阅读说明：本技术 PH中性β-乳球蛋白饮料的制备 (Preparation of pH neutral beta-lactoglobulin beverage ) 是由 S·B·尼尔森 T·C·雅格 K·B·劳里德森 K·桑德加 G·D·M·马西尔 H·贝特尔 于 2019-06-26 设计创作，主要内容包括：本发明涉及一种新的经过包装、热处理的饮料制品,其pH值在5.5-8.0的范围内。此外,本发明涉及一种生产经过包装、热处理的饮料制品的方法,并且涉及该经过包装、热处理的饮料制品的不同用途。(The present invention relates to a new packaged, heat-treated beverage product having a pH in the range of 5.5 to 8.0. Furthermore, the present invention relates to a method of producing a packaged, heat-treated beverage product, and to different uses of the packaged, heat-treated beverage product.)

1. A packaged heat-treated beverage product having a pH in the range of 5.5-8.0, said beverage comprising:

-protein in a total amount of 1 to 20% w/w of the weight of the beverage, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG),

-optionally a sweetener and/or a flavouring.

2. The packaged heat-treated beverage product according to claim 1, wherein the product is at least pasteurized.

3. The packaged heat-treated beverage product of claim 1, wherein the product is at least sterile.

4. A packaged heat-treated beverage product according to any preceding claim, wherein the protein component of the beverage product has a color value Δ b, measured at room temperature, in the range-0.10 to +0.51 of the CIELAB color scale, wherein,

Δb*＝b_{Samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}*。

5. A packaged heat-treated beverage product according to any preceding claim, wherein the beverage product has a colour value Δ b, measured at room temperature, in the range-0.10 to +0.51 on the CIELAB colour scale, wherein,

Δb*＝b_{samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}*。

6. The packaged heat-treated beverage product according to any one of the preceding claims, wherein the total amount of Na, K, Mg and Ca is at most 400 mM.

7. A packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product comprises at most 100mg phosphorus per 100g protein and at most 700mg potassium per 100g protein.

8. A packaged heat-treated beverage product according to any one of the preceding claims, wherein the pH of the product is in the range of 6.5-7.5, preferably in the range of 6.5-7.0.

9. A packaged heat-treated beverage product according to any preceding claim, wherein the turbidity of the product is at most 200 NTU.

10. The packaged heat-treated beverage product according to any preceding claim, wherein the product has a turbidity of greater than 200 NTU.

11. A packaged heat-treated beverage product according to any preceding claim, wherein the product has a viscosity of at most 200cP measured at 22 ℃ at a shear rate of 100/s.

12. A packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product comprises a total amount of protein of from 1 to 10% w/w, relative to the weight of the beverage product.

13. A packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product comprises a total amount of protein of 10 to 20% w/w relative to the weight of the beverage product.

14. A packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product further comprises carbohydrates in an amount of 0 to 95% of the total energy content of the product.

15. A packaged heat-treated beverage product according to any preceding claim, wherein the beverage product further comprises a lipid content of from 0 to 50% of the total energy content of the product.

16. A packaged heat-treated beverage product according to any preceding claim, wherein the beverage product comprises BLG isolate.

17. The packaged heat-treated beverage product according to any one of the preceding claims, wherein each major non-BLG whey protein is present in a weight percentage relative to the total amount of protein of at most 20%, preferably at most 15%, more preferably at most 10%, especially preferably at most 6%, most preferably at most 4% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

18. Packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product comprises at least 50% w/w protein nanogel relative to the total amount of protein.

19. The packaged heat-treated beverage product according to any one of claims 1 to 17, wherein the beverage product comprises at least 60% w/w soluble whey protein aggregates relative to the total amount of protein.

20. A method of producing a packaged heat-treated beverage product having a pH of 5.5 to 8.0 comprising the steps of:

a) providing a liquid solution comprising:

-a total amount of protein of 1 to 20 wt%, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG),

-optionally a sweetener and/or a flavouring agent,

b) Packaging the liquid solution, wherein the liquid solution is packaged,

wherein the liquid solution in step a) and/or the packaged liquid solution in step b) is subjected to a heat treatment comprising at least pasteurization.

21. The method of claim 20, wherein the heat treatment comprises sterilization.

22. The method of any one of claims 20 to 21, wherein the sterilizing comprises a temperature in the range of 120 to 150 ℃ for 4 to 30 seconds.

23. The method of claim 20, wherein the pasteurization comprises heating to a temperature of 85 ℃ to 95 ℃ for 1 to 3 minutes.

24. Use of a protein solution comprising a total amount of protein of from 1 to 20% w/w by weight of the solution in controlling whiteness of a sterile beverage product, wherein at least 85 w/w% of the protein is β -lactoglobulin (BLG), the pH of the sterile beverage product being in the range 5.5-8.0.

25. A packaged heat-treated beverage product according to any one of claims 1 to 19, wherein the beverage product is for use in a method of treating a disease associated with protein malabsorption.

26. Use of the packaged heat-treated beverage product of any one of claims 1-19 as a dietary supplement.

27. Use of a packaged heat-treated beverage product according to claim 24, wherein the beverage product is ingested before, during or after exercise.

Technical Field

The present invention relates to a novel packaged heat-treated beverage product having a pH in the range of 5.5 to 8.0. The invention also relates to a method of producing a packaged heat-treated beverage product, and to different uses of the packaged heat-treated beverage product.

Background

Beverages used for sports nutrition are particularly likely to contain whey proteins, which are added for their unique nutritional benefits to athletes. Some medical and therapeutic nutritional beverages also contain whey proteins because they provide a large number of essential amino acids for protein synthesis, digestibility, and health benefits.

Whey protein can be isolated from whey (milk serum) or whey. Whey typically comprises a mixture of beta-lactoglobulin (BLG), alpha-lactalbumin (ALA), serum albumin, and immunoglobulins, with BLG being the predominant. Whey Protein Concentrate (WPC) therefore comprises a mixture of these proteins. Whey Protein Isolate (WPI) has a lower fat and lactose content than WPC.

Whey products may be yellow in color. Therefore, many attempts have been made in the past to remove or reduce the yellow color of whey products.

The traditional whitening/bleaching method for opalescence is to use hydrogen peroxide (HP, H)₂O₂) The whey is chemically bleached. These methods may have negative effects on taste and may enhance Unfolding and aggregation of whey proteins (Kramer et al, 2017. "effects of Oxidation and Protein Unfolding on Cross-Linking of beta-Lactoglobulin and alfa-Lactoglobulin", J.Agric.food chem.2017,65, 10258-.

WO2005/004616 a1 describes a method of bleaching or whitening a dairy product comprising adding Lipoxygenase (LOX) to the dairy product. The method can be used for whitening whey and dairy products.

Other methods of whitening whey include the addition of chlorophyll or the use of titanium dioxide (TiO) in milk products₂). Titanium dioxide is an inorganic, inert white pigment used in cheese milk, candy, chewing gum, toothpaste, etc. and has been approved by the FDA for food grade use.

WO2018/115520 a1 discloses a method for producing an edible isolated beta-lactoglobulin composition and/or a composition comprising crystallized beta-lactoglobulin based on crystallization of BLG in salinization mode. The crystallized BLG may then be separated from the remaining mother liquor.

WO2011/112695 a1 discloses nutritional compositions and methods of making and using the nutritional compositions. The nutritional composition comprises whey protein micelles and leucine and provides a sufficient amount of leucine to improve protein synthesis in the human body while also maintaining a low viscosity fluid matrix and acceptable organoleptic properties.

WO2011/051436 a1 discloses at least partially transparent compositions for human or animal consumption and relates to packaging of such compositions. One embodiment of the present invention relates to an at least partially transparent container comprising an at least partially transparent aqueous non-alcoholic composition. The container includes at least one polarizer that makes visible the liquid crystals present in the composition.

WO2004/049819 a2 discloses a method for improving a functional property of globulin, the method comprising the steps of: providing a solution of one or more globulins, wherein the one or more globulins are at least partially aggregated in the fibrils; and performing one or more of the following steps in random order: increasing the pH; increasing the salt concentration; concentrating the solution; and changing the solvent quality of the solution. Preferably, the solution of one or more globulins is provided by heating at low pH or by addition of a denaturant. Also disclosed are the protein additives so obtained, their use in food and non-food applications and in food and non-food products containing the protein additives.

WO2010/037736 a1 discloses the isolation of whey proteins and the preparation of whey products and whey isolates. In particular, the invention relates to the isolation of a beta-lactoglobulin product and an alpha-enriched whey protein isolate from whey obtained from animals. The alpha-enriched whey protein isolate provided by the invention has high alpha-lactalbumin and immunoglobulin G besides low beta-lactoglobulin.

FR 2296428 discloses protein compositions for dietary and therapeutic use based on whey proteins (lacosaerum proteins) obtained by any known separation method. The composition can be used for treating or preventing digestive system diseases (such as diarrhea) in infants and adults, increasing resistance to intestinal infections and treating certain metabolic diseases (such as hyper-anthocyanidinaemia). They may also be used dermatologically or cosmetically and may form part of a low protein diet.

Disclosure of Invention

The present inventors have observed that the degree to which the colour of a whey protein-containing beverage is neutral or white affects the perception of the whey protein-containing beverage by the consumer. Clear beverages with a yellow appearance or milky beverages with a yellowish color are not attractive to consumers.

It is an object of the present invention to provide a packaged, pH neutral, heat-treated beverage product having a more neutral color than conventional whey-containing beverages.

Another object is to utilize a softer yellow reduction process. Another object is that it should not negatively affect the stability of the beverage.

