunt and Morrison (1966) after successive 5 extractions of soybean hulls found in addition galactomannans, a xylan. This xylan is insoluble in water, consisting of β- (1 -> 4) glycosidic bonds characterized by its low proportion of side chains. Xylans with this structure are associated with cellulose in lignified tissues.

Cipriani et al. (2009) isolated from soybean meal a

arabinogalactan type I. Its main chain is composed of β-Dgalactopyranose- (1-4) -linked, substituted at 0-3 by α-Larabinofuranose units, which in turn are substituted at 0-5, 0-3 , 5 and 0-2.5. This structure is probably linked in 0-4 of some type I1 ramnogalacturonan ramnosyl moieties formed by repeating (-> 4) -αD-galacturonic acid- (1-> 2) -αL-ramnopyranose- (1->) groups .

Biological potential of soy polysaccharides

Koga and Kikuchi (1993) isolated from soybean meal a fraction of polysaccharides capable of increasing glycolysis, macrophage interleukin 120 production and splenocyte proliferation. Liao et al. (2001) isolated black soybean polysaccharides, with characteristics of (1-6) -aD-glucan, capable of stimulating macrophages and T lymphocytes, suggesting that these polysaccharides may inhibit the proliferation of U397 leukemic cells, resulting in a modifying biological response. and immuno-enhancing effect. 25 In subsequent studies, Liao, Chen and Yang (2005) concluded that in addition to the activities already described, polysaccharides extracted from black soybeans, in addition to restoring leukocyte counts, also enhanced bone marrow cell formation.

Chen et al. (2010) tested the effects of soybean oligosaccharides on serum lipid, glucose and oxidative stress levels in obese rats. Results showed a reduction in blood glucose levels, serum lipids and oxidative stress at doses of 150, 300 and 450 mg / kg indicating ability to reverse cardio-cerebrovascular diseases.

Cipriani et al. (2009) isolated from soybean meal a type I arabinogalactan 5 capable of inhibiting ethanol-induced gastric lesions in rats, indicating a potential gastroprotective effect. The possible mechanisms described for this type of polysaccharide activity lie in the mucosal surface binding abilities acting as a protective layer; decreased secretory acid and pepsin activity and mucosal protection by increasing mucus synthesis and / or cleaning radicals (MATSUMOTO; MORIGUCHI; YAMADA, 1993; NERGARD et al., 2005; YAMADA, 1994).

Mateos-Aparicio et al. (2010) isolated from okara (byproduct resulting from the extraction of soy milk) pectic polysaccharides with potential antioxidant activity.

Tsai et al. (1983) tested the effects of polysaccharide ingestion on

soybean in gastrointestinal functions, nutritional balance, steroid excretion, glucose tolerance, serum lipid levels and blood parameters in humans. After 17 days of supplementation with soy polysaccharides, there were no significant changes in blood parameters and levels of 20 serum lipids, however studies have shown a significant increase in fecal content weight and water content, and also showed potential hypoglycaemic ability. Takahashi et al. (1999) in their work demonstrated that water-soluble soy polysaccharides have the ability to reduce gastrointestinal transit time due to the high presence of fibers in their composition.

US 2005/0107452 relates to a nutraceutical composition containing up to 99% soy polysaccharides capable of increasing blood cell counts. These polysaccharides are formed by a α- (1-> 6) -linked glucose backbone and one or more mannose, galactose and glucose residues and are obtained from extraction in organic solvents followed by protein precipitation with sulfate. calcium (WANG, SY et al. Black soybean polysaccharides. US 2005/0107452, Dec. 27, 2004).

CN 101537023 discloses the administration of soy polysaccharides in conjunction with other substances for treatment and

Control of cancer cell hyperplasia, diffusion and metastasis (PENG, Y. Medicine for treating cancer. CN 101537023 (A) 1 Sep. 23, 2009).

Anti-adhesion properties of soybean have been studied. Neeser, Koellreutter and Wuersch (1986) tested soy oligo-, polysaccharides and glycopeptides containing mannose for the inhibitory potential of E. coli type 1 fimbriae-mediated bacterial adherence. Results showed that small glycopeptides containing high mannose contents were able to inhibit hemagglutination in pig erythrocytes and bacterial adhesion in human oral cells. Kiers et al. (2002) investigated the inhibitory effects of tempeh on Escherichia coli (ETEC) K88. Tempeh is a fermented food made from boiled soybean inoculated with fungus, usually of the genus Rhizopus. Tempeh extracts and cooked soybean extracts inhibited haemagglutination in Escherichia coli (ETEC) K88-induced hamster erythrocytes in vitro. This activity is given as a result of the presence of analog aggregating components and / or adhesins and / or analog receptors. Becker et al. (2007) and Becker and Galletti (2008) investigated tempeh and its in vitro binding capacity to different bacterial species (Salmonella sp., E. coli, Lactobacillus spp.). Good tempeh binding capacity results were identified for E. coli K88, E. coli ATCC 25922 and S. enterica CIDC 510 species.

Polysaccharide Extraction

Several articles demonstrate the extraction of soy polysaccharides. Some report the extraction of neutral polysaccharides with chelating agents (90 ° C, 0.5% ammonium oxalate and NaOH) (KAWAMURA; NARASAKI, 1961), hot alkaline extraction (0.1 N NaOH) (MORITA, 1965), alkaline extraction at 30 ° C under pressure (0.5% NaOH, 120 ° C, 1 h) (LABAVITCH; FREEMAN; ALBERSHEIM, 1976) or acidic hot extraction (pH 2-6, 40-120 ° C, 0.5 - 6 h) (Furuta et al., 1998). In common they all suggest that soy polysaccharides are composed of arabinose, galactose and galacturonic acid. The presence of these monosaccharides indicates a considerable amount of pectins (HUISMAN; SCHOLS; VORAGEN, 1998).

Below is a description of patent documents

related to the extraction of soy polysaccharides, but these do not coincide with our proposal and therefore do not affect the originality of the rearing.

JP 03-067595 reports the extraction of polysaccharides from insoluble soy fibers. To this end, the fibers undergo atomization process by applying shear stress in aqueous medium, followed by fiber and protein hydrolysis and subsequent fractionation (YUICHI, M. et al. Production of water-soluble polysaccharide. JP 03-067595 , Mar. 22, 1991).

JP 11-279203 deals with a method of extracting soluble soy polysaccharides obtained from crude soy extraction or already processed material, with gelling and stabilizing properties. The material first undergoes an extraction step at pH 1-4 (hydrochloric acid, oxalic acid, phosphoric acid), 30-80 ° C 2-6 h. After the material undergoes centrifugation and purification (alcohol precipitation, reverse osmosis, ultrafiltration, gel filtration 20 and dialysis using a polar solvent) (HITOSHI, F. et al. Water soluble soybean polysaccharide, and its production and use thereof. JP 11 - 279203, Oct. 12, 1999).

