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Title:
HIGHLY DIGESTIBLE PROTEIN-RICH NUTRITIONAL COMPOSITIONS, USES THEREOF, AND METHODS FOR PREPARING THE SAME
Document Type and Number:
WIPO Patent Application WO/2019/170707
Kind Code:
A1
Abstract:
The invention relates to highly digestible protein-rich nutritional compositions, and the use thereof in methods for maintaining or increasing muscle mass. Provided is the use of a nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a non-coagulating protein and a coagulating protein in a relative weight ratio in the range of from 22:78 to 70:30, wherein the non- coagulating protein comprises whey proteins and globular serum proteins and coagulating protein comprises casein or caseinate, and wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein the remainder fraction of said non-coagulating protein is non-bound, for : a) preventing or reducing coagulation in the upper gastro-intestinal tract; b) increasing the rate of gastric emptying; c) enhancing protein digestion and amino acid absorption; d) increasing the blood serum concentration of free essential amino acids, preferably leucine; and/or e) enhancing muscle protein synthesis, increasing muscle mass, strength and function, in a subject.

Inventors:
GANZEVLES, Renate Antonia (6700 AE Wageningen, 6700 AE, NL)
KUDLA, Urszula (6700 AE Wageningen, 6700 AE, NL)
NIEUWENHUIJSE, Johannes Andries (6700 AE Wageningen, 6700 AE, NL)
TIMMER-KEETELS, Christina Josephina Antonia Maria (6700 AE Wageningen, 6700 AE, NL)
VAN DIJCK, Willemina Gerharda Frederika Maria (6700 AE Wageningen, 6700 AE, NL)
ARRUDA PEREIRA VALENÇA DE SOUSA, Joana (6700 AE Wageningen, 6700 AE, NL)
VAN DE VEN, Martinus Johannes Maria (6700 AE Wageningen, 6700 AE, NL)
Application Number:
EP2019/055502
Publication Date:
September 12, 2019
Filing Date:
March 06, 2019
Export Citation:
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Assignee:
FRIESLANDCAMPINA NEDERLAND B.V. (Stationsplein 4, 3818 LE Amersfoort, 3818 LE, NL)
International Classes:
A23L33/19; A23J3/08; A23C21/06
Attorney, Agent or Firm:
FRIESLANDCAMPINA NEDERLAND B.V. (Bronland 20 P.O. Box 238, 6700 AE Wageningen, 6700 AE, NL)
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Claims:
Claims

1. The use of a nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a non-coagulating protein and a coagulating protein in a relative weight ratio in the range of from 22:78 to 70:30, wherein the non coagulating protein comprises whey proteins and globular serum proteins and coagulating protein comprises casein or caseinate, and wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein the remainder fraction of said non-coagulating protein is non-bound, for :

a) preventing or reducing coagulation in the upper gastro-intestinal tract; b) increasing the rate of gastric emptying;

c) enhancing protein digestion and amino acid absorption;

d) increasing the blood serum concentration of free essential amino acids, preferably leucine; and/or

e) enhancing muscle protein synthesis, increasing muscle mass, strength and function,

in a subject.

2. Use according to claim 1, wherein at least 50w% of the non-bound non coagulating protein in the composition is in a native state.

3. Use according to claim 2, wherein the native, non-bound non- coagulating protein is present in an amount of at least 0.1 wt%, preferably at least 0.3 wt%, most preferably at least 0.5% based on the total weight of the composition.

4. Use according to claim 2 or 3, wherein the native-non-bound non coagulating protein is present in an amount of at least 1.5%, preferably at least 5%, more preferably at least 10% by weight of total protein in the composition.

5. Use according to any one of the preceding claims, wherein the weight ratio of bound non- coagulating protein to coagulating protein is at least 1 to 20, preferably at least 1 to 10, more preferably at least 1 to 8, and wherein the remainder part of non- coagulating protein is not bound and native.

6. Use according to any one of the preceding claims, wherein the nutritional composition comprises a mixture of non- coagulating protein and coagulating protein in a relative weight ratio in the range of from 25:75 to 60:40, preferably from 30:70 to 60:40, and more preferably from 30:70 to 55:45.

7. Use according to any one of the preceding claims, wherein the whey protein in the composition comprises whey proteins from milk, from cheese whey, from acid casein whey or from milk serum.

8. Use according to any one of the preceding claims, wherein the casein or caseinate in the composition is selected from the group consisting of micellar casein, non-micellar casein, sodium caseinate, calcium caseinate, potassium caseinate and magnesium caseinate.

9. Use according to any one of the preceding claims, wherein the coagulating protein is micellar casein and wherein the non- coagulating protein is globular serum protein.

10. Use according to any one of the preceding claims, wherein the bound fraction of said non- coagulating protein is obtained by exposure of a mixture of said non- coagulating and said coagulating protein to a process involving heat- treatment, preferably high temperature pasteurization, or high pressure treatment, or homogenization.

11. Use according to any one of the preceding claims, wherein the non-bound fraction of said non- coagulating protein is obtained by adding a source of non coagulating protein to a composition comprising non- coagulating protein that is bound to coagulating protein, wherein said source comprises at least 50w%, preferably at least 60w%, non- coagulating protein in a native state.

12. Use according to any one of the preceding claims, wherein said mixture of coagulating protein and non-coagulating protein makes up at least 70% by weight of total protein in the composition.

13. Use according to any one of the preceding claims, wherein the nutritional composition is a fermented or an acidified nutritional composition, preferably selected from the group consisting of sour milk products and/or acidified fresh products, like yoghurt, fermented milk, viili, fermented cream, sour cream, quark, butter milk, kefir, and dairy shot drinks.

14. Use according to any one of the preceding claims, wherein the composition is in the form of a drinkable or spoonable liquid, a chewable substance, or a powder.

15. A method for providing a nutritional composition comprising at least 4 wt% of protein, comprising a non- coagulating protein and a coagulating protein, wherein the non- coagulating protein comprises whey proteins and globular serum proteins and coagulating protein comprises casein or caseinate, and wherein the weight ratio of non- coagulating protein to coagulating protein is in the range of 22:78 to 70:30, comprising the steps of

(i) heating a mixture comprising a coagulating protein and a non

coagulating protein under conditions to obtain protein denaturation; and

(ii) adding to said heated mixture a source of non- coagulating protein, wherein at least 50w% of said non- coagulating protein is in a native state, to obtain a composition wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein another fraction of said non-coagulating protein is native and non-bound.

16. Method according to claim 15, wherein the coagulating protein is selected from the group consisting of micellar casein, non-micellar casein, sodium caseinate, calcium caseinate, potassium caseinate and magnesium caseinate and/or wherein the non-coagulating whey proteins and globular serum proteins are derived from milk, from cheese whey, from acid casein whey or from milk serum.

17. Method according to claim 15 or 16, wherein step (i) comprises heating to a temperature of 85°C for at least 5 minutes, preferably at least 10 minutes.

18. Method according to any one of claims 15-17, wherein no UHT treatment or similar severe heat treatment is performed after step (ii).

19. Method according to any one of claims 15-18, further comprising following step (i) or (ii) a step of fermenting the composition.

20. A nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a non- coagulating protein and a coagulating protein in a relative weight ratio in the range from 22:78 to 70:30, and wherein a fraction of said non coagulating protein is bound to said coagulating protein and wherein another fraction of said non- coagulating protein is non-bound, for use in a method of treatment and/or prevention of a disease or condition linked to a loss of muscle mass and/or strength.

