MXPA00001480A - Methods for producing sialyloligosaccharides in a dairy source - Google Patents

Abstract

The present invention provides methods for producing sialyloligosaccharides i(in situ) in dairy sources and cheese processing waste streams, prior to, during, or after processing of the dairy source during the cheese manufacturing process. The methods of the present invention use the catalytic activity of a(2-3) i(trans)-sialidases to exploit the high concentrations of lactose and a(2-3) sialosides which naturally occur in dairy sources and cheese processing waste streams to drive the enzymatic synthesis of a(2-3) sialyllactose. a(2-3) sialyloligosaccharides produced according to these methods are additionally encompassed by the present invention. The invention also provides for recovery of the sialyloligosaccharides produced by these methods. The invention further provides a method for producing a(2-3) sialyllactose. The invention additionally provides a method of enriching for a(2-3) sialyllactose in milk using transgenic mammals that express an a(2-3) i(trans)-sialidase transgene. The invention also provides for recovery of the sialyllactose contained in the milk produced by this transgenic mammal either before or after processing of the milk. Transgenic mammals containing an a(2-3) i(trans)-sialidase encoding sequence operably linked to a regulatory sequence of a gene expressed in mammary tissue are also provided by the invention.

Description

METHODS FOR PRODUCING ALKALID SULPHALINES IN A DAIRY SOURCE 1. INTRODUCTION This invention relates to methods for producing (2-3) sialyl oligosaccharides in a milk source or cheese processing waste stream by contacting the milk source or the waste stream from the cheese processing with a catalytic amount of at least one (2-3) trans-sialidase. In the preferred embodiments, the methods of the invention are applied to produce (2-3) sialillactose in a milk source or waste stream from cheese processing. Methods for isolating a (2-3) sialyl oligosaccharides produced according to the methods of the invention are also provided. The invention further relates to a method for producing (2-3) sialillactose in milk using a transgenic mammal that contains the coding sequence of an α (2-3) trans-sialidase operably linked to a regulatory sequence of a expressed gene in breast tissue. 2. BACKGROUND OF THE INVENTION 2.1. SIALILOLIOGOSACÁRIDOS IN RESIDUAL CURRENTS OF CHEESE The serum is a main byproduct of the cheese making that, for environmental reasons, presents a problem to dispose the waste. In the United States alone, fluid serum is being produced at a rate of approximately 62.6 billion pounds per year. Typically, the whey is composed of about 5% by weight of lactose, 1% by weight of protein and about 0.5% by weight of salts, where the difference of the mixture is water. At present, many cheese-producing countries are making a major effort to develop uses for this commodity, which was previously considered a residual product of cheese processing. Although the protein concentrate obtained by ultrafiltration of whey has become a valuable commodity in the food industry and has found applications in animal feed, fertilizers, fermentation - and food filler, most of the lactose-rich ultrafiltered permeate, resulting , it is still considered a disposable fraction. At present, some sialyl oligosaccharides have been found with valuable application as pharmaceuticals. See, for example, U.S. Patent No. 5,270,462 to Shimatani et al. It has been shown that sialillactose neutralizes the enterotoxins of some pathogenic microbes including Escherichia coli, Vibrio cholerae and Salmonella. See, for example, U.S. Patent No. 5,330,975 to Hiro et al. It has also been shown that (2-3) sialillactose (-Neu5Ac- (2-3) -Gal-ß- (1-4) -Glc) interferes with the colonization of Helicobacter pylori and by this means prevents or inhibits gastric ulcers and duodenals. See, for example, U.S. Patent No. 5,514,660 to Zopf et al. Furthermore, it has been proposed that sialillactose inhibits the formation of the immune complex by disrupting the occupation of the Fc carbohydrate binding site in IgG and is useful in the treatment of arthritis. See, for example, U.S. Patent No. 5,164,374 to Rademacher et al. To date, the sialyl oligosaccharides that are commercially available are very expensive due to their low amount in natural sources. For example, a (2-3) sialillactose and a (2-3) sialillactose isolated from bovine colostrum is marketed at $ 75.60 and $ 83.30 per milligram, respectively (Sigma Chemical Company, 1997). An effort has been focused on the cultivation of sialyl oligosaccharides from a vast supply of serum available as a residual product of cheese processing. The processes to isolate the sialiloligosacáricos have used techniques such as ultrafiltration, ion exchange resins and phase partition chemistry. U.S. Patent No. 4,001,198 to Thomas and U.S. Patent No. 4,202,909 to Pederson; U.S. Patent No. 4,547,386 to Chambers et al .; U.S. Patent No. 4,617,861 to Armstrong; U.S. Patent Nos. 4,971,701 and 4,855,056 to Harju et al .; U.S. Patent No. 4,968,521 to Mclnychyn; U.S. Patent No. 4,543,261 to Harmon et al .; U.S. Patents Nos. 5,118,516 and 5,270,462 to Shimatani; JP Kokai 01-168,693; JP Kokai 03-143,351; JP Kokai 59-184,197; JP Kokoku 40-1234; JP Kokai 63-284,199 and the Japanese Patent Publication No. 21234/1965, each of which is incorporated herein by reference in its entirety. Yields of up to 6 grams of the sialyl oligosaccharide to (2-3) sialillactose per kilogram of the waste stream from cheese processing have been reported. U.S. Patent No. 5,575,916 to Brian et al. which is incorporated in the present as a reference in its integrity. 2. 2 SIALIDASES AND SIALILTRANSFERASAS Sialic acids are carboxylated sugars of 9 carbons that are generally found as the terminal monosaccharides in oligosaccharide chains, in mammalian cells, sialic acids are very frequently linked to ß-galactose with a link to (2) -3), and to N-acetylglucosamine and N-acetylgalactosamine with a bond to (2-6). Cross et al., 1993, Annu. Rev. Microbiol. 47: 385- Sialidases catalyze the elimination of sialic acid residues [sic] from the oligosaccharide chain. Due to the wide range of substitutions that can occur in different carbons of sialic acid molecules, there are at least 39 different species of sialic acids. Colli,., 1993, FASEB J 7: 1257-1264. In general, the sialidases present specificity for the substrate for the specific forms of sialic acid bonds. Viral sialidases split bonds to (2- 3) glycosides more efficiently than bonds to (2-3), but bacterial sialidases are not as specific.

Cross et al., 1993, Annu. Rev. Microbiol. 47: 385-411 (Citing Corfield et al., 1982, Sialic Acids: Chemistry, Metabolism and Function, Vol 10, New York: Springer-Verlag, pp. 195-261). At low concentrations of enzymes, bacterial sialidases have a preference for unfolding the glycosidic linkages to (2-3) or to (2-6). Cross et al., 1993, Annu. Rev. Microbiol. 47: 385-411. The CMP-sialyltransferases catalyze the transfer of the cytidine monophosphate-cialic acid residues (CMP-sialic acid) to the acceptor molecules. Although many sialidases have at least some specificity for the substrate, the CMP-sialyltransfereses act on specific substrates. Mammalian CMP-sialyltransferases are generally found in the Golgi, however, there is evidence that there may be CMP-sialyltransferases associated with the cell surface as well. Cross et al., 1993, Annu. Rev. Microbiol. 47: 385-411. (Citing Roth et al., 1971, J. Cell Biol. 51: 536-547; Sur, 1991, Glycobíology 1: 563: 575; Yogeeswaran et al., 1974, Biochem. Biophys. Res. Commun. 59: 591- 599). 2. 3. g (2-3) TRANS-SIALIDASE OF TRIPANOSOMA CRUZI Trypanosoma cruzi (order quinetoplástida) is the intracellular parasite responsible for Chagas disease, in Ibero-American countries. Chagas disease mainly affects nerve and muscle cells. A serious manifestation of Chagas disease is progressive, chronic fibrotic myocarditis. Colli, 1993, FASEB J. 1257-1264. Approximately 16-18 million people are infected with T. cruzi. Colli, 1993, FASEB J. 1257-1264. T. cruzi invades a wide variety of host cells, and a considerable amount of research has been focused on surface molecules to determine the molecules that may be involved in the parasite / host interaction. Colli, 1993, FASEB J. 1257-1264. A surface molecule that has generated great interest is (2-3) trans-sialidase. This molecule has the ability to catalyze the elimination of sialic acid from a donor molecule containing saccharide (sialidase activity) and catalyze the transfer of sialic acid to an acceptor molecule containing saccharide (trans-sialidase activity). Schenkman et al., 1992, J. Exp. Med. 175: 567-575. The gene encoding trans-sialidase from T. cruzi has been cloned or characterized at the molecular level. The a (2-3) trans-sialidase from T. cruzi catalyzes the transfer of sialic acid from a ß-galactosyl sialoglyconjugate donor terminal to a terminal β-galactose in an acceptor molecule. Colli,. 1993, FASEB J. 7: 1257-1264. The a (2-3) trans-sialidase of T. cruzi does not use CMP-sialic acid as a substrate and prefers sialyl to (2-3) residues bound to β-galactosyl as donor molecules of sialic acid on sialic acid bound to the positions a (2-6), a (2-8) - ya (2-9) -. Schenkman et al., 1994, Annu. Rev.; Microbiol. 48: 499-523. In addition, the a (2-3) trans-sialidase of T. cruzi can not use free sialic acid as a substrate. Vandekerckhove et al. 1992, Glycobiology 2: 541-548. The a (2-3) t ans-sialidase of T. cruzi has a broad optimum pH centered on 7.0. Cross et al., 1993, Annu Rev. Microbiol. 47: 385-411. More detailed analyzes of the a (2-3) trans-sialidase have revealed that the amino-terminal portion of the protein is responsible for the activity of a (2-3) trans-sialidase.

Campetella et al., 1994, Mol. Biochem. Parasi tol. 64: 337-340; Schenkman et al., 1994, J. Biol. Chem. 269: 7970-7975. It has also been determined that there are at least two crucial amino acid residues: Tyr 342 and Pro231 of the a (2-3) trans-sialidase that appear necessary for the complete activity of a (2-3) trans-sialidase. Cremona et al., 1995, Gene 160: 123-25. The importance of Tyr 342 is demonstrated by the fact that the natural variants of a (2-3) transsialidase of T. cruzi that have a substitution of Tyr 342- > His, lack the activity of ^ a (2-3) trans-sialidase. Uemura et al., 1992, EMBO J. 11: 3837-3844. Trans-sialidase activity has also been discovered in Trypanosoma brucei, the causative agent of African sleeping sickness, Endotrypanum spp, and in Pneumocystis carinii. Like the a (2-3) trans-sialidase from T. cruzi, trans-sialidase from T. brucei has an optimum pH of 7.0. However, unlike T. cruzi trans-sialidase, which is expressed during the trypomastigote stage, trans-sialidase from T. brucei seems to be expressed during the procyclic stage of the parasite's life cycle, when the parasite resides in the midgut of its insect vector (Glossina spp., the "tsetse fly"). Cross et al., 1993, Annu i? Ev. Microbiol. 47: 385-411. 2. 4. SIALILLACTOSE PRODUCTION Numerous methods have been described for producing sialylated oligosaccharides by means of enzymes. U.S. Patent No. 5,374,541 to Ong et al., Discloses a method for producing sialyl oligosaccharides. According to this method, β-galactosidase is used to form β-galactosyl glucosides in the presence of CMP-sialic acid and α (2-3) - or α (2-6) -CMP-sialyltransferases to form sialylated oligosaccharides. This method does not use (2-3) trans-sialidase. U.S. Patent No. 5,409,817 to Ito et al. Describes a process of three enzymes to produce (2-3) sialylgalatosides. According to this process, the CMP-sialyltransferases transfer sialic acid from CMP-sialic acid to acceptor molecules, these acceptor molecules become donor molecules for a (2-3) trans-sialidase from Trypanosoma cruzi, and the CMP- is regenerated. sialic acid in the system by the action of the CMP-sialicic acid synthetase and free sialicic acid added. The process described in U.S. Patent No. 5,409,817 to Ito et al. Specifically requires the addition of free sialic acid. The free sialic acid is converted to CMP-sialic acid by the CMP-sialicic acid synthetase, and the sialic acid portion is transferred to an acceptor molecule by means of the CMP-sialyltransferase.

