Nutritional compositions

Abstract

The invention provides a process for making a nutritional composition comprising an aggregate of a casein protein and a second protein, the process comprising the following separate steps, in any order: a) a pasteurisation step, in which a temperature of at least 60oC is applied for a duration capable of pasteurising the aqueous composition; and b) a high pressure processing (HPP) step comprising applying a pressure of about 500 MPa to 750 MPa to the aqueous composition; wherein the process allows a covalent bond to form between a thiol group on a casein protein, which thiol group is only exposed in an at least partially fragmented casein micelle and a thiol group on the second protein to form the aggregate. The invention also provides nutritional compositions produced by the process and the use of such compositions.

Description

[0001] NUTRITIONAL COMPOSITIONS

[0002] Field

[0003] The present invention relates to nutritional compositions and methods for their production and uses thereof.

[0004] Background

[0005] Proteins are made up of amino acids and these provide health benefits, for example through synthesis of muscle, providing calcium and phosphate, aiding cognition and aiding in recovery and sleep.

[0006] Milk proteins are the first source of protein consumed by humans as neonates, either in the form of human milk or infant formula. Milk proteins are an important source of protein in the human diet. Milk primarily comprises 2 distinct types of protein - whey protein and casein.

[0007] Proteins need to be digested into free amino acids, and di- and tripeptides to be absorbed into the bloodstream and be utilised for tissue protein synthesis. The digestion process starts in the mouth, where mastication takes place, and the product is mixed with saliva. Although there are no proteolytic enzymes present in saliva and protein hydrolysis does thus not take place during oral digestion, the oral digestion can have an important role on overall protein digestion in the digestive tract. Following oral digestion, the product reaches the gastric phase of digestion, where it is mixed with gastric juice. In terms of protein digestion, two aspects of the gastric juice are crucial, including the low pH (1-2 for adults, 3-5 for infants) and the presence of the protease pepsin. Pepsin is an endopeptidase that can digest proteins into peptides; however, pepsin cannot hydrolyse proteins into small enough hydrolytic products for uptake into the blood. The action of pepsin can also lead to the coagulation of the casein fraction of milk and consequently lead to gastric coagulation of milk. The low pH of the gastric fluid can also impact protein stability, when proteins start approaching their isoelectric point. While the pH of the gastric fluid is low, particularly for adults, its buffering capacity is also low. Hence, when gastric fluid is mixed with products, rapid increases in pH are usually observed, particularly when products contain buffering compounds, such as milk. This depends on the concentration of buffering compounds in the product and the product volume ingested. For milk, for instance, the pH of the stomach contents can increase to >6 when a glass of milk is consumed on a fasted stomach, and it cantake hours for the pH to decrease to that of the original value. Hence, in addition to the dynamics of protein digestion, pH in the stomach is variable which also affects protein stability and digestion in the stomach.

[0008] Following gastric digestion, the product continues to the intestinal phase for further hydrolysis and absorption. The rate at which the product enters the intestine is determined by the rate of gastric emptying. Careful control of gastric emptying is important and there are various routes by which the rate of gastric emptying is controlled. First and foremost, there is a restriction in volume flow from the stomach to the intestine. Next to this, there are also hormonal feedback loops and feedback loops based on energy content and / or concentrations of specific nutrients of material emptying into the intestine, limiting maximum gastric emptying timescale. Furthermore, there is also a limitation on particle size. Only particles smaller than ~2 mm will be emptied from the stomach. Hence, for solid foods, breakdown of particles via the combined action of pepsin, acid and gastric motility is important and a rate determining step for gastric digesta entering the intestine. Once emptied into the intestine, the proteins and peptides are further cleaved by the collective action of the intestinal proteases and peptidases, including trypsin, chymotrypsin and brush border peptidases. Together, these can break down proteins and peptides to free amino acids, and di- and tripeptides, which can be absorbed into the bloodstream.

[0009] Whey protein accounts for 20% of milk proteins and is characterised by a rapid rate of digestion and a rapid but transient peak aminoacidemia. The remaining 80% of milk protein exists as casein, which is slowly digested, and results in a more sustained but lower peak aminoacidemia. This has led to the classification of whey proteins as fast proteins and caseins as slow proteins.

[0010] The differences between caseins and whey proteins in terms of blood amino acid kinetics have been related to their difference in behaviour during the gastric phase of the digestion process. Native whey proteins are, in general, hardly affected by the gastric phase of digestion. In contrast, the casein fraction in milk, which occurs mainly in the form of so-called casein micelles, is highly susceptible to gastric coagulation. These casein micelles are spherical particles consisting of tens of thousands of casein molecules but also contain about 6% calcium phosphate on a dry matter basis. This allows the casein micelles to act as a carrier of calcium and phosphate, in a stable and bioavailable form.

[0011] The specific structure of the casein micelle allows controlled destabilization by both the action of enzymes and lowering of the pH, which can result in coagulation of casein micelles. In the stomach, pepsin and the acidity of the gastric secretions both drive coagulation ofcasein micelles, but it has been established that the enzymatic destabilization of the casein micelles is the primary cause of gastric coagulation. Casein-based coagula that form during the gastric phase of digestion of milk can readily exceed 10 mm and are thus far too large for gastric emptying, which typically requires particles less than about 2 mm. Therefore, further breakdown of these coagula is required before they can be released from the stomach. This occurs partly due to further hydrolysis by pepsin but also breakdown due to gastric motility. Once the coagulum particles, sufficiently small, are emptied into the intestine, they are hydrolysed further by the intestinal proteases and peptidases. Ileal digestibility measurements in humans, pigs and rats show that milk proteins are digested to varying extents in the intestine. Therefore, aminoacidemia upon consumption of milk can be influenced by the rate of the gastric transit.

[0012] Proteins are consumed in different forms, both as whole food as well as in the form of ingredients. Milk proteins may be consumed as whole milk or as, for example concentrates or isolates of milk protein, micellar casein, whey protein or caseinates. In addition, hydrolysed proteins may be used to improve digestion in products such as infant formula. In these products, protein structures can differ notably due to processing steps applied in the production of foods or protein ingredients, such as heat treatment, changes in pH or the addition or removal of salts. As a result of these processing steps, various aspects of protein digestion, for example protein hydrolysis, but also gastric coagulation of proteins can be affected, leading to changes in protein digestion and amino acid absorption kinetics. Likewise, transforming milk into widely consumed products such as yoghurt or cheese also affects protein structures as well as digestion behaviour and amino acid appearance in blood and subsequent tissue protein synthesis rates.

[0013] While milk has many health benefits, it may cause gut inflammation and intolerance in some people. There exists a need to provide nutritional compositions which are sustained in the stomach for longer than proteins such as milk to provide improved digestion and greater amino acid and calcium bioavailability than the protein in its naturally occurring form, with sustained protein release and improved satiety.

[0014] There particularly exists a need to provide nutritional compositions which are sustained in the stomach for longer than milk to provide improved digestion, greater amino acid and calcium bioavailability and less intolerance than milk, with less gut inflammation and sustained protein release and improved satiety.

[0015] An aim of an embodiment of the invention to provide a nutritional composition with greater nutritional value than a natural composition comprising proteins, such as pasteurisedwhole or skimmed cow’s milk, particularly with at least one of the properties selected from the group consisting of being sustained in the stomach for longer, providing improved digestion, providing greater amino acid availability, providing greater calcium bioavailability, causing less intolerance, causing less gut inflammation, providing greater sustained protein release and improved satiety.

[0016] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0017] Summary

[0018] A first aspect provides a process for making a nutritional composition comprising an aggregate of a first protein which is a casein protein and a second protein comprising a cysteine residue, the process comprising subjecting an aqueous composition comprising a casein micelle comprising the first protein which is a casein protein and a second protein comprising a cysteine residue, to the following separate steps, in any order:

[0019] a) a pasteurisation step, in which a temperature of at least 60°C is applied for a duration capable of pasteurising the aqueous composition; and

[0020] b) a high pressure processing (HPP) step comprising applying a pressure of about 500 MPa + / - 10% to 750 MPa + / - 10% to the aqueous composition at a temperature and for a duration capable of at least partially fragmenting the casein micelle; wherein the aqueous composition has a pH of greater than about 6 prior to step a) or step b), the temperature of the aqueous composition at pressure does not exceed 85°C and the process at least partially denatures the second protein to allow a covalent bond to form between a thiol group on a casein protein, which thiol group is only exposed in the at least partially fragmented casein micelle, and a thiol group on the second protein to form the aggregate and increases the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements of the aggregate and increases the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements of the aggregate, when measured by Fourier Transform Infrared spectroscopy compared to an aqueous composition comprising the casein protein and the second protein to which the process has not been applied.In an alternative to the process of the first aspect, the HPP step is performed on a pasteurised aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein comprising a cysteine.

