Technical field

The present invention relates to a method for obtaining a mixture of biological factors isolated from colostrum, through one or more microdiafiltration steps, which makes it possible to separate large-size proteins, such as caseins and lactalbumins and fat globules from the permeate, in which serum-soluble proteins, including the biological factors of interest, remain. The mixture can also be obtained in sterile form, by eliminating cells and pathogenic microorganisms by means of a sterilizing filtration.

The present invention also relates to a mixture of biological factors isolated from colostrum that comprises the proteins IgG, IgA, IGF-1 and TGF βΐ, in the following quantities, indicated per mg of dry product: 0.15 - 0.35 mg of IgG; 0.01 - 0.05 mg of IgA; 0.5 - 3.0 ng of IGF-1 ; 30 - 90 pg of TGF βΐ .

A further object of the invention is a nutraceutical formulation that comprises the mixture of biological factors isolated from colostrum.

Moreover, the invention relates to a supplement for milk for newborns, or for follow-on milk or growth milk, or for drinking milk, comprising the mixture of the invention.

Finally, the invention relates to milk for newborns, or follow-on milk or growth milk, both powdered and reconstituted, or drinking milk, supplemented with the mixture of the invention.

Breastfeeding consists in feeding a newborn or an infant directly on the breast of a woman and not from a feeding bottle or other container. Babies have a sucking reflex that allows them to suck and swallow the milk. Most mothers can feed their own newborn (or their own newborns in the case of twins and multiple births) by breastfeeding for the first six months, or even longer, without supplement of formula or solid food.

The alternatives to breastfeeding comprise:

β maternal milk, produced by the mother of the newborn herself; β natural milk, produced by a healthy wet nurse or coming from a human milk bank;

^• a substitute of natural milk, given with a small cup, which is a safer method than the use of a feeding bottle and of a teat. Background art

In most situations, natural milk is the best source of nourishment for human newborns, preventing diseases, promoting health and reducing health care costs (apart from situations in which the mother is taking particular drugs or is infected by tuberculosis or HIV). Experts disagree on how long to breastfeed in order to obtain the maximum benefits and on the risks associated with the use of milk substitute formulations.

The World Health Organization (WHO) recommends a minimum of two years of breastfeeding and exclusive breastfeeding for the first six months of life. The American Academy of Pediatrics (AAP) recommends at least one year of breastfeeding and exclusive breastfeeding for the first six months of life. Exclusive breastfeeding for the first six months of life "gives continuous protection against diarrhea and infections of the respiratory tract", which are more common in newborns fed with formula. Both the World Health Organization (WHO) and the American Academy of Pediatrics (AAP) stress the value of breastfeeding for mothers and children.

During breastfeeding, nutrients and antibodies pass to the child and the maternal bond may also be strengthened. Research has demonstrated a variety of benefits associated with breastfeeding of a newborn.

For example, a study conducted by the University of Wisconsin has found that adult women who were breastfed during their childhood could have a lower risk of developing breast cancer than their counterparts who had not been breastfed. Moreover, in children at risk of atopy (defined as at least one parent or one sibling with atopy), the atopy syndrome may be prevented or delayed by exclusive breastfeeding for four months, although these benefits might no longer be present after four months of age. Atopic dermatitis, which is the most common form of eczema, can be reduced by exclusive breastfeeding beyond 12 weeks, in subjects who have a history of familiar atopy.

Moreover, it seems that breastfeeding, by introducing gluten in the diet, may reduce the risk of developing coeliac disease, although it is not yet clear whether it simply slows down the symptoms or provides protection for life.

Newborns who are exclusively breastfed also have a lower likelihood of developing type 1 diabetes mellitus than counterparts with a shorter duration of breastfeeding and earlier exposure to bovine milk and solid food. It appears that breastfeeding also protects against type 2 diabetes mellitus, at least partly, thanks to its effects on the weight of the child; breastfeeding in fact seems to be associated with a reduced risk of obesity in children from 39 to 42 months.

Breastfeeding, moreover, ensures beneficial effects with respect to some common disorders of newborns and children.

For example, it provides protection against diarrhea; in comparison to counterparts fed with formula, the percentage of deaths due to diarrhea in breastfed newborns is lower. Moreover, necrotizing enterocolitis, which affects mainly premature newborns, is six to ten times more common in newborns fed exclusively with formula and three times more common in newborns fed with a mix of natural milk and formula than in newborns who are exclusively breastfed. In newborns born at more than 30 weeks, necrotizing enterocolitis was twenty times more common in newborns fed exclusively with formula.

A longer duration of some types of middle ear infections (otitis media with effusion) in the first two years of life is associated with a shorter period of breastfeeding. A reduced intensity and duration of any infection from otitis media has been associated with breastfeeding.

It seems that breastfeeding reduces the symptoms of upper respiratory tract infections in premature newborns up to seven months after discharge from the hospital. It also reduces the risk of acquiring infections of the urinary tract in newborns up to seven months after birth.

