WO2018115340A1

WO2018115340A1 - Infant formula for cow's milk protein allergic infants

Info

Publication number: WO2018115340A1
Application number: PCT/EP2017/084198
Authority: WO
Inventors: Anne THEVENIER; Giulia MARCHESINI; Susanne SCHUH
Original assignee: Nestec S.A.
Priority date: 2016-12-22
Filing date: 2017-12-21
Publication date: 2018-06-28

Abstract

The present invention provides an infant formula comprising potato protein 5 microparticles, carbohydrate and fat. Also provided is the use of potato protein microparticles in the preparation of an infant formula, and a process for preparing an infant formula.

Description

INFANT FORMULA FOR COW'S MILK PROTEIN ALLERGIC INFANTS

FIELD OF THE INVENTION The present invention relates to processes for preparing infant formulas. In particular, the invention relates to processes for preparing infant formulas that are suitable for infants with cow's milk protein allergy.

BACKGROUND TO THE INVENTION

Human breast milk and breast feeding are considered to be the optimal form of nutrition for healthy infants during the first months of life. However, there is a need for nutritional sources that can be used in addition to breast milk. Furthermore, not all infants can be breast fed and the needs of more vulnerable infants, such as preterm infants, cannot be achieved by their mother's milk, so there is also a need for alternatives to breast milk.

Nutritional compositions that satisfy the nutritional requirements of infants may be used as a substitute for or complement to human breast milk. Preferably, infant formulas should have an acceptable taste, and be hypoallergenic when targeted to infants who are allergic or at risk of allergy.

Infant formulas are typically formulated with cow's milk protein. For example, bovine whey protein and/or casein are often used as the protein source in infant formulas. However, some infants exhibit an allergy to cow's milk proteins, making such formulas unsuitable. Allergies to cow's milk and to infant formulas containing cow's milk protein may be due to the differences between the proteins in cow's milk and those in human milk. The principal recognised cow's milk allergens are alpha-lactalbumin (aLA), beta- lactoglobulin (bLG) and bovine serum albumin (BSA).

To reduce allergenicity, cow's milk proteins may be hydrolysed (e.g. enzymatically) either partially or, in the case of products intended for the management of Cow's Milk Protein Allergy (CMPA), extensively. However, such proteins must be highly processed to provide sufficient hydrolysis to reduce the risk of an allergic reaction. Such processing may be viewed unfavourably with an increasing tendency to provide more natural diets, and a strong hydrolysis process also tends to have a negative impact on taste. In addition, the extensive processing increases the cost of the product formulas.

Alternatives to cow's milk protein may be used in nutritional compositions, for example soy and rice proteins. However, soy-based nutritional compositions are not recommended by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) for infants (0-12 months), because of the risk of a cross allergic response. Rice-base nutritional compositions require the addition of numerous free amino acids to provide the correct amino acid profile for infant formulas, due to the incomplete natural amino acid distribution in rice proteins. This increases cost and may provide the resulting formula with a less palatable taste. Furthermore, rice proteins are generally insoluble and require at least partial hydrolysis for solubilisation.

Infant formulas may be formulated entirely from free amino acids for infants with severe cases of multiple allergies. However, ESPGHAN guidelines indicate that such formulas should not be used as a first line solution in the case of cow's milk protein allergic infants. Furthermore, overprescription of amino acid based formulas adds to the cost burden on national health systems as amino acid based formulas are even more expensive than extensively hydrolysed formulas.

Accordingly, there is a significant need for nutritional compositions for infants that comprise less potential allergens, and preferably which require minimal processing, have good taste and have low cost. In particular, there is a need for infant formulas that are suitable for administration to infants with cow's milk protein allergy. There is also a related need for processes for preparing such infant formulas.

SUMMARY OF INVENTION

The inventors have developed an infant formula based on potato protein, which is naturally absent in the major allergens found in milk and soy. Accordingly, the invention may provide a naturally hypoallergenic infant formula that is suitable for infants with cow's milk protein allergy. In particular, the inventors have developed an improved process for producing an infant formula comprising potato protein, wherein the process utilises potato protein in the form of potato protein microparticles. This enables the processing during production of mixtures comprising an increased Total Solids concentration and provides improved spray drying.

The use of potato protein in an infant formula is advantageous as it has a well balanced amino acid profile, which is closer to that of human milk than rice or soy protein. Accordingly, less addition of free amino acids is required to provide a composition with the required nutritional profile, which renders the resulting product more cost effective and gives it a more palatable taste.

Moreover, as a result of their lower allergen profile, the potato protein components do not require extensive hydrolysis, which provides significant benefits in terms of cost and for the development of the infant, because the intact or slightly hydrolysed proteins facilitate improved gut maturation.

Furthermore, the need for an emulsifier may be reduced or removed, because the potato protein itself may provide any necessary emulsifier properties. In addition, use of potato protein provides for good acceptance, for example in terms of taste and texture of the infant formula.

In one aspect, the invention provides an infant formula comprising potato protein microparticles, carbohydrate and fat.

By "potato protein microparticles" is meant a particle that is obtained by heat-treatment and subsequent homogenisation of non-aggregated potato protein, for example as described below with regard to the process of the invention.

In one embodiment, the infant formula is in the form of a powder. In one embodiment, the infant formula is in the form of a reconstituted infant formula (i.e. a liquid infant formula that has been reconstituted from the powdered form). Preferably, the infant formula is in the form of a powder. In a preferred embodiment, the major source of protein in the infant formula is potato protein and the remaining protein is plant protein. The term "major source of protein is potato protein" means that the largest fraction of the total protein by weight in the infant formula originates from potato protein.