The present inventors have now found that such packaged heat-treated beverages can be provided in a wide neutral pH range of 5.5-8.0 and a wide protein concentration of 1-20 wt%, while still having a low viscosity, a stable and a more neutral color. The present invention provides two beverages, both of which are transparent and in other embodiments opaque.

Accordingly, one aspect of the present invention relates to a packaged heat-treated beverage product having a pH in the range of 5.5 to 8.0, the beverage comprising:

-protein in a total amount of 1 to 20% w/w by weight of the beverage, wherein at least 85% w/w of the protein is beta-lactoglobulin (BLG),

-optionally, a sweetener and/or a flavoring agent.

Another aspect of the invention relates to a method of producing a packaged heat-treated beverage product having a pH of 5.5 to 8.0, the method comprising the steps of:

a) providing a liquid solution comprising:

-the total amount of protein is between 1 and 20 wt%, wherein at least 85% of the protein is beta-lactoglobulin (BLG),

-optionally, sweeteners and/or flavouring agents,

b) the liquid solution is packed in a bag,

wherein the liquid solution of step a) and/or the packaged liquid solution of step b) is subjected to a heat treatment comprising at least pasteurization.

Another aspect of the invention relates to the use of a protein solution comprising 1 to 20% w/w of the total amount of protein, relative to the weight of the solution, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG), for controlling the whiteness of a sterile beverage preparation having a pH in the range of 5.5-8.0.

Yet another aspect of the present invention relates to a packaged heat-treated beverage product as defined herein for use in a method of treating a disease associated with protein deficiency.

Another aspect of the present invention relates to the use of a packaged heat-treated beverage product as defined herein as a dietary supplement.

Drawings

FIG. 1 is a graph showing a milky BLG sample heated at 94 ℃ for 14 minutes at pH 6.0.

Figure 2 is a graph of a gelled WPI sample heated at 94 ℃ for 14 minutes at pH 6.0.

Fig. 3 shows the amounts of insoluble protein, native whey protein, soluble whey protein aggregates and protein nanogels, a (BLG 98.2 w/w%), B (BLG 95.9 w/w%) and c (wpi) in the exemplary whey protein beverage of example 9.

FIG. 4 shows semi-dynamic in vitro digestion of exemplary beverages A (BLG 98.2 w/w%), B (BLG 95.9 w/w%) and C (WPI).

The top of fig. 5 shows: during the semi-dynamic in vitro digestion of the samples, SDS-PAGE analysis of protein aliquots taken at selected time points (17.5-105 min); the following shows: pH during digestion of the sample.

Fig. 6 shows a simulation of acidification in the stomach. The gel strength was measured during acidification of three beverages containing mainly soluble whey protein aggregates (beverage a and WPI) or protein nanogels (beverage B).

FIG. 7 shows a liquid beverage having a protein content of 10-16 w/w% after heat treatment at 90 ℃ for 5 minutes.

FIG. 8 shows the measurement results of viscosity with a protein content of 10-16 w/w% after heat treatment at 90 ℃ for 5 minutes.

FIG. 9 shows the particle size of heat-treated beverages with a protein content of 10-16 w/w%.

FIG. 10 shows on the left a 10 w/w% beverage at pH 6.0 and pH 7.0 using BLG powder A and on the right a 12 w/w% beverage at pH 6.0 using BLG powder A (according to Table 9), respectively.

Detailed Description

Definition of

In the context of the present invention, the term "beta-lactoglobulin" or "BLG" relates to beta-lactoglobulin from a mammalian species, e.g. occurring in native, unfolded and/or glycosylated form, and includes naturally occurring genetic variants. The term also includes aggregated BLG, precipitated BLG and crystallized BLG. When referring to the amount of BLG, reference is made to the total amount of BLG including aggregated BLG. The total amount of BLG was determined according to example 1.31. The term "aggregated BLG" relates to BLG that is at least partially unfolded and which typically aggregates with other denatured BLG molecules and/or other denatured whey proteins by hydrophobic interactions and/or covalent bonds.

BLG is the most predominant protein in bovine whey and is present in several genetic variants, the major variants in cow milk being labeled a and B. BLG is a lipoprotein that binds many hydrophobic molecules, suggesting a role in their transport. BLG has also been shown to be able to bind iron via siderophores and may play a role in combating pathogens. Human breast milk lacks homologs of BLG.

Bovine BLG is a relatively small protein of about 162 amino acid residues and has a molecular weight of about 18.3-18.4 kDa. Under physiological conditions, it is predominantly dimeric, but dissociates to monomers below about pH 3, retaining its native state as determined using nmr spectroscopy. Conversely, BLG can also exist in tetrameric, octameric, and other multimeric forms under a variety of natural conditions.

In the context of the present invention, the term "non-aggregated β -lactoglobulin" or "non-aggregated BLG" also relates to β -lactoglobulin from a mammalian species, which is present in native, unfolded and/or glycosylated form and includes naturally occurring genetic variants. However, the term does not include aggregated BLG, precipitated BLG, or crystallized BLG. The amount or concentration of non-aggregated BLG was determined according to example 1.6.

By calculating (m)_{Total BLG}-m_{Non-aggregated BLG})/m_{Total BLG}X 100% to determine the percentage of non-aggregated BLG relative to the total BLG. m is_{Total BLG}Is the concentration or amount of BLG, m, determined according to example 1.31_{Non-aggregated BLG}Is the concentration or amount of non-aggregated BLG determined according to example 1.6.

In the context of the present invention, the term "crystal" refers to a solid material whose constituent elements (such as atoms, molecules or ions) are arranged in a highly ordered microstructure to form an omnidirectionally extending lattice.

In the context of the present invention, the term "BLG crystals" relates to protein crystals comprising mainly non-aggregated and preferably native BLG, arranged in a highly ordered microstructure, forming an omnidirectionally extended lattice. The BLG crystal may be, for example, monocrystalline or polycrystalline, or may be, for example, a whole crystal, a fragment of a crystal, or a combination thereof. Crystal fragments are, for example, fragments formed by subjecting the intact crystal to mechanical shear during processing. The crystal fragments also have a highly ordered crystal microstructure, but may lack uniform surfaces and/or uniform edges or corners of the intact crystals. See fig. 18 for PCT application number PCT/EP2017/084553 illustrating a number of intact BLG crystals and fig. 13 for PCT application number PCT/EP2017/084553 illustrating BLG crystal fragments. In both cases, the BLG crystal or crystal fragment can be visually identified as a well-defined, compact and coherent structure using an optical microscope. The BLG crystals or crystal fragments are typically at least partially transparent. Furthermore, protein crystals are known to be birefringent, and this optical property can be used to identify unknown particles with a crystal structure. Amorphous BLG aggregates, on the other hand, typically exhibit an open or porous mass with poorly defined boundaries, opacity, and irregular size.

In the context of the present invention, the term "crystallization" relates to the formation of protein crystals. Crystallization may occur, for example, spontaneously or by addition of seed crystals.

In the context of the present invention, the term "edible composition" refers to a composition that is safe for human consumption and use as a food ingredient and does not contain problematic amounts of toxic ingredients (e.g., toluene or other undesirable organic solvents).

In the context of the present invention, the term "ALA" or "alpha-lactalbumin" relates to alpha-lactalbumin from a mammalian species, for example, which occurs in its native and/or glycosylated form and includes naturally occurring genetic variants. The term also includes aggregated ALA and precipitated BLG. When referring to the amount of ALA, reference is made to the total amount of ALA including, for example, aggregated ALA. The total amount of ALA was determined according to example 1.31. The term "aggregated ALA" relates to ALA which is typically at least partially unfolded and which is typically aggregated with other denatured ALA molecules and/or other denatured whey proteins by hydrophobic interactions and/or covalent bonds.

Alpha-lactalbumin (ALA) is a protein that is present in the milk of almost all mammalian species. ALA forms the regulatory subunit of the Lactose Synthase (LS) heterodimer, whereas β -1, 4-galactosyltransferase (β 4Gal-T1) forms the catalytic component. These proteins together allow LS to produce lactose by transferring the galactose moiety to glucose. One of its main structural differences with β -lactoglobulin is that ALA does not have any free thiol groups that can act as starting points for covalent aggregation reactions.

In the context of the present invention, the term "non-aggregated ALA" also relates to ALA from a mammalian species, which is present in native, unfolded and/or glycosylated form and comprises naturally occurring genetic variants. However, the term does not include aggregated ALA or precipitated ALA. The amount or concentration of non-aggregated BLG was determined according to example 1.6.

By calculating (m)_{Total ALA}-m_{Non-aggregating ALA})/m_{Total ALA}X 100% to determine the percentage of non-aggregated ALA relative to total ALA. m is_{Total ALA}Is the concentration or amount of ALA determined according to example 1.31, and m_{Non-aggregating ALA}Is the concentration or amount of non-aggregated ALA determined according to example 1.6.

In the context of the present invention, the term "macrocasein" or "CMP" relates to the hydrophilic peptide, residue 106-169, which originates from the hydrolysis of "kappa-CN" or "kappa-casein" in mammalian species by aspartic proteases (e.g.chymosin), e.g.which is present in native and/or glycosylated form and includes naturally occurring genetic variants.

In the context of the present invention, the term "BLG isolate" refers to a composition comprising at least 85% w/w BLG relative to total protein. The total protein content of the BLG isolate is preferably at least 30% w/w, preferably at least 80% w/w, relative to total solids.

In the context of the present invention, the term "BLG isolate powder" relates to BLG isolate in powder form, and is preferably a free flowing powder.

In the context of the present invention, the term "BLG isolate liquid" relates to BLG isolate in liquid form, preferably an aqueous liquid.