According to the description of Ito et al., The formation of these sialylated acceptor molecules is required to drive forward the reaction of a (2-3) trans-sialidase. In addition to free sialic acid, the method of Ito et al. Also requires the presence of three enzymes including CMP-sialic acid synthetase and CMP-sialyltransferase. In addition, milk sources and waste streams from cheese processing do not contain CMP-sialic acid synthetase. 2. 5 EXPRESSION OF TRANSGENES IN MILK Numerous foreign proteins have been successfully expressed transgenically in cattle milk. Most of this work has focused on the expression of proteins that are foreign to the mammary glands. Colman, A., 1996, Am. J. Clin. Nutr. 63: 639S-645S. To date, the specific expression of transgenic cattle milk has been achieved by means of regulatory sequences of milk-specific protein genes that are operably linked to the sequence of the gene encoding the target protein, micro-injecting these genetic constructions in the pro-nuclei of fertilized embryos, and implanting the embryos in recipient females. See, for example, right et al., 1991, Biotechnology (NY) 9: 830-834; Carver et al., 1993, Biotechnology (NY) 11: 1263-1270; Paterson et al., 1994, Appl. Microbiol Biotechnol. 40: 691-698. Proteins that have been successfully expressed in milk of transgenic animals include: l-antitrypsin (right et al., 1991, Biotechnology (NY) 9: 830-834; Carver et al., 1993, Biotechnology (NY) 11: 1263- 1270, Factor IX (Clark et al., 1989, Biotechnology (NY) 7: 487: 492), protein C (Velander et al., 1992, Proc Nati Acad Sci USA, 89: 12003-12007); tissue plasminogen activator (Ebert et al., 1991, Biotechnology (NY) 9: 835-838) and fibrinogen, although most of these transgenes express for proteins that complement the milk composition, very few, if any some of the expressed proteins interact directly with the components of the milk to modify the natural composition of the milk.There is a need for methods that provide large-scale production of a (2-3) sialyl oligosaccharides, such as a -3) sialillactose, which have commercial and / or therapeutic value. 3. SUMMARY OF THE INVENTION The present invention greatly advances the field of commercial production of sialyl oligosaccharides by providing methods for producing the sialyl oligosaccharides in situ in milk sources and waste streams of cheese processing. The methods of the invention have specific applications in the production of a (2-3) sialyllactose in a milk source before, during or after the processing of the milk source during the cheese making process, greatly increasing by this means the recoverable yield of (2-3) sialyllactose from the milk source. Dairy sources and waste streams from cheese processing are known to contain high concentrations of lactose and numerous (2-3) sialosides, such as, for example, k casein, and gangliosides. Applicants are the first to provide a method for producing (2-3) sialyllactose in a milk source or a waste stream from cheese processing. More specifically, the method of the present invention utilizes the catalytic activity of a (2-3) trans-sialidases to exploit the high concentrations of lactose and (2-3) sialosides found naturally in milk sources, for boost the enzymatic synthesis of a (2-3) sialillactose. This catalytic activity does not require the presence of CMP-sialic acid synthetase, CMP-sialyltransferase and / or free sialic acid to drive the sialylation of a (2-3) sialillactose and others to (2-3) sialyl oligosaccharides. Accordingly, the invention offers a novel method for producing (2-3) sialyl oligosaccharides, and specifically, (2-3) sialillactose (a-Neu5Ac- (2-3) -Gal-β- (1-4) - Glc), in a milk source or residual cheese processing stream catalyzing lactose sialidation (Gal-ß- (1-4) -Glc). In specific embodiments, the method of the invention is applied to the milk source before or during processing. In another specific embodiment, the method of the present invention is applied after the processing of the milk source (for example, to a waste stream from cheese processing). The present invention offers a method for producing sialyl oligosaccharides in a milk source. This method consists in contacting a catalytic amount of at least one a (2-3) trans-sialidase with a milk source to form a milk / rans-sialidase mixture and incubating the milk / trans-sialidase mixture under conditions suitable for activity of a (2-3) trans-sialidase. The a (2-3) sialyl oligosaccharides produced according to this method are further comprised by the present invention. The invention also provides for the recovery of the sialyl oligosaccharides contained in the incubated milk mixture / trans-sialidase or otherwise, in compositions formed after the processing of the incubated milk / rans-sialidase mixture (eg, a waste stream from cheese processing ), using techniques that include, but are not limited to, ultrafiltration, diafiltration, nanofiltration, electrodialysis, phase partitioning, and ion exchange chromatography. The present invention also provides a method for producing sialyl oligosaccharides in a waste stream for cheese processing. This method consists in contacting a catalytic amount of at least one a (2-3) trans-sialidase with a residual stream from the cheese processing to form a residual current / rans-sialidase mixture and incubating the residual current mixture / rans. -sialidase under conditions suitable for the activity of a (2-3) trans-sialidase. The a (2-3) sialyl oligosaccharides produced according to this method are further comprised by the present invention. The invention also provides for the operation of the sialyl oligosaccharides contained in the incubated dairy / trans-sialidase mixture using techniques that include, but are not limited to,, ultrafiltration, diafiltration, nanofiltration, electrodialysis, phase separation and ion exchange chromatography. The invention further provides a method for producing (2-3) sialyllactose. This method consists of contacting a catalytic amount of at least one a (2-3) trans-sialidase with lactose and one a (2-3) sialyl oligosaccharide, in the absence of CMP-sialyltransferase, to form a mixture, and incubate this mixing under conditions suitable for the activity of (2-3) trans-sialidase. The a (2-3) sialillactose produced according to this method is further comprised by the present invention. The invention also provides for the recovery of the sialillactose contained in this incubated mixture using techniques including, but not limited to, ultrafiltration, diafiltration, nanofiltration, electrodialysis, phase partitioning and ion exchange chromatography. The invention further provides an enrichment method for a (2-3) sialillactose in milk using transgenic mammals expressing for a transgene of (2-3) trans-sialidase. According to this method, a transgene consisting of a sequence encoding a (2-3) trans-sialidase is operably linked to a regulatory sequence of a gene expressed in breast tissue and this transgene a (2-3) trans-sialidase / regulatory sequence is then introduced into the germline of a mammal to produce a transgenic mammal. Milk produced by a transgenic mammal demonstrating (2-3) trans-sialidase activity in mammary tissue contains enriched concentrations of a (2-3) sialillactose. The invention also suggests the recovery of the sialillactose contained in the milk produced by this transgenic mammal before or after processing the milk. Transgenic mammals containing a coding sequence for a (2-3) trans-sialidase operably linked to a regulatory sequence of a gene expressed in mammary tissue are also provided by the invention. Importantly, a milk source, waste stream from cheese processing and the transgenic mammal can be used to produce enriched concentrations of a (2-3) sialyllactose. As used herein, "trans-sialidase" refers to a compound that catalyzes the transfer of sialic acid from a saccharide-containing molecule (eg, oligosaccharide, polysaccharide, glycoprotein or glycolipid) to another molecule containing saccharide and which does not require the presence of free sialic acid, CMP-sialic acid, synthetase and / or CMP-sialyltransferase in the reaction mixture for its activity. As used herein, "trans-sialidase activity" refers to the catalytic reaction in that an enzyme catalyzes the elimination of a sialic acid from a molecule containing saccharide and the transfer of sialic acid to another molecule containing saccharide, covalently binding the sialic acid to the acceptor molecule through a glycosidic bond. used in the present, a "catalytic amount" of enzyme to (2-3) trans-sialidase refers to the amount of enzymes sufficient to cause the trans of a sialic acid from a molecule containing saccharide to another molecule containing saccharide. As used herein, "conditions suitable for trans-sialidase activity" comprise the appropriate conditions (e.g., temperature, pH-and incubation time) sufficient to allow the enzymatic removal of a sialic acid from a molecule containing saccharide and the transfer of sialic acid to another molecule containing saccharide. As used herein, "a (2-3) sialyl oligosaccharides" refers to sugars in which a sialic acid is covalently attached to the 3 'carbon of a β-galactose moiety by a glycosidic linkage. In the methods of the present invention, a (2-3) sialoligosaccharides comprises saccharides with any form of sialic acid covalently linked to 3'-β-galactose. As used in this"Milk source" refers to a lactation product in a mammal, a substance prepared by the product or a by-product thereof. As used herein, "milk source" includes, but is not limited to, milk, colostrum, cheese processing mixture, and a composition that simulates milk. As used herein, "milk processing mixture" is a compilation of dairy processing ingredients at any stage during dairy processing (eg, pasteurization, fermentation or cheese making) in addition to the waste stream from cheese processing . As used herein "a milk simulating composition" is a solution lacking one or more of the following: CMP-sialyltransferase, CMP-synthetase and / or free sialic acid, but containing at least a (2-3 ) sialosides to act as donors of trans-sialidase, lactose and, optionally, suitable buffering agents to maximize the activity of a (2-3) transsialidase when it is added to the solution. As used herein, "cheese processing waste stream" refers to a byproduct of cheese making and includes, but is not limited to, the complete serum, permeated from demineralized whey, the regeneration stream from the permeate of demineralized serum, serum permeate, crystallized lactose, dry lactose by aspersion, sperm powder, edible lactose and lactose. Serum containing sialic acids is a by-product obtained when rennet cheese or casein is produced from milks such as cow's milk, goat's milk and sheep's milk. For example, acid whey is generated by separating the solids when the skim milk is coagulated to form cottage cheese. Acid whey is characterized by a high content of lactic acid. When this cheese is prepared from the whole milk, the remaining liquid is sweet whey, which can also be processed by evaporation to form the dry whey powder. The sweet serum can also be dried, demineralized and evaporated to form demineralized serum permeate. The sweet whey can also be subjected to ultrafiltration to generate a serum permeate and a whey protein concentrate. The permeate of serum can also be processed by crystallizing lactose to form lactose and a mother liquor. The mother liquor resulting from the crystallization of lactose from a permeate of whey is known in the art as "Delac". The a (2-3) trans-sialidase used according to the method of the present invention comprises trans-sialidases from quinetoplastide, trans-sialidases from Trypanosoma, Endotrypanum, and Pneumocystis and includes trans-sialidases from Trypanosoma cruzi, Trypanosoma brucei, Endotrypanum spp. and Pneumocystis carinni. The trans-sialidases that can be used according to the method of the present invention are further defined in section 5.1. 4. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Complete nucleotide sequence of a (2-3) trans-sialidase from Trypanosoma cruzi (Genbank D50685). Figure 2. Deduced amino acid sequence of a (2-3) trans-sialidase from Trypanosoma cruzi (Genbank D50685). Figure 3. Nucleotide sequence of an a (2-3) trans-sialidase functional Trypanosoma cruzi devoid of amino acid repeats (Genbank L26499). Figure 4. Deduced amino acid sequence of a Trypanosoma cruzi functional a (2-3) trans-sialidase devoid of amino acid repeats (Genbank L26499). Figure 5. Effect of pH on enrichment of a (2-3) sialillactose in mozzarella serum. The concentration of a (2-3) sialillactose (μg / ml) is shown as a function of the incubation time of 0.1% lysate of a (2-3) trans-sialidase at 25 ° C. The tables represent pH 4.0; clear diamonds represent pH 5.0; the circles represent pH 6.0; the triangles represent pH 7.0; the crossed squares represent pH 8.0; the shaded diamonds represent pH 9.0. The enrichment of a (2-3) sialillactose was observed at all tested pHs, with only minimal enrichment observed after 20 minutes at pH 4.0. Figure 6. Enrichment of a (2-3) sialillactose in skimmed milk. The concentration of (2-3) sialillactose (μg / ml) is shown as a function of the incubation time with 0.1% trans-sialidase lysate at 22 ° C. Figure 7A-B Enrichment of a (2-3) sialillactose in mozzarella serum. The concentration of a (2-3) sialillactose (μg / ml) is shown as a function of the incubation time with 0.1% of the trans-sialidase lysate at 25 ° C.

(FIGURE 7A) and 23 ° C (FIGURE 7B). Figure 8 Enrichment of a (2-3) sialillactose in Swiss cheese whey. The concentration of (2-3) sialillactose (μg / ml) is shown as a function of the incubation time with 0.1% lysate of a (2-3) trans-sialidase at 23 ° C for 43 hours. Figure 9 Enrichment of a (2-3) sialillactose in a solution containing 20 μg / ml of lactose, 5 μg / ml of casein k at 23 ° C for 22 hours.

. DETAILED DESCRIPTION OF THE INVENTION The invention relates to methods for producing sialyl oligosaccharides in a milk source, particularly, at (2-3) sialyllactose, by contacting a catalytic amount of (2-3) trans-sialidase with a milk source for forming a dairy / trans-sialidase mixture and incubating this mixture under conditions suitable for the activity of the (2-3) trans-sialidase. The invention also relates to methods for recovering (2-3) sialylated oligosaccharides from this incubated milk / trans-sialidase mixture, or otherwise from the compositions formed after the processing of the dairy / trans-mixture. sialidase (for example, a residual stream of cheese processing). In a specific embodiment, a (2-3) sialillactose is recovered from the composition processed by ultrafiltration and ion exchange chromatography. The invention further provides methods for producing 0. (2-3) sialyl oligosaccharides in a residual stream of cheese processing by contacting a catalytic amount of trans-sialidase with a residual stream of cheese processing to form a residual current / rans mixture. -sialidase and incubating this mixture under conditions suitable for the activity of a (2-3) transsialidase. The invention also relates to methods for recovering (2-3) sialyl oligosaccharides from this incubated residual / trans-sialidase mixture. The methods of the present invention can be used to produce (2-3) sialyl oligosaccharides in any reaction mixture containing siallylated saccharide (2-3) compositions (eg oligosaccharides, polysaccharides, glycoproteins and glycolipids) and lactose. The starting materials can, therefore, be obtained from all milk sources (eg, human and animal milk, serum and colostrum) or otherwise, a mixture of lactose and coapositions of (2-3) sialylated saccharides that simulate a milk source. . 1. (2-3) TRANg-SIALIDASE The a (2-3) trans-sialidase used according to the method of the invention is an a (2-3) trans-sialidase, or derivative (including fragments or proteins of fusion), or analogs thereof, which can catalyze the separation of sialic acid from a molecule containing saccharides and catalyze the transfer of sialic acid to a second molecule containing saccharide. The a (2-3) trans-sialidases which can be used according to the method of the invention include, but are not limited to, a (2-3) kinestoplastid trans-sialidase from a species of the genus Trypanosoma, Endotrypanum. and Pneumocystis such as, for example, (2-3) trans-sialidase from Trypanosoma cruzi, (2-3) trans-sialidase from T. brucei (Pontes de Carvalho et al., 1993, J. Exp. Med. 177: 465-474), trans-sialidase from Pneumocystis carinni (L, Trimbal, N. Pavia &MEA Pereira, unpublished information as mentioned in Schenkman et al., 1994, Annu Rev. Microbiol. 48: 499- 523), and trans-sialidase from Endotrypanum spp. (Medina-Acosta et al., 1994, Mol Biochem Parasitol Nucleic Trans-sialidase nucleic acid sequences are known (eg, Genbank Sequence L26499, SPTREMBL: Q26964 (Ue ura), SPTREMBL: Q26965 (Uemura), SPTREMBL: Q26966 (Uemura), Q26969 (Cremona et al.), Genbank D540685 (Uemura) In specific embodiments, a polypeptide containing or comprising a fragment of at least 50 (continuous) amino acids of a (2-3) ) trans-sialidase are used according to the method of the invention In other embodiments, the fragment consists of at least 100, 150, 200, 250, 300, 350, 400, 450, 500 or 550 amino acids of the a (2 3) trans-sialidase In other specific embodiments, such fragments are not larger than 500, 400, 300, 200 or 100 amino acids the derivatives or analogues of a (2-3) trans-sialidase, include but are not limited to , those molecules that catalyze the transfer of sialic acid from a molecule containing saccarid or, (for example, an oligosaccharide, polysaccharide, glycoprotein or glycolipid) to another molecule containing saccharide and which are encoded by a DNA sequence that hybridizes to the complement of a DNA sequence encoding a (2-3) trans -sialidase, as it may be for example, those mentioned above, under conditions of high severity, delayed high severity or low severity. As an example and not as a limitation, the procedures that use the conditions of low severity are as follows (see also Shilo and Weinberg, 1981, Proc. Nati, Acad. Sci. USA 78: 6789-6792): the filters that contain DNA are pretreated for 6 hours at 40 ° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1 % BSA and 500 μg / ml of denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.01% Ficoll, 0.2% BSA, 100 μg / ml salmon sperm DNA, 10% (weight / volume) dextran sulfate, and 5-20 x 10 6 cpm of 32P labeled probe is used. The filters are incubated in the hybridization mixture for 18-20 hours at 40 ° C and then washed for 1.5 hours at 55 ° C in a solution containing 2X SSC, 25mM Tris-HCl (pH 7.4) 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60 ° C. The filters are subjected to analysis, dried and placed for autoradiography. If necessary, the filters are washed a third time at 65-68 ° C and re-exposed to the film. Other conditions of low severity that can be used are well known in the art. By way of example and not as limitation, the procedures that use conditions of high severity are as follows: The pre-hybridization of DNA-containing filters is carried out during 8 hours overnight at 65 ° C in buffer solution composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 μg / ml denatured salmon sperm DNA. The filters are hybridized for 48 hours at 65 ° C in the mixture for pre-hybridization containing 100 μg / ml of denatured salmon sperm DNA and 5-20 x 10 cpm of P-labeled probe. performed at 37 ° C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50 ° C for 45 minutes before autoradiography. It is also possible to use other conditions of high severity known in the art. For example and not as a limitation, the procedures that use conditions of moderately high severity are as follows: filters containing DNA are pretreated for 6 hours overnight at 55 ° C in buffer solution composed of 6X SSC, 5X 0.5 Denhart SDS, 100 μg / ml salmon sperm DNA. Hybridizations are performed in g the same solution with the addition of 5-20 x 10 cpm of the P-labeled probe and incubated for 8-48 hours at 55 ° C. The filters are washed at 60 ° C in IX SSC, 0.1% SDS, with two changes after 30 minutes. Other conditions for screening with moderately high severity are known in the art. For further guidance related to hybridization conditions, see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. The invention also relates to a (2-3) trans-sialidase derivatives or analogs made by modifying the sequence of a (2-3) trans-sialidase by substitutions, additions or deletions that offer molecules with a (2) activity. -3) trans-sialidase (i.e., catalyzes the transfer of sialic acid from one molecule containing saccharide to another). Thus, derivatives of a (2-3) trans-sialidase include polypeptides which contain, as a primary amino acid sequence, all or part of the amino acid sequence of a (2-3) trans-sialidase including modified sequences in which functionally equivalent amino acid residues are substituted by residues within the sequence giving rise to a polypeptide that is functionally active (i.e., a polypeptide that possesses the activity of trans-sialidase). For example, one or more amino acid residues within the secence can be replaced by another amino acid of a similar polarity that acts as a functional equivalent, giving rise to a silent modification. Conservative substitutions for an amino acid within the sequence can be selected from other members of the class to which the amino acids belong. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Neutral polar amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The amino acids with positive charge (basic) include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. Such derivatives of α (2-3) trans-sialidase can be made by chemical synthesis of peptides or by recombinant production from nucleic acids encoding the (2-3) trans-sialidase that have been mutated. Any technique for known mutagenesis can be used, including but not limited to chemical mutagenesis, site-directed mutagenesis, in vitro, (Hutchinson et al., 1978, J. Biol. Chem 253: 6551), using the ® linkers. (Pharmacia), etcetera. Trans-sialidase or functionally active derivatives (including fragments of fusion proteins), or analogues used according to the method of the present invention can be obtained by purification from biological tissue or cell culture, or they can be produced by recombinant or synthetic techniques known in the art.