21. Nutritional composition for use in a method according to claim 20, wherein the disease or disorder is selected from the group consisting of a decline of lean body mass, muscle decline, bone decline, sarcopenia, cachexia, osteoporosis and osteosarcopenia.

22. A method for

a) preventing or reducing coagulation in the upper gastro-intestinal tract; b) increasing the rate of gastric emptying;

c) enhancing protein digestion and amino acid absorption;

d) increasing the blood serum concentration of free essential amino acids, preferably leucine; and/or

e) enhancing muscle protein synthesis, increasing muscle mass, strength and function,

in a subject, comprising administering to the subject a nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a non- coagulating protein and a coagulating protein in a relative weight ratio in the range of from 22:78 to 70:30, wherein the non-coagulating protein comprises whey proteins proteins and the coagulating protein comprises casein or caseinate, and wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein the remainder fraction of said non-coagulating protein is non-bound.

23. Method according to claim 22, wherein the subject suffers from a disease or disorder selected from the group consisting of a decline of lean body mass, muscle decline, bone decline, sarcopenia, cachexia, osteoporosis and osteosarcopenia.

24. Method according to claim 22 or 23, wherein at least 50w% of the non bound non-coagulating protein in the composition is in a native state.

25. Method according to any one of claims 22-24, wherein the native, non bound non-coagulating protein is present in an amount of at least 0.1 wt%, preferably at least 0.3 wt%, most preferably at least 0.5% based on the total weight of the composition.

26. Method according to any one of claims 22-25, wherein the native-non bound non-coagulating protein is present in an amount of at least 1.5%, preferably at least 5%, more preferably at least 10% by weight of total protein in the composition.

Description:
Title: Highly digestible protein-rich nutritional compositions, uses thereof, and methods for preparing the same.

FIELD OF THE INVENTION

The invention relates to highly digestible protein-rich nutritional compositions, and to uses thereof, e.g. in methods for maintaining or increasing muscle mass.

BACKGROUND TO THE INVENTION

It is well established that ingestion of a meal-like amount of protein in the form of free amino acids, milk protein, or beef strongly stimulates skeletal muscle protein synthesis. This postprandial muscle protein synthetic response depends, amongst others, on the quantity, timing and the type of protein that is ingested. Previous work suggests that whey protein ingestion results in greater

postprandial protein retention than does casein ingestion. See for example Dangin et al. (J Nutr. 2002;132:3228S-33S; J Physiol 2003;549:635-44).

Milk of all mammals contains caseins, globular proteins and in addition some non-protein N-containing components (NPN) - in cows milk about 5% of the total N. Of the true protein in cows milk, typically 82-83% is casein, and 17-18% is globular proteins, also called‘globular serum proteins’. Caseins are non- globular proteins having little secondary structure. Hence they cannot be denatured by heat. In milk of all mammals, they are associated into particles containing some 10-30 000 molecules, the so-called casein micelles; association is by weak interactions, mainly electrostatic and hydrophobic. Caseins can be separated from the other milk constituents by renneting, acidification or microfiltration. The resulting high-casein product is referred to as, respectively, cheese-curd, acid-casein and Micellar-Casein-Isolate. The remaining liquid is called cheese whey, acid-casein whey and milk serum, respectively. The wheys and milk serum are rich in globular serum proteins, often called‘whey protein’, and in NPN. The proteins can be separated from whey or milk serum using ultrafiltration, yielding Whey Protein Concentrate (WPC) or Serum Protein Concentrate (SPC). Most of the proteins in WPC and SPC are globular proteins, also called‘globular serum proteins’. Cheese whey and whey protein concentrate from cheese whey further contain Casein Macropeptide (CMP) which is not a globular protein. The globular proteins from milk do not coagulate at the conditions in the stomach of humans, if they are in the native (folded) state. However, many standard processes as applied in the food (dairy) industry, e.g. high-pasteurisation or high-pressure treatment, result in the denaturation and aggregation of the globular proteins. Denatured and aggregated globular proteins coagulate at the conditions in the stomach of humans. Caseins always coagulate at the conditions in the stomach of humans; they cannot be denatured. Caseins in cheese-curd are already coagulated at the moment of consumption. As used herein, ‘globular serum protein’ is also referred to as‘whey protein’.

The greater anabolic properties of whey protein than of casein are mainly attributed to the amino acid composition and the faster digestion and absorption kinetics of the globular proteins. The latter results in a greater increase in postprandial plasma amino acid availability and thereby a greater increase in muscle protein synthesis. In particular, nutritional compositions mainly containing casein and/or caseinate tend to coagulate in the stomach. Hence, casein is often referred to as‘’coagulating, or slow protein” whereas whey protein is an example of a‘’non- coagulating, or fast protein”.

Besides differences in protein digestion and absorption kinetics, whey protein and casein also markedly differ in their amino acid composition. Whey protein has a considerably higher leucine content. The latter may also contribute to the proposed greater anabolic properties of whey protein than of casein, because leucine has been identified as the main nutritional signal responsible for stimulating postprandial muscle protein accretion. This is in line with the study by Luiking et al. (Nutr J. 2014; 13: 9), reporting that a specifically designed nutritional supplement high in whey protein and enriched in leucine is more effective than a conventional dairy product to stimulate postprandial muscle protein synthesis overall in healthy older subjects. This effect was attributed to the higher plasma levels of leucine and essential amino acids after intake of the high whey protein, leucine-enriched supplement. Hence, it is clear that the intake of dairy protein, especially whey protein, is highly effective for the maintenance or growth of skeletal muscle mass. In order to enhance the stimulatory effect on muscle protein synthesis, increasing the whey protein content of a consumer product by adding whey protein would be desirable. However, whey protein fractions are relatively expensive. In addition, pasteurizing heat treatments that are typically required for bacteriological safety induce gelling and sedimentation of whey protein, resulting in highly viscous products with limited processability. Thus, for heat-treated products it is technically challenging to include markedly higher whey protein concentrations.

It would therefore be desirable to provide improved protein-rich nutritional compositions that do not suffer from the above drawbacks. The present inventors especially sought for the manufacture of protein-rich compositions that are easily digestible, thereby allowing for increased levels of plasma leucine, without sacrificing the viscosity of the composition. Among others, they aimed at providing a fermented, e.g. yoghurt-type, liquid nutritional composition for use as dietary or therapeutical protein supplement for subjects wherein muscle growth/recovery is desired, including athletes, older persons, infants and ill or malnourished patients.

It was surprisingly found that at least some of the above goals could be met by the steering of certain process parameters, including the pretreatment of different ingredients and the relative order of adding the ingredients during the manufacturing process. More specifically, a novel process was designed wherein a coagulating protein (casein) is heated in the presence of one part of a non coagulating protein (whey protein or globular serum protein), followed by the addition of another part of the non- coagulating protein as raw or mildly pasteurized ingredient such that these proteins remain essentially native.

Herewith, a fraction of the non- coagulating protein becomes bound/aggregates onto the coagulating protein, while the other fraction of non- coagulating protein remains non-bound or native. The 2- step procedure of“combined heating” and “post-heating addition” is optionally followed by a fermentation step. As an alternative to fermentation, acidification with mineral acids / organic acids / acidulants (e.g. glucono delta lactone; GDL) is also possible. The resulting protein dense product displays desirable processability properties combined with a surprisingly high digestion rate, presumably by tuning the interactions between the coagulating and non-coagulating protein ingredients.