The term "whey" relates to the liquid phase remaining after the casein in milk has been precipitated and removed. The casein precipitation is for example done by acidification of milk and/or using rennet. There are several types of whey, such as "sweet whey" which is a whey product produced by rennet-based precipitation of casein, and "acid whey" or "acid whey" which is a whey product produced by acid-based precipitation of casein. Acid-based precipitation of casein may be achieved, for example, by addition of food acids or by bacterial culture.

The term "whey" refers to the liquid that remains when casein and milk fat globules are removed from milk, for example by microfiltration or macrofiltration. The whey may also be referred to as "ideal whey".

The term "milk albumin" or "serum protein" relates to proteins present in milk albumin.

In the context of the present invention, the term "whey protein" relates to proteins found in whey or milk serum. Whey protein may be a subset of the protein species found in whey or whey, even a single whey protein species, or it may be a complete set of protein species found in whey or/and whey.

In the context of the present invention, the main non-BLG proteins of standard whey protein concentrates from sweet whey are ALA, CMP, bovine serum albumin, immunoglobulins, osteopontin, lactoferrin and lactoperoxidase. In the context of the present invention, the weight percentage of the major non-BLG whey proteins of a standard whey protein concentrate from sweet whey is:

the ALA content was 18% w/w relative to the total protein content,

the CMP content was 18% w/w relative to the total amount of protein,

the BSA content was 4% w/w relative to the total protein,

the content of casein species relative to the total amount of protein is 5% w/w,

the content of immunoglobulins is 6% w/w relative to the total amount of protein,

the osteopontin content was 0.5% w/w relative to the total amount of protein,

the content of lactoferrin was 0.1% w/w relative to the total amount of protein

The amount of lactoperoxidase was 0.1% w/w relative to the total amount of protein.

The term casein relates to casein found in milk and includes native micellar casein, casein species alone and caseinate found in raw milk.

In the context of the present invention, the term "mother liquor" relates to the whey protein solution remaining after the BLG has been crystallized and the BLG crystals have been at least partially removed. The mother liquor may still contain some BLG crystals, but typically only escape the separated small BLG crystals.

In the context of the present invention, a liquid that is "supersaturated" or "supersaturated with respect to BLG" comprises a concentration of dissolved, unaggregated BLG that is above the saturation point of unaggregated BLG in the liquid under given physical and chemical conditions. The term "supersaturation" is well known in the Crystallization art (see, for example, the gram Coquerella, "Crystallization of molecular systems from solution: phase diagrams, supersaturation and other basic concepts", Chemical Society Reviews, page 2300, 7 th edition, 2014) and supersaturation can be determined by many different measurement techniques (e.g., by spectroscopy or particle size analysis). In the context of the present invention, supersaturation with respect to BLG is determined by the following procedure.

The step of testing whether the liquid is supersaturated with respect to BLG under a particular set of conditions:

a) 50ml of the liquid sample to be tested was transferred into a centrifuge tube (VWR catalog No. 525-0402) having a height of 115mm, an inner diameter of 25mm and a capacity of 50 ml. In steps a) -h), care should be taken to keep the sample and its subsequent fractions under the initial physical and chemical conditions of the liquid;

b) the sample was immediately centrifuged at 3000g for 3.0 minutes, accelerated for a maximum of 30 seconds and decelerated for a maximum of 30 seconds;

c) immediately after centrifugation, transfer as much supernatant as possible (without disturbing the pellet if it has already formed) to a second centrifuge tube (of the same type as in step a);

d) a sub-sample of 0.05mL of supernatant (sub-sample a) was taken;

e) adding 10mg BLG crystals (at least 98% pure relative to total solids, non-aggregated BLG) having a particle size of up to 200 microns to a second centrifuge tube and stirring the mixture;

f) the second centrifuge tube was left at the initial temperature for 60 minutes;

g) immediately after step f), the second centrifuge tube is centrifuged at 500g for 10 minutes, and then another 0.05mL aliquot of the supernatant (subsample B) is taken;

h) recovering the centrifuged pellet of step g), if any, resuspended in MilliQ water and immediately examined for the presence of microscopically observable crystals in the suspension;

i) The concentration of non-aggregated BLG in subsamples a and B was determined using the method outlined in example 1.6-results are expressed as% BLG w/w relative to the total weight of the subsample. The concentration of non-aggregated BLG of the subsample A is called C_BLG，AThe concentration of non-aggregated BLG of the subsample B is called C_BLG，B；

j) If C is present_BLG，BLower than C_BLG，AAnd crystals are observed in step i), the liquid from which the sample of step a) is taken is supersaturated (under certain conditions).

In the context of the present invention, the terms "liquid" and "solution" include both compositions that do not contain particulate matter and compositions that comprise liquid and solid and/or semi-solid particles (e.g., protein crystals or other protein particles). Thus, a "liquid" or "solution" may be a suspension, or even a slurry. However, "liquids" and "solutions" are preferably pumpable.

In the context of the present invention, the terms "whey protein concentrate" (WPC) and "serum protein concentrate" (SPC) relate to dry or aqueous compositions comprising between 20 and 89% w/w of the total amount of protein, relative to the total amount of solids.

The WPC or SPC preferably comprises:

20-89% w/w protein relative to the total solids,

15-70% w/w BLG relative to the total amount of protein,

(ii) 8-50% w/w ALA relative to total protein, and

0-40% w/w CMP relative to protein.

Optionally, but also preferably, the WPC or SPC may comprise:

20-89% w/w protein relative to the total solids,

15-90% w/w BLG relative to the total amount of protein,

4-50% w/w ALA relative to total protein, and

0-40% w/w CMP relative to protein.

Preferably, the WPC or SPC comprises:

20-89% w/w protein relative to the total solids,

15-80% w/w BLG relative to the total amount of protein,

4-50% w/w ALA relative to total protein, and

0-40% w/w CMP relative to protein.

More preferably, the WPC or SPC comprises:

70-89% w/w protein relative to the total solids,

30-90% w/w BLG relative to the total amount of protein,

4-35% w/w ALA relative to total protein, and

0-25% w/w CMP relative to protein.

SPC typically contains no CMP or only trace amounts of CMP.

The terms "whey protein isolate" (WPI) and "serum protein isolate" (SPI) relate to dry or aqueous compositions comprising 90-100% w/w of the total amount of protein relative to the total amount of solids.

The WPI or SPI preferably comprises:

90-100% w/w protein relative to the total solids,

15-70% w/w BLG relative to the total amount of protein,

(ii) 8-50% w/w ALA relative to total protein, and

0-40% w/w CMP relative to the total amount of protein.

Optionally, but also preferably, the WPI or SPI may comprise:

90-100% w/w protein relative to the total solids,

30-95% w/w BLG relative to the total amount of protein,

4-35% w/w ALA relative to total protein, and

0-25% w/w CMP relative to the total amount of protein.

More preferably, the WPI or SPI may comprise:

90-100% w/w protein relative to the total solids,

30-90% w/w BLG relative to the total amount of protein,

4-35% w/w ALA relative to total protein, and

0-25% w/w CMP relative to the total amount of protein.

SPI typically contains no or only trace CMP.

In the context of the present invention, the term "other protein" refers to a protein that is not BLG. Other proteins present in whey protein solutions typically comprise one or more non-BLG proteins found in milk serum or whey. Non-limiting examples of such proteins are alpha-lactalbumin, bovine serum albumin, immunoglobulin, Casein Macropeptide (CMP), osteopontin, lactoferrin, and milk fat globule membrane protein.

The term "consisting essentially of … …" means that the claim or feature in question covers the named material or step as well as those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

In the context of the present invention, the phrase "Y and/or X" means "Y" or "X" or "Y and X". The phrase "n" according to the same logic₁，n₂，...，n_i-1And/or n_i"represents" n₁"or" n₂"or_i-1"or" n_i", or n₁，n₂，...n_i-1And n_iAny combination of these ingredients.

In the context of the present invention, the term "dry" or "dried" means that the composition or product in question comprises at most 10% w/w water, preferably at most 6% w/w, more preferably even less water.

In the context of the present invention, the term "physical microbial reduction" relates to a physical interaction with a composition which results in a reduction of the total amount of viable microorganisms of the composition. The term does not include the addition of chemicals that result in killing of microorganisms. The term also does not include the heat exposure to which the atomized droplets of liquid are exposed during spray drying, but includes possible preheating prior to spray drying.

In the context of the present invention, the pH of the powder refers to the pH of 10g of powder mixed into 90g of demineralized water and measured according to example 1.16.

In the context of the present invention, unless otherwise specified (e.g., total solids or total protein), the weight percent (% w/w) of a component of a certain composition, product or material refers to the weight percent of that component relative to the weight of the particular composition, product or material.

In the context of the present invention, both the processing step "concentration" and the verb "concentration" relate to the concentration of proteins, including the concentration of proteins by total amount of solids and the concentration of proteins by total weight. This means that, for example, the concentration does not necessarily require an increase in the absolute concentration w/w of protein in the composition, as long as the content of protein relative to the total amount of solids is increased.

In the context of the present invention, the term "weight ratio" between component X and component Y means the ratio obtained by calculating m_X/m_YA value obtained wherein m_XIs the amount (by weight) of component X, and m_YIs the amount (by weight) of component Y.

In the context of the present invention, the term "at least pasteurization" relates to a heat treatment with a microbiological kill equal to or higher than a heat treatment at 70 ℃ for 10 seconds. The reference for determining the bactericidal effect was E.coli O157: H7(E.coli O157: H7).

In the context of the present invention, the term "whey protein feed" relates to a whey protein source from which the liquid BLG isolate is derived. The BLG content of the whey protein feed, typically WPC, WPI, SPC or SPI, relative to the total amount of protein, is lower than that of the liquid BLG isolate.

