RU2815764C2 - Complexes of proteins with high isoelectric point with casein - Google Patents

Abstract

FIELD: biotechnology; food industry.

SUBSTANCE: disclosed is a method of producing complexes of a protein having an isoelectric point of at least 7 and selected from lactoferrin, lactadherin and lysozyme, with casein. Method includes the following stages: a. providing a protein solution having an isoelectric point of at least 7, and casein at a pH in range of 6.5 to 7.5, in which the solution provided in step a) contains a protein having an isoelectric point of at least 7, and casein in weight ratio of more than 1:10 and less than 10:1; b. linear increase in the temperature of the solution provided at step a) from the denaturation temperature of the protein having an isoelectric point of at least 7 to a temperature in range of 85 to 95 °C; c. holding the solution of step b) at temperature of 85 to 95 °C for at least 30 seconds; and d. linear reduction of temperature of the solution of step c) from 85–95 °C to temperature below 5 °C for a period of at least 30 minutes; e. holding the solution of step d) at temperature below 5 °C for at least 15 minutes. Disclosed is a protein complex having an isoelectric point of at least 7 s β-casein; food product; use of the complex for the production of a medicinal agent; use of the product for the manufacture of a medicinal agent.

EFFECT: invention enables to obtain complexes which are more stable than complexes of the previous level of technology, namely, to heating, pH modifications, mechanical stress and presence of surfactants.

18 cl, 1 tbl, 14 dwg, 6 ex

Description

Scope of the invention

The present invention provides a method for preparing complexes of proteins with a high isoelectric point with casein, including a certain sequence of stages of heating, cooling and aging. In addition, complexes obtained in this way and their use, in particular their use in therapeutic methods, are proposed.

Prerequisites for creating the invention

Several bioactive proteins, such as lactoferrin, lysozyme or lactadherin, have been identified as having beneficial effects on health, and therefore attempts have been made to use such proteins in nutritional products or pharmaceutical products. However, such proteins are very sensitive to aggressive processing, such as high temperature, and the incorporation of such proteins into industrial products has proven difficult. Therefore, there is a need to develop stabilized forms of such proteins.

For example, lactoferrin is a component of human breast milk. It is also present in foods such as infant formula, but in significantly lower concentrations than in human breast milk. In this regard, supplementation of infant formula with lactoferrin obtained from cattle has been previously described. Bovine lactoferrin (bLf) is an iron-binding glycoprotein of approximately 77 kDa and consists of a single polypeptide chain of approximately 700 amino acids. Human lactoferrin is an 80 kDa glycoprotein that exhibits high affinity for iron. The sequence homology between human Lf and bovine Lf is about 70%, and their three-dimensional (3D) structure is very similar but not identical.

Lf is a highly cationic protein with a high isoelectric point (pI) of approximately 8.9. At neutral pH (~pH 7), Lf is known to bind to other anionic proteins such as β-lactoglobulin, casein and albumin. For this reason, when lactoferrin is added during wet mixing of infant formula, the proteins will be denatured by unfolding and refolding by temperature and pH, resulting in protein instability and increased viscosity. This phenomenon makes it very difficult to add lactoferrin in a wet state (i.e. before drying) to a product such as infant formula.

Thus, it has been described adding lactoferrin to products, such as infant formula, in solid form by dry blending after preparing the powder base of the product. However, this method is problematic because lactoferrin added in solid form must be sterile. To achieve the required level of sterility, lactoferrin must be subjected to sterilization techniques. Most sterilization techniques involve the use of high temperature, which can lead to denaturation of lactoferrin. Other sterilization techniques, such as membrane filtration, are available, but they can be expensive and require specialized equipment. In addition, special and precise aseptic dispensing equipment is required to add sterile lactoferrin to the final product.

Thus, to address these issues, it would be highly desirable to develop alternative forms of lactoferrin that would allow lactoferrin to be added along with other infant formula ingredients to wet formula and subjected to aseptic processing or spray drying of the infant formula composition.

As an alternative to addition in native form, addition of lactoferrin to foods in the form of complexes has been described. WO2012/045801 describes coacervates of complexes containing lactoferrin and at least one other protein with an isoelectric point below pH 7.0, which can be used to slow the enzymatic breakdown of protein and to improve metabolic reactions, inflammatory bowel response, satiety and food intake in person. However, this document does not address the issue of stabilizing lactoferrin during processing of a product such as infant formula, and in particular does not indicate that the coacervates described will be stable enough to be added to wet formula during the production of infant formula.

Oppositely charged proteins in solution (which are colloids) at certain pH, ionic strength, and stoichiometry spontaneously interact to form complexes. Such complexes may remain soluble or cause separation of liquid phases in solution. In the latter case, coacervates of complexes are formed. “The more concentrated phase in the colloidal component is the coacervate and the other phase is the equilibrium solution” (IUPAC, 1997). Both phases remain in equilibrium and are therefore considered incompatible. Both soluble complexes and coacervates are very sensitive to the properties of the environment and are essentially reversible by changes in pH, ionic strength or temperature, and therefore can be destroyed during food processing. Such reversibility of protein complexes is described in the literature. For example, Anema et al. Coacervates of lysozyme and β-casein; Journal of Colloid and Interface Science 398 (2013): 255–261 report that complexes of lactoferrin or lysozyme and β-casein are reversible, namely by changes in pH and ionic strength (e.g., addition of NaCl).

It has been described how to increase the stability of coacervate complexes through the use of cross-linking agents. However, cross-linking agents would not be advisable to be added to products for sensitive consumers, such as infants and young children, where the ingredients that can be used are limited by very strict regulations.