JP 10-036405 patents the process of extracting water-soluble polysaccharides from vegetables. The method consists of extraction with 25 hydrochloric acid, pH 4.5, 120 ° C for 1.5 h. After centrifugation, the obtained supernatant is adjusted to pH 7.0 and protease is added (40 ° C 2 h). At the end of the enzymatic reaction the material is purified on activated carbon column. The product obtained has low foaming property and is capable of forming a high transparency aqueous solution (HITOSHI, F. et al. 30 Production of water-soluble polysaccharide. JP 10-036405, Feb. 02, 1998). JP 10-036404 reports obtaining polysaccharides rich in soybean hull acid. The extraction method consists of an aqueous solution containing soybean hulls, pH adjusted to 3-6 with hydrochloric acid and citric acid, subsequent heating under pressure (70-130 0C1 10 5 atm, 0.5 - 5 h), followed by filtration, neutralization and precipitation with organic solvent (KENJI, N.; SHIN, S. Manufacture of polysaccharide containing uronic acid. JP 10-036404, Feb. 10, 1998).

JP 06-256402 deals with an extraction of water-soluble polysaccharides with thickening, gelling and hypocholesterolemic properties. Extraction is performed with residues obtained from protein and grain oil extraction using hexametaphosphoric acid as the extracting agent, pH between 3-7 and 80 ° C (FUMIHIDE, Y. et al. Production of watersoluble polysaccharide. JP 06-256402, Sep. 13, 1994).

JP 07-188301 discloses a method of extracting soybean polysaccharides suitable for beverage formulation. Extraction of the polysaccharides is performed in a hot aqueous solution at pH 6-9 with the addition of a reducing agent (sodium or potassium hyposulfite, sodium or potassium bisulfite) or oxidizing agent (sodium hypochlorite, calcium hypochlorite, calcium hydroxyhypochlorite). and hydrogen peroxide), followed by the use of a protease (pepsin, papain, subtilisin, bromelain). High molecular weight flocculating agent may be added to the system to separate and remove the precipitate. The polysaccharides obtained show good turbidity and low rate of temporal deterioration (HITOSHI, F. et al. Production of water soluble polysaccharide. JP 07-188301, Jul. 25, 1995).

WO 2007139057 reports on obtaining soluble soy polysaccharides

with capabilities to stabilize dairy drink dispersions. Polysaccharides are obtained from okara in the presence of sequestering agent under neutral conditions and high pressure (NANAE, F .; JUNKO1 T.; AKIHIRO, N. Novel. Water-soluble polysaccharides. 2007139057 (Al), Dec. 06, 2007). Similarly, WO 2008149738 patents a crosslinking water soluble soy polysaccharide capable of stabilizing proteins near their isoelectric point (NANAE, F. et al. Method for producing water-soluble polysaccharide. WO 2008149738 (Al), Dec. 11, 2008).

US 4,119,435 shows soy polysaccharides produced from

alkaline extraction (ph> 11), temperature between 40-60 ° C whether or not containing an organic solvent. The final extract obtained contains a maximum of 13% protein. The present invention does not require advanced technology, is inexpensive, and makes use of organic solvents to prevent water absorption by the grain, reducing the amount of water needed in the process (NAKAO, Y. et al. Process for preparing soybean polysaccharides, US 4,119,435, Oct. 10, 1978).

Pl 9600244-1 reports the process for producing aqueous soybean extract for the preparation of food products, involving the steps of grain selection, flour preparation and alkaline extraction (sodium hydroxide, pH> 10.7), washing with water. and drying (JATOBÁ, SS Process for the production of aqueous soy extract. Pl 9600244-1 (A), Jan. 29, 1996).

CN 102232500 relates to a method of extracting soluble soy polysaccharides from soybean powder. The steps involved in this process 20 consist of: protein elimination in alkaline medium; enzymatic hydrolysis; enzyme inactivation (100 ° C, 10 min), filtration, ethanol precipitation (4 x, 12 h), evaporation and concentration, centrifugation (5000 rpm, 15 min) and lyophilization (YAO, L .; ZHENYU, W .; HUA, Z. One kind withdraws the soluble soybean polysaccharide from the big bean cake the method (CN 25 1 02232500, Nov. 09, 2011).

CN 1827651 deals with a method of preparing soluble soy polysaccharides from soy residues. The method involves sequential alkaline extractions with 7% recovery rate. The invention does not require major technologies, has a reasonable throughput rate and makes little energy use (MA, F. Q. Process for preparing water soluble soybean polysaccharide with bean pulp and bean dreg raw materials. CN

1827651 (A), Sep. 06, 2006).

WO 2010044255 reports the production of a new dispersion stabilizer from soybean. The stabilizer consists of water-soluble phosphorylated polysaccharides 5, the main constituent of which is an acid sugar and a bivalent metal ion capable of stabilizing milk proteins at pH near the isoelectric point (NANAE, F .; AKIHIRO, N .; RYUJI, Y. Process for producing phosphorilated water-soluble polysaccharide (WO 2010044255 (Al), Apr. 22,2010).

CN 1970578 refers to the production of soluble soy polysaccharides,

using 1-3% hexametaphosphate solution for 1-50 min in microwave followed by ethanol precipitation and acetone washes (YIN, GW Microwave method for extracting water-soluble soybean polysaccharide from bean dregs. CN 1970578 (A), May 30, 2007).

CN 101619095 deals with the preparation of polysaccharides and proteins of

soy from soy residues. This preparation comprises the following steps: extraction in hot water; cellulase hydrolysis; concentration, drying and conditioning. This invention effectively utilizes soybean residues promoting value addition (LI, X. Preparation method of soybean polysaccharide protein in soybean dregs. CN 101619095 (A), Jan. 06,

2010).

CN 101671400 reports a method of preparing soluble soy polysaccharides from enzymatic hydrolysis, weak acid extraction at high temperatures, centrifugation, precipitation, filtration and drying. The invention has the advantage of improving extraction yield, lowering production costs, reducing environmental pollution and is easily industrially reproducible (TU, Z. et al. Method of preparing soluble soybean polysaccharide. CN 101671400 ( A), Mar. 17, 2010).

CN 101880336 reports the obtaining of pectic polysaccharides by microfiltration, ultrafiltration and nanofiltration of a soluble soy polysaccharide extract with pH between 4.5 and 6.5, having as advantage the easy control of the ash and protein content (HUA, Y. et al Membrane method for preparing pectic substance soybean water-soluble polysaccharide in grades (CN 101880336 (A), Nov. 10, 2010). Similarly, CN 101693746 discloses a method for ultrafiltration separation of soy polysaccharides 5 after cooking at acid pH. In the end the polysaccharides undergo alcohol precipitation and spray-drying. The method has the following advantages: energy-saving, non-polluting, time-saving, process can be expanded and molecular weight classified soy polysaccharides can be applied in food industries (Ql, J. Method for using 10 membrane separation and classification to prepare soybean polysaccharides with different molecular weights (CN 101693746 (A), Apr. 14, 2010).

The techniques presented often involve high cost or multistage processes until the final product is obtained. Processes involving enzymes are not industrially suitable for this type of product, as they increase the cost of production due to the price of enzymes, usually require a long period of time, delaying the process, and adding another step to the process due to need for enzymatic inactivation. Processes involving micro-, ultra- and nanofiltration while providing products with select molecular sizes are also costly. Membranes have high cost and relatively short shelf life due to pore clogging. Processes involving high energy expenditure, such as microwave use, multiple steps to obtain the final product, steps with long lead times, need for purification and use of various chemical reagents are difficult to employ industrially.

This paper, in a different way, has developed a low-cost, fast, and commercially more convenient method for industrial scales, while making use of an oil industry by-product, adding value and reducing waste. The extraction process described here has been unprecedentedly combined to produce products that are economically viable technological alternatives that have been shown to be more effective in preventing gastrointestinal disease than commercially available products.