[0021] In another alternative to the process of the first aspect, the pasteurisation step is performed on an HPP treated aqueous composition comprising an at least partially fragmented casein micelle comprising a first protein which is a casein protein and a second protein comprising cysteine.

[0022] A second aspect provides a nutritional composition produced by the processes of the first aspect.

[0023] In one embodiment of the second aspect, the nutritional composition produced by the processes of the first aspect comprises an aggregate of a first protein which is a casein protein and a second protein comprising a cysteine residue, in which the aggregate comprises a casein protein bonded to the second protein by a covalent bond and the aggregate comprises, as a proportion of its total secondary structure elements, at least about 30% structural elements having a wavelength of 1675-1698 cm'1and at least about 11% of structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy.

[0024] A third aspect provides a method for improving digestibility of an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein, the method comprising subjecting the aqueous solution to the processes of the first aspect.

[0025] A fourth aspect provides a method for improving release of one or more amino acids from an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein, the method comprising subjecting the aqueous composition to the processes of the first aspect.

[0026] In one embodiment of the fourth aspect the amino acids are essential amino acids, optionally branched chain amino acids, optionally selected from leucine and isoleucine. A fifth aspect provides a method for reducing intolerance and gut discomfort when administering an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein in a subject in need thereof, the method comprising subjecting the aqueous composition to the processes of the first aspect.

[0027] A sixth aspect provides a method for reducing an inflammatory, immune or allergic response when administering an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein to a subject, the method comprising subjecting the aqueous composition to the processes of the first aspect.A seventh aspect provides a nutritional composition comprising an aggregate of a first protein which is a casein protein and a second protein comprising a cysteine residue, in which the aggregate comprises a casein protein bonded to the second protein by a covalent bond and the aggregate comprises, as a proportion of its total secondary structure elements, at least about 30% structural elements having a wavelength of 1675-1698 cm'1and at least about 11% of structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy.

[0028] The aggregate of the second or seventh aspects may also include additional casein proteins and second proteins.

[0029] Without wishing to be bound by theory, the inventors believe that the covalent bond between a casein protein and the second protein allows the formation of an aggregate characterised by an increase in measurable structural elements indicative of aggregation, which aggregates are relatively stable compared to those formed by weak, generally reversible reactions between casein proteins and second proteins. Such aggregates, as present in the nutritional composition of the second or seventh aspect, provide a nutritional composition with greater nutritional value than a natural composition comprising casein and other proteins, such as cow’s milk or man-made compositions comprising casein and other proteins, in which a casein protein is not bonded to a second protein by a covalent bond and which do not have increased structural elements indicative of aggregation, particularly with at least one of the properties selected from the group consisting of being sustained in the stomach for longer, providing improved digestion, providing greater amino acid availability, providing greater calcium bioavailability, causing less intolerance, causing less gut inflammation, providing greater sustained protein release and improved satiety.

[0030] In one embodiment of second aspect, the nutritional composition is more readily hydrolysed by human gastric enzymes than a composition in which the casein protein is not bonded to the second protein by a covalent bond and in which the proportion of its total secondary structure elements having a wavelength of 1675-1698 cm'1and structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy is not increased by the process of the first aspect.

[0031] In one embodiment of the seventh aspect, the nutritional composition is more readily hydrolysed by human gastric enzymes than a composition in which the casein protein is not bonded to the second protein by a covalent bond and which comprises as a proportion of its total secondary structure elements less than about 30% structural elements having awavelength of 1675-1698 cm'1and less than about 11% of structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy.

[0032] In one embodiment, the nutritional composition of the second or seventh comprises at least 90% insoluble protein at pH4.6.

[0033] In one embodiment of second aspect, the nutritional composition has greater water holding capacity than a composition in which the casein protein is not bonded to the second protein by a covalent bond and in which the proportion of its total secondary structure elements having a wavelength of 1675-1698 cm'1and structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy is not increased by the process of the first aspect.

[0034] In one embodiment of the seventh aspect, the nutritional composition has greater water holding capacity than one in which the casein protein is not bonded to the second protein by a covalent bond and which comprises an aggregate which comprises as a proportion of its total secondary structure elements less than about 30% structural elements having a wavelength of 1675-1698 cm'1and less than about 11% of structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy.

[0035] In one embodiment of second aspect, the nutritional composition releases more essential amino acids after gastric digestion than a composition in which the casein protein is not bonded to the second protein by a covalent bond and in which the proportion of its total secondary structure elements having a wavelength of 1675-1698 cm'1and structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy is not increased by the process of the first aspect.

[0036] In one embodiment of the seventh aspect, the nutritional composition releases more essential amino acids after gastric digestion than one in which the casein protein is not bonded to the second protein by a covalent bond and which comprises an aggregate which comprises as a proportion of its total secondary structure elements less than about 30% structural elements having a wavelength of 1675-1698 cm'1and less than about 11% of structural elements having a wavelength of 1689-1698 as determined by Fourier Transform Infrared spectroscopy.

[0037] An eighth aspect uses the nutritional composition of the second or seventh aspects or the method of any one of the third to sixth aspects for any one or more of:

[0038] a) maintaining or increasing muscle protein synthesis,

[0039] b) maintaining or increasing muscle mass,

[0040] c) preventing or reducing loss of muscle mass,d) maintaining or increasing growth,

[0041] e) preventing or decreasing muscle catabolism,

[0042] f) preventing or treating cachexia,

[0043] g) preventing or treating sarcopenia,

[0044] h) modulating blood sugar levels,

[0045] i) increasing insulin response to raised blood glucose concentration,

[0046] j) increased satiety,

[0047] k) reduced satiation,

[0048] l) reducing food intake,

[0049] m) reducing calorie intake,

[0050] n) improving glucose metabolism,

[0051] o) increasing rate of recovery following surgery, injury or exercise, and

[0052] p) increased sports performance.

[0053] A ninth aspect provides a method of treating a muscle wasting disorder or condition involving abnormal muscle synthesis such as cachexia and muscular dystrophy or for treatment of obesity, the method comprising administering the nutritional composition of the second or seventh aspects.

[0054] An alternative to the ninth aspect uses the nutritional composition of the second or seventh aspects in the manufacture of a medicament for treating a muscle wasting disorder or condition involving abnormal muscle synthesis such as cachexia and muscular dystrophy or for treating obesity.

[0055] The casein protein of the aqueous composition of the first aspect or the aggregate of the second or seventh aspect may be one selected from the group consisting of aSl-CN, aS2-CN, P-CN and K-CN or combinations thereof. In one embodiment the casein protein of the aqueous composition of the first aspect or the aggregate of the second or seventh aspect may be one selected from the group consisting of aSl-CN, aS2-CN and P-CN or combinations thereof.

[0056] The casein protein of the aggregate of the second or seventh aspect may be recombinant, synthetic or naturally occurring or is provided, for example, by a caseinate salt such as sodium caseinate or calcium caseinate or by casein proteins derived from animal sources.

[0057] The casein micelle of the aqueous composition subjected to the process of the first aspect or used to provide the casein protein of the aggregate of the second or seventh aspect may be provided animal sources, such as by a milk or a milk fraction, which milk or milkfraction is fresh or reconstituted whole milk, raw milk, skim milk, homogenised milk, nonhomogenised milk, including a retentate or a permeate of a milk that has been subjected to filtration, optionally ultrafiltration, nanofiltration or microfiltration, which retentate or permeate comprises casein micelles. It may be provided, for example, by animal milk fractions, including milk protein concentrate (MPC), milk protein isolate (MPI), a composition comprising casein micelles derived from any milk processing stream, or derived from the breakthrough or adsorbed fractions obtained by chromatographic separation of any milk processing stream or a full or partial hydrolysate of any of these compositions or a mixture thereof comprising casein micelles. It may be provided by micellar casein.

[0058] In one embodiment of the first aspect the aqueous composition comprises a casein micelle from an animal milk or a fraction or a product thereof or isolated from milk or a milk fraction.

[0059] In one embodiment of the first aspect the aqueous composition comprises a second protein from an animal milk or a plant “milk” or a fraction or product thereof or isolated from an animal milk or a plant “milk” or a fraction or a product thereof.

[0060] In one embodiment of the first aspect the aqueous composition comprises a casein micelle and a second protein both conveniently provided by an animal milk or a fraction or product thereof.

[0061] The second protein referred to in the first and seventh aspects may be recombinant or provided from animal sources, for example, by a milk or a milk fraction, which milk or milk fraction is fresh or reconstituted whole milk, raw milk, skim milk, homogenised milk, nonhomogenised milk, including a retentate or a permeate of a milk or full or partial hydrolysate thereof, which milk fraction or full or partial hydrolysate thereof has been subjected to filtration, optionally ultrafiltration, nanofiltration or microfiltration, which retentate or permeate comprises said second protein. It may be provided, for example, by milk protein concentrate (MPC), milk protein isolate (MPI), whey protein isolate (WPI), whey protein concentrate (WPC), a whey protein fraction, a composition comprising said second protein derived from any milk processing stream, or derived from the breakthrough or adsorbed fractions obtained by chromatographic separation of any milk processing stream or a mixture thereof comprising said second protein.