Maternal milk has a strong effect against infections, because it includes factors such as lactoferrin (which bonds with iron and inhibits the growth of intestinal bacteria) and immunoglobulins G and A, which provide protection against microorganisms.

Maternal milk also contains the ideal ratio among the amino acids cysteine, methionine and taurine for supporting the development of the central and peripheral nervous system.

By comparing the pathologies described above with the activities of some active factors contained in maternal milk, it is evident that the main cause of these pathologies is associated with the total absence of these active factors in milk for children.

These factors, IFN γ (interferon γ), TNF a (Tumor Necrosis Factor a), lactoferrin, TGF βΐ (Transforming Growth Factor βΐ), IGF-1 (Insulin-like growth factor 1), IgG and IgA immunoglobulins, complement factors, micronutrients and prebiotic substances, are present in different concentrations in the biological fluids of all mammals depending on the species, race, type of fluid and geographical origin. Milk and colostrum of herbivores are very rich in IgG antibodies, because they do not pass through the placental filter and therefore must be consumed only through breastfeeding. Cows of the Holstein and Frisian breeds are the richest in active factors.

Many of the diseases that affect newborns and children in the first months of life are due to the fact that their immune system is not developed completely and susceptibility to bacterial and viral infections is high during childhood. During this period, an allergic sensitization to environmental allergens and food allergens also occurs commonly. At the same time, there is a close immunological interaction between the mother and her child during pregnancy and the period of breastfeeding.

Colostrum and milk in fact contain various immunomodulators ("colostrum factors"), which not only can act directly to neutralize the noxious effects of pathogens encountered by the newborn but can also affect the maturation of the immune system of the newborn itself.

A description of the functions mediated by these active factors will be able to show better their essential physiological role. Among the many potentially active immunological components that have been identified in colostrum, cytokines are of particular importance. Cytokines are small soluble proteins that act in an autocrine/paracrine manner, by bonding to specific cell receptors and orchestrating the development and function of the immune system. Various types of cells can produce cytokines: the cells of inborn immunity and acquired immunity such as monocytes, macrophages, T and B lymphocytes but also non-immune cells, including fibroblasts and mammary epithelial cells.

Cytokines act at low concentrations, up to 1 pg/ml, and there is increasing evidence that they are present in human colostrum and bovine colostrum in such quantities as to have an effect in vivo on the newborn. In order to have a biological effect on offspring, colostrum factors must pass from the mother to the children in an active form and must be present in physiologically significant concentrations in the intestinal lumen of the newborn.

Although cytokines can be detected in human and bovine milk throughout the nursing period, the highest concentrations have been found in colostrum. Infant formula does not contain cytokines.

Among these proteins, TNF a and IFN γ are particularly noteworthy and are prototypes of innate immunity and acquired immunity respectively.

IFN γ plays an important role in boosting the activation of cell- mediated immunity: it activates neutrophils, macrophages and "natural killer" cells. High levels of this cytokine in the intestinal mucosa have been correlated to the defense of the intestinal tract against microorganisms. Moreover, IFN γ has an inhibiting role on the synthesis of immunoglobulins E (IgE) and it has been suggested, therefore, that a correct quantity of this cytokine may be of help in preventing the onset of food allergies and environmental allergies.

The main physiological function of TNF a is to recruit neutrophils and monocytes to the sites of infection and activate them in order to eliminate microbes. This cytokine is considered a proinflammatory cytokine which, although essential at a low concentration, may have noxious effects if produced in excess for a long period of time. The neonatal immune system produces extremely low levels of TNF; however, low levels of TNF allow the development of allergic reactions.

Lactoferrin is a glycoprotein that binds the iron and is synthesized by specific granules in polymorphonuclear cells and glandular epithelial cells. It is a ubiquitous biglobular protein which is present in milk, on the surfaces of mucosae, in plasma, saliva, tears, seminal fluid, endocervical fluid, vaginal secretions, synovial fluids, cerebrospinal fluids and other biological fluids. Under inflammatory conditions, its production is increased in the peripheral regions by neutrophils. In milk, it plays an essential role in the defensive mechanisms of the mammary gland of nursing animals as well as of the intestinal microbial infections of newborns.

Bacteria need iron in order to grow and lactoferrin, by chelating iron in particular conditions, may inhibit their proliferation. Moreover, lactoferrin may kill directly certain bacterial strains or may weaken bacterial resistance by adhering to their surface and compromising their bond to the host cells. For these reasons, lactoferrin is considered a fundamental element in the innate defensive system of the host.

Lactoferrin is also responsible for the fungistatic effect of human milk and it has been shown that it is active against various viruses, including the herpes virus, adenovirus, rotavirus and poliovirus.