In WO2012/045801, coacervates of complexes of lactoferrin and a protein having an isoelectric point below 7 are induced by pH. However, complexes such as those described herein are not stable enough to withstand harsh processing conditions such as high temperature, making them unsuitable for processing with other ingredients of a product such as infant formula.

Moreover, Anema et al. Co-acervates of lactoferrin and caseins; Soft Matter 8 (2012): 4471–4478, the formation of coacervates of lactoferrin and caseins, namely β-casein, induced by adjusting the pH to 6.55 at different temperatures was investigated. However, this document does not address at all the issue of reversibility of the resulting coacervates, which may be damaged when subjected to aggressive processing such as sterilization or drying.

In the work of Li Quanyang et al. Formation of lactoferrin/sodium caseinate complexes and their adsorption behavior at the air/water interface; Food Chemistry, 232 (2017); 697–703, a method is described that involves heating followed by instantaneous cooling of a solution of sodium caseinate and lactoferrin. The disadvantage of the method described in this document is that it is not suitable for the formation of complexes with micellar casein. It is desirable to provide a method that also allows the formation of complexes with micellar casein.

The work of Pan et al. Self-assembly of β-casein and lysozyme; Journal of Colloid and Interface Science, 316(2) (2007), 405–412, describes a method for forming β-casein and lysozyme complexes involving adjusting the pH to a suitable range of 4.0 to 6.0 or 9.0 to 12 ,0 followed by heating at 80 °C for 30 minutes and cooling. This method results in the formation of nanoparticles that are unstable at pH 3.0 and 12.0 and aggregate in the presence of 0.15 M NaCl. Only grafting of dextran onto β-casein via the Maillard reaction allowed the nanoparticles to be stabilized at neutral pH and saline. It would be desirable to provide a method for producing more stable complexes that can withstand such conditions without adding an additional component, such as dextran, to the complex. This is especially important in infant formula, where it is desirable to keep the list of ingredients as short as possible, and regulatory restrictions are very strict.

Lactoferrin and/or other sensitive proteins, such as lysozymes, are preferably provided in a form that provides them with sufficient resistance to harsh processing conditions, such as heat sterilization or drying, thereby avoiding the need for alternative sterilization and aseptic dosing methods that complicate the production of products with sensitive proteins similar to lactoferrin and increasing its cost. Additionally, it is desirable to be able to form complexes with all types of casein, including micellar casein.

An advantage of the present invention is that it solves the above problems.

Summary of the invention

In a first aspect, the invention provides a method for preparing complexes of a protein having an isoelectric point of at least 7 with casein, comprising the steps of:

a. providing a solution of protein having an isoelectric point of at least 7 and casein at a pH in the range of 6.5 to 7.5;

b. a linear increase in temperature from the denaturation temperature of a protein having an isoelectric point of 7 or more to a temperature in the range of 85 to 95 °C;

c. keeping the solution at a temperature of 85 to 95 °C for at least 30 seconds; And

d. a linear decrease in temperature from 85–95 °C to a temperature below 5 °C over a period of at least 30 minutes;

e. keeping the solution at a temperature below 5 °C for at least 15 minutes.

In a second aspect, the invention provides complexes of a protein having an isoelectric point of at least 7 with casein, produced or produced by the method of the invention.

In a third aspect, the invention provides a product containing the complexes of the invention.

In a fourth aspect, the invention provides a method for producing the product of the invention.

In a fifth aspect, the invention provides complexes of the invention for use in therapy.

In a fifth aspect, the invention relates to complexes of the invention for use in a method

a) preventing, reducing and/or treating infections;

b) modulating, stimulating and/or maintaining the immune response;

c) stimulating and/or maintaining the growth of beneficial intestinal flora;

d) stimulation and/or maintenance of cognitive function;

f) stimulating and/or maintaining healthy growth of an infant or young child; or

f) stimulating softer stools in the infant.

Brief description of graphic materials

Fig. 1. Image of coacervates obtained in example 1 using confocal laser scanning microscopy (CLSM). The photographic image was obtained by mapping the sample slices along the Z axis using 63 objective lenses. Coacervates have the form of homogeneous spherical particles with a porous structure.

Fig. 2. A: CLSM image of coacervates obtained in example 1 after centrifugation. Image of a bead after centrifugation, obtained using 20 objective lenses. B: Image of the coacervate solution after centrifugation. The precipitate of coacervates is clearly visible.

Fig. 3. CLSM image of the coacervates obtained in Example 1 before (A) and after (B) cooling for 24 hours, using 20 objective lenses. Coacervates are clearly visible in both images, although the coacervates are smaller in size after cooling overnight.

Fig. 4. CLSM image of the composition obtained in example 2 (comparative), using 20 objective lenses. No coacervates are visible in the supernatant (A) or sediment (B).

Fig. 5. CLSM image of the composition obtained in example 3 (comparative), using 20 objective lenses. No coacervates are visible in the supernatant (A) or sediment (B).

Fig. 6. CLSM image of the composition obtained in example 4 (comparative), using 20 objective lenses. No coacervates are visible in the supernatant (A) or sediment (B).

Fig. 7. Light microscopy image of the coacervates from Example 5 after dilution of the coacervates in sodium dodecyl sulfate (SDS). The coacervates are still visible as homogeneous spherical particles. Coacervates are enlarged 10 times.

Fig. 8. Light microscopy image of the coacervates from Example 5 after they have been subjected to high speed centrifugation. The coacervates are still visible as homogeneous spherical particles. Coacervates are enlarged 10 times.

