BACKGROUND OF THE INVENTION It is known that in addition to essential nutrients, human milk contains several types of bioactive factors, including growth hormones, enzymes, antimicrobial factors, anti- inflammatory agents, transporters and peptide and non-peptide hormones (Kunz et al., 1999). Many of these factors appear in high concentrations in the colostrum, and can have multiple functional roles. The gastrointestinal tract of a newborn infant undergoes pronounced growth, morphological changes and functional maturation postnatally, and many of these milk factors have beneficial effects on this (Xu, 1996).

It is commonly recommended that newborn infants should be breast fed if possible.

However, for various reasons many infants are fed by infant milk formulas. The reason may be that the mother is not capable of producing enough milk, the mother may be ill, the infant may be unable to suckle, the infant is separated from his or her mother etc. Although various kind of infant formulas are commercially available, it is often found that the children do not tolerate the formulas well. The children may have various gastrointestinal symptons. The symptoms are explained to be due to the immaturity of the gastrointestinal tract of the children.

Although it is known that breast milk is generally better for a newborn infant compared to infant milk formulas, it is not known which factors of breast milk may be of most benefit.

In other words it is not known, how to improve the infant milk formulas to better correspond to breast milk.

It is an aim of the present invention to eliminate the problems associated with the prior art and to provide an infant milk formula composition which contains a factor present on high

level in normal breast milk after childbirth and has an effect in maturation of the gastrointestinal tract of a newborn infant.

The carbonic anhydrases (CAs) are an expanding family of zinc-containing enzymes, which classically participate in the maintenance of pH homeostasis in the human body, catalyzing the reversible reaction: C02 + H20 HCO3-+ H+. Carbonic anhydrase VI (CA VI) is the only known secreted isoenzyme of this family (Fernley et al., 1979, Feldstein and Silverman, 1984 and Murakami and Sly 1987), which has been detected to date only in the saliva secreted by the serous acinar cells of mammalian parotid and submandibular glands (Parkkila et al., 1990).

In this invention it was surprisingly found that CA VI is secreted to milk and that its concentration is approximately eight times higher in the colostrum than in the mature milk, the latter levels corresponding to those reported earlier for adult human saliva (Kivela et al.

1997). Thus, during the early postnatal period, CA VI is transferred to the infantile alimentary tract in the milk in concentrations which exceed several fold the levels found in adult human saliva.

Finnish Patent No. 103089 suggests the production of a nutritional product based on colostrum, from which fats and casein has been removed and creatin and L-carnitin added.

The patent publication does not mention CA VI.

High amount of carbonic anhydrase CA VI is supplied to the adult gastrointestinal tract daily in the saliva (Parkkila et al., 1993) whereas the newborn infant's saliva secretion is low due to the immaturity of the salivary glands (Davidson, 1982 and Scott, 1979).

It has been proposed that CA VI may participate in protecting the teeth from-caries (Kivela et al. 1999) and in neutralizing of excess acid in the mucous layer covering the esophageal and gastric epithelium (Parkkila et al., 1997). Thatcher et al (1998) identified gustin, a salivary factor implicated in taste bud growth as a developmental factor, as CA VI.

The inventors have as first suggested that CA VI could be added to infant milk formula composition and in this way improve the quality of infant milk formulas.

SUMMARY OF THE INVENTION One object of this invention is to provide a new infant milk formula composition, which comprises carbonic anhydrase isoenzyme VI (CA VI). The infant milk formula composition comprises CA VI preferably in an amount typical for normal breast milk about up to six months, preferably up to three months from childbirth.

More specifically, the infant milk formula composition is mainly characterized by what is stated in the characterizing part of claim 1.

The composition may comprise CA VI 10 mg-150 mg/1, preferably the amount is 30 mg - 100 mg/1, more preferably 60 mg-100 mg/1 ready for use infant milk formula composition.

Carbonic anhydrase isoenzyme VI (CA VI) may be added to ready for use infant milk formula composition before the use or it may be added during production process of the infant milk formula composition.

The infant milk formula composition may be in liquid form or in powder form. If it is in powder form, ready for use product is made by adding a suitable amount of liquid, typically water, to the powder.

According to one preferred embodiment of this invention CA VI is isolated from milk.

