MX2007006749A - Method of growing bacteria to deliver bioactive compounds to the intestine of ruminants - Google Patents

Abstract

Methods for increasing the resistance to rumen inactivation of a cultured Gram positive bacteria strain useful for gastrointestinal delivery of bioactive compounds to ruminants, which includes the steps of growinga culture of the bacteria strain through at least one passage in a growth medium containing an amount of lysozyme effective to induce the growth of bacterial cell walls resistant to protozoal predation;and recovering the bacteria strain from the lysozyme-containing medium. Rumen-bypass feed supplements produced by the inventive method are also disclosed, as well as methods for supplement­ing the diet of a ruminant with the rumen bypass feed supplements and an in vitro method for evaluating the resistance of Gram positive bacteria strains to rumen inactivation in vivo.

Description

METHOD FOR GROWTH OF BACTERIA TO ADMINISTER BIOACTIVE COMPOUNDS TO THE INTESTINE OF RUMINANTS RECIPROCAL REFERENCE WITH RELATED REQUEST The present invention claims priority under the benefit of 35 U.S.C. §119 (e) of the Provisional Application of E.U.A. Serial Number 60 / 633,611 filed December 6, 2004, the description of which is incorporated herein by reference.

TECHNICAL FIELD This invention relates to a method for identifying microorganisms useful for the gastrointestinal administration of bioactive compounds to ruminants that are inherently resistant to inactivation in the rumen, as well as a method for the growth of useful microorganisms less resistant to inactivation so that these are more resistant to inactivation in the rumen. Microorganisms, when administered orally to ruminants, are capable of administering whole cells gastrointestinally, and the nutrients and bioactive compounds contained within the cells, to ruminants. The present invention also includes the growth of microorganisms more resistant to inactivation in the rumen that are useful for administration Gastrointestinal of bioactive compounds a ??? ruminants and methods to supply the diets of ruminants with these.

TECHNICAL BACKGROUND Probiotic cultures based on Bifidobacterium, Propionibacterium and Lactobacillus have been used increasingly to maintain intestinal function in monogastric farm animals and in humans. The claimed benefits include increased dysregulation, improved immune function and a reduction in gastrointestinal discomforts. Although probiotics, with yeast and fungal probiotics as selected examples, are used in ruminants, the difficulty of ensuring that probiotics pass through the rumen and into the small and large intestines has limited interest in functional intestinal probiotics in ruminants. . The rumen acts as a major barrier to the passage of bacteria in ruminants and experiments have suggested that less than 10% of a bacterial culture added to the diet can be recovered by leaving the rumen. The englobamiento and digestion of bacteria by protozoos are responsible for the majority of the bacterial decomposition in the rumen. The first step and the limiting step in the bacterial decomposition by the protozoa in the rumen is the degradation of the bacterial cell wall. Previous studies have shown that this decomposition is strongly affected by the composition and integration of the cell wall bacterial and that in fact by the growth of the bacterium in the presence of a continuous stress from the degradation of the cell wall or enzyme lysozyme it is possible to "harden" the bacterial cell wall making it more resistant to predation by protozoa. In ruminants, the ingested food enters the reticulum-rumen, the first of the multiple compartments of the stomach possessed by ruminants. Within the reticulum-rumen, the ingested food is pre-digested or degraded by microbial fermentation. Considerable amounts of ingested protein are degraded in the reticulo-rumen to soluble peptides and amino acids. A proportion of these peptides and amino acids become wastes towards ammonia and are no longer useful for ruminants. The remnant is used by the rumen microorganisms and incorporated into its own biomass. When the content of the rumen passes to the abomasum and the intestine, a portion of the microbial biomass of the rumen passes out from the reticulum-rumen with the rest of the contents of the rumen. This microbial biomass is subsequently digested in the small intestine, providing nutrients to the ruminant. However, a significant portion of the bacteria present in the reticulum-rumen is consumed and digested by the population of protozoa residing in the reticulum-rumen. This is a waste process for the host ruminant because the bacterial cells and nutrients contained within the cells do not pass outside the rumen and do not contribute to the nutrition of the ruminant.

Similarly, the animals are fed by bacterial preparations that adhere to the intestinal epithelium thus improving the growth rate of the animal and the conversion of the feed. (U.S. Patent No. 4,980,164, U.S. Patent No. 5,256,425). However, in ruminants, bacterial preparations also have a low survival ratio when they pass through the rumen. To overcome the loss in viability with oral administration, Batich (U.S. Patent No. 6,242,230) describes a process for encapsulating the bacteria within a gel matrix so that they can be administered to the small intestine of the animals. The purpose of Batich is to prevent the host animal from generating an immune response to the bacteria, thus reducing its ability to survive. Batich is only designed to overcome the immune response of the host and does not transmit any resistance to the digestion of the protozoa and therefore the hydrolytic conditions of the rumen can result in the degradation of the encapsulating matrix. This is also an expensive process and uses chemicals that can reduce the viability of certain microorganisms. Because yeasts are many times larger than bacteria, they are not susceptible to predation by protozoa in the rumen as are bacteria but are typically susceptible to lysis within the rumen. Shiozaki et. to the. (U.S. Patent No. 4,562,149) describes a method for the growth of a yeast, Saccharomyces cerevisiae, such that the cell is enriched between 10 and 20% S-adenosil methionine This invention is an attempt to use yeast, instead of bacteria, to synthesize methionine. Although it is novel, it is not economically possible since yeasts are less efficient than bacteria to synthesize said amino acids. Additionally, no evidence is provided to indicate that this method produces a product that is resistant to the degradation of methionine within the rumen. Similarly, Ohsumi et al., Biosci. Biotech Biochem. 58, 1302-1305 (1994) describes a method for yeast growth that is enriched for the lysine content. Although the lysine content is increased above what is normally observed in wild-type yeast, the process has not been considered economical as a method for the production of lysine with rumen deviation. Strauss et al., Can. J.

