Formulation usable as a substitute for breast milk for children with cow's milk protein allergy

Abstract

A hypoallergenic food formulation comprises donkey milk mixed with a plant- derived proteins hydrolysate, fats and carbohydrates. This formulation can be used as a substitute for breast milk for children with cow's milk protein allergy (CM A) and advantageously used as a sole source of nourishment, that is, as a complete formulation, intended for infants and children with CMA.

Description

[0001] “FORMULATION USABLE AS A SUBSTITUTE FOR BREAST MILK FOR CHILDREN WITH COW’S MILK PROTEIN ALLERGY”

[0002] FIELD OF THE INVENTION The embodiments described here concern a formulation which can be used as a substitute for breast milk for children with cow’s milk protein allergy (CMA). In particular, the embodiments concern a food formulation based on donkey milk, more specifically a hypoallergenic food formulation for feeding infants and children. The formulation according to the present description can be advantageously used as a sole source of nourishment, that is, as a complete formulation, intended for infants and children with CMA.

[0003] BACKGROUND OF THE INVENTION

[0004] It is known that cow’s milk protein allergy (CMA) affects about 2-3% of children under 2 years of age (Luyt D et al.; “BSACI guideline for the diagnosis and management of cow’s milk allergy.” Clin Exp Allergy. 20I4;44(5): 642-72) and that in most cases children overcome it between the age of 3 and 5 (Koletzko S et al.; European Society of Pediatric Gastroenterology, Hepatology, and Nutrition. “Diagnostic approach and management of cow’s-milk protein allergy in infants and children: ESPGHAN GI Committee practical guidelines.” J Pediatr Gastroenterol Nutr. 2012 Aug;55(2):221-9.). The immune response triggered by intact cow’s milk proteins can be immunoglobulin E (IgE) mediated, non-IgE- mediated, or mixed. One of the reasons this allergy is triggered in newborns is the early introduction of cow’s milk proteins into the diet when exclusive breastfeeding is no longer possible. Cow’s milk can trigger the allergy in predisposed subjects because its protein fraction has a very different composition from that of breast milk. Historically, beta-lactoglobulins have been considered the major allergen in cow’s milk, since they are not present in human milk. Today, however, scientific evidence shows that the casein fraction contains most of the allergens that cause CMA. At the structural level, the cow’s milk proteins that most frequently trigger an allergic response are alphasi-casein, alphaS2-casein, beta-casein, gammai-casein, gamma?- casein, gammas-casein, kappa-casein (Bos d 8 family of allergens), alphalactalbumin (Bos d 4 allergen), beta-lactalbumin (Bos d 5 allergen), immunoglobulins (Bos d 7), serum bovine albumin (Bos d 6), and lactoferrin (Restani et al.; “Molecular aspects of milk allergens and their role in clinical events”; Anal Bioanal Chem (2009) 395:47-56).

[0005] Symptoms of CMA can be immediate (within two hours of ingestion, mainly related to the IgE-mediated reaction) or delayed (after 48 hours and beyond from ingestion, also related to the non- IgE-mediated reaction). Milk protein allergy can affect the digestive system (for example dysphagia, regurgitation, vomiting), the respiratory system (for example nasal congestion, cough), the skin (for example urticaria, eczema) or cause a generalized reaction (anaphylactic shock) (Koletzko S et al.; European Society of Pediatric Gastroenterology, Hepatology, and Nutrition. “Diagnostic approach and management of cow’s milk protein allergy in infants and children: ESPGHAN GI Committee practical guidelines.” J Pediatr Gastroenterol Nutr. 2012 Aug;55(2):221-9.).

[0006] If CMA is suspected, the allergen is eliminated from the diet. Depending on the type of reaction manifested, one continues with the IgE analysis or with the “oral challenge” (controlled administration of the allergen and measurement of the child’s response). Another test used to evaluate the child’s response is the “skin prick test”: a drop of cow’s milk is deposited on the child’s skin and the diameter of the rash that forms at the site of deposition is measured. The larger the rash, the greater the immunological reaction to the allergen.

[0007] In the event CMA is diagnosed and it is impossible to continue with exclusive breastfeeding, the formula with intact cow’s milk proteins has to be replaced with a specific formulation in which the protein source is hypoallergenic.

[0008] In the event of a mild or moderate reaction, the child can take a formula based on extensively hydrolyzed cow’s milk proteins (generally caseins or beta lactoglobulins), in which the epitopes most likely to trigger the immune response have been “cut” through an enzymatic process. The peptides thus obtained are less likely to cause an immunological response. In the event of a severe reaction, the child can take a formula based on free amino acids, which by definition does not cause an allergic response. In this case, there are no peptides or protein portions that could cause an immune response.

[0009] A hypoallergenic formulation containing amino acids and suitable for the nutrition of infants suffering from cow’s milk protein allergy has to comply with the legislative and nutritional requirements for complete infant formulas. This implies that the formulation has to be nutritionally complete (presence of macro and micronutrients) so that it can be used as the only source of nutrition in the child. In addition, in formulations for children with severe allergic reactions, the protein content is entirely represented by free amino acids, which are safe for use in children with allergy to cow’s milk proteins.

[0010] Formulas based on extensive hydrolysates of cow’s milk proteins and based on amino acids generally have an unpleasant taste with accentuated bitter notes, and children often reject hypoallergenic products because of their taste (Polidori et al.; “Use of Donkey Milk in Cases of Cow’s Milk Protein Allergies”; International Journal of Child Health and Nutrition, 2015, 4, 174-179). In addition, a cow’s milk and derivatives exclusion diet, in addition to the use of hypoallergenic formulas, causes feeding difficulties in many patients suffering from CMA, not only in the first years of life but also in later ages (Maslin et al.; “Cows’ milk exclusion diet during infancy: Is there a long term effect on children’s eating behavior and food preferences?”; Pediatr. Allergy Immunol., 2016,27,141-146).