DETAILED DESRIPTION OF THE INVENTION

Accordingly, disclosed is a nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a coagulating protein and a non coagulating protein in a relative weight ratio in the range of 22:78 to 70:30, and wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein the remainder fraction of said non- coagulating protein is non-bound, for use in any one or more of the following:

a) preventing or reducing coagulation in the upper gastro-intestinal tract; b) increasing the rate of gastric emptying;

c) enhancing protein digestion and amino acid absorption;

d) increasing the blood serum concentration of free essential amino acids, preferably leucine;

e) enhancing muscle mass and/or muscle strength

in a subject.

In particular, the invention provides the use of a nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a non- coagulating protein and a coagulating protein in a relative weight ratio in the range of from 22:78 to 70:30, wherein the non-coagulating protein comprises whey proteins and globular serum proteins and coagulating protein comprises casein or caseinate, and wherein a fraction of said non- coagulating protein is bound to said coagulating protein and wherein the remainder fraction of said non- coagulating protein is non-bound, for :

a) preventing or reducing coagulation in the upper gastro-intestinal tract; b) increasing the rate of gastric emptying;

c) enhancing protein digestion and amino acid absorption;

d) increasing the blood serum concentration of free essential amino acids, preferably leucine; and/or e) enhancing muscle protein synthesis, increasing muscle mass, strength and function,

in a subject.

Accordingly, also provided herein is a method for

a) preventing or reducing coagulation in the upper gastro-intestinal tract; b) increasing the rate of gastric emptying;

c) enhancing protein digestion and amino acid absorption;

d) increasing the blood serum concentration of free essential amino acids, preferably leucine; and/or

e) enhancing muscle protein synthesis, increasing muscle mass, strength and function,

in a subject, comprising administering to the subject a nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a non- coagulating protein and a coagulating protein in a relative weight ratio in the range of from 22:78 to 70:30, wherein the non-coagulating protein comprises whey proteins and the coagulating protein comprises casein or caseinate, and wherein a fraction of said non- coagulating protein is bound to said coagulating protein and wherein the remainder fraction of said non- coagulating protein is non-bound.

In one embodiment, the protein-rich composition finds its use in medical applications. For example, provided is a nutritional composition comprising at least 4 wt% of protein, comprising a mixture of a coagulating protein and a non coagulating protein in a relative weight ratio in the range of 22:78 to 70:30 (preferably 22:78 to 50:50), wherein the non-coagulating protein comprises whey proteins and globular serum proteins and coagulating protein comprises casein or caseinate, and wherein a fraction of said non- coagulating protein is bound to said coagulating protein and wherein another fraction of said non- coagulating protein is non-bound, for use in a method of treatment and/or prevention of a disorder or conditions linked to a loss of muscle mass and/or strength. In one aspect, the disorder is selected from the group consisting of a decline of lean body mass, decline of muscle function, bone mass decline, sarcopenia, cachexia, muscle loss with decreased activity or any form of inactivity, osteoporosis, and any

combination thereof. A further embodiment of the invention relates to a method for providing a nutritional composition comprising at least 4 wt% of protein, wherein the non coagulating protein comprises whey proteins and globular serum proteins and coagulating protein comprises casein or caseinate, comprising a non- coagulating protein and a coagulating protein and wherein the ratio of non- coagulating protein to coagulating protein is in the range of from 22:78 to 70:30, preferably from 22:78 to 50:50, comprising the steps of

(i) heating a mixture comprising a coagulating protein and a non coagulating protein under conditions to achieve binding of said non-coagulating protein to said coagulating protein; and

(ii) adding to said heated mixture a source of non-coagulating protein wherein at least 50w% of non- coagulating protein in said source is in a native state, to obtain a composition wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein the remained fraction of said non-coagulating protein is native and non-bound.

Nutritional compositions comprising mixtures of heat-treated coagulating and non-coagulating (dairy) proteins are known in the art. For example,

W02010/131952 relates to a method for reducing digestive coagulation of proteins. Disclosed is the use of anti- coagulating protein in the manufacture of a nutritional composition that further comprises coagulating protein, for use in preventing or reducing coagulation in the upper gastro-intestinal tract of a subject of said coagulating protein in said nutritional composition. According to W02010/131952, the preferred coagulating protein comprises caseinate and the preferred anti-coagulating protein comprises pea protein or soy protein or both. W02010/131952 is silent about any combined heat treatment and/or adding of non-coagulating protein prior and after heating.

W02014/011039 relates to a process of producing a composition comprising at least two different proteins, of which at least one is a coagulating protein preferably a casein, and at least one is an anti-coagulating protein, preferably pea, soy whey- protein or a combination thereof, comprising the steps of: a) heat- sterilising a first liquid component comprising said coagulating protein, b) heat- sterilising a second liquid component comprising said anti- coagulating protein, and c) mixing said first component with said second component to obtain a mixture thereof. The obtained mixture is useful as a food constituent having reduced coagulation in the upper gastro-intestinal tract. In contrast to the present invention, the heat- sterilisation steps a) and b) of the process of

W02014/011039 are performed such that the first liquid component and the second liquid component are not mixed with each other during heating steps a) and b). Whereas according to W02014/011039 a further protein may be added to the mixture as third component, this third protein is distinct from the

coagulating protein and the anti- coagulating protein. Additionally, the third protein is preferably heat-sterilized as well.

W02016/097308 relates to yoghurt products of good quality, both with regard to physical and sensory properties, comprising high amounts of proteins including high amounts of native whey proteins, and methods for manufacturing such yoghurt products. Disclosed is a process comprising mixing a casein-rich fraction and a native whey protein concentrate and thereby obtaining a mixture of casein and native whey protein wherein the whey protein to casein ratio is increased compared to the ratio of whey protein to casein in the raw milk; and subjecting the mixture of casein and native whey protein to heat treatment and adding a starter culture to the heat treated mixture of casein and whey protein to provide a fermented mixture. According to W02016/097308, the heat treatment is carried out by heating the mixture of casein and native whey protein at a temperature and for a time period sufficient to obtain denaturation of only 30 to 70 % of the native whey protein of the mixture. Thus, whereas W02016/097308 also relates to heating a mixture of casein and whey, this heat treatment is milder and the denaturation degree is lower as compared to a process of the present invention. Moreover, W02016/097308 fails to teach or suggest the subsequent step of adding native/undenatured whey to the heated mixture.

W02010/123351 relates to whey proteins and the use thereof in (milk- based) compositions for body weight management. Disclosed is a high (8wt%) protein yoghurt to which whey proteins are added separately after a heat treatment of skim milk. However, in contrast to the present invention, the whey protein sources used are either Hiprotal60MP or Vivinal Alpha, both comprising predominantly denatured (i.e. non-native) whey proteins.

US2016/262412 pertains to a high protein, fruit flavoured beverage comprising fruit flavouring agents and high protein denatured whey protein compositions, and to a method of producing the beverage. Nothing is mentioned about digestibility properties or specific applications relating thereto, e.g.

treatment and/or prevention of a disease or condition linked to a loss of muscle mass strength. US2016/262412 discloses a yoghurt base comprising whey proteins and casein, to which a fruit mixture comprising whey protein is added. The whey protein in the fruit mixture is predominantly present as thermally denatured whey protein in the form of insoluble whey protein particles in the size range 0.5-10 micron.