[0062] As used herein, animal milk refers to the liquid produced by the mammary glands of mammals. Milk is an emulsion or colloid of butterfat globules within a water-based fluid that contains dissolved carbohydrates, proteins and minerals.The animal milk or fraction or product thereof may be from a cow, sheep, goat, camel, pig, water buffalo, yak, horse, donkey, llama or human source or a mixture thereof. The animal milk or fraction or product thereof may be derived from fresh or reconstituted whole milk, raw milk, skim milk, homogenised milk, non-homogenised milk or colostrum or a mixture thereof.

[0063] The second protein referred to in the first and seventh aspects may be a plant protein or fraction or product thereof. The plant protein may be soy, pea, oat, walnut, hemp seed, spelt, macadamia, flax, hazelnut, sunflower seed, pistachio, pumpkin seed, sesame seed, banana, peanut, rice, almond, coconut, cashew, potato or quinoa protein or a mixture thereof.

[0064] In one embodiment of the first and seventh aspects the second protein is provided by a plant “milk. In one embodiment the plant “milk” is an aqueous solution comprising soy, pea, oat, walnut, hemp seed, spelt, macadamia, flax, hazelnut, sunflower seed, pistachio, pumpkin seed, sesame seed, banana, peanut, rice, almond, coconut, cashew, potato or quinoa protein or a mixture thereof.

[0065] In one embodiment of the first and seventh aspects the second protein is an animal protein, for example collagen, gelatin, albumin or an enzyme or a whey protein.

[0066] The enzyme may be plasmin, an acid phosphatase, an alkaline phosphatase, a protease, a lipase, a catalase or a peroxidase for example.

[0067] The whey protein may be selected from the group consisting of P-lactoglobulin (BLG), a-lactalbumin (ALA), glycomacropeptide (GMP), bovine serum albumin (BSA) or other non-bovine animal milk albumins, immunoglobulin, lactoferrin, lactoperoxidase, lysozyme, proteose peptone or a mixture thereof, each of which whey proteins comprises at least one thiol group or at least one free thiol.

[0068] The aggregate of the nutritional composition of the second and seventh aspect may further comprise a calcium cation, optionally provided as a calcium salt, optionally as calcium phosphate, and optionally as colloidal calcium phosphate. In one embodiment at least some of the calcium cation is provided by a fragmented casein micelle, optionally from milk or a milk fraction as defined herein.

[0069] The casein micelle of the aqueous composition used in the process of the first aspect to provide the first protein which is a casein protein, may further provide a calcium cation, optionally provided as a calcium salt, optionally as calcium phosphate and optionally as colloidal calcium phosphate.

[0070] In one embodiment of the second and seventh aspect in which the aggregate comprises a calcium cation, at least one of the first protein which is a casein protein, a second protein orcalcium cation are derived from an animal milk or a milk fraction or milk product thereof, optionally at least two of the first protein which is a casein protein, a second protein or calcium cation are derived from an animal or plant milk or a milk fraction or milk product and optionally, all the first protein which is a casein protein, the second protein and the calcium cation are derived from an animal milk or a milk fraction or milk product. In this embodiment the nutritional composition may be more readily hydrolysed by human gastric enzymes than an animal milk or plant “milk” or fraction or product in which a casein protein is not bonded to the second protein by a covalent bond.

[0071] In one embodiment of the first aspect in which the aggregate comprises a calcium cation, the calcium cation is associated with the casein micelle and at least one of the casein micelle and the second protein of the aqueous composition is derived from an animal milk or a milk fraction. In one embodiment, the aqueous composition is an animal milk or a milk fraction or milk product comprising a casein micelle comprising calcium, optionally as calcium phosphate, optionally as colloidal calcium phosphate, and at least one whey protein.

[0072] In this embodiment of the first, second or seventh aspects the nutritional composition may be more readily hydrolysed by human gastric enzymes than an animal milk or milk fraction or milk product to which the process of the first aspect has not been applied, particularly than an animal milk or milk fraction in which a casein protein is not bonded to the second protein by a covalent bond.

[0073] A tenth aspect provides a method for improving the bio-accessibility of calcium when administering an aqueous composition comprising a casein micelle comprising a first protein which is casein and calcium, optionally as calcium phosphate, optionally as colloidal calcium phosphate, and a second protein to a subject in need thereof, the method comprising subjecting the aqueous composition to the processes of the first aspect.

[0074] An eleventh aspect uses the nutritional composition of the second or seventh aspects in which the aqueous composition comprises calcium, optionally as calcium phosphate, optionally as colloidal calcium phosphate for increasing calcium absorption.

[0075] Increased calcium absorption may benefit several health conditions, including: osteoporosis, osteopenia, hypocalcemia, rickets and osteomalacia, preeclampsia and may assist with cardiovascular health.

[0076] Osteoporosis involves weakened bones that are more prone to fractures. Enhanced calcium absorption may help maintain bone density and strength.

[0077] Osteopenia is a precursor to osteoporosis and is characterised by lower-than-normal bone density. Better calcium absorption may help prevent its progressionHypocalcemia involves low levels of calcium in the blood, which can lead to muscle cramps, spasms, and abnormal heart rhythms. Improved calcium absorption may help maintain normal calcium levels.

[0078] Rickets and osteomalacia are conditions caused by vitamin D deficiency which result in soft and weak bones. Enhanced calcium absorption, often aided by vitamin D, may help in the treatment and prevention of these conditions.

[0079] Preeclampsia is a pregnancy complication characterised by high blood pressure and signs of damage to other organ systems. Adequate calcium intake and absorption may help reduce the risk of preeclampsia.

[0080] Some studies suggest that calcium, along with vitamin D, may help in maintaining cardiovascular health by regulating blood pressure.

[0081] Ensuring adequate calcium intake and absorption is crucial for overall health, especially for bone health and preventing related conditions.

[0082] In addition, calcium plays a significant role in various aspects of appearance and physique. It can assist with skin health, hair strength, nail health, oral health and bone structure.

[0083] Calcium is essential for maintaining healthy skin. It helps in the renewal of skin cells, preventing dry and flaky skin. Adequate calcium levels can also reduce the appearance of wrinkles and improve skin texture.

[0084] Calcium contributes to the strength and health of hair. It helps in the secretion of hormones and enzymes that are vital for hair growth and maintenance.

[0085] Calcium is crucial for strong and healthy nails. It helps prevent brittle nails and promotes nail growth.

[0086] Calcium is a major component of teeth and is essential for maintaining their strength and health. It helps prevent tooth decay and supports overall oral health.

[0087] While not directly related to appearance, strong bones contribute to a healthy posture and physique. Adequate calcium intake helps maintain bone density and prevents conditions like osteoporosis.

[0088] The method of the tenth aspect may provide a positive impact or improve on these aspects of appearance and overall health

[0089] The nutritional composition of the second or seventh aspect or the aqueous composition subjected to the process of the first aspect may include one or more additives including vitamins, particularly vitamin D, minerals, flavouring agents; colouring agents; salts; sugars and / or sweeteners; stabilisers and / or thickeners; enzymes; probiotics; prebiotics;postbiotics; creatine, coenzyme Q, additional protein, including animal and plant protein, for example soy, pea, rice, almond, or oat proteins, optionally provided by plant milk comprising such protein; additional amino acids, branched chain amino acids, beta alanine, beta-hydroxy beta-methylbutyrate, fatty acids including conjugated linoleic acid, carnitine, extracts and any further constituents known in the art of food technology or beverage production to those skilled in the art.

[0090] In a particular embodiment, the process of the first aspect comprises subjecting an aqueous composition which is cow’s milk or a fraction thereof comprising a casein micelle comprising one or more casein proteins, calcium cation and one or more whey proteins to, in any order:

[0091] a) a pasteurisation step, in which a temperature of at least 60°C is applied for a duration capable of pasteurising the aqueous composition; and

[0092] b) a high pressure processing (HPP) step comprising applying a pressure of about 500 MPa + / - 10% to 750 MPa + / - 10% to the aqueous composition at a temperature and for a duration capable of at least partially fragmenting the casein micelle; wherein the aqueous composition has a pH of greater than about 6 prior to step a) or step b), the temperature of the aqueous composition at pressure does not exceed 85°C and the process at least partially denatures the second protein to allow a covalent bond to form between a thiol group on a casein protein, which thiol group is only exposed in the at least partially fragmented casein micelle and a thiol group on the second protein to form an aggregate and increases the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements of the aggregate and increases the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements of the aggregate, when measured by Fourier Transform Infrared spectroscopy compared to an aqueous composition comprising the casein protein and the second protein to which the process has not been applied.

[0093] The aggregate produced by this embodiment of the first aspect may also include additional casein proteins and whey proteins.