[0011] The CMA diet can induce macro and micronutrient imbalances that have to be avoided thanks to an adequate formulation of the powdered products (the only source of nutrition in children before weaning and the main liquid component from weaning onward) and a targeted dietary support. Some of the most important formulation aspects of the formulas to be used in the first year of age concern the amount of protein (a high amount of protein is correlated to growth problems and obesity in later stages of the child’s development), the amount and quality of fats (high importance of essential fatty acids and DHA), the presence of lactose in the formulation, the correct enrichment in micronutrients (for example, a deficiency of vitamin D, calcium and phosphorus can have effects on bone mineral density). For this reason, it is essential to formulate hypoallergenic products in accordance with current legislation and with the most recent indications regarding nutrition in the first years of the child’s life. The GNPD database document [online] Mintel “Donkey Milk and Sicilian

[0012] Almonds Drink”, Database accession no. 10876532, describes a beverage consisting of water, donkey milk, Sicilian almonds, rice maltodextrin. Maltodextrins are water-soluble complex carbohydrates. Moreover, as far as the protein part is concerned, the formulation of this known beverage has proteins that essentially derive from donkey milk and probably from almonds. Furthermore, the formulation of this known beverage cannot be a complete food from a nutritional point of view, because it does not have the trace minerals / elements / vitamins part, and because it does not respect the macronutrient levels for formulations for newborns or infants (infant / follow on formula) and present in the reference legislation.

[0013] The legislative requirements relating to the composition of infant and early childhood formulas are set out in European Regulation 2016 / 127. In particular, this regulation states that:

[0014] Art. 1: Infant formulas and follow-on formulas may only be placed on the market if they comply with this Regulation.

[0015] Art. 2: Infant formulas shall comply with the compositional requirements set out in Annex I, taking into account the values for the indispensable and conditionally indispensable amino acids set out in Annex III.

[0016] It should be noted that Annex I of the above regulation defines the macro and micronutrients that have to be present in infant formula.

[0017] The formula of the known document “Donkey Milk and Sicilian Almonds Drink” mentioned above cannot, therefore, be defined as a complete infant formula from a nutritional point of view, since it does not meet the legislative requirements for macronutrients (see table below where the values that do not meet the requirements are highlighted in bold and underlined) and micronutrients (not present in the formulation described in this known document).

[0018] 100 ml 100 kcal 100 ml 100 kcal

[0019] Energy kcal 54 100 60 / 70

[0020] Fats g 1.7 3.15 4.4-6.0

[0021] Carbohydrates g 9 16.67 9.0-14.0 Proteins g 0.7 1.30 1.86-2.8

[0022] It is known to use donkey milk in the case of newborns or infants with cow’s milk allergies or intolerances. In general, it is known that donkey milk, like other types of milk of animal origin, is characterized by a good organoleptic profile that does not need to be mitigated. Donkey milk, in fact, is sweet thanks to its natural lactose content, therefore donkey milk itself has no sensory notes that need masking.

[0023] Document EP 2 653 040 Bl discloses a fortified donkey milk-based food product for preterm infants and / or term infants with allergies or intolerance to cow’s milk proteins, and a method for its preparation. This document aims to exploit the nutritional and hypoallergenic properties of donkey milk and compensate for the deficiency of the lipid and caloric portion, which would make it unsuitable for feeding a newborn, by supplementing it with the addition of a mixture of triglycerides derived from vegetable oils in which 35-55% of the total content of palmitic acid (Cl 6:0) is esterified in the 0 (sn-2) position in the glycerol molecule, vegetable oils rich in essential fatty acids (omega-3 and omega-6) and long-chain polyunsaturated fatty acids (LC-PUFA), in order to achieve a composition closer to that of breast milk.

[0024] Document US 2022 / 312799 Al discloses a non-dairy beverage containing rice and pea proteins, such as to provide an appropriate nutritional profile and a suitable taste. This document describes that hydrolyzed rice proteins are characterized by very unpleasant organoleptic properties and have, for example, bitter, hydrolyzed, burnt and astringent flavors. This problem is even more relevant when such plant proteins are used as the sole protein source in the composition. This known document proposes the use of pea proteins to improve the sensory properties of a non-dairy beverage comprising hydrolyzed rice proteins and optionally free amino acids. This document explains that a mixture of pea and hydrolyzed rice proteins, optionally with free amino acids, has a more balanced flavor and taste profile than pea or rice proteins alone. In fact, hydrolyzed rice proteins and pea proteins both give a strong and characteristic flavor and taste to the non-dairy beverages to which they are added. This document proposes a combination of pea proteins with hydrolyzed rice proteins, with the pea protein present from a minimum of 10% up to 60% by weight based on the total weight of the protein component, having a positive impact on the flavor and therefore on the acceptance of the non-dairy beverage, significantly mitigating the unwanted sensory attributes originating from the individual protein sources. It is the specific combination of hydrolyzed rice and pea proteins that reduces the unpleasant taste given by plant proteins to the nondairy beverage disclosed in this known document. The formulation described in this known document does not contain, in any case, donkey milk or any other milk of animal origin characterized by a good organoleptic profile that does not need to be mitigated. There is therefore the need to perfect a food formulation that can overcome at least one of the disadvantages of the state of the art.

[0025] In particular, one purpose of the present invention is to provide a food formulation that overcomes the disadvantages of hypoallergenic formulations, both those based on extensive hydrolysates of cow’s milk proteins, and also those based on amino acids.

[0026] Another purpose is to provide a food formulation that is suitable for feeding infants and children, in particular usable as a complete formulation intended for infants and children suffering from CMA.

[0027] The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

[0028] SUMMARY OF THE INVENTION

[0029] The present invention is set forth and characterized in the independent claim.

[0030] The dependent claims describe other characteristics of the present invention or variants to the main inventive idea.

[0031] In accordance with the above purposes, the embodiments concern a nutritionally complete hypoallergenic food formulation comprising donkey milk mixed with a hydrolysate of plant-derived proteins.

[0032] As used here, the term “complete formulation” or “nutritionally complete formulation” refers to the fact that the formulation is usable as a sole source of nutrition because it contains all the macro and micronutrients necessary for the nutrition of the consumer, in this case children, newborns or infants.

[0033] In possible embodiments, the formulation comprises fats and carbohydrates. The formulation described here can be a breast milk substitute for children with cow’s milk protein allergy.

[0034] In possible embodiments, the fats comprise saturated fatty acids and unsaturated fatty acids, in particular monounsaturated fatty acids and polyunsaturated fatty acids.