Lactoferrin is particularly resistant to proteolytic degradation in the digestive tract, is absorbed by the intestine by means of specific receptors located on the microvilli and, if taken orally, stimulates both local immune response and systemic immune response. Lactoferrin also has a role in the absorption of nutrients: it can transfer metallic ions such as iron, manganese and zinc and helps the absorption of sugars. Moreover, it stimulates the proliferation of intestinal endothelial cells and the growth of lymphatic follicles associated with the intestine. This property suggests the possibility of administering lactoferrin to premature newborns and to subjects with damaged intestinal mucosa. Moreover, lactoferrin regulates the appropriate composition of the intestinal microflora, suppressing the growth of pathogenic bacteria and at the same time promoting the multiplication of the non-pathogenic microflora (for example Lactobacillus and Bifidobacterium) that protects the intestinal mucosa. Finally, lactoferrin provides protection from the toxicity of oxygen radicals. This propriety can be particularly important if food for children, based on modified cow's milk, contains mineral iron, which can be a source of harmful free radicals.

Colostrum and milk are particularly rich in growth factors, among which the Transforming Growth Factor β (TGF β) and Insulin-like Growth Factor I (IGF-I) have a fundamental function.

TGF β has a critical effect on the production of immunoglobulins A

(IgA), which are the main humoral effector of mucosal immunity. Colostrum and maternal milk in the first period of nursing contain an abundant quantity of TGF β, which might start the production of IgA in newborns.

TGF β also has an inhibiting role: it inhibits proliferation of thymocytes, T cells, B cells and "natural killer" cells. Moreover, it inhibits some differentiating functions of lymphocytes, including the production of immunoglobulins by human B lymphocytes. TGF βΐ, moreover, can interact with other lymphokines and modulate selectively IgA response.

IGF-I regulates the growth of beta cells in the islets of Langerhans, their survival and their metabolism and provides protection against diabetes type 1. IGF-I stimulates glucose transport in human muscles and the synthesis of muscular proteins.

Insulin and IGF-I seem to be capable of stimulating cell proliferation and differentiation; because of this capacity of modulating proliferation as well as adhesion and migration of cells, the IGF-I growth factor plays an important role in many pathologies, such as cancer and psoriasis.

The antibodies (known also as immunoglobulins, or Ig) are gammaglobulins present in blood and in other biological fluids of vertebrates, used by the immune system to identify and neutralize foreign substances, such as bacteria and viruses. They are constituted typically by basic structural units, each having two larger "heavy" polypeptide chains and two smaller "light" polypeptide chains, joined to form, for example, monomers with a single unit, dimers with two units or pentamers with five units.

Antibodies are produced by a type of white blood cells known as B cells. There are different types of heavy chains, which identify different isotypes of immunoglobulins. In mammals, five different isotypes of antibodies (IgA, IgD, IgE, IgG and IgM) are known and differ in their biological and functional proprieties and in their ability to recognize different antigens. Recognition of an antigen by an antibody marks it for attack by other components of the immune system. Antibodies may also neutralize their targets directly, for example by means of the bond to a part of a pathogenic agent that is necessary to trigger an infection.

Antibodies that bind to the surface of the bacteria attract the first component of the complement cascade and start the activation of the "classic" complement system, which determines suppression of the bacteria.

Immunoglobulins G (IgG) are involved mainly in the secondary antibody response that occurs approximately one month after recognition of the antigens. IgG can bind to many types of pathogenic agents, for example viruses, bacteria and fungi, and protect the organism by causing the agglutination and immobilization of these pathogens, activating the complement (classic path), by opsonizing them for phagocytosis and neutralizing their toxins. They play also an important role in antibody- dependent cell-mediated cytotoxicity. IgG provide most of antibody-based immunity.

IgG is the only isotype that can pass through the human placenta, thus supplying protection to the fetus inside the uterus. Together with IgA secreted in maternal milk, IgG absorbed through the placenta provide the newborn with humoral immunity before his immune system develops.

It has been found that immunointegration with antibodies of bovine milk protects against diseases of the gastrointestinal tract. The most abundant immunoglobulins in colostrum are IgG.

Immunoglobulins A (IgA) are synthesized by B lymphocytes, more precisely by plasma cells, or effector B cells. They may be synthesized in monomer form or dimer form. They have two important functions: they ensure mucosal immunity and neonatal immunity. The role of IgA in mucosae is extremely important, because most microbes enter the body through the only two systems that communicate with the external environment: the digestive system and the respiratory system.

IgA are extremely important also because they are transmitted as a first defense system from the mother to the child, who is not capable of producing antibodies during the first six months. Maternal milk is in fact particularly rich in IgA, which protect his digestive system against microbes. The complement system is represented by a plurality of proteic factors that work together to eliminate the pathogens that infect the body, after their recognition by antibodies. The complement plays a main role in the defensive mechanism against microbes, being involved in both, specific immunity and in non-specific immunity. Suppression of microorganisms, removal of immune complexes, induction and intensification of antibody responses constitute the main biological functions of the complement.