Salmonellosis

There is a great need from

food / feed and human and animal medicine to create methods of defense against enterobacterial disease, as enteric infections are one of the global causes of mortality, reaching more than 2 million deaths / year (Wilson et al. 2002 ; PATON; MORONA; PATON, 2006; RASKO; SPERANDIO, 2010; GANNER et al., 2010).

Enterobacteria are gram-negative bacteria mainly causing gastrointestinal diseases. They are among the main genera: Escherichia, Salmonella, Clostridium and Helicobacter.

The Center for Disease Control and Prevention (CDC-USA) in 2011, 15 released some data on the problem of foodborne disease (OTD) in the United States. Each year, one in six Americans gets sick from eating contaminated food, representing a total of 48 million sick Americans and 3,000 dead a year (CDC, 2011). The CDC points to a halving of the number of 20 infections caused by E. coli 0157 over the past 15 years. However, this does not happen with infections caused by Salmonella sp., They continue to grow, accounting today for $ 365 million in medical expenses.

The Secretary of Health Surveillance released in 2011 - a survey containing the epidemiological data of salmonellosis in Brazil. Between 2000 and 2011 there were 1660 outbreaks caused by Salmonella sp, of which 19 outbreaks in 2011 (SVS, 2011).

Salmonellosis is transmitted mainly through poultry (29% meat; 18% - eggs), pork (12%), beef (8%). Together these sources account for approximately 70% of the contamination chain (CDC, 2011). These data reflect that in addition to a human health problem, enteric diseases are one of the biggest problems in livestock (PATON; MORONA; PATON, 2006). Thus by implementing prophylactic measures against gastrointestinal infections in animals, the risk of transmission through the food chain will be reduced.

The process of infection by Salmonella happens in three stages:

bacterial colonization, invasion and multiplication. The adhesion of pathogens to the surface of the intestinal epithelium (colonization) is the first critical stage of the infection process (OFEK; MIRELMAN; SHARON, 1977; LINDHORST; KIEBURG; WENZEL, 1998; Wilson et al., 2002; OFEK; HASTY; SHARON, 10 2003; MORAN, 2011). It is mediated by microbial adhesion factors called adhesins. These adhesins can be protein or carbohydrates.

There are three main types of adhesin-receptor interactions: Iectin Carbohydrates; protein proteins and protein hydrophobins (OFEK; HASTY; DOYLE, 2003). The first type of interaction is the binding of 15 carbohydrate Iectins. Iectins are carbohydrate-specific binding proteins present on the surface of pathogenic bacteria, more specifically in fimbriae (LINDHORST; KIEBURG; WENZEL, 1998). The second type of interaction involves recognition between bacterial proteins and complementary proteins located on the surface of the intestinal mucosa. The third type 20 is characterized by the interaction between proteins and lipids (OFEK; HASTY; DOYLE, 2003).

Lectin-carbohydrate interactions are widespread in most pathogens, and are important for cell-cell communication in various physiological processes, playing a crucial role in the pathogenic event 25 (LINDHORST; KIEBURG; WENZEL, 1998; OFEK; DOYLE, 2003). Lectins, in gram-negative bacteria, are located in the fimbriae, being able to recognize and bind to complementary carbohydrates available on the surface of the host gastro-intestinal tissue, thus initiating the bacterial adhesion process (LINDHORST; KIEBURG; WENZEL, 1998; 30 MORAN, 2011). The fimbriae are protein filaments that protrude from the bacterial surface and have adhesion capacity due to the presence of lectins. The pathogenesis of Salmonella enterica infections requires adhesion to various host cell surfaces, and a large number of adhesive structures 5 can be found. Depending on the serotype (serovar) genes for various types of fimbria adhesins are identified (WAGNER; HENSEL, 2011). Control of contamination by Samonella sp.

In livestock growth-promoting antibiotics (GrowthPromoting Antibiotics) have been used frequently in recent decades to control intestinal pathogens (reducing mortality) and as promoters of performance or animal health (BOROWSKY, 2009; GANNER1 2011; FERKET, 2011). One of the most important consequences of prolonged use of these antibiotics has been the emergence of bacteria resistant to this type of treatment, in addition to causing several side effects (SHARON; OFEK, 2000; SPRING; PRIVULESCU, 2012; HAJATI; REZAEI, 2010; GANNER, 2011). Consequently, research on alternatives to antibiotic use has been intensified (SHARON; OFEK, 2000; BIGGS; PARSONS; FAHEY, 2007; GANNER, 2011). Natural alternatives such as essential oils, yeasts, prebiotics, probiotics, oligosaccharides, polysaccharides, glycoconjugates are the focus of interest and are increasingly being studied (MORAN, 2011; GANNER, 2011; KEMPERMAN, RA; MELLEMA, M. Prebiotic use of water soluble soybean polysaccharide WO 2011088943 (Al), Dec. 21, 2010; ZHAN, Y. A method of preventing animals from diseases and improving immune function of animals (EP 0826303 (A1), Mar. 04,1998).

Currently one of the commercially available alternatives to the use of proven antibiotics is a cell wall product of the Saccharomyces cerevisiae yeast. The yeast cell wall is predominantly composed of β-glucans (35-55%) and 30 mannoproteins (30-50%) (KLIS; BOORSMA; GROOT, 2006; KOGAN; KOCHER, 2007). Immunomodulatory activities have been described for βglucans (ΒΟΗΝ; BeMILLER1 1995; KOGAN; KOCHER, 2007; BUSSIERE,

2010) and mannoproteins are being correlated to bacterial adsorption activities (OFEK; HASTY; SHARON, 2003; MORAN, 2011). Thus the characteristic of manooligosaccharides in increasing the resistance of

against enteric diseases, and promote growth is given because of its ability to: 1) inhibit colonization of enteric pathogens by blocking bacterial adhesion (OFEK; HASTY; SHARON, 2003; SPRING; PIVUULESCU, 2012; MORAN, 2011) ; 2) increase immunity (FERKET,

2011); 3) and increase the mucus barrier in the intestinal lumen by producing immunoglobulin A - IgA (MCKAY; PERDUE, 1993; BIGGS;

PARSONS; FAHEY, 2007).

Research with different alternatives is being published in the last decade. Trichosporon mycotoxinivorans cell wall adhered to enterobacteria in vitro (GANNER et al., 2010). Lactobacillus johnsonii was able to compete for the same receptors as enteropathogenic bacteria (NEESER et al. 2000). Lactobacillus aeidophilus interfered with the adhesion process of Salmonella sp. (NAKAZATO et al., 2011). Microalgae sulfated polysaccharides inhibited the cytoadhesion process by Helicobaeter pylori (GUZMAN-MURILLO; ASCENCIO, 2000). Milk and its components 20 (MARTÍN-SOSA; MARTÍN; HUESO, 2002; MARTÍN; MARTÍN-SOSA; HUESO,

2002), cranberry (eranberry) (BURGUER et al., 2000), carrot, coffee, mango (BECKER et al., 2007; BECKER; GALLETTI, 2008) and soybean (NEESER; KOELLREUTTER; WUERSCH, 1986; KIERS et al. , 2002; KIERS et al., 2003; BEGKER et al., 2007; BECKER; GALLETTI, 2008) were able to adsorb enterobacteria in vitro.