In one embodiment, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, preferably at least about 75%, by weight of the total protein in the infant formula is potato protein.

In one embodiment, 100% by weight of the total protein in the infant formula is potato protein. Thus, in one embodiment, the sole source of protein is potato protein.

In one embodiment, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the potato protein in the infant formula is in the form of potato protein microparticles.

In a preferred embodiment, the protein (in particular, the potato protein) is intact protein. Preferably, the protein has not been subjected to artificial hydrolysis.

In another embodiment, the protein (in particular, the potato protein) is partially hydrolysed protein.

In one embodiment, the infant formula further comprises free amino acids.

In one embodiment, the infant formula does not comprise a further emulsifier. The potato protein may provide sufficient function as an emulsifier.

In one embodiment, the infant formula further comprises lactose. In one embodiment, the infant formula does not comprise lactose.

In one embodiment, the infant formula further comprises probiotics. In one embodiment, the infant formula does not comprise probiotics. In one embodiment, the infant formula further comprises nucleotides. In one embodiment, the infant formula does not comprise nucleotides.

In one embodiment, the infant formula comprises:

(a) 1 .8-3.2 g protein per 100 kcal;

(b) 9-14 g carbohydrate per 100 kcal; and

(c) 4.0-6.0 g lipids per 100 kcal.

In another aspect, the invention provides the use of potato protein microparticles in the preparation of an infant formula. In one embodiment, the potato protein microparticles used in the preparation of the infant formula have a particle size distribution whereby the particle size of 90% of the particles is in the range of from 0.1 to 20 μιτι, preferably 0.1 to 10 μιτι, In a preferred embodiment the potato protein microparticles used in the preparation of the infant formula have a an average particle size of 0.5 to 10 μιτι, preferably from 1 to 10 μιτι, or from 1 to 8 μιτι. The infant formula may be an infant formula as described above with regard to the first aspect of the invention.

In a further aspect, the invention provides a process for preparing an infant formula, comprising the steps of: (a) providing a dispersion of potato protein in water; (b) heating the dispersion to a temperature of between 80 and 90°C for between 10 and 30 minutes and subsequently homogenising the dispersion to form potato protein microparticles; (c) combining the potato protein microparticles with at least one carbohydrate and at least one fat to form a mixture; (d) spray drying the mixture to form an infant formula. Optionally a concentration/evaporation step may be carried out on the mixture prior to spray drying.

In one embodiment, the process comprises the step of adjusting the pH of the dispersion to between 4.5 and 6.5 prior to heating in step (b); preferably to between 5.0 and 6.0; preferably to between 5.2 and 5.6; preferably to about 5.4.

In one embodiment the dispersion is preferably cooled prior to homogenisation, preferably to a temperature below 60°C, or below 40°C, for example to between 20°C and 40°C. In one embodiment, the homogenisation in step (b) of the process is carried out at a pressure of between 50 and 600 bar, for example between 50 and 400 bar. In one embodiment, the potato protein microparticles formed in step (b) of the process have a particle size distribution whereby the particle size of 90% of the of particles is in the range of from of from 0.1 to 20 μιτι, preferably from 0.1 to 10 μιτι. In one embodiment, the potato protein microparticles formed in step (b) of the process have an average particle size of from 0.5 to 10 m

In one embodiment, the process comprises the step of homogenising the mixture prior to spray drying.

In one embodiment, step (b) of the process comprises heating the dispersion to a temperature of between 83 and 85°C; preferably about 85°C.

In one embodiment, the mixture formed in step (c) comprises particles having a particle size distribution whereby the particle size of 90% of the of particles is in the range of from 1 and 20 μιτι and/or an average particle size of from 2 to 7 μιτι prior to spray drying.

In one embodiment, the mixture comprises at least 40% Total Solids prior to spray drying. In another aspect, the invention provides an infant formula obtainable by the process of the invention.

In one embodiment, the infant formula comprises between 2 and 8 wt.% potato protein, preferably between 3 and 5 wt.% potato protein; preferably between 3.5 and 4.5 wt.% potato protein.

In a further aspect, the invention provides a method of feeding an infant comprising administering to the infant the infant formula of the invention. In yet a further aspect, the invention provides an infant formula for use in feeding an infant having cow's milk protein allergy, wherein the infant formula is an infant formula of the invention as defined herein. DESCRIPTION OF THE DRAWINGS

Figure 1

Comparison of essential amino acid levels between potato and rice protein, and FAO 2013 recommendations.

Figure 2

Figure 3

Comparison of histidine levels between potato and rice protein, and Institute of Medicine of the National Academies recommendations (assuming an infant formula intake of 1000 mL per day, with a minimum of 1 .8 g protein per 100 kcal for infants of 6 months of age (or 12.6 g protein per day)).

Figure 4

Comparison of isoleucine, leucine, lysine and tryptophan levels between potato and rice protein, and Institute of Medicine of the National Academies recommendations (assuming an infant formula intake of 1000 mL per day, with a minimum of 1 .8 g protein per 100 kcal for infants of 6 months of age (or 12.6 g protein per day)).

Figure 5

Comparison of branched-chain amino acid (BCAA) levels between potato and rice protein, and whole milk. Figure 6

Comparison of mean levels of threonine and lysine between potato and rice protein, and FAO 2013 recommendations.

Figure 7

Comparison of mean levels of combined aromatic amino acids between potato and rice protein, and whole milk.