Micronutrients and oligosaccharides can perform important functions as prebiotic substances during the growth and development of the newborn. Prebiotic substances can be defined as non-digestible food ingredients that influence the body in a beneficial way due to the selective stimulation of the growth and/or activity of one bacterium or of a limited number of bacteria in the colon, which can improve the health of the host.

The concentration of micronutrients between colostrum and natural milk (2 months after birth) is affected by variations, including a reduction in the content of iron, zinc, potassium and sodium and an increase in the content of calcium and phosphorus, probably in order to meet the nutritional requirements of the newborn. The range of micronutrients is affected by maternal age, by the weight increase of the mother during pregnancy, by the interval between pregnancies, by the gestational age of the newborn and by birth weight.

Oligosaccharides and glycoconjugates are some of the most important bioactive components of milk. Their main role seems to be to provide protection against pathogens by acting as competitive inhibitors for binding sites on the epithelial surfaces of the intestine. It has also been proven that some of these components act as growth promoters for the beneficial microflora of the colon. The maximum concentration of these molecules is present in colostrum.

The active factors described are among the main components that make maternal milk different from formula and thus influence in a positive manner the health of the newborn. For example, formula does not contain cytokines (in particular IFN γ and TNF a), growth factors (TGF βΐ and IGF- 1 ), immunoglobulins or complement factors and it therefore limits the development of the immune system and the correct execution of the biological and growth functions of newborns.

Lactoferrin is present in artificial formulations based on bovine milk modified commercially in much lower quantities than the average concentration in human milk; formula based on soy used for children who suffer from lactose intolerance does not contain it. At the same time, bovine milk is not suitable for human newborns, because the relative concentrations of the active factors and of the other serum proteins are significantly different between human milk and bovine milk.

Only bovine oligosaccharides and glycoconjugates can be used advantageously in milk-based products as bioactive components for human nutrition, because their chemical structure is similar to that of the prebiotic substances contained in human milk.

In summary, it is evident that natural human milk offers the best nutritional value for newborns.

The active factors described previously are present in and can be obtained from many biological fluids and organs of mammals such as blood, milk, saliva, liver, or can be obtained by genetic engineering. However, each one of these sources has safety and/or cost problems that cannot be managed yet; on the other hand, colostrum, besides having no safety problems, is the richest source of active factors and therefore the most advantageous.

Colostrum is usually produced in quantities that exceed the needs of the newborn. Technical and hygiene problems related to the collection and treatment of bovine colostrum, however, have limited its large-scale use by the dairy and food industry so far. The low coagulation temperatures of colostrum interfere with the pasteurization and the high protein content causes problems in industrial processes.

Many researches have shown that bovine colostrum is easily absorbed by human beings, is up to 40 times richer in immune factors than human colostrum, and that only bovine colostrum contains particular glycoproteins and protease inhibitors that are extremely effective in protecting the active components of colostrum itself against destruction by the digestive enzymes and acids contained in the stomach of adult human beings. Bovine colostrum is therefore safe for consumption by human beings.

There are many products prepared from bovine colostrum (bovine colostrum concentrates) sold in Europe. In most cases, however, the clinical indications are not clear or are not supported by clinical data and the products themselves do not always meet the appropriate qualitative standards for experimental medicine. It is essential that reports on the proprieties of colostrum concentrates provide information on the active components present, including concentration of the active factors. This information is required in order to meet the legal requirements applicable to the evaluation of effectiveness tests.

The general approach for the use of colostrum for human nutrition is to use colostrum collected up to 24 hours after birth; it should be noticed, however, that the composition of colostrum changes rapidly even during the first hours after birth and in particular the concentration of the active factors undergoes an exponential decrease, until it becomes so low that the factors themselves cannot be measured; the concentration would be, at the same time, too low for any biological activity.

Moreover, the use of colostrum encompasses some industrial problems that have not been solved yet. Colostrum contains, among its components, some strong growth factors; it is capable of promoting the growth of human cells in culture 240 times more than any method of culture without causing mutations or damage to the cells. This surprising property makes human utilization more complicated. Colostrum is sterile in the mammary gland, but during collection and storage it comes into contact with bacteria and viruses which, despite care, are present in non-sterile environments. The bacteria and viruses, even in very small quantities in the initial stage, proliferate very quickly during freezing, thawing (rather long steps because of the composition of colostrum) and industrial treatment and reach values that are unacceptable for laws currently in force and are also dangerous for human beings.