The ability to block bacterial adhesion represents an ideal strategy to combat bacterial pathogenesis due to its importance at the beginning of the infectious process. It is also suitable for application as a prophylactic strategy to prevent infection, since preventing the colonization of pathogens in the human and animal intestines reduces the number of gastrointestinal infections and the risk of transmission through the food chain (Becker, 2005; Becker et al. al, 2007; GANNER, 2011; GANNER et al, 2010).

The main disadvantage of anti-adhesion therapy is that most pathogens have genes encoding more than one type of adhesin, so that during the infectious process more than one of these adhesins can be expressed by the pathogen population. Adherence may also involve factors other than adhesin receptors, such as hydrophobic interactions or other non-specific interactions. For anti-adhesion therapy to be completely effective, it is necessary to use multiple agents that specifically inhibit each type of adhesin of the infecting pathogen or a single agent with broad spectrum of anti-adhesion activity (BECKER; GALLETTI, 2008).

In an attempt to reduce the number of enteric diseases and based on these concepts of anti-adherence therapy, many producers are including mannose-rich materials in animal diets (Molist et al, 2011). The anti-adhesion mechanism is not fully elucidated (SPRING; PRIVULESCU, 2012; HAJATI; REZAEI, 2010), however some theories are described. The most widespread is evidenced by the use of analogous receptors, where enterobacteria (eg Salmonella sp.), Through the Iectins located in the fimbriae, will bind to free mannose (adsorption phenomenon) due to its high availability and concentration, thus avoiding adhesion and infection process (OFEK; MIRELMAN; SHARON, 1977; MORAN, 2011). The bacteria bound to the mannose-rich material will then be flushed out of the intestine. This mechanism is known as the “Oligosaccharide Decoy Mechanism”, ie oligosaccharide trap mechanism (FIRON et al, 1987; MORAN, 2011).

Another theory regarding anti-adhesion therapies is based on the use of analogous adhesins. These isolated adhesin molecules bind to receptors and competitively block bacterial adhesion (OFEK; HASTY; DOYLE, 2003). However, the use of adhesin analogues for anti-adhesion therapy may be impractical, as they are typically macromolecules that are not readily available and need to be employed in high concentrations. In addition, careful attention should be given to their toxicity and immunogenicity (BEACHEY, 1981; OFEK; HASTY; SHARON,

2003).

Saccharides are therefore ideal as anti-bacterial adhesion agents.

pathogenic because they are non-toxic, non-mutagenic and non-bactericidal, thus avoiding the risk of resistance (SHARON; OFEK, 2000). It is noteworthy that information about their sources of obtaining, accessibility, concentration and possible structures of receptors for bacterial adhesion 10 are not yet explicit. Further studies are needed to elucidate such information (BECKER; GALLETTI, 2008).

There are several documents reporting carbohydrate activity in the treatment and prevention of infectious diseases caused by bacteria.

US 6,001,819, US 5,736,533 and WO 9640169 refer to a method of treating respiratory infections caused by S. pneumoniae, H. influenzae, H. parainfluenzae, Pseudomonas cepacia bacteria by administering an effective binding inhibitor whose formulation contains oligosaccharides. Oligosaccharide units contain: galactose, glucose, Nacetylglucosamine, N-acetylgalactosamine, fucose and N-acetylneuraminic acid. These can be obtained from enzymatic synthesis, chemical synthesis or degradation of natural or isolated oligosaccharides, glycolipids and glycopeptides from human milk (SIMON, OM et al. Bacterial inhibition with an oligosaccharide compound. US 6,001,819, Dec. 14, 1999; SIMON, OM Bacterial inhibition with an oligosaccharide compound US 5,736,533, Apr 07, 1998; SIMON, OM; BARTHELSON, RA; JOHNSON, KF Bacterial inhibition with an oligosaccharide compound WO 9640169 (A1), Dec. 19 , 1996).

WO 2007138047 reports the use of tri and tetraoligosaccharides as binding agents for pathogens. In particular it reports how to use these oligosaccharides to improve the flora of the animal's gastrointestinal tract followed by specific excretion of enteric pathogens, improving weight gain, reducing food conversion and thereby increasing animal health and welfare (BRUGGEMAN, DESCHEPPER, K. Tri and tetraoligo saccharides suitable as agglutination agents for enteric pathogens (WO 2007138047 (A2), Dec. 06, 2007).

WO 0165949 reports the use of carbohydrates (oligosaccharides) not

digestible in the manufacture of a composition for treating and preventing pathogenic bacteria in the large intestine of domestic animals. The raw materials that give rise to these carbohydrates are: coconut fiber, beet pulp, chicory, rice bran, locust bean or gum arabic (BAILLON, ML; 10 GIFFARD, CJ Treatment of infection in animals. WO 0165949 (Al) , Sep. 13, 2001).

WO 0051644 relates to a composition and method for treating diarrhea, especially those mediated by enteropathogenic E. coli (EPEC), using oligosaccharide compositions. The oligosaccharide sequences involved in EPEC binding are mainly composed of: Nacetylgalactosamine, difucosyl lactose, lacto-A / fucopentose isomers; GM1, GM2 and GM3 gangliosides and glycoprotein-linked lactosamine (VANMAELE, R. P.; ARMSTRONG, G. D. Treatment or diarrhea caused by enteropathogenic Escherichia coli. WO 0051644 (A1), Sep. 08, 2000).

EP 0171026 reports a feed used in livestock containing inoculum

oligosaccharides wherein one to five fructose units are linked by β- (1- »2) bonds. This food can be used as a method of promoting growth and minimizing recurrent diarrhea during the growing season. Inulooligosaccharides are easily obtained from 25 Helianthus tuberosa inulin (HIDAMAKA, H .; EIDA, T .; HAMAYA, T.. Livestock feed containing inulooligosaccharides and breeding from Iivesotock by using the same. EP 0171026 (B2) , Oct. 02, 1991).

EP 0549478 is to provide of a method of preparing glucogalactooligosaccharides consisting essentially of glucose and galactose (1: 1), obtained by heating lactose in the presence of an inorganic acid and water in a small amount (20%) at a temperature of 120-200 ° C. 0C for 5-20 s in an extruder. Another objective of this same invention is the creation of a livestock feed, preferably used during the growing season, enabling the reduction of diarrhea and pasty feces (KATTA, Y. et al. Method for preparing galacto-oligosaccharides and feed for livestock containing the EP 0549478 (B1), Sep. 17, 1997).

WO 2010120682 is a feed or pharmaceutical composition containing one or more milk-derived oligosaccharides, or a glycoconjugate with the property of treating animal infections (MORROW, AL; NEW-BURG, DS; RUIZ-PALACIOS, GM Milk oligosaccharide 10 compositions and use thereof in treating infection in animals (WO 2010120682 (Al), Oct. 21, 2010).

WO 03045401 reports a method for improving the health and production of ruminants by administering effective amounts of undigested oligosaccharides from the gluco- and / or isomalto15 oligosaccharide group. These oligosaccharides are able to decrease the incidence of diarrhea and increase the lactation of animals (LANE, R. L. Compositions and methods for animal treatment. WO 03045401 (Al), Jun. 05, 2003).

WO 03028738 reports the use of linear or branched chain neutral oligosaccharides to prevent pathogen invasion and infection of mammalian cells. The invention reports its use in foods, diet products and pharmaceutical agents. Oligosaccharides consist of a backbone composed of [Gal-HexNAcj-Gal-Glc, joined together by β- (1 -> 3) or β- (1—> 6) glycosidic bonds (STAHL, B. et. al Antiinfectious carbohydrates (WO 03028738 (A2), Apr. 10, 2003).