According to another preferred embodiment CA VI is prepared by genetic engineering methods.

According to a highly preferred embodiment of this invention, a suitable host, a microorganism, such as yeast, other fungi, bacteria or cell of a suitable mammalian cell line, is transferred to express human CA VI, and CA VI is isolated and purified from the growth medium or from the cells. Alternatively an effectively milk producing (non-human) animal, such as cow, is transferred to express human CA VI and in this way produced human CA VI is isolated from the milk of the transgenic animal. Human CA VI may effect in maturation of the gastrointestinal tract of a newborn infant more effectively than CA VI from other sources.

One object of this invention is to provide a new method for producing infant milk formula compositions, which comprises the steps of : - purifying CA VI from a suitable source, such as from milk or from the growth medium or host cells of genetically engineered microorganisms or cell lines transferred to express CA VI; - preparing infant milk formula composition by conventional methods; - adding CA VI to infant milk formula composition during the composition preparation process or to a ready for use product in an amount normal breast milk comprises CA VI at a certain point of time after childbirth; and - recovering the ready for use infant milk formula composition.

More specifically, the method for producing infant milk formula composition is mainly characterized by what is stated in the characterizing part of claim 13.

The nutritional supplement product intended to be added to infant milk formula composition is mainly characterized by what is stated in the characterizing part of claim 21.

It is highly advantageous that infant milk formula compositions of this invention comprise CA VI in concentrations typical for normal breast milk after childbirth. The composition is useful for reducing the symptoms caused by the immaturity of the gastrointestinal tract of an infant.

Other features, aspects and advantages of the present invention will become apparent from the following description and appended claims.

FIGURES Figure 1. A: Western blot of the saliva of the human newborn infant (N saliva), human colostral milk (milk), human adult saliva (A saliva) and purified human salivary CA VI (CA VI) using anti-human CA VI antibody (anti-CA VI) and normal rabbit serum (NRS).

The anti-human CA VI antibody recognized the 42-kDa polypeptides of glycosylated CA VI in all the samples. In addition, the 36-kDa polypeptide of the deglycosylated form of

CA VI was visible in the purified human salivary CA VI sample. Control stainings using NRS were negative. B: Western blot of rat milk (milk), rat mammary glands and purified rat salivary CA VI (S CA VI) using antibody raised against rat CA VI. The antibody recognized in all the samples similar CA VI 42-and 36-kDa polypeptides. From three different mammary gland specimens, the positive signal was strongest in the lactating gland (lact.), moderate in the gland from pregnant animal (preg.), and faintest in the resting gland (rest.).

Figure 2. SDS-PAGE and Colloidal Coomassie Blue staining of total colostral milk (milk), CA purified from colostral milk (M CA VI; 0.2 gg left lane, 0.6 gag right lane) and CA purified from saliva (S CA VI). The polypeptides of about 42 kDa mass are seen in CAs purified from both milk and saliva. A polypeptide of similar size is also visible in the total milk sample.

Figure 3. PNGase F treatment of human salivary and milk CA VI followed by SDS-PAGE and Colloidal Coomassie Blue staining. Without PNGase F treatment (-), the 42 kDa polypeptides for both salivary (S) and milk (M) CA VI are seen, corresponding to the glycosylated form of CA VI, but after digestion (+) the 36 kDa polypeptides for both samples are seen, indicating that the two glycopolypeptides have polypeptide cores of similar sizes.

Figure 4. Mean concentrations of CA VI in human colostrum and mature milk.

Figure 5. Immunohistochemical staining of rat mammary glands using the anti-rat CA VI antibody (A, C, D) and NRS (B). Milk inside the alveoli (arrows) stained strongly for CA VI in the lactating gland (A) and moderately in the gland of the pregnant animal (C). No staining was seen inside the alveoli of the resting gland (D). The cytoplasm of the alveolar epithelium showed a faint staining intensity (D, arrowheads) in all the glands. Control staining of a lactating mammary gland with NRS was negative (B).

Figure 6. SDS-PAGE analysis of CA VI preparation purified from cow's milk.