Anim. Sci., (2004), used Pichia pastoris, another species of yeast to demonstrate that when this organism is genetically designed, it can be used to administer certain recombinant proteins to the small intestine of ruminants. However, Pichia pastoris is not considered safe to feed livestock. Bolla et al (U.S. Patent No. 6,737,262) discloses a method for the incorporation of fungi or other microorganisms into the forage by means of which the organism has been genetically transformed to produce peptides of at least two amino acids, instead of individual amino acids. Additionally, the inventors state that the encapsulation additional may be necessary to ensure that peptides that deviate from the rumen environment. In all the aforementioned cases, manipulation of the yeast cells is required, either by intense selection or genetic manipulation. Typically; Saccharomyces cerevisiae are provided to livestock to provide available nutrients to the rumen and are not particularly suitable for the production of large amounts of compounds that could be bioactive in the small intestine. Although bacteria can be used commercially to produce a wide range of biologically active compounds and nutrients compared to yeast, the goal is to have the compounds or excrete them out of the cell to make the compounds easier to isolate. There is no known method invented by which bacterial preparations grow, either by a selection of intense strain or via culture conditions, so that the bacteria and the bioactive compounds contained therein, are protected from rumen degradation. In the production of bacterial preparations, nutrients and other compounds that have bioactive properties, which are intended for administration to ruminants, it is important to protect the active ingredients against the microbial degradation that occurs in the rumen. It is well known that the speed of production of meat, wool and / or milk can be increased if the sources of essential amino acids that limit growth and other bioactive compounds are protected from the alteration by the rumen microorganisms and are subsequently available for absorption by the animal subsequently in the gastrointestinal tract. Numerous inventions exist for the manufacture of biologically active compounds and stable nutrients within the rumen by encapsulation with a coating or by embedding the compound within a chemical matrix. The Patent of E.U.A. No. 3,959,493, teaches stable products in the rumen that comprise biologically active substances protected with aliphatic fatty acids. The Patent of E.U.A. No. 3,655,864, issued to Grass et al., Teaches veterinary compositions that allow post-ruminal or ruminal administration of biologically active additives for forage, in which the compositions are embedded or covered with a matrix of glyceryl tristearate with a unsaturated higher fatty acid liquid. The Patent of E.U.A. No. 4,473,545, issued to Drake et al., Teaches an animal foraging additive comprising a composition of a relatively insoluble binder, a material soluble in particle and an active material. The particulate material is such that it is readily soluble under a particular range of pH conditions. The dissolution of the materials in the particle returns to the permeable binder in water thus releasing the active material. The Patent of E.U.A. No. 4,533,557 teaches an additive to forage for ruminants comprising a mixture in the form of a tablet or in the form of a granule of at least one biologically active ingredient, chitosan and a Protective material of long chain fatty acids. The Patent of E.U.A. No. 6,238,727 and the U.S. Patent. No. 5,885,610 describes the preparation of insoluble mineral salts of essential amino acids so that they are insoluble in the rumen and therefore are not available for microbial degradation but are subsequently available for absorption in the small intestine. Klose (U.S. Patent No. 6,013,286) discloses a composition of matter and a method for the administration of a bioactive compound to ruminants so that the compound does not enter the rumen directly but passes intact into the small intestine. This method requires that the material have a specific gravity between about 0.3 and 2.0 and that the particles comprise a core of bioactive substance with a hydrophobic coating that completely encapsulates the core. In addition, a surfactant is applied to the surface of the hydrophobic coating to ensure that the particles do not float in the rumen. In all inventions when bioactive compounds are encapsulated or embedded within matrices designed to protect them from rumen degradation, the compound is required to be produced initially by microbial synthesis or fermentation or chemical synthesis, then purified and subjected to the process of encapsulation This multi-step process is a costly and inefficient method for the production of ruminally protected bioactive compounds. At each step, there is a loss of product and a loss of bioactivity in the recovered element. L-lysine is produced by fermentation with L-lysine-producing strains Corynebacterium glutamicum. The productivity of C. glutamicum can be improved by selection of the strain, improvements in fermentation technology (eg agitation, oxygen supply, composition of the nutrient medium). Also, recombinant DNA technology methods have been used to improve L-lysine production of C. glutamicum strains by amplifying individual genes for biosynthesis. In this way, an increased production of L-lysine was obtained by amplifying a DNA fragment that confers resistance to aminoethyl cysteine (EP 88 166), reaction-resistant aspartate kinase (EP 387 527), amplification of dihydrodipicolinate synthase (EP 197 335), aspartate aminotransferase (EP 219 027), aspartate phosphoenolpyruvate carboxylase (EP 143 195 and EP 358 940), semialdehyde dehydrogenase (EP 219 027) and pyruvate carboxylase (DE 198 31 609). In the industrial production of L-lysine, it is necessary to separate the L-lysine product from the bacterial cell to improve the efficiency in L-lysine synthesis by the bacterium. It has been discovered that the LysE gene is responsible for exporting L-lysine out of the cytoplasm of C. glutamicum and making the medium and is crucial for efficient industrial production of L-lysine (Tryfona et al., Process Biochem (2004) ). The increased activity of Vehicle that exports L-LysE LysE promotes the production of lysine (DE 195 48 222). The problem that exists is that there is no means to protect bacteria and other microorganisms from degradation in the rumen so that they can be diverted from the rumen and administered intact to the small intestine. Similarly, the bio-active compounds they produce can be excreted from the bacterial cells so that they can be purified. Once purified, bioactive compounds must be protected against rumen degradation by encapsulation or embedding technology.