WO 9702829 claims a method for reducing the duration of diarrhea and

recurrent episodes in humans through the administration of undigested oligosaccharides or fructooligosaccharides. These oligosaccharides may be produced from enzymatic synthesis, chemical synthesis or be isolated from plants. Its form of administration may be through candies, tablets, gums, milk, yoghurts, fermented products and nutraceuticals (DOHNALEK, M.I.H.; OSTROM, K.M.; HILTY, M..D. Use of indigestible oligosaccharides to prevent and treat diarrhea. WO

9702829 (A2), Jan. 30, 1997).

US 5,776,524 reports the process of producing an animal feed useful for reducing the amount of bacteria harmful to the small intestine. The composition of the animal feed contains, based on a dry matter, from 0.2 to 1.5% fructooligosaccharides and is given to a pet such as dogs, cats or horses. These fructooligosaccharides occur naturally and can be found in a variety of fruits and vegetables including bananas, garlic, honey, onions, brown sugar, barley, tomatoes and asparagus. There are three varieties of 10 fructooligosaccharides: 1-kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4), which are either extracted from plants or chemically synthesized (REINHART, GA). Jul. 07, 1998).

WO 8903218, WO 9107181, US 4,902,674 and US 4,987,124 claim a fructooligosaccharide composition that inhibits the growth of Salmonella in the gut preventing infections. The composition is fermented by other microflora that compete with Salmonella, producing metabolites that inhibit Salmonella growth. Fructooligosaccharides are characterized by sucrose molecules with 1-8 fructose residues (SPEIGHTS, RM; PERNA, PJ. Method for inhibiting growth of Salmonella. WO 8903218 (Al), Apr. 20, 1989; SPEIGHTS, RM; PERNA, PJ. ; DOWNING, SL Method for inhibiting the growth of Salmonella WO 9107181 (A1), May 10, 1991; SPEIGHTS, RM Method for inhibiting the growth of Salmonella US 4,902,674, Feb. 20, 1990; SPEIGHTS, RM; PERNA, PJ; DOWNING, SL Method for inhibiting the growth of Salmonella (US 4,987,124, Jan. 22, 1991).

US 5688777 involves the use of undigested oligosaccharides to treat and prevent infections caused by C. difficile (GAELEB, A. A. Inhibition of C. difficile infections by indigestible oligosaccharides. US 5688777, Nov. 18, 1997). WO 9826662 reports compositions and methods for treating and preventing bacterial and viral diseases. Such compositions comprise polymer-linked oligosaccharides, more specifically, dendrimer-linked, inhibiting the binding of pathogenic microorganisms to their receptors located on host cells. The source of oligosaccharides include glucoproteins, glycosaminoglycans and glycolipids (SCHENGRUND, C.L.; THOMPSON, J. Compounds and methods for treating and preventing bacterial and viral disease. WO 9826662 (Al), Jun. 25, 1998).

US 7,893,041 deals with specific oligosaccharides or combinations of 10 oligosaccharides that can be used to treat or prevent infections caused by various agents associated with enteric disorders, respiratory infections, vaginal infections, urinary tract infections, eye infections or oral cavity infections. Oligosaccharides may be administered in mono (one) or polyvalent (two or more) forms. In monovalent form, free oligosaccharides may be administered alone or in combination. Oligosaccharides are composed primarily of a α (1-2), α- (1-3), α- (1-4) linked galactose fucose group (MORROW, A. L .; NEWBURG, D.

S.; RUIZ-PALACIOS, G. M. Oligosaccharide compositions and use thereof in the treatment of infection. US 7,893,041 (B2), Feb. 22, 2011).

WO 2011073112 reports α-Manp derivatives as antagonists

Efficiencies of bacterial adhesion, preventing and treating infections (ERNST, B.; HEROLD, J. Mannose derivatives as antagonist of bacterial adhesion. WO 2011073112 (A2), Jun. 23, 2011).

JP 8173055 is a pet food containing a mixture of polysaccharides with mannose, which presents excellent productivity and effective prevention against Salmonella. Polysaccharides are obtained from an enzymatic treatment of coconut pulp residues (SHINICHI, H.; TAKASHI, K.; MITSURU, I. Mannose-based polysaccharid-containing feed. JP 8173055 (A), Jul. 09, 1996).

US 5,703,060 deals with a method of prevention and treatment of

infections and infestations in animals by administering an effective amount of an acetylated mannan derivative. This manna is extracted from Aloe mucilage, its main chain is composed of p-D-Manp- (1-4) -linked units. Galactose residues are present in a 20: 1 mannose: galactose ratio (MCANALLEY, B. H .; CARPENTER, R. H .;

MCDANIEL, H. R. Uses Aloe products in the prevention and treatment of infections and infestations. US 5,703,060, Dec. 30, 1997).

WO 2009144070 reports a premix for animal feed capable of inhibiting colonization of gram-negative bacteria and promoting animal growth, comprising: polymers with 10-100% mannose content, a β-mananase enzyme and an anti- adhesion capable of inhibiting 20% of bacterial bonds (BRUFAU, JB et al. An enzymatic pre-mixture against gram negative bacteria coionization in the intestinal animal tract. WO 2009144070 (A2), Dec. 03, 2009).

JP 8099884 is a polysaccharide hydrolyzate capable of removing gram-negative bacteria from mammals, birds and animal products such as eggs, thereby preventing the proliferation of these bacteria. Polysaccharide hydrolyzate is obtained from enzymatic hydrolysis (galactomannanase or pectinase) of guar gum, tamarind gum, pectins and xanthan gum (TSUTOMU, O .; YUKIKO, N.; SHIGEMITSU, A. Supressant for 20 profiferation of gram- negative bacterium (JP 8099884 (A), Apr. 16, 1996).

US 6,126,961 claims an animal feed composition for reducing colonization by Salmonella and other pathogenic bacteria in animal intestines. Such composition includes natural polysaccharides containing mannose units and derivatives, galactose and derivatives, galactomannans, galactosamine, fucose and arabinose. These polysaccharides are incorporated into the feed and bind to pathogens adhesins (KROSS, R. D. Compositions and method for reducing the colonization of animal intestines by Salmonella and other bacterial pathogens. US 6,126,961, Oct. 03, 2000).

EN 1357917, WO 02060452 and EP 1698341 report the use of non-digestible carbohydrates by the digestive tract in the treatment of intestinal infections in animals caused by bacteria of the genera Salmonella, Escherichia and Shigella. These consist of 1-OaD-glucopyranosyl-Dsorbitol, 6-OaD-glucopyranosyl-D-sorbitol, lactobionic acid and maltobionic acid (MUNIR, M. et al. Use of carbohydrates to eliminate intestinal infections in animals. EN 1357917 (E), Dec. 17, 2001;

KINGINGBERG, M. et.al. Use of carbohydrates for eliminating intestinal infections in animals. WO 02060452 (A2), Aug. 08, 2002; KLINGEBERG, M. Use of carbohydrates for eliminating intestinal infections in animals. EP 1698341 (A2), Sep. 06, 2006).