DETAILED DESCRIPTION OF THE INVENTION By"infant milk formula composition"we mean here any nutritional formula or substitute composition, which is aimed for the feeding of an infant instead of breast milk. The infant milk formula composition may be any commercially available infant milk formula composition based for example on cow milk or soy milk. It may be based also on another mother's milk. Usually another mother's milk comprises lower levels of CA VI, because it is not available immidiately or shortly after the birth of a child.

By"nutritional supplement products"is meant here products, which are aimed to be added to infant milk formula compositions and which are in liquid or in solid form. Such products may be for example products which contain purified CA VI and optionally buffers and factors, which stabilize the product. The product is preferably sterilized by filtering because heating could inactivate the enzyme. The product may be provided in dropping bottles such as vitamins (Vitamin A for example) and may be stored in refrigerator before use.

By"enzyme preparation comprising CA VI"is here meant an enzyme preparation comprising as the major activity CA VI. The preparation may comprise in addition small amounts of non-harmful components still left after the purification process of the enzyme.

If the enzyme is purified from milk, some amount of normal milk ingredients may still be left. The amount of milk ingredients may be 0.1-10 w-%, preferably it is 0.1-5 w-% of the enzyme preparation. The enzyme preparation of this invention may comprise CA VI from various sources especially in a case, when a milk-producing animal is transferred to express human CA VI. Alternatively the animal is transferred not to express natural CA VI or the desired CA VI is separated from the enzyme preparation.

The infant milk formula composition of this invention comprises CA VI preferably"in an amount typical for normal breast milk up to six months, preferably up to three months from childbirth". In connection of this invention it has been shown that breast milk comprises after child birth the highest level of CA VI as can be seen in Figure 4. The amount of CA VI decreases gradually and is after 6 months post partum on the level of saliva, but is on lower level already after 3 months post partum. According to this invention the infant milk

formula should contain CA VI in an amount which corresponds to the amount of CA VI in breast milk of a mother having a child of the same age.

The infant milk formula composition should comprise CA VI preferably 10 mg-150 mg/1, preferably 30 mg-100 mg/1, more preferably 60-100 mg/1 ready for use infant milk formula composition. Ready for use infant milk formula composition means here a liquid composition or a powder, into which liquid has been added before use.

According to a preferred embodiment of this invention, CA VI is added to the infant milk formula composition in an amount which corresponds to the amount of CA VI in the breast milk of a mother having a child of the same age. Different infant milk products can be prepared comprising of gradually decreasing amounts of CA VI. Alternatively the proportioning of CA VI is adjusted to the age of the child. This is preferably carried out by adding CA VI by the parents or nurses of a child to a ready for use infant milk product in an amount recommended for a child of that age.

The infant milk formula composition may be in liquid or powder form. In the latter case, liquid, normally water, is added to the powder product in an amount removed from the composition during the production process.

The infant milk formula composition of this invention is recommended for children up to 6 months. However, it may be used also for elder children, if they have gastrointestinal disorders.

Carbonic anhydrase isoenzyme VI Carbonic anhydrases maintain pH homeostasis in various tissues of the human body by catalyzing the reversible reaction C02 + H2O <=> HCO3-+ H+. Carbonic anhydrase isoenzyme VI (CA VI) is secreted into human saliva by the serous acinar cells of the parotid and submandibular glands. CA VI is the only known secreted isoenzyme of the CA gene family, and has several properties that distinguish it from the well-characterized cytoplasmic isoenzymes.

Its reported molecular weight is 39-46 kDa (Feldstein & Silverman 1984, Kadoya et al.

1987, Murakami & Sly 1987, Fernley 1991, Parkkila S et al. 1991b, Ogawa et al. 1992).

The-enzyme--molecule has two N-linked oligosaccharide-chains,-whieh-ean-be-eleaved by endo- (3-N-acetylglucosaminidase F but not by endo- (3-N-acetylglucosaminidase H, indicating that the oligosaccharides are of a complex type (Murakami & Sly 1987).