Preparations of a (2-3) native trans-sialidase can be obtained from different sources. Normal methods for protein purification can be used to isolate and purify or partially purify a (2-3) trans-sialidases from any known source that contains or produces a. { 2-3) desired trans-sialidase, for example, T. cruzi or T. brucei. Such standard protein purification techniques include but are not limited to chromatography (eg, ion exchange, affinity chromatography, gel filtration / gel filtration and high performance liquid phase reverse phase chromatography (RP-HPLC)), centrifugation, differential solubility and electrophoresis (for a review of protein purification techniques see Scopes, Protein Purification, Principies and Procedure, 2nd ed., CR Cantor, Editor, Springer Verlag, New York, New York (1987), and Parvez et al., Progress in HPLC, vol. 1, Science Press, (1985) Utrecht, The Netherlands). For example, antibodies to trans-sialidases can be generated using known techniques, and can be used to prepare an affinity chromatography column to purify the respective trans-sialidases by well known techniques (see for example, Hudson &May , 1986, Practical Immunology, Blackwell Scientific Publications, Oxford, United Kingdo). . 1.1 RECOMBINANT PRODUCTION OF RANS-SIALIDASE The recombinant expression techniques can be applied to obtain the a (2-3) trans-sialidases, derivatives and analogues used according to the method of the invention (see for example, Sambrook et al., 1989, Molecular Cloning, A. Laboratory Manual, Cold Spring Harbor Laboratory, 2nd ed., Cold Spring Harbor, New York, Glover, DM (ed.), 1985, DNA cloning: A Practical Approach, MRDL Press, Ltd., Oxford , UK, vol. I, II). The nucleic acid sequences of a (2-3) trans-sialidases co or can be, for example, those described above, are known and can be isolated using well-known techniques, such as genomic DNA library screening or cDNA, chemical synthesis or polymerase chain reaction (PCR). In addition, given these known sequences it is possible to clone other (2-3) trans-sialidases using known routine recombinant techniques, for example, PCR and hybridization for the complement of the known nucleic acid sequence under highly stringent conditions, with moderately high severity and with low severity, in combination with the assays which select the known biochemical properties of the (2-3) trans-sialidase of interest, or in general, to catalyze the transfer of sialic acid from a molecule containing donor saccharide to an acceptor molecule containing saccharide. The gene sequence of the cloned a (2-3) trans-sialidase can be modified by any of the various strategies known in the art, to recombinantly produce an a (2-3) trans-sialidase, derivative or analog, a nucleic acid sequence encoding the a (2-3) trans-sialidase derivative or analog is operably linked to a promoter such that the a (2-3) trans-sialidase, derivative or analog is produced from of this sequence. For example, it is possible to introduce a vector into a cell, within which cell the vector or a portion thereof is expressed, producing an a (2-3) trans-sialidase or a part thereof. In a preferred embodiment, the nucleic acid is DNA if the source of RNA polymerase is RNA polymerase directed to DNA, but the nucleic acid can also be RNA if the polymerase source is RNA polymerase directed to RNA or if it is present in the transcriptase cell reverse or is provided to produce DNA from RNA. Such a vector can remain episomal or can be integrated into the chromosome, as long as it can be transcribed to produce the desired RNA. Vectors thus can be constructed by the methods of recombinant DNA technology normal in the art. It is possible to use a series of host-vector systems to express the coding sequence of the protein. These include, but are not limited to, mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus); insect cell systems infected with viruses (eg, baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophages, DNA, plasmid DNA or cosmid DNA. The vector expression elements vary in their resistances and specificities and it depends on the host-vector system used it is possible to employ any of a series of suitable transcription and translation elements. The expression of an a (2-3) trans-sialidase derivative or analog can be controlled by any promoter / enhancer element known in the art. Such promoters include, but are not limited to, the early SUB 40 promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the 3 'long terminal repeat of the Rous sarcoma virus (Yamamoto et al. ., 1980, Cell 22: 787-797), HSV-1 (herpes simplex virus-1), thymidine kinase promoter (Wagner et al., 1981, Proc. Nati. Acad. Sci. USA 78: 1441-1445 ), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42); prokaryotic expression vectors such as ß-lactamase (Vílla-Kamaroff et al., 1978, Proc. Nati, Acad. Sci. USA 75: 3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Nati, Acad. Sci. USA 80: 21-25); see also "Useful Proteins from Recombinant Bacteria" in Scientific American, 1980, 242: 74-94; expression vectors in plants consisting of the promoter region of nopaline synthetase (Herrera-Estrella et al., 1983, Nature 303: 209-213) or the 35S RNA promoter of the cauliflower mosaic virus (Gardner et al., 1981, Nucí, Acids Res. 9: 2871), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310: 115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter and the following animal transcriptional control regions, which exhibit tissue specificity and have been used in transgenic animals: the control region of the elastase I gene that is active in pancreatic acidic cells (Swift et al., 1984, Cell 38: 639-646, Ornitz et al., 1986, Cold Spring Harbor Symp. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); the control region of the insulin gene that is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122), the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318, 533-538; Alexander et al., 1987, Mol. Cell, Biol. 7: 1436-1444), the control region of mouse mammary tumor virus that is active in testicular, breast, lymphoid and chewing cells. (Leder et al., 1986, Cell 45: 485-495), the control region of the albumin gene that is active in liver (Pinkert et al., 1987, Genes and Devel. 1: 268-276), the region of control of the alpha-fetoprotein gene that is active in the liver (Krumlauf et al., 1985, Mol Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); the control region of the alpha 1-antitrypsin gene that is active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), the control region of the beta-globin gene that is active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340); the control region of the myelin basic protein gene that is active in oligodendritic cells in the brain (Readhead et al., 1987, Cell 48: 703-712); the control region of the myosin light chain -2 gene that is active in skeletal muscle (Sani, 1985, Nature 314: 283-286), and the control region of the gonadotropic releasing hormone gene that is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378). The promoter element that is operably linked to the nucleic acid encoding the trans-sialidase, derivative or analog, can also be a bacteriophage promoter with the source of the bacteriophage RNA polymerase expressed from a gene for RNA polymerase on a separate plasmid, for example, under the control of an inducible promoter, eg, nucleic acid encoding transialidase, derivative or analog, operably linked to the promoter of the T7RNA polymerase with a separate plasmid encoding the T7RNA polymerase . In a preferred embodiment of the invention, the expression of a (2-3) trans-sialidase, derivative or analog is controlled by a regulatory sequence of a gene expressed in mammary tissue, such as, for example, the regulatory sequence of a gene coding for a milk-specific protein (see for example, Wright et al., 1991, Biotechnology (NY) 9: 830-834; Carver et al., 1993, Biotechnology (NY) 11: 1263-1270); Clark et al., 1989, Biotechnology (NY) 7: 487-492; Velander et al., 1992, Proc. Nati Acad. Sci. USA, 89: 12003-12007; Ebert et al, 1991, Biotechnology (NY) 9: 835-838). It is also possible to choose the strain of the host cells that modulate the expression of the inserted sequences, or modify and process the genetic product in the specific form desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, it is possible to control the expression of a (2-3) trans-sialidase, derivatives or analogs designed by genetic engineering. In addition, different host cells have specific characteristics and mechanisms for the processing and modification of translation and posttranslation (eg, glycosylation, protein phosphorylation). Suitable cell lines or host systems can be chosen to ensure the desired modification and processing of the expressed foreign protein. For example, it is possible to use expression in a bacterial system to produce a non-glycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can be used to ensure "native" glycosylation of a heterologous protein. In addition, different vector / host expression systems can affect the processing reactions to different degrees. The nucleic acid sequence encoding the (2-3) trans-sialidase can be used in vitro or in vivo, -to create and / or destroy the translation, initiation and / or termination sequences, or to create variations in the coding regions. Any of the techniques for known mutagenesis can be used, including but not limited to, site-directed mutagenesis, in vitro (Hutchinson et al., 1978, J. Biol. Chem. 253: 6551), the use of ® linkers ( Pharmacia) and so on. The experimentation included in the mutagenesis consists mainly of site-directed mutagenesis followed by proof of the phenotype of the modified gene product. Some of the most commonly used site-directed mutagenesis protocols take advantage of vectors that can provide single-stranded as well as double-stranded DNA, as necessary. In general, the mutagenesis protocol with such vectors is as follows. A mutagenic primer is synthesized, that is, a primer complementary to the sequence to be changed, but consisting of one or a small number of modified, added or deleted bases. The primer is extended in vitro by a DNA polymerase and, after some additional manipulations, the double-stranded DNA is now transfected into bacterial cells. Next, by a series of methods, the desired DNA is identified, and the desired protein is purified from clones containing the sequence used. For larger sequences, additional cloning steps are often required because long inserts (longer than 2 kilobases) are unstable in these vectors. The protocols are known to those skilled in the art and kits for site-directed mutagenesis are available to biotechnology supplier companies, for example, from Amersham Life Science, Inc. (Arlingon Heights IL) and Stratagene Cloning Systems (The Jolla, CA). In specific embodiments, the a (2-3) trans-sialidase, derivative or analogue used according to the method of the invention is generated by site-directed mutagenesis of the DNA encoding a (2-3) trans-sialidase not functional. In a specific embodiment, the codon encoding the amino acid at position 342.