The use of blocking agents is not only forbidden in food supplements, but is also devoid of any usefulness because the capacity of proliferation mediated by the active factors exceeds the possibility of blocking them efficiently. The easiest alternative is to irradiate colostrum. The European Union has allowed this treatment in food supplements only for a short time. However, the labeling requirement as irradiated product on the packaging is an unappealing commercial message, even though the product is not actually radioactive but harmless. However, one can suppose that very few mothers would give an irradiated product to their children. Moreover, irradiation with γ rays can be performed with an intensity of 1 to 25 kGy. The colostrum collected in the first hour after birth is very rich in active factors and extremely viscous. The dose for making colostrum sterile is 10 to 25 kGy, depending on the viscosity of the batch, on the bacterial load and on the packaging: the two first factors are occasional variables and therefore it is preferable to work with the maximum quantity of radiation allowed (25 kGy). With 25 kGy, the two cytokines, interferon γ and TNF a, are destroyed; the other active factors are reduced by 80%. Some active factors such as IgG undergo unacceptable modifications in their molecular structure. Moreover, the allergens are maintained and often undergo modifications in their three-dimensional structure that make them even more allergenic. The method is excessively variable, unstable and unsafe, with commercial problems, and therefore not applicable.

The method most commonly used for products that are already marketed entails sterilization of the active factors by means of heat, followed by freeze-drying. The thermal conditions, however, eliminate the possibility of keeping the factors active in the final product: the drastic thermal shock in fact would cause denaturation of the proteins and loss of activity of these factors. In particular, the complement is destroyed at 54°C and as the temperature increases, the other factors are degraded gradually, up to 122°C (temperature applied to formula), when all the factors are destroyed, while casein and lactalbumin preserve their allergenic load. For this reason, the content of active factors is not stated in commercial products obtained after sterilization.

Another problem related to the purification of active factors from bovine colostrum is the necessary preliminary elimination of caseins, which may cause many allergic problems and hinder the separation of less abundant and smaller proteins. Caseins can be removed after their coagulation (precipitation), followed by a filtration, but the conditions required to provide coagulation, particularly a low value of pH, cause the denaturation of many proteins, including the active factors of interest.

For example, patent application WO2008/1 19186 relates to the enrichment of commercial colostrum in TGF β; in accordance with the described method, the step of casein coagulation may be performed with two types of polysaccharides, pectin or carrageenan λ. In the first case, the casein is separated by means of microfiltration (with 0.1 μιη pores) and the resulting serum-colostrum enriched in TGF β is precipitated with acids at pH 5.1 for recovery of the growth factors. It is probable, however, that such an acid value of pH causes denaturation of the structure of TGF β. Moreover, the pores are small enough to block even pathogenic agents (it is thus a sterilizing microfiltration) but can be easily blocked by agglomerates of casein. Once the pores have been blocked, therefore, other proteins, even if they are smaller, cannot pass through the membrane and the resulting permeate is hardly enriched in growth factors. For precipitation with carrageenan λ, vice versa, the serum portion of colostrum must be discarded, because TGF β coprecipitates with the polysaccharide. The casein precipitate enriched with growth factors, however, does not undergo any sterilization treatment for the elimination of pathogenic agents.

Finally, said patent application does not provide any information on the concentration of growth factors in the final product nor on their biological activity.

Patent application WO2007/074135 relates to a nutritional composition which comprises a product derived from colostrum and prebiotic components, which ensures an improvement in the regulation and stimulation of immune system development. Although enriched with serum proteins, the product derived from colostrum still contains caseins, in a ratio with respect to the serum proteins comprised between 50:50 and 80:20. The caseins are partially eliminated by enzyme hydrolysis followed by their separation. The resulting product thereafter undergoes microfiltration. However, little information is given in relation to the method for obtaining the product derived from colostrum and the content of the active factors present therein. The only available quantity, as far as the active factors of interest are concerned, is given for lactoferrin, which may be added optionally to the composition.

It is therefore evident that there is the need, shared also by the regulatory authorities, which acknowledge the superiority of breastfeeding, to make formula safer and more similar to natural human milk. In view of this, to supplement formula with the active factors mentioned above means to be capable of giving to the newborn, to the preterm child and to the growing child a nutrient that is extremely similar to maternal milk.

In particular, there is the need to provide a sterile mixture derived from colostrum that comprises the active factors IgG, IgA, IGF 1, TGF βΐ, and preferably also lactoferrin, transferrin, C4A, IL-2, IFN γ and TNF a, in defined concentrations, which can be used in the production of formula, so as to increase the similarity of the milk thus obtained to maternal milk. There is the further need to provide a nutraceutical formulation that comprises the cited sterile mixture derived from colostrum. Although said nutraceutical formulation may be used in nutrition in general, it is preferable to use it in order to supplement the conventional formula for feeding newborns. Preferably, the cited nutraceutical formulation may exist as liquid or powdered formulation, ready to be added to conventional formula in defined quantities, before reconstitution of milk for children, with water. Disclosure of the invention

An object of the present invention is therefore to provide a mixture of biological factors isolated from colostrum that comprises the active factors IgG, IgA, IGF-1 , TGF βΐ and preferably also lactoferrin, transferrin, C4A, IL-2, IFN γ and TNF a, particularly adapted for human nutrition. In view of this aim, an object of the invention is to provide a mixture of biological factors derived from colostrum that contains said active factors in specific and defined quantities. Another object is to provide a formulation for nutraceutical use that comprises the mixture of biological factors described above. This formulation may be used, for example, as a supplement for formula for children and newborns.