Figure 8

Particle size distribution comparison of preparations with and without potato protein microparticles.

Figure 9

Micrographs of a potato protein microparticle dispersion, before the addition of other ingredients, prepared according to the process of the invention. Figure 9 (A, B) Scale bar: 10 μιτι. Figure 9 (C, D, E) Scale bar: 1 μιτι.

Figure 10 Comparison of the average rating for the sensorial attribute powdery given by trained technical tasters for reconstituted liquid formula prepared from a spray dried powder where the powder has been prepared using potato protein microparticles and reconstituted liquid formula prepared from a spray dried powder where the powder has been prepared using standard potato protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an infant formula comprising potato protein microparticles, carbohydrate and fat. The present invention also provides the use of potato protein microparticles in the preparation of an infant formula, together with a process for preparing an infant formula comprising potato protein microparticles.

The process of the invention comprises the steps of: (a) providing a dispersion of potato protein in water; (b) heating the dispersion to a temperature of between 80 and 90°C for between 10 and 30 minutes and subsequently homogenising the dispersion to form potato protein microparticles; (c) combining the potato protein microparticles with at least one carbohydrate and at least one fat to form a mixture; (d) spray drying the mixture to form an infant formula.

The inventors have found that the combination of a heat treatment followed by a homogenisation step produces potato protein microparticles having particular properties of size and solubility. These properties advantageously improve the production of infant formulas, as compared to the use of regular potato protein, by (i) enabling processing of infant formula intermediates at a higher Total Solids concentration prior to spray drying, (ii) reducing viscosity of infant formula intermediates and so increasing the efficiency of spray drying, and (iii) improving texture of the infant formula product (reduced powdery texture).

Without wishing to be bound by theory, the inventors believe that the particle size and solubility properties of the potato protein microparticles provide an increase in their heat stability, reducing the formation of interactions during subsequent concentration and heat treatment, improving emulsion stability.

A typical process known in the art for preparing an infant formula comprises combining protein, carbohydrate and fat in water to form a mixture (or intermediate), optionally homogenising the mixture, followed by spray drying of the mixture.

The inventors have found that when a standard process is applied to the preparation of an infant formula comprising potato protein - which does not lead to the formation of potato protein microparticles - the potato protein can gel or coagulate, making it extremely difficult to process infant formula intermediates comprising greater than 35% Total Solids.

However, the inventors have found that the use of potato protein microparticles avoids these negative effects and enables the processing of infant formula intermediates of at least 40% Total Solids prior to spray drying. This enables an infant formula comprising potato protein to be processed at a higher Total Solids concentration than would otherwise be possible. In addition, the reduction in viscosity provides for improved spray drying.

This therefore provides a significant improvement over standard preparation processes. The inventors have also observed structural differences between reconstituted liquid formulas prepared from spray dried powders where the powder has been prepared using potato protein microparticles (such as according to the process of the invention) and reconstituted liquid formulas prepared from spray dried powders where the powder has been prepared using standard potato protein (not microparticles).

The process of the invention comprises the step of providing a dispersion of potato protein in water. As used herein, the term dispersion means that potato protein is dispersed, e.g. through mixing, in water. At least some of the potato protein may dissolve in the water.

A suitable dispersion may be obtained by dispersing potato protein in water, e.g. demineralised water, and mixing. The water may be at a temperature of between 15 and 25°C, for example about 20°C. The dispersion may be mixed for a period of time to allow for hydration of the potato protein. In one embodiment, the dispersion is mixed for between 30 and 120 minutes, for example about 60 minutes. The inventors have found that particularly advantageous results may be achieved if the pH of the potato protein dispersion is adjusted to have an acidic pH (i.e. less than pH 7) prior to the formation of the potato protein microparticles. In one embodiment, the pH of the dispersion is adjusted to between 4.5 and 6.5 (as determined at 20°C) prior to heating in step (b) of the process; preferably to between 5.0 and 6.0; more preferably to between 5.2 and 5.6; even more preferably to about 5.4. The pH of the dispersion may be adjusted using standard techniques known in the art. By way of example, the pH of the dispersion may be adjusted using citric acid (e.g. a 30% aqueous solution) or potassium hydroxide (e.g. a 30% aqueous solution).

The process of the invention comprises the step of heating the dispersion to a temperature of between 80 and 90°C (e.g. about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 or 90°C; or between 82 and 88°C, or between 84 and 86°C, or between 83 and 85°C) for between 10 and 30 minutes (e.g. between 15 and 30 minutes, or between 15 and 20 minutes; or for about 10, 15, 20, 25 or 30 minutes) and subsequently homogenising the dispersion to form potato protein microparticles.

In one embodiment the dispersion is cooled prior to homogenisation, e.g. to a temperature below 80°C, e.g. below 60°C.

Heating and cooling of the dispersion may be carried out using any suitable means and apparatus known in the art.

Homogenisation of the heated dispersion may be carried out using any suitable means and apparatus known in the art. In one embodiment, the homogenisation is carried out at a pressure of between 50 and 600 bar, for example between 100 and 500 bar, or between 50 and 400 bar, for example between 200 and 400 bar (e.g. between 250 and 400 bar). In one embodiment, the homogenisation is carried out in two stages. A two-stage homogenisation may be carried out at two different pressures, for example a 200 bar first stage followed by a 50 bar second stage.

Without wishing to be bound by theory, the inventors believe that the combination of heating of the dispersion followed by a subsequent homogenisation leads to the formation of potato protein microparticles having particularly advantageous size and stability properties.