[0094] In one embodiment of the first aspect, no microorganisms selected from the group consisting of E.coli spp., Salmonella spp. and Listeria spp. are added to the milk or milk fraction before, during or after the process of the first aspect.

[0095] Detailed DescriptionGeneral Definitions

[0096] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art.

[0097] The present disclosure may refer to the contents of certain documents being incorporated herein by reference. In the event of any inconsistent teaching between the teaching of the present disclosure and the contents of those documents, the teaching of the present disclosure takes precedence.

[0098] It is to be understood that if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art.

[0099] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0100] As used herein, the word “comprise” and other forms of the word, such as “comprising” and “comprises” and “includes” or “including” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

[0101] As used herein, the term “and / or”, e.g., “X and / or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0102] As used herein, the term “about”, unless stated to the contrary, refers to + / - 5% or + / -10%, of the designated value.

[0103] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to "the" includes a single as well as two or more and so forth.

[0104] Unless otherwise indicated, terms such as "first," "second," etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements onthe items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and / or a higher-numbered item (e.g., a “third” item).

[0105] As used herein, the phrase “at least one of’, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0106] As used herein, relative terms such as “greater”, improved”, “more” or “increased” or “increases” should be understood to define properties that are “greater”, improved”, “more” or “increased” or “increases” by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or about 50% or more compared to their comparator.

[0107] As used herein, relative terms such as “less” or “decreased” or “decreases” should be understood to define properties that are “less”, “decreased” or “decreases” by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or about 50% or more compared to their comparator.

[0108] Each embodiment of the present disclosure described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise or required otherwise by context.

[0109] Food processing of liquid milk and milk products generally involves heat treatment to pasteurise or sterilise the product for food safety and to extend its shelflife.

[0110] The Applicant’s previous invention, as described in PCT / AU2016 / 050579, published as WO 2017 / 004668 Al describes a process which uses high pressure processing instead of heat treatment to deactivate pathogens in milk for food safety purposes and to extend the shelf life of milk. Importantly in the process described the temperature of the milk is kept below 60°C to avoid or minimise degradation of the milk in terms of nutritional value and / or organoleptic properties.

[0111] The inventors have unexpectedly found that applying high pressure processing and pasteurisation to cow’s milk produced an aggregate of casein proteins, calcium and at least partially denatured whey proteins with increased structural elements indicative of aggregationwhen compared to unprocessed cow’s milk or cow’s milk processed by other methods. This aggregate was more readily hydrolysed by human gastric enzymes than pasteurised whole milk.

[0112] In essence the inventors found that applying high pressure processing and pasteurisation to cow’s milk they could increase the proportion of structural elements indicative of an aggregated structure in the processed cow’s milk. This processed cow’s milk has improved nutritional properties compared to unprocessed cow’s milk or processed cow’s milk with less structural elements indicative of aggregation.

[0113] The inventors consider that the process would increase the proportion of structural elements indicative of aggregation in other aqueous compositions comprising casein micelles and a second protein and would provide similar improvements in the nutritional properties of these aqueous compositions if applied.

[0114] Accordingly, in a further aspect the invention provides a method of increasing the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements in an aqueous composition and increasing the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements in an aqueous composition, when measured by Fourier Transform Infrared spectroscopy, wherein the aqueous composition comprises a casein micelle comprising a first protein which is a casein protein and a second protein comprising a cysteine residue, the method comprising subjecting the aqueous composition to, in any order:

[0115] a) a pasteurisation step, in which a temperature of at least 60°C is applied for a duration capable of pasteurising the aqueous composition; and

[0116] b) a high pressure processing (HPP) step comprising applying a pressure of about 500 MPa + / - 10% to 750 MPa + / - 10% to the aqueous composition at a temperature and for a duration capable of at least partially fragmenting the casein micelle; wherein the aqueous composition has a pH of greater than about 6 prior to step a) or step b), the temperature of the aqueous composition at pressure does not exceed 85°C and the process at least partially denatures the second protein to allow a covalent bond to form between a thiol group on the casein protein, which thiol group is only exposed in the at least partially fragmented casein micelle and a thiol group on the second protein to form an aggregate and increases the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements of the aggregate and increases the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements in an aqueous composition, when measured by Fourier Transform Infrared spectroscopy comparedto an aqueous composition comprising the casein protein and the second protein to which the process has not been applied.

[0117] The inventors found that when they applied the process of the further aspect to milk, they produced an aggregate comprising increased structural element having a wavelength of 1675-1698 cm'1and 1689-1698 cm'1(which are indicative of aggregation) and reduced native secondary structure elements with wavelengths between 1612-1675 cm'1. These aggregates are insoluble at pH4.6. The aggregates were found to create, in the stomach, a porous, fine-stranded, elastic curd capable of holding a large amount of water, leading to a feeling of fullness (satiety). The resultant coagulum that is formed in the gastric phase has a modified composition leading to softer curds which are more favourably digested. Upon passing through into the intestine, the proteins are further hydrolysed into peptides and amino acids which are absorbed by the body. In addition, calcium and phosphate, usually contained within the casein micelle are associated with the aggregate leading to improved absorption by the body for enhanced nutrition.

[0118] Without wishing to be bound by theory, it is thought that the combination of pasteurisation and high pressure processing of milk at a temperature and for a duration that provides at least partially denatured whey proteins and fragmented casein micelles in which thiol groups normally within the casein micelle are exposed, allowing aggregates of the denatured whey proteins and casein proteins to form through hydrophobic interactions and covalent bonding of exposed thiol groups on the caseins with free thiol groups provided by cysteines in the denatured whey proteins, the aggregates comprising increased structural element having a wavelength of 1675-1698 cm'1and 1689-1698 cm'1(which are indicative of aggregation) and the reduction of native secondary structure elements with wavelengths between 1612-1675 cm'1.

[0119] In one embodiment of each of the aspects the nutritional composition is an aqueous composition, including a suspension or emulsion.

[0120] Although the invention was determined using milk and milk proteins - casein and whey proteins; it is entirely plausible that aggregates of other proteins comprising cysteine can be formed with fragmented casein micelles or indeed with recombinant or synthetic or isolated natural casein proteins.

[0121] Specific Definitions

[0122] A nutrition composition as used herein, is a composition for use to provide at least some of the nutritional needs of a subject.An aggregate as used herein, is a mass of two or more components associated with each other. Protein aggregation is a phenomenon in which disrupted or denatured proteins accumulate and clump together through the formation of bonds. The aggregate contains a second protein and a casein protein component.

[0123] The covalent bond between the casein protein and the second protein is preferably a disulphide bond or disulphide bridge.

[0124] The second protein must contain a cysteine. The invention extends to second proteins or peptide or polypeptide fragments thereof, provided such peptide or polypeptide comprises a cysteine.

[0125] Denatured as used herein, refers to a protein which is no longer in its native form due to breaking of bonds leading to partial or complete unfolding of the protein. It can include unfolding of the secondary, tertiary or quaternary structure.

[0126] Percentage denaturation refers to the proportion of proteins in the composition that are at least partially denatured.

[0127] As used herein, a protein that is least partially denatured means that at least 10% of the proteins in the composition are denatured.

[0128] In one embodiment of one or more of the aspects, if the second protein is or comprises P-lactoglobulin, it is at least 60% denatured, at least 70% denatured, at least 80% denatured, at least 90% denatured, at least 95% denatured or at least 97% denatured.

[0129] In one embodiment of one or more of the aspects, if the second protein is or comprises a-lactalbumin, it is at least 35% denatured, at least 45% denatured; at least 50% denatured, at least 55% denatured, or at least 60% denatured.

[0130] In one embodiment of one or more of the aspects, if the second protein is or comprises bovine serum albumin, it is at least 38% denatured, at least 50% denatured, at least 60% denatured, at least 70% denatured.

[0131] A fragmented casein micelle as used herein is a casein micelle in which the micelle structure has been disrupted by the disassociation of covalent bonds or other interactions including hydrophobic interactions, electrostatic interactions and hydrogen bonds to expose thiol groups that are not normally on the surface of the micelle. Such thiol groups are then able to form covalent bonds with free thiol groups in cysteine residues in the denatured second protein.

[0132] The fragmentation of the casein micelle exposes casein fragments and thiol groups on casein proteins that would otherwise remain trapped within the micelle and may release calcium in the form of calcium phosphate.As used herein, a casein micelle that is at least partially fragmented means that at least 10% of the casein population has been fragmented or separated from the native casein micelle as it exists in nature.

[0133] In one embodiment of the first aspect or further aspect, the pH of the aqueous composition is greater than about pH 6.1.6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, pH 7.0. pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8 or more during at least the pasteurisation step or the high pressure processing (HPP) step.

[0134] In one embodiment of the first aspect or further aspect, the aqueous composition has not been subjected to pasteurisation prior to the high pressure processing step.