W02014/011040 relates to a protein and lipid comprising composition with reduced digestive coagulation. Disclosed is a process of producing a composition comprising at least two different proteins, of which at least one is a casein and at least one is an anti-coagulating protein, preferably a non-dairy protein such as pea or soy protein. The process comprising the steps of: a) heat- sterilizing a first liquid component comprising casein in an amount of at least 85wt% of the total protein content of the first component, b) heat- sterilizing a second liquid component comprising the anti- coagulating protein, and c) mixing the first component with the second component to obtain a mixture thereof. Whereas whey protein may be combined and heat- sterilized with the soy or pea protein, W02014/011040 is completely silent about adding a source of native whey to this mixture. In contrast, W02014/011040 teaches that any optional further protein must also be heat-sterilized. Furthermore, the resulting product is heat sterilized and will therefore not contain any substantial amount of native whey.

Thus, a method for providing a composition of the invention and the methods and uses of the composition as herein disclosed are not derivable from the prior art. Uses of the composition

Based on the unique and beneficial properties relating to gastric emptying, digestibility and amino acid release of a composition comprising bound and non bound non-coagulating protein, a person skilled in the art will recognize and appreciate the wide variety of possible therapeutic and non-therapeutic uses thereof.

In one aspect, the use is of non-medical or non-therapeutic nature. For example, the composition is used in any one or more of the following:

a) preventing or reducing coagulation in the upper gastro-intestinal tract;

b) increasing the rate of gastric emptying;

c) enhancing protein digestion and amino acid absorption;

d) increasing the blood serum concentration of free essential amino acids, preferably leucine;

e) maintaining or enhancing muscle mass, and/or muscle strength, or attenuating muscle mass loss and/or muscle strength loss ;

in a subject, optionally wherein said use is not for the purpose of carrying out therapy on the animal or human body.

In one embodiment, the subject is a healthy subject, like an infant, child, adolescent, adult or aged subject. The subject may be a physically active subject, e.g. an athlete. In a specific aspect, the composition is for use in a subject in need of enhanced muscle growth and/or enhanced muscle recovery. For example, it is used in a subject involved in weight training in order to promote muscle growth. In this situation, the combination of lifting heavy weights and protein feeding is used to build new (contractile) muscle tissue. For endurance athletes, protein intake might also be important. In addition to high quality protein foods (e.g. fish/poultry/dairy/meat/eggs etc), protein supplements are an useful addition to the endurance athlete’s toolkit. Protein intake will not only help the muscle recover but may also increase the mitochondrial protein synthesis, which in turn will improve the energy metabolism (i.e. more efficient use of energy) of the muscle. Indeed, a protein-rich composition as provided herein which is high in the essential amino acid leucine (the key trigger to activate the process of muscle protein synthesis) and is often digested quicker than whole foods, thereby providing an efficient recovery strategy.

The nutritional composition for use as herein disclosed can be consumed at any time, e.g. as addition to a meal, as snack in between meals, before, during and after exercise, as well as prior to sleep as an extra opportunity for protein intake during the day.

In another embodiment, the invention provides a composition for use in the treatment and/or prevention of a condition linked to loss of muscle mass and/or strength in a subject. In yet another embodiment, the invention provides a method for treatment and/or prevention of a condition linked to loss of muscle mass and/or strength in a subject, comprising administering a composition of the invention to the subject. The subject may be an older or sick person,

malnourished, or someone subjected to inactivity (e.g. injury, illness, space flight, cast, immobilisation, hospitalization). For example, the subject, optionally an elderly subject, suffers from a decline of lean body mass, muscle wasting, muscle decline, bone decline, sarcopenia, cachexia, osteoporosis and/or osteosarcopenia.

The combined effect of osteoporosis and sarcopenia is a serious threat to (frail) older people. Musculoskeletal decline is associated with decreased independency and quality of life as well as excess mortality due to a decline in muscle mass. Muscle weakness is a contributor to increased fall risk and thereby age-related fracture risk. The loss of muscle mass contributes to reduction in mechanical loading, which usually stimulates bone formation. Older people with osteosarcopenia are more likely to experience impaired mobility and have the highest prevalence of atraumatic fractures. Women are more likely to experience osteosarcopenia, as they usually have lower lean muscle mass and strength, and lower bone mineral density (BMD) compared to men of the same age.

Nutritional Composition

A nutritional composition for use according to the invention is protein-rich to ensure an adequate supply of proteins / amino acids to a subject.

It comprises at least 4 wt% of protein, i.e. at least 4 g per 100 g, of the

composition. The composition can be in a solid, liquid, or semi-liquid (e.g. spoonable) form. For example, a protein-dense liquid nutritional composition for use according to the invention comprises at least 4 g of protein per 100 ml of the composition. The total protein that is present in the nutritional composition, i.e. the combination of all proteins present, may also be referred to as the“protein fraction” of the nutritional composition. The nutritional composition thus comprises a protein fraction of 4 wt% (i.e. at least 4 g per 100 g) of the

composition. In a preferred embodiment the composition comprises a protein fraction of at least 5 wt%, more preferably of at least 6 wt%, most preferably 7 wt% or higher. The upper limit of the protein concentration is not critical, and can be up to about 60 wt% e.g. for a powdered product. Typically, the protein content is up to about 50 wt%, preferably up to about 40 wt%.

In a preferred embodiment of the invention, the protein content is in the range from about 5 to 18 wt%. In a more preferred embodiment of the invention, the protein content is in the range from 6 to 15 wt%. In another preferred embodiment of the invention, the protein content is in the range from 6 to 18 wt%. For example, in one aspect the protein content is in the range from 6 to 9 wt% or from 7 to 9 wt%. In another embodiment of the invention, the protein content is in the range from 6.5 to 18 wt% . For example, the protein content may be in the range from 7 to 17 wt%. In still another preferred embodiment of the invention, the protein content is in the range from 7 to 15 wt%. In a more preferred embodiment of the invention, the protein content is in the range from 7 to 13 wt% . In another preferred embodiment of the invention, the protein content is in the range from 7 to 12 wt%. In yet another preferred embodiment of the invention, the protein content is in the range from 6 to 12 wt%, and in a most preferred embodiment of the invention, the protein content is in the range from 6 to 10 wt%.

Other preferred compositions comprise a protein fraction of at least 6 g, preferably at least 7 g, like 8 g or more, 9 g or more of protein per 100 g or per 100 ml of the composition. In a specific aspect, the composition is a liquid composition comprising 4-20 g protein per 100 ml of the composition, preferably 6-15 g, more preferably 6-12 g per 100 ml of the composition. In another specific aspect, the composition is a solid composition, for example or a sports bar, and comprises 4-20 g protein per 100 g of the composition, preferably 4-15 g, more preferably 5-12 g per 100 g of the composition. In yet another aspect, the composition is semi- solid, for example a yogurt, and comprises 4-20 g protein per 100 g of the composition, preferably 4-15 g, more preferably 5-12 g per 100 g of the composition. In another specific aspect, the composition is provided as a powder that is to be rehydrated prior to use, and is formulated to provide 4-20 g protein per 100 g of the final reconstituted composition, preferably 4-15 g, more preferably 5-12 g per 100 g of the final composition. For example, the powdered composition comprises 20 to 60w% protein on the basis of total solids. In a preferred embodiment, the powder comprising 30 tot 55 w % protein (TS).