In the context of the present invention, the term "BLG-enriched composition" relates to a BLG-enriched composition obtained by separating BLG from a whey protein feed. BLG-enriched compositions typically comprise the same whey protein as whey protein feed, but BLG is present in significantly higher concentrations relative to the total amount of protein than in whey protein feed. BLG-enriched compositions may be prepared from whey protein feed, for example, by chromatography, protein crystallization, and/or membrane-based protein fractionation. The BLG-enriched composition comprises at least 85% w/w, preferably at least 90% w/w BLG relative to the total amount of protein. In some cases, the BLG-enriched composition may be used directly as a liquid BLG isolate. However, additional processing is typically required to convert the BLG-enriched composition into a liquid BLG isolate.

In the context of the present invention, the term "whey protein solution" is used to describe a specific aqueous whey protein composition which is supersaturated in a salination mode relative to BLG and which can be used to prepare BLG crystals.

In the context of the present invention, the term "sterile" means that the sterile composition or product in question does not contain any living microorganisms and therefore does not grow during storage at room temperature. The sterilized composition is sterile.

Liquids such as beverage products typically have a shelf life of at least six months at room temperature when sterilized and aseptically packaged in sterile containers. The sterilization process kills spores and microorganisms that may cause the liquid to deteriorate.

In the context of the present invention, the term "energy content" refers to the total content of energy contained in a food product. The energy content may be measured in kilojoules (kJ) or kilocalories (kcal) and is referred to as calories per serving, e.g., kcal per 100 grams serving. An example is a beverage with an energy content of 350kcal per 100 g of beverage.

The total energy content of a food product includes energy contributions from all the macronutrients present in the food product, such as energy from proteins, lipids and carbohydrates. The energy distribution of the macronutrients in the food product can be calculated based on the amount of macronutrients in the food product and the contribution of the macronutrients to the total energy content of the food product. The energy distribution can be expressed as a percentage of energy (E%) of the total energy content of the food product. For example, for a beverage comprising 20E% protein, 50E% carbohydrate and 30E% lipid, this means that 20% of the total energy is from protein, 50% of the total energy is from carbohydrate and 30% of the total energy is from fat (lipid).

In the context of the present invention, the term "nutritionally complete nutritional supplement" is understood to be a food product comprising proteins, lipids and carbohydrates and further comprising vitamins, minerals and trace elements, wherein the nutritional content of the beverage matches a perfectly healthy diet.

In the context of the present invention, the term "nutritionally incomplete supplement" refers to a food product comprising one or more macronutrients and optionally also vitamins, minerals and trace elements. A nutritionally incompetent beverage may contain protein as the only nutrient or may, for example, contain protein and carbohydrate.

The term "Food for Special Medical Purposes (FSMP)" or "medical food" is a food for oral or tube feeding (tube feeding) for specific medical disorders, diseases or conditions having special nutritional requirements and used under medical supervision. The medical food can be a supplement/beverage with complete nutrition or a supplement/beverage with incomplete nutrition.

The term "nutrient" refers to a substance used by an organism to survive, grow, and reproduce. The nutrient may be a macronutrient or a micronutrient. Macronutrients are nutrients that provide energy when consumed, such as proteins, lipids and carbohydrates. Micronutrients are nutrients like vitamins, minerals and trace elements.

The term "instant beverage powder" or "instant beverage powder product" refers to a powder that can be converted to a liquid beverage by the addition of a liquid, such as water.

In the context of the present invention, the terms "beverage product" and "product" as used as an entity (a substentive) refer to any water-based liquid that can be ingested as a beverage, e.g. by pouring, sipping or tube feeding.

In the context of the present invention, the term "protein component" relates to the protein in the composition in question, for example in a powder or beverage preparation.

In the context of the present invention, the term "astringency" relates to mouthfeel. Astringency feels like contraction of the cheek muscles, resulting in increased salivation. Therefore, astringency is not a taste per se, but a physical mouth feel in the mouth and a feeling of change with time.

In the context of the present invention, the term "dry mouth feel" relates to the sensation in the mouth, which is perceived as dry mouth and teeth and results in a minimization of saliva production.

Thus, dry mouth feel is not a taste, but physical mouth feel and time-dependent sensation in the mouth.

In the context of the present invention, the term "mineral" as used herein, unless otherwise indicated, refers to any of a major mineral, a trace mineral or a trace mineral, other minerals, and combinations thereof. The main minerals comprise calcium, phosphorus, potassium, sulfur, sodium, chlorine, magnesium. The trace or micro-minerals comprise iron, cobalt, copper, zinc, molybdenum, iodine, selenium, manganese, and the other minerals comprise chromium, fluorine, boron, lithium, and strontium.

In the context of the present invention, unless otherwise indicated, the terms "lipid", "fat" and "oil" as used herein are used interchangeably and refer to a lipid material derived from a plant or animal or processed. These terms also include synthetic lipid materials, so long as such synthetic materials are suitable for human consumption.

In the context of the present invention, the term "transparent" includes beverage products that have a distinctly clear appearance and which allow light to pass through and display a clear image. The turbidity of the clear beverage was at most 200 NTU.

In the context of the present invention, the term "opaque" includes beverage products which have a distinctly opaque appearance and which have a turbidity of more than 200 NTU.

One aspect of the present invention relates to a packaged heat-treated beverage product having a pH of 5.5 to 8.0, said beverage comprising:

-protein in a total amount of 1 to 20% w/w by weight of the beverage, wherein at least 85% w/w of the protein is beta-lactoglobulin (BLG),

-optionally, a sweetener and/or a flavoring agent.

One advantage of the present invention is that drinkable beverages can be produced that have a neutral pH and a low viscosity.

For a number of reasons, it is highly beneficial that at least 85% w/w of the protein in the packaged heat-treated beverage product is BLG.

The advantage is that the packaged heat-treated beverage product according to the invention is more stable and less coloured than a similar WPI beverage.

This is achieved by the packaged heat-treated beverage product of the present invention. Thus, it was surprisingly found that the packaged heat-treated beverage was less coloured even when a high protein concentration was applied compared to a pH neutral WPI beverage with a heat treatment of a more yellowish colour.

Advantageously, therefore, no bleaching or additional whitening is required to remove or reduce the yellow coloration due to the inventive composition of the packaged heat-treated beverage of the present invention.

In some preferred embodiments of the packaged heat-treated beverage product of the invention, at least 85% w/w of the protein is BLG. Preferably, at least 88% w/w of the protein is BLG, more preferably at least 90% w/w of the protein is BLG, even more preferably at least 91% w/w of the protein is BLG, most preferably at least 92% w/w of the protein is BLG.

Even higher relative amounts of BLG are possible and desirable, and therefore in some preferred embodiments of the invention at least 94% w/w of the protein in the packaged heat-treated beverage product is BLG, more preferably at least 96% w/w of the protein is BLG, even more preferably at least 98% w/w of the protein is BLG, most preferably about 100% w/w of the protein is BLG.

For example, the packaged heat-treated beverage product preferably comprises at least 97.5% w/w BLG relative to the total amount of protein, preferably at least 98.0% w/w BLG, more preferably at least 98.5% w/w BLG, even more preferably at least 99.0% BLG, and most preferably at least 99.5% w/w BLG relative to the total amount of protein, e.g. about 100.0% w/w BLG relative to the total amount of protein.

The protein in the packaged heat-treated beverage product is preferably made from mammalian milk, and preferably from ruminant milk (e.g., milk from cattle, sheep, goats, buffalo, camels, llamas, horses, and/or deer). Particularly preferred are proteins derived from bovine milk. Thus, the protein of the packaged heat-treated beverage product is preferably milk protein.

The protein of the packaged heat-treated beverage product is preferably whey protein or milk albumin, even more preferably bovine whey protein or milk albumin.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product is at least pasteurized.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product is sterile.

For both clear and opaque beverages, the visual appearance of the beverage product is important to the consumer. The inventors have found that it is advantageous to be able to control the colour of a beverage, or to control the lack of colour of a beverage, particularly for clear aqueous beverages or white creamy beverages.

However, even if a dedicated colorant is added during the production of the beverage, the inventors have found that it is advantageous to be able to avoid an additional color source to avoid undesired changes or alterations in the visual appearance of the beverage. The present inventors have found that the high BLG protein profiles described herein are neutral/colorless in color and have less color change than conventional WPI. Conventional WPI has a yellowish appearance which can be somewhat attenuated by the addition of oxidizing agents such as bleach. However, the addition of an oxidizing agent is generally undesirable and is even no longer necessary for the present invention.

The CIELAB color scale as described in example 1.9 was used to determine the color of the beverage. For example, a positive Δ b value indicates a more yellow color than demineralized water, while a negative Δ b value indicates a more blue beverage than demineralized water. Therefore, consumers generally prefer that the color difference b should be close to 0 in order to obtain a beverage that is neither yellow nor blue.

In some preferred embodiments of the invention, the packaged heat-treated beverage product has a color value Δ b in the range of-0.10 to +0.51 of the CIELAB color scale, particularly if the product has a turbidity of at most 200NTU, more preferably at most 40 NTU.

In a further preferred embodiment of the invention, the packaged heat-treated beverage product has a color value Δ b in the range of 0.0 to 0.40, preferably in the range of +0.10 to +0.25 of the CIELAB color scale.

For beverage products that are opaque (e.g., have a turbidity of greater than 200NTU, preferably greater than 1000 NTU), the color value ab of the packaged heat-treated beverage product is preferably in the range of-6 to-1.7, preferably in the range of-5.0 to-2.0 of the CIELAB color scale.

In some preferred embodiments of the invention, the color value ab of the protein component of the packaged heat-treated beverage product is in the range of-0.10 to +0.51 of the CIELAB color scale, in particular if the turbidity of the product is at most 200NTU, more preferably at most 40 NTU.