BRIEF DESCRIPTION OF THE INVENTION Methods for identifying Gram-positive bacterial strains useful for gastrointestinal administration of bioactive compounds to ruminants that are resistant to rumen inactivation have now been discovered. Other methods for increasing resistance to rumen inactivation of cultured bacterial strains useful for gastrointestinal administration of bioactive compounds to ruminants have been discovered, no matter how inherently resistant the bacterial strain can be to inactivation in the rumen. Therefore, in accordance with one aspect of the present invention, an in vitro method is provided to evaluate the strength of a bacterial strain to inactivation in ruméh in vivo, wherein the method comprises: culturing in vitro, a Gram-positive bacterial strain useful for gastrointestinal administration of a bioactive compound to ruminants in a nutrient medium containing natural or synthetic ruminal fluid; and measuring the degradation of the protein in bacterial cultures as a function of time. The ruminal fluid is selected at the approximate conditions of the rumen to be found by the bacterial strain to be administered. The natural ruminal fluid is taken from the contents of the rumen of a healthy ruminant within the first twenty-four hours after feeding. Synthetic ruminal fluid is a mixture of materials selected to stimulate conditions in the rumen, including one or more protozoan species that consume microorganisms in the rumen. Said protozoa species are easily identified by one skilled in the art. Preferred methods according to the present invention test the release of C14-labeled leucine to measure the degradation of the protein according to the method of Wallace et al., Br. J. Nutr., 58, 313-323 (1987). , the description of which is incorporated in the present invention as a reference. The results are expressed as a rate described as% of remaining bacteria present that are degraded per hour. For purposes of the present invention, the strains Bacteria with a degradation rate of less than 8% per hour are defined as resistant to inactivation in the rumen. Strains having a degradation rate of less than 6% per hour are preferred for the administration of the bioactive compound to ruminants, with strains having a degradation rate of less than 4% per hour being more preferred. Correspondingly, strains that are resistant to inactivation in the rumen will have more than 20% of the dose of the bacteria provided to an animal per day administered through the intact reticulum-rumen. Preferred strains will have more than 50% of the dose of the bacteria provided to an animal per day administered through the intact reticulum-rumen and most preferred will have more than 80% of the dose of the bacteria provided to an animal per day administered through the intact reticle-rumen. Accordingly, one embodiment of this aspect of the invention further includes the step of identifying bacterial strains resistant to inactivation in the rumen that have a degradation rate of less than 8% per hour as measured by the release of C14 labeled leucine. conformity with the method of Wallace et al. According to another embodiment of this aspect of the invention, the useful bacterial strain is a lysine-producing bacterial strain, preferably a strain of Cornyebacterium glutamicum, and more preferably a strain of C. glutamicum known to be overproduced. of lysine, including strains of C. glutamicum genetically modified to overproduce lysine. However, this method can be applied essentially to any bacterial species that are suitable for gastrointestinal administration of a bioactive compound to a ruminant for which an evaluation of the resistance to inactivation in the rumen is desired. For purposes of the present invention, "gastrointestinal administration" is defined as the inclusion of administration to the abomasum, small intestine and large intestine of a ruminant. Exactly where the bioactive compound is administered depends on the nature of the bioactive compound to be administered, which is understood by one skilled in the art who seeks to administer the compound. The present invention does not modify the location of administration but protects the bioactive compound from inactivation in the rumen, as desirable. The method according to this aspect of the invention provides the ability to select bacterial strains with reduced degradation in the rumen that can be used to administer gastrointestinally specific bacteria, and bioactive compounds contained therein to a ruminant, wherein the cell wall bacterial serves to provide protection to cellular contents by deviation of the rumen. The selected bacterial strains may have adequate resistance to degradation in the rumen to allow the ingestion of the bacterial biomass useful to ruminants without further modification.

Strains that have been discovered to have adequate resistance to rumen modification that allow ingestion of cellular contents to ruminants without further modification include C. glutamicum strains ATCC 13058, 13825, 14066, 14067, 14068, 21 127 and 700239. Therefore, in accordance with another aspect of the present invention, a forage supplement with rumen deviation containing the biomass of a strain of C. glutamicum containing lysine selected from the group consisting of strains of C. glutamicum ATCC 13058, 13825, 14066, 14067, 14068, 21 127 and 700239. The present invention also provides a method by which bacterial strains can become more resistant to inactivation in the rumen. The method according to this aspect of the invention can be used to increase the resistance to inactivation in the rumen of bacterial strains identified as resistant to inactivation in the rumen and those that are not. Therefore, in accordance with another aspect of the invention, a method is provided for increasing the resistance of a bacterial strain grown to inactivation in the rumen, wherein the bacterial strain is a gram positive bacterial strain that is nutritionally beneficial to the ruminants, and the method includes the steps of: growing a culture of the bacterial strain through at least one passage in the growth medium containing an amount of lysozyme effective to induce the growth of bacterial cell walls resistant to predation of protozoa; and recovering the bacterial strain from the medium containing lysozyme. In accordance with one embodiment of this aspect of the invention, the concentration of the lysozyme in the growth medium is between about 1 and about 100 ug / ml. According to another embodiment of this aspect of the invention a plurality of growth passages are used, with the preferred number of passages being between about 2 and about 20. In accordance with even another embodiment of this aspect of the invention, the passage of Recovery takes place after the last passage, after which the bacterial biomass recovers, in which the bacterial cell walls are resistant to degradation in the rumen. Then, the biomass is preferably dehydrated and concentrated for ingestion to a ruminant by conventional means. In another embodiment of this aspect of the invention the bacterial strain is a lysine-producing bacterial strain, preferably a strain of Cornye-bacterium glutamicum, and more preferably a strain of C. glutamicum known to overproduce lysine, including strains genetically modified to overproduce lysine. However, this method can be applied similarly essentially to all bacterial species useful for gastrointestinal administration of bioactive compounds to ruminants for which an increase in resistance to inactivation in the rumen is desired. The present invention also includes forage supplements with rumen deviation containing bacterial biomass useful for gastrointestinal administration of bioactive compounds to ruminants that are resistant to rumen inactivation obtained by any method in accordance with the present invention and methods for supplements for the diet of a ruminant with supplements for forage with rumen deviation. When included in animal fodder and offered to ruminants, the bacterium functions as a system for the gastrointestinal administration of bioactive compounds to ruminants. The foregoing objects and other objects, features and advantages of the present invention are more readily apparent from the detailed description of the preferred embodiments set forth below, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the rate of degradation of two strains of C. glutamicum that do not grow in the presence of lysozyme compared to S. ruminantium Z108; Figure 2 illustrates the rate of degradation of the same two strains of C. glutamicum grown in the presence of lysozyme compared to S. ruminantium Z108; Figure 3 illustrates the amount of decomposition in the rumen fluid of strains of C. glutamicum ATCC 13869, 700239 and 31269 grown in the presence and absence of lysozyme compared to S. bovis ES1; Figure 4 illustrates the decomposition rate in the rumen fluid for the same strains of C. glutamicum grown in the presence and absence of lysozyme compared to S. bovis ES1; and Figure 5 illustrates the decomposition in the rumen fluid from the cattle of Bifidobacter longum, Propionibacterium freudenreichii, Lactobacillus raffinolactis, Lacto. Fermentum, Lactobacillus lactis, Lactobacillus pentosus and Propionibacter acidipropionici grown in the presence and absence of lysozyme compared to S. bovis ES1.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES To impart resistance to degradation in the rumen, useful bacteria are grown in nutrient medium in the presence of lysozyme, preferably under fermentation conditions which are ideal for the growth of the specific organism in commercial quantities and optimized for synthesis of the bioactive compound of interest. . Examples of suitable nutrient media include Lennox medium (Kumagai et al.