A composition of the invention is further characterized in that it comprises a mixture of a coagulating protein and a non-coagulating protein and wherein the weight ratio of non-coagulating protein to coagulating protein is in in the range of from 22:78 to 70:30.

According to the invention, a fraction of the non-coagulating protein is bound to said coagulating protein and a remainder fraction of said non

coagulating protein is in a non-bound state, e.g. as free or native protein.

In a preferred embodiment, in order to provide improved digestibility properties, at least 50w% of the non-bound non-coagulating protein in the composition is in a native state. For example, at least 60w%, or better at least 70w%, or even better at least 75w%, of the non-bound non-coagulating protein in the composition is in a native state. In one embodiment, the native, non-bound non-coagulating protein is present in an amount of at least 0.1 wt%, preferably at least 0.3 wt%, most preferably at least 0.5% based on the total weight of the composition. For example, in one embodiment the composition comprises at least 0.1 w % of native, globular serum proteins.

In one aspect, the weight ratio of bound non- coagulating protein to coagulating protein is at least 1 to 20, preferably at least 1 to 10, more preferably at least 1 to 8, and the remainder part of non- coagulating protein is not bound and native.

Good results are obtained with a composition wherein the native, non bound non-coagulating protein is present in an amount of at least 1.5%, preferably at least 5%, more preferably at least 10% by weight of total protein in the composition.

In a composition for use according to the present invention, the coagulating protein is a dairy or milk protein, in particular casein or caseinate. In one embodiment, the composition comprises one or more coagulating protein selected from the group consisting of micellar casein, sodium caseinate, calcium caseinate, potassium caseinate and magnesium caseinate.

A non-coagulating protein is herein defined as a protein that does not coagulate in the stomach of a human person under normal digestive conditions. The coagulation capacity of a given protein can be established by in vitro methods known in the art. For example, for a non- coagulating protein this means that at least 85%, preferably at least 90% stays in the liquid phase in a stomach digestion model starting in the presence of artificial digestive juice as defined in example 1 after about 10-60 minutes at 37°C. According to the present invention, the non-coagulating protein comprises or consists of whey protein and globular serum protein. In a preferred embodiment, the coagulating protein is micellar casein and the non- coagulating protein is globular serum protein.

The globular proteins in milk consist of a mixture of beta-lactoglobulin (— 65%), alpha-lactalbumin (— 20%), bovine serum albumin (~6%), and

immunoglobulins. These are soluble in their native forms, independent of pH. As explained herein above, the non-coagulating proteins for use in the present invention can be obtained from milk using different processes. For example, they are obtained, typically by microfiltration, or from cheese whey that resulted from the renneting process. Hence, in one embodiment the non- coagulating protein is the protein in a Whey Protein Concentrate (WPC) from cheese whey. In another embodiment, it is the protein from a WPC derived from acid- casein whey that is obtained by acidification of milk. In yet another embodiment, the non

coagulating protein is the protein in a Serum Protein Concentrate (SPC) obtained by microfiltration of milk. To ensure that the resulting composition possesses advantageous digestibility properties, sources of whey proteins that have not been subjected or exposed to protein denaturing conditions, such as high temperature (e.g. UHT) treatment or calcium sequestration, are clearly preferred. Microparticulated whey proteins (MPW or mpWPC) refer to whey proteins that have undergone thermal and mechanical treatment to denature whey proteins and create particles similar to the size of fat globules in milk.

Thus, the use of microparticulated whey protein is not in line with the present invention.

The whey protein for use as non-coagulating protein according to the present invention can be intact or it can be (partially) hydrolysed. The intact whey protein may for example be a whey protein concentrate (WPC), whey protein isolate (WPI), serum protein concentrate (SPC) or serum protein isolate (SPI). As indicated above, WPC, WPI, SPC and SPI may be obtained by processes known in the art, such as the processing of sweet whey or acid whey,

ultrafiltration or microfiltration processes. Whey protein concentrate (WPC) typically comprises about 35 to about 80 wt.% protein, based on dry matter.

Whey protein isolate (WPI) and Serum protein isolate (SPI) typically comprise about 85 wt.% or more protein, based on dry matter. In one embodiment, the non-coagulating protein comprises or consists of a protein hydrolysate. For example, (partially) hydrolyzed protein obtained from acid whey protein, sweet whey proteins, a whey protein concentrate, whey protein isolate or a

demineralized whey powder. For example, the hydrolysate is a partially hydrolysed whey protein, a partially hydrolysed beta-lactoglobulin and/or partially hydrolysed alpha-lactalbumin.

The composition for use as herein described comprises a mixture of a non coagulating protein and a coagulating protein in a relative weight ratio in the range of from 22:78 to 70:30, preferably 22:78 to 65:35. In a preferred

embodiment of the invention, the weight ratio of non- coagulating to coagulating protein is from 25:75 to 60:30, more preferably from 30:70 to 40:60, and most preferably from 30:70 to 55:45. For example, the composition comprises whey protein and casein in a relative weight ratio in the range of from 22:78 to 70:30, preferably 25:75 to 60:40, more preferably from 30:70 to 60:40, and most preferably from 30:70 to 55:45.

An exemplary composition for use according to the present invention has a content of protein in the range from 4 % to 20 % (w/w), and a weight ratio of whey protein (i.e. native and denatured whey proteins and globular serum proteins) to casein in a range from 22:78 to 50:50 (w/w).

A nutritional composition as provided herein can have any suitable form. For example, in one embodiment the invention provides a composition in the form of a drinkable or a spoonable liquid. In another embodiment, it is a chewable substance, e.g. a recovery protein bar, cookie or the like for use as pre- or post-workout snack. It may furthermore comprise complex carbohydrates such as oats.

In yet another embodiment, the nutritional composition can be provided as a powder, for instance a dry powder which is particularly useful as a beverage component to provide a protein fortified beverage. Accordingly, in another aspect of the invention, there is provided a beverage comprising the composition of the invention, such as a high protein health or sports drinks and shakes for use by athletes, recreatively active people, older people or sick people.

In a specific aspect, the nutritional composition is a fermented nutritional composition, preferably selected from the group consisting of sour milk products and/or acidified fresh products, like yoghurt, fermented milk, villi, fermented cream, sour cream, quark, butter milk, kefir, and dairy shot drinks. In one aspect, the fermented nutritional composition is a yoghurt or a quark.

In one embodiment, it is a yoghurt, which yoghurt can be set or stirred. Set yoghurt is a type of yoghurt fermented and cooled in the final package and characterised by a three-dimensional gel matrix giving it a firm jelly like texture. In a preferred embodiment, the yoghurt is a stirred yoghurt. Stirred yoghurt is a type of yoghurt fermented in a tank and the final coagulum is broken by stirring prior to cooling and packing. In stirred yoghurt, the three-dimensional gel matrix is no longer visible. Stirred yoghurt is a weak gel system consisting of weakly associated protein clusters. For example, it is a yoghurt comprising 5-8wt% of total protein and wherein the whey protein/casein ratio is in the range 30:70 to 60:40, preferably 40:60 to 55:45.