In one embodiment, the potato protein microparticles formed in step (b) of the process, i.e. following heating of the dispersion and subsequent cooling and homogenisation, have an average particle size of from 0.5 to 10 μιτι, preferably from 1 to 10 μιτι, preferably from 1 to 8 μιτι, for example from 2 to 7 μιτι.

In the present context average particle size refers to the volume based mean diameter value D(4,3) of the microparticles as determined using laser diffraction.

By "particle size distribution" is meant the range of particle size that the microparticles exhibit, as determined using laser diffraction. Particle size can be measured with conventional means that will be familiar to a skilled person, for example by using the equipment and method mentioned in the present Examples.

Particle size may be determined by laser diffraction using a laser granulometer, for example using a laser with a wavelength of 633 nm, such as a Mastersizer 2000 (Malvern Instruments, UK) as used in the present examples.

Optionally, the potato protein microparticles obtained in step (b) of the process (in the form of a dispersion in water) may heated prior to step (c) of the process to a temperature of between 50 and 65°C; preferably between 56 and 60°C; more preferably about 58°C.

Following step (b) of the process, in step (c) of the process the potato protein microparticles obtained (in the form of a dispersion in water) are combined with at least one carbohydrate (as defined below) and at least one fat (as defined below) to form a liquid mixture. The mixture is subsequently spray dried to form an infant formula.

Optionally, the potato protein microparticles and the at least one carbohydrate may be combined first, with the at least one fat added to the mixture subsequently.

Vitamins and minerals may also be added to form part of the mixture. If desired, additional emulsifiers may be included. Any lipophilic vitamins, emulsifiers and the like may be dissolved in the fat source prior to combining it with the other components of the other mixture.

The potato protein microparticles, at least one carbohydrate and at least one fat are combined together in step (c) of the process in appropriate proportions as necessary for an infant formula.

The step of combining the potato protein microparticles with at least one carbohydrate and at least one fat may be carried out at a temperature of between 50 and 65°C, preferably between 56 and 60°C, more preferably about 58°C, to aid dispersal of the ingredients.

In one embodiment, the process of the invention further comprises the step of homogenising the mixture (i.e. the mixture obtained in step (c) of the process) prior to spray drying. In one embodiment, the pH of the mixture is adjusted to between 5.0 and 7.0 (as measured at 58°C), preferably 5.0 to 6.5, prior to homogenisation and/or spray drying.

The step of homogenising the mixture may be carried out at a pressure of between 50 and 500bar, e.g. between 200 and 400 bar (e.g. between 250 and 400 bar). The homogenisation of the mixture may be carried out in two stages. A two-stage homogenisation may be carried out at two different pressures, for example a 200 bar first stage followed by a 50 bar second stage. In one embodiment, the mixture formed in step (c) of the process comprises potato protein microparticles having a particle size distribution whereby the particle size of 90% of the of particles is in the range of from 1 to 20 μιτι and/or an average size of from 0.5 to 10 m (e.g. from 1 to 10, or from 1 to 8, e.g. from 2 to 7, e.g. from 3 to 7 μιτι) prior to spray drying.

The mixture may comprise at least 40% Total Solids prior to spray drying (e.g. at least 40%, 45% or 50%). Spray drying is carried out using a suitable spray drying apparatus to convert the liquid mixture to a powder. Preferably, the powder has a moisture content of less than about 5% by weight.

Infant

The term "infant" refers to a child under the age of 12 months, e.g. between 0 and 6 months.

Infant formula

The term "infant formula" may refer to a foodstuff intended for particular nutritional use by infants during the first year of life and satisfying by itself the nutritional requirements of this category of person, as defined in European Commission Directive 2006/141/EC of 22 December 2006.

Infants can be fed solely with infant formulas or the infant formula can be used as a complement of human milk.

The term "infant formula" includes hypoallergenic infant formulas. A hypoallergenic composition is a composition which is unlikely to cause allergic reactions.

The infant formula of the invention may be in the form of a powder. The infant formula may be in the form of a reconstituted infant formula (i.e. a liquid infant formula that has been reconstituted from the powdered form). Preferably, the infant formula is in the form of a powder.

The powder is preferably capable of being reconstituted into a liquid composition suitable for feeding an infant, for example by the addition of water.

In one embodiment, the infant formula has an energy density of about 60-70 kcal per 100 ml_, when formulated as instructed. Allergy

An infant formula produced according to the process of the invention may be suitable for infants having cow's milk protein allergy. The term "allergy" refers to a hypersensitivity of the immune system to a substance which is normally tolerated. The allergy may be an allergy detected by a medical doctor.

Infant formulas are typically formulated with cow's milk protein. For example, bovine whey protein and/or casein are often used as the protein source in infant formulas. However, some infants exhibit an allergy to cow's milk proteins, making such formulas unsuitable.

Allergies to cows' milk and to infant formulas containing cow's milk protein may be due to the differences between the proteins in cows' milk and those in human milk. The principal recognised cow's milk allergens are alpha-lactalbumin (al_A), beta- lactoglobulin (bl_G) and bovine serum albumin (BSA).

Protein

The term "protein" refers to polymers of amino acids, and includes polypeptides and peptides. The term "protein" does not encompass free amino acids, which may also be present in the infant formula of the invention. In one embodiment, the protein content of the infant formula of the invention is preferably in the range 1 to 4g per l OOkcal, preferable 1 .8-3.2 g protein per 100 kcal. In a preferred embodiment, the protein content of the infant formula of the invention is preferably in the range 1 .8-2.8 g protein per 100 kcal.