Bioscience, Biotechnology, and Biochemistry, 69, 2051-2056 (2005)), CGXII medium (Keilhauer et al., 1993. J Bacteriol 175: 5595-5603), Luria Bertani broth (Lennox, ES 1955. Virology 1: 190-206 ) and the complex medium described by Broer & Kramer (J. Bacteriol., 1990, 172, 7241-7248). The lysozyme is added to the nutrient medium at an effective concentration to strengthen the resistance to lysine in the rumen. The concentration of lysozyme should not be too low, so that there is no statistically or commercially significant improvement in the performance of degradation in the rumen, or too high for cell growth to inhibit unacceptably. Accordingly, the concentration of lysozyme is preferably between about 0.1 and about 100 ug / ml, and more preferably between about 1 and about 10 ug / ml. Preferred methods employ a plurality of serial passages in growth medium containing lysozyme. Methods that employ between about 2 and about 10 serial passages are more preferred. The bacterium is grown in each passage for between about 12 and about 48 hours with a total growth time in the presence of lysozyme between about 1 and about 20 days, preferably. The bacterial cells are then harvested by filtration and / or centrifugation, concentrated and / or dried and packaged in a commercially acceptable manner.

The method of the present invention that employs lysozyme to impart resistance to inactivation in the rumen can be applied to any bacterial species useful for gastrointestinal administration of bioactive compounds to ruminants. Examples of such species include, but are not limited to, Bifidobacterium infantis, Lactobacillus reuteri, Bifidobacterium longum, Leuconostoc mesenteroides, Bacillus coagulans, Bifidobacterium thermophilum, Pediococcus acidilactici, Bacillus lentus, Lactobacillus acidophilus, Pediococc. cerevis. (Damnosus), Bacillus licheniformis, Lactobacillus brevis, Pediococcus pentosaceus, Bacillus pumi-lus, Lactobacillus bulgaricus, Propionibacter. freudenreichii, Bacillus subtilis, Lactobacillus casei, Propionibactehum shermanii, Bacteroides amylophilus, Lactobacillus cellobiosus, capillosus Bacteroides, Lactobacillus curvatus, cremoirs Streptococcus, Bacteroides ruminicola, Lactobacillus delbrueckii, Streptococcus diacetilactis, Bacteroides suis, Lactobacillus fermentum, Streptococcus faecium, Bifidobacterium adolescentis, Lactobacillus helveti- cus, Streptococcus intermedius, Bifidobacterium animalis, Lactobacillus lactis, Streptococcus lactis, Bifidobacterium bifidum, Lactobacillus plantarum and Streptococcus thermophilus. The resulting additives for forage also confer useful benefits to monogastric animals, including humans, even though deworming properties from the rumen are not required. The invention is particularly well suited for use with gram positive bacteria due to its thick peptidoglycan cell wall.

Examples of gram-positive bacteria include mycobacteria, nocardia, lactobacillus, streptococcus, Bacillus and Corynebacteria. Many commercially useful lysine producing strains of C. glutamicum have been developed which are suitable for use with the present invention such as strains ATCC 13058, 13825, 14066, 14067, 14068, 21 127 and 700239. Strains of Corynebacteria glutamicum are preferred. capable of synthesizing high concentrations of L-lysine such as strains ATCC 21127 and 700239. Strains of Corynebacteria glutamicum that are deficient in the LysE exporter gene are also preferred. Bioactive compounds that can be administered by bacterial species grown in the presence of lysozyme include nutrients such as amino acids, derivatives thereof, hydroxy amino acid homologs, proteins, carbohydrates, fats, vitamins, and animal drugs, alone or as a mixture of two or more. Illustrative examples of bioactive compounds include amino acids such as lysine, methionine, tryptophan, threonine, etc .; amino acid derivatives such as N-acylamino acids, calcium N-hydroxymethylmethionine salt, HCI lysine, etc .; homologues of hydroxy amino acids such as 2-hydroxy-4-methylmercapto-butyric acid and salts thereof, etc .; carbohydrates such as starch, sucrose, glucose, etc .; fats such as polyunsaturated fatty acids, omega-3 fatty acids, omega-6 fatty acids, trans fatty acids, etc .; and vitamins and substances with a similar function to vitamins such as vitamin A, vitamin A acetate, vitamin palmitate A, B vitamins such as thiamine, thiamine HCI, riboflavin, nicotinic acid, nicotinamide, calcium pantothenate, choline or pantothenate, pyridoxine HCl, choline chloride, cyanocobalamin, biotin, folic acid, etc., p-aminobenzoic acid, vitamins D2 and D3, vitamin E, etc. In addition to the nutrients, the bioactive compounds also include therapeutic compounds including hormones such as estrogen, stilbestrol, hexestrol, protein-shot, goitrogen, growth hormone, etc. Therapeutic bioactive compounds also include peptides and therapeutic proteins, including enzymes such as amylase, protease, xylanase, pectinase, cellulase, lactase, lipase, etc .; hormonal proteins such as growth hormone, somatotropin, etc.; carbohydrates for microbial binding such as mannan- and fructo-oligosaccharides and anti-microbial peptide compounds such as bacteriocins. Accordingly, the method of the present invention, in addition to being useful with both strains naturally occurring bacteria and strains produced by intensive selection processes, can also be applied to recombinantly produced bacterial strains. A recombinant bacterial strain genetically engineered to produce a therapeutically desired peptide or protein can then be modified by the method of the invention to allow recombinant bacterial cells to safely deviate from the rumen for gastrointestinal administration of the peptide or protein.