In a specific aspect, it is a Greek style yoghurt comprising 7-12w% total protein having a whey/casein ratio in the range of 30:70 to 50:50. In another embodiment, it is a quark. For example, it is a low-fat quark comprising 8-12w% total protein comprising at least 0.5wt% of the native, non bound whey protein based on the total weight of the composition.

Method of manufacturing

As mentioned herein above, the bound fraction of said non-coagulating protein is suitably obtained by a process causing the denaturation majority of the proteins, for example upon exposure of (a solution of) a mixture of said non coagulating and said coagulating protein to a process involving heat-treatment, preferably (high temperature) pasteurization, or high pressure treatment, or homogenization. In a separate, subsequent step, a source of non-coagulating protein adding to said heated mixture, wherein majority of said non-coagulating protein is in a native state, to obtain a composition wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein another fraction of said non- coagulating protein is native and non-bound.

A further embodiment of the invention therefore relates to a method for providing a nutritional composition comprising at least 4 wt% of protein, comprising a non-coagulating protein and a coagulating protein, wherein the non coagulating protein comprises whey proteins and globular serum proteins and coagulating protein comprises casein or caseinate, and wherein the weight ratio of non- coagulating protein to coagulating protein is in the range of 22:78 to 70:30, comprising the steps of

(i) heating a mixture comprising a coagulating protein and a non

coagulating protein under conditions to obtain protein denaturation; and

(ii) adding to said heated mixture a source of non-coagulating protein wherein at least 50w% of said non- coagulating protein is in a native state, to obtain a composition wherein a fraction of said non-coagulating protein is bound to said coagulating protein and wherein another fraction of said non-coagulating protein is native and non-bound.

Preferably, in step (i) a heating process is used wherein a mixture of non coagulating protein and coagulating protein is exposed to an elevated

temperature for a sufficient period to induce denaturation of at least 60, or preferably 80% of the protein, resulting in the aggregation of at least part of the non-coagulating protein onto the coagulating protein. Herewith, the coagulating protein no longer coagulates in the stomach, thereby making the coagulating protein "faster” in terms of digestibility. The heating process preferably comprises high pasteurization, e.g. at least 6 minutes, in particular at least 10 minutes at 85°C, or shorter or longer depending on the temperature. In the process of the present invention, heat- sterilisation is performed with any suitable method, such as retort sterilisation, ultra-high temperature (UHT) treatment or direct-steam injection (DSI). Heat- sterilisation comprises heating to a

temperature of at least 80°C, preferably at least 90°C.

The mixture preferably comprises casein or caseinate, more preferably selected from the group consisting of micellar casein, non-micellar casein, sodium caseinate, calcium caseinate, potassium caseinate and magnesium caseinate and whey proteins, preferably globular serum proteins. The weight ratio of non coagulating protein to coagulating protein in the mixture to be heated (i.e. prior to adding native non-coagulating protein) is at least 5:95 or preferably at least 10:90 to 20:80. For example, a (skimmed) milk is used having a globular serum proteimcasein weight ratio of 17:83. The milk may be raw or it may be

pretreated. For example, milk is suitably thermized (or pasteurized) and/or bactofuged according to standard procedures to minimize the load of viable bacteria and/or bacterial spores so as to ensure safety of consumption.

To increase the protein content, the milk may be concentrated or topped up with additional (milk) protein, for instance with nonfat dry milk or a skim milk powder (SMP).

In step (ii) of a method for providing a nutritional composition according to the invention, the non-bound fraction of said non- coagulating protein is obtained by adding a source of native non-coagulating protein to a composition comprising non-coagulating protein which is bound to a coagulating protein. In order to prevent unwanted protein denaturation, the source of native, non-coagulating whey protein has not been treated or exposed to high temperature treatment or other denaturing conditions. However, it may have been subjected to a mild heat treatment (mild heat pasteurization at adequate pH, time and temperature conditions to prevent denaturation) in order to enhance the shelf life while maintaining a good viscosity and high protein digestibility of the final

composition. For example, native globular serum protein which has been treated at about 72°C for 10-60 seconds, preferably 15-30 seconds, is suitably used. In another embodiment, raw whey is used. The native non-coagulating protein is suitably added while mixing. It may be added as a powder, as a liquid, or a combination thereof.

As a result of the 2- step process of the invention comprising "combined heating” and "post-heating addition”, a fraction of the non- coagulating protein is bound to the coagulating protein and the remainder fraction of the non

coagulating protein is non-bound. Hence, the non-coagulating protein is present in the composition in the denatured form as well as in the native form. This provides an optimal combination of protein availability and protein digestibility. Accordingly, it is preferred that no UHT treatment or similar severe heat treatment is performed after step (ii) .

As said herein above, the invention provides in a preferred embodiment a protein-rich, highly digestible fermented product. For example, provided is a fermented nutritional composition, preferably selected from the group consisting of sour milk products and/or acidified fresh products, like yoghurt, fermented milk, viili, fermented cream, sour cream, quark, butter milk, kefir, and dairy shot drinks.

To that end, the process of combined heating and post-heating addition is followed by a step of fermenting the composition by methods known in the art, typically using a suitable bacterial culture, referred to in the art as "starter culture”, to provide lactic acid fermentation. The starter culture is added in conventional amounts, after which the fermentation is carried out in a time sufficient to obtain proper acidification, typically to obtain pH about 4.6 or lower, e.g. pH 4.5. The fermentation is performed at a conventional temperature, often in the range of about 35 to about 45°C. In one embodiment, fermentation is carried out for about 10-15 hours at a temperature of about 38-40°C. In one embodiment, the heated mixture is fermented and then dried to obtain a powder, followed by adding additional native serum protein powder by means of dry blending. Alternatively, native serum protein is added after the heating step, then fermented and then dried

Exemplary methods of the invention include the following processes:

Combined heating and post addition of whey proteins/globular serum protein followed by fermenting, and optionally drying.

Combined heating, fermenting, than post addition of whey proteins globular serum proteins, and optionally drying

Combined heating, fermenting, optionally a concentration step (e.g.

centrifugation or filtration), than post addition of whey proteins/globular serum proteins in dry form, or in a neutral or acidified liquid form, and optionally drying.

Combined heating, fermenting and drying and than post addition of whey protein / globular serum protein powder (dry blending)

Micro-organisms which can be used in the fermentation step are well known in the art. For example, Streptococcus and Leuconostoc species are used for sour cream and buttermilk production while Lactobacillus bulgaricus can also be used for Bulgarian buttermilk production. Streptococcus species are predominantly used for cheese production often in conjunction with other species such as Penicillium, Brevibacterium or Lactobacillus species. For yoghurt production, micro-organisms used include Lactobacillus species such as L.

bulgaricus or L. acidophilius or streptococci such as Strep thermophilus or Strep lactis. For making kefir, a typical starter culture comprises a mixture of Strep lactis, Strep cremoris, several yeast species and other lactic acid bacteria.

LEGEND TO THE FIGURES

Figure 1: Analysis of different test yoghurts in an in-vitro stomach model. For details see Example 2.

Figure 2: Visual illustration of the production processes for product A, B and C, D of Example 3. Figure 3: Illustration that addition of whey protein can be done in the form of a solution or a dry powder

Figure 4: Total essential amino acids (EEA) concentrations in blood over time, after ingestion of a yoghurt with added whey protein in either denatured form (triangles; comparative sample) or as native protein (circles; according to the invention). For details see Example 4.