Neuraminidase has no effect on the endo-ß-N-acetylglucosaminidase F-digested protein, suggesting that CA VI has no 0-linked oligosaccharide which contains neuraminidase- sensitive sialic acid residues (Murakami & Sly 1987). The complete amino acid sequence of ovine CA VI was determined by Fernley et al. (1988), and the complete nucleotide sequence for human CA6 cDNA by Aldred et al. (1991). The human CA6 gene is located on chromosome 1 (Aldred et al. 1991). The human CA VI protein has a sequence identity of 35 % to human CA II, while residues involved at the active site of the enzyme are conserved. Human CA VI has three potential N-glycosylation sites and two cysteine residues (Cys25 and Cys207) (Aldred et al. 1991), the latter presumably forming a disulphide bond, as in the ovine enzyme (Fernley et al. 1988).

Carbonic anhydrase VI is the only secretory isoform in the CA gene family, having been identified previously in the saliva of several mammalian species. It is demonstrated here for the first time that CA VI is also present in milk. The specific antibodies to salivary CA VI recognized polypeptides of similar molecular mass (42 kDa) in human and rat saliva and milk, the polypeptide was effectively purified from human milk on CA inhibitor affinity chromatography, and the entire amino acid sequence of the 42-kDa polypeptide obtained from MALDI-MS (40% coverage) was identical to salivary CA VI. Moreover, digestion of the purified human milk 42-kDa polypeptide with PNGase F reduced its molecular mass to 36 kDa, indicating that it is a glycopolypeptide by nature and that its polypeptide backbone is probably identical to that of salivary CA VI.

Immunohistochemical staining of a rat mammary gland showed that the enzyme is present in both the alveolar milk and the glandular epithelia. The fairly faint epithelial staining is likely to be due to the rapid secretion rate of the epithelia during late pregnancy and lactation. The immunoblots revealing the major 42-kDa polypeptide in rat mammary gland samples confirmed that the immunohistochemical results are specific to CA VI.

Quantification of CA VI in milk using a time-resolved immunofluorometric assay revealed an approximately eight times higher concentration in human colostrum than in mature milk, the latter corresponding to the levels previously detected in human saliva. The high concentration in the colostrum in particular, its functional and structural stability in an acidic milieu, and its growth-supporting role in the taste buds suggest that milk CA VI is an essential factor in normal growth and development of the infant alimentary tract.

In this invention, CA VI was also isolated from mature cow's milk. SDS-PAGE analysis of the purified CA VI fraction showed three polypeptide bands: 29 kDa, 32 kDa and 35 kDa.

The 32 kDa band was strongest of these, suggesting that the polypeptide from cow's milk is smaller than in human milk.

Recent findings reported in the literature are in accordance with the conceptual CA VI functions as a growth factor. Henkin et al. (1999a) described a clinical disorder in which patients had decreased salivary CA VI concentrations associated with a loss and distortion of taste and smell after an influenza-like illness. The morphological chances in the taste buds presented apoptotic-like features (Henkin et al. (1999a). Treatment with zinc normalized the CA VI concentrations and the senses of taste and smell in some cases (Henkin et al. 1999b). The taste bud morphology was also normalized in these patients, suggesting that CA VI may function as a trophic factor for the taste bud stem cells (Henkin et al. 1999b). Furthermore, CA VI has been shown to have characteristics similar to nerve growth factor (NGF) (Henkin et al. (1988). There is also evidence that CA VI activates calmodulin dependent bovine brain cAMP phosphodiesterase, which is a factor involved in taste function (Law et al. 1987) and that CA inhibitors can cause taste distortions in clinical use (Hansson, 1961, Graber and Kelleher, 1988 and Miller and Miller, 1990).

Many milk glycoproteins have a multifunctional nature and CA VI as a glycoprotein and an acid-base modulating enzyme may also possess multifunctional properties, such as antimicrobial (Hooper et al. 1995), anti-inflammatory, immunomodulating and mucosa- protecting effects (Parkkila et al. 1997).

Source for CA VI The enzyme preparation of this invent may be purified from milk or from the culture medium or from the cells of a recombinant microorganism or cell line transferred to express CA VI enzyme.

According to a one preferred embodiment of this invention CA VI is produced by recombinant technology in a suitable host organism. cDNA for human CA VI is available on public domain (EMBL Accession number J05305, and is disclosed also in Grubb (1999). cDNA can be isolated from a human salivary gland cDNA, mammary gland cDNA or genomic DNA libraries. The CA VI cDNA can be integrated into an appropriate expression vector that allows the large-scale production of CA VI in a suitable host, such as yeast, other fungi, bacterium or mammalian host cells, such as CHO cells according to conventional methods such as described in Sambrook et al. (1989) and Ausubel et al.