Fig. 9. Light microscopy image of coacervates from Example 5 after dilution with water and pH adjustment to 3.5. The coacervates are still visible as homogeneous spherical particles. Coacervates are enlarged 10 times.

Fig. 10. Light microscopy image of coacervates from Example 5 after dilution with water and pH adjustment to 3.5 and subsequent heating at 70°C for 15 minutes. The coacervates are still visible as homogeneous spherical particles. Coacervates are enlarged 10 times.

Fig. 11. Light microscopy image of the coacervates from Example 5 after dilution with water and pH adjustment to 11. The coacervates are still visible as uniform spherical particles. Coacervates are enlarged 10 times.

Fig. 12. Light microscopy image of coacervates from Example 5 after dilution with water and pH adjustment to 10 and subsequent heating at 70°C for 15 minutes. The coacervates are still visible as homogeneous spherical particles. Coacervates are enlarged 10 times.

Fig. 13. Light microscopy image of coacervates from Example 5 after dilution with 100 mM NaCl. The coacervates are still visible as homogeneous spherical particles. Coacervates are magnified 10-fold (A) and 20-fold (B), respectively.

Fig. 14. Light microscopy image of coacervates from Example 6. Coacervates are visible as homogeneous spherical particles. In the larger image, the coacervates are magnified 10x, and in the smaller image they are presented at 4x magnification.

Detailed Description of the Invention

Definitions

As used herein, the following terms have the definitions below.

The term "infant" means a child under 12 months of age.

The expression "young child" means a child between one and seven years of age.

The expression “nutritional composition” means a composition that is fed to a subject. The nutritional composition is typically for oral or intravenous administration and typically includes a lipid or fat source and a protein source.

In a specific embodiment, the composition of the present invention is a hypoallergenic nutritional composition. The expression “hypoallergenic nutritional composition” means a nutritional composition that has a low likelihood of causing allergic reactions.

In a specific embodiment, the composition of the present invention is an “artificial nutritional composition.” The expression “artificial nutritional composition” means a formula produced by chemical and/or biological means that may be chemically identical to the naturally occurring formula found in mammalian milk (i.e., the artificial composition is not breast milk).

The expression "infant formula" as used in this document refers to a food product which is specifically intended for consumption by infants during the first months of life and which in itself meets the nutritional needs of this category of persons (Article 2(c) of European Commission Directive 91/ 321/EEC 2006/141/EC of 22 December 2006 on infant and complementary formulas). It also refers to a nutritional composition intended for use by infants as defined in the Codex Alimentarius (Codex STAN 72-1981) and Infant Specialties (including the article Food for Special Medical Purpose). The expression "infant formula" covers both "infant formula" and "follow-up formula" or "weaning formula".

“Follow-up formula” or “formula for complementary infants” is given starting from the 6th month. It constitutes the main liquid element in the gradually increasing variety of diets for this category of people.

The expression "infant food" means a food product specifically intended for consumption by infants or young children during the first years of life.

The expression “cereal-based infant composition” means a food product specifically intended for consumption by infants or young children during the first years of life.

The term "fortifier" refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula.

The expressions “days/weeks/months/years” and “days/weeks/months/years after birth” can be used interchangeably.

“Mother's milk” refers to the mother's breast milk or colostrum.

An "oligosaccharide" is a saccharide polymer containing a small number (usually three to ten) simple sugars (monosaccharides).

The term “prebiotic” means a substrate selectively utilized by host microorganisms that confer health benefits (Consensus: International Scientific Association for Probiotics and Prebiotics (ISAPP) Joint Opinion on the Definition and Scope of Prebiotics, Nature Reviews Gastroenterology & Hepatology, 2017, 14, 491–502).

The term “probiotic” refers to live microorganisms that, when administered in appropriate quantities, provide a health benefit to the host (World Food Organization (FAO)/World Health Organization (WHO), 2002). Microbial cells are essentially bacteria or yeast.

Unless otherwise stated, all percentages are by weight.

Method for obtaining complexes

The present invention provides a specific method for preparing casein-protein complexes having an isoelectric point of at least 7. The resulting complexes may be soluble complexes or coacervates. Preferably the complexes are coacervates.

The method includes a certain sequence of stages of heating, holding and cooling. The complexes obtained by this particular method are particularly stable and can be maintained despite changes in the environment and can be subjected to harsh conditions such as pH changes, heat, application of mechanical stress, changes in ionic strength and the addition of surfactants. as will be shown in the examples.

Any protein having an isoelectric point of at least 7 can form complexes with casein using the method of the present invention. In a preferred aspect of the invention, the protein has an isoelectric point of at least 8, preferably at least 8.5, most preferably an isoelectric point of 8.9. Such proteins are preferred because the higher the isoelectric point, the more cationic they will be at neutral pH, and therefore the more easily they will interact with caseins at natural pH.

In particular, advantageous proteins having an isoelectric point of at least 7 include lactoferrin, lactadherin and lysozyme. Such proteins are particularly preferred due to the health benefits associated with their consumption. For example, lactoferrin has been described as having a beneficial role in preventing, reducing and/or treating infections, modulating, stimulating and/or maintaining the immune response, stimulating and/or maintaining the growth of beneficial intestinal flora, stimulating and/or maintaining cognitive function, stimulating and/or or maintaining the healthy growth of an infant or young child; or stimulation of soft stools in an infant.

All types of caseins are suitable for the purpose of the present invention. However, β-casein, κ-casein and α-s1-casein are preferred for addition to foods for infants and young children, such as infant formula, infant formula, infant cereals, baby food and formula for children aged 1 year and older. up to 3 years of age as they are naturally present in human breast milk. whereas α-s2-casein is found in cow's milk but not in breast milk.