These beverages have less yellow colour than beverages comprising WPI with higher Δ b values and more yellow colour.

In some other preferred embodiments of the present invention, the color value Δ b of the protein component of the packaged heat-treated beverage product is in the range of 0.0 to 0.40, preferably in the range of +0.10 to +0.25 of the CIELAB color scale.

a-values indicate green-red components, green in the negative direction and red in the positive direction. It is generally preferred that the color difference a should be about zero so that the beverage is neither red nor green.

It is generally preferred that the colour value Δ a of the protein component in the packaged heat-treated beverage product is in the range-0.2 to 0.2 of the CIELAB scale, particularly if the product has a turbidity of at most 200NTU, more preferably at most 40 NTU.

Preferably, the packaged heat-treated beverage product has a color value Δ a in the range of-0.15 to 0.15, preferably in the range of-0.10 to 0.10 on the CIELAB color scale.

The inventors have found that it may be advantageous to control the mineral content to achieve certain desired characteristics of the packaged heat-treated beverage product.

The present inventors have unexpectedly found that when using a BLG isolate as defined herein, beverage products with high mineral concentrations can be produced without compromising viscosity and avoiding gelation (see e.g. example 2). This provides the possibility that a packaged heat-treated beverage product with a high mineral content can be produced and that a beverage of a nutritionally complete nutritional supplement or a nutritionally incomplete supplement can be produced.

In some embodiments of the present invention, the packaged heat-treated beverage product comprises a plurality of minerals. In an exemplary embodiment, the packaged heat-treated beverage product comprises at least four minerals. In one embodiment, the four minerals are sodium, potassium, magnesium and calcium.

In some preferred embodiments of the invention, the total amount of Na, K, Mg and Ca in the packaged heat-treated beverage product is in the range of 0 to 400mM, preferably in the range of 10-200mM or preferably in the range of 20-100 mM.

In other preferred embodiments of the invention, the total amount of Na, K, Mg and Ca in the packaged heat-treated beverage product is in the range of 0 to 100mM, more preferably in the range of 5-50mM, even more preferably in the range of 10-35 mM.

In some preferred embodiments of the invention, the total amount of Na, K, Mg and Ca in the packaged heat-treated beverage product is at most 400 mM.

In a further preferred embodiment of the invention the total amount of Na, K, Mg and Ca in the packaged heat-treated beverage product is at most 300mM, preferably at most 200mM, or preferably at most 100mM, or preferably at most 80mM or preferably at most 60mM or preferably at most 40mM or preferably at most 30mM or preferably at most 20mM or preferably at most 10mM or preferably at most 5mM or preferably at most 1 mM.

In some preferred embodiments of the invention, the total amount of Mg and Ca in the packaged heat-treated beverage product is at most 75mM, more preferably at most 40mM in the packaged heat-treated beverage product, and more preferably at most 20mM in the packaged heat-treated beverage product.

In other preferred embodiments of the present invention, the total amount of Mg and Ca in the packaged heat-treated beverage product is at most 10mM, more preferably at most 8.0mM in the packaged heat-treated beverage product, more preferably at most 6.0mM in the packaged heat-treated beverage product, even more preferably at most 4.0mM in the packaged heat-treated beverage product, and most preferably at most 2.0mM in the packaged heat-treated beverage product.

In another exemplary embodiment of the invention, the packaged heat-treated beverage product comprises a plurality of minerals selected from the group consisting of: calcium, iodine, zinc, copper, chromium, iron, phosphorus, magnesium, selenium, manganese, molybdenum, sodium, potassium, and combinations thereof.

In other preferred embodiments of the invention, the heat-treated beverage product is a low mineral beverage.

In the context of the present invention, the term "low mineral" relates to a composition, e.g. a liquid, a beverage, a powder or another food product, having at least one, preferably two, even more preferably all of the following:

-ash content up to 1.2% w/w relative to total solids is transparent,

-the total content of calcium and magnesium is at most 0.3% w/w relative to the total solids,

-the total content of sodium and potassium is at most 0.10% w/w relative to the total solids,

-a total content of phosphorus of at most 100mg phosphorus per 100g protein.

Preferably, the low mineral composition has at least one, preferably two or more, even more preferably all of the following:

-ash content of at most 0.7% w/w relative to total solids,

-the total content of calcium and magnesium is at most 0.2% w/w relative to the total solids,

-the total content of sodium and potassium is at most 0.08% w/w relative to the total solids,

-a total phosphorus content of at most 80 mg phosphorus per 100g protein.

Even more preferably, the low mineral composition has at least one, preferably two or more, even more preferably all of the following:

-ash content of at most 0.5% w/w relative to total solids,

-the total content of calcium and magnesium is at most 0.15% w/w relative to the total amount of solids,

-the total content of sodium and potassium is at most 0.06% w/w relative to the total solids,

-a total phosphorus content of at most 50mg phosphorus per 100g protein.

It is particularly preferred that the low mineral composition has the following characteristics:

-ash content of at most 0.5% w/w relative to total solids,

-the total content of calcium and magnesium is at most 0.15% w/w relative to the total amount of solids,

-the total content of sodium and potassium is at most 0.06% w/w relative to the total solids,

-a total phosphorus content of at most 50mg phosphorus per 100g protein.

In another exemplary embodiment of the invention, the packaged heat-treated beverage product comprises a plurality of minerals selected from the group consisting of: calcium, iodine, zinc, copper, chromium, iron, phosphorus, magnesium, selenium, manganese, molybdenum, sodium, potassium, and combinations thereof.

The present inventors have found that the present invention allows the preparation of packaged heat-treated beverage products having very low levels of phosphorus and other minerals, such as potassium, which is advantageous for patients suffering from kidney disease or suffering from reduced kidney function.

The packaged heat-treated beverage product is preferably a low phosphorous beverage product.

The packaged heat-treated beverage product is preferably a low potassium beverage product.

The packaged heat-treated beverage product is preferably a low phosphorous and low potassium beverage product.

In the context of the present invention, the term "low phosphorus" relates to compositions, such as liquids, powders or other food products, having a total content of phosphorus of at most 100 mg per 100g of protein. Preferably, the low phosphorus composition has a total phosphorus content of at most 80mg phosphorus per 100g protein. More preferably, the low phosphorus composition may have a total phosphorus content of at most 50mg phosphorus per 100g protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of up to 20mg phosphorus per 100g protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of at most 5mg phosphorus per 100g protein. The low phosphorous composition according to the invention may be used as a food ingredient for the production of a food product for a patient population with reduced renal function.

Thus, in some particularly preferred embodiments of the present invention, the packaged heat-treated beverage product comprises at most 80mg of phosphorus per 100g of protein. Preferably, the packaged heat-treated beverage product comprises at most 30mg of phosphorus per 100g of protein. More preferably, the packaged heat-treated beverage product comprises at most 20mg of phosphorus per 100g of protein. Even more preferably, the packaged heat-treated beverage product comprises at most 10mg of phosphorus per 100g of protein. Most preferably, the packaged heat-treated beverage product contains at most 5mg of phosphorus per 100g of protein.

The phosphorus content is related to the total amount of elemental phosphorus in the composition in question and is determined according to example 1.19.

In the context of the present invention, the term "low potassium" relates to compositions, such as liquids, powders or other food products, having a total potassium content of at most 700mg potassium per 100g protein. Preferably, the low potassium composition has a total potassium content of up to 600mg potassium per 100g protein. More preferably, the low potassium composition may have a total potassium content of up to 500mg potassium per 100g protein. More preferably, the low potassium composition may have a total potassium content of up to 400mg potassium per 100g protein. More preferably, the low potassium composition may have a total potassium content of up to 300mg potassium per 100g protein. Even more preferably, the low potassium composition may have a total potassium content of up to 200mg potassium per 100g protein. Even more preferably, the low potassium composition may have a total potassium content of up to 100mg potassium per 100g protein. Even more preferably, the low potassium composition may have a total potassium content of up to 50mg potassium per 100g protein, and even more preferably, the low potassium composition may have a total potassium content of up to 10mg potassium per 100g protein.

The low potassium composition according to the invention can be used as a food ingredient for the production of a food product for a patient population with reduced renal function.

Thus, in some particularly preferred embodiments of the present invention, the packaged heat-treated beverage product comprises at most 600mg potassium per 100g protein. More preferably, the packaged heat-treated beverage product comprises at most 500mg potassium per 100g protein. More preferably, the packaged heat-treated beverage product comprises at most 400mg potassium per 100g protein. More preferably, the packaged heat-treated beverage product comprises at most 300mg potassium per 100g protein. Even more preferably, the packaged heat-treated beverage product comprises at most 200mg potassium per 100g protein. Even more preferably, the packaged heat-treated beverage product comprises at most 100mg potassium per 100g protein. Even more preferably, the packaged heat-treated beverage product comprises at most 50mg potassium per 100g protein, and even more preferably, the packaged heat-treated beverage product comprises at most 10mg potassium per 100g protein.

The amount of potassium is related to the total amount of elemental potassium in the composition in question and is determined according to example 1.19.

In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises at most 100mg phosphorus/100 g protein and at most 700mg potassium/100 g protein, preferably at most 80mg phosphorus/100 g protein and at most 600mg potassium/100 g protein, more preferably at most 60mg phosphorus/100 g protein and at most 500mg potassium/100 g protein, more preferably at most 50mg phosphorus/100 g protein and at most 400mg potassium/100 g protein, or more preferably at most 20mg phosphorus/100 g protein, at most 200mg potassium/100 g protein, even more preferably at most 10mg phosphorus/100 g protein, at most 50mg potassium/100 g protein. In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises at most 100mg of phosphorus per 100g of protein and at most 340mg of potassium per 100g of protein.