The infant formula of the invention comprises potato protein as the major protein source.

In one embodiment, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% by weight of the total protein is potato protein.

The remaining protein in the infant formula of the invention may be any protein which is suitable for use in infant formula. Preferably, the infant formula does not comprise dairy protein. Accordingly, in a preferred embodiment 100% by weight of the total protein is non-dairy protein.

In a preferred embodiment, 100% by weight of the total protein is plant protein. Example plant proteins that may optionally be used in the infant formula of the invention, in addition to the potato protein, include pea, rice, quinoa, oat, sunflower or coconut proteins, or combinations thereof.

Further example non-dairy proteins for use in the infant formula of the invention include algal protein or leaf protein.

In a preferred embodiment, the major source of protein in the infant formula is potato protein and the remaining protein is plant protein. In a preferred embodiment, 100% by weight of the total protein is potato protein.

Potato protein for use in the infant formulas of the invention is readily accessible or available, for example as concentrates or isolates, for example from commercial sources. Potato protein may be extracted from potato tuber juice, which may itself be separated from potato solids by any of a number of suitable techniques known in the art. Chromatographic techniques may be used to purify potato proteins from the tuber juice in a similar manner to the isolation of milk proteins. Once isolated, the potato protein may be concentrated and subjected to temperature treatment and/or pH adjustment. Further steps may for example include removal of triglycoalkaloids, spray drying and/or UV treatment. Suitable potato protein sources include complete potato protein extract (i.e. extract not subjected to fractionation by molecular mass); and potato protein fractionated by molecular mass, for example a high molecular mass fraction (e.g. greater than 35 kDa); or a low molecular mass fraction (e.g. less than 35 kDa). In a preferred embodiment, the potato protein source is a low molecular mass potato protein fraction of less than 35 kDa.

The protein may be, for example, intact protein or hydrolysed protein (e.g. partially hydrolysed protein). Preferably, the protein is intact protein. Hydrolysis of protein may in general be termed "partial" or "extensive" depending on the degree to which hydrolysis is carried out. Protein hydrolysates may have an extent of hydrolysis that is characterised by NPN/TN%, which refers to the non-protein nitrogen divided by the total nitrogen χ 100. The non-protein nitrogen refers to amino nitrogen that is free to react with a reagent such as trinitrobenzenesulfonic acid (TNBS). NPN/TN% may be measured as described in Adler-Nissen (Adler-Nissen, J. (1979) J. Agric. Food Chem. 27: 1256-1262).

The term "extensive hydrolysis" may refer to hydrolysis that provides protein that has a NPN/TN% greater than 95%. The term "partial hydrolysis" may refer to hydrolysis that provides protein that has a NPN/TN% in the range 70-85%.

In one embodiment, the protein has an NPN/TN% between 5-90%, 70-90% or 70- 85%, preferably between 70-85%. In another embodiment, the protein has an NPN/TN% between 5-25% or 15-25%. In another embodiment, the protein has an NPN/TN% between 25-55%, 25-50% or 50-55%.

In one embodiment, 60-70% of the protein population has a molecular mass of less than 5000 Da.

In another embodiment, the protein has an NPN/TN% greater than 95%. These are "extensive" hydrolysates. In one embodiment, 60-70% of the protein population has a molecular mass of less than 3000 Da. In one embodiment, at least 95% of the protein population has a molecular mass of less than 3000 Da.

Proteins for use in the infant formula of the invention may be hydrolysed by any suitable method known in the art. For example, proteins may enzymatically hydrolysed, for example using a protease.

For example, protein may be hydrolysed using alcalase (e.g. at an enzyme:substrate ratio of about 0.5-5% by weight and for a duration of about 1 -5 hours).

Free amino acids

The infant formulas of the invention may further comprise free amino acids. Such free amino acids provide a protein equivalent source.

Free amino acids may be incorporated in the infant formulas of the invention to supplement the amino acids comprised in the protein. The levels of free amino acids may be chosen to provide an amino acid profile that is sufficient for infant nutrition, in particular an amino acid profile that satisfies nutritional regulations (e.g. European Commission Directive 2006/141 /EC). Example free amino acids for use in the infant formula of the invention include histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, valine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, carnitine, taurine and mixtures thereof. Carbohydrate

The carbohydrate content of the infant formula of the invention is preferably in the range 9-14 g carbohydrate per 100 kcal.

The carbohydrate may be any carbohydrate which is suitable for use in infant formula.

Example carbohydrates for use in the infant formula of the invention include lactose, saccharose, maltodextrin and starch. Mixtures of carbohydrates may be used.

In one embodiment, the carbohydrate comprises maltodextrin. In one embodiment, at least 40%, 50%, 60% or 70% by weight of the total carbohydrate is maltodextrin. In one embodiment, the carbohydrate comprises lactose. In one embodiment, at least 40%, 50%, 60% or 70% by weight of the total carbohydrate is lactose. In another embodiment, 100% by weight of the total carbohydrate is lactose.

In one embodiment, the carbohydrate comprises lactose and maltodextrin. Fat

The fat content of the infant formula of the invention is preferably in the range 4.0-6.0 g lipids per 100 kcal. The fat may be any lipid or fat which is suitable for use in infant formula.

Example fats for use in the infant formula of the invention include sunflower oil, low erucic acid rapeseed oil, safflower oil, canola oil, olive oil, coconut oil, palm kernel oil, soybean oil, fish oil, palm oleic, high oleic sunflower oil and high oleic safflower oil, and microbial fermentation oil containing long chain, polyunsaturated fatty acids.