[0135] Persons skilled in the art would be aware of the process termed Fourier Transform Infrared spectroscopy or FTIR. The application of FTIR to milk proteins to show changes in protein structure induced by heat treatment, is described in Grewal et al., (2018) Food Hydrocolloids 80; 160-167.

[0136] As used herein, reference to “structural elements indicative of aggregation” refers to those elements indicted by a wavelength of 1675-1698 cm'1when analysed by FTIR according to the method of Grewal et al, (2018) supra, as applied in accordance with the examples. These structural elements may comprise intramolecular beta sheets (indicted by a wavelength of 1675-1688 cm'1when analysed by FTIR according to the method of Grewal et al, (2018) supra.) as applied in accordance with the examples and aggregated beta sheets (indicted by a wavelength of 1689-1698 cm'1when analysed by FTIR according to the method of Grewal et al, (2018) supra, as applied in accordance with the examples) but the edges of these definitions is poorly defined and may not be reliable. This terminology is poorly defined in the art and the descriptions may not be entirely reliable; it is the wavelengths that are relevant, not the terminology used.

[0137] In one embodiment, the process of the first aspect and further aspect increases the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements of the aggregate when measured by Fourier Transform Infrared spectroscopy compared to an aqueous composition comprising the casein protein and the second protein to which the process has not been applied by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, or about 300% or more.In one embodiment, the process of the first aspect and further aspect increases the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements of the aggregate when measured by Fourier Transform Infrared spectroscopy compared to an aqueous composition comprising the casein protein and the second protein to which the process has not been applied by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 205%, 210%, 215%, 220%, 225%, 230%, 235%, 240%, 245%, 250%, 255%, 260%, 265%, 270%, 275%, 280%, 285%, 290%, 295%, or about 300% or more.

[0138] In an embodiment, the process of the first aspect or further aspect increases the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements of the aggregate when measured by Fourier Transform Infrared spectroscopy from 16.2% in raw milk to 61.4% in raw milk treated by the process of the first aspect.

[0139] In an embodiment, the process of the first aspect or further aspect increases the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements of the aggregate when measured by Fourier Transform Infrared spectroscopy from 6.2% in raw milk to 21.9% in raw milk treated by the process of the first aspect.

[0140] Other milk processing techniques may provide milk comprising structural elements having a wavelength of 1675-1698 cm'1but these are at a lower proportion of total secondary structure elements when measured by Fourier Transform Infrared spectroscopy. Notably the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements in milk that has been pasteurised, when measured by Fourier Transform Infrared spectroscopy is only 23.6%, in UHT treated milk is only 25.0%, in ultrafiltered milk is only 17.7% and in raw milk subjected to cold HPP (not heated) is only 24.6%.

[0141] Other milk processing techniques may provide milk comprising structural elements having a wavelength of 1689-1698 cm'1but these are at a lower proportion of total secondary structure elements when measured by Fourier Transform Infrared spectroscopy. Notably the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements in milk that has been pasteurised, when measured by Fourier Transform Infrared spectroscopy is only 8.2%, in UHT treated milk is only 7.8%, in ultrafiltered milk is only 1.4% and in raw milk subjected to cold HPP (not heated) is only 7.1%.While in raw milk treated in accordance with the method described in PCT / AU2016 / 050579 the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements is 42.6% and the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements is only 10.3%, compared to 21.9% in milk treated by the process of the first aspect.

[0142] In an embodiment of the second and seventh aspects the aggregate comprises, as a proportion of its total secondary structure elements, at least about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%.39% , 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or more structural elements having a wavelength of 1675-1698 cm'1as determined by Fourier Transform Infrared spectroscopy.

[0143] In an embodiment of the second and seventh aspects the aggregate comprises, as a proportion of its total secondary structure elements, at least about 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%.39% , 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or more structural elements having a wavelength of 1689-1698 cm'1as determined by Fourier Transform Infrared spectroscopy.

[0144] In one embodiment, in the nutritional composition of the second aspect or produced according to the process of the first aspect, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of casein micelles are in a fragmented or disassociated state.

[0145] In one embodiment, in the nutritional composition of the second aspect or the seventh aspect or produced according to the process of the first aspect, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of calcium or calcium phosphate, if present, is released from the casein micelle before association with the aggregate.

[0146] “More readily hydrolysed by human gastric enzymes” as used herein, in relation to the nutritional composition compared to equivalent compositions in which a casein protein is not bonded to the second protein by a covalent bond, means that when the protein is exposed to enzymes at a concentration usually present in the human digestive system, the soluble nitrogen content is greater at any given time period, representative of typical gastric & / or intestinal digestion.The nutritional composition of the second or seventh aspect may produce more porous gel structures when acidified to pH 4.6, or another method that simulates human gastric digestion, the coagulum formed has a more open structure and greater elasticity, than that of the native form. Methods that simulate human gastric digestion will be known to those skilled in the art.

[0147] Acid induced gelation, or coagulation may be used to assess the gelling behaviour and rheological properties of the curd (or coagulum, or clot, or gel), that is formed during gastric digestion. The pH at gelation point, and the physical and viscoelastic properties of the coagulum indicate a composition’s digestive properties, which in turn can inform the rate & extent of digestion.

[0148] Some of the important clot properties in predicting digestibility behaviour include: 1) the accessibility of the coagulum to digestive enzymes - such properties as the physiological structure of the curd, whether dense or open and porous, can determine this accessibility,

[0149] 2) the composition of the curd vs the serum (i.e. the serum is the soluble portion that does not precipitate as a curd and which passes quickly through the gastric lining into the intestinal phase), including the whey / casein / aggregate composition of the curd; several methods can be used to interpret composition, such as protein composition of bulk vs pH4.6 soluble vs centrifuge soluble, SDS page composition, HPLC identification of native / denatured proteins,

[0150] 3) the viscoelastic properties of the curd,

[0151] 4) how the clot forms during acidification - i.e. pH at gelation point, which is dependent on the properties of the individual proteins contained within the aggregate, such as their isoelectric points; a higher pH at gelation signifies a different aggregate composition incorporating whey proteins along with caseins, and

[0152] 5) hydration and water holding capacity of the curd.

[0153] Some of the important measurements of digestibility - both rate and extent of digestion - include:

[0154] 1) hydrolysis, which may be measured in several ways including % soluble nitrogen over total nitrogen, and

[0155] 2) quantification of proteins, peptides and / or amino acids at various stages of digestion.

[0156] Other aspects of differentiation during digestion include:1) satiety, which can be implied by the type and proportion of proteins within the clot compared to the serum, the rate of breakdown of the clot, and the water holding capacity of the clot,

[0157] 2) calcium bio accessibility, which is governed by the concentration of calcium in the serum which passes through the gastric lining into the intestine which is then available for absorption into the blood - calcium is most bio accessible at lower concentration so calcium that is bound within the aggregate and precipitated within the curd delivers slower release and greater bioacessibility,

[0158] 3) Amino acid bioacessibility, wherein individual amino acids can be quantified via HPLC at various stages of digestion, and

[0159] 4) Digestibility, where the unfolded whey proteins, dissociated caseins or aggregates are more susceptible to digestive enzymes.

[0160] The nutritional composition of the second or seventh aspect may, upon acidification to pH 4.6, form a more hydrated gel with greater water holding capacity compared to the native form (untreated aqueous composition).

[0161] “More hydrated” in relation to gel structures as referred to herein, means that the gels have a greater water holding capacity (WHC), where WHC is measured as absorbed water within an acidified coagulated gel, by quantifying the weight of the sample compared to the dry weight of the same sample (gH2O / g DS). Such analytical methodology will be known to those skilled in the art.

[0162] The nutritional composition of the second or seventh aspect may, upon exposure to human gastric enzymes, forms a curd that is more quickly & / or more completely digested.

[0163] “More quickly digested” as used herein, refers to the rate of hydrolysis during digestion, and can be measured as the proportion of soluble nitrogen as a % of total nitrogen at selected timepoints during gastric and intestinal digestion compared to the native form. More completely digested as used herein, means more hydrolysed at the end of digestion, and that the proportion of soluble nitrogen as a % of total nitrogen is greater at the end of intestinal digestion compared to the native form. Such analytical method(s) will be known to those skilled in the art.

[0164] The nutritional composition of the second or seventh aspect may have a pH at gelation point during acidification that is higher than that of a composition in which a casein protein is not bonded to a second protein by a covalent bond.The nutritional composition of the second or seventh aspect may form a gel upon acidification to pH 4.6 that is more elastic than produced by that of a composition in which a casein protein is not bonded to a second protein by a covalent bond.

[0165] “More elastic” as used herein, means the gel formed upon acidification has a higher storage modulus and / or lower viscosity over shear rate at 37°C. Analytical methods to determine this will be known to those skilled in the art.

[0166] The nutritional composition of the second or seventh aspect may release more free amino acids during digestion compared to a composition in which a casein protein is not bonded to the second protein by a covalent bond.