(1988). Grubb (1999) has disclosed the isolation of CA VI encoding cDNA from human genomic DNA using PCR synthesis and the expression of the enzyme from yeast Pichia pastors expression system (Invitrogen K1710-01).

The host should preferably be capable of glycosylating, and it is of advantage, if the host is capable of secreting the enzyme into the culture medium of the host. The secreted CA VI can be purified from the medium using inhibitor affinity chromatography as described below (Parkkila et al., 1990). The enzyme is purified from culture medium or host cells preferably to homogeneity or the preparation may contain small amounts of non-harmful components still left after the purification process of the enzyme. The enzyme preparation may contain 0.1-5 w-% of the enzyme preparation ingredients from host cells or from the medium.

According to one other preferred embodiment of this invention the enzyme preparation is purified from milk, because milk is a natural and safe source for CA VI. The enzyme may be purified from milk to homogeneity or near to homogeneity, which means that the enzyme preparation may contain other milk ingredients 0.1-5 w-% of the enzyme prepa- ration. However, in normal case milk components, such as proteins and fats, may still be left in the preparation, because usually they do not have harmful effect to the infant milk

formula. The amount of milk ingredients may be 0.1-10 w-%, preferably it is 0.1-5 w-% of the enzyme preparation.

The milk which is used as the source for CA VI may be the milk of a (non-human) animal producing efficiently milk such as cow, sheep or goat or related, or preferably closely related animals. Isolation of the enzyme from human milk is naturally possible, but not practical due to its limited availability. Because cow's milk is usually available in higher amounts, the milk is preferably cow's milk. The milk may be mature non-manufactured milk, but preferably it is colostrum, because the amount of CA VI is clearly higher in colostrum. The milk should not be treated with methods, for example sterilization, which potentially inactivate the enzyme, such as heating or pasteurization.

By colostrum is here meant milk, which is produced by human or a milk-producing animal, such as cow, a few days after parturition. It is known that the amount of proteins in cow colostrum decreases rapidly within the first two days after parturition leveling out by ten days. The amount of immunoglobulins is decreased within two days (Powell et al. (1984).

Colostrum means in this invention milk from human or milk from a milk producing animal, preferably cow, 0-10 days, preferably 0-5 days, most preferably 0-2 days after parturition.

It is also possible that a milk-producing animal, such as cow, is transferred to overexpress and secrete endogenous CA VI, or express and secrete CA VI from another milk- producing animal, or specifically human CA VI into its milk. In that case the normal mature milk of the cow would be suitable source for the purification of CA VI. Another advantage of this embodiment is that the transgenic animal can be transferred to express human CA VI, which may be more suitable than the natural CA VI of the transgenic animal for infants generally, or at least for some individuals.

Methods of transferring animals, such as cow, to express or overexpress desired nucleotide sequences are available and well-known to a person skilled in the art (for example EP 471832).

Purification of CA VI Purification of CA VI from milk, preferably colostrum can be carried out essentially as described by Parkkila et al. (1990). The method comprises the steps: - centrifuging milk and collecting the fatfree supernatant; - carrying out one or more affinity purification steps; - removing displacing agents used for dissociation of the enzyme from the affinity matrix; and - recovering the enzyme.

Milk, preferably colostrum, is centrifuged to remove epithelial cells, particles, bacteria or cell debris and fat, preferably by 35 000 x g at 4°C for 30 min. The clear fatfree supernatant is collected and mixed with buffer, preferably ice-cold 0.1 M Tris-S04 buffer, pH 8.7, containing a protease inhibitor, such as 1 mM benzamidine and the solution is subjected to affinity purification. The inhibitor affinity chromatography is performed using CM Bio-Gel A or a corresponding matrix coupled to enzyme inhibitor, preferably p- aminomethyl benzenesulfonamide. After dissociation of the enzyme from the affinity matrix, the displacing agent is removed by a suitable method, such as dialysis. Further purification is possible by gel filtration or HPLC, but further purification is normally not needed. The purification of the enzyme can be carried out by various different protein chemistry methods well known to a person skilled in the art. However, the method described here and in the examples is useful in large scale and fast, the yield is high and there are only negligible amount of impurities left in the enzyme preparation.