Try 342 of Genbank L26499) is mutated to code for a tyrosine residue. In another specific embodiment, a more active (2-3) trans-sialidase is generated by site-directed mutagenesis of DNA encoding a less active a (2-3) transsialidase by mutating the codon coding for the amino acid in the position 231 of the a (2-3) trans-sialidase less active (relative to Pro 231 of Genbank L26499) to encode a proline residue. In other specific embodiments, the derivative or analog of a (2-3) trans-sialidase can be expressed as a function of a chimeric protein product (consisting of the protein, fragment, analog or derivative linked by a peptide bond to the sequence of the heterologous protein (of a different protein)). Such a chimeric product can be prepared by ligating suitable nucleic acid sequences coding for the desired amino acid sequences to each other by methods known in the art, in the proper coding framework, and expressing the chimeric product by methods commonly known in the art. technique (see, for example, section 5.6). . 1.2. CHEMICAL SYNTHESIS OF TRAWg-SIALIDASE In addition, (2-3) trans-sialidases derivatives (including fragments and chimeric proteins) and analogs can be synthesized chemically, see for example, Clark-Lewis et al., 1991, Biochem . 30: 3128-3135 and Merrifield, 1963, J. AMER. Chem. Soc. 85: 2149-2156. for example, the a (2-3) trans-sialidases derivatives and analogues can be synthesized by solid base techniques, split from the resin and purified by preparative high performance liquid chromatography (for example, see Creighton, 1983, Proteins, Structures and Molecular Principles, WH Freeman and Co., NY pp. 50-60). The a (2-3) trans-sialidases, and the derivatives and analogues thereof can also be synthesized by the use of a peptide synthesizer. The composition of the synthetic peptides can be confirmed by analysis or sequencing of the amino acids (for example, the Edman degradation procedure, see Creighton, 1983, Proteins, Structures and Molecular Principles, WH Freeman and Co., NY pp. 34- 49). Furthermore, if desired, it is possible to introduce non-traditional amino acids or analogous chemical amino acids as a substitution or addition in the a (2-3) trans-sialidase, derivative or analogue. Non-traditional amino acids include, but are not limited to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, α-Albu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulin, cysteic acid, T-butylglycine, T-butyl alanine, phenyl glycine, cyclohexyl alanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylaminoaceids, C-methylamino acids, N α -methylamino acids and amino acids in general. In addition, the amino acid may be D (dextrorotatory) or L (levorotatory). By way of example, but not as a limitation, the proteins (including the peptides) of the invention can be chemically synthesized and purified as follows: the a (2-3) trans-sialidases, derivatives and analogs can be synthesized using the Na -9-fluorenylmethyloxycarbonyl or the chemistry of Fmoc solid phase peptide synthesis using a Rainin Symphony multiplex peptide synthesizer. The normal cycle used to couple an amino acid to a growing peptide-resin chain generally includes: (1) washing the peptide-resin three times for 30 seconds with N, N-dimethylformamide (DMF); (2) separating the Fmoc protecting group on the amino terminus by deprotection with 20% piperidine in DMF by two washes of 15 minutes each, during which process mixing takes place [sic] by bubbling nitrogen through the reaction vessel for one second every ten seconds to avoid sedimentation of the peptide-resins; (3) wash the peptide-resin three times for 30 seconds with DMF; (4) coupling the amino acid to the peptide-resin by adding equal volumes of a 250 mM solution of the Fmoc derivative to the appropriate amino acid and an activator mixture consisting of 400 mM N-methylmorpholine and (2- (1 H-benzotriazole hexafluorophosphate -1-4)) -1, 1, 3, 3-tetramethyluronium (HBTU) 250 mM in DMF; (5) leave the solution mixed for 45 minutes; and (6) washing the peptide-resin three times for 30 seconds of ~ [sic] DMF. This cycle can be repeated as necessary with the appropriate amino acids in sequence to produce the desired polypeptide. The exceptions to this cycle program are the coupling of the amino acids predicted as difficult by the nature of their hydrophobicity or the expected inclusions within a helical formation during the synthesis. For these situations, the aforementioned cycle can be modified by repeating step 4 a second time immediately after the completion of the first 45 minute coupling step for a "double coupling" of the amino acid of interest. In addition, in the first step of coupling in the synthesis of polypeptides it is possible to allow the resin to swell for a more effective coupling by increasing the mixing time in the washings with initial DMFs up to 3 washes of 15 minutes instead of 3 washes of 30 minutes. seconds. After synthesis of the polypeptide, the polypeptide can be cleaved or dissociated from the resin as follows: (1) washing the polypeptide-resin three times for 30 seconds with (DMF); (2) separating the Fmoc protecting group at the amino terminus by washing it twice for 15 minutes in 20% piperidine in DMF; (3) washing the polypeptide-resin three times for 30 seconds with DMF; and (4) mixing a dissociation cocktail consisting of 95% trifluoroacetic acid (TFA), 2.4% water, 2.4% phenol and 0.2% triisopropylsilane with the polypeptide-resin for 2 hours, then filtering the peptide in the dissociation cocktail to separate it from the resin, and precipitate the peptide from the solution by the addition of two volumes of ethyl ether. To isolate the polypeptide, it is possible to allow the ether-peptide solution to stand at -20 ° C for 20 minutes, then centrifuge at 6000 x G for 6 minutes to pack the polypeptide, and the polypeptide can be washed three times with ethyl ether to separate the ingredients of the residual dissociation cocktail. The final polypeptide product can be purified by reverse phase, high resolution liquid chromatography (RP-HPLC) with the primary solvent consisting of TFA at 0. 1% and the eluent buffer consisting of 80% acetonitrile and 0.1% TFA. The purified polypeptide can then be lyophilized to a powder. . 2. TESTS FOR THE ACTIVITY OF a (2-3) RANS-SIALIDASE The invention is based on parts in the discovery that the addition of a (2-3) trans-sialidase to a milk source in sufficient quantities catalyses the transfer of sialic acids from the population of sialyl oligosaccharides from the milk source will favor the sialylation of lactose due to the high concentration of lactose in milk sources. Thus, the ability of a (2-3) trans-sialidases, and derivatives and analogs thereof to catalyze the transfer of sialic acid from a donor source of (2-3) sialyl oligosaccharides to an oligosaccharide acceptor having a portion of β-galactose in its non-reducing termination is indicative of the utility of these proteins, derivatives and analogs in the production of sialyl oligosaccharides in a milk source or waste stream from cheese processing according to the methods of the present invention. The trans-sialidases that can be used according to the methods of the invention comprise all the sequences of proteins with functional (2-3) trans-sialidase activity. The a (2-3) trans-sialidases, therefore, are defined by their catalytic activity in which a (2-3) trans-sialidase directs the transfer of a sialic acid from a molecule containing saccharides (e.g. oligosaccharide, polysaccharide, glycoprotein o-glycolipid) to another. Assays for the activity of a (2-3) trans-sialidase are known in the art and can be applied in accordance with the present invention, both to identify the a (2-3) trans-sialidases, derivatives and analogs by demonstrating the necessary catalytic activity, as well as for the optimization of the reaction parameters (e.g., concentration, temperature, pH and incubation time) to incubate the a (2-3) trans-sialidase, derivatives or analogues with the milk source or the residual current of cheese processing. In one embodiment, the activity of a (2-3) transsialidase is measured using the method described in Vandekerckhove, et al., 1992, Glycobiology 2: 541-548. In brief, a (2-3) trans-sialidase is incubated in 20 mM Hepes buffer (Sigma H-3375) at pH = 7.2 in the presence of a (2-3) sialillactose and [D-glucose-1- C] lactose (60 mCi / mmol) (Amersham, Arlington Heights, IL). The reactions are interrupted by the addition of 20 μl of ethanol. The resulting compounds are analyzed by thin layer chromatography (TLC) on silica gel plates (EM Science, TLC Fertigplatten Kieselgel 60F254, 10 x 10 cm) and subjected to chromatography in ethanol-N-butanol-pyridine-water-acid acetic [100: 10: 10: 30: 3 (v / v)]. The molecules containing sialic acid are visualized by staining with resorsinol against the standards of a (2-3) sialillactose and a (2-6) sialillactose Neu5Ac, MU-Neu5Ac. Other assays for the activity of a (2-3) trans-sialidase are known and can be used according to the present invention to test and / or to optimize the activity of (2-3) trans-sialidase. In addition, assays for glycosyltransferase activity known in the art can also be routinely modified to test and / or optimize the activity of a (2-3) trans-sialidase. . 3. ENRICHMENT OF a (2-3) ACALYOLISOLISOLISES INCLUDED (2-3) SIALILLACTOSE The invention provides methods for producing sialyl oligosaccharides, particularly (2-3) sialyllactose in a milk source or in a residual stream from cheese processing. In one embodiment, the present invention provides a method for producing sialyl oligosaccharides in a milk source. This method consists in contacting a catalytic amount of at least one a (2-3) trans-sialidase with a milk source to form a milk / trans-sialidase mixture., and incubate the dairy / trans-sialidase mixture under conditions suitable for the activity of the (2-3) trans-sialidase. In another embodiment, the present invention provides a method for producing sialyl oligosaccharides in a waste stream from cheese processing. This method consists in contacting a catalytic amount of at least one (2-3) trans-sialidase with a residual stream of the cheese processing to form a residual current / trans-sialidase mixture and incubating the residual current / trans-sialidase mixture. under conditions suitable for the activity of a (2-3) trans-sialidase. In a further embodiment of the present invention, sialyl oligosaccharides are produced and recovered from a milk source by a method consisting of contacting a catalytic amount of at least one (2-3) trans-sialidase with a milk source for forming a dairy / trans-sialidase mixture, incubating the milk / trans-sialidase mixture under conditions suitable for the activity of the (2-3) trans-sialidase and recovering the sialyl oligosaccharides from the incubated milk / rans-sialidase mixture. The present invention also provides a method for producing sialyl oligosaccharides in a milk source which is subsequently processed to make cheeses, followed by recovery of the sialyl oligosaccharides from the residual stream of cheese processing. When producing sialyl oligosaccharides from a dairy source, processed for cheese making and recovering from the residual stream of cheese processing by the method of the present invention, the method consists in contacting a catalytic amount of at least one a (2- 3) trans-sialidase with a milk source to form a milk mixture / rans-sialidase incubate the milk mixture / trans-sialidase under conditions suitable for the activity of the (2-3) trans-sialidase and process the milk incubated mixture / trans-sialidase using a known protocol for the manufacture of cheeses, and recover the sialyl oligosaccharides from the residual stream from the processing of cheeses obtained from the incubated milk / trans-sialidase mixture. In another embodiment of the present invention, the sialyl oligosaccharides are produced and recovered from the waste streams of the cheese processing by a method consisting of contacting a catalytic amount of at least one (2-3) trans-sialidase with a Residual stream of cheese processing to form a residual current / trans-sialidase mixture, incubate the residual current / rans-sialidase mixture under conditions suitable for the activity of the (2-3) trans-sialidase, and recover the sialyl oligosaccharides from the incubated residual stream / rans-sialidase. In each embodiment of the methods of the present invention, a (2-3) trans-sialidases consist of molecules with enzymatic activity, wherein a sialic acid is transferred from a saccharide-containing molecule to another molecule containing saccharide. The saccharide-containing molecules can be oligosaccharides, polysaccharides, glycoproteins or glycolipids. The a (2-3) trans-sialidases used according to the methods of the present invention are further defined in Sections 3.1 and 5.1. The a (2-3) trans-sialidase used according to the methods of the present invention. invention can be a (2-3) purified trans-sialidase, derivative or the like; a (2-3) trans-sialidase, derivative or analog, partially purified; or a crude or filtered eukaryotic or bacterial lysate (e.g., E.Coli) containing the activity of ot (2-3) trans-sialidase. The optimal enzyme concentrations used according to the methods of the present invention can be determined routinely using known techniques. In specific embodiments, the concentration of a (2-3) trans-sialidase used according to the methods of the invention is at least 0.001, 0.005, 0.01, 0.05, 0.075, 0.10 or 0.4 units / ml (wherein one unit is defined as the concentration of enzyme necessary to produce 1 umol of NAN-a (2-3) -Gal-ß (1-4) -GlcNAc-ß-Gal-ß (1-3) -Gal-ß- (1 -4) -Glc (LST-d) / min in a standard assay using a (2-3) sialillactose and Gal-β (1-4) -GlcNAc-β- (1-3) -Gal-β (1- 4) -Glc (lacto-N-neotetraose, LNnT) as substrates). The milk sources used in the methods according to the present invention include, but are not limited to, milk, colostrum, cheese processing mixture or a milk simulating composition. As used herein, the phrase cheese processing mixture refers to a collection of dairy processing ingredients at any stage during the processing of dairy products (eg, cheese making) in addition to the waste stream from the processing of dairy products. cheeses A milk simulant composition is a solution lacking one or more of CMP-sialyltransferase, CMP-synthetase and / or free sialic acid, but containing at least (2-3) sialosides to act as donors for the a (2- 3) trans-sialidase, lactose and, optionally, suitable buffering agents to maximize the activity of the (2-3) trans-sialidase when it is added to the solution. Otherwise, a milk simulation composition is a solution that contains at least (2-3) sialosides to act as donors for a (2-3) trans-sialidase and lactose, and where the presence of sialic acid Free, CMP-sialyltransferase and / or CMP-synthetase is not required to boost lactose sialylation by a (2-3) trans-sialidase. A residual stream of cheese processing is the portion of cheese making not retained by the cheese after curd or cottage cheese formation. The waste stream from cheese processing usually refers to the drained fluid from cottage cheese, which is often discarded. A residual stream of the cheese processing of the present invention includes, but is not limited to, the complete, permeated serum of demineralized serum, stream of demineralized permeate regeneration, serum permeate, crystallized lactose, spray-dried lactose, whey powder, edible lactose and lactose. In each embodiment of the present invention, the a (2-3) trans-sialidase is contacted with the milk source or the residual stream of the cheese processing and the resulting mixture can be stirred, mixed or subjected to any other method of combination. If (2-3) trans-sialidase is added to the colostrum, milk or a mixture of cheese processing, a milk simulation composition, or milk that has been subjected to some processing, it can dictate the amount of agitation or mixing that it may be necessary Although milk is relatively fluid, processed milk, such as milk being processed for cheese, can become very viscous and require more agitation, mixing or the like for effective enzymatic activity to occur. In the same way, a waste stream from the cheese processing may be a viscous solution and may require similar forms of stirring, mixing or the like for efficient enzymatic activity. Suitable conditions for producing particular sialyl oligosaccharides at (2-3) sialyllactose, in a milk source or waste stream from cheese processing by the methods of the present invention can be determined and optimized by known routine techniques. In a modality, the milk source or the residual stream of cheese processing is initially cooled to 2-20 ° C. The oum time to incubate the dairy mixture / transsialidase, generated according to the present invention can be determined routinely by known techniques. In -the specific modalities, the dairy / trans-sialidase mixture is incubated for a period of at least 0.5, 1.0, 5.0 or 10.0 hours. In a preferred embodiment, the dairy / trans-sialidase mixture is incubated for 12-30 hours. In a more preferred embodiment, the dairy / trans-sialidase mixture is incubated for 20-25 hours. The oum temperature for incubation of the dairy / trans-sialidase mixture, generated according to the methods of the present invention can be determined routinely by known techniques. In specific embodiments, the dairy / trans-sialidase mixture is incubated at about 0-30 ° C or 2-20 ° C. In preferred embodiments, the dairy / trans-sialidase mixture is incubated at 5-15 ° C or 8-12 ° C. In embodiments where the milk source is a milk simulating composition, the milk / trans-sialidase mixture can be incubated at about 0.45 ° C, 10-45 ° C or 20-40 ° C. The oum pH for incubation of the milk mixture / transsialidase according to the present invention can be determined routinely by known techniques. In specific embodiments, the milk / trans-sialidase mixture is incubated at a pH of about pH 5-9, more preferably at about pH 6-8, and most preferably the pH is at about 7. Other conditions to oize the incubation of the milk mixture / trans-sialidase will be apparent to those skilled in the art and are within the scope of the present invention. In a specific embodiment, the dairy / trans-sialidase mixture can be stirred, stirred, shaken, mixed or the like to assist in the uniform distribution of the enzyme within the mixture. In one embodiment of the invention, the exogenous (2-3) sialyl oligosaccharides are added to the milk / trans-sialidase mixture. The supplemented exogenous (2-3) sialyl oligosaccharides may contain a single homogenous population of α (2-3) sialyl oligosaccharides, or may otherwise consist of a mixture of different (2-3) sialyl oligosaccharides. The a (2-3) sialyl oligosaccharide supplemented during this incubation step should be selected to minimize possible negative effects with the taste, texture, appearance or quality of the dairy product (eg, cheese). After incubation, the milk can be pasteurized by any pasteurization method known in the art, including, but not limited to, HTST (sterilizer / high temperature pasteurizer, short time) at 161 ° F (72 ° C) for 18 seconds and cooled to 80 ° F (27 ° C). The sialyl oligosaccharides, including, but not limited to, a (2-3) sialyllactose can be recovered from the incubated milk / trans-sialidase mixture, or from the pasteurized / trans-sialidase mixture, by the methods described in section 5.4 . When the dairy / trans-sialidase mixture is to be used to make cheese, the dairy / trans-sialidase mixture is collected and processed to make the cheese. Otherwise, the milk can be pasteurized in batches (a protocol used is the elevation and decrease of a complete batch to 160 ° F (71 ° C) or by the HTST pasteurizer / heat exchanger (rapid rise to 71 ° C, maintain for 2 minutes, quick cooling to 27 ° C.) Milk can also be sterilized by UHT (ultra high temperature sterilization) (rapid rise to 132 ° C, hold for 6 seconds, rapid cooling to 27 ° F.) Depending on the process Subsequently, this method of sterilization can utilize heat exchange or clean steam injection In an alternative embodiment of the invention, the milk source is processed for the manufacture of cheeses and the sialyl oligosaccharides are recovered from the residual stream of the cheese processing by the methods described in section 5.5 In another embodiment of the invention, at least one a (2-3) trans-sialidase is contacted with a residual stream from the processing of The optimal time for incubating the residual current / trans-sialidase mixture according to this embodiment can be determined routinely by known techniques. In the specific modalities, the residual current / trans-sialidase mixture is incubated for a period of at least 0.5, 1.0, 5.0 or 10.0 hours. In a preferred embodiment, the residual current / trans-sialidase mixture is incubated for 5-45 hours. In a more preferred embodiment, the residual current / trans-sialidase mixture is incubated for 10-35 hours. In a more preferred embodiment, the residual current / trans-sialidase mixture is incubated for 10-35 hours.