The fat may also be in the form of fractions derived from these oils, such as palm olein, medium chain triglycerides and esters of fatty acids such as arachidonic acid, linoleic acid, palmitic acid, stearic acid, docosahexaeonic acid, linolenic acid, oleic acid, lauric acid, capric acid, caprylic acid, caproic acid, and the like.

Further example fats include structured lipids (i.e. lipids that are modified chemically or enzymatically in order to change their structure). Preferably, the structured lipids are sn2 structured lipids, for example comprising triglycerides having an elevated level of palmitic acid at the sn2 position of the triglyceride.

Oils containing high quantities of preformed arachidonic acid and/or docosahexaenoic acid, such as fish oils or microbial oils, may also be added.

Long chain polyunsaturated fatty acids, such as dihomo-Y-linolenic acid, arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, may be added. Willemsen et al. showed that the addition of such fatty acids supported epithelial barrier integrity and reduced IL-4 mediated permeability (Willemsen, L.E. et al. (2008) Eur. J. Nutr. 47: 183-91 ).

Structured lipids may be added or may be omitted. Medium chain triglycerides may be added or may be omitted.

Further ingredients

The infant formula of the invention preferably also contains all vitamins and minerals understood to be essential in the daily diet in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals.

Example vitamins, minerals and other nutrients for use in the infant formula of the invention include vitamin A, vitamin B1 , vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine and L- carnitine.

Minerals are usually added in their salt form. The infant fornnula of the invention may also comprise at least one probiotic. The term "probiotic" refers to microbial cell preparations or components of microbial cells with beneficial effects on the health or well-being of the host (Salminen, S. et al. (1999) Trends Food Sci. Technol. 10: 107-10).

In particular, probiotics may improve gut barrier function (Rao, R.K. (2013) Curr. Nutr. Food Sci. 9: 99-107). Preferred probiotics are those which as a whole are safe, are L(+) lactic acid producing cultures and have acceptable shelf-life for products that are required to remain stable and effective for up to 24 months.

Examples of probiotic micro-organisms for use in the infant formula of the invention include yeasts, such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis; and bacteria, such as the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.

Specific examples of suitable probiotic microorganisms are: Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus casei Shirota, Lactobacillus curvatus, Lactobacillus delbruckii subsp. lactis, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus (Lactobacillus GG), Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus acidilactici, Pediococcus halophilus, Streptococcus faecalis, Streptococcus thermophilus, Staphylococcus carnosus and Staphylococcus xylosus. Preferred probiotic bacterial strains include Lactobacillus rhamnosus; Lactobacillus rhamnosus LPR (CGMCC 1 .3724); Bifidobacterium lactis BL818 (CNCM 1 -3446) sold inter alia by the Christian Hansen company of Denmark under the trade mark BB 12; and Bifidobacterium longum BL999 (ATCC BAA-999) sold by Morinaga Milk Industry Co. Ltd. of Japan under the trade mark BB536.

The infant formula of the invention may also contain other substances which may have a beneficial effect such as human milk oligosaccharides, prebiotics, lactoferrin, fibres, nucleotides, nucleosides and the like.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

EXAMPLES

Example 1

Nutritional comparison between potato protein and rice protein Potato protein contains higher levels of the following essential amino acids compared to rice protein (Figures 1 and 2): valine; isoleucine; leucine; lysine; threonine and aromatic amino acids. The concentrations of tryptophan and the sulphur-containing amino acids are similar between potato and rice proteins.

However, rice protein contains higher concentrations of histidine than potato protein. Overall, the essential amino acid concentrations in potato protein are better than rice protein, and may require lower levels of additional amino acid fortification.

Potato protein contains more essential amino acids in compliance with the FAO 2013 recommendations compared to rice protein (Table 1 ).

Table 1. Amino acid concentrations in potato and rice proteins that are compliant with the FAO 2013 recommendations for 0-6 month-old infants. Although the levels of histidine are lower in potato protein than rice protein, and are lower than the FAO 2013 recommendations for 0-6 month-old infants, potato protein will still deliver histidine levels that are compliant with the 214 mg/d histidine suggested by Institute of Medicine of the National Academies Al [Adequate Intake] for 0-6 month- old infants (Figure 3).

Furthermore, although the concentrations of isoleucine, leucine, lysine and tryptophan are lower in potato compared to the FAO 2013 recommendations, these levels are similar or higher than the levels in rice. Additionally, potato protein will meet the Institute of Medicine of the National Academies Al recommendations for these amino acids, while rice protein will not meet the recommendations for isoleucine and lysine (Figure 4).

The concentrations for isoleucine, leucine and lysine taken from the supplier data indicates that the levels of these amino acids will be compliant with WHO 2007, 2013 and EC Directive 2006/141 /EC, and codex standard (CODEX STAN 72-1981 ), in addition to Institute of Medicine of the National Academies Al recommendations.

Branched-chain amino acids (BCAA)

Branched-chain amino acids (BCAA; leucine, isoleucine and valine) have an important role in protein synthesis. Leucine is an activator of mTOR, and promotes protein synthesis and suppress protein catabolism, resulting in maintenance of muscle protein during restricted dietary intake. Children with food allergies follow dietary restrictions, therefore they are at risk of developing malnutrition, hence consumption of plant protein with high levels of BCAAs may help maintain muscle proteins. Additionally, the best food sources of BCAAs are meat, fish, dairy products and eggs, which may not be consumed at all, or at least consumed in smaller amounts by infants and small children with food allergies. Figure 5 shows that the sum of BCAA (%AA) in potato is closer to that in milk and therefore provides an advantage to children with cow's milk protein allergy. Accordingly, providing a protein source with higher levels of BCAAs may benefit this paediatric population.