[0167] The nutritional composition of the second or seventh aspect may provide more sustained protein release than a composition in which a casein protein is not bonded to the second protein by a covalent bond.

[0168] The nutritional composition of the second or seventh aspect may be more tolerated (or cause less intolerance and gut discomfort) than a composition in which a casein protein is not bonded to the second protein by a covalent bond, because proteins in milk which are thought to cause intolerance such as, for example, casein and P-lactoglobulin, if present are at least partially unfolded and the more porous curds in the stomach are more readily digested, reducing discomfort and bloating.

[0169] Pasteurisation is a heat treatment that destroys pathogenic microorganisms in certain foods and beverages. As used herein, pasteurisation encompasses all heat treatments above 60°C applied to milk or milk fractions as defined herein, including HTST pasteurisation, ultra-pasteurisation, ultra-high temperature (UHT) pasteurisation and sterilisation.

[0170] Pasteurisation of milk generally requires temperatures of about 63°C (145°F) maintained for about 30 minutes or, alternatively, heating to a higher temperature, optionally, 72°C (162°F), and holding for about 15 seconds (and yet higher temperatures for shorter periods of time). The times and temperatures are those determined to be necessary to destroy Mycobacterium tuberculosis and other, more heat-resistant, disease-causing microorganisms found in milk. The treatment also destroys many of the microorganisms that cause spoilage and so prolongs the storage time of milk.

[0171] Ultra-high temperature (UHT) pasteurisation involves heating the composition to 138— 150 °C (280-302 °F) for one or two seconds.

[0172] In one embodiment of the first aspect, the temperature of the aqueous composition does not exceed 85°C, 84°C, 83°C, 82°C, 81°C, 80°C, 79°C, 78°C, 77°C, 76°C, 75°C, 74°C, 73°C, 72°C, 71°C, 70°C, 69°C, 68°C, 67°C, 66°C, 65°C, 64°C or 63°C during the process.In one embodiment of the first aspect, the temperature of the aqueous composition exceeds ambient temperature prior to and / or after the HPP step. Ambient temperature is used herein to refer to a temperature of typically 18-25°C.

[0173] In one embodiment of the first aspect, the temperature of the aqueous composition does not exceed 84°C, 83°C, 82°C, 81°C, 80°C, 79°C, 78°C, 77°C, 76°C,75°C, 74°C, 73°C, 72°C, 71°C, 70°C, 69°C, 68°C, 67°C, 66°C, 65°C, 64°C, 63°C, 62°C, 61°C, 60°C, 59°C, 58°C, 57°C, 56°C, 55°C, 54°C, 53°C, 52°C, 51°C, 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41°C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C or 22°C at pressure during the high pressure processing step.

[0174] In one embodiment of the first aspect, the temperature of the aqueous composition is at least 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C or at least 84°C, during the HPP step (at pressure).

[0175] High Pressure Processing (HPP) as defined herein is the application of high pressure to a substrate comprising a casein micelle, optionally milk or a milk fraction comprising a casein micelle, at a pressure and for a temperature and a duration to at least partially fragment the casein micelle.

[0176] In one embodiment of the first aspect, the HPP step comprises applying a pressure of about 500 MPa to about 750 MPa, optionally between about 500 MPa and about 650 MPa and optionally about 600 MPa. In one embodiment the pressure is 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820 or 825 MPa

[0177] In one embodiment of the first aspect, the pressure is applied for a duration ranging from about 1 minute to about 10 minutes, optionally for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes.

[0178] In certain embodiments of the first aspect, the aqueous composition is transferred to one or more sealed containers prior to either the pasteurisation step or the high pressure processing step.

[0179] In certain embodiments of the first aspect, the pasteurisation step or the high pressure processing step is performed before the aqueous composition is transferred to one or more sealed containers.In certain embodiments of the first aspect, the pasteurisation step or the high pressure processing step occurs after the aqueous composition is transferred to one or more sealed containers.

[0180] In certain embodiments of the first aspect, both the pasteurisation step and the high pressure processing step occur before the aqueous composition is transferred to one or more sealed containers.

[0181] In certain embodiments of the first aspect, both the pasteurisation step and high pressure processing step occurs after the aqueous composition is transferred to one or more sealed containers.

[0182] In certain embodiments of the first aspect, the high pressure processing step includes immersing the one or more sealed containers in a fluid, wherein the fluid is subsequently pressurised thereby subjecting the aqueous composition to the elevated pressure.

[0183] In certain embodiments of the first aspect, the aqueous composition that has been transferred to the one or more sealed containers in a fluid is heated, or maintained such that the temperature of the aqueous composition in the one or more sealed containers is at or near the temperature of the fluid.

[0184] In one embodiment of the first aspect, the temperature of the aqueous composition is heated or maintained by placing the one or more sealed containers in a gas or liquid medium which is separate from the fluid of the high pressure processing step.

[0185] In one embodiment of the first aspect, the aqueous composition is heated or maintained by placing the one or more sealed containers in a liquid medium, such as water.

[0186] In certain embodiments of the first aspect, the materials and / or equipment associated with the process of the first aspect are pre-heated and / or stabilised prior to the pasteurisation step or the high pressure processing step or both steps.

[0187] In one embodiment of the first aspect, the materials and equipment include one or more components selected from the sealed containers, any vessel holding the sealed containers during the pasteurisation and / or high pressure processing step, or any fluid contained within the heating and / or high pressure vessel. In a further form, the materials and equipment include the heating or high pressure processing equipment. In one form, the materials and equipment are pre-heated in a liquid or gas medium. In a preferred form, the liquid medium is water.

[0188] In certain embodiments of the further aspect, the process further includes the step of cooling the aqueous composition to a temperature of about 4°C after either the pasteurisation step or the high pressure processing step or both steps.In certain embodiments, the process of the first aspect further includes subjecting the aqueous composition to any one or more of the following process steps prior to the high pressure processing step: clarification, bactofugation, filtration and centrifugation. In one form, filtration includes any one or more of the following process steps: microfiltration, ultrafiltration, nanofiltration, diafiltration and reverse osmosis.

[0189] In certain embodiments, the process of the first aspect further includes the step of subjecting the aqueous composition to UV treatment and / or ozone treatment, prior to the pasteurisation step or the high pressure processing step or prior to both steps.

[0190] In certain embodiments, the process of the first aspect further includes the step of standardizing the aqueous composition to a certain fat content prior to the high pressure processing step.

[0191] In certain embodiments, the first aspect further comprises applying a homogenisation step to the aqueous composition either before or after the pasteurisation step or the high pressure processing step.

[0192] In certain embodiments, the first aspect does not include the application of a homogenisation step to the aqueous composition either before or after the pasteurisation step or the high pressure processing step.

[0193] In one embodiment of the first aspect, the nutritional composition is not produced by addition of enzymes for hydrolysing proteins.

[0194] The nutritional composition of the second and seventh aspects is more readily hydrolysed by gastric enzymes than a composition in which a casein protein is not bonded to the second protein by a covalent bond, when administered to subjects, particularly human subjects.

[0195] The process of the first aspect improves one or more attributes of an aqueous compositions when administered to subjects, particularly human subjects when compared to compositions to which the process of the first aspect has not been applied.

[0196] Improves, as used herein is defined as at least a 5%, 10%, 20%, 30%, 40% or 50% or more improvement in said attribute when compared to a composition in which a casein protein is not bound to a second protein by a covalent bond.

[0197] The nutritional composition of the second or seventh aspects may be administered to a subject in an effective amount for maintaining or increasing muscle protein synthesis, maintaining or increasing muscle mass, preventing or reducing loss of muscle

[0198] mass, maintaining or increasing growth, preventing or decreasing muscle catabolism, preventing or treating cachexia, preventing or treating sarcopenia, modulating blood sugarlevels, increasing insulin response to raised blood glucose concentration, increasing satiety, reducing satiation, reducing food intake, reducing calorie intake, improving glucose metabolism, increasing rate of recovery following surgery, injury or exercise, or increased sports performance.

[0199] The term “effective amount” refers to an amount of the nutritional composition, of the present invention effective to yield a desired response. The "effective amount" will, obviously, vary with such factors as the particular condition being addressed, the physical condition of the subject, the type of subject being treated, the duration of the treatment and more.

[0200] The nutritional composition of the second or seventh aspects may be used for non-therapeutic therapies, including those selected from the group consisting of improving appearance, strength or endurance and weight loss to improve appearance.

[0201] The nutritional composition of the second or seventh aspects may be used for therapeutic therapies, including those selected from the group consisting of maintaining or increasing muscle protein synthesis, maintaining or increasing muscle mass, preventing or reducing loss of muscle mass, maintaining or increasing growth, preventing or decreasing muscle catabolism in subjects with a muscle wasting disorder or condition involving abnormal muscle synthesis such as cachexia and muscular dystrophy or for treatment of obesity.