Figure 5: Analysis of different test quarks in an in-vitro stomach model. Circles represent a reference quark having a casein/whey protein ratio of 20:80. Squares represent a whey protein enriched quark of the invention (casein/whey protein ratio 50:50). The curve shows the % of protein in liquid phase as a function of time. For details see Example 5. Figure 6: Analysis of the different Greek style yoghurts tested (both 8.8% protein and whey protein/casein ratio of 40:60); circles represent the native enriched sample and squares represent the denatured enriched reference sample in an in- vitro stomach model (for details see example 6). The graph illustrates the % of protein in the liquid phase as a function of time.

EXPERIMENTAL SECTION

Example 1: Gastric digestion model system

Protein digestion data were obtained using an established in vitro digestion stomach model. Given that the transit time of liquids through the stomach is much faster than that of (semi)solids, the amount of protein in the - fast emptying - liquid fraction (or liquid phase) is key for a fast digestion.

Gastric phase

- Prepare 380 mL Simulated Gastric Fluid:

o 243.2 mL of SGF electrolyte stock solution;

o 60.8 mL porcine pepsin stock solution of 25,000 U/mL made up in SGF electrolyte stock solution (pepsin from porcine gastric mucosa, 3,200-4,500 U/mg protein, Sigma);

o 190 pL of 0.3 M CaCL;

o 1900 pL of 1 M HC1 to reach pH3.0;

o 73.9 mL of water.

- Start the Applikon ADI1010 (Controller) and ADI1025 (fermenter) and pipet 350 mL of the sample, standardised to a protein concentration of 3.3 wt%, in the fermenter. Heat the sample to 37°C. Stirring bar can be set at 100 rpm.

- Add 350 mL SGF to the 350 mL sample.

- Take the first sample and start pH profile. This gives the start signal for the ADI1010 to add 1 M HC1 following the set pH curve as depicted in Table 1. The slope of the decrease is the same for all the tested samples. Make sure the amount of HC1 added is recorded.

Table 1: pH curve

Table 2: Test set-up of the adult in vitro digestion model

During digestion, samples (typically 1 * 50 ml per sampling point) are taken for analysis during the test series. Since pH can differ a little, samples are taken based on time points, not on pH. Pictures of the fermenter are taken at the same time the 50 ml samples are taken. The samples are immediately filtered after the sampling on a filter paper. Observations are written down in a logbook. The sample is divided for different analyses. Enzymes present in the samples were inactivated as follows:

1) filtrated 50 ml sample is cooled on ice. 2) from each sample the protein content is analysed using the Kjeldahl nitrogen analysis method.

Example 2: Digestibility of exemplary yoghurts

Materials:

Skim milk powder (SMP) was obtained from Promex (FrieslandCampina

Milkpowder)

Whey protein concentrate (Nutriwhey 800F) is an acid WPC with 80% protein on dry matter obtained from FrieslandCampina DMV

The starter culture used was Yomix 860 (Dupont)

Methods:

A series of yoghurts was produced from the compositions indicated in Table 3 (all amounts indicated in percentages ) :

Table 3 : Yoghurt compositions

The details of the production processes for the individual samples are summarised below:

For the production of yoghurt samples 1, 2 and 5:

· Standardize skimmed milk to 0% fat; 3.4% protein

• Weigh the milk into a bucket and place it in the water bath at 7°C

• Add the SMP (for sample 1 and 2, 3 and 4) or WPC (sample 5) to the milk gently with sufficient stirring to avoid lump formation

• Mix for about lh until all powders are well dissolved and hydrated

· Pasteurise at 85°C for 10 min

• Cool to 5°C

• Inoculate the milk with the starter culture using a sterile pipette

• Ferment at 39°C for 12-15 hours until pH < 4.3

• Gently break the yoghurt gel structure first with a spatula

· Stir the yogurt with a propeller stirrer at 700-800rpm while cooling to 7°C.

Cooling time around 30min

• Fill into 500ml PE beakers

For the production of sample 3 and 4:

· Standardize skimmed to milk 0% fat; 3.4% protein • Weigh the milk into a bucket and place it in the water bath at 7°C

• Add the SMP to the milk gently with sufficient stirring to avoid lump

formation

• Mix for about lh until all powder are well dissolved and hydrated

• Pasteurise at 85°C for lOmin

• Start cooling to 5°C

• In a separate bucket, dissolve the 3.6% WPC in the 36.1% water (gently with sufficient stirring to avoid lump formation) and mix this solution for lh to allow for hydration of the protein (for sample 3 this solutions is used as such, while for sample 4 it is heated to 85°C for 10 minutes)

• While cooling the milk, at < 39°C add the (unheated for sample 3, heated for sample 4) WPC solution to the milk and stir gently

• Inoculate the milk with the starter culture using a sterile pipette

• Ferment at 39°C for 12-15 hours until pH < 4.3

• Gently break the yoghurt gel structure first with a spatula

• Stir the yogurt with a propeller stirrer at 600-700rpm while cooling to 7°C.

Cooling time around 30min

• Fill into 500ml PE beakers

Yoghurt samples were prepared for analysis in the in-vitro stomach model.

Compositions of the samples are indicated in Table 4.

Table 4: Compositions of the samples prepared for analysis in the in-vitro stomach model

Of each of the 5 samples above, 350 ml was analysed in the in-vitro stomach model. The samples were standardized to a protein concentration of 3.3 wt%

The data in Figure 1 show that the high native whey protein yoghurt (sample 3) results in the highest fraction of protein in the liquid phase in the (in-vitro) stomach. Since liquids have a shorter transit time through the stomach, it is likely that this composition leads to a faster increase in protein in the small intestine and hence a faster digestion. Increasing the leucine content of the yoghurt by adding whey to the milk either before heating (sample 5), or separately adding denatured whey after heating (sample 4) leads to a lower amount of protein in the liquid phase; even lower than the reference yoghurts (samples 1 and 2) with a 20:80 whey proteimcasein ratio. Furthermore, the yoghurts comprising denatured whey protein (samples 4 and 5) have an undesirable hard and gel-like consistency. Example 3: Digestibility of different yoghurts

Sample preparation

In this example, the digestibility of two fermented nutritional compositions of the invention (samples C and D) was compared with two known protein-rich yoghurts (reference samples A, and B). The materials used are described in Example 2, compositions are indicated in Table 5.

Table 5: Compositions

For the production of samples A and B (see also figure 2):

• Standardize skimmed milk to 0% fat; 3.4% protein

• Weigh the milk into a bucket and place it in the water bath at 7°C

• Add the SMP (for sample A) or SMP and WPC (sample B) gently to the milk with sufficient stirring to avoid lump formation

• Mix for about lh until all powders are well dissolved and hydrated

• Pasteurise at 85°C for lOmin

• Cool to 5°C • Inoculate the milk with the starter culture using a sterile pipette

• Ferment at 39°C for 12-15 hours until pH < 4.3

• Gently break the yoghurt gel structure first with a spatula

• Stir the yogurt with a propeller stirrer at 700-800rpm while cooling to 7°C.