Another object of the invention is to provide a new method for treating colostrum in order to obtain the sterile mixture of biological factors cited above, so that it is suitable for human nutrition, even for people who are allergic to milk proteins. Within this scope, an object is to provide a method for treating colostrum so as to obtain said mixture, in which the biological factors are present in specific and defined quantities.

Finally, another object of the invention is to provide a method that is highly reliable, can be provided rather simply and is economically competitive for treating colostrum, said method being capable of preserving at the same time the presence and the activity of the biological factors contained in colostrum.

This aim and these and other objects that will become better apparent hereinafter are achieved by a method for obtaining a mixture of biological factors isolated from colostrum, comprising the steps of:

a) microdiaflltration of whole colostrum, previously diluted with water or with a saline aqueous solution comprising pharmaceutically acceptable ions so as to yield a volume VI, said microdiaflltration comprising at least one step of filtering the diluted whole colostrum through a cylindrical ceramic membrane with a porosity comprised between 2 and 6 microns, at a temperature comprised between 2°C and 20°C, and at a transmembrane pressure (tmp) comprised between 0.2 bars and 2 bars, yielding a retentate fraction and a permeate fraction;

b) collecting the resulting permeate until its total volume V2 amounts to at least 40% of the volume VI of initial diluted whole colostrum;

c) optional sterilizing filtration of the permeate; and

d) optional removal of the water contained in the permeate.

It is preferable that the total volume V2 of permeate obtained during step b) amounts to at least 50% of the volume VI of the initial diluted whole colostrum.

Ways of carrying out the invention

In a preferred embodiment of the method of the present invention, step a) may be repeated cyclically, subjecting again the retentate obtained in a microdiaflltration cycle to a further microdiaflltration pass; in this case one obtains in each instance a successive retentate fraction and a successive permeate fraction, which is joined to the previously obtained permeate fractions, until the total volume V2, obtained as the sum of the joined fractions of the permeate obtained in step b), amounts to the desired percentage of the volume VI of initial diluted whole colostrum (at least 40% or at least 50% of the volume VI).

More preferably, the microdiaflltration according to step a) of the method can be performed continuously, reconstituting partially or completely the volume of the retentate fraction by continuous addition of water or of a saline aqueous solution comprising ions that are acceptable from a pharmaceutical point of view; the method of continuous microdiafiltration can be performed until the total volume V2 of permeate obtained in step b) amounts to the desired percentage of the volume VI of initial diluted whole colostrum (at least 40% or at least 50% of the volume VI).

As mentioned previously, the concentration of active factors in colostrum decreases exponentially immediately after birth: the colostrum collected during the first hour after birth is therefore the richest in active factors that can be obtained. For this reason, it constitutes the preferred starting material for the present invention. On the other hand, it should be noted that the colostrum of the first hour after birth is more difficult to treat than the subsequent colostrum because of its higher viscosity. The fact that the present invention allows the treatment of colostrum of the first hour and attainment of a sterile mixture of its active factors is therefore an important result.

In particular, as mentioned earlier, the problem of the high viscosity of first-hour colostrum can now be reduced by dilution of the colostrum with an appropriate medium before subjecting it to microdiafiltration.

Preferably, whole colostrum can be diluted with at least 10 parts per volume of water or with a saline aqueous solution comprising ions that are acceptable from a pharmaceutical point of view, yielding the volume designated as VI . By way of example, a saline solution that can be used to dilute colostrum may be constituted by 0.03 moles of KCl and 0.02 moles of

NaCl per liter of water.

In a preferred application of the case described previously, in which the retentate obtained by a microdiafiltration pass is then subjected to further microdiafiltration passes according to step a), it is preferable to reconstitute said retentate with water or with a saline aqueous solution comprising ions that are acceptable from a pharmaceutical point of view, thus reconstituting partially or completely the volume VI of the initially diluted colostrum.

The microdiafiltration according to the present invention makes it possible to remove for the most part from whole colostrum (i.e., without initial skimming) the lipid fraction, the caseins and the lactalbumins, which constitute a very relevant percentage of colostrum proteins and which it is desirable to eliminate from the final product in order to avoid allergy problems. In the method according to the present invention, the combination of the values of the transmembrane pressure, of the dimensions of the pores of the cylindrical ceramic membrane and of the dilution of the colostrum makes it possible to retain the lipid fraction, the caseins and the lactalbumins of the whole colostrum (unskimmed) in the retentate, while the other soluble proteins, the active biological factors in particular, and the oligonutrients pass through the membrane into the permeate. The size of the pores of the membrane is a crucial point of the process, because casein forms a colloid that makes filtration of colostrum impossible on membranes with a lower porosity.