The purified enzyme preparation may be used as such or it may comprise also stabilizers, such as glycerol, casein, sucrose or lactose.

Similar methods are used when purifying CA VI from the cells or from the culture medium of a microbial host or cell line genetically engineered to produce CA VI.

Infant milk formula compositions Conventional infant milk formulas are often based on cow milk proteins like casein or mixtures of casein and whey. For children suffering from allergy or metabolic disorders,

the protein source may be different. Soy based formulas have been developed for infants that cannot tolerate lactose or are allergic to milk proteins. Casein hydrolysates may be used for infants who do not digest protein well or who suffer from malabsortion or poor eating habitats to provide adequate nutrition. In those cases soy proteins or protein hydrolysates may be used. For example European Patent Application EP 0951 842 describes a typical infant milk formula (EP 0951 842 Table 1). The formula was based on 40-60 % whey and 40-60 % of casein. The formula provides energy about 67-70 kcal/100 ml. Typical protein levels in commercially available infant formula are 1.2-1.8 g/100 ml. The other ingredients of the formula are typically: Arg is 3.0-3.5 % (=g/100g protein), Trp 1.3-1.5 %, BCAA 22-25 g/lOOg protein, Tyr + Phe 7-9, Thr 5-7, Cys 1.0-1.6, carbohydrates=CHO 4-7.5 (g/100m1), Lactose 80-100 (% =g/100g CHO), maltodextrins 0-20 (%), folic acid 15 (ug/100kcal), Vitamin B6 60 (ug/100kcal), Vitamin B12 0.3 (ug/100kcal), Zinc 0.6 mg/100 kcal, vitamin B2 150 ug/100kcal.

Conventional infant formula is formed by mixing 60 % sweet whey and 40 % casein, the ratio of the amounts of tryptophan to the total amount of large neutral amino acids (Phe, Tyr, Val, Ile, Leu) is about 4.4-4.7/100 ml Other examples of infant formula compositions are described in United States Patent No.

5,902,617. Commercially available infant milk formulas may comprise in 1000 g of the product: energy 676-670 cal, protein 15.0-19.0 g depending on the source, which may be cow's milk, reduced mineral whey and milk, soy protein isolate or casein hydrolysate containing Cys, Tyr, and Trp. The infant milk formula may comprise fat 36. 3-38 g, the source of which may be soy and coconut oils or medium chain triglycerides. The infant formula composition may furthermore comprise polyunsaturated fatty acids 11-14 g, saturated fatty acids 16-18.2 g, linoleic acids 7400-10816 mg, carbohydrates 68.9- 72.3 g. The source of carbohydrates may be lactose or corn syrap and sucrose or sucrose and starch. In addition commercial infant formulas may comprise minerals, vitamins, other nutrients and 900 g water.

Other formulas based on a variety of protein and carbohydrate sources are also available.

The formulas may be in the form of liquid in a ready to feed concentration or a concentrate or dehydrated or lyophilized.

A method for preparing an infant formula composition Infant formula is prepared for example by a process described in International Patent Publication No. WO 97/35488 : a) pasteurised milk (skimmed, evaporated or whole milk) is standardised by the addition of whey protein concentrate, minerals, water-soluble vitamins, trace elements and carbohydrates at high temperatures, for example at 60 °C ; b) vegetable oil, oil-soluble emulsifiers, oil-soluble vitamins and anti-oxidants are mixed at high temperatures, for example at 60 °C ; c) the oil mixture obtained from b) (an oil phase) is added to the standardised milk obtained from a) (a water phase) with sufficient agitation to allow mixing; d) the mixture obtained above is homogenised in two stages at high temperature and pressure, for example at 60 °C at 150 and 30 bar; e) the emulsion obtained above is cooled to a low temperature, for example to 5 °C f) if desired, water-soluble vitamins, minerals and trace emulsions are added to the cooled emulsion; g) the emulsion from e) is sterilised on-line at ultra high temperature (UHT) and/or in appropriate containers to obtain a formula in the form of a sterile liquid; or h) the emulsion from e) is pasteurised and spray-dried to give a dry powder which is filled into appropriate containers; i) if desired, other dry ingredients, e. g. vitamins, minerals, trace elements, whey protein concentrate and carbohydrates can be added to the spray-dried powder.