Cooling time around 30min

• Fill into 500ml PE beakers

For the production of sample C (see also figure 2):

• Standardize skimmed to milk 0% fat; 3.4% protein

• Weigh the milk into a bucket and place it in the water bath at 7°C

• Add the SMP to the milk gently with sufficient stirring to avoid lump

formation

• Mix for about lh until all powder are well dissolved and hydrated

• Pasteurise at 85°C for lOmin

• Start cooling to 5°C

• In a separate bucket, dissolve the WPC in the water (gently with sufficient stirring to avoid lump formation) and mix this for lh to allow for hydration of the protein

• While cooling the milk, at < 39°C add the WPC solution to the milk and stir gently

• Inoculate the milk with the starter culture using a sterile pipette

• Ferment at 39°C for 12-15 hours until pH < 4.3

• Gently break the yoghurt gel structure first with a spatula

• Stir the yogurt with a propeller stirrer at 600-700rpm while cooling to 7°C.

Cooling time around 30min

• Fill into 500ml PE beakers

For the production of sample D (see also figure 2):

• Standardize skimmed to milk 0% fat; 3.4% protein

• Weigh the milk into a bucket and place it in the water bath at 7°C

• Add the SMP to the milk gently with sufficient stirring to avoid lump

formation • Mix for about lh until all powder are well dissolved and hydrated

• Pasteurise at 85°C for lOmin

• Start cooling to 5°C

• While cooling, at < 39°C add the WPC to the milk gently with sufficient stirring to avoid lump formation. Be careful not to incorporate air.

• Mix for about lh until all powder are well dissolved and hydrated

• Inoculate the milk with the starter culture using a sterile pipette

• Ferment at 39°C for 12-15 hours until pH < 4.3

• Gently break the yoghurt gel structure first with a spatula

· Stir the yogurt with a propeller stirrer at 600-700rpm while cooling to 7°C.

Cooling time around 30min

• Fill into 500ml PE beakers

Test Setup

All tested samples (see table 6) were incubated under stomach conditions in the in vitro stomach model (see Example 1) in order to follow the amount of protein in the liquid phase as a function of time in the (in-vitro) stomach. The samples were standardized to a protein concentration of 3.3 wt%.

Table 6: Composition of the casein samples for 450 mL

The results are shown in Figure 3. It illustrates that when preparing a yoghurt with increased whey protein by just adding additional non-coagulating globular protein from whey to the milk (B), the amount of protein in the liquid phase decreases compared the reference high protein yoghurt (A). In contrast, when keeping the added whey protein native, the amount of protein in the liquid phase clearly increases (C and D). Clearly, for samples C and D according to the invention the amount of protein in solution is higher than for the reference samples at all time points. Thus, it makes no difference whether the native, non coagulating protein is added as dry powder or as a solution.

Example 4: Digestibility of exemplary yoghurt compositions

This example shows the analysis of amino acid profiles in blood after ingestion of high protein yoghurt compositions.

To that end, a randomized, single-blinded cross-over trial was performed in healthy men and women (aged 18-65y) in order to compare postprandial blood amino acid concentrations after ingestion of a fixed amount of protein (25 grams) comprised in 417 grams of either the product B (comparative sample) or D (of the invention) according to Example 3 herein above.

After ingestion of the products, 283 grams of water was consumed to match a total volume of 700 ml

Blood samples were collected in the fasting state and 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240 and 300 min after ingestion of the product. Blood serum samples were later analysed for 20 amino acid concentrations using the

Phenomenex EZ:faast amino acid kit

(https://www.phenomenex.com/Products/AminoAcidDetail/EZfaast ). Data are shown in Figure 4.

Surprisingly, product D according to the invention comprising native whey protein was found to give a higher peak in essential amino acids in blood, compared to reference product B, while both have the same protein composition. Repeated measurements of the amino acid kinetics in blood confirmed a significant (p<0.05) difference between sample B and D for total essential amino acids (EEA; see Fig. 4) as well as total amino acids (data not shown).

Example 5: In-vitro digestion of exemplary quark compositions

Conventional low-fat (plain) quark contains about 10w% protein, with a whey protein to casein ratio of 20:80. This example demonstrates that the invention is also applicable for quark compositions. A quark enriched with native whey protein having a final whey protein to casein ratio of 50:50 was prepared as follows:

1. A low-fat commercial natural quark ( in tubes of 50 mL was centrifuged at 5000 x g for 20 min at 20°C to isolate the acid whey.

2. A native whey protein concentrate (WPC80) was dissolved in the acid whey to a final level of 10w% protein. To that end, 12,5 g of WPC80 powder was gradually added to 87,5 g of acid whey under stirring with a magnetic stirrer at room temperature. 3. 75 g of this WPC suspension was mixed with 125 g of the original natural quark in order to obtain the same final protein content but having an increased whey protein/casein ratio .

Pure original quark was used as control sample

The two samples thus obtained,

Reference quark 20:80 - low-fat quark; 10% protein, whey protein/casein ratio 20:80

Enriched quark 50:50 - Whey protein enriched quark; 10% protein, whey protein/casein ratio 50:50,

were subjected to the in-vitro digestion analysis of Example 1.

Figure 5 shows that the amount of protein in the liquid phase (i.e. the liquid that can pass through the filter) is higher than that of the quark without added native whey protein during at least the first 30 minutes of digestion.

Example 6: In-vitro digestion of exemplary Greek style yoghurt compositions

Greek style yoghurt is a yoghurt that is concentrated such that it has a high protein (8.77 w%) concentration (at a standard whey protein/casein ratio of 20:80). In this example, two whey protein enriched compositions are prepared from this Greek style yoghurt; one in which the added whey protein is native, and one in which the added whey protein is denatured.

Standard Greek style yoghurt was prepared by:

pasteurising skimmed milk (approximately 3.5w % protein) for 6 minutes at 92°C, cooling to a fermentation temperature of 42°C, adding yoghurt culture, and fermenting until a pH 4.4

concentrating this yoghurt with a separator (centrifuge to remove serum) until a protein content of 8.8 w % and subsequently cooling and filling the product.

for this example, the serum is stored to be used for the whey protein enrichment For this example the Greek style yoghurt was enriched with whey protein as follows:

1. Dissolving WPC80 in the serum (acid whey) at a level of 8.8% protein. To that end, 54.6 g of WPC80 powder was gradually added to 445 g of the serum (retained from centrifugation of the yoghurt) while stirring with a magnetic stirrer at room temperature. This mixture was allowed to hydrate for at least 2 hours.

2. Mixing 250 g of this WPC solution with 750 g Greek style yoghurt in order to obtain an increased whey protein/casein ratio of 40:60, at the same protein content of 8.8w%.

3. Splitting the mixture obtained at point 2 in two halves; using the one half - as such - as the‘native enriched’ sample and the other to be heated to become the ‘denatured enriched’ sample.

4. For the‘denatured enriched’ sample, water bath was set at 95°C, bottles with product immersed in the water bath once it was hot, and product was kept at a temperature above 83°C for 5 minutes.

The two samples obtained,

Native enriched Greek style yoghurt; 8.8 w % protein, whey protein/casein ratio 40:60

Denatured enriched Greek style yoghurt; 8.8 w % protein, whey

protein/casein ratio 40:60,

were subjected to the in-vitro digestion of example 1. Figure 6 shows that the amount of protein in the liquid phase (that is able to pass through the filter paper) is higher for the native enriched sample than for the denatured enriched example, during the complete course of digestion.