[0035] In possible embodiments, the fats comprise one or more plant-derived oils, in particular palm kernel / palm heart, sunflower or rapeseed oil.

[0036] In possible embodiments, the food formulation described here can be in powder, concentrated or semi-solid form, or ready-to-eat liquid form. In possible embodiments, the donkey milk can be in powder or liquid form.

[0037] In possible embodiments, the plant protein hydrolysate can be a partial hydrolysate or an extensive hydrolysate.

[0038] In possible embodiments, the plant-derived proteins can be rice proteins.

[0039] In possible embodiments, the donkey milk, expressed in relation to its dry matter, is present from 15% to 57% on the dry weight of the formulation.

[0040] In possible embodiments, the liquid donkey milk is present from 20% to 75% by weight of the formulation, in the event it is liquid.

[0041] In possible embodiments, the formulation contains fats from 21.8% to 29.9% on the dry weight of the formulation. In possible embodiments, the formulation contains carbohydrates from 51.9% to 64.1% on the weight of the formulation expressed in dry matter.

[0042] In possible embodiments, the formulation contains proteins from 9.9% to 14.5% on the weight of the formulation expressed in dry matter.

[0043] In possible embodiments, the protein part of the formulation provides at least 20% w / w of proteins derived from whole donkey milk and up to approximately

[0044] 80% w / w of proteins derived from the plant-derived proteins hydrolysate, for example rice-protein.

[0045] In possible embodiments, the formulation contains one or more of either minerals, trace elements, vitamins, oligonucleotides, fibers, probiotics and essential amino acids.

[0046] According to one aspect, the formulation described here is intended to feed infants and children suffering from CMA, and it is a nutritionally complete formulation. It can be in powder, concentrated or semi-solid form, or ready-to-eat liquid form, or in powder form which is reconstituted by dilution with water or a water-based liquid.

[0047] In some embodiments, which can be combined with all the embodiments described here, there is provided a nutritionally complete hypoallergenic food formulation for newborns or infants comprising fats from 21.8% to 29.9% on the dry weight of the formulation, carbohydrates from 51.9% to 64.1% on the weight of the formulation expressed in dry matter, proteins from 9.9% to 14.5% on the weight of the formulation expressed in dry matter, one or more minerals, trace elements, vitamins, oligonucleotides, fibers, probiotics and essential amino acids, the formulation comprising donkey milk mixed with a plant-derived proteins hydrolysate, wherein the protein part of the formulation provides at least 20% w / w of proteins derived from donkey milk and up to approximately 80% w / w of proteins derived from the protein hydrolysate.

[0048] According to another aspect, there is provided a ready-to-eat liquid food product which comprises, or is based on, the food formulation. This liquid food product can be produced directly in liquid form, or be obtained by reconstituting a powdered food formulation as described here with water or a water-based liquid.

[0049] It is understood that elements and characteristics of one embodiment can be conveniently incorporated into other embodiments without further clarifications. DESCRIPTION OF SOME EMBODIMENTS

[0050] We will now refer in detail to the possible embodiments of the invention. We must clarify that the phraseology and terminology used here is for the purposes of providing non-limiting examples.

[0051] Unless otherwise defined, all the technical and scientific terms used here and hereafter have the same meaning as commonly understood by a person with ordinary experience in the field of the art to which the present invention belongs.

[0052] Even if materials and substances similar or equivalent to those described here can be used in practice and in the trials of the present invention, the materials and substances are described hereafter as an example. In the event of conflict, the present application shall prevail, including its definitions. The materials and examples have a purely illustrative purpose and shall not be understood restrictively.

[0053] Moreover, all percentages, fractions and ratios between the different components shall be understood as fractions by weight (w / w) with respect to the total composition, even if not explicitly indicated, unless otherwise indicated. All percentage ranges indicated here are given with the provision that the sum with respect the overall composition is 100%, unless otherwise indicated. All ranges reported here shall be understood to include the extremes, including those that report a range “between” two values, unless otherwise indicated.

[0054] The present description also includes the ranges that derive from uniting or overlapping two or more ranges described, unless otherwise indicated.

[0055] The present description also includes the ranges that can derive from the combination of two or more values taken at different points, unless otherwise indicated.

[0056] To the extent that the publications referenced here provide a definition for a term that conflicts with the definition implicitly or explicitly employed in the present description, the definition employed in the present description shall prevail.

[0057] The embodiments described here concern a food formulation based on donkey milk.

[0058] The formulation described here can be used as a breast milk substitute for infants with cow’s milk protein allergy (CM A). The formulation described here can be provided in powder, concentrated, liquid or semi-solid form.

[0059] In possible implementations, the donkey milk used can be powdered or liquid.

[0060] In particular, the formulation described here comprises, or consists of, donkey milk, for example in powder or liquid form, mixed with a plant-derived proteins hydrolysate, such as for example, but not limited to, rice proteins.

[0061] The protein hydrolysate can be partial or extensive hydrolysate. Generally, a partial hydrolysate is understood as a hydrolysate with a degree of hydrolysis comprised between 8 and 14% and with at least 50% of the peptides comprised between 500 and 5000 Da. With regard to one of the two main components of the formulation described here, that is, donkey milk, whether powdered or liquid, it is noted that the donkey milk has, advantageously, a protein profile that, unlike cow’s milk, is more similar to human milk in terms of protein quantity and of the serum’s caseins / proteins ratio. This is due to the fact that the caseins present in donkey milk are lower than in cow’s milk. The hypoallergenicity of the protein fraction of donkey milk can be explained both by the similarities with breast milk proteins and also by the differences with cow’s milk proteins (Bertino et al.; “Detailed proteomic analysis on DM: Insight into its hypoallergenicity”; Frontiers in Bioscience E2, January 1,

[0062] 2010, 526-536). Having analyzed six different protein species identified in donkey milk, an explanation of hypoallergenicity is given for each below:

[0063] - Donkey milk alphas i -casein, one of the major components of the casein fraction, can be present both in the phosphorylated form (the same post-translational modification present in cow’s milk) and also in the glycosylated form (post- translational modification not present in cow’s or breast milk). The exact sequence of the protein is not yet known, but it is assumed to be similar to horse’s alphas i- casein that shares a good similarity (42.9%) with the corresponding breast milk protein; - AlphaS2-casein is a very small component of donkey milk. This protein is absent in breast milk while it represents about 10% of the casein fraction of cow’s milk and is one of its major allergens;