The bacterial cells themselves may also have value for gastrointestinal administration of compounds contained on the cell surface of bacteria. In addition, cells without nutritional value that function to compete with pathogens in the intestine (eg, competitive exclusion) are also included within the scope of the definition of "bioactive compounds" for purposes of the present invention. A separate in vitro method is also provided to identify useful bacterial strains that are either inert to the inactivation in the rumen or that must grow in a growth medium containing lysozyme to render them inert to inactivation in the rumen. This method of evaluating bacterial strains useful for resistance to inactivation in the rumen can be applied to the useful bacterial species identified above. The method serves to identify strains that are inherently resistant to inactivation in the rumen and that have utility as forage supplements with deviation of the rumen without initially growing them in a medium containing lysozyme and strains to which resistance must initially be imparted to the inactivation in the rumen when cultivating in the presence of lysozyme. The in vitro method cultivates a Gram-positive bacterial strain useful for gastrointestinal administration of a bioactive compound to ruminants in a growth medium containing natural or synthetic ruminal fluid and measures the degradation of the protein as a function of time. The growth medium will contain about 80 and about 99% by volume of a nutrient medium and of about 1 and about 20% by volume of ruminal fluid. Examples of suitable nutrient media include Dehority medium (Scott and Dehority, J. Bacteriol., 89, 1169-1175 (1965)), Hobson's M2 medium (Hobson, Methods Microbiol., 3B, 133-149 (1969)) and CRT media (Wallace et al., Int. J. Syst, Evol Microbiol., 53, 965-970 (2003)). Many other suitable means are described in the books by Hungate (Hungate R E 1966. The rumen and its microoes, Academic Press, New York, NY) and Hobson & Stewart (The Rumen Microbial Ecosystem, Chapman and Hall, London). The ruminal fluid was selected at approximate rumen conditions. The natural ruminal fluid is obtained from the contents of the rumen of healthy ruminants. For example, the fluid can be removed from ruminants with rumen-fistula. The fluid is preferably obtained from the same species of ruminants, and preferably from ruminants subject to the same feeding conditions as ruminants to which the bacterial strain will be administered. The fluid is preferably obtained within the first 24 hours after feeding, and more preferably within about one to about three hours after the morning feeding. The rumen fluid must be filtered to remove unwanted particle matter. Synthetic ruminal fluid is prepared from materials that stimulate the conditions to be found in the rumen. The fluid will contain one or more species of predatory protozoa that consume microorganisms in the rumen and nutrients for growth of the bacteria that include sugars, phosphate and bicarbonate pH regulators, mineral salts, volatile fatty acids and vitamins. Examples of synthetic media are well described by Hobson & Stewart (The Rumen Microbial Ecosystem, Chapman and Hall, London). Examples of ruminal protozoa responsible for bacterial degradation include the species of Epidinium, Eudiplodinium, Isotricha Dasythcha, Entodinium and Polyplastron (Ivan et al 2000. J Anim Sci 78, 750-759, Ivan et al 2000b J Dairy Sci 83, 776-787). The bacterial strain useful to be evaluated is grown in the growth medium under the temperature conditions to be found in the rumen., for example, between about 36 and about 40 ° C. The incubation time can be selected to be approximate to the amount of time in which the bacterial strain will reside in the rumen, typically between about one and about 48 hours. A longer time can be used to obtain a greater amount of protein degradation data, for example, from 12 to about 48 hours. The amount of protein degradation expected for the amount of time that the bacterial strain will actually be consumed in the rumen can be extrapolated from these data. The degradation of protein for the bacterial strain is measured in terms of the weight of the product or degradation products produced as a function of time. A preferred method according to the present invention tests the release of C14-labeled leucine to measure the degradation of the protein according to the method of Wallace et al., Br. J. Nutr., 58, 313-323 (1987), the description of which is incorporated herein by reference. The results are expressed as a ratio described as% of remaining bacteria present that are degraded per hour. For purposes of the present invention, bacterial strains with a degradation rate of less than 8% per hour are defined as resistant to inactivation in the rumen. Strains having a degradation rate of less than 6% per hour are preferred for the administration of the bioactive compound to ruminants, with strains having a degradation rate of less than 4% per hour being more preferred. Bacterial strains after evaluation are not considered resistant to degradation in the rumen, for example, strains that have a degradation rate greater than 8% per hour, can then be grown in the presence of lysozyme to improve resistance to the degradation in the rumen. The improvement can be measured by re-evaluating the bacterial strain after exposure to lysozyme with the in vitro evaluation method of the present invention using natural or synthetic ruminal fluid. The degree of improvement can be expressed as the percentage reduction in the rate of degradation compared to the same unit of time after exposure to lysozyme. However, the degree of improvement is not as important as having the rate of degradation falling below the threshold required for the bacterial strain to be considered resistant to degradation in the rumen, is defined by the present request. That is, a large increase in resistance may still be insufficient while a small increase may be more than adequate. Useful bacterial strains identified as resistant to degradation in the rumen, or resistant to degradation in the rumen when grown in the presence of lysozyme, can be grown (with lysozyme required for resistance to degradation in the rumen) and The biomass can be harvested in commercial quantities with commercial fermentation equipment including equipment for batch cultivation, batch entry and continuous cultivation. The biomass can optionally be mixed with acceptable fillers, binders, flavoring additives, and the like, to form a forage supplement with rumen deviation, or the biomass itself can serve as the forage supplement to be mixed with a feed ration. to the ruminant. Alternatively, the biomass and other additives can be dissolved or suspended in an aqueous medium to form a forage supplement with deviation of the rumen that is sprinkled on the forage ration. The formation of any dosage form is essentially conventional and well known to one skilled in the art. Other known nutritional ingredients for ruminants can be added to any form of forage supplement with rumen deviation. Harvested bacterial cells resistant to degradation in the rumen can conveniently be provided to a ruminant mixed with a conventional forage for ruminant. The forages typically are edible plant materials by ruminants, such as legume hay, grass hay, corn silage, grass silage, legume silage, corn grain, oats, barley, distiller grain, beer grain, bean flour of soybean and cottonseed meal and are included in an amount as is typically recommended by a nutritionist for animal husbandry, which ordinarily does not exceed 5% by weight of the content of dry forage solids. For a supplement for protected rumen lysine forage, such as a forage supplement containing the biomass of C. glutamicum, the amount of supplement to be added to the dry forage content of the forage it must be an effective amount to provide a daily average of between about 5 and about 150 mg of metabolically available lysine per kg of body weight of the ruminant. A quantity of metabolically available lysine of between about 15 and about 75 g per kg of body weight of the ruminant is preferred. The metabolically available lysine can be measured by determining the flow of lysine from an in vitro rumen simulation system; measuring the flow of lysine to the small intestine in adapted animals with abomasal and / or intestinal cannulas or by measuring the increase in the percentage of milk protein and / or production in female ruminants fed a diet designed to be deficient in lysine metabolically available. There are numerous permutations of these methods known to those experts in the art that can be used to determine the metabolically available lysine. Forage supplements with rumen deviation of the present invention can be provided to any ruminant in need of nutritional supplementation, including livestock, research animals and exhibition animals in zoos and other wildlife exhibits. Examples of ruminants include cattle, oxen, sheep and goats. Forage supplements with rumen diversion can be supplied to livestock that are raised for production of meat, milk, skin, hair or wool, or ruminants used as farm animals. The following non-limiting examples which are set forth in the present invention below illustrate certain aspects of the invention.