The heat-treated beverage product comprising low amounts of phosphorus and potassium, which preferably also comprises carbohydrates in a total amount of 30-60%, preferably 35-50E%, of the total energy content of the beverage, and lipids in a total amount of 20-60%, preferably 30-50E%, of the total energy content of the beverage, may advantageously be supplemented with carbohydrates and lipids.

In one embodiment of the present invention, the packaged heat-treated beverage product comprises a plurality of vitamins. In an exemplary embodiment, the packaged heat-treated beverage product comprises at least ten vitamins. In an exemplary embodiment, the substantially transparent liquid nutritional composition comprises a plurality of vitamins selected from the group consisting of: vitamin a, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin K, riboflavin, pantothenic acid, vitamin E, thiamin, niacin, folic acid, biotin, and combinations thereof.

In one embodiment of the invention, the packaged heat-treated beverage comprises a multivitamin and a multimineral.

In some embodiments of the invention, the packaged heat-treated beverage product comprises one or more food acids selected from the group consisting of citric acid, malic acid, tartaric acid, acetic acid, benzoic acid, butyric acid, lactic acid, lactobionic acid, fumaric acid, succinic acid, ascorbic acid, adipic acid, phosphoric acid, and mixtures thereof.

In some preferred embodiments, the packaged heat-treated beverage product optionally comprises a sweetener, a sugar polymer, and/or a flavoring agent.

In one embodiment of the present invention, the packaged heat-treated beverage product comprises a flavoring agent selected from the group consisting of salt, flavoring, flavor enhancer, and/or aroma. In a preferred embodiment of the invention, the flavoring agent comprises chocolate, cocoa, lemon, orange, lime, strawberry, banana, argan fruit flavors or combinations thereof. The choice of taste may depend on the beverage to be produced.

In some preferred embodiments of the present invention, the pH of the packaged heat-treated beverage product is in the range of 6.5 to 7.5. Most preferably, the pH used is from 6.5 to 7.0 or from 6.8 to 7.2.

With respect to appearance, it was surprisingly found that the use of a whey protein beverage wherein at least 85% w/w of the protein is BLG provides an improvement in visual sensory (colour and turbidity) and viscosity compared to heat treated WPI beverages.

The pH of the packaged heat-treated beverage product is preferably in the range of 5.5 to 6.2, optionally the pH of the packaged heat-treated beverage product is in the range of 6.2-8.0.

Optionally, the packaged heat-treated beverage product has a pH in the range of 6.8 to 8.0, more preferably the packaged heat-treated beverage product has a pH in the range of 6.2-8.0.

The packaged heat-treated beverage product of the present invention is preferably clear and transparent, having a low viscosity in the pH range of 6.2-8.0, preferably in the pH range of 6.3-7.6, more preferably in the pH range of 6.5-7.2.

The packaged heat-treated beverage product of the present invention preferably has a low viscosity and a creamy appearance in the range of pH 5.5-8.0, preferably in the range of pH 5.7-6.8, more preferably in the range of pH 5.8-6.0.

In some preferred embodiments of the present invention, it has been found that the beverage product of the present invention is preferably heat-treated in the range of pH 5.6-6.2, preferably in the range of pH 5.6-8.0, optionally mixed with carbohydrate, fat, mineral and vitamin sources, adjusted to a preferred pH of 6.2-8.0, and subjected to a second heat treatment (UHT).

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a turbidity of at most 200 NTU.

The visual appearance of the packaged heat-treated beverage product is of interest to consumers. Transparency is a parameter used by consumers to evaluate products. One way to determine the clarity of a beverage product is by measuring the turbidity of the beverage as described in example 1.7.

In some embodiments of the packaged heat-treated beverage product, it is advantageous that the beverage product is transparent. This may be advantageous, for example, when the beverage is used as a sports drink or in "protein water", in which case it is beneficial for the beverage to resemble water in appearance.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a turbidity of at most 200NTU, such beverage being clear and/or transparent.

The inventors have surprisingly found that by heat treating a beverage product according to the invention, a clear heat treated beverage product having a turbidity of at most 200NTU can be obtained.

This is found when the heat treatment applied is both sterilisation and pasteurisation.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a turbidity of at most 150NTU, or preferably a turbidity of at most 100NTU, or preferably a turbidity of at most 80NTU, or preferably a turbidity of at most 60NTU, or more preferably a turbidity of at most 40NTU, or a turbidity of at most 30NTU, preferably a turbidity of at most 20NTU, more preferably a turbidity of at most 10NTU, more preferably a turbidity of at most 5NTU, even more preferably a turbidity of at most 2 NTU.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a turbidity of greater than 200NTU, such beverage being opaque.

In some embodiments of the packaged heat-treated beverage product, it is advantageous that the beverage product is opaque. This is advantageous, for example, when the beverage should resemble milk and have a milky appearance. The appearance of nutritionally complete nutritional supplements is also not generally transparent.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a turbidity of greater than 250 NTU. Preferably, the packaged heat-treated beverage product has a turbidity of greater than 300NTU, more preferably, a turbidity of greater than 500NTU, more preferably, a turbidity of greater than 1000, preferably a turbidity of greater than 1500NTU, and even more preferably a turbidity of greater than 2000 NTU.

The amount of insolubles in the heat treated beverage product is a measure of the instability of the beverage and the extent to which settling of the precipitate occurs over time. Beverages with high amounts of insolubles are generally considered unstable.

In the context of the present invention, a whey protein beverage preparation is considered "stable" if at most 15% of the total amount of protein in the heated sample settles after centrifugation at 3000g for 5 minutes. See the analytical method in example 1.10.

It was surprisingly found that when BLG with a content of at least 85 w/w% is used as protein source, the protein component contains at most 15% insolubles after centrifugation at 3000g for 5 minutes, compared to WPI with a lower BLG content used as protein source, indicating that the beverage product is stable.

Thus, in some preferred embodiments of the present invention, the protein component of the heat-treated beverage product contains up to 15% insolubles.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product comprises at most 15% insolubles.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product preferably comprises at most 12% insolubles, more preferably at most 10% insolubles, even more preferably at most 8% insolubles, most preferably at most 6% insolubles.

Even lower levels of insolubles are generally preferred, and in some preferred embodiments, the packaged heat-treated beverage product contains at most 4% insolubles, preferably at most 2% insolubles, more preferably at most 1% insolubles, and most preferably no detectable insolubles at all.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a viscosity of at most 200cP (centipoise), measured at a shear rate of 100/s at 22 degrees Celsius.

Consumers prefer that the heat-treated beverage be a liquid rather than a gel.

One method of determining the viscosity of a beverage product is by measuring the viscosity of the beverage as described in example 1.8.

In some embodiments of the packaged heat-treated beverage product, it is advantageous for the beverage product to have a low viscosity. This is advantageous when the beverage is used as a sports drink or, in some embodiments, as a nutritionally complete beverage.

The inventors have surprisingly found that beverage products having a neutral pH and which have been subjected to a heat treatment such as pasteurization or even sterilization have a viscosity of at most 200 centipoise, measured at a shear rate of 100/s at 22 degrees celsius.

Thus, in some preferred embodiments of the present invention, the packaged heat-treated beverage product has a viscosity of at most 200 cP.

Preferably, the packaged heat-treated beverage product has a viscosity of at most 150cP, preferably at most 100cP, more preferably at most 80cP, even more preferably at most 50cP, most preferably at most 40 cP.

Even lower viscosities are generally preferred, and thus in some preferred embodiments of the invention, the viscosity of the packaged heat-treated beverage product is at most 20cP, preferably at most 10cP, more preferably at most 5cP, even more preferably at most 3cP, even more preferably at most 2cP, even more preferably at most 1 cP.

In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises a total amount of protein of from 2 to 18% w/w relative to the weight of the beverage product.

In other preferred embodiments of the invention the packaged heat-treated beverage product comprises a total amount of protein of from 3 to 20% w/w, more preferably from 3 to 18% w/w, even more preferably from 3 to 15% w/w, most preferably from 3 to 10% w/w, based on the weight of the beverage.

In some embodiments of the invention, it is advantageous that the packaged heat-treated beverage product has a total amount of protein of from 1.0 to 10.0% w/w of the weight of the beverage.

In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises a total amount of protein of from 1 to 10% w/w of the weight of the beverage product.

The packaged heat-treated beverage product preferably comprises a total amount of protein of from 2.0 to 9.0% w/w of the weight of the beverage, or the packaged heat-treated beverage product preferably comprises a total amount of protein of from 3.0 to 8.0% w/w of the weight of the beverage, or the packaged heat-treated beverage product preferably comprises a total amount of protein of from 5.0 to 7.5% w/w of the weight of the beverage, or the packaged heat-treated beverage product preferably comprises a total amount of protein of from 4.0 to 6.0% w/w of the weight of the beverage.

Most preferably, the packaged heat-treated beverage product comprises a total amount of protein of from 4.0 to 6.0% w/w by weight of the beverage. This protein range is particularly relevant when the heat-treated beverage product is a sports beverage. However, this range is also relevant for certain medical applications of beverages.

In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises a total amount of protein of 10 to 20% w/w relative to the weight of the beverage product.

In some embodiments of the invention, the packaged heat-treated beverage product preferably comprises a total amount of protein of 10 to 18% w/w by weight of the beverage, or preferably comprises a total amount of protein of 12.0 to 16.0% w/w by weight of the beverage, or preferably comprises a total amount of protein of 13.0-15.0% w/w by weight of the beverage.