Casein, and β-casein in particular, has unique properties that allow it to form complexes. In particular, the highly hydrophilic N-terminal domain and highly hydrophobic C-terminal domain of the β-casein sequence allow it to form surfactant-like aggregates. This particular contrast in the sequence of β-casein gives this protein properties particularly useful for the purpose of the present invention. Since one-tenth of the amino acids at the N-terminus of a protein contain one-third of the total charge, and at a pI of 5, a high density of negative charges is formed at the N-terminus at neutral pH, giving this protein the ability to bind strongly to proteins that are cationic at that pH.

In a preferred aspect, the casein is in the form of micellar casein. In nutritional compositions such as infant formula and milk formulas for children aged 1 to 3 years, micellar casein has particular advantages over caseinate. The behavior of micellar casein is different compared to caseinate, such as sodium caseinate. A particular advantage of the method of the present invention is that it allows the formation of complexes with all types of casein, as defined above, including micellar casein.

In a preferred aspect of the invention, the protein having an isoelectric point greater than 7 is lactoferrin and the casein is β-casein.

In the first step of the method of the invention (step (a)), a solution of a protein having an isoelectric point of at least 7 and casein is provided. This solution is prepared in a manner known to one skilled in the art by dissolving both proteins in an aqueous medium. In a preferred aspect of the invention, the solution obtained in step a) contains a protein having an isoelectric point of at least 7, and casein in a weight ratio of greater than 1:10 to less than 10:1. In a preferred aspect of the invention, the weight ratio of the protein having an isoelectric point is at least 7, to casein is at least 1:9, preferably at least 1:8, more preferably at least 1:7, even more preferably at least 1:6 and most preferably at least 1:5 In another preferred aspect of the invention, the weight ratio of protein having an isoelectric point of at least 7 to casein is at most 9:1, preferably at most 8:1, more preferably at least 7:1, even more preferably at least 6:1 and most preferably a maximum of 5:1. For example, the weight ratio of protein having an isoelectric point of at least 7 to casein may advantageously be from 1:9 to 9:1, preferably from 1:8 to 8:1, more preferably from 1 : 7 to 7: 1, even more preferably from 1: 5 to 5: 1. In a particularly preferred embodiment, the weight ratio of protein having an isoelectric point of at least 7 to casein is from 1: 5 to 4: 1, preferably from 1:5 to 3:1, more preferably 1:5 to 2:1, most preferably 1:5 to 1:1.

Preferably, the pH of the protein solution is adjusted to a value in the range of 6.5 to 7.5. Preferably the pH is from 6.5 to 7.3, preferably it is from 6.5 to 7.2, preferably it is from 6.6 to 7.1, more preferably it is from 6.7 to 7.0, more preferably 6.7 to 6.9, most preferably 6.8.

In a preferred aspect of the invention, the solution has a total protein content of from 3 to 10% by weight, based on the total weight of the solution.

In an optional step, before further processing the solution, the proteins can be conditioned at the denaturation temperature of a protein having an isoelectric point of at least 7. This optional conditioning step is performed by heating the protein solution at the denaturation temperature of a protein having an isoelectric point of at least 7, and by maintaining the solution at this temperature for at least 2 minutes, preferably at least 3 minutes. Although this step is not necessary for the formation of complexes, their formation is stimulated by the partial denaturation of a protein having an isoelectric point of at least 7, achieved during this conditioning phase. During this stage, under the influence of the unfolding of the protein structure, various groups become available for interaction with casein. In a preferred aspect, this conditioning step is performed at a temperature of from 60 to 70°C. This temperature range, in particular, is selected for lactoferrin. The most preferred temperature for conditioning lactoferrin is 65°C.

In the second essential step of the method, the solution is heated to a temperature in the range from 85 to 95°C, preferably in the range from 86 to 94°C, more preferably in the range from 87 to 93°C, more preferably in the range from 88 to 92°C, even more preferably in the range of 89 to 91°C and most preferably 90°C. The time required to heat the solution to this temperature is not critical to the successful formation of complexes. In principle, the longer the ramp time, the better the complex formation. Thus, the maximum time for the temperature ramp stage is determined by the best cost-effectiveness ratio. Such optimal ramp time may vary, for example, depending on the volume being processed, and can be determined by a person skilled in the art through basic trial and error. However, optimal results are obtained by increasing the temperature linearly over a period of 1 minute to 1 hour, preferably 1 to 30 minutes.

The solution is then maintained at this temperature for a period of at least 30 seconds, preferably at least 1 minute. It is preferable not to keep the solution at this temperature for more than 10 minutes, more preferably for more than 5 minutes.

After this holding period, the temperature is reduced linearly to below 5°C. To ensure complex formation, it is important to decrease the temperature linearly over a sufficiently long period of time. A period of at least 30 minutes is acceptable. Preferably, the temperature is reduced linearly over a period of 1 hour. Preferably, the temperature is reduced linearly over a period of maximum 10 hours, more preferably over a period of maximum 5 hours, even more preferably over a period of maximum 3 hours. This limitation of the duration of this ramp-down stage within the process is intended to maintain the economic viability of the process. The temperature is reduced linearly to a temperature of 5°C or lower, preferably less than 5°C, more preferably to a temperature of 4°C or lower. However, at this stage of aging, negative temperatures are unsuitable, since the aqueous solution of proteins will freeze and the process of complex formation will stop. In addition, freezing will concentrate the ionic environment around the proteins, leading to undesirable additional denaturation or structural changes of the proteins. Thus, the temperature is preferably reduced linearly to a temperature of from 5 to 1°C, preferably from less than 5°C to 1°C, more preferably from less than 5°C to 2°C, most preferably from 4 to 2°C.