All parts and percentages are by weight unless otherwise mentioned, and all temperatures are in degrees Celsius.

EXAMPLES EXAMPLE 1 Susceptibility of C. glutamicum strains to ruminal degradation via predation by protozoa The degradation of C. glutamicum and S. ruminantium (which represents an "average" rumen bacterium) was determined in the rumen according to the method described by Wallace et al. , Br. J. Nutr., 58, 313-323 (1987). Strains of Corynebacteria glutamicum (ATCC 13761 and ATCC 13869 were grown in Wallace and McPherson aerobic medium, S. ruminantium Z108 was grown in Wallace and McPherson's anaerobic medium, Wallace and McPherson's medium and preparation are described by the journal article referred to above Wallace et al.The cultures were grown overnight at 39 ° C. The cells were harvested by centrifugation at 1000 g x 10 minutes at room temperature.The cells were resuspended in Cole-man's anaerobic pH regulator containing 5 mM L-leucine C14 and incubated overnight (OD = 1.0) to label the bacterial protein, a sample (1 ml) was removed and placed in 0.25 ml of 25% TCA for the protein determination Two 50-ul aliquots were placed into scintillation fluid to determine the amount of radioactivity added The rumen fluid was removed from three sheep 2 hours after the filtration and filtered through muslin. Unlabeled L-leucine (5 mM) was added and the filtered rumen fluid (SRF) remained hot. A sample (1 mL) was added to 1 mL of 4% formalin for the protozoa counts. The labeled bacterial cell suspension (0.5 ml) was added to 4.5 ml of SRF or pH regulator and incubated at 39 ° C. The samples (0.5 ml) were removed at 0, 1, 2 and 3 hours and placed inside 0.125 ml of TCA. The Samples were centrifuged at 14,000 rpm for 3 minutes and 200 ul of supernatant were counted to determine the radioactivity released, determined by the bacterial protein degraded by the protozoa. Based on the release of radioactivity, determined by the bacterial protein degraded by the protozoa, the percentage degradation was determined at each time point. The data were adjusted to the equation (Mehrez and Orskov, 1987): Y = a + (c - a) * 1- (exp [-kd * x]); where Y = degradation at a specified time x, hour; a = initial degradation; c = maximum degradation; kd = rate of degradation, hr "1; Effective degradation was determined for the selected ruminal ratios of passage in accordance with the equation: Y = a + (c * kd) / (Kd + Kp) where Kp = ratio of ruminal replacement The results are shown in Figure 1. Both strains of C. glutamicum showed rapid degradation compared to S. ruminantium Z108. The disappearance during the 3-hour incubation period was 71.2 and 83.1% for strains 10336 and 13869 respectively compared to 21.9% for S. ruminantium Z108. Effective degradation at a rumen turnover rate of 0.07 hr "1 was 71.2 and 53.8% for strains 13761 and 13869 respectively.

EXAMPLE 2 Growth of C. glutamicum in the presence of low levels of lysozyme Wallace and McPherson medium without C14 (24 x 7 ml) was prepared.

To each of the 4 series of tubes, 0.5 ml of sterilized lysozyme was added through a filter (0.1, 1.0, 10, 100, or 1000 ug / ml). To a final series of 4 tubes, 0.5 ml of Coleman's medium D (Control) was added. The tubes were inoculated with cultures of strain C. glutamicum ATCC 13761 and incubated at 39 ° C for 48 hours and OD (650 nm) was measured for each organism at 24 and 48 hours. Strain 13761 grew well at the two lower levels of exposure to lysozyme (0.1 and 1 ug / ml). Growth decreased by half with 100 ug / ml and was completely inhibited at 1000 ug / ml.

EXAMPLE 3 Growth effect of C. glutamicum strains in the presence of lysozyme on susceptibility to predation by protozoa The methodology was essentially the same as in experiment 1, except that the treatments consisted of the strains C. glutamicum ATCC 13761 and ATCC 13869 grown as in experiment 1 or in the presence of lysozyme (0.5 ml 0.25 μg / ml lysozyme; 16.7 ug / ml of final concentration). As in experiment 1, S. ruminantium Z108 was used as an organism for monitoring. The results are shown in figure 2. The degradation was lower in this experiment than in experiment 1 for all organisms. When strains were grown without lysozyme (native), the disappearance during the 3-hour incubation period was 37.9 and 42.8% for strains 13761 and 13869 respectively, compared to 13.3% for S. ruminantium Z108. However, when they were grown in the presence of lysozyme, the degradation after 3 hours was reduced to 15.2% for strain 13869 but had no effect for strain 13761 (36.3%). The effective degradation at a rumen turnover rate of 0.07 hr "1 was 53.8 and 64.7% for strains 13761 and 13869 respectively when they were not grown in the presence of lysozyme and 36.1 and 24.5% for the respective strains when they were grown. in the presence of lysozyme.