WO 0054788 is a pharmaceutical composition for veterinary use containing lactic acid, undigestible carbohydrates, calcium and aluminum salts. This composition regenerates the intestinal flora during diarrhea or dyspeptic syndromes, acting in the treatment of dysmicrobism after antibiotic use in digestive diseases of animals. (GALLI, G. Pharmaceutical composition for medical and veterinary use for intestinal regeneration 15 WO in diarrhea and ordyspeptic syndrome. WO 0054788 (A1), Sep. 21, 2000).

WO 9956754 deals with compositions containing bound fucose (1 -> 2) residues. These compositions may be used in the treatment and prevention of gastrointestinal infections caused by Escherichia coli and Vibrio choierae (PRIETO, PA; RUIZ-PALACIOS, GM Compositions containing a1,2-Fucose Iinkage and uses thereof. WO 9956754 (Al), Nov. 11, 1999).

EP 0089940 discloses a therapeutic composition used in the treatment of gastrointestinal infections caused by gram-negative bacteria, especially E. coli K88 +. The active compound functions as an analogous receptor for the bacterium (LINDBERG, A. Compositions for therapeutic or diagnostic use containing oligosaccharides. EP 0089940 (A1), Set. 28, 1983).

WO 8102520 claims compositions used in the treatment of bacterial urinary infections containing as active constituent αD-Galp- (1- * 4) -D-Gal terminal units (KELLENIUS, G. et al. Compositions for therapeutic or diagnostic use in connection with bacterial infections, and their use (WO 8102520 (A1), Set. 17, 1981). PT 103983 refers to compositions containing one or two sulfated polysaccharides obtained from macro and microalgae with antiviral and antibacterial activity. Polysaccharides were obtained from Phorphyridium sp., For this purpose the cell cultures were centrifuged (10000 5 rpm, 20 min), filtered, ethanol precipitated and dialyzed against distilled water (VIEIRA, VV; MORAIS, RMSC. polysaccharides with anti-viral activity and anti-bacterial adhesion, their formulations, process for making them and their uses (PT 103983 (B), Feb. 02, 2008).

US 5,474,986 is a method for treating infections caused by

by uropathogenic bacteria, mainly E. coli, by modified galabiosis derivatives at the 3 'and anomeric positions. Investigations indicate that modifications to more than one hydroxyl group result in a drastic reduction in glycoside's ability to inhibit agglutination of red cells by E. coli mutants (MAGNUSSON, HG; KIHLBERG, JO). (US 5,474,986, Dec. 12, 1995).

US 7,682,631 is directed to compositions that prevent or treat infections caused by a variety of pathogens. Biofunctional materials include glycolipids, glycoproteins, peptides, polypeptides, lipids, monosaccharides and polysaccharides. These are identical to receptors located on the surface of host cells and can be recognized and bound by pathogen adhesins. Thus, these compositions can be used to prevent enteric infections, once the pathogen is bound, it is easily removed from the gastrointestinal tract (STUTZENBERGER, FJ et al. Adhesin-specific nanoparticles and process for using same. US 7,682,631 (B2), Mar. 23, 2010).

The object of the present invention is to provide a more specific active product from soybean meal extract by improving the effects on the microbial ecosystem of the gastrointestinal tract. More particularly, the invention aims to provide a protein and carbohydrate extract that selectively controls the microbial ecosystem of an animal's gastrointestinal tract by selecting enteropathogens and causing their excretion after binding to fimbria adhesins.

Description of the technical problem approach

Gastrointestinal disorders caused by enterobacteria are continuously causing morbidity and mortality in humans, other mammals, and other animals such as birds. Problems associated with frequent drug use in the treatment of these diseases include lack of efficacy and / or specificity, toxicity, and development of bacterial resistance. Thus, non-toxic and therapeutically efficient agents are required for the treatment of these diseases. In this problem, the focus was to find an alternative to the use of antibiotics.

Carbohydrates and proteins have been shown to act as enterobacterial-like receptors so that pathogens recognize them, and host tissue adhesion is inhibited resulting in a lack of ability to initiate infection. The present invention relates to compositions for treating and preventing bacterial diseases, in particular carbohydrate derivatives and proteins that inhibit receptor binding in cells. While the complete mechanism is not fully understood the present invention may be limited to some mechanisms by which the benefit is achieved. It is then contemplated that the carbohydrate / protein derivatives in the present invention mimic the glycid / protein portion of the cell receptors and thus provide an alternative object for the microorganism. The present invention contemplates that such inhibitors may be used for both treatment and prevention of diseases caused by enterobacteria (also as prevention of the spread of infection in an individual with the emerging disease).

Therefore, the main object of this invention is to produce an extract from soybean meal with the potential to adsorb enterobacteria according to the processes and steps described herein. The first object of this invention deals with the chemical processes of preparation and production which enable the production of a product from soybean meal containing high protein and modified carbohydrates. The present invention, unlike the state of the art, is shown as a method: (A) inexpensive - use of low value chemicals in small quantities; (B) fast - small number of steps; (C) that does not require mandatory additional purifications; (D) easily scaled; (E) uses low-cost, large-scale raw material available; (F) results in value added to the oil industry byproduct.

The second object of this invention deals with the use of said extracts of

soybean meal in the prevention and / or treatment of gastrointestinal diseases in mammalian and / or poultry animals mainly caused by enterobacteria, in particular Salmonella enterica sv. Typhimurium.

Figure quote

Next, reference is made to the drawing attached to the text for better

understanding of the examples and embodiments presented:

Figure 1: This is a flowchart illustrating the production process of soybean meal extract with bacterial adsorption capacity. The numbers represent the following operations: 1- dilution, pH adjustment, autoclaving and filtration; 2-pH adjustment and centrifugation; 3- autoclaving, dialysis and lyophilization. The acronyms: SOB, PPT, RES represent supernatant, precipitate and residue, respectively.

Detailed Description of the Invention The process proposed in the present invention is depicted in Figure 1 in flowchart form. Next, the series of steps of this process will be detailed, from the initial preparation of the raw materials to the final obtainment of a soybean meal extract with bacterial adsorption capacity.

The raw material used is soybean meal, obtained after mechanical and / or chemical extraction of oil from soybeans. The characteristics of the raw material used are fundamental and may interfere in the generation of the final product. Soybean meal must have a maximum of 10% residual oil, be free from the presence of organic solvents used during processing for oil extraction and dry with moisture content between 5 20%. Sometimes the raw material is not compliant for use and needs to undergo previous treatments. Various types of suitability may be included at the discretion of the operator. Options include particle size adjustment, drying, homogenization and sterilization.

In a first step, extraction is performed in aqueous medium, buffered or not, in a concentration of 100 - 500 g / L, with a pH lower than

9, preferably between (0-3) and (6-8). The pH adjustment is made with the use of acids, preferably hydrochloric acid, or alkaline agents, preferably sodium hydroxide. The use of any of these chemical compounds may be sufficient, however the combination of two or more types of compounds may be used for pH adjustment.

The temperature ranges from 100 - 130 ° C, preferably between 115 - 125 150 ° C. In the present invention, heat treatment should preferably be carried out under the application of relative pressure of less than 1.5 atm for 30-120 minutes. Time periods of less than 30 minutes do not give rise to active extracts due to the fact that they do not cause partial hydrolysis of the material; time periods greater than 120 minutes at these temperatures may result in excessive hydrolysis of the material. The temperature should be monitored and kept constant throughout the process.