Lysine and threonine Lysine and threonine are the first and second most limiting amino acids, respectively, for protein synthesis in human subjects consuming a predominantly cereal-based diet such as wheat and rice. The main roles of lysine and threonine are in protein synthesis. Unlike other plant proteins sources such as rice and wheat proteins, potato protein has higher levels of these two amino acids, with lysine levels close to the requirement set by the FAO 2013 recommendations and threonine levels exceeding it (Figure 6).

The best food sources of threonine and lysine are soy, dairy products, nuts, and fish, beef or chicken. These food sources may not be consumed at all, or at least consumed in smaller amounts by infants and small children with food allergies. Therefore providing a non-animal source of protein with high concentrations of these two amino acids will benefit this paediatric population.

Aromatic amino acids

Phenylalanine is a precursor for tyrosine, the neurotransmitters dopamine, norepinephrine, and adrenaline, and the skin pigment melanin. Potato protein exceeds the requirements set by the FAO 2013 recommendations for 0-6 month-old infants, while rice does not meet the recommended level.

The best food sources of phenylalanine are eggs, chicken, liver, beef, milk and soybeans. These food sources may not be consumed at all, or at least consumed in smaller amounts by infants and small children with food allergies. However, the combined levels of phenylalanine and tyrosine in potato protein are similar to those in milk (Figure 7), which provides an advantage to infants and children with cow's milk protein allergy.

Example 2 Infant formula formulation

An infant formula may be formulated according to the recipe in Table 2. Caloric density 0.6-1 .0 kcal/mL

Protein 1 .8-3.2 g/100 kcal

Carbohydrate 9-14 g/100 kcal

Fat 4.0-6.0 g/100 kcal

Other solids (e.g. free amino 0-2.0%

acids)

Ash 2-3%

Water <3%

Optional probiotics

Table 2.

The infant formula may optionally contain lactose. Example 3

Two example infant formula liquid mixtures (concentrates) were prepared, one using potato protein microparticles and one using standard potato protein (non- microparticulated), according to the recipes illustrated in Table 3, .

Tripotassium citrate 1 .0 1 .0

monohydrate

Tricalcium dicitrate 0.80 0.80

tetrahydrate (micronised)

Ca Phosphate tribasic 0.31 0.31

Trisodium citrate dihydrate 0.70 0.70

Magnesium Oxyde 0.07 0.07

Citric acid 0.02 0.02

Water 2.50 2.50

Table 3

To obtain potato protein microparticles, a dispersion of 4 wt.% potato protein in reverse osmosis water was prepared and the pH adjusted to 5.4 at 20 °C 30% potassium hydroxide solution. The dispersion was subsequently heated by means of a plate heat exchanger to a temperature of 85 °C and kept at this temperature for 30 minutes, and homogenized in a two-stage homogeniser at 200 bar pressure followed by 50 bar pressure and cooled at 58 °C. Fat, minerals and carbohydrates were added to the protein microparticles dispersion and the pH was standardized to 5.3 with 30% citric acid solution. The ingredient mix was homogenized in a two-stage homogeniser at 200 bar pressure followed by 50 bar pressure. The concentrated infant formula had a total solid value (TS%) of 21 % and viscosity of 10 mPa.s (70 °C and 200 1/s). The concentrate was spray dried.

To obtain the standard potato protein formula, a dispersion of 4 wt.% potato protein was prepared in reverse osmosis water. Fat, minerals and carbohydrates were added to the standard potato protein dispersion and the pH was standardized to 5.3 with 30% citric acid solution. The ingredient mix was homogenized in a two-stage homogeniser at 200 bar pressure followed by 50 bar pressure. The concentrated infant formula had a total solid value (TS%) of 24 and viscosity of 40 mPa.s (70 °C and 200 1/s). The concentrate was spray dried. The viscosity may be determined by methods known to a skilled person, e.g. by using a rheometer (Haake Rheostress 6000 coupled with UMTC TM-PE-P temperature controller) equipped with a plate/plate geometry (60 mm diameter) with 2 mm gap.

The viscosity of the reconstituted powders at the below mentioned total solid value (TS%) was determined under the following conditions in order to mimic a standard process at high total solid:

1 . Flow curves at controlled shear rate ramp from 0 - 300 s-1 (linear increase) were obtained at 25 °C +/- 0.1 . (simulating dissolving/mixing of ingredients) [Table 4];

Table 4

Time test at shear rate of 50 s-1 between 0 and 1800 seconds were obtained at 70 °C with gentle stirring for 1 hour (simulating storage time prior to spray drying) [Table 5];

Table 5 3. Temperature ramp at a shear rate of 200 s-1 between 48 - 87 °C (linear increase

0.13 °C/s) were obtained (simulating a heating step prior to spray drying) [Table 6];

Table 6

Flow curves at controlled shear rate ramp from 0 - 800 s-1 (linear increase) were obtained at 75 °C +/- 0.1 for 1 hour (simulating conditions during spray drying with different shearing forces) [Table 7].

Table 7 Each of Tables 3-6 presents the results for (a) standard potato protein, and (b) potato protein microparticles. In each case, the example mixture prepared using potato protein microparticles demonstrated improved viscosity properties.