[0202] As used herein, the terms “treatment” or “treating” include curing a condition, as well as alleviation of or reduction of symptoms associated with a condition. The term treating also includes slowing the progression of a condition. The terms “treating”, “treatment” and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacologic and / or physiologic effect. It may include one or more of a) inhibiting the condition, i.e., arresting its development, or (b) relieving or ameliorating the effects of the condition, i.e., cause regression of the effects of the condition.

[0203] As used herein, the terms “prevention” or “preventing” include prophylaxis, and include reducing the likelihood of having a condition or a symptom thereof. The effect may be prophylactic in terms of completely or partially preventing a condition or sign or symptom thereof.

[0204] The nutritional composition is for administering enterally (e.g. orally).

[0205] The nutritional composition of the seventh aspect may be prepared by any suitable method, including the process of the first aspect or by a modification of that process.The nutritional composition of the second or seventh aspect or prepared by the process of the first aspect may be used in the preparation of a food, drink, ready to drink nutritional composition, food additive, drink additive, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical or a cultured product, a yoghurt or cheese or an intermediate in the preparation thereof, It may be in the form of a powder or a flowing solid or semi-solid product.

[0206] In one embodiment of the process of the first aspect, the process further comprises processing the nutritional composition into a food, drink, ready to drink nutritional composition, food additive, drink additive, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical or a cultured product, a yoghurt or cheese or an intermediate in the preparation thereof. It may also further comprise formulating the nutritional composition into a powder or a flowing solid or semi-solid product.

[0207] Examples

[0208] The invention will now be described in detail by way of reference only to the following non-limiting examples.

[0209] Example 1

[0210] Raw milk was collected from a dairy farm located in the Sunshine Coast, Queensland, Australia and maintained at <5°C until processed. The milk was prepared to remove impurities and disperse fat, by heating the milk to about 55°C and passing through a centrifugal separator and homogeniser, then immediately cooling to 4°C.

[0211] One portion of the milk (SI) was heated to about 72°C for about 15 seconds, then cooled to 4°C and bottled. A second portion of the milk (S2) was heated to about 72°C for about 15 seconds, then cooled to about 40°C, bottled and subjected to high pressure at 600MPa for 3 minutes, then cooled to 4°C. The temperature of this sample at pressure did not exceed 85°C. A third portion of the milk (S3) was heated to about 140°C for about 5 seconds, then cooled to 4°C and bottled. A fourth portion of the milk (S4) remained unprocessed.

[0212] Protein determination by Kjeldah (see ISO, E. (2014). ISO 8968-1: 2014 (IDF 20-1: 2014) milk and milk products: Determination of nitrogen content-Part 1 : Kjeldahl principle and crude protein calculation) showed the proportion of insoluble protein at pH4.6 was 77%, 91%, 89% and 75% for SI -4 respectively.HPLC analysis showed the proportion of denatured P-lactoglobulin was 4.1%, 97.7%, 88.4% and 0.0% for Sl-4 respectively and the proportion of denatured a-lactalbumin was 0.5%, 49.7%, 43.9% and 0.0% for Sl-4 respectively.

[0213] FTIR was performed according to the method of Grewal et al., (2018) Food Hydrocolloids 80; 160-167. Sample spectra were acquired in the range of 4000-6000 cm-1 using a PerkinElmer Frontier FTIR spectrometer (PerkinElmer, Torrence, MA, USA) with a resolution of 4 cm-1 and averaging 16 scans for each spectrum. The bulk samples were defatted by centrifugation prior to assessment. Approximately 0.5 mL of sample was added to an attenuated total reflectance (ATR) cell. The spectra of three sub samples of each sample were taken by refilling the ATR cell. Background spectrum was scanned at the beginning of the measurements with a blank Diamond ATR cell using same instrumental conditions as for the sample spectra acquisition. The FTIR spectra of all samples were exported to Unscrambler software (Version 10.2, CAMO AS, Trondheim, Norway). Spectra were baseline corrected by subtracting with water as a background. The spectra were and smoothed using Savitzky-Golay smoothing to remove the vibrations resulting from the noise of derivation and for better identification of the peaks. The second derivative spectra were applied only in the region 1700-1600 cm 1 (Amide I) for enhancing the resolution of the peaks related to structural changes of proteins.

[0214] Analysis of the secondary structural elements by FTIR showed that, as a proportion of the total secondary structure elements the proportion of structural elements having a wavelength of 1689-1698 was 8.2%, 21.9%, 7.8% and 6.2% for Sl-4 respectively and the proportion of structural elements having a wavelength of 1675-1698 cm'1was 23.6%, 61.4%, 25.0% and 16.2% for Sl-4 respectively. These results demonstrate structural differences between the proteins in pasteurised milk, milk that is pasteurised and subjected to high pressure processing, milk subjected to pasteurisation under UHT conditions and raw milk. The results indicate that S2 exhibited more whey protein denaturation and aggregation than any other treatment, and the aggregates displayed a different secondary structure to the other treatments.

[0215] Example 2

[0216] The milk samples as described in Example 1 were subjected to in-vitro digestion using an INFOGEST digestion protocol such as described by Brodkorb et al., 2019, INFOGEST static in vitro simulation of gastrointestinal food digestion (2019) Nature Protocols, 14, 991-1014.The change in soluble nitrogen was measured as a proportion of total nitrogen obtained by centrifugation of the samples at 14,000g upon pH adjustment to pH 4.6 using an acetate buffer. The change in soluble N % at the end of gastric digestion was 28.3%, 58.5%, 44.1% and 17.7%, and at the end of intestinal digestion was 57.7%, 77.9%, 64.3% and 50.5% for SI -4 respectively.

[0217] The amino acid release was measured. At the end of digestion the average essential amino acid release was 56.9%, 65.7%, 57.5% and 56.8%, leucine release was 76.4%, 88.1%, 78.91% and 69.8%, and isoleucine release was 81.9%, 98.0%, 84.4% and 82.3% for Sl-4 respectively. These results indicate that S2 is more readily hydrolysed during digestion in this model, which mimics the action of gastric enzymes in human digestion.

[0218] Example 3

[0219] The milk samples as described in Example 1 were assessed for their digestive properties, including the formation of clots and the structure of any clot formed.

[0220] The water holding capacity was assessed by acidifying to pH 4.6 with a sodium acetate buffer and centrifuging at 1250xg for lOmins at 20C. The expelled whey was drained and weighed and the water holding capacity calculated as g ftO / g DM. The results were 3.51, 4.81, 3.60 and 3.18 for Sl-4 respectively.

[0221] The results indicate that S2 had a greater water holding capacity, indicating a more porous and open clot structure. This was supported by visual assessment of the freeze-dried clots.

[0222] Example 4

[0223] Raw milk was collected from a dairy farm located in the Sunshine Coast, Queensland, Australia and maintained at <5°C until processed. One portion of the milk (SI) was heated to about but not exceeding 40°C, immediately bottled and subjected to high pressure at 600MPa for 3 minutes, then cooled to 4°C. A second portion of the milk (S2) was heated to about 72°C and held for about 15 seconds, cooled to about 40°C, bottled and subjected to high pressure at 600MPa for 3 minutes, then cooled to 4°C. For both samples, the temperature at pressure did not exceed 85°C.

[0224] Both samples were defatted to remove cream by centrifugation at 4°C for 20 min at 3225 *g (Avanti J-26XP, Beckman instrument Australia Pty. Ltd, Gladesville, NSW, Australia), and the skim milk portion analysed, thus eliminating any differences in fat content or structure.Protein determination by Kjeldahl supra, showed the proportion of insoluble protein at pH4.6 was 87%, 91% for Sl-2 respectively.

[0225] HPLC analysis showed the proportion of denatured a-lactalbumin was 22.4% and 49.7% for Sl-2 respectively. FTIR analysis of the secondary structural elements carried out in the same way as Example 1 showed that, as a proportion of the total secondary structure elements, the proportion of structural elements having a wavelength of 1675-1698 cm'1was 42.6% and 61.4% for Sl-2 respectively and that the proportion of structural elements having a wavelength of 1689-1698 was 10.3% and 21.9% for Sl-2 respectively.

[0226] These results indicate that S2 exhibited more whey protein denaturation and aggregation compared to SI.

[0227] Example 5

[0228] Raw milk was bottled at 4°C, then High Pressure processed at 600MPa for 3min (SI). A second portion of the milk (S2) was heated to about 72°C for about 15 seconds, then cooled to about 40°C, bottled and subjected to high pressure at 600MPa for 3 minutes, then cooled to 4°C (S2). Meanwhile, a batch of UHT milk was obtained, having been through a UHT pasteurisation step then cooled. The first half was transferred into bottles with no additional processing (S3), while the second half was transferred into bottles and heated to about 40°C then High Pressure processed at 600MPa for 3 min before cooling to 4°C (S4). Finally, a batch of ultrafiltered milk was obtained, having been through an ultrafiltration step to concentrate proteins, and a pasteurisation step at about 72°C for about 15 seconds. The first half was transferred into bottles with no further processing (S5), while the second half was transferred into bottles and heated to about 40°C then High Pressure processed at 600MPa for 3 min (S6). All batches were immediately cooled to 4°C after processing. For each sample subjected to High Pressure processing, the temperature at pressure did not exceed 85°C.