EXAMPLES 4 - 6 Effect of the growth of the strains of C. glutamicum 13869, 700239 and 31269 in the presence of lysozyme on susceptibility to predation by protozoa Strains of C. glutamicum 13869, 700239 and 31269 were obtained from the American Type Culture Collection (ATCC). The cultures were revived on nutrient agar and transferred to the broth nutrient according to ATCC instructions. When healthy growth was observed (after passages of 3 x 24 hours through the nutrient broth at 39 ° C) the cultures were grown for an additional 3 x 24 hours at 39 ° C in nutrient broth plus or minus 20 μg / ml lysozyme. egg white chicken. S. bovis ES1 was previously isolated from the rumen of a sheep and remained within the culture collection of the Institute of Rural Sciences, University of Wales, Aberystwyth. Bacteria were labeled by growing the cultures for 24 hours at 39 ° C in a Wallace and McPherson medium containing rumen fluid with ammonia cysteine and L- [U-14C] leucine as the only sources for addition of N, and with 20 ug / ml chicken egg white lysozyme that was added to previously grown cultures in the presence of lysozyme. The cell was harvested by centrifugation and washed once with Coleman's Saline D solution (Coleman, 1978) before being incorporated with filtered ruminal fluid. The ruminal fluid was removed 2 hours after the morning feeding from 3 animals of cattle with fistulas in the rumen that received a ration based on a lawn silage. The fluid was filtered through 4 layers of muslin cloth. The apparent degradation of the protein was measured by the release of [14C] in the trichoroacetic acid soluble material during the 3 hour incubations. Unlabelled L-leucine was included in all incubations at a final concentration of 5 mmol / L.

The decomposition of the different bacteria during the three-hour incubation in the rumen fluid is shown in figure 3. An initial comparison of the decomposition rate of the bacteria (as% / h) shows that the strain of C. glutamicum 700239 decomposed significantly slower than any of the other strains of C. glutamicum or rumen S. bovis bacteria (5.63, 2.58, 8.27, 6.88% / h of SED 1.287 for C. glutamicum strains 13869, 700239 and 31269 and S. bovis ES1 respectively, figure 4). When data were examined for the strains of C. glutamicum alone it was evident that although again there was a significant difference in the rate of decomposition between the strains used there was no improvement for these strains after pre-incubation with lysozyme (Table 1) .

TABLE 1 Effect of growth on the presence and absence of lysozyme on the decomposition of strains of C. glutamicum 13869, 700239 and 31269 in the rumen fluid The assay used in the present invention was based on that described by Wallace et al., Wherein the release of leucine C 4 from the bacteria in the rumen fluid in the presence of an excess of unlabeled leucine is used to measure the decomposition of the bacteria in the rumen. For the three bacterial strains, the growth of the cultures in lysozyme had no significant effect on the rate of decomposition in the rumen fluid, despite the fact that there is a numerical decrease of about 16%. Two possible reasons for this lack of effect are presented below: Time in culture: in the current experiment of C. glutamicum was incubated with lysozyme for a total of 96 hours (3 passages from 24 to 20 ug / ml in nutrient broth plus one in the middle Wallace and McPherson used to mark the cells). It is possible that insufficient time allows crops to change their cell structure in response to lysozyme. The low degradation of cultures even in the absence of lysozyme: in the current experiment the degradation of C. glutamicum strains grown in the absence of lysozyme ranged from 9 to 3% / h. This is considerably lower than the values previously recorded with other strains of C. glutamicum for example (12 and 14% h with the strains 10334 and 10337) and much lower from the figures registered with B. fibrisolvens (about 30% / h) where the initial observations were made that lysozyme could reduce degradation. In contrast, the figures for S. bovis recorded in the present invention are very similar to those previously observed. It is possible that the reason why lysozyme conferred little protective effect was that the cells were already relatively resistant to attack by protozoa. It is notable that when C. glutamicum 700239 was grown in the presence of lysozyme, the rate of degradation was about 2% / h. Assuming that when added to the rumen C. glutamicum 700239 it could leave the rumen at a relatively modest rate of 10% / h in the liquid phase, then for a period of about 24 hours to 77% of the C. glutamicum 700239 could be diverted from the rumen with less than 15% being degraded. At a more realistic speed of 15% / h of liquid refill compared to 85% it could be diverted from the rumen with less than 12% being degraded. In the current experimental growth in the presence of lysozyme, the strains of C. glutamicum investigated against degradation in the rumen were apparently not protected. However, C. glutamicum 700239 strain remarkably resistant to rumen decomposition is expected to provide a suitable vector for the passage of amino acids such as lysine through the rumen.

EXAMPLES 7-13 Effect of the growth of strains of Bifidobacterium longum, Propionibacerium freudenreichii, Lactobacillus raffinolactis, Lactobacillus fermentum, Lactobacillus lactis, Lactobacillus pentosus and Propionibacerium acidipropionici in the presence of lysozyme on susceptibility to predation by protozoa The effect on the decomposition in the rumen fluid of seven potentially different probiotic organisms, different from C. glutamicum, was investigated by the growth of organisms in the presence of lysozyme in vitro, using as a control a typical rumen bacterium, Streptococcus. bovis, as in examples 1-6. Bifidobacterium longum, P. freudenreichii and P. acidipropionici were obtained from the National Collection of Industrial and Marine Bacteria (NCIMB) and the National Collection of Food Bacteria, Aberdeen. Lactobacillus raffinolactis, Lactobacillus fermentum, Lactobacillus lactis and Lactobacillus pentosus were obtained from Dr Kevin Hillman, Gutbugs, UK. The bacteria were grown and labeled and the apparent decomposition of the protein was measured as in Examples 4-6, including S. bovis ES1. The decomposition of the different materials during the three hours of incubation in the rumen fluid is shown in figure 5. The growth in the presence of lysozyme decreased the decomposition of S. bovis, P. freuden-reichii and L. raffinolactis in more 70% The decomposition of L. pentosus, B. longum and L. fermentum decreased between 40 and 50% while there was no effect on the decomposition of L. lactis or P acidipropionici (Table 2).