Example 4

Particle size distribution (PSD) was determined for two example infant formula liquid mixtures (concentrates) prior to fat addition, one using potato protein microparticles prepared according to the process of the invention and one using standard potato protein.

The size of the particles, expressed in micrometers (μιτι) was measured using Malvern Mastersizer 2000 granulometer (laser diffraction unit, Malvern Instruments, Ltd., UK). Ultra pure and gas free water used to disperse the liquid sample was prepared using Honeywell water pressure reducer (maximum deionised water pressure: 1 bar) and ERMA water degasser (to reduce the dissolved air in the deionised water).

Measurement settings used were a refractive index of 1 .52 for proteins and 1 .33 for water at an absorption of 0.01 . All samples were measured at an obscuration rate of 2.0 - 2.5%.

The measurement results were calculated in the Malvern software based on the Mie theory. The volume based mean diameter value D(4,3) is reported.

The mean particle diameter D(4,3) of the ingredient mix before oil addition prepared using standard potato protein was 72.037 μιτι [Figure 8 (a)]. Average particle size for the mixture prepared using potato protein microparticles was 5.970 μιτι [Figure 8 (b)]. PSD for the two mixtures is as shown in Figures 8 (a) and (b).

Example 5

Method for microscopy picture:

Samples of potato proteins microparticleswere fixed in 3.7 % paraformaldehyde in PBS buffer (pH: 7.3). Fixed samples were embedded in 4% aqueous agar solution and solidified on ice. Small cubes of 1 mm3 were cut and then the samples were dehydrated in graded series of ethanol solution, from 30% to 100%, 30 minutes each bath, and 3 times 1 hour for the 100% ethanol solution. Samples were then gradually infiltrated in graded LR White resin series prior to final infiltration in pure resin 3 times for 1 hour each. The polymerization was carried out at 60°C for 48 hours. After polymerization, ultrathin sections of 70 nm were sliced and collected on 100 mesh nickel grids. Samples were imaged with a Tecnai Spirit BioTWIN using a LaB6 filament at 80kV electron microscope (FEI, Netherlands).

The results are shown in the micrograms depicted in Figure 9. (A, B) Scale bar: 10 μιτι. (C, D, E) Scale bar: 1 μηη. As depicted in Figure 9, a large proportion of large agglomerates was observed in dispersions prepared from spray dried powders obtained using the potato protein microparticles.

Example 6 Three samples of infant formula (2 containing potato protein microparticles and a control without microparticles) have been reconstituted by adding 14 g of powder in 90 g of Vittel (94 ppm calcium) water at 40 °C and stirred until complete dissolution.

The samples have been tasted with a panel of 7 internal trained technical tasters and the attribute "powdery" has been rated from 0 (no powdery) to 3 (very powdery). The definition of powdery used by the panel is the following: tactile sensation in the mouth provoked by the presence of tiny particles like undissolved powder or flour. Especially perceivable on teeth surface, the upper front teeth and in the throat while chewing.

The results are shown in Figure 10. Figure 10 shows the average rating for the attribute powdery given by the trained technical tasters. A 2-sample student t-test has been carried out on the data showing that:

- Between sample 1 and 2: p-value = 0.75 (>0.05), not significantly different

- Between sample 1 and Reference: p-value = 0.01 (<0.05), significantly different

- Between sample 2 and Reference: p-value = 0.03 (<0.05), significantly different This shows that the samples where the potato protein have been pretreated to form microparticles are less powdery than the same formulation produced without forming potato microparticles prior to formulating and spray drying. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions, uses and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.

Claims

1 . An infant formula comprising potato protein microparticles, carbohydrate and fat.

2. The infant formula of claim 1 , wherein the infant formula is in the form of a powder or a liquid reconstituted from said powder.

3. Use of potato protein microparticles in the preparation of an infant formula.

4. The use of claim 3, wherein the potato protein microparticles have a mean particle diameter D(4,3) of 0.5 to 10 μιτι.

5. A process for preparing an infant formula, comprising the steps of:

(a) providing a dispersion of potato protein in water;

(b) heating the dispersion to a temperature of between 80 and 90°C for between 10 and 30 minutes and subsequently homogenising the heated dispersion to form potato protein microparticles;

(c) combining the potato protein microparticles with at least one carbohydrate and at least one fat to form a mixture;

(d) spray drying the mixture to form an infant formula.

6. The process of claim 5, wherein the infant formula comprises between 3 and 5 wt.% potato protein; preferably between 3.5 and 4.5 wt.% potato protein.

7. The process of claim 5 or claim 6, comprising the step of adjusting the pH of the dispersion to between 4.5 and 6.5 prior to heating in step (b); preferably to between 5.0 and 6.0; preferably to between 5.2 and 5.6.

8. The process of any one of claims 5 to 7, wherein the homogenisation in step (b) of the process is carried out at a pressure of between 50 and 400 bar.

9. The process of any one of claims 5 to 8, comprising the step of homogenising the mixture prior to spray drying.

10. The process of any one of claims 5 to 9, wherein step (b) comprises heating the dispersion to a temperature of between 83 and 85°C; preferably about 85°C.

1 1 . The process of any one of claims 5 to 10, wherein step (b) comprises heating the dispersion for between 15 and 45 minutes;.

12. An infant formula obtainable by a process according to any one of claims 5 to 1 1 .

13. A method of feeding an infant comprising administering to the infant the infant formula of any one of claims 1 , 2 or 12.

14. An infant formula for use in feeding an infant having cow's milk protein allergy, wherein the infant formula is as defined in any one of claims 1 , 2 or 12.