[0229] Protein determination by Kjeldahl supra, showed the proportion of insoluble protein at pH4.6 was 82% and 91% for Sl-2 respectively.

[0230] The proportion of denatured P-LG as determined by RP-HPLC was 38.9%, 97.7%, 83.7%. 98.0%, 79.7% and 98.9% for Sl-6 respectively. The proportion of denatured a-lactalbumin by RP-HPLC was 0.1%, 49.7%, 37.0% and 67.2% for SI, 2, 5-6 respectively. The proportion of denatured BSAby RP-HPLC was 29.0%, 57.7%, 58.9% and 69.7% for SI, 2, 5-6 respectively.FTIR analysis carried out in the same way as Example 1, showed that as a proportion of the total secondary structure elements, the proportion of structural elements having a wavelength of 1675-1698 cm'1was 24.6%, 61.4%, 25.7%, 37.7%, 17.7% and 52.4% for Sl-6 respectively and that the proportion of structural elements having a wavelength of 1689-1698 was 7.1%, 21.9%, 8.2%, 11.8%, 1.4% and 12.7% for Sl-6 respectively.

[0231] Thus, all products treated according to the methods described herein exhibited greater P-LG and a-La denaturation than those using alternative heat or pressure treatments, with higher proportion of secondary aggregation structures.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A process for making a nutritional composition comprising an aggregate of a first protein which is a casein protein and a second protein comprising a cysteine residue, the process comprising subjecting an aqueous composition comprising a casein micelle comprising the first protein which is a casein protein, and a second protein comprising a cysteine residue, to the following separate steps, in any order:a) a pasteurisation step, in which a temperature of at least 60°C is applied for a duration capable of pasteurising the aqueous composition; andb) a high pressure processing (HPP) step comprising applying a pressure of about 500 MPa + / - 10% to 750 MPa + / - 10% to the aqueous composition at a temperature and for a duration capable of at least partially fragmenting the casein micelle; wherein the aqueous composition has a pH of greater than about 6 prior to step a) or step b), the temperature of the aqueous composition at pressure does not exceed 85°C and wherein the process at least partially denatures the second protein to allow a covalent bond to form between a thiol group on a casein protein, which thiol group is only exposed in the at least partially fragmented casein micelle and a thiol group on the second protein to form the aggregate and increases the proportion of structural elements having a wavelength of 1675-1698 cm'1to total secondary structure elements of the aggregate, and increases the proportion of structural elements having a wavelength of 1689-1698 cm'1to total secondary structure elements of the aggregate, when measured by Fourier Transform Infrared spectroscopy, compared to an aqueous composition comprising the casein protein and the second protein to which the process has not been applied.

2. A nutritional composition produced by the process of claim 1.

3. A method for improving digestibility of an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein, the method comprising subjecting the aqueous composition to the processes of claim 1.

4. A method for improving release of one or more amino acids from an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein, the method comprising subjecting the aqueous composition to the processes of claim 1.

5. A method for reducing intolerance and gut discomfort in a subject in need thereof when administering an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein, the method comprising subjecting the aqueous composition to the processes of claim 1.

6. A method for reducing an inflammatory, immune or allergic response when administering an aqueous composition comprising a casein micelle comprising a first protein which is a casein protein and a second protein to a subject, the method comprising subjecting the aqueous composition to the processes of claim 1.

7. Use of the nutritional composition of claim 2 or the method of any one of claims 3 to 6 for any one or more of:a) maintaining or increasing muscle protein synthesis,b) maintaining or increasing muscle mass,c) preventing or reducing loss of muscle mass,d) maintaining or increasing growth,e) preventing or decreasing muscle catabolism,f) preventing or treating cachexia,g) preventing or treating sarcopenia,h) modulating blood sugar levels,i) increasing insulin response to raised blood glucose concentration,j) increased satiety,k) reduced satiation,l) reducing food intake,m) reducing calorie intake,n) improving glucose metabolism,o) increasing rate of recovery following surgery, injury or exercise, andp) increased sports performance.

8. A method of treating a muscle wasting disorder or condition involving abnormal muscle synthesis such as cachexia and muscular dystrophy or for treatment of obesity, in a subject in need thereof, the method comprising administering the nutritional composition of claim 2.

9. The nutritional composition of claim 2 or the process of claim 1 or the method of any one of claims 3 to 6 or the use of claim 7, in which the casein protein is one or more selected from the group consisting of aSl-CN, aS2-CN, P-CN and K-CN or combinations thereof or aSl-CN, aS2-CN and P-CN or combinations thereof.

10. The nutritional composition of claim 2 or the process of claim 1 or the method of any one of claims 3 to 6 or the use of claim 7, or the nutritional composition, process, method or use of claim 9, in which the second protein is an animal protein selected from the group consisting of collagen, gelatin, albumin, an enzyme or a whey protein.

11. The nutritional composition, process, method or use of claim 10, in which the whey protein is selected from the group consisting of P-lactoglobulin, a-lactalbumin, glycomacropeptide, bovine serum albumin or other non-bovine animal milk albumins, Immunoglobulin, lactoferrin, lactoperoxidase, lysozyme, proteose peptone or a mixture thereof.

12. The process of claim 1 in which the casein micelle further comprises a calcium cation, optionally as a calcium salt, optionally as calcium phosphate, optionally as colloidal calcium phosphate, and the aggregate comprises said calcium cation.

13. The nutritional composition of claim 2 in which the aggregate further comprises a calcium cation, optionally provided as a calcium salt, optionally as calcium phosphate, and optionally as colloidal calcium phosphate.

14. The process of claim 1 or the method of any one of claims 3 to 6 or claim 8 or the use of claim 7, in which the aqueous composition is cow’s milk or a fraction or product thereof comprising a casein protein and a whey protein.

15. A nutritional composition produced by the processes of claim 14.

16. A method for improving the bio-accessibility of calcium when administering an aqueous composition comprising a casein micelle comprising a first protein which is casein and calcium, optionally as calcium phosphate, optionally as colloidal calcium phosphate, anda second protein to a subject in need thereof, the method comprising subjecting the aqueous composition to the processes of claim 14.

17. Use of the nutritional composition of claim 15 or produced by the process of claim 12 or claim 14 for increasing calcium absorption in a subject in need thereof and optionally for treating osteoporosis, osteopenia, hypocalcemia, Rickets and osteomalacia, preeclampsia and improving cardiovascular health or for improving skin health, hair strength, nail health, oral health and bone structure.

18. The process of claim 1, 12 or claim 14 in which the pasteurisation step comprises applying a temperature of about 63°C (145°F) for about 30 minutes or a temperature of about 72°C (162°F) for about 15 seconds, and yet higher temperatures for shorter periods of time, including applying a temperature of 138-150 °C (280-302 °F) for one or two seconds.

19. The process of claim 1, 12 or claim 14, wherein the temperature of the aqueous composition does not exceed 85°C, 84°C, 83°C, 82°C, 81°C, 80°C, 79°C, 78°C, 77°C, 76°C or 75°C during the process.

20. The process of claim 1, 12 or claim 14, wherein the temperature of the aqueous composition is at least about 25°C, or at least about 35°C, or at least about 45°C, or at least about 55°C, or at least about 65°C during the HPP step when at pressure or is below about 45°C, or below about 55°C, or below about 65°C, or below about 75°C or below 84°C during the HPP step when at pressure.

21. The process of claim 1, 12 or claim 14, wherein the HPP step comprises applying a pressure of about 500 MPa to about 750 MPa, optionally between 500 MPa and 650 MPa and optionally about 600 MPa for a duration ranging from about 1 minute to about 10 minutes, optionally for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes.

22. The nutritional composition of claim 2, 13 or claim 15 in the form of a food, drink, ready to drink nutritional composition, food additive, drink additive, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical or a cultured product, a yoghurt or cheese or an intermediate in the preparation thereof.

23. The nutritional composition of claim 2, 13, 15 or claim 22 in the form of a powder or a flowing solid or semi-solid product.

24. The process of claim 1, 12 or claim 14, further comprising formulating the nutritional composition as a food, drink, ready to drink nutritional composition, food additive, drink additive, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical or a cultured product, a yoghurt or cheese or an intermediate in the preparation thereof.

25. The process of claim 1, 12 or claim 14, further comprising formulating the nutritional composition as a powder or a flowing solid or semi-solid product.

26. The nutritional composition of claim 2, 13, 15 or 22 or the process of claim 2, 12, 14 or claim 23, in which the nutritional composition is more readily hydrolysed by human gastric enzymes than a composition in which a casein protein is not bonded to second protein by a covalent bond.