The optimum temperature for incubating the residual waste / trans-sialidase mixture according to the present invention can be determined routinely by known techniques. In specific embodiments, the residual current / trans-sialidase mixture is incubated at about 2-40 ° C, preferably 15-37 ° C, most preferably 22-27 ° C. The optimum pH for incubating the milk source / trans-sialidase mixture according to the present invention can be determined routinely by known techniques. In the specific embodiments, the residual current / trans-sialidase mixture is incubated at a pH of about 4-9, more preferably at a pH of about 6-8, and more preferably the pH is at about pH 7. Other conditions to optimize the Incubation of the residual current / trans-sialidase mixture will be apparent to those skilled in the art and are within the scope of the present invention. In specific embodiments, the residual current / trans-sialidase mixture can be stirred, stirred, shaken, mixed or the like to aid in the uniform distribution of the enzyme within the mixture. In one embodiment of the invention, they are added to exogenous (2-3) sialyl oligosaccharides to the milk source / trans-sialidase mixture. The complemented exogenous (2-3) sialyl oligosaccharides may contain a single homogenous population of a (2-3) sialyl oligosaccharides, or otherwise, may consist of a mixture of different (2-3) sialyl oligosaccharides. After incubation of the residual current / trans-sialidase mixture, the sialyl oligosaccharides, including but not limited to a (2-3) sialyllactose can be recovered from the incubated residual stream / trans-sialidase by the methods described in section 5.4 . . 4 RECOVERY OF OLIGOSACCHARIC SALTS The sialyl oligosaccharides produced according to the methods of the present invention can be recovered from the milk source before or during processing (e.g., pasteurization, fermentation and / or one or more of the other processing steps included in the manufacture of cheese or other dairy products). Otherwise, the syllable oligosaccharides produced according to the methods of the present invention can be recovered after the processing of the milk source (eg, a waste stream from cheese processing). The sialyl oligosaccharides produced according to the methods of the invention can be recovered using methods known in the art, including, but not limited to, ultrafiltration, diafiltration, electrodialysis, ion exchange chromatography, and phase partition chemistry. In the specific embodiments of the invention, the a (2-3) sialyl oligosaccharides produced according to the methods of the invention are recovered from a residual stream of cheese processing (i.e., any waste stream or by-product generated during the process of cheese making). Serum containing sialic acids is a by-product obtained when rennet cheese or casein is produced from milks such as cow's milk, goat's milk and sheep's milk. For example, acid whey is generated by separating the solids when the denatured milk coagulates to form Cottage cheese. Acid whey is characterized by a high lactic acid content. When the cheese is prepared from whole milk, the remaining liquid is sweet whey, which can also be processed by evaporation to form dry whey powder. The sweet serum can also be dried, demineralized and evaporated to form demineralized serum permeate. The sweet whey can also be subjected to ultrafiltration to generate a serum permeate and a whey protein concentrate. The serum permate can also be processed by crystallizing lactose to form lactose and a mother liquor. The mother liquor resulting from the crystallization of lactose from a permeate of whey is known in the art as "Delac". When an a (2-3) trans-sialidase is contacted with a milk source before or during the manufacture of cheeses and sialyl oligosaccharides are recovered from a waste stream from cheese processing, the waste streams from the processing of suitable cheeses include, but they are not limited to, complete serum, permeate of demineralized serum, the regeneration current from the demineralized serum permeate, serum permeate, crystallized lactose, spray-dried lactose, whey powder, edible lactose and lactose. Preferably, the aqueous mother liquor material resulting from the crystallization of lactose (ie, Delac) is used. When (2-3) trans-sialidase is contacted with a residual stream of cheese processing and then the sialyl oligosaccharides are recovered, the residual streams from the processing of suitable cheeses include colostrum, milk, milk powder, whole serum, permeate of demineralized serum, the regeneration current from the permeate of demineralized serum, permeate of serum and whey powder. Ordinarily, the fluid cheese whey is dried to produce a non-hygroscopic, highly dispersible powder. The fresh fluid serum is clarified by passing it through a clarifier type desenlodador. The whey is separated to separate the fat, then concentrated in double or triple effect evaporators for a solids content of approximately 62% by weight. The solids can be removed by separation at room temperature, or more preferably, the concentrated whey is cooled before the solids are removed. When the waste stream from the cheese processing to be processed is the solids obtained from the drying of whey, the solids may first be dissolved in water, preferably in an amount of about 1 to 620 g, preferably 50 to 200 g. , more preferably about 100 g of solids per liter of water. The dissolution of the solids obtained from the drying of the cheese whey can be carried out at room temperature or at elevated temperatures to accelerate the dissolution process and increase the amount of dissolved solids. Preferably, temperatures are suitable from 20 ° -80 ° C, otherwise, the solids can be processed directly by extraction with a solvent. In one embodiment of the invention, the sialyl oligosaccharides produced according to the methods of the invention are recovered from a milk source or from the residual stream of cheese processing by a method consisting of: adjusting the pH of the milk source or the current residual of cheese processing to form an acid mixture; contacting this acid mixture with a cation exchanger; and concentrate and desalt the eluent. See, for example, Shimatani et al., U.S. Patent No. 5,270,462, the content of which is incorporated herein by reference in its entirety. In another embodiment of the invention, the sialyl oligosaccharides produced according to the methods of the invention are recovered from a milk source or from the residual stream of cheese processing by a method consisting in: subjecting a milk source or waste stream to the processing of cheeses at ultrafiltration, fractionation from 20,000 to 500,000 daltons at pH 4.0, to 6.0 to form an ultrafiltrate and subject the resulting ultrafiltrate to a second ultrafiltration, fractionating to 1,000 to 10,000 daltons at a pH of 6.0 to 8.0 under 0.2 to 2.0 MPa, to eliminate impurities like proteins. See, for example, JP Kokai 01-168,693, the content is hereby incorporated herein by reference in its entirety. In another embodiment of the invention, the sialyl oligosaccharides produced according to the methods of the invention are recovered from a milk source or waste stream from cheese processing by a method consisting of: desalting the milk source or the waste stream from the cheese processing and passing the desalted solution through an anion exchange column.

See, for example, JP Kokai 59-184,197, the content of which is incorporated herein by reference in its completeness. Other methods that can be used during the recovery of the sialyl oligosaccharides produced according to the methods of the present invention include ultrafiltration (see, for example, U.S. Patent No. 4,001,198, to Thomas and U.S. Patent No. 4,202,909 to Pederson); the concentration and addition of a divalent cation (see, for example, US Pat.

No. 4,547,386 to Chambers et al.); separation and fermentation (see, for example, U.S. Patent No. 4,617,861 of Armstrong); demineralization using an electrolytic cell (see, for example, U.S. Patent No. 4,971,701 and 4,4,855,056 to Harju et al); separation on a bed of strongly acidic cation exchange resin (see, for example, U.S. Patent No. 4,543,261 to Harmon et al.); electrodialysis or an ion exchange using a cation exchange resin and a strongly basic anion exchange resin, or electrodialysis and ion exchange using a cation exchange resin and strongly basic anion exchange resin to desalt the permeate (see, for example, US Pat. No. 5,118,516 to Shimatani). The descriptions of each of the references mentioned in this paragraph are incorporated as a reference in their strengths. In a preferred embodiment, the sialyl oligosaccharides produced according to the methods of the invention are recovered from a milk source or waste stream from the cheese processing using an anion exchange resin. Accordingly - with this embodiment, the milk source or waste stream from the cheese processing is optionally pretreated to remove the positively charged materials using known techniques (see, for example, DeWitt et al., 1986, Neth. Milk Dairy J. 40: 41-56; and Ayers et al., 1986, New Zealand J. Dairy Sci. &Tech. 21: 21-35; JP Kokai 52: 151200 and 63: 39545; and JP 2-104246 and 2- 138295). Suitable cation exchange resins can be prepared by conventional techniques known to those skilled in the art. For example, a suitable cation exchange resin can be produced from a monofunctional and polyfunctional monomer mixture polymerizable by radical emulsion polymerization techniques, then they can be functionalized with acidic groups such as carboxylic acid groups or sulfonic acid groups that exist in protonated form. The degree of crosslinking in the cation exchange resin can be chosen, depending on the operating conditions of the cation exchange column. A highly crosslinked resin offers the advantage of durability and a high degree of mechanical integrity, however, it has decreased porosity and a drop in mass transfer. A resin with low crosslinking is more brittle and tends to swell by absorption of the mobile phase. A suitable resin can have from 2 to 12% crosslinking or crosslinking, preferably 8% crosslinking. The particle size of the cation exchange resin is selected to allow efficient flow of the milk source or waste stream from the cheese processing while effectively separating the positively charged materials. A suitable particle size for a column of 30 x 18 cm is 100-200 mesh. Suitable cation exchange resins include, but are not limited to CM-Sephadex, SP-Sephadex, CM-sepharose, S-sepharose, CM-cellulose, cellulose phosphate, sulfoxyethyl cellulose, Amberlite, Dowex-50W, Dowex HCR- S, Dowex Macroporus resin, Duolit C433, SP Trisacryl plus-M SP Trisacryl plus-LS, Oxycellulose, AG MP-50 Resin, Bio-Rex 70. The most preferred suitable resins are DOWEX TM 50 x 8 (a bound aromatic sulfonic acid to a cross-linked polystyrene resin from Dow Chemical) and AMBERLYST TM-15 AMBERLITE TM IR-120 and AMBERLITE TM-200, acid resins. The milk source or waste stream from the cheese processing can be contacted with a cation exchange resin in any suitable form, which will allow the positively charged materials to be absorbed into the cation exchange resin, preferably the cation exchange resin. it is loaded into a column, and the milk source or waste stream from the cheese processing passes through the column, to remove the positively charged materials. An amount of cation exchange resin is selected to effect the separation of the positively charged materials, and will vary greatly depending on the milk source or the waste stream from the cheese processing in question. Usually, if a serum permeate is being treated, the charge ratio of the residual stream from the cheese processing to the cation exchange resin can be from 5 to 20, preferably from 8-15 of greater preference from 9 to 12: 1 v / v When the contact is made in a column, the milk source or the residual stream of the cheese processing is preferably passed at a speed from 1 to 70 cm / min, preferably from 2 to 15 cm / min. , more preferably at a speed of 4.6 cm / min. A suitable pressure can be selected to obtain the desired flow rate. Usually, a pressure from 0 to 100 PSIG is selected. Suitable flow rates can also be obtained by applying a negative pressure to the eluent end of the column, and collecting the eluent. It is also possible to use a combination of positive pressure and negative pressure. The temperature used for the contact of the milk source or waste stream from cheese processing with the cation exchange resin is not particularly limited, provided that the temperature is not high enough to cause decomposition of the components of the milk source or the current residual. In general, room temperature of 17 to 25 ° C is used. Otherwise, positively charged materials can be separated by techniques such as electrodialysis, ultrafiltration, reverse osmosis or saline precipitation. After the optional processing of the milk raw or the residual stream of the cheese processing to separate the positively charged materials, the milk source or the residual stream of the cheese processing is brought into contact with an anion exchange resin. Suitable anion exchange resins can be prepared by conventional techniques known to those skilled in the art. For example, a suitable anion exchange resin can be produced from a monofunctional and polyfunctional monomer mixture polymerizable by radical emulsion polymerization techniques, then functionalized with strongly basic groups such as quaternary ammonium groups. The degree of crosslinking in the anion exchange resin can be chosen, depending on the operating conditions of the anion exchange column. A suitable resin can have from 2 to 12% crosslinking, preferably 8% crosslinking. The particle size of the anion exchange resin is selected to allow efficient flow of the milk source or the waste stream from the cheese processing, while effectively separating the negatively charged materials [sic]. A suitable particle size for a column of 30 x 18 cm is 100-200 mesh. Suitable anion exchange resins include, but are not limited to, DEAE Sephadex, QAE Sephadex, DEAE Sepharose, Q Sepharose, DEAE Sephacell / DEAE Cellulose, Ecteola cellulose, PEI cellulose, QAE cellulose, Amberlite, Dowex 1-X2, Dowex 1 -X4, Dowex 1-X8, Dowex 2-X8, Dowex Macroporus Resins, Dowex WGR-2, DEAE Trisacryl Plus-M DEAE Trisacryl Plus-LS, Amberlite LA-2, AG 1-X2, AG 1-X4, AG 1 -X8 AG 2-X8, AG MP-1 Resin, AG 4-X4, AG 3-X4, Bio-Rex 5 and ALIQUAT-336 (tricaprylmethylammonium chloride from Henkel Corp.). The most particularly suitable anion exchange resins are DOWEX ™ 1 x 8 (a methylbenzylammonium bonded to a crosslinked polystyrene resin from Dow Chemical) and AMBERLYSTE ™ A-26, AMBERLITE TM IRA 400. AMBERLITE TM IRA 400, AMBERLITE TM IRA 416 AND AMBERLITE TM IRA 910, strongly basic resins. A milk source or waste stream from the cheese processing may be contacted with the anion exchange resin in any suitable manner that allows the negatively charged materials to be absorbed onto the anion exchange resin. Preferably, the anion exchange resin is loaded onto a column, and the milk source or the waste stream from the cheese processing passes through the column, to absorb the negatively charged materials on the resin. An amount of anion exchange resin is selected to effect absorption of the negatively charged materials and will vary greatly depending on the milk source or the waste stream from the cheese processing in question. Usually, when the waste stream is permeated with whey, the ratio of the charge of the waste stream from the cheese processing to the anion exchange resin is from 5 to 200, preferably from 8 to 15, most preferably from 9 to 15. to 12: 1 v / v. When the contact is made in a column, the milk source or the residual stream of the cheese processing preferably passes at a speed from 1 to 70 cm / min, preferably 2 to 15 cm / min, more preferably at a rate of 4.6 cm / min. It is possible to select a suitable pressure to obtain the desired flow velocity. Usually, a pressure from 0 to 100 PSIG is selected. Suitable flow rates can also be obtained by applying a negative pressure to the eluent end of the column, and collecting the eluent. A combination of positive and negative pressure can also be used. The temperature used for the contact of the milk source or the residual stream of the cheese processing with the anion exchange resin is not particularly limited, provided that the temperature is not high enough to cause decomposition of the components of the milk source or the residual current. The pH of the serum stream can also be adjusted in addition to the temperature. In general, ambient temperature of 17 to 25 ° C and pH of 4 to 9 are used. 7AX to contact the eluent with the anion exchange resin, the negatively charged components of the milk stream or the residual current of the cheese processing they are absorbed on the anion exchange resin. The materials absorbed in the anion exchange resin are negatively charged materials from a milk source or waste stream from the processing of cheeses, which include, but are not limited to, sialyl oligosaccharides such as a (2-3) sialyllactose, a (2- 6) sialillactose and (2-6) sialillactosamine. The resulting liquid, after contact with the anion exchange resin, which contains mainly water and lactose can be dried and discarded as animal feed, fertilizer or as a food supplement. Then, the anion exchange resin is purged of the sialyl oligosaccharide by eluting with an aqueous solution of a suitable salt such as sodium acetate, ammonium acetate, sodium chloride, sodium bicarbonate, sodium formate, ammonium chloride or a sodium salt. lithium such as lithium acetate, lithium bicarbonate, lithium sulfate, lithium formate, lithium perchlorate, lithium chloride and lithium bromide as an eluent. The cleaning of an anion exchange resin with an aqueous salt can be carried out by conventional means known to those skilled in the art. Those known to those skilled in the art. The sialyl oligosaccharides can also be separated from the anion exchange resin with an aqueous alkaline solution, although the concentration of the aqueous alkali must be diluted so as not to destroy the structure of the sialyl oligosaccharide. The proper desorption conditions can be determined by routine experimentation. When eluting with an aqueous solution of lithium salts, desalination by reverse osmosis is not necessary. The complete eluent can be concentrated and dried, then the remaining solids washed with an organic solvent. The lithium salts dissolve and the lithium salt of the sialyl oligosaccharide remains as a solid. Specifically, the lithium salts of a (2-3) sialillactose, a (2-6) sialillactose and a (2-6) sialillactosamine have very low solubility in organic solvents. The lithium salts used in the eluent must be freely soluble in water, and have a high solubility in an organic solvent. In the context of the present invention, a high solubility in an organic solvent is = 1 g of lithium salt per ml of organic solvent, preferably >; 5 g / ml, more preferred > 10 g / ml at the temperature of the solids being washed. Lithium salts found to be freely soluble in water and having a high solubility in organic solvents include lithium acetate, lithium bicarbonate, lithium sulfate, lithium formate, lithium perchlorate, lithium chloride, and lithium bromide. The organic solvent used to wash the concentrated eluent should dissolve the lithium salt eluted, and still have a low solvation effect on the lithium salt of a sialyl oligosaccharide. In the context of the present invention, a low solvating effect on the lithium salt of a sialyl oligosaccharide is when the solubility of the lithium salt of the sialyl oligosaccharide is < 0.5 g per ml of the organic solvent, preferably = 0.25 g / ml, more preferably < 0.1 g / ml at the temperature of the solids being washed. Suitable solvents include, but are not limited to acetone, methyl ethyl ketone, 3-pentanone, diethyl ether, t-methyl butyl ether, methanol, ethanol, and a mixture thereof. The organic solvent preferably contains < 0.1% by weight, more preferred < 0.01% by weight of water, more preferably the organic solvent is anhydrous. The use of an organic solvent containing high concentrations of water gives rise to the dissolution of the lithium salts of the sialyl oligosaccharides. The temperature of the organic solvent is not particularly limited, however, preferably the organic solvent is at room temperature or lower, more preferably from 0 to 5 ° C. Due to the high hygroscopicity of the lithium salts of the sialyl oligosaccharide, the washing of the solids is carried out under conventional conditions which are known to those skilled in the art, to limit the absorption of atmospheric moisture. For example, the washing can be carried out under an inert atmosphere, in a drying box or using a Schlenk type apparatus. When purging the anion exchange resin with an eluent, a suitable purge solution is 50 mM. The pH is the eluent preferably adjusted to be from 4 to 9, more preferably from 5 to 6. Generally 2 to 5, preferably 4 column volumes of the purge solution are used to separate the sialyl oligosaccharides from the resin of anion exchange, preferably carried out at room temperature. Preferably, the lithium acetate is used to purge the anion exchange resin of the sialyl oligosaccharides. The sodium salt of the sialyl oligosaccharide can be obtained by conventional ion exchange techniques, known to those skilled in the art. When a different eluent of the lithium salt is used to separate the sialyl oligosaccharides from the anion exchange resin, the eluent containing the sialyl oligosaccharides and the salt can be concentrated and desalted, subjecting the eluent to reverse osmosis to remove the salt of the sialyl oligosaccharide. The reverse osmosis can be performed by a membrane with a molecular weight cut-off of 100 to 700 daltons, preferably a cut of 400 daltons. The reverse osmosis is preferably carried out at a pressure of from 300 to 1,600 psi, more preferably from 400 to 600 psi, even more preferably at a pressure of 450 psi.