[0064] - Beta-casein is the predominant form of caseins of donkey milk, similar to breast milk. The major site of antibody recognition of bovine beta-casein lies between amino acids 17 and 21. This sequence is different in donkey milk;

[0065] - Kappa-casein is present in very low amounts in donkey milk, similar to breast milk. In cow’s milk, kappa-casein is one of the minor allergens;

[0066] - Alpha-lactalbumin is a protein found in donkey milk, breast milk and cow’s milk. It is considered a minor allergen in cow’s milk, and the specificity of binding to the antibodies of some allergic subjects lies mainly in the characteristics of the region comprised between Lysine 5 and Tyrosine 18. The comparison of this region of the human protein and of the donkey protein shows 100% similarity;

[0067] - Beta lactoglobulins are the major component of the proteins of donkey milk serum and appear to share a high similarity in sequence and structure with human glycodelin. This similarity is not shared by bovine beta lactoglobulins.

[0068] The use of donkey milk for beneficial purposes for health has been known since ancient times (Mansueto P. et al.; “Ass’s milk in allergy to cow’s milk protein: a review”; Acta Medica Mediterranea, 2013, 29: 153). In recent decades, donkey milk has generated more and more interest among allergists thanks to its nutritional and hypoallergenic characteristics, and since the ‘90s it has been successfully used in some clinical trials and case reports involving patients suffering from CMA and multiple food allergies (Polidori et al.; “Use of donkey milk in Cases of Cow’s Milk Protein Allergies”; International Journal of Child Health and Nutrition, 2015, 4, 174-179). The main limitations of these studies remain the fact that they were not designed according to the AAP guidelines (AAP guidelines, Pediatrics 2000; 106; 346 Committee on Nutrition Hypoallergenic Infant Formulas - AAP, 2000), that they were conducted on small groups of patients and that the products used for the treatment were donkey milk as such or incomplete formulations containing donkey milk, therefore products that could not be used as the only source of nutrition in the first year of life.

[0069] In addition, the Applicant has experimentally found that donkey milk contains, in addition to the casein and beta-lactoglobulin fraction, several other proteins, such as serum albumin, lactoferrin, lysozyme, immunoglobulins. These proteins give donkey milk antimicrobial properties (mainly due to the lysozyme and lactoferrin) useful for reducing the incidence of gastrointestinal infections in children with CMA.

[0070] Donkey milk contains a high amount of polyunsaturated fats and has a low omega-6 / omega-3 fat ratio, both of which are very important characteristics for the treatment of CMA (Mansueto et al.; “Ass’s milk in allergy to cow’s milk protein: a review”; Acta Medica Mediterranea, 2013, 29: 153). Despite this, due to the low total amount of fat, donkey milk as such cannot be used as the only source of nutrition in the first year of a child’s life.

[0071] Donkey milk, like breast milk, is very rich in lactose, which makes it sweet and palatable. In addition, the high lactose content promotes the proliferation of intestinal flora, inhibits the growth of harmful bacteria, promotes the intestinal absorption of calcium, phosphorus and other minerals, increasing bone mineralization in children who take donkey milk (Ling Li et al.; “The nutritional ingredients and antioxidant activity of donkey milk and donkey milk powder”; Food Sci Biotechnol, 2018, 27(2):393-400).

[0072] Donkey milk is considered a food with high digestibility, mainly due to the reduced amount of caseins. To demonstrate this, in vitro studies have been conducted which have demonstrated a rapid digestibility of the casein fraction in conditions similar to gastric ones, and a good digestibility of the betalactoglobulins in conditions similar to intestinal ones (Marietta et al.; “Donkey milk proteins; digestibility and nutritional significance”; Milk Proteins - From structure to biological properties and health aspects; 2016).

[0073] In relation to the other main component of the formulation described here, the plant-derived proteins hydrolysate, it should be noted that these proteins are advantageously chosen among plant-derived proteins that are not considered allergens, and that can therefore be used for the production of hypoallergenic formulations. For example, if these proteins are rice, the raw materials obtained from rice are used for the production of food for newborns and children, especially during the weaning phase. Rice proteins are not counted among allergens and are also used, in their hydrolyzed version, for the production of hypoallergenic formulas for children with CMA, highlighting an increasing trend toward the use of plant-derived proteins that can be beneficial to human health (Dupont et al.;

[0074] “Hydrolyzed rice protein-based formulas, a vegetal alternative in cow’s milk allergy”; Nutrients 2020, 12, 2654).

[0075] The Applicant has found that the content of essential amino acids in many plant proteins is lower than in animal-derived proteins. For example, in the case of rice proteins, lysine is deficient. In addition, the digestibility of plant proteins (70-90%, 93% for rice proteins) is lower than the digestibility of animal proteins (90-99%). To compensate for these deficiencies, known hydrolyzed rice protein-based formulas generally have a slightly higher amount of protein than hydrolyzed cow’s milk-based formulas and have essential amino acids added to them. Thanks to these measures, children with CMA who use hydrolyzed rice protein-based hypoallergenic formulas have normal growth (Dupont et al.; “Hydrolyzed rice protein-based formulas, a vegetal alternative in cow’s milk allergy”; Nutrients 2020, 12, 2654).

[0076] Hydrolyzed rice protein-based hypoallergenic formulas have been shown to have a good degree of acceptability (Dupont et al.; “Hydrolyzed rice protein-based formulas, a vegetal alternative in cow’s milk allergy”; Nutrients 2020, 12, 2654).

[0077] Advantageously, therefore, the food formulation described here is a hypoallergenic formulation that overcomes the disadvantages of hypoallergenic formulations, both based on extensive hydrolysates of cow’s milk proteins, and also based on amino acids, as well as based on the use of donkey milk as such.

[0078] In addition, the hypoallergenic food formulation described here is suitable for feeding infants and young children, in particular it can be used as a complete formulation intended for infants and children suffering from CMA.

[0079] In some embodiments, the formulation described here comprises fats, for example a mixture of vegetable fats (e.g. palm kemel / heart of palm, sunflower, rapeseed).