Purified CA VI is added to the infant milk formula preparation at a suitable stage of the preparation. Advantageously CA VI is added at a relatively late stage in the preparation.

This is because high temperatures are used at several points in the preparation, which could result in inactivation of CA VI. If CA VI is added after step e), the formula preparation should be filtered in order to prepare a sterile product instead of heating or pasteurizing.

CA VI is preferably added after sterilization in step g) or pasteurization in step h).

Alternatively CA VI enzyme preparation is added to a ready for use infant milk formula composition before use.

The method of this invention comprises the following steps: - CA VI is purified from a suitable source; - infant milk formula composition is prepared according to conventional methods; - CA VI is added to infant milk formula composition in an amount normal breast milk comprises CA VI at a certain point of time after childbirth; and - the infant milk formula composition is recovered from the production process.

Addition of CA VI at the time of feeding It is also possible to add CA VI as a nutritional supplement product or concentrate. The enzyme may be packaged in a buffered solution, filtered with for example 0.2 micron filter to sterilize the concentrate and the concentrate may be refrigerated when the container is opened. The formulation may be added as drops per volume. The CA VI concentration of the product or concentrate should be calculated to be suitable to provide CA VI 10 mg- 150 mg/1 ready for use infant milk formula composition. The addition can be made at the time of feeding.

The present invention will further be illustrated by way of Examples.

Example 1 Antisera The production and characterization of the antisera to human and rat CA VI have been described earlier (Parkkila et al. 1990). Both antisera have been characterized by western blots in which they have shown high isoenzyme specificity.

Collection of milk, saliva and tissue samples Colostral milk samples were obtained from 9 mothers on the 2-4th days post partum, and saliva samples were obtained at the same time from two 3-day-old infants prior to nursing.

Mature milk samples were obtained from four mothers on the 90th day post partum. The procedures were carried out according to the provisions of the Declaration of Helsinki and informed consent was obtained from each mother. Rat milk samples were collected from the rats of Spraque-Dawley strain anesthetized with fentanyl-fluanisone (3ml/kg, Janssen Pharmaceutica, Beerse, Belgium) on the second day post partum and pooled. All the milk

samples were stored frozen prior to use in the experiments. Mammary gland specimens were taken from a sexually mature non-pregnant female rat, and from another two days before and two days after parturition. The tissue samples were homogenized in ice-cold 0.1 M Tris-S04 buffer, pH 8.7, containing 1 mM PMSF, 1 mM benzamidine and 1 mM o- phenanthroline as protease inhibitors and used for western blotting, or fixed in Carnoy's fluid and embedded in paraffin as described (Karhumaa et al. 2000).

Purification of CAs Human colostrum (15 ml) was centrifuged (35 000 x g) at 4°C for 30 min and the clear supernatant (10 ml) was collected and mixed with 30 ml of ice-cold 0.1 M Tris-S04 buffer, pH 8.7, containing 1 mM benzamidine as a protease inhibitor and subjected to affinity purification. The inhibitor affinity chromatography was performed using CM Bio-Gel A coupled to p-aminomethyl benzenesulfonamide as described (Parkkila et al. 1990).

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting The milk samples were centrifuged (15 000 x g) at 4°C for 10 minutes and the supernatants were recovered. Samples of CAs purified from human colostrum (0.2 and 0.6 pg), human and rat purified salivary CA VI (1 Rg), human colostrum (10 ltl), human saliva (10 gel), rat milk (30 ug) and rat homogenized mammary gland (30 jig) were subjected to SDS-PAGE under reducing conditions according to Laemmli (1970). All the reagents for SDS-PAGE were from Bio-Rad Laboratories (Richmond, CA) or Sigma (St. Louis, MO). The western blotting was carried out as described (Karhumaa et al. 2000).

Protein sequence analysis The protein sequencing of trypsin-digested polypeptides was carried out by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) followed by analysis with the ProFound and PeptideSearch programs. The sequencing was performed in the HHMI Biopolymer/W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University.