After said heat treatment, the mixture is processed using suitable media such as cloth strainer, filter paper, vacuum filtration system, wire mesh, etc. or by centrifugation. The residue then undergoes a drying or lyophilizing process to remove moisture and can be used in a new extraction cycle, resulting in increased final yield.

The second step begins with neutralization of the supernatant obtained after the filtration process. This neutralization (pH 6.0 - 8.0) is carried out in the same manner as in the previous step by the use of acids or alkaline agents. After neutralization the material should be centrifuged. The residue obtained can be lyophilized and added to the residue obtained in the first step.

In a third step the obtained supernatant may be subjected to an optional substep which is a new heat treatment cycle, similar to the process already described, with a temperature between 100 - 130 ° C under the application of relative pressure less than 1.5 atm. for 10 - 45 minutes. Temperature should be monitored and kept constant throughout the process. Following the completion of the additional substep, volume reduction should be carried out. The extract is then dried, resulting in a soybean meal extract 10 capable of adsorbing enterobacteria.

The product then obtained consists of: 20 - 70% protein and 10 60% carbohydrate. Further analysis has shown that these carbohydrates are mainly composed of β-D-galactopyranose units joined by β- (1-4) bonds.

To improve extraction and recovery processes, the material can be

be subjected to known physical or chemical processes such as the use of sonication, homogenization, use of surfactants, etc.

The degree of purification does not influence the increase of bacterial adsorption activity, so the crude soybean meal extract does not necessarily require further purification steps. However such subsequent purification processes have been envisaged and can be performed on soybean meal extract. The use of proteolytic enzymes such as protease, peptidase (pepsin, trypsin, papain, subtilisin, bromelain, etc.) for protein hydrolysis and strong acids (trichloroacetic acid) or ammonium sulfate solutions that cause protein precipitation. Polar organic solvents (ethanol, methanol, isopropanol, n-propanol, acetone, etc.), preferably 1: 5 (v / v) ethanol, may be added for the purpose of precipitating macromolecules. After purification the product thus obtained is composed of: 5-40% protein and 25-70% carbohydrate. Further analysis showed that the carbohydrates present do not change and remain mainly composed of bonded β-D-galactopyranose units (1-4).

The determination of enterobacteria adsorption capacity was performed by in vitro bioassays. In the bioassays, the products resulting from the process were diluted in an appropriate proportion to reach the final concentration established.

The examples of this invention serve to prove that extraction products and subsequent purification steps (optional) are effective in producing an extract capable of adsorbing enterobacteria.

To determine the adsorption capacity, the adaptation of the methodologies proposed by Becker et al. (2007) and Ganner et al. (2010). These methodologies determine in vitro the ability to bind to enteropathogenic bacteria through the optical density (OD) x colony forming units (CFU) / mL ratio.

The tests performed with the extracts were statistically valid and significantly different from the binding control (this works as a negative control). The extracts were similar in capacity to the positive control. Under these circumstances, the results obtained prove that the proposed process steps are effective for producing an extract capable of adsorbing enterobacteria.

This invention also relates to a method of treating diseases caused by enterobacteria, especially in the case of salmonellosis, by administering an effective amount of soybean meal extract. Thus, the extract may be used in the prevention and treatment of gastrointestinal disorders.

The extract can be used in vivo, in combination with feed, to treat infected animals suspected of being infected with enterobacteria. This makes it possible to eliminate or prevent transmission to humans from animals. It is emphasized that the final composition must necessarily include an effective amount of any of the proposed process products. The present invention is explained in more detail by the following examples, but is not limited by said examples.

Example 1 - Obtaining a soybean meal extract capable of adsorbing enterobacteria

Soybean meal diluted in distilled water (150 g / L) had its pH

adjusted to 2.0 with 1 M HCl, and was subjected to a relative pressure of 1 atm, 121 ° C for 1 h. After this treatment the material was filtered, neutralized with 0.5 M NaOH and centrifuged. The supernatant was again autoclaved for a further 15 min, then concentrated, dialyzed against distilled water, concentrated again, lyophilized and labeled as EA. The composition of the soybean meal extract obtained is shown below (Table 1). In the 13 C nuclear magnetic resonance (NMR) spectrum it is possible to identify the presence of characteristic carbohydrate (110-60 ppm) and protein (10 to 55, 130, 170 to 180 ppm) signals. The anomeric region (110 - 90 ppm) has a major signal at 104.4 ppm relative to the C-1 of β-D-Galp units (FRANSEN et al., 2000; ClPRIANI et al., 2004; LIMA, 2008) . Signs are observed in 77.6; 74.5; 73.5; 72.1; 60.9 ppm, corresponding to C-4, C-5, C-3, C-2 and C-6, respectively, of the β-D-Galp-O-> 4) -linked units (GORIN; MAZUREK, 1975; FRANSEN et al., 2000; LIMA, 2008; CIPRIANI et al., 2009).

TABLE 1 - CHEMICAL ANALYSIS AND MONOSACARIDIC COMPOSITION OF THE OBTAINED FRACTION

Fraction Yield PTN Carbohydrates Composition Solid monosac (mol% Ld (%) a (%) b Totals (%) c Rha Fuc Ara Xyl Man Gal I Glc UAe EA 13.4 59.8 30.8 3.5 1.2 19 .8 6.2 1.2 31.7 I 5.5 30.8 PTN: Proeins; UA: Uronic Acids; Rha: Ramnose; Fuc: Fucose; Ara: Arabinose; Xyl: Xylose; Man: Manose; Gal: Galactose; Glc: Glucose; Yield to dry matter bran

initial soybean; LOWRY hey there. (1951); DUBOIS ef al. (1956); Neutral monosaccharides quantified in the form of GLC alditol acetates, and FILIZETTI-COZZI; CARPITA (1991). 10

15

Example 2 - Obtaining a polysaccharide-rich soybean meal extract capable of adsorbing enterobacteria

The soybean meal extract obtained in example 1 was dissolved (2.5% w / v) in 50 mM phosphate buffer, pH 7.0 and a protease - Pronase E (1% w / w) was added. The mixtures were then incubated at 37 ° C for 24 h. After the incubation period, the enzyme was inactivated (80 ° C, 20 min), the suspension was centrifuged and the supernatants were dialyzed against distilled water in and lyophilized, resulting in the EAte fraction. The composition of the soybean meal extract obtained is shown below (Table 2). The 13 C NMR spectrum of the EAte fraction shows a similar profile to that of the EA fraction. In the anomeric region (110 - 90 ppm) an intense signal can be seen at 104.4 ppm for β-D-Galp units (FRANSEN et al. 2000; CIPRIANI et al. 2004; LIMA 2008). Chemical shifts are also observed in 77.6; 74.5; 73.5; 72.1; 60.9 ppm, corresponding to C-4, C-5, C-3, C-2 and C-6, respectively, of the pD-Galp- (1-4) -linked units (GORIN; MAZUREK, 1975). ; FRANSEN et al, 2000; LIMA, 2008; CIPRIANI et al, 2009).

TABLE 2 - CHEMICAL ANALYSIS AND MONOSACARDIC COMPOSITION OF THE OBTAINED FRACTION

Fraction Yield PTN Carbohydrates Monosaccharide composition mol% td (%) a (%) b Totals (%) c Rha Fuc Ara xyi Man Gai Gic UAe 32.1 8.8 65.5 7.1 1.6 7.6 7.6 7 .8 1.4 38.8 0.9 34.9 PTN: Proelnas; UA: Uronic Acids; Rha: Ramnose; Fuc: Fucose; Ara: Arabinose; Xyl: Xylose; Man: Mannose; Gal: Galactose; Glc: Glucose; a Yield relative to initial dry matter of soybean meal b LOWRY etal. (1951); dUBOIS et al (1956); d Neutral monosaccharides quantified in the form of GLC alditol acetates; 0 FILIZETTI-COZZI; CARPITA (1991).