[0080] In particular, in possible embodiments, the aforementioned formulation contains fats from 21.8 % to 29.9 % on the weight of the formulation expressed in dry matter.

[0081] In some embodiments, the aforementioned fats comprise saturated fatty acids and unsaturated fatty acids, in particular monounsaturated fatty acids and polyunsaturated fatty acids.

[0082] For example, the formulation described here comprises long-chain polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA) and docosahexaenoic acid (DHA).

[0083] In some embodiments, the formulation described here comprises carbohydrates; these may derive in part from the donkey milk present in the formulation.

[0084] In particular, in possible embodiments, the aforementioned formulation contains carbohydrates from 51.9 % to 64.1 % on the weight of the formulation expressed in dry matter.

[0085] In some embodiments, the formulation described here can be fortified with one or more of either minerals, trace elements, vitamins, oligonucleotides, prebiotic fibers, probiotics and essential amino acids.

[0086] In some embodiments, the formulation described here contains proteins from 9.9% to 14.5% on the weight of the formulation expressed in dry matter.

[0087] The method for producing the formulation described here provides to mix the ingredients provided. Packaging is usually done in special containers, such as jars and bottles for example.

[0088] The formulation described here can be a powdered, concentrated, semi-solid, or ready-to-eat liquid formulation, and can be used as the sole source of nutrition, that is, as a complete formulation. Possibly, if the formulation is in powder form, it is used after reconstitution with water or a water-based liquid. For example, a specific recommended dilution is 13 g of powder + 90 ml of water.

[0089] The nutritional profile of the formulation described here responds to the most recent guidelines in the field of infant nutrition, and complies with current European legislation in terms of composition of infant formulas (EU Regulation 2016 / 127). Furthermore, the fact that the formulation described here contains at least a part of the protein fraction in the form of intact proteins with high biological value (source of essential amino acids) is of high nutritional importance.

[0090] Donkey milk, in powder or liquid form, and the plant-derived proteins hydrolysate present in the example formulation described here represent peculiar aspects of the present invention. In particular, the donkey milk, having hypoallergenic characteristics, can for example be provided in powder form in an amount equal to 20% on the dry weight of the formulation and in liquid form in an amount equal to 25%, in order to achieve a primary effect on the palatability and on nutritional aspects of the formulation. In possible implementations, the dry donkey milk can range from 15% to 57%, or more specifically from 17% to 30% on the dry weight of the formulation. In possible implementations, the liquid donkey milk can range from 20% to 75%, or more specifically from 22% to 39% on the weight of the formulation. In addition, the plant-derived proteins, for example rice proteins, that have been selected here represent a hypoallergenic source of plant protein that can be used in the context of CMA, which can minimize the risk of response in treated children, unlike hydrolysates of animal origin or other plant sources.

[0091] Furthermore, in the event that the hydrolysate is partial, the mild hydrolysis profile by means of which this partial hydrolysate of plant-derived proteins, such as rice proteins, is obtained has a positive effect on the taste of the formulation. The Applicant has found that a partial hydrolysate of plant-derived proteins, for example rice proteins, in itself gives the raw material a better organoleptic profile; in fact, the Applicant has found that partial, that is non-extensive, hydrolysis results in longer peptides, which correspond to a better taste. Nothing prevents one from being able to use other types of plant hydrolysates as an alternative or in combination with rice proteins, provided that when combined with donkey milk they guarantee good nutritional and organoleptic aspects. One example can be partial or extensive hydrolyzed soy proteins. The latest ESPGHAN guidelines clearly state that it is possible to use hypoallergenic formulas based on soy protein in children with cow’s milk protein allergies (Vandenplas Y et al 2024 - An ESPGHAN). Some examples with peculiar characteristics of hypoallergenicity, palatability and nutritional composition according to the present invention are described in detail below.

[0092] In particular, a peculiar aspect in terms of hypoallergenicity lies in the characteristics of the protein part of the formulation according to the embodiments described here: 20% w / w of the proteins can derive from whole donkey milk, while up to about 80% w / w of the proteins can derive from the plant-derived proteins hydrolysate. A minimal part of the protein share (around 1%) is given by essential free amino acids that can be added to the formulation in order to reach the minimum legislative requirement. Both the donkey milk as well as the hydrolyzed plant-derived proteins, for example rice proteins, are chosen so as to be hypoallergenic sources that, for the first time, are combined here in a formulation for the treatment of CMA.

[0093] In order to demonstrate its hypoallergenicity, the formulation described here was tested in vitro by the Applicant using a cellular model that mimics the immune response of children suffering from CMA. This test showed that the formulation described here with donkey milk and plant-derived proteins hydrolysate, for example rice proteins, does not induce the response of the engineered cells, therefore it was promising for the following steps of the clinical study with experimental purposes planned by the Applicant. For the first time, a nutritionally complete formulation with donkey milk was subjected to a multicenter clinical study based on AAP guidelines (AAP, 2000). Previous studies in the literature have only considered “case reports” or studies conducted on very particular patients (preterm infants, very serious cases) and have involved the use of donkey milk only, or of formulations that are not complete from a nutritional point of view. The experimental clinical study of the formulation according to the present description is a prospective randomized, double-blind, study with the control group aimed at assessing the hypoallergenicity and acceptability of the formulation.

[0094] In some embodiments, the hydrolyzed plant-derived proteins used in the formulation according to the present disclosure can be, for example, partially hydrolyzed, that is, they may have a low degree of hydrolysis and, consequently, are only slightly bitter. This, combined with the sweetness of naturally lactose-rich donkey milk, allows to have a better and more balanced organoleptic profile compared to standard hypoallergenic formulations. It has been experimentally assessed that an amount of powdered donkey milk in a range between 15% and 57% on the dry weight, in particular between 17% and 30% on the dry weight, for example equal to 20% on the dry weight of the formulation is sufficient to mitigate the bitter notes given by the hydrolyzed plant-derived proteins and to optimize the palatability of the formulation described here. The same result is achieved by adding an amount of liquid donkey milk to the formulation in a range between 20% and 75% on the weight of the formulation, if it is liquid, in particular from 22% to 39%, for example equal to 25% on the weight of the formulation.