A particular advantage of linear temperature reduction over an extended period of time, as described above, is that it allows the creation of casein complexes in all forms, and in particular also with micellar casein. Slow cooling is essential to break up the micelles and make the proteins available for interaction with a protein having an isoelectric point of at least 7. The flash cooling method is less advantageous than the present method as it is not suitable for complexing micellar casein and will not work. only with caseinate.

Then, after decreasing the temperature linearly to the above values, the solution is maintained at a temperature to allow time for complex formation to complete. The solution is preferably maintained at this temperature for at least 20 minutes, preferably for 30 minutes. This minimum duration is necessary for successful complex formation. There is no specific limitation on the time the solution is kept at these temperatures. As will be demonstrated in the examples, the solution can be stored in the refrigerator for several days. However, in practice, the length of holding time is limited by economic considerations. Excessive duration of the aging stage will make this method economically unviable. Thus, it is preferable to keep the solution at this temperature for a maximum of 1 day, preferably a maximum of 10 hours, more preferably a maximum of 5 hours, most preferably a maximum of 3 hours.

Complexes

The complexes, preferably coacervates, obtained or obtained by the method of the present invention differ from the complexes obtained by the standard method, which consists only of adjusting the pH of the protein solution at a certain temperature, ionic strength and stoichiometric ratio. The successive steps of heating, cooling and holding protein compositions at specific temperatures for specific periods of time, as described above, lead to significant changes in the structure of the complexes, which makes the complexes more stable compared to prior art complexes, namely, heat, pH modifications , mechanical stress and the presence of surfactants. Thus, complexes obtained or obtained by the method in accordance with any of the above-described embodiments are also the subject of the present invention.

Preferred complexes according to the present invention are complexes, preferably coacervates, of lactoferrin and casein, even more preferably complexes of lactoferrin and β-casein, most preferably coacervates of lactoferrin and β-casein.

Products containing complexes

The complexes of the present invention can advantageously be added to all types of products to which it is desirable to add casein and/or a protein having an isoelectric point of at least 7, such as, for example, lactoferrin, lactadherin and/or lysozyme.

The product may be any type of product into which complexes may be included, such as a product in the form of a food or drink product, an animal nutrition product, a nutritional supplement for humans or animals, a pharmaceutical composition or a cosmetic composition. The product may be in solid, liquid or semi-liquid form.

Food and drink products include all products intended for human consumption orally for nutrition and/or pleasure. This may, for example, be a nutritional composition such as a composition for infants and/or young children, for pregnant or lactating women or women wishing to become pregnant, for people who require special nutrition due to an adverse health condition, or for the elderly of people. More preferably, the nutritional composition is selected from infant formula, infant cereal, follow-on formula, infant formula for children 1 to 3 years of age, or dairy products for pregnant and lactating women or women wishing to become pregnant. Other examples of food and drink products include dairy products such as milk-based products or yoghurts, soups, sauces, sweet and savory snacks, powdered beverages and cereal products. In general, dairy products advantageously contain the complexes of the present invention because casein is already naturally present in such ingredients.

The product may also be presented in the form of an animal food or animal nutritional supplement. Preferably the animal is a mammal. Examples of animals include primates (eg, humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like.

Nutritional supplements are typically available in liquid, gel, powder, or tablet or capsule form. Powdered additives generally cover additives intended to be dissolved in water or sprinkled on a food product or added to a beverage. Such supplements are intended to provide the user with additional nutrients and/or health benefits, as well as other beneficial ingredients such as, for example, lactoferrin, lactadherin and/or lysozyme. The additive in accordance with the present invention can be used to provide nutrients and/or effects beneficial to the health of humans as well as animals, as defined above. Nutritional supplements include, for example, powdered supplements for addition to breast milk, for example for premature or low birth weight infants. They also include supplements for women who are pregnant or breastfeeding, or for women wanting to become pregnant.

Pharmaceutical products include, for example, products in the form of drops, syrups, powder, tablets or capsules intended for the treatment and prevention of an adverse medical condition in a subject in need thereof.

Cosmetic compositions are generally intended to have an aesthetic effect on the body and may be for topical or oral administration.

The product of the present invention preferably contains the complexes of the present invention in a therapeutically effective amount.

In a preferred embodiment, the product of the present invention is an infant formula, infant cereal composition, follow-up formula or infant formula for children 1 to 3 years of age containing complexes, preferably coacervates, of lactoferrin and casein, more preferably complexes of lactoferrin and β-casein , most preferably coacervates of lactoferrin and β-casein. In such products, the complexes are preferably present in an amount providing from 0.5 to 10 g, preferably from 1 to 10 g, more preferably from 2 to 7 g of lactoferrin per liter of product.

All types of products in accordance with the invention can be prepared and produced according to the knowledge of a person skilled in the art. An advantage of the complexes of the present invention is that they are stable enough to be processed together with other product ingredients.

For example, when producing a spray-dried product such as infant formula, 1- to 3-year-old infant formula, or a follow-on formula in powder form, the complexes are stable enough to be added to a wet formula and spray-dried with other product ingredients. This is advantageous from a process economic point of view, as it eliminates the need for aseptic dosing and dry mixing of sensitive proteins like lactoferrin, lactadherin and/or lysozyme. Additionally, the introduction of complexes into the wet mixture is advantageous in terms of product structure. In particular, the complexes will mix homogeneously with other ingredients. In contrast, the use of dry blended powders may result in non-uniform products due to differences in the properties of the blended powders, such as differences in density or particle size. Heterogeneity can lead to inaccurate protein dosing.