The microdiafiltration technique facilitates the progressive elimination of the lipid fraction, of the caseins and of the lactalbumins, by preventing them from clogging the pores of the membrane. In order to ensure the effectiveness of the filtration process it is in fact important to avoid blocking the pores of the membrane, not only because of the consequent drop in permeation and thus the decrease in process efficiency, but also because of the fact that the active biological factors are adsorbed on the layer of lipids, caseins and lactalbumins retained on the surface of the membrane, creating an unwanted loss of these active substances in the permeate.

Microdiafiltration must be performed at a temperature comprised between 2°C and 20°C; in particular, both the diluted whole colostrum solution that must undergo microdiafiltration and the resulting permeate (or permeates, if successive microdiafiltration steps are provided) must be kept at a temperature comprised within this interval throughout the duration of the process. Cooling is necessary because the high rate of flow and the high pressure value at which the filtration system works produce a heating that might cause thermal degradation of the active biological factors to be preserved, in addition to an unwanted increase in any bacterial load.

At the end of the microdiafiltration, the content of caseins and lactalbumins is reduced substantially with respect to their initial concentration in the whole colostrum, collected for example during the first hour after birth; moreover, other large aggregates are filtered, such as fat globules, at least 90% of which are retained in the filter.

In a preferred embodiment of the present invention, the permeate obtained during step b) undergoes a further step c) of sterilizing filtration, performed with a filter that has a porosity equal to 0.22 microns. This second filtration allows an elimination of all the cells present in the colostrum, including therefore pathogenic microorganisms. In this manner a sterile solution is produced which is safe for human uses. Optionally, before performing the sterilizing filtration, the solution is passed through a prefilter with a nominal porosity of 1.2 - 1.5 microns, for retaining any large molecules and aggregates still present in the solution, avoiding blockage of the small pores of the sterilizing membrane. Essentially, the optional prefiltration is used to extend the life time of the 0.22-micron sterilizing filter.

Advantageously, therefore, this method makes it possible to obtain a mixture of active biological factors isolated from whole colostrum, without the allergenic components and completely sterile from a microbiological point of view. The sterile solution thus obtained can be used as such in different applications. However, the production and handling of sterile solutions, for example introduced in vials, is complicated and laborious from the industrial point of view and scarcely practical for the end user. It is therefore preferable to eliminate the water from the permeate obtained at the end of the microdiafiltration b) or from the permeate obtained at the end of the sterilizing filtration c) by subjecting it to lyophilization.

Lyophilization is performed in a controlled environment, generally at a maximum temperature of 20°C. It is particularly useful also because it is a "gentle process" that eliminates the water at temperatures not exceeding 30°C, without stressing the product and without the consequent risk of denaturing the components.

Lyophilization is thus the more convenient solution, which moreover produces a product (a lyophilized mixture of biological factors isolated from colostrum) that is easier to handle than the sterile solution. The solid that results from the solution filtered in a sterile manner, after lyophilization, has a controlled bacterial load; moreover, a lyophilized compound is "quiescent" from a microbiological point of view, because it is in the powder state in the absence of water. It can thus be stored at room temperature for a long time, can be transported easily without particular precautions and is completely soluble.

Preferred embodiments of the present invention provide for the use of colostrum of human origin or from domestic animals selected from bovines, ovines, equines and goats. Bovine colostrum, for example, offers the advantages of being available in abundant quantities, of containing high concentrations of active factors and of being compatible with the biological functions of the human body, once the allergenic proteins have been eliminated; moreover, colostrum collected from multiparous cows is richer than that of first-calf cows. In order to ensure richness in biological factors in the final product, moreover, it is preferable to collect the colostrum within the first hour after birth.

It should be noted that the method according to the invention offers the advantage of ensuring the attainment of a mixture of biological factors isolated from colostrum in a biologically effective form because it avoids the use of any treatment that may cause denaturation of the proteins and modification of the three-dimensional structure of these factors (such as high temperatures or low values of pH).

Another aspect of the invention relates to a mixture of biological factors isolated from colostrum, which comprises IgG, IgA, IGF-1 and TGF βΐ in the following quantities, indicated per mg of dry product:

• 0.15 - 0.35 mg of IgG;

· 0.01 - 0 .05 mg of IgA;

• 0.5 - 3.0 ng of IGF-1 ;

• 30 - 90 pg of TGF pl .

Preferably, the mixture of biological factors isolated from colostrum may also contain, in addition to the factors cited above, at least one or more of the following factors, selected from the group consisting of: lactoferrin, transferrin, complement factor C4A, interleukin 2 (IL-2), interferon γ (IFN γ) and TNF a, in the following quantities, indicated per mg of dry product:

• 0.5 - 3.5 μg of lactoferrin;

• 0.3 - 1.3 μg of transferrin;

· 5.0 - 15.0 ng of C4A;

• 2.0 - 5.0 pg of IL-2;

• 3.0 - 9.0 pg of IFN y;

• 2.0 - 7.0 pg of TNF .