[0095] According to one possible example embodiment, the formulation described here, whether it is in powder form reconstituted with water or produced directly in liquid form, has a protein content of 1.6 g / 100 ml, lower than hydrolyzed rice protein-based formulas reconstituted with water available on the market (see Table 1 below) and in line with the most recent nutritional indications of protein content reduction in infant formulas.

[0096] Table 1

[0097] Per 100 ml

[0098] Protein source g Proteins / 100 kcal of product

[0099] Intact breast Breast milk 0.9- 1.2 milk proteins

[0100] Formulation 1 / 3 donkey milk, according to the 2 / 3 Hydrolyzed 1.6 2.4 present invention rice proteins

[0101] Risolac Hydrolyzed rice

[0102] 2.1 3.1

[0103] (Plasmon)* proteins

[0104] Hydrolyzed rice

[0105] Novalac Riz* 1.8 2.6 proteins

[0106] *Data obtained from Dupont et al.; “Hydrolyzed rice protein-based formulas, a vegetal alternative in cow’s milk allergy”; Nutrients 2020, 12, 2654 The reduction of the proteins in the formulation described here is advantageously made possible by the presence of at least 20% of the protein share deriving from the donkey milk, which has a greater degree of digestibility and compensates for the limits of the hydrolyzed plant-derived proteins, for example rice proteins. Furthermore, the formulation described here brings a balanced profile of essential amino acids that exceeds the minimum requirement defined in European Regulation 2016 / 127 by at least 10%, and which is given by the sum of the essential amino acids of the plant-derived proteins hydrolysate, for example rice proteins, of the donkey milk and, in minimal part, by any added free amino acids.

[0107] The following Table 2 shows a comparison between an aminogram of the present formulation and an aminogram indicating the minimum essential amino acids that have to be present in order to meet the regulatory requirements for formulations for children (European Regulation 2016 / 127 Annex III).

[0108] Table 2 grams / 100 Kcal

[0109] Formulation European Regulation 2015 / 127 aminogram Annex III B

[0110] L- Alanine 0.14

[0111] L- Arginine 0.19 0.069

[0112] L-Aspartic Acid 0.25

[0113] L-Cysteine 0.04 0.024

[0114] L-Glutamic

[0115] 0.47

[0116] Acid

[0117] L-Glutamine 0.00

[0118] Glycine 0.09

[0119] L-Histidine 0.06 0.045

[0120] L-Isoleucine 0.09 0.072

[0121] L-Proline 0.13 L-Serine 0.14

[0122] L-Phenylalanine 0.12 0.029

[0123] The presence of the intact donkey milk proteins in the formulation described here has countless positive effects on the body, for example it stimulates the development of the immune system and promotes the optimal absorption of nutrients, and could lead to an improvement in the nutritional and growth status of children who take the formulation described here.

[0124] As disclosed above, in order to obtain a nutritionally complete formulation, it is possible to use ingredients capable of completing the profile of the donkey milk and of the hydrolyzed proteins.

[0125] In possible embodiments, the aforementioned formulation contains fats from 21.8 % to 29.9 % on the dry weight of the formulation. In particular, according to possible embodiments, plant-derived oils (for example palm kemel / palm heart, sunflower, rapeseed) can be added to the formulation in order to reach the optimal amount of fats and a balanced profile of saturated, monounsaturated and polyunsaturated fats suitable for the nutritional needs of infants. This blend of oils brings, among others, linoleic acid and alpha linolenic acid, which are considered essential fatty acids.

[0126] In addition, Schizochytrium sp. microalgae oil and oil extracted from the Mortierella alpina mushroom can be added, which allow to carry DHA and ARA, respectively, for children’s optimal cognitive development. Furthermore, the formulation can contain carbohydrates, for example in the form of lactose and maltodextrins. In particular, in possible embodiments, the aforementioned formulation contains carbohydrates from 51.9 % to 64.1 % on the weight of the formulation expressed in dry matter. For example, lactose is contained in quantities of 6.4 g / 100 kcal, of which 45% is derived from the donkey milk contained in the formulation itself. The amount of lactose greater than 4.5 g / 100 kcal and of intact proteins allows to improve the absorption of calcium present in the formulation. In addition, the formulation can have all the micronutrients necessary for the correct growth of patients, even in conjunction with a diet free of cow’s milk and dairy products.

[0127] Below is an example of the nutritional profile of the formulation described here, on 100 ml:

[0128] - energy: 66 kcal

[0129] - proteins: 1.6 g

[0130] - fats: 3.5 g

[0131] - carbohydrates: 6.9 g

[0132] This nutritional profile is in line with the European guidelines regarding complete foods used as formulations for newborns or infants to be used as complete products, that is, as the only source of nutrition.

[0133] The calorie content of complete formulations for infants and young children is calculated on the ready-to-eat product. If the product is powdered, the energy value is calculated by considering the product reconstituted with water according to the manufacturer’s preparation instructions. This is defined in Article 2 of Regulation 2016 / 127 concerning the requirements for the composition of infant formulas. The values set out in Annexes I and II to this Regulation apply to ready-to-eat infant formulas and follow-on formulas, marketed as such or after preparation in accordance with the manufacturer’s instructions. Only the addition of water is required for such preparation.

[0134] The formulation in accordance with the embodiments described here resolves the limits of the hypoallergenic formulas based on plant hydrolysates that have a higher protein content than the formulation described here, and it is not possible to reduce it, as is required by the most recent nutritional guidelines, since plant proteins are low in some essential amino acids for which there are specific requirements to be met. In the formulation described here, the protein content given by the donkey milk covers at least 20% of the amount of protein and allows, together with the possible addition of individual amino acids, to make up for the deficiencies of the rice protein hydrolysate, allowing to meet the new recommendations relating to the lowering of the protein content of formulations for newborns or infants.

[0135] EXAMPLES An example of a recipe of the formulation, expressed on 100 g of powder, is shown below in Table 3.

[0136] Table 3 g / 100 g powder

[0137] Powdered donkey milk 20

[0138] Hydrolyzed rice proteins 10.85

[0139] Free essential ammino acids 0.15

[0140] Powder containing carbohydrates, fats, ARA, DHA and minerals

[0141] Table 4 below shows an example of nutritional composition for 100 g of powder and for 100 ml of ready-to-eat liquid product, which can be obtained by reconstituting the powdered food formulation described here with water, or made directly liquid on the basis of the food formulation. The complete formulation described here also meets these requirements in accordance with EC Directive (2016), EFSA (2014).