Thus, a method of producing a product selected from an infant formula, a follow-on formula, or an infant formula for children 1 to 3 years of age in powder form, comprising preparing a product concentrate containing the complexes of the present invention and said spray-dried product concentrate, also is the subject of the present invention. Preparation of the wet mixture and spray drying are carried out according to the general knowledge of one skilled in the art.

In another aspect, the invention provides a method for preparing a product selected from a liquid infant formula, a follow-up formula, or an infant formula for children 1 to 3 years of age, comprising mixing the complexes of the present invention with a liquid product base and aseptically treating the product base containing the complexes. Preparation of the liquid product base and aseptic processing are carried out according to the general knowledge of a person skilled in the art.

First medical use

The advantage of complexes, preferably coacervates, or a product containing complexes, preferably containing coacervates, as described above, is that they can be used in therapy. Thus, the invention also provides such complexes and such products for use in therapy. In a particularly preferred aspect, the invention provides complexes, preferably coacervates of lactoferrin, lactadherin or lysozyme and casein, and products containing such complexes or coacervates, for use in therapy. Even more preferably, such complexes are complexes, preferably coacervates, of lactoferrin and casein, preferably lactoferrin and β-casein.

As used herein, therapy refers to the treatment or prevention of a disease or dysfunction, and also includes prophylactic treatment, ie, the prevention of an adverse medical condition. In this document, therapy also includes the implementation of medical and veterinary therapy.

In other words, the present invention relates to a method of treating a subject, comprising administering to the subject a therapeutically effective amount of a complex or product in accordance with the present invention.

In another embodiment, the invention relates to the use of complexes or product in accordance with the present invention for the production of a medicinal product.

Additional Medical Uses

The present invention provides complexes, preferably coacervates, of lactoferrin and casein or a product containing such complexes as described above, for use in the following specific therapeutic modalities:

a) preventing, reducing and/or treating infections;

b) modulating, stimulating and/or maintaining the immune response;

c) stimulation and/or maintenance of the growth of beneficial intestinal flora;

d) stimulation and/or maintenance of cognitive function;

f) promoting and/or maintaining healthy growth of an infant or young child; or

f) stimulation of soft stools in the infant.

In a preferred embodiment, the invention provides complexes, preferably coacervates, of lactoferrin and β-casein or a product containing such complexes as described above, for use in the following specific therapeutic modalities:

a) preventing, reducing and/or treating infections;

b) modulating, stimulating and/or maintaining the immune response;

c) stimulation and/or maintenance of the growth of beneficial intestinal flora;

d) stimulation and/or maintenance of cognitive function;

f) promoting and/or maintaining healthy growth of an infant or young child; or

f) stimulation of soft stools in the infant.

In a preferred embodiment, the invention provides an infant formula, infant formula or infant formula for children aged 1 to 3 years, containing complexes, preferably coacervates, of lactoferrin and β-casein, as described above, for use in the following specific therapeutic modalities:

a) preventing, reducing and/or treating infections;

b) modulating, stimulating and/or maintaining the immune response;

c) stimulation and/or maintenance of the growth of beneficial intestinal flora;

d) stimulation and/or maintenance of cognitive function;

f) promoting and/or maintaining healthy growth of an infant or young child; or

f) stimulation of soft stools in the infant.

The present invention will now be described in more detail using the following examples.

Example 1: Preparation and detection of complexes according to the present invention

Stock solutions of lactoferrin (source: Westland Milk Products (HY03; 98.9% protein; from Westand) and micellar β-casein (source: Ultranor Beta (Y044; 75% protein; from Kerry) at a protein concentration of 5.5% wt. /wt was prepared and left to solubilize for 2 hours while stirring using a Stuart Stirrer Multiposition SB 162-3 magnetic stirrer at speed 3. Then, to obtain a mixture in a mass ratio of 1: 1 (β-cn: Lf), the corresponding volumes of each The stock solution was weighed and mixed together for 2 minutes using a Pasteur pipette.The pH was then adjusted to 6.8 using 0.1 M HCl/NaOH, and the solution was directly placed into a rheometer (AR2000ex, from TA Instruments) for heat treatment. 28 g (±0.05 g) of the mixture was weighed into the rheometer sample cup.The sample was then subjected to a Statistical Process Control (SPC) heat-cool-hold mode as described below in Table 1.

Table 1. Log of processing using SPC in the heating - cooling - holding mode

1. Conditioning stage Initial temperature: 65.0°C
Normal force: 0
Pre-shear angular velocity: 16.78 (rad/s) for 1 minute
Equilibrium state: 2 minutes 2. Peak hold stage 1 Temperature: 65°C
Duration: 1 minute and 20 seconds
Angular velocity: 16.78 (rad/s) 3. Temperature ramp stage 1 Ramp from 65 to 87°C
Ramp duration: 2 minutes and 4 seconds
Angular velocity: 16.78 (rad/s) 4. Temperature ramp stage 2 Ramp from 87 to 90°C
Ramp duration: 1 minute and 20 seconds
Mechanical impact (Pa): 0.1080 5. Peak hold stage 2 Temperature: 90°C
Duration: 1 minute
Angular velocity: 16.78 (rad/s) 6. Temperature ramp stage 3 Ramp from 90 to 4°C
Ramp duration: 1 hour
Angular velocity: 16.78 (rad/s) 7. Peak hold stage 3 Temperature: 4°C
Duration: 30 minutes
Angular velocity: 16.78 (rad/s)

The resulting solution was analyzed by confocal laser scanning microscopy (CLSM) using a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Heildelberg GmbH, Mannheim, Germany). A small amount of sample was placed on a glass slide. Samples were stained by pipetting 10 μL of Nile green dye (0.001 g/100 mL) onto the surface of the sample. A cover glass was placed on top. 3D images were obtained by mapping slices of the complexes along the z axis. Z-slice maps are obtained by visualizing multiple sample planes, resulting in a 3D image.