Example 3 - Evaluation of Enterobacterial Adsorption Ability by Soybean Meal Extracts

The tests were performed with Salmonella enterica sv. Typhimurium ATCC 14028, maintained in BHI medium.

The first step of the protocol is to make a Uog Ion (Colony Forming Units) x detection time graph. The detection time corresponds to the time required to reach the optical density of 0.1 upon bacterial growth. Detection time was determined from nonlinear regression as described in Becker et al. (2007).

For this, the bacteria were cultured in BHI (Brain Heart 5 Infusion) medium for 24 h at 37 ° C. The bacterial suspension had its optical density set to 0.1. From this suspension, successive dilutions were made in PBS (10 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0). From each dilution, 0.1 ml was used for plating and 0.1 ml for reading optical density. Plating was performed at 10'6, 10'7, 10'108 dilutions in triplicate in BHI-Agar by depth, followed by incubation at 37 ° C and subsequent CFU counting.

For optical density readings, high-adherence BSA-blocked polystyrene plates filled with 200 μΙ_ TSB (Tryptic Soy Broth) medium incubated at 37 ° C for 8 h in 15 microplate reader were used, providing readings optical densities at 690 nm every 15 min. Wells were blocked by incubating 300 µl of 1% BSA solution for 1 h at 40 ° C, followed by washing with 300 µl PBS 3 times. Each dilution was plated in quintuplicate.

At the end a curve was plotted, where Iog UFC is on the ordinate axis and detection time on the abscissa axis.

Sample solutions at a concentration of 1% were dissolved in vortexed PBS1 and homogenized in an ultrasonic bath for 30 seconds three times. Then 300 pL of this suspension was transferred in quintuplicate to the wells of the high-adherence polystyrene plate. After incubation at 40 ° C for 16-18 h, the plate was washed three times with 300 µl PBS per well.

Blocking of spaces not occupied by the product and prevention of nonspecific binding occurred with 300 µl 1% BSA, 40 ° C for 1 h, followed by washing with 300 µl PBS three times. After bacterial growth and proper dilution, 300 µl of the bacterial suspension was added to the wells, which were incubated for 60 min at 37 ° C to allow their binding to the test product. After washing six times with 300 μΙ_ of PBS1 to remove non-binding bacteria, 200 µl TSB1 and one drop of paraffin oil were added so that the medium did not evaporate. Thus the plate was incubated in a microplate reader at 37 ° C with readings every 15 min at 690 nm for 16 h.

Binding control consisted of wells containing only BSA blockade and bacteria without adding the product to be tested. Growth control consisted of wells previously blocked with BSA containing a bacterial solution in TSB medium so that it could be monitored.

some problem during the growth of bacteria. Whites from each sample were also plated, these consisted of wells previously blocked with BSA containing the product to be tested without addition of bacteria. As a positive control a commercial product from S. cerevisiae yeast was used.

The amount of adhesion numbers (CFU / 3mg) between bacteria and

samples of interest are listed in Table 3. They were calculated based on the linear regression obtained from the standard curve (y = -0.9854x + 9.559 R2 = 0.9925) so that 99.25% of the results are represented by this curve.

20

TABLE 3 - DETECTION TIME AND BACTERIA QUANTITY CONNECTED BY 3 MG OF TESTED FRACTION_

Fractions toD = 0.1 (h) CFU / 3mg EA 4.25 2.36 x 105aA EN 4.34 1.93 x 105aA Between 4.65 9.53 x 104aB Positive Control 4.88 5.61 x104aB EAte 4.92 5.19 x 104aB Negative Control (BSA) 6.19 2.85 X 103Bb toD = o, i: Detection time for optical density of 0.1; EA: Soluble fraction obtained from acid extraction; EN: Soluble fraction obtained from neutral extraction; EAte: Fraction 25 obtained after proteinase AE treatment; ENte: Fraction obtained after treatment with proteinase EN; a'b Means followed by different letters indicate significant difference (Tukey test: P <0.05). A, B Averages followed by different letters indicate significant difference (Tukey test: P <0.01). The EA fraction had the largest number of bound cells / 3 mg (mass of the fraction tested per plate well), 2.36 x 105 CFU; followed by the EN, Ente and EAte fractions that showed bound bacterial cell amounts between 1.93 x 105 and 5.19 x 104 CFU. All fractions differed significantly from the negative control (BSA). The results showed no significant difference to the positive control, but are important, since they presented results statistically comparable to the binding capacity of the tested commercial product. These fractions are similar, with galactose as the major sugar even after enzymatic treatment.

Claims (19)

1. Soybean meal extract as a bacterial inhibitor and process for the production of soybean meal, characterized by the use of soybean meal in the extraction of compounds containing proteins and carbohydrates, with adsorptive capacity to gram-negative bacteria. Process for producing soybean meal extract according to claim 1, characterized in that the solubilization of soybean meal in water or buffered medium at a concentration of 100-500 g / l. Process for producing soybean meal extract according to claim 1, characterized in that the extraction is carried out at a pH lower than 9. Process for producing soybean meal extract according to claim 1, characterized in that the extraction is carried out at a temperature between 110-130 ° C. Production process of soybean meal extract according to claim 1, characterized in that the extraction is carried out at a relative pressure of less than 1.5 atm. Process for producing soybean meal extract according to claims 2 to 5, characterized in that it is carried out between 30-120 minutes. Processes according to any one of claims 1 to 6, which are used to obtain soybean meal extract. Processes according to Claim 7, characterized in that they comprise subsequent purification steps such as dialysis, chromatography, ultrafiltration and use of proteolytic enzymes, strong acids and polar organic solvents, comprising any combination thereof. Processes according to claims 7 and 8, characterized by separation of the extract, comprising filtration, centrifugation or pressing steps, or any combination thereof. Processes according to Claim 9, characterized in that the soybean meal extract is dried or lyophilized. 11. A process for producing soybean meal extract comprising extracting soybean meal with water in a solution of pH less than 9 and temperature between 110-130 ° C for 30-120 minutes generating a protein extract between 20-70% and carbohydrates between 10-60%. Soybean meal extract according to Claim 10, characterized in that it comprises 20-70% protein content. Soybean meal extract according to Claim 10, characterized in that it comprises carbohydrate content between 10-60%. Extract obtained from soybean meal according to claims 2 to 13, characterized in that they are applicable to the treatment and prevention of gastrointestinal diseases. Extract obtained from soybean meal according to claims 2 to 13, characterized in that they are applicable in the feed industry, used in the preparation of feed or its ingredients or used as food supplements or in drinking water. 16. Feed composition that reduces intestinal colonization in animals caused by Salmonella or other enteropathogenic bacteria, characterized by an extract obtained from soybean meal containing protein and carbohydrates and an animal feed in combination with these extracts. Use according to claim 16, wherein the animal is a monogastric animal. Use according to claim 16, wherein the animal is a ruminant animal. Use according to claims 16 to 18, wherein the animal is a pig, bird or cattle.