A content of biological factors comprised within these intervals is such as to ensure beneficial effects for the organism that will consume the mixture. It is important to stress the fact that these values refer to the content of biological factors in their active and functional form; in fact, in order to perform their physiological role in the body, they must not be denatured, because the loss of structure causes a subsequent loss of functionality. Colostrum is the natural source that is richest in the biological factors of interest and, since it is the first food of newborns, is also rich in oligo- elements and prebiotic substances. Preferably, therefore, the mixture of biological factors isolated from colostrum may also comprise calcium (Ca), magnesium (Mg), manganese (Mn), sodium (Na), potassium (K), copper (Cu), zinc (Zn) and iron (Fe).

A further aspect of the invention relates to a nutraceutical formulation that comprises the mixture of biological factors described previously.

Since colostrum is the first food of newborns, a formulation that comprises biological factors usually present therein is particularly useful in supplementing milk for food use.

The present invention relates, therefore, in another aspect, to a supplement for milk for newborns, for follow-on milk or growth milk, or for drinking milk, that comprises the mixture of biological factors described previously.

Lastly, a further aspect of the invention relates to milk for newborns, follow-on milk or growth milk, both in powder form and reconstituted, or drinking milk, with the addition of the mixture of biological factors isolated from colostrum.

In conclusion, it has been shown that the method according to the invention fully achieves the intended aim and objects, because it is capable of providing a mixture of biological factors isolated from colostrum in their active and functional form from the physiological point of view, particularly adapted for human nutrition and, if required, sterile. The invention achieves its aim also because it provides a new method for obtaining a mixture of colostrum factors that is suitable for human consumption even by people who are allergic to milk proteins.

EXAMPLE

In a 500 1 reactor, different cans of bovine colostrum collected during the first hour after birth from multiparous cows, for a final weight of 50 kg, were mixed. The mixture was diluted with 450 1 of a saline solution that had a concentration 0.03 M of KC1 and 0.02 M of NaCl .

The diluted mixture then underwent a microdiafiltration through a membrane with a porosity equal to 4 μιη, with a filtering surface having an extension of about 1 m ² , the parameters of the apparatus being set so that the value of the transmembrane pressure (TMP) in the filtration cartridge is comprised between 0.8 bar and 1.4 bar (specifically, arrangements have been made to maintain it between 0.9 bar and 1.0 bar). At the input of the cartridge there is a coarse-mesh prefilter and an exchanger that allows maintaining the temperature of the product at a temperature value lower than 16°C.

The mixture was microdiafiltered under the conditions described above until a volume of permeate equal to 250 1 was collected. The volume of the retentate was then restored to the initial value of 500 1 by addition of 250 1 of saline solution 0.03 M of KC1 and 0.02 M of NaCl .

The microdiafiltration continued under the same conditions as described above, by addition of the permeate to the one obtained in the first filtration cycle, until another 410 1 of permeate were collected.

The total volume of permeate collected amounted therefore to 660 1. The permeate of the microdiafiltration at this point was concentrated on a nanofiltration membrane by use of a cartridge with a cut-off equal to 400 Da and by application of a transmembrane pressure (TMP) of 14 bars, until a final volume of 150 1 was reached. 25 1 of permeate were then passed through the cartridge again. This concentration pass by nanofiltration is not essential for the success of the process, but allows a considerable increase in productivity of the subsequent lyophilization step.

The concentrated mixture thus obtained, which was still rather turbid, was filtered by means of a filter press loaded with cards with a porosity of 1.5 μπι, yielding an opalescent solution. A further 35 1 of the nanofiltration permeate were passed through this last filtration apparatus. It should be noted that this filtration, too, is not a critical step of the process, but it makes it possible to clear the solution, avoiding the blocking of the 0.5-μηι prefilter used in the subsequent pass.

The final volume of concentrated product thus obtained is 210 1.

The prefiltered product was then loaded into a freeze-dryer by filtering through two apparatuses arranged in series and equipped with a pre-filter with a porosity of 0.5 μηι and a sterilizing filter with a porosity of 0.22 μηι.

The cycle used for lyophilization is shown in the following table:

The content of ten biological factors was then assessed by means of commercially available kits, which determine the quantity of factors that are present in active form.

The measured biological factors and the kits used for their assessments are listed hereinafter:

C4 A I ELISA human C4A kit (BD - OptEIA) The analytical results obtained on the lyophilized product by means of the kits listed above are reported in the following list:

Lactoferrin: 1.952 μg for 1 mg of lyophilized product.

Transferrin: 0.830 g for 1 mg of lyophilized product.

IgA: 0.030 mg for 1 mg of lyophilized product.

IgG: 0.260 mg for 1 mg of lyophilized product.

IGF- 1 : 1.630 ng for 1 mg of lyophilized product.

IL - 2: 3.700 pg for 1 mg of lyophilized product.

IFN γ: 6.160 pg for 1 mg of lyophilized product.

TNF a: 4.600 pg for 1 mg of lyophilized product.

C4A: 10.800 ng for 1 mg of lyophilized product.

TGF βΐ : 64.000 pg for 1 mg of lyophilized product.