TABLE 2 Effect of growth on the presence and absence of lysozyme on probiotic organisms in the rumen fluid The following examples and description of the preferred embodiments should be taken as illustrative, rather than limiting of the present invention as defined by the claims. Numerous combinations of the above-stated characteristics can be used without departing from the present invention as set out in the claims. Said variations are not considered as a deviation from the spirit and scope of the invention, and it is intended that all such modifications be included within the scope of the following claims.

Claims (14)

NOVELTY OF THE INVENTION CLAIMS

1 .- A method for increasing the resistance to inactivation in the rumen of a gram positive bacterial strain producing lysine comprising the steps of: growing a culture of the bacterial strain through at least one passage in the growth medium that contains an amount of lysozyme effective to induce the growth of bacterial cell walls resistant to predation by protozoa; and recovering the bacterial strain from the medium containing lysozyme.

2. The method according to claim 1, further characterized in that said bacterial strain is a strain of Corynebacteria glutamicum.

3. The method according to claim 2, further characterized in that said strain of Corynebacteria glutamicum is an ATCC strain selected from the group consisting of 13058, 13825, 14066, 14067, 14068, 21 127 and 700239.

4.- The method according to claim 2, further characterized in that said strain of Corynebacteria glutamicum overproduces lysine.

5. - The method according to claim 4, further characterized in that said strain of Corynebacteria glutamicum is genetically modified to overproduce lysine.

6. - E! method according to claim 1, further characterized in that the concentration of lysozyme in said growth medium is between about 0.01 and about 100 ug / ml

7. The method according to claim 6, further characterized in that said concentration of lysozyme is between about 0.1 and about 10 ug / ml.

8. The method according to claim 1, further characterized in that a plurality of passages are used in the growth medium containing lysozyme.

9. - The method according to claim 8, further characterized in that between approximately 2 and approximately 10 passages are used.

10. - A forage supplement with rumen-protected lysine comprising a lysine-producing bacterial strain grown by the method of claim 1.

11. - Lysine forage supplement protected in the rumen according to claim 10, further characterized in that said bacterial strain is a strain of Corynebacteria glutamicum.

12. - The forage supplement with Protein protected in the rumen according to claim 11, further characterized in that said strain of Corynebacteria glutamicum overproduces lysine.

13. - The forage supplement with protected lysine in the rumen according to claim 12, further characterized in that said strain of Corynebacteria glutamicum is genetically modified to overproduce lysine.

14. - The rumen-protected lysine forage supplement according to claim 10, further characterized in that said bacterial strains have a rumen degradation rate of less than about 8% per hour as measured by leucine release marked with C14 in accordance with the method of Wallace et al. 5. - The lysine forage supplement protected in the rumen according to claim 14, further characterized in that said rate of degradation is less than about 6% per hour. 16. - A forage supplement with rumen-protected lysine comprising a lysine-producing bacterial strain having a rumen degradation rate of less than about 8% per hour as measured by the release of C14 labeled leucine. conformity with the method of Wallace et al. 17. - The lysine forage supplement protected in the rumen according to claim 14 or claim 16, further characterized in that the rate of degradation in the rumen is such that more than 20% of the dose of the bacteria provided to a ruminant per day is administered through the intact reticulum-rumen. 18. - The forage supplement with protected lysine in the rumen according to claim 14 or claim 16, further characterized in that said bacterial strain is a strain of Corynebacteria glutamicum ATCC selected from the group consisting of 13058, 13825, 14066, 14067, 14068, 21 127 and 700239. 19. - A method for increasing the metabolically available lysine content of a fodder ration for ruminant comprising the addition to said fodder ration of an effective amount of the lysine forage supplement protected in the rumen of claim 10 or claim 16. 20. The method according to claim 19, further characterized in that said forage supplement is added to said ration of forage in an amount effective to provide between about 5 and about 150 mg of metabolically available lysine per kg of body weight of the ruminant. twenty-one . - The method according to claim 19, further characterized in that said ruminant is a milk producing cow. 22. An in vitro method for evaluating the resistance of a lysine-producing bacterial strain to rumen inactivation in vivo, comprising the steps of: culturing in vitro, a gram positive bacterial strain producing lysine in a nutrient medium containing natural or synthetic ruminal fluid; and measuring the degradation of protein in the bacterial culture as a function of time. 23. - The method according to claim 22, further characterized in that said bacterial strain is a strain of Corynebacteria glutamicum. 24. - The method according to claim 23, further characterized in that said strain of Corynebacteria glutamicum is an ATCC strain selected from the group consisting of 13058, 13825, 14066, 14067, 14068, 21 127 and 700239. 25. - The method according to claim 23, further characterized in that said strain of Corynebacteria glutamicum overproduces lysine. 26. - The method according to claim 25, further characterized in that said strain of Corynebacteria glutamicum is genetically modified to overproduce lysine. 27. - The method according to claim 22, further characterized in that said step that measures the degradation of the protein comprises the measurement of the release of leucine labeled with C14 according to the method of Wallace et al. 28. - The method according to claim 22, further characterized in that said method further includes the step of identify bacterial strains resistant to inactivation in the rumen that have a degradation rate of less than 8% per hour. 29. The method according to claim 22, further characterized in that said ruminal fluid is natural ruminal fluid. 30. The method according to claim 22, further characterized in that said ruminal fluid is synthetic ruminal fluid. 31. - The method according to claim 28, further characterized in that said method further includes the step of identifying bacterial strains resistant to inactivation in the rumen that have a rate of degradation in the rumen in such a way that more than 20% of the dose of the bacteria supplied to a ruminant per day is administered through the intact reticulum-rumen. 32. - The method according to claim 31, further characterized in that the strains identified have a rate of degradation such that more than 50% of said dose is administered intact.