[0142] Table 4

[0143] Per 100 g of Per 100 ml of powder product

[0144] Energy kcal 504 66

[0145] Proteins g 12.3 1.60

[0146] L-Alanine g 0.69 0.09

[0147] L-Arginine g 0.96 0.12

[0148] L- Aspartic Acid g 1.26 0.16

[0149] L-Cystine g 0.20 0.03

[0150] L-Glutamic Acid g 2.35 0.30 Glycine g 0.48 0.06

[0151] L-Histidine g 0.28 0.04

[0152] L-Isoleucine g 0.47 0.06

[0153] L-Leucine g 1.00 0.13

[0154] L-Lysine g 0.67 0.09

[0155] L-Methionine g 0.24 0.03

[0156] L-Proline g 0.65 0.08

[0157] L-Serine g 0.72 0.09

[0158] L-Threonine g 0.47 0.06

[0159] L-Tryptophan g 0.20 0.03

[0160] L-Tyrosine g 0.52 0.07

[0161] L-Valine g 0.69 0.09

[0162] L-Phenylalanine g 0.63 0.08

[0163] Carbohydrates g 53.0 6.9

[0164] Of which sugars g 34.6 4.5

[0165] Of which lactose g 32.5 4.2

[0166] Of which g

[0167] 17.2 2.2 maltodextrins

[0168] Fats g 26.8 3.5 Of which saturated g 10.9 1.4

[0169] Of which g

[0170] 9.9 1.3 monounsaturated

[0171] Of which g

[0172] 5.8 0.8 polyunsaturated

[0173] Linoleic acid (n6) g 4.9 0.6

[0174] Alpha-linolenic acid g

[0175] 0.50 0.07

[0176] (n3)

[0177] Arachidonic acid mg

[0178] 100 13.0

[0179] ARA (n6)

[0180] Docosahexaenoic mg

[0181] 115 15.0 acid DHA (n3)

[0182] Sodium mg 216.828 28.188

[0183] Potassium mg 730.260 94.934

[0184] Calcium mg 622.700 80.951

[0185] Phosphorus mg 449.800 58.474

[0186] Magnesium mg 72.800 9.464

[0187] Chlorine mg 308.710 40.132

[0188] Iron mg 4.590 0.597

[0189] Zinc mg 4.850 0.631

[0190] Copper mg 0.51 1 0.066 Manganese mg 0.245 0.032 L-Carnitine mg 12.000 1.560

[0191] Choline mg 188.900 24.557

[0192] Taurine mg 40.000 5.200

[0193] Inositol mg 80.000 10.400

[0194] Adenosine 5'- mg monophosphate 5.213 0.678

[0195] (AMP)

[0196] Cytidine 5'- mg monophosphate 8.688 1.129

[0197] (CMP)

[0198] Guanosine 5'- mg monophosphate 1.738 0.226

[0199] (GMP)

[0200] Inosine 5'- mg monophosphate 3.475 0.452

[0201] (IMP)

[0202] Uridine 5'- mg monophosphate 6.082 0.791

[0203] (UMP)

[0204] An example of the energy distribution of the macronutrients in the formulation is shown below in Table 5. The complete formulation described here also meets these energy requirements in accordance with EC Directive (2016), EFSA (2014).

[0205] Table 5

[0206] % on g / 100 energy kcal total Proteins 2.4 10 %

[0207] EXPERIMENTAL DATA

[0208] Objective

[0209] The Applicant conducted a clinical study to demonstrate the hypoallergenicity of a complete formulation according to the present description, containing donkey milk and plant proteins hydrolysate, for example rice proteins, in children with a proven IgE-mediated allergy to cow’s milk proteins.

[0210] The aim was to determine whether a complete formulation containing donkey milk and plant proteins hydrolysate, for example rice proteins, such as the one described for example in Tables 3 and 4 as above, is well tolerated by children affected by cow’s milk protein allergy.

[0211] The safety and tolerability of the formulation were proven following the guidelines of the American Academy of Pediatrics (AAP) (AAP, 2000). In accordance with these guidelines, it was necessary to conduct a double-blind clinical trial with a control group in which the complete formulation containing donkey milk and plant proteins hydrolysate, for example rice proteins, according to the present description was administered to children with allergy to cow’s milk proteins. In this experimental setting, demonstrating safety requires that no allergic reactions are recorded in 90% of children with a 95% confidence interval, while demonstrating tolerability requires that children are able to take a certain amount of the formulation during a 7-day period (7-day open challenge).

[0212] Study design

[0213] The study design was: multicenter, randomized, double-blind, with control group (placebo).

[0214] Infants and children under the age of 10 with proven allergy to cow’s milk proteins were recruited from 4 centers in Europe (3 in Italy and 1 in Spain) between September 2020 and November 2023.

[0215] The subjects performed a double-blind, placebo-controlled food challenge (DBPCFC) to the complete formulation containing donkey milk and rice proteins hydrolysate (test formula) and to the formulation containing amino acids (control formula) following a cross-over modality. Patients received an incremental amount of test formula and control formula (first a small taste followed by dosages of 1 ml, 5 ml, 20 ml, 40 ml, 80 ml, 120 ml at 20 min intervals). The food challenge was considered passed if the subjects were able to take a minimum of 100 mL of formulations.

[0216] The subjects who successfully passed the food challenge, both with the test formula as well as with the control formula, then continued with a one-week 7-day open challenge with only the complete formulation containing donkey milk and plant proteins hydrolysate, for example rice proteins, to verify its tolerability, the degree of satisfaction and to confirm the absence of delayed reactions. Parents of patients were instructed to administer a minimum of 120 ml / day to children up to 2 years of age and a minimum of 240 ml / day to children over 2 years of age over a period of one week (minimum 7 days). A consumption of 100 ml a day, every day for a period of 7 days was considered adequate to verify the tolerability of the formulation.

[0217] An evaluation on a scale of 1 to 10 was completed by the parents regarding the degree of the child’s satisfaction with the complete formulation containing donkey milk and plant proteins hydrolysate, for example rice proteins, (<5 = below average satisfaction, 5 = average satisfaction, >5 = above average satisfaction).