In some preferred embodiments, the packaged heat-treated beverage product comprises a total amount of protein of from 1.0 to 6.0% w/w by weight of the beverage, and in other preferred embodiments, the packaged heat-treated beverage product comprises a total amount of protein of from 6.0 to 12.0% w/w by weight of the beverage.

Or in other preferred embodiments, the packaged heat-treated beverage product comprises a total amount of protein of from 12.0 to 20.0% w/w by weight of the beverage.

All proteins in the beverage are preferably whey proteins and/or milk albumin.

The packaged heat-treated beverage product of the invention is particularly useful as a sports beverage, in which case it preferably optionally comprises only a limited content of lipids and/or optionally also a limited content of carbohydrates.

In some preferred embodiments of the invention the preparation is particularly useful as a sports drink and comprises, for example, protein in a total amount of 1-20% w/w, preferably 2-15% w/w, or preferably 2-10% w/w, most preferably 2-6% w/w, based on the weight of the drink.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly suitable for use as a nutritional supplement for nutritional incompletion and comprises, for example, protein in a total amount of 2-20% w/w, preferably 3-10% w/w, by weight of the beverage.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly suitable for use as a nutritionally complete nutritional supplement and comprises, for example, protein in a total amount of 4-20% w/w, or preferably in a total amount of 5-18% w/w, by weight of the beverage.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly advantageous for patients suffering from kidney disease or having poor kidney function.

In some preferred embodiments of the invention, for example, the packaged heat-treated beverage product comprises a total amount of protein of 2-20% w/w by weight of the beverage, or preferably a total amount of protein of 3-12% w/w by weight of the beverage, or preferably a total amount of protein of 3-10% w/w by weight of the beverage.

It is particularly preferred that the packaged heat-treated beverage product comprises BLG isolate, e.g. in combination with other protein sources, preferably as the main protein source, possibly even as the sole protein source.

Protein naturalness depends on many factors, including protein concentration, pH, temperature, and heat treatment time.

Intrinsic tryptophan fluorescence emission (I330nm/I350nm) is a measure of the extent of deployment of the BLG, and the inventors have found that at high BLG, tryptophan fluorescence emission correlates with low or no deployment of the BLG, and measured according to example 1.1 (I330nm/I350 nm).

In some preferred embodiments of the invention, the BLG isolate powder has an intrinsic tryptophan fluorescence emissivity (I330nm/I350nm) of at least 1.11.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission rate (I330nm/I350nm) of the BLG isolate powder is at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, and most preferably at least 1.19.

If the BLG isolate contains a large amount of non-protein material, it is preferable to isolate the protein fraction before measuring the intrinsic tryptophan fluorescence emission rate. Thus, in some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission rate of the protein component of the BLG isolate powder is at least 1.11.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission rate (I330nm/I350nm) of the protein component of the BLG isolate powder is at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

Protein components can be separated from BLG isolate powder by dissolving the BLG isolate powder in demineralized water and then subjecting the solution to dialysis or ultrafiltration-based diafiltration using a protein-retaining filter.

Protein denaturation can also be described by another assay method than Trp fluorescence. This process is described in example 1.3. The principle of this method is as follows:

it is known that the solubility of denatured whey protein at pH4.6 is lower than that at pH values below or above pH4.6, and therefore, the denaturation of a whey protein composition is determined by measuring the amount of soluble protein at pH4.6 relative to the total amount of protein at pH at which the protein in solution is stable.

Thus, the protein denaturation degree D of the whey protein composition is calculated as follows:

D＝((P_{pH 7.0 or 3.0}-S_{pH 4.6})/P_{pH 7.0 or 3.0})×100％

Wherein (P)_{pH 7.0 or 3.0}) Is the total protein content at pH 7.0 or 3.0, and (S)_{pH 4.6}) Is the total protein content in the supernatant at pH 4.6. See example 1.3.

In some preferred embodiments of the invention, the protein denaturation degree of the BLG isolate powder is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 3%, even more preferably at most 1%, most preferably at most 0.2%.

In some embodiments of the invention, when the protein component and/or beverage product has been subjected to, for example, high temperature heat treatment, then the protein denatures by greater than 10%, preferably greater than 20%, preferably greater than 30%, preferably greater than 40%, or preferably greater than 50%, or preferably greater than 70%, or preferably greater than 80%, or preferably greater than 90%, or preferably greater than 95%, or preferably greater than 99%.

The packaged heat-treated beverage products of the present invention may contain other macronutrients in addition to protein. In some embodiments of the present invention, the packaged heat-treated beverage product further comprises a carbohydrate. The total carbohydrate content of the heat-treated beverage product of the present invention depends on the intended use of the heat-treated beverage product.

In some embodiments of the present invention, the packaged heat-treated beverage product further comprises at least one source of carbohydrates. In an exemplary embodiment, the at least one carbohydrate source is selected from the group consisting of: sucrose (sucrose), beet sugar (saccharose), maltose, dextrose, galactose, maltodextrin, corn syrup solids, sucralose, glucose polymers, corn syrup, modified starch, resistant starch, rice-derived carbohydrates, isomaltulose, white sugar, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohols, fructooligosaccharides, soy fiber, corn fiber, guar gum, konjac flour, polydextrose, fiber sol (Fibersol), and combinations thereof. In some embodiments of the invention, the packaged heat-treated beverage product comprises indigestible carbohydrates, and if polysaccharides, the fructans comprise inulin or fructooligosaccharides.

In some preferred embodiments of the present invention, the packaged heat-treated beverage products and liquid solutions comprise sugar polymers, i.e., oligosaccharides and/or polysaccharides.

In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises carbohydrates which represent 0 to 95% of the total energy content of the product, preferably in the range of 10 to 85% of the total energy content of the product, preferably 20 to 75% of the total energy content of the product, or preferably 30 to 60% of the total energy content of the product.

Generally even lower carbohydrate contents are preferred, and therefore in some preferred embodiments of the invention it is preferred that the carbohydrate content is in the range between 0 and 30% of the total energy content of the product, more preferably in the range between 0 and 20% of the total energy content of the product, even more preferably 0-10% of the total energy content of the product.

In some preferred embodiments of the present invention, the carbohydrate content of the packaged heat-treated beverage product comprises at most 3% of the total energy content of the product, more preferably at most 1% of the total energy content of the product, even more preferably at most 0.1% of the total energy content of the product.

In some preferred embodiments of the invention, the preparation is particularly useful as a sports drink and comprises, for example, a total amount of carbohydrates of at most 75%, preferably at most 40E%, preferably at most 10E% or preferably at most 5E% of the total energy content (E) of the drink.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product is particularly suitable for use as a nutritionally incomplete nutritional supplement and, for example, comprises a total amount of carbohydrates between 70 and 95%, preferably 80-90E%, of the total energy content (E) of the beverage.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product is particularly useful as a nutritionally complete nutritional supplement and, for example, comprises between 30-60%, preferably between 35-50E% of the total energy content of the beverage in total carbohydrate content.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly advantageous for patients suffering from kidney disease or reduced kidney function.

In some preferred embodiments of the invention, for example, the total amount of carbohydrates contained in the packaged heat-treated beverage product is between 30-60%, preferably between 35-50E% of the total energy content of the beverage.

In one embodiment of the present invention, the packaged heat-treated beverage product further comprises at least one additional ingredient selected from the group consisting of: vitamins, flavors, minerals, sweeteners, antioxidants, food acids, lipids, carbohydrates, prebiotics, probiotics, and non-whey proteins.

The further ingredients ensure that the packaged heat-treated beverage product comprises the required nutrients, i.e. nutrients which are particularly suitable for patients suffering from protein deficiency or athletes wishing to strengthen muscles.

In one embodiment of the invention, the liquid solution further comprises at least one high intensity sweetener. In one embodiment, the at least one high intensity sweetener is selected from the group consisting of: aspartame, cyclamate, sucralose, acesulfame salt, neotame (neotame), saccharin, stevia extracts, steviol glycosides (e.g., rebaudioside a), or combinations thereof. In some embodiments of the present invention, it is particularly preferred that the sweetener comprises or even consists of one or more High Intensity Sweeteners (HIS).

HIS is present in both natural and artificial sweeteners, and typically has a sweetness intensity that is at least 10 times that of sucrose.

If used, the total amount of HIS is generally in the range of 0.01-2% w/w. For example, the total amount of HIS may be in the range of 0.05-1.5% w/w. Alternatively, the total amount of HIS may be in the range of 0.1-1.0% w/w.

The choice of sweetener may depend on the beverage to be produced, for example, a high intensity sweetener (e.g., aspartame, acesulfame potassium or sucralose) may be used in beverages that do not require the sweetener to provide energy, while for beverages with natural characteristics, a natural sweetener (e.g., steviol glycosides, sorbitol or sucrose) may be used.

It may furthermore be preferred that the sweetener comprises or even consists of one or more polyol sweeteners. Non-limiting examples of useful polyol sweeteners are maltitol, mannitol, lactitol, sorbitol, inositol, xylitol, threitol, galactitol, or combinations thereof. If used, the total amount of polyol sweetener is typically in the range of 1-20% w/w. For example, the total amount of polyol sweetener may be in the range of 2-15% w/w. Alternatively, the total amount of polyol sweetener may be in the range of 4-10% w/w.

The packaged heat-treated beverage products of the present invention may contain other macronutrients in addition to protein. In some embodiments of the invention, the packaged heat-treated beverage product further comprises a lipid. The total lipid content in the heat-treated beverage products of the present invention depends on the intended use of the heat-treated beverage product.

In some preferred embodiments of the invention, the lipid content of the packaged heat-treated beverage product is from 0 to 50% of the total energy content of the product, or preferably from 0 to 40% of the total energy content of the product, or preferably in the range between 0 and 30% of the total energy content of the product, or preferably in the range between 0 and 20% of the total energy content of the product, or preferably in the range between 0 and 10% of the total energy content of the product, or preferably in the range between 0 and 5% of the total energy content of the product.