CLSM analysis revealed that complexes of β-casein and lactoferrin were formed. In FIG. Figure 1 shows a photographic image obtained by mapping the sample slices along the Z axis using 63 objective lenses. Coacervates form spherical particles of uniform shape. Coacervates are also characterized by a porous structure with many vacuoles, which are indicated in the figure by white arrows.

The sample was then centrifuged at relative centrifugal force (RCF) 8000 for 5 minutes at 20°C. A clear separation can be seen between the sediment layer (coacervate) and the supernatant layer (equilibrium dilution phase), as shown in FIG. 2B. Both the sediment (pellet) and the supernatant were subjected to CLSM analysis as described above. In FIG. Figure 2A shows a CLSM image of a granule with 20 objective lenses. The coacervates are again clearly visible as spherical particles.

The composition was then subjected to 24 hour cooling at 4°C. The sediment layer containing coacervates was analyzed by CLSM before (Figure 3A) and after (Figure 3B) the cooling day. Although both images show distinct and distinctly shaped coacervates, there is a clear difference in size, indicating that the coacervates decrease in size as the time at low temperature increases. This decrease in size is associated with a decrease in the frequency of vacuoles within the coacervate structure.

As will be shown in the following comparative examples, the formation of coacervates is the result of a combination of successive steps of heating, slow cooling and holding at low temperature. Subjecting the β-casein and lactoferrin composition to slow cooling and holding at 4°C alone, as well as heat treatment alone, did not successfully produce coacervates.

Example 2: Lactoferrin and β-casein composition without heating or cooling treatment (comparative)

Stock solutions of lactoferrin (source: Westland Milk Products (HY03; 98.9% protein) and micellar β-casein (source: Ultranor Beta (Y044; 75% protein) at a protein concentration of 5.5% w/w were prepared by dissolving protein in water. A solution of lactoferrin and β-casein in a mass ratio of 1:1 was prepared by mixing the two stock solutions. The pH was then adjusted to 6.8. This solution was conditioned at 65 °C for approximately 3 minutes.

The resulting solution was analyzed using confocal laser scanning microscopy (CLSM). A small amount of sample was placed on a glass slide. Samples were stained by pipetting 10 μL of Nile green dye (0.001 g/100 mL) onto the surface of the sample. A cover glass was placed on top.

Complexes of lactoferrin and β-casein were not formed. In particular, as seen in FIG. 4, no coacervates were found in the sample.

Example 3: Slow cooling of lactoferrin and β-casein composition (comparative)

Stock solutions of lactoferrin (source: Westland Milk Products (HY03; 98.9% protein) and micellar β-casein (source: Ultranor Beta (Y044; 75% protein) at a protein concentration of 5.5% w/w were prepared by dissolving protein in water. A solution of lactoferrin and β-casein in a mass ratio of 1:1 was prepared by mixing the two stock solutions. The pH was then adjusted to 6.8. This solution was conditioned at 65 °C for approximately 3 minutes.

The temperature was then decreased linearly from 65°C to 4°C over 1 hour. The solution was then kept at 4 °C for 30 minutes. The resulting solution was analyzed using confocal laser scanning microscopy (CLSM). A small amount of sample was placed on a glass slide. Samples were stained by pipetting 10 μL of Nile green dye (0.001 g/100 mL) onto the surface of the sample. A cover glass was placed on top.

Complexes of lactoferrin and β-casein were not formed. In particular, as seen in FIG. 5, no coacervates were found in the sample.

Example 4: Heat treatment of lactoferrin and β-casein composition (comparative)

Stock solutions of lactoferrin (source: Westland Milk Products (HY03; 98.9% protein) and micellar β-casein (source: Ultranor Beta (Y044; 75% protein) at a protein concentration of 5.5% w/w were prepared by dissolving protein in water. A solution of lactoferrin and β-casein in a mass ratio of 1:1 was prepared by mixing the two stock solutions. The pH was then adjusted to 6.8. This solution was conditioned at 65°C for approximately 3 minutes.

The temperature was then increased linearly from 65°C to 90°C. Subsequently, the solution was kept at 90 °C for 1 min, and then the temperature was linearly decreased from 90 to 10 °C over 5 min. It was then kept at 10°C for 5 minutes. The resulting solution was analyzed using confocal laser scanning microscopy (CLSM). A small amount of sample was placed on a glass slide. Samples were stained by pipetting 10 μL of Nile green dye (0.001 g/100 mL) onto the surface of the sample. A cover glass was placed on top.

Complexes of lactoferrin and β-casein were not formed. In particular, as seen in FIG. 6, no coacervates were found in the sample.

Example 5: assessing the stability of complexes

The stability of the coacervates prepared as described in Example 1 was assessed by exposing such coacervates to various treatments and then assessing whether the coacervates were still present in light microscope images.

In the first experiment, the coacervate granule obtained in example 1 was diluted in a 0.1% sodium dodecyl sulfate (SDS) solution. In FIG. Figure 7 shows that even after contact with this ionic surfactant, the coacervates remained stable.

In the second experiment, the coacervate granule obtained in example 1 was diluted with 0.02 N. HCl solution and then centrifuged at RCF 20,000 for 20 minutes at 4°C. Despite acid treatment and centrifugation, the coacervates remained stable, as can be seen in Fig. 8, which shows the coacervates present in the granule.