[0218] Test formula and control formula

[0219] The test formula used was: complete formula containing donkey milk and rice protein hydrolysate, as described in Tables 3 and 4 above.

[0220] The control formula used was: complete formula for the nutritional management of children with cow’s milk protein allergy containing free amino acids as the only protein source.

[0221] Both products meet the macro and micronutrient requirements for infant formula (up to one year of age) and follow-on formula (from one year of age onward) defined by European Regulation 2016 127.

[0222] Statistical analysis

[0223] The safety of the test product (complete formula containing donkey milk and plant proteins hydrolysate, for example rice proteins) was verified by statistical analysis, using a non-inferiority test for a proportion, assuming a binomial distribution. For the one-week (7-day) open food challenge, descriptive statistics were used to summarize the consumption of the formulation and any clinical symptoms.

[0224] Results

[0225] Between September 2020 and November 2023, 39 children were recruited at 4 sites in Europe (3 in Italy and 1 in Spain).

[0226] Fig.l attached is a schematic representation of child recruitment. CM=cow’s milk; OFC=oral food challenge; FAS=full analysis set; PPS=per protocol set.

[0227] As shown in fig. 1, of the 39 children recruited, 36 had a proven diagnosis of allergy to cow’s milk proteins, while 3 children were excluded because they had a negative result during the initial confirmatory oral food challenge phase. The 36 children enrolled were randomized to either the test group or the control group in order to carry out the DBPCFC: 19 took the test formula first and then the control, 17 took the control first and then the test formula. 29 children completed the DBPCFC for both products and continued with the 7-day open food challenge. All children were following a cow’s milk protein exclusion diet before entering the study.

[0228] Primary Objective: Safety

[0229] None of the children had an allergic reaction to the complete formula containing donkey milk and plant proteins hydrolysate, for example rice proteins (test formula), which can therefore be defined as hypoallergenic according to the AAP criteria (AAP, 2000).

[0230] Secondary Objective: Tolerability

[0231] A total of 29 children completed the 7-day open food challenge at home. The median dose of product consumed during this trial was 180 ml / day (interquartile range 120-150 ml / day). A minimum consumption of 100 ml / day was considered adequate when consumed daily for the 7-day food challenge. The degree of satisfaction reported by parents was higher than the average (>5) for 17 (58.6%) children. Of the 29 children who completed the entire study, 14 (48.3%) children continued to consume the product even after the end of the study, for a period ranging from 3 months to 1 year. No serious adverse events were recorded during the entire study period.

[0232] Discussion

[0233] These data demonstrate that the complete formula containing donkey milk and plant proteins hydrolysate, for example rice proteins, in accordance with the present description, as described for example in Tables 3 and 4 above, is hypoallergenic and safe for children with cow’s milk protein allergy. In addition, the results clearly highlight the fact that the formulation in accordance with the present description has a good organoleptic profile, since about 59% of parents judged the formulation to be above average for a hypoallergenic formula. The degree of acceptability of the formulation is further confirmed by the fact that 48% of the children who completed the study continued to take the formulation beyond the end of the study, for a period ranging from 3 months to 1 year. In conclusion, the hypoallergenicity of the complete formulation containing donkey milk and plant proteins hydrolysate, for example rice proteins, in accordance with the present description has been confirmed by DBPCFC testing in children with cow’s milk protein allergy according to the guidelines for hypoallergenic formulas (AAP, 2000). This study provides evidence that the complete formulation containing donkey milk and plant proteins hydrolysate, for example rice proteins, can be prescribed for the treatment of infants and children suffering from cow’s milk protein allergy. The study also demonstrates that this new formulation has a high satisfaction rating, indicated by the parents, which was confirmed by the high percentage of children who continued to take the product after the study had ended.

[0234] It is clear that modifications and / or additions of ingredients or components may be made to the food formulation as described heretofore, without thereby departing from the field and scope of the present invention, as defined by the claims.

[0235] It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art will be able to achieve other equivalent forms of liquid food product, having the characteristics as set forth m the claims and hence all coming within the field of protection defined thereby.

Claims

CLAIMS1. Nutritionally complete hypoallergenic food formulation for newborns or infants comprising fats from 21.8% to 29.9% on the dry weight of the formulation, carbohydrates from 51.9% to 64.1% on the weight of the formulation expressed in dry matter, proteins from 9.9% to 14.5% on the weight of the formulation expressed in dry matter, one or more minerals, trace elements, vitamins, oligonucleotides, fibers, probiotics and essential amino acids, said formulation comprising donkey milk mixed with a plant-derived proteins hydrolysate, wherein the protein part of said formulation provides at least 20% w / w of proteins derived from donkey milk and up to approximately 80% w / w of proteins derived from the protein hydrolysate.

2. Formulation as in claim 1, wherein said fats comprise saturated fatty acids and unsaturated fatty acids, in particular monounsaturated fatty acids and polyunsaturated fatty acids.

3. Formulation as in claim 1 or 2, wherein said fats comprise one or more plant- derived oils, in particular palm kernel / palm heart, sunflower or rapeseed oil.

4. Formulation as in any claim hereinbefore, wherein said donkey milk, expressed with respect to its dry matter, is present from 15% to 57% by weight referring to the dry matter of the formulation.

5. Food formulation as in any claim hereinbefore, wherein the donkey milk is in powder or liquid form.

6. Food formulation as in any claim hereinbefore, wherein said protein hydrolysate is a partial hydrolysate.

7. Food formulation as in any previous claim from 1 to 5, wherein said protein hydrolysate is an extensive hydrolysate.

8. Food formulation as in any claim hereinbefore, wherein said plant-derived proteins are, or comprise, rice proteins.

9. Food formulation as in any claim hereinbefore, wherein said plant-derived proteins are, or comprise, soy proteins.

10. Formulation as in any claim hereinbefore, for feeding infants and children, in particular as a complete formulation intended for infants and children suffering from CM A.

11. Formulation as in any claim hereinbefore usable as breast milk substitute forchildren with cow’s milk protein allergy.

12. Liquid food product comprising a formulation as in any claim hereinbefore reconstituted by dilution with water or a water-based liquid.

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