The content of lipids is according to ISO 1211: 2010 (determination of fat content)Gravimetric).

In some preferred embodiments of the invention, the lipid content of the packaged heat-treated beverage product comprises at most 3% of the total energy content of the product, more preferably at most 1% of the total energy content of the product, even more preferably at most 0.1% of the total energy content of the product.

In some preferred embodiments of the invention, the preparation is particularly useful as a sports drink and comprises, for example, a total amount of lipids of at most 10E%, preferably at most 1E%.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly suitable for use as a nutritionally incomplete nutritional supplement and comprises, for example, a total amount of lipids of at most 10%, preferably at most 1E%, of the total energy content of the beverage.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly useful as a nutritionally complete nutritional supplement and comprises, for example, a total amount of lipids that is 20-50%, preferably 30-40E%, or more preferably 25-40E% of the total energy content.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly advantageous for patients suffering from kidney disease or reduced kidney function.

In some preferred embodiments of the invention, for example, the packaged heat-treated beverage product comprises a total amount of lipids in the range of 20-60%, preferably in the range of 30-50E% of the total energy content.

In one embodiment of the invention the packaged heat-treated beverage product comprises food-grade fats, such as rapeseed oil and/or MCT (medium chain triglycerides), preferably in an amount of 2-10 wt%. Preferably, these fats contain a substantial proportion, e.g. at least 40%, preferably at least 60%, of unsaturated fatty acids, most preferably polyunsaturated fatty acids. Most preferably, the beverage is in emulsified form and the lipid is preferably present in the aqueous phase of the beverage product in the form of emulsified droplets.

The beverage product typically comprises a total amount of water in the range of 50-99% w/w, preferably 45-97% w/w, more preferably 40-95% w/w, even more preferably in the range of 35-90% w/w, most preferably in the range of 30-85% w/w.

In some preferred embodiments of the invention, the beverage product comprises a total amount of water in the range of 55-90% w/w, preferably 57-85% w/w, more preferably 60-80% w/w, even more preferably in the range of 62-75% w/w, most preferably in the range of 65-70% w/w.

In some preferred embodiments of the invention, the beverage product comprises a total amount of water in the range of 90-99% w/w, preferably 92-98.5% w/w, more preferably 94-98% w/w, even more preferably in the range of 95-98% w/w, most preferably in the range of 96-98% w/w. For example, these embodiments are useful for clear aqueous beverages.

In some preferred embodiments of the invention, the beverage product is non-alcoholic, meaning that it contains at most 1.0% w/w ethanol, more preferably at most 0.5% w/w, even more preferably at most 0.1% w/w, and most preferably no detectable ethanol.

The beverage product typically comprises total solids in the range of 1-45% w/w, preferably 5-40% w/w, more preferably 10-35% w/w, even more preferably in the range of 12-30% w/w, most preferably in the range of 16-25% w/w.

In some preferred embodiments of the invention the beverage product comprises a total amount of solids in the range of 10-45% w/w, preferably 15-43% w/w, more preferably 20-40% w/w, even more preferably in the range of 25-38% w/w, most preferably in the range of 30-35% w/w.

In some preferred embodiments of the invention, the beverage product comprises a total amount of solids in the range of 1-10% w/w, preferably 1.5-8% w/w, more preferably 2-6% w/w, even more preferably in the range of 2-5% w/w, most preferably in the range of 2-4% w/w. For example, these embodiments are useful for clear aqueous beverages.

The part of the beverage product that is not solid is preferably water.

In some preferred embodiments of the invention the sum of alpha-lactalbumin (ALA) and Caseinomacropeptide (CMP) comprises at least 40% w/w of the non-BLG proteins in the beverage, preferably at least 60% w/w, even more preferably at least 70% w/w, most preferably at least 90% w/w of the non-BLG proteins in the beverage.

In some preferred embodiments of the invention ALA comprises at most 80% w/w of the non-BLG proteins in the beverage product, preferably at most 60% w/w, even more preferably at most 40% w/w, and most preferably at most 30% w/w of the non-BLG proteins in the beverage product.

Even lower ALA content may be preferred, and therefore in some preferred embodiments of the invention ALA comprises at most 20% w/w of non-BLG proteins in the beverage product, preferably at most 15% w/w, even more preferably at most 10% w/w, most preferably at most 5% w/w of non-BLG proteins in the beverage product.

In a further preferred embodiment of the invention each major non-BLG whey protein is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Even lower concentrations of major non-BLG whey proteins may be desirable. Thus, in a further preferred embodiment of the invention, each major non-BLG whey protein is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

The inventors have found that a reduction of lactoferrin and/or lactoperoxidase is particularly advantageous for obtaining a color neutral whey protein product.

Thus, in some preferred embodiments of the invention, lactoferrin is present in a weight percentage relative to the total amount of protein which is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoferrin may be desirable. Thus, in a further preferred embodiment of the invention, lactoferrin is present in a weight percentage relative to the total amount of protein which is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Similarly, in some preferred embodiments of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% relative to the total amount of protein in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoperoxidase may be desirable. Thus, in a further preferred embodiment of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Lactoferrin and lactoperoxidase were quantified according to example 1.29.

In one embodiment of the present invention, the packaged heat-treated beverage product is a nutritionally complete nutritional supplement.

In one embodiment of the present invention, the packaged heat-treated beverage product is a nutritionally incomplete nutritional supplement.

In one embodiment of the present invention, the packaged heat-treated beverage product is a sports beverage.

In one embodiment of the invention, the packaged heat-treated beverage product is a low-phosphorous and low-potassium beverage suitable for patients suffering from kidney disease or reduced kidney function.

The packaged heat-treated beverage product of the invention is particularly useful as a sports beverage, in which case it preferably comprises only a limited amount of lipids and/or optionally also a limited amount of carbohydrates.

In some preferred embodiments of the invention, the article is particularly useful as a sports beverage and comprises, for example:

-the total amount of protein is 1-20% w/w relative to the weight of the beverage, preferably 2-15% w/w relative to the weight of the beverage, or 2-10% w/w relative to the weight of the beverage, most preferably 2-6% w/w relative to the weight of the beverage.

-the total amount of carbohydrates represents at most 75%, preferably at most 40E%, preferably at most 10E% or preferably at most 5E% of the total energy content of the beverage (E), and

-the total amount of lipids is at most 10E%, preferably at most 1E%.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product is particularly useful as a nutritional supplement for nutritional incompetence, including, for example:

-the total amount of protein is 2-20% w/w relative to the weight of the beverage, or preferably 3-10% w/w relative to the weight of the beverage,

-the total amount of carbohydrates represents between 70 and 95%, preferably 80-90E%, of the total energy content (E) of the beverage, and

-the total amount of lipids represents at most 10%, preferably at most 1E% of the total energy content of the beverage.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product is particularly useful as a nutritionally complete nutritional supplement and includes, for example:

-the total amount of protein relative to the weight of the beverage is in the range of 4-20% w/w, or preferably in the range of 5-18% w/w,

-the total amount of carbohydrates is between 30 and 60%, preferably between 35 and 50E%, of the total energy content of the beverage, and

the total amount of lipids represents 20-50%, preferably 30-40E% or preferably 25-45E% of the total energy content.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly advantageous for patients suffering from kidney disease or reduced kidney function. The content of phosphorus and other minerals (e.g., potassium) in the beverage product is very low.

In some preferred embodiments of the present invention, packaged heat-treated beverage products include, for example:

-the total amount of protein is 2-20% w/w relative to the weight of the beverage, or preferably 3-12% w/w relative to the weight of the beverage, or preferably 3-10% w/w relative to the weight of the beverage,

-the total amount of carbohydrates is between 30 and 60%, preferably between 35 and 50E%, of the total energy content of the beverage, and

the total amount of lipids represents 20-60%, preferably 30-50E% of the total energy content.

The inventors have seen evidence that beverages with a high content of protein nanogels, relative to the total amount of protein, reach the stomach with a reduced viscosity compared to beverages containing large amounts of soluble whey protein aggregates (see example 9). Soluble whey protein aggregates are typically formed after sterilization of beverages containing conventional whey protein isolates. The ability of food to develop viscosity and/or texture in the stomach has previously been linked to Satiety (Halford et al; Satiety products for appetite control: science and regulations for functional food for weight management); Proceedings of the Nutrition Society (2012),71, 350-. This is very advantageous for people who do not have or have poor appetite but who need high energy nutrition to recover and/or maintain muscle mass or other bodily functions.

In the context of the present invention, the term "protein nanogels" or "protein nanogels" relates to submicron sized particles of denatured whey proteins, which are generally spherical or nearly spherical in shape. Protein nanogels are also known as whey protein micelles and their micellar properties are discussed, for example, in WO2007/110421a2, but are questionable. The amount of soluble whey protein aggregates was quantified according to example 1.32. Protein nanogels have an opaque milky appearance when suspended and are therefore well suited for opaque beverages.

In the context of the present invention, the term "soluble whey protein aggregates" refers to small aggregates of denatured whey proteins, which are capable of forming a firm gel (much stronger than native whey proteins) when acidified to pH 4.6, and which typically have a linear, worm-like, branched or chain-like shape, typically with submicron dimensions. Soluble whey protein aggregates are well known to the person skilled in the art and, for example, as described in WO2007/110421a2, they are referred to as linear aggregates. The amount of soluble whey protein aggregates was quantified according to example 1.32. Soluble whey protein aggregates generally form clear solutions when dissolved in water and are therefore very suitable for use in clear beverages