After the salts have been separated by reverse osmosis, the resulting material can be concentrated to provide a solid material containing sialyl oligosaccharides such as (2-3) sialillactose and (2-6) sialillactose, which can be crystallized to from a mixture of water and organic solvents. Preferably, the solvents for the precipitation are selected from the group of ethanol, acetone, methanol, isopropanol, diethyl ether, t-butyl methyl ether, ethyl acetate, hexane, tetrahydrofuran and water. In addition, the eluent, from the anion exchange column, which contains a mixture of sialyl oligosaccharides including (2-3) sialillactose, a (2-6) sialillactose and (2-6) sialillactosamine, can be subjected to separation of the sialiloligosaccharides contained therein, by column chromatography on DOWEX 1 x 2 anion exchange resin, at pH 4 to 6 using a buffer solution a suitable salt such as sodium acetate, ammonium acetate or a lithium salt such as lithium acetate, lithium perchlorate, lithium chloride and lithium bromide as eluent. A lithium acetate solution is preferred. Suitable anion exchange resins can be prepared by conventional techniques known to those skilled in the art as already described. The degree of crosslinking of the anion exchange resin can be chosen, depending on the operating conditions of the anion exchange column. A suitable resin can have from 2 to 12% crosslinking, preferably 2% crosslinking. The particle size of the anion exchange resin is selected to allow the efficient flow of the milk source or the waste stream from the cheese processing, while efficiently effecting the chromatographic separation of the negatively charged materials. A suitable particle size for a 20 x 100 cm column is 200-400 mesh. Suitable anion exchange resins include, but are not limited to, DEAE Sephadex, QAE Sephadex, DEAE Sepharose, Q Sepharose, DEAE Sephacell, DEAE Cellulose, Ecteola cellulose, PEI cellulose, QAE cellulose, Amberlite, Dowex 1-X2, Dowex 1 -X4, Dowex 1-X8, Dowex 2-X8, Dowex Macroporus Resins, Dowex WGR-2, DEAE Trisacryl Plus-M DEAE Trisacryl Plus-LS, Amberlite LA-2, AG 1-X2, AG 1-X4, AG 1 -X8 AG 2-X8, AG MP-1 Resin, AG 4-X4, AG 3-X4, Bio-Rex 5 and ALIQUAT-336 (tricaprylmethylammonium chloride from Henkel Corp.). The preferred resins are DOWEX ™ 1 x 2 (a trimethylbenzylammonium bonded to a crosslinked polystyrene resin from Dow Chemical) and the basic resins AMBERLYST and AMBERLITE. The mixture of sialyl oligosaccharides to be separated is subjected to column chromatography on an anion exchange resin. An amount of anion exchange resin is selected to effect the separation of the different sialyl oligosaccharides. Typically, the charge ratio of the sialyl oligosaccharide to the anion exchange resin is from 0.1 to 5, preferably from 0.2 to 4, most preferably 1 gram of material per liter of resin at a loading concentration from 0 to 10 mM of salt. Chromatography is carried out at a speed of from 1 to 20 cm / h, preferably 4.6 cm / h of superficial velocity. A suitable pressure can be selected to obtain the desired flow rate. Usually a pressure is selected from 0 to 22 PSIG. It is also possible to obtain adequate flow rates by applying a negative pressure at the eluent end of the column, and collecting the eluent. It is also possible to use a combination of positive and negative pressure. It is possible to use any temperature to contact the milk source or the residual stream of the cheese processing with an anion exchange resin, as long as the temperature is not too high to cause the decomposition of the components of the sialyl oligosaccharides. Generally, room temperature of 17 to 25 ° C is used.

When the eluant buffer is a lithium salt, the individual sialyl oligosaccharides can be isolated by concentrating the eluent to form a solid and washing the lithium salts with an organic solvent. The isolation of the lithium salt of a sialyl oligosaccharide from a lithium salt eluent is as already described. The sodium salt of the sialyl oligosaccharide can be obtained by conventional ion exchange techniques, known to those skilled in the art. - When eX eluent buffer is not a lithium salt, the individual sialyl oligosaccharides can be isolated by reverse osmosis techniques. In accordance with another embodiment of the present invention, a milk source or waste stream from cheese processing can be treated without using an anion exchange column and without using reverse osmosis. According to this embodiment, a milk source or waste stream from the cheese processing is contacted with a solvent, where the sialyl oligosaccharides are extracted. - The sialyl oligosaccharides that are extracted include, but are not limited to, (2-3) sialyllactose, (2-6) sialillactose and (2-6) sialillactosamine. - A milk source or waste stream from the cheese processing can be contacted with a solvent in any suitable form to effectively extract by solubilization, the sialyl oligosaccharides. For example, solid lactose, in powder form, can be packed in a column, and a solvent passed through the packed column. As the solvent passes through the column, the sialyl oligosaccharides are extracted from the solid lactose. To improve the solubilization of the sialyl oligosaccharide, the solvent can be recirculated through the column, until an equilibrium concentration of the sialyl oligosaccharide is obtained in the solvent. To improve the solubilization of the sialyl oligosaccharide, the solvent can be recirculated at elevated temperature, below the thermal decomposition point of the sialyl oligosaccharides, preferably from 27 ° C to 80 ° C, more preferably from 60 ° C to 75 ° C, ambient pressure A milk source or waste stream from cheese processing can also be contacted with a solvent, such as a slurry or slurry from the milk source or waste stream from cheese processing in the solvent. The milk source or the residual stream of the cheese processing is mixed with the solvent, preferably in a ratio of 1: 4 v / v, more preferably 1: 3 v / v. The slurry or suspension is then stirred until the sialyl oligosaccharides are solubilized in the solvent. The ratio of the milk source or the residual stream from the cheese processing to the solvent is selected to maximize the amount of sialyl oligosaccharide recovered and to deduct the minimum amount of solvent used. Due to the high solubility of the sialyl oligosaccharides in the chosen solvent, the amount of solvent is usually much less than the volume of the milk source or the residual stream of the cheese processing. Therefore, when lactose is being processed it is not necessary that the lactose be completely dissolved. The suspension can be stirred at any temperature, below the point of thermal decomposition of the sialyl oligosaccharides, preferably from 4 ° C to 80 ° C, more preferably from 4 to 27 ° C, at ambient pressure. Suitable solvent systems are water, C [1- 5] alcohols, such as methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec-butanol, tert-butanol, tertiary amyl alcohol and Isoamyl alcohol and a mixture of these. The amount of water in the solvent system of alcohols of C [1- 5] will vary depending on the alcohol used. Preferably, the solvent contains from 0 to 75% water (v / v), more preferably from 20 to 70% water (v / v), more preferably from 44 to 66% water. A particularly preferred solvent system is an aqueous ethanol solvent containing from 44 to 66% water. If elevated temperature is used, it is preferred to separate the solvent from the column, slurry or suspension after reaching the maximum concentration of the sialyl oligosaccharide, followed by cooling of the separated solvent. With the cooling of the separated solvent, the solubilized lactose will crystallize and can be separated from the solvent containing the sialyl oligosaccharide by conventional means such as filtration, centrifugation and decantation. An aqueous solution of lactose, such as the mother liquor obtained by the crystallization of lactose, can also be treated with a solvent at elevated temperature, preferably from 60 to 75 ° C, preferably from 68 to 72 ° C, followed by the cooling and precipitation of the lactose from the solution. The separation of the lactose precipitated from the solvent and the concentration of the solvent provide the sialyl oligosaccharide. The aqueous lactose solution and the solvent are mixed in a ratio of about 1: 3 v / v, preferably 1: 2 v / v, most preferably 1: 1 v / v. A suitable solvent for the treatment of an aqueous lactose solution is a C [1- 5] alcohol. The separated solvent or column eluent can be concentrated to produce high purity sialyl oligosaccharide. This material can also be purified by recrystallization from aqueous ethanol and a suitable organic solvent, to remove the impurities from the lactose.

In another embodiment of the column treatment technique, slurry or suspension, a portion of the extraction solvent can be separated and passed through an anion exchange column and the solvent can be returned to the system. In this embodiment, the sialyl oligosaccharide can be concentrated on the anion exchange column. The solvent that passes through the anion exchange resin can be separated continuously or in batches. Once the anion exchange column has been saturated with the sialyl oligosaccharide, the column can be separated from the system and purged to obtain the sialyl oligosaccharide. A suitable purge solution is 120 mM LiOAc. Generally, from 2 to 5, preferably 4 column volumes of the purge solution are used to separate the negatively charged materials from the anion exchange resin, carried out at room temperature. Suitable anion exchange resins, contact conditions and purge conditions have been previously described. Sialyloligosaccharides can also be extracted from residual waste streams using supercritical C02 extraction techniques in a method analogous to the methods used to extract caffeine from coffee seeds, a technique for extracting caffeine from the seeds of coffee. coffee using supercritical, wet C02 is described in U.S. Patent Nos. 3,806,619, and 4,260,639. In general, the supercritical C02 extraction method consists of contacting the lactose or an aqueous solution of lactose with supercritical C02, under conditions to effect the solubilization of the sialyl oligosaccharides by the supercritical CO2. The supercritical CO, containing the sialyl oligosaccharides, is separated from the lactose or the aqueous lactose solution, then the CO2 is separated by evaporation, leaving behind the extracted sialyl oligosaccharides. Whey containing sialic acids is a by-product obtained when cheese or rennet casein is produced from milks such as cow's milk, goat's milk and sheep's milk. Due to the fat in the milk sources and the small amount of curd or fat that often remain in the whey, it is preferable that the fat content of these compositions generated according to the method of the invention be previously removed by a cream separator or clarifier. In order for the whey proteins, such as beta-lactoglobulin, to be efficiently adsorbed in a cation exchanger, the milk source or whey may be previously concentrated in an ultrafiltration device. In addition, the milk source or serum can be previously desalted with an electric dialyzer and / or an ion exchange resin. The milk source or serum is adjusted to pH 2-5 before being subjected to the cation exchanger. As materials to adjust the pH it is possible to use any kind of materials. For example, these include an acid such as hydrochloric acid, sulfuric acid, acetic acid, tactical acid [sic] and citric acid. Otherwise, the acidified whey that has been desalted with the resin to have a pH of about 1-4, can be used to adjust the pH, so that the whey contains a high content of sialic acids. In the milk source or serum that have been adjusted to pH 2-5, the sialic acids are negatively charged, although most of the milk source or whey protein is positively charged. When this milk source or serum is contacted with the cation exchanger, the milk source or whey protein is selectively adsorbed to the cation exchanger and, as a result, the sialic acids are selectively recovered as a solution passed through the exchanger. If the pH of the milk source or serum is greater than 5, the sialic acids and most of the milk source or whey protein are negatively charged. So, the separation is not effective, although these two can be separated with an anion exchanger using the difference in adsorption. If the pH of the milk source or serum is less than 2, the sialic acids decompose and therefore the process is not practical. The solution passed through the cation exchanger obtained according to this embodiment can optionally be concentrated, desalted and / or dried using the known techniques. In addition, a mother liquor obtained after the solution is passed through the exchanger, concentrated and then crystallized to separate lactose can be used as a material having a high content of sialic acids. The concentration can be done by an evaporator. The crystallization can be done by cooling or by the addition of a seed crystal. To obtain a composition with a much higher content of sialic acids, it is preferable that the pH of the solution passing through the exchanger and / or its mother liquor be adjusted before these are concentrated and / or desalted. The concentration can be carried out by evaporation or by ultrafiltration. Desalting can be done by electrical dialysis, ion exchange, ultrafiltration or diafiltration. Diafiltration is a technique to further increase the protein content, where a liquid, which has been concentrated to some degree, is ultrafiltered while at the same time adding water to it and removing a solution that has passed. When the solution that has passed through the exchanger and / or its mother liquor is adjusted to a pH of 4 or higher, the concentration can be performed by ultrafiltration using an ultrafiltration membrane having a molecular weight for the cut of 2,000 approximately equal to 50,000 dalton . The concentration can also be carried out by ultrafiltration using an ultrafiltration membrane having a cut-off molecular weight of 10,000 at a pH of 4 or less. In other words, the kappa-casein glucomacropeptide (GMP) as a sialic acid is present as a monomer at a pH of 4 or lower, while associating in a multimonomer at a pH above 4. As materials to adjust the pH is possible use any kind of materials. These include alkalis such as sodium hydroxide, potassium hydroxide, calcium hydroxide, potassium carbonate, sodium citrate, and so on. The concentrate thus obtained is a composition having a high content of sialic acids such as GMP. By the way, alpha-lactalbumin, which is commonly found in whey together with sialic acids, can be separated from sialic acids, for example, by ultrafiltration of the solution passing through the exchanger. or its mother liquor at a pH of 4 or higher using an ultrafiltration membrane with a cut-off molecular weight of 2,000 to 50,000 daltons. . 5. TRANSGENIC MAMMALS PRODUCERS OF MILK ENRICHED TO (2-3) SIALILLACTOSE The content of a (2-3) sialillactose in milk can also be enriched by expressing a (2-3) trans-sialidase, derivatives and analogs (see section 5.1) in transgenic mammals. In one embodiment, the transgenic mammals of the invention contain a coding sequence for a (2-3) trans-sialidase that has been operably linked to a regulatory sequence of a gene expressed in breast tissue. In the same way, the invention offers the methods for enrichment for a (2-3) sialillactose in milk comprising the steps of introducing a transgene containing a coding sequence for (2-3) trans-sialidase operably linked to a regulatory sequence of a gene expressed in mammary tissue in the germline of a mammal to produce a transgenic mammal; select the transgenic mammal demonstrating the activity of a (2-3) trans-sialidase; and obtain the milk of the selected transgenic mammal. Transgenes of a (2-3) trans-sialidase introduced into the transgenic animals of the invention contain the nucleotide sequences encoding a (2-3) trans-sialidase, derivatives or analogues (as described in section 5.1) operably linked to the regulatory sequences (ie, promoters, enhancers, inducible and non-inducible operators and other elements that drive and / or control the expression) of a gene expressed in breast tissue. The coding sequence of the nucleotide used to produce the transgenic animals of the invention can be controlled by any suitable regulatory sequence, but preferably are promoter and / or regulatory nucleotide sequences of the mammalian milk protein. of milk-specific protein that can be used to boost the expression of the target sequence, include, but are not limited to, promoters obtained from: serum acid protein, β-lactoglobulin, α-lactalbumin, α-sl-casein and β Casein See, for example, Colman, A., 1996, Am J. Clin. Nutr. 63: 639S-645S (cited Houdebine, 1994, "Biotechnol., 43: 269-87). Many nucleotide sequences of regulatory sequences from genes expressed in mammary tissue are known (See, for example, (Houdebine, 1994, J. Biotechnol, 43: 269-87) Otherwise, the regulatory sequences contained in the nucleotide sequences. Genomic genes known to be expressed in mammary tissue can be identified using known techniques, for example, the genomic nucleotide sequences located upstream of the coding sequence of the gene expressed in breast tissue can be cloned adjacent to a reporter gene, such as , for example, a chloramphenicol acetyltransferase (CAT) gene.The genomic sequence / reporter gene construct is then introduced into a mammal using known techniques (see, for example, section 5.5.1) and the presence of regulatory sequences in the genomic sequence construction / reporter gene is indicated by the activity of the reporter gene, which is rehearsed using the known techniques. To define more precisely the regulatory elements, it is possible to generate deletion mutants and perform the tests for the reporter gene activity. The regulatory sequences of the transgene to (2-3) trans-sialidase can include all, or any portion of, the promoters, enhanced or their corresponding genes. For example, the (2-3) trans-sialidase / transgene construct of the regulatory sequence of the invention may contain the nucleotide coding sequence for the mammalian milk protein, or any portion thereof, fused within the framework of Correct coding to the nucleotide sequence coding for a (2-3) trans-sialidase. The expression of these chimeric constructs can be regulated by the regulatory sequence of the mammalian milk gene component of the chimeric or in another way, by the regulatory sequence of another gene that is expressed in mammary tissue.

In addition, the nucleotide regulatory sequences of the gene constructs transgene of a (2-3) trans-sialidase include but are not limited to all, or any portion of the promoter of the endogenous milk protein of the founder animal into which it is introduced. the gene of a (2-3) trans-sialidase. Regulatory nucleotide sequences can be obtained from mammalian milk protein genomic DNA using known techniques, including, but not limited to, PCR and sieving by hybridization of genomic libraries, as further described in section 5.1. For a review of the techniques that can be used, see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Springs Harbor Press, N. Y). These techniques can also be applied to generate the a (2-3) trans-sialidase / transgene regulator of the invention and to design chimeric gene constructs that use the regulatory sequences in addition to the regulatory sequences of the mammalian milk protein. In addition, methods that have been applied to construct transgenes that have successfully expressed proteins in the milk of transgenic mammals can be modified in a routine manner to generate the transgenic mammals with a (2-3) trans-sialidase of the invention. See, for example, Wright et al., 1991, Biotechnology (NY) 9: 830-834; Carver et al., 1993, Biotechnology (NY) ll: 1263-1270); Clark et al., 1989, Biotechnology (NY) 7: 487-492; Velander_et al., 1992, Proc. Nati Acad. Sci. USA, 89: 12003-12007; Ebert et al, 1991, Biotechnology (NY) 9: 835-838, the contents of each of which are incorporated herein by reference in their entireties. . 5.1.Production of transgenic animals Mammals of any species, including but not limited to sheep, goats, pigs and cows and non-human primates, for example, monkeys, baboons and chimpanzees, can be used to generate transgenic animals for (2-3) trans-sialidase of the invention. Any known technique can be used to introduce the transgene into the animals to produce the founder lines of the transgenic animals. These techniques include, but are not limited to, pronuclear microinjection (Paterson et al., 1994, Appl Microbiol Biotechnol., 40: 691-698; Carver et al., 1993, Biotechnology (NY) 11: 1263-1270; et al., 1991, Biotechnology (NY) 9: 830-834; and Hoppe et al., 1989, U.S. Patent No. 4,873,191); the transfer of the gene mediated by retroviruses in germ lines (Van der Putten et al., 1985, Proc Nati, Acad Sci USA, 82: 6148-6152), blastocysts and embryos; genetic targeting in embryonic cells (Thompson et al., 1989, Cell 56: 313-321); electroporation of cells or embryos (Lo, 1983, Mol Cell, Biol 3: 1803-1814); introduction of nucleic acid constructs in stem cells, pleuripotent, embryonic and transferring the stem cells back to the blastocysts; and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57: 717-723); etc. For a review of these techniques see Gordon, 1989, "Transgenic animals", Jntl. Rev. Cytol. 115, 171-229, which is incorporated herein by reference in its entirety. Any of the known techniques can be used to produce transgenic clones containing a gene for trans-sialidases, for example, nuclear transfer in enucleated oocytes of nuclei from cultured embryonic, fetal or adult cells, induced at rest (Campell et al., 1996, Na ture 380: 64-66, Wilmut et al., 1997, Nature 385: 810-813). In addition, it is possible to achieve expression of the transgene for a (2-3) trans-sialidase by removing the mammary secretory epithelium of the animals, transfecting the epithelial cells with a transgenic construct and reintroducing the transfected epithelial cells in the animal during the delivery period. so that the target gene is expressed in the subsequent lactation period. See, for example, Bremel et al., 1989, J. Dairy Sci. 72: 2826-2833. Although this method has the disadvantage of providing transient expression of a (2-3) sialyllactose, as the mammary secretory epithelium is released during the period of drought, this technology provides a method by which the objective of enriching concentrations of a ( 2-3) sialillactose in milk without the significant investment of time creating transgenic animals. Once the founding animals are produced, they can be procreated, procreated within the same race, procreated with another race or crossed to produce colonies of the particular animal. Examples of breeding strategies include, but are not limited to, mixing breeds of founder animals with more than one integration site to establish separate lines; procreation within the same race of separate lines to produce transgenic compounds that express the transgene at higher levels due to the additive expression effects of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to increase expression and eliminate the need for animal screening by DNA analysis; crossing of separate homozygous lines to produce lines of heterozygotes or compound heterozygotes.

The present invention deals with the transgenic animals that carry the transgene in all its cells, as well as animals that carry the transgene in some, but not in all its cells, that is, mosaic animals. The transgene can be integrated as a single transgene or in concata, for example, tandems from head to head or tandem from head to tail. . 5.2 Monitoring of transgenic animals The transgenic animals that are produced according to the procedures detailed in section 5.1.1 are preferably sifted and evaluated to select those animals that can be used as suitable milk producers containing enriched concentrations of a (2- 3) sialillactose compared to non-transgenic animals. The initial screening can be performed by Southern blot analysis or PCR techniques to analyze animal tissues to verify that the integration of the transgene has been achieved. The level of mRNA expression of the transgene in the tissues of the transgenic animals can also be assessed using techniques that include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis and reverse transcriptase -PCR (rt-PCR) and the like. Transgenic animals that express for a protein of a (2-3) trans-sialidase detected by immunocytochemical analysis, using antibodies directed against a (2-3) trans-sialidase) at easily detectable levels can serve as suitable producers of milk containing concentrations enriched with a (2-3) sialillactose. . 3.3 Enrichment of a (2-3) sialillactose in the milk of transgenic mammals The invention is responsible for a method for the enrichment of (2-3) sialillactose in milk, which consists of the steps of: introducing a transgene containing a sequence coding for a (2-3) trans-sialidase operably linked to a regulatory sequence of a gene expressed in mammary tissue in the germline of a mammal to produce a transgenic mammal; selecting a transgenic mammal demonstrating a (2-3) trans-sialidase activity; and obtain the milk of the selected transgenic mammal. The a (2-3) sialillactose produced according to this invention is also comprised by the invention. The a (2-3) sialyllactose enriched in accordance with the method of the invention can be recovered using the known techniques as well as those described infra (see section 5.4). In specific embodiments, a (2-3) sialillactose is recovered from the milk of the selected transgenic mammal before, during or after processing of the milk. In a preferred embodiment, a (2-3) sialillactose is recovered from the milk of the selected transgenic mammal after the milk has been subjected to processing (eg, a waste stream from the cheese processing obtained from this milk). The invention is further illustrated with reference to the following examples. It will be apparent to those skilled in the art that multiple modifications, both to materials and methods, may be practiced without departing from the purpose and scope of this invention. . 6 EXAMPLE: ISOLATION AND CLONING OF A DNA SEQUENCE THAT CODIFIES FOR THE ACTIVITY OF a (2-3) trans-sialidase In this example, a chimeric DNA sequence was cloned using the polymerase chain reaction (PCR) and as DNA template of a (2-3) trans-sialidase from Trypanosoma cruzi. Site-directed mutagenesis was applied to modify this sequence to encode a tyrosine at position 342 in place of the histidine initially encoded at this position. The mutated sequence was cloned into a pGEX expression plasmid, transformed into a host cell, and expressed as a glutathione-S-transferase fusion protein having (2-3) trans-sialidase activity. Two sets of oligonucleotide primers (for example PCR Primer Set # 1 and PCR Primer Set # 2) were synthesized for use in PCR to enzymatically amplify a coding sequence for a (2-3) trans-sialidase from T. cruzi genomic DNA. The PCR Primer Set # 1 was designed to amplify a region of the nucleic acid sequence that codes for the amino terminal region of the trans-sialidase of T. Cruzi that maintains the active domain for the activity of a (2-3) trans -sialidase. In order to then directionally clone the amplified fragment, primer 5 was designed to include a recognition sequence for a single restriction endonuclease (i.e., Xba I) at the 5 'end of the amplified fragment. The sequences of the 5 'primer and the 3' primer of the # 1 series were: tslxba-5 ': 5'-TTTTCTAGAATGCTGGCACCC £ GATCGAGC-3' tSl-3 ': 5' -CTGTGCGACAAAAAGCCAACAAGACCAACC-3 ' The PCR Primer Set # 2 was designed to amplify a region of the nucleic acid sequence that encodes the carboxyl terminal region of the (2-3) trans-sialidase of T. Cruzi and that overlaps the amplified fragment by the PCR Primer Set # 1. The 3 'primer was designed to include a recognition sequence for another unique restriction endonuclease (i.e., Xho I) at the 3 r end of the amplified fragment. The sequences of the 5 'primer and the 3' primer of the # 2 series were: ts2-5f: 5 '-ACTGAACCTCTGGCTGACGGATAACCAGC-3' tS2XHO-3 '5'-TTTCTCGAGTCAGGCACTCGTGTCGCTGCT-3' The known PCR techniques were used to amplify the DNA fragments generated using each of these series of primers and as genomic DNA template of T. cruzi. The DNA fragments generated from the PCR of the primers of set # 1 and the primers of set # 2 were ligated to generate the full-length fragment of the (2-3) trans-sialidase using the following procedure. The DNA fragment generated from the PCR of the primers of set # 1 was digested with the restriction endonucleases Xbal and PstI. The DNA fragment generated from the PCR of the primers of set # 2 was digested with Xhol and PstI. The PstI site was contained in a region that both PCR products had in common. The digestion of the PCR products generated "sticky ends" in the products. The Xbal site at the 5 'end of the DNA fragment generated from the PCR of the primers of set # 1 and the Xhol site at the 3' end of the DNA fragment generated from the _PCR of the primers of set # 2 were designed to be used in the directional cloning of the entire sequence in the appropriate expression plasmid. Both PCR products were then ligated together in the digested Xbal / XhoI-pGEX plasmid (Pharmacia, Piscataway, NJ) which contains the same restriction endonuclease sites in its polylinker region. The nucleotide sequence of a (2-3) trans-sialidase was directionally cloned in the framework of the pGEX fusion gene, glutathione-S-transferase. The trans-sialidase construct pGEX was then transformed into host cells using well-known techniques. The DNA sequence analysis revealed that the procedure described in this example gave rise to the cloning of a (2-3) trans-sialidase containing a Tyr 342-Hα mutation and was thus inactive (hereinafter, this clone it will be mentioned as "pGEX-TS / His"). Therefore, a site-directed mutagenesis protocol that changed His342- »Tyr was followed. The one of mutagenisis directed to the site was used realizing the following method. A series of oligonucleotide primers ("mut-5 '" and "mut-3'") were designed to mutate His 342 in Tyr342 to generate an active trans-sialidase. The mut-5 'and mut-3' sequences were: mut-5 ': 5'-GGGCAAGTATCCATTGGTGATGAAAATTCCGCCTACAGCT-3' mut-3 ': 5'-TACAGCTTATCATCCTTGTACAGGACGGAGCTGTAGGCGG-3' The mut-5 'and mut-3' primers were used together with the PCR primers from the PCR Primer Sets # 1 and # 2 to amplify the overlapping DNA fragments encoding the a (2-3) trans -sialidase using pGEX-TS / His as a template. The primers were designed to amplify two fragments that are superimposed by 65 nucleotides and to include the mutations directed by the PCR of the His 342 codon that would eventually code for a Try 342. The new overlapping fragments were purified by gel and used in a PCR reaction as primer and template. That is, the two fragments were mixed together, denatured with heat and re-tuned in a PCR reaction without the PCR primer (which allows the tempered fragments to be filled at the end). The primer of the 5 'end of the PCR Primer Set # 1 and the primer of the 3' end of the PCR Primer Set # 2 were then added to the mixture to amplify the coding fragment for the mutated trans-sialidase., full length. The new fragment was then ligated into pGEX as already described and used to transform E. coli BL21 cells (hereinafter this clone will be referred to as "pGEX-TS / Tyr"). The E. coli carrier of pGEX-TS / Tyr were expressed and assayed for the activity of a (2-3) trans-sialidase using known techniques. Clones expressing for (2-3) trans-sialidase activity were isolated and used in the lysate preparations used to generate the data to be presented in Figures 5-10 and sections 5.7 and 5.8. . 6.1 Preparation of used a (2-3) trans-sialidase of t. cruzi A single colony of E. coli BL21 cells carrying pGEX-TS / Tyr was inoculated in 2 ml of LB medium (tryptone, yeast extract, NaCl) containing a suitable antibiotic and the bacterial culture was incubated at 37 ° C in an incubator with agitation during the night. The following day, one liter of LB medium containing the appropriate antibiotic was inoculated with 50 μl of bacterial culture that was left overnight, and the culture was incubated at 37 ° C in a shaking incubator until the DOβoo = 1-0 of the culture. Bacteria in the 1 liter culture were induced to express a (2-3) trans-sialidase by the addition of isopropylthio-β-D-galactoside (IPTG) at a final concentration of 100 μM. The liter of induced culture was then placed in an incubator with shaking at 20 ° C, overnight. The next day, the bacterial cells were harvested by centrifugation, and a bacterial lysate was prepared using an APV Gaulin homogenizer. The homogenizer uses high pressure (10,000 psi) to lyse the cells. Otherwise, it is possible to use a French press DynoMIll, cavitation with N2, Dounce homogenizer, freeze-thaw or similar tool. Otherwise, the enzyme (2-3) trans-sialidase can be expressed using other methods known in the art, including, but not limited to, secretory expression in E. coli,. Expression in fungi and expression in insect cells . 7 EXAMPLE: ENRICHMENT AND ISOLATION OF a. { 2-3) SIALILLACTOSE IN MILK BEFORE THE MANUFACTURE OF CHEESES In this example. The addition of (2-3) trans-sialidase from T. Cruzi to untreated milk prior to the use of this milk in the manufacture of a rennet cheese is shown to result in an enrichment of (2-3) sialyllactose. . 7.1 Materials and methods Sixty-seven gallons of fresh milk, without treatment, were collected in seven milk cans and cooled to 37 ° F (3 ° C). Twenty thousand units of a (2-3) trans-sialidase were added to each milk can. A unit of a (2-3) trans-sialidase = 1 μmol of NAN-a (2-3) -Gal-ß- (1-4) -GlcNAc-ß- (1-3) -Gal-ß- ( 1-4) Glc (LST-d) produced / min in the standard assay using a (2-3) sialillactose and Gal-β- (1-4) -GlcNAc-β- (1-3) -Gal-β- (1-4) -Glc (lacto-N-neotetraose, LNnT) as substrates. In this particular example a filtered / frozen / thawed lysate of E. coli containing 400 units / ml of a (2-3) trans-sialidase was used. The milk containing the a (2-3) trans-sialidase was incubated at 50 ° F (10 ° C) for 12 hours. Samples were collected from two of the milk cans at 0, 1, 2, 3 and 11 hours of incubation and analyzed for the content of a (2-3) sialillactose. - - After 12 hours of incubation, the milk was pasteurized by continuous HTST at 161 ° F (70 ° C), for 18 seconds and cooled to 80 ° F (27 ° C) according to the standard procedure. See Kosikowski, Frank V .; 1977, Cheese and Fermented Foods, 2nd edition Edwards Brothers publication, Ann Arbor, MI. The milk was collected in a "double circle" tub and processed for white cheddar cheese according to the standard procedure. . 7.1.1 Protocol for the manufacture of cheese In time = 0, 10 grams of a 33 g culture of freeze-dried lactic acid (EZAL EZ100 5000 #, Lot No. 93053A, Rhone Polenc) were added to the pasteurized milk and the temperature was raised to 88F (31 ° C). 3 ounces of 0.02% CaCl2 (Rhone Polenc) were added. The culture was constantly stirred at 12 rpm and the temperature was kept constant at 31 ° F for 1 hour. The acidity of the solution was 0.17. In time = 1 hour and 15 minutes, the milk coagulator was added. Two ounces of a 50,000 MCU / ml solution of Chymax were added to 480 # of fermented milk (3 oz. Of Chymax solution are recommended / 1000 #) Pfizer brand contains chymosin, NaCl and propylene glycol). The solution was mixed for 30 seconds and then left to stand for 30 minutes. During this stage it was observed that the milk formed clots forming, forming a consistency with the appearance of yogurt / sour cream and titratable acidity dropped to 0.10. At time = 1 hour and 45 minutes, the cottage cheese was cut into 1 cm cubes using two knives for a period of 10 minutes. - In time = 1 hour and 55 minutes the temperature was elevated 2 ° F every 5 minutes until the temperature reached 102 ° F (39 ° C). The temperature was maintained at 102 ° F with constant stirring. After 30 minutes of cooking, the stirring blades were replaced with stirring rakes and the serum was drained. 8 liters of serum were collected for analysis. The pH started to fall from the level of 6.5 and the titratable acidity began to rise from 0.10. At time = 6 hours and 20 minutes, the pH of the serum had dropped to 5.85. The acidity had risen to 0.285 and the pH of the serum had dropped to 5.85. Although lower pH and higher acidity are desired (up to 0.6), it is considered that the use of a lactic acid culture dried by freezing, old was the reason for higher pH levels and lower acidity observed in this example. The rennet was salted (1.25 # of salt in 44 # of rennet), stirred and then compressed overnight at room temperature. The rennet was stored at 50 ° F (10 ° C) for 6 months. . 7.1.2 Analysis of the dry weight of the sample Five ml of each milk or serum sample were placed in aluminum weighers previously weighed and placed in a vacuum oven (<3 mmHg) at 85 ° C, during the night, Samples were then weighed and returned to the furnace for an additional 2 hours. The weighing process was repeated every 2 hours until 2 consecutive, consistent readings were obtained. The net weight of the dry sample was expressed in terms of percent weight by volume of the sample. The results are shown in Table 1. . 7.1.3 Reaction of a (2-3) fcran = r-sialidase The milk and serum samples were frozen immediately after collection. For the analysis, frozen samples were rapidly thawed, boiled to coagulate the remaining protein and centrifuged at 10,000 x g in a microcentrifuge for 10 minutes. The supernatant was collected and filtered through a 10,000 MW filter to separate the (2-3) sialillactose from the remaining higher molecular weight compounds as a preparation for high performance liquid chromatography (HPLC). The amount of a (2-3) sialillactose in the milk samples was quantified by HPLC and the values were expressed in terms of total dissolved solids. . 7.2 RESULTS As demonstrated by the data presented in Table 1, the treatment of a (2-3) trans-sialidase gave rise to a significant increase in a (2-3) sialillactose during the first hour of inoculation. This level of activity was most likely maintained throughout the 11-hour incubation period. The fluctuation in a (2-3) sialillactose levels after the first hour was attributed to the difficulty in obtaining homogenous samples from the milk cans. Interestingly, a large increase in the concentration of a (2-3) sialyllactose in the serum was observed after the milk curd was obtained. The final result was that treatment with a (2-3) trans-sialidase in the milk before the production of cheddar cheese gave rise to a double to quadruple increase in a (2-3) sialillactose compared to other serum samples of the milk source from which it had not previously been treated with a (2-3) trans-sialidase. The addition of a (2-3) trans-sialidase to the milk without treatment has no effect on the flavor or quality of the cheese generated using the milk treated with a (2-3) trans-sialidase. . 8 EXAMPLE: ENRICHMENT OF a (2-3) SIALILLACTOSA IN DAIRY SOURCES AND RESIDUAL CURRENTS OF THE PROCESSING OF CHEESES This example investigates the enrichment of a (2-3) sialyllactose in milk sources and waste streams from the processing of cheeses that have been put in contact with bacterial used obtaining activity of a (2-3) tifens-sialidase. The bacterial Wastes were prepared as stated below in section 5.6. The methods to produce and test for a (2-3) sialillactose were essentially as set forth in section 5.3. As shown in Figure 5, the addition of a (2-3) trans-sialidase increased the concentrations of a (2-3) sialyllactose over a pH range of 4.0-9.0 in the incubation mixture. The increased concentrations of a (2-3) sialyllactose from 2 to 5 times were observed in the milk sources and residual streams from the processing of tested cheeses including: mozarella serum (see Figures 5 and 7A-7B); skimmed milk (see Figure 6); Swiss cheese whey (see Figure 8); and in a milk simulator composition (see Figure 9). The present invention is not limited in scope by the specific embodiments described which are proposed as illustrations of the individual aspects of the invention, and functionally equivalent methods and components are within the scope thereof. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the aforementioned description and the accompanying drawings. These modifications are proposed to fall within the scope of the appended claims.

Claims (46)

1. A method for producing sialyl oligosaccharides in a milk source consists of: (i) contacting a catalytic amount of at least one a (2-3) trans-sialidase with a milk source to form a dairy / trans-sialidase mixture; and (ii) incubating the milk / trans-sialidase mixture under conditions suitable for the activity of the (2-3) transsialidase.

2. The method of claim 1 further comprising recovering the sialyl oligosaccharides from the incubated milk / trans-sialidase mixture.

3. The method of claim 1 further comprising the steps of: (iii) processing the dairy / trans-sialidase mixture for cheese making to form a waste stream from cheese processing; and (iv) recover sialyl oligosaccharides from the residual stream of cheese processing.

4. The method of claim 1, wherein the a (2-3) trans-sialidase is a quinetoplastid trans-sialidase.

The method of claim 1, wherein the a. { 2-3) trans-sialidase is encoded by a gene isolated from a species of the genus selected from the group consisting of: Trypanosoma, Endotrypanum and Pneumocystis.

6. The method of claim 1, wherein wherein a (2-3) trans-sialidase is produced recombinantly.

7. The method of claim 1, wherein the milk source consists of a member selected from the group comprising milk, colostrum and mixtures of cheese processing.

The method of claim 1, wherein the milk source / trans-sialidase mixture is incubated for at least 1 hour.

The method of claim 1, wherein the milk source / trans-sialidase mixture is incubated at a temperature of about 5 ° C to about 45 ° C.

The method of claim 1, wherein the milk source / trans-sialidase mixture has a pH of from about 6 to about 8.

The method of claim 3, wherein the residual stream of the cheese processing comprises a member selected from the group consisting of: complete serum, permeate of demineralized serum, a regeneration stream from the demineralized serum permeate, serum permeate, crystallized lactose, spray-dried lactose, whey powder, edible lactose and lactose.

The method of claim 2, wherein the recovery step comprises ultrafiltration of the milk source / trans-sialidase incubated mixture to form an ultrafiltrate.

13. The method of claim 3, wherein the recovery step comprises ultrafiltering the waste stream from the cheese processing to form an ultrafiltrate.

The method of claim 12 or 13, wherein the recovery step further comprises contacting the ultrafiltrate with an ion exchanger resin.

15. The method of claim 14, wherein the ion exchange resin is an anion exchange resin.

16. The method of claim 14, wherein the anion exchange resin is a cation exchange resin resin.

The method of claim 2, wherein the recovery step comprises: (a) contacting the milk source / rans-sialidase incubated mixture from step (ii) with a solvent, and extracting the sialyl oligosaccharides with the solvent for forming a solvent containing sialyl oligosaccharides; (b) separating the solvent containing sialyl oligosaccharides from the milk source / trans-sialidase incubated mixture; and (c) isolating the sialyl oligosaccharides from the solvent containing sialyl oligosaccharides.

18. The method of claim 3, wherein the recovery step comprises: (a) contacting the waste stream from the cheese processing with a solvent and extracting sialyl oligosaccharides with the solvent to form a solvent containing sialyl oligosaccharides; (b) separating the solvent containing sialyl oligosaccharides from the residual stream of cheese processing; and (c) isolating the sialyl oligosaccharides from the solvent containing sialyl oligosaccharides.

19. A method for producing sialyl oligosaccharides in a waste stream from cheese processing comprises: (i) contacting a catalytic amount of at least one (2-3) trans-sialidase with a waste stream from the cheese processing to form a mixture residual current / trans-sialidase; and (ii) incubating the residual dairy / trans-sialidase mixture under conditions suitable for the activity of the (2-3) trans-sialidase.

20. The method of claim 19 further comprises recovering sialyl oligosaccharides from the incubated residual stream / rans-sialidase mixture.

The method of claim 19, wherein the a (2-3) trans-sialidase is a kinetoplastid trans-sialidase.

22. The method of claim 19, wherein the a (2-3) trans-sialidase is encoded by a gene isolated from a species of the genus Trypanosoma, Endotrypanum or Pneumocystis.

23. The method of claim 19, wherein the a (2-3) trans-sialidase is produced recombinantly.

The method of claim 19, wherein the residual current / rans-sialidase mixture is incubated for at least 1 hour.

25. The method of claim 19, wherein the residual / rans-sialidase mixture is incubated at a temperature of about 5 ° C to about 45 ° C.

The method of claim 19, wherein the residual waste / trans-sialidase mixture has a pH of about 5 to about 8.

The method of claim 19, wherein the residual stream of the cheese processing comprises an element selected from the group consisting of: complete serum, permeate of demineralized serum, regeneration current of demineralized serum permeate, serum permeate and whey powder.

The method of claim 20, wherein the recovery step comprises ultrafiltering the residual stream / trans-sialidase incubated mixture to form an ultrafiltrate.

29. The method of claim 28, wherein the recovery step further comprises contacting the ultrafiltrate with an ion exchange resin,

30. The method of claim 29, wherein the ion exchange resin is an exchange resin. anionic

31. The method of claim 29, wherein the ion exchange resin is a cation exchange resin resin.

32. The method of claim 20, wherein the recovery step comprises: (a) contacting the incubated residual stream / trans-sialidase from step (ii) with a solvent, and extracting the sialyl oligosaccharides with the solvent to form a solvent containing sialyl oligosaccharides; (b) separating the solvent containing sialyl oligosaccharides from the residual stream / trans-sialidase incubated mixture; and (c) isolating the sialyl oligosaccharides from the solvent containing sialyl oligosaccharides.

33. The method of claim 17, 18 or 32, wherein the solvent is selected from the group consisting of water, C [1- 5] alcohol and mixtures thereof.

34. The method of claim 3 or 19, wherein the residual stream of the cheese processing is the mother liquor obtained by crystallization of lactose from the cheese whey.

35. The method of claim 1 or 19, wherein the exogenous (2-3) sialyl oligosaccharides are added during the incubation step.

36. A method for producing (2-3) sialyllactose comprises: (i) contacting a catalytic amount of at least one a (2-3) trans-sialidase with lactose and one (2-3) sialyl oligosaccharide, in the absence of CMP-sialyltransferase, to form a mixture; and (ii) incubating the mixture under conditions suitable for the activity of (2-3) trans-sialidase.

37. A transgenic mammal that contains a coding sequence for (2-3) trans-sialidase operably linked to a regulatory sequence of a gene expressed in breast tissue.

38. The transgenic animal of claim 37, wherein the regulatory sequence is derived from a gene encoding a specific milk protein.

39. The method of claim 38, wherein the regulatory sequence is obtained from a gene encoding a protein selected from the group consisting of: serum protein, β-lactoglobulin, α-lactalbumin, α-sl-casQ and β-casein.

40. The transgenic mammal of claim 37, wherein the coding sequence for a (2-3) trans-sialidase encodes a quinetoplastid trans-sialidase.

41. The transgenic mammal of claim 37, wherein the coding sequence of a (2-3) trans-sialidase hybrid under conditions of high stringency to a gene of a (2-3) trans-sialidase selected from the group consisting of Trypanosoma cruzi , Trypanosoma brucei, Endotrypanum spp. and Pneumocystis carinii.

42. The transgenic mammal of claim 37, in which the transgenic mammal is a cow, sheep, pig or goat.

43. A method for the enrichment of a (2-3) sialyllactose in milk comprises: (i) introducing a transgene containing a coding sequence for (2-3) trans-sialidase operably linked to a regulatory sequence of a gene expressed in mammary tissue in the germline of a mammal to produce a transgenic mammal; (ii) selecting a transgenic mammal demonstrating a (2-3) trans-sialidase activity; and (ii) obtaining milk from the selected transgenic mammal.

44. The method of claim 43 further comprises recovering (2-3) sialillactose from the milk.

45. An a (2-3) sialyllactose formed by the process consisting of contacting a catalytic amount of at least one a (2-3) trans-sialidase with a milk source to form a dairy / trans-sialidase mixture; and incubating the dairy / trans-sialidase mixture under conditions suitable for the activity of the (2-3) trans-sialidase.

46. An a (2-3) sialyllactose formed by the process consisting in contacting a catalytic amount of at least one a (2-3) trans-sialidase with a residual stream from the cheese processing to form a residual current mixture. / trans-sialidase; and incubating the residual current / trans sialidase mixture under conditions suitable for the activity of a (2-3) tra s-slalides. SUMMARY OF THE INVENTION The present invention consists in the methods for producing sialyl oligosaccharides in situ in milk sources and waste streams in the cheese processing, before, during or after the processing of the milk source in the cheese making process. The methods of the present invention utilize the catalytic activity of a (2-3) trans-sialidases to take advantage of the high concentrations of lactose and the a (2-3) sialosides found in milk sources and the waste streams of the cheese processing to direct the enzymatic synthesis of (2-3) sialillactose. The (2-3) sialyl oligosaccharides produced by these methods are also encompassed by the present invention. The invention also offers the recovery of the sialyl oligosaccharides produced by these methods. In addition, the invention provides a method for producing (2-3) sialyllactose. The invention further provides an enrichment method for a (2-3) sialillactose in milk using transgenic mammals expressing for a transgene of (2-3) trans sialidase. The invention also provides for the recovery of the sialillactose contained in the milk produced by this transgenic mammal before or after processing of the milk. Transgenic mammals containing an α (2-3) trans-sialidase encoding the unit sequence operably to a regulatory sequence of a gene expressed in mammalian tissue are also provided by the invention.