Deglycosylation studies Purified milk and salivary CA VI (1 ug) were digested with PNGase F as described earlier (Petaja-Repo et al. 1991), and the deglycosylated and non-deglycosylated proteins were

subjected to SDS-PAGE followed by Colloidal Coomassie staining (Novex, San Diego, CA).

Fluoroimmunoassay The competitive time-resolved fluoroimmunoassay for CA VI was performed on the human milk samples (colostrum n=9, mature milk n=4) as described in detail previously for saliva samples (Parkkila et al 1993). The mean intra-assay coefficient of variation (CV) of the present series was 9.4-% and the interassay CV determined in three assays was 9.7- %.

Immunocytochemistry Mammary gland samples fixed in Carnoy's fluid were sectioned at 5 um and placed on gelatin-coated microscope slides. Immunohistochemical staining was performed using the biotin-streptavidin complex method as described (Karhumaa et al. 2000). The stained sections were examined and photographed with a Nikon Eclipse E600 microscope (Tokyo, Japan).

Documentation of the presence of CA VI in human and rat milk Western blotting of samples of human colostrum, saliva, and purified salivary CA VI with specific antibody to human salivary CA VI revealed a major 42-kDa polypeptide band in all cases. In addition, a minor 36-kDa band, representing the deglycosylated form of the enzyme, was detected in the purified salivary CA VI (Fig. 1A). A major 42-kDa band and minor 36-kDa polypeptide band were also obtained from rat milk and purified salivary CA VI samples subjected to immunoblotting with specific antibody to rat salivary CA VI (Fig.

1B). The 42-kDa polypeptide was also effectively purified from human colostrum in CA inhibitor affinity chromatography (Fig. 2). PNGase F digestion of the purified human milk and salivary CA VI reduced their molecular size from 42-kDa to 36-kDa, indicating that both glycopolypeptides have a similar-sized polypeptide core (Fig. 3). To confirm that the human milk CA is isoenzyme VI, the purified colostral isoenzyme was isolated from a SDS-gel followed by trypsin digestion and sequencing with MALDI-MS. The analysis of the sequence data with the ProFound and PeptideSearch databases revealed a 100% identity with human salivary CA VI. The sequenced polypeptides covered 40% of the full- length CA VI.

CA VI concentrations in human colostral and mature milk The mean concentrations of CA VI were 34.7 mg/1 (range 10.0-78.4 mg/1, n=9) in colostrum and 4.5 mg/1 (2.6-6.9 mg/1, n=4) in mature milk (Fig. 4). The CA VI levels in the saliva of two infants were 1.9 and 3.6 mg/1, about half of the concentration in adults (Parkkila et al 1993).

Detection of CA VI in the rat mammary gland by immunohistochemistry The mammary glands of a sexually mature nonpregnant female rat showed a faint positive reaction for CA VI in the alveolar epithelia, and the same was observed prior to and after parturition (Fig. 5A, C, D). A strong reaction was seen in the alveolar milk after parturition, the reaction being slightly weaker prior to parturition (Compare figures 5A and C). No reaction was seen in the alveolar lumen of the resting gland (Fig. 5D). Western blotting of mammary gland homogenates showed the most intense band to be in the lactating gland, a moderate band in glandular tissue prior to parturition, and a faint band in the resting gland (Fig. 1B).

Example 2 CA VI was purified from fresh non-manufactured mature cow's milk. The purification was carried out essentially as described in example 1. Samples of CAs purified from cow's milk were subjected to SDS-PAGE under reducing conditions as described in example 1.

The SDS-PAGE analysis gave three polypeptides bands: 29 kDa, 32 kDa, and 35 kDa (Figure 6). The 32 kDa band was strongest, which indicated that the polypeptide is smaller than CA VI from human milk.

Example 3 Preparation of an infant milk formula composition CA VI is purified from the culture medium of a microorganism transferred to express CA VI or from cow's colostrum after 2 days from calving. The purification is carried out as described in example 1.

An infant milk formula composition is prepared by conventional methods as described for example in International Patent Publication WO 97/35488. To the ready for use infant milk formula is added purified CA VI to give a concentration between 10-150 mg/1.

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