In the third experiment, the stability of the coacervates in an acidic environment was assessed by diluting the coacervate granule obtained in Example 1 in water and adjusting the pH to 3.5. In FIG. Figure 9 shows that coacervates are still present. In FIG. 10 also shows that coacervates are still preserved after subsequent heat treatment at 70°C for 15 minutes at pH 3.5.

In the fourth experiment, the effect of basal pH was examined. The coacervate granule obtained in Example 1 was diluted in water and adjusted to pH 10. In FIG. Figure 11 shows that the coacervates remain stable. In FIG. 12 shows that this also occurs when the coacervates are heated to 70°C for 15 minutes after adjusting the pH.

In the fifth experiment, the effect of ionic strength on the stability of coacervates was assessed by diluting the coacervate granule obtained in Example 1 in 100 mM NaCl. Under these conditions, the coacervates also remained stable, as shown in Fig. 13. In FIG. 13A the coacervates are magnified 10 times, and in FIG. 13B - 20 times.

The results of these experiments indicate that coacervates produced by the method of the present invention are substantially more stable than coacervates formed as described in the prior art literature. Indeed, it has been described that such coacervates of the prior art, which are formed by adjusting the pH of a solution of lactoferrin and β-casein, are unstable, in particular when heated or under the influence of a change in pH. The present example demonstrates that the coacervates of the present invention surprisingly remain stable over a wide pH range and can withstand heat, severe mechanical stress, and do not dissolve in the presence of surfactants.

Example 6: Effect of the mass ratio of lactoferrin and β-casein on the formation of complexes

Example 1 was repeated using as a starting solution a solution of lactoferrin and β-casein in a mass ratio of 1:5 and a solution of lactoferrin and β-casein in a mass ratio of 1:10. Except for changing this ratio, the method was identical to that described in Example 1.

In FIG. 14 shows that coacervates are formed at a mass ratio of lactoferrin to β-casein of 1:5, even though they are smaller in size than those obtained at a mass ratio of 1:1. In FIG. 14, a small photograph shows a 4x magnification of the area indicated by the black arrow.

On the contrary, at a mass ratio of lactoferrin to β-casein of 1:10, no coacervates were formed.

Claims (35)

1. A method for producing complexes of a protein having an isoelectric point of at least 7 and selected from lactoferrin, lactadherin and lysozyme with casein, comprising the steps of: a. providing a solution of a protein having an isoelectric point of at least 7 and casein at a pH ranging from 6.5 to 7.5, wherein the solution provided in step a) contains a protein having an isoelectric point of at least 7 and casein in a mass ratio of more than 1: 10 and less than 10: 1; b. linearly increasing the temperature of the solution provided in step a) from the denaturation temperature of a protein having an isoelectric point of at least 7 to a temperature in the range of 85 to 95°C; c. keeping the solution of step b) at a temperature of from 85 to 95°C for at least 30 seconds; And d. linear reduction in the temperature of the solution of step c) from 85-95°C to a temperature below 5°C over a period of at least 30 minutes, e. keeping the solution of step d) at a temperature below 5°C for at least 15 minutes. 2. The method according to claim 1, which is a method for producing coacervates of a protein having an isoelectric point of at least 7 with casein. 3. The method according to claim 1 or 2, wherein the step of conditioning the solution from step a) at the denaturation temperature of a protein having an isoelectric point of at least 7 is carried out for at least 2 minutes before ramping up the temperature in step b). 4. The method according to any of the preceding claims, wherein the protein has an isoelectric point of at least 8. 5. The method of claim 4, wherein the protein has an isoelectric point of at least 8.5. 6. The method of claim 5, wherein the protein has an isoelectric point of at least 8.9. 7. The method according to any of the preceding claims, wherein the casein is α-casein or β-casein. 8. The method according to any of the preceding claims, in which the solution obtained in step a) contains a protein having an isoelectric point of at least 7, and casein in a mass ratio of from 1: 5 to 5: 1. 9. The method according to any of the preceding claims, wherein the solution obtained in step a) has a pH of 6.6 to 7.4. 10. The method according to any of the preceding paragraphs, in which in step e) the solution of step d) is kept at a temperature of from 2 to 5°C after a linear decrease in temperature. 11. The method of claim 1, wherein the protein having an isoelectric point of at least 7 is lactoferrin. 12. A protein complex having an isoelectric point of at least 7 with β-casein, obtained or obtained by the method according to any one of paragraphs. 1–11. 13. The complex according to claim 12, which is a coacervate. 14. Food product containing the complex according to claim 12 or 13. 15. Use of the complex according to claim 12 or 13 for the production of a medicinal product. 16. Use according to claim 15, where the medicinal product is intended for: a) preventing, reducing and/or treating infections; b) modulating, stimulating and/or maintaining the immune response; c) stimulating and/or maintaining the growth of beneficial intestinal flora; d) stimulation and/or maintenance of cognitive function; f) stimulating and/or maintaining healthy growth of an infant or young child; or f) stimulation of soft stools in the infant. 17. Use of a product containing the complex according to claim 12 or 13 for the production of a medicinal product. 18. Use according to claim 17, where the medicinal product is intended for: a) preventing, reducing and/or treating infections; b) modulating, stimulating and/or maintaining the immune response; c) stimulating and/or maintaining the growth of beneficial intestinal flora; d) stimulation and/or maintenance of cognitive function; f) stimulating and/or maintaining healthy growth of an infant or young child; or f) stimulation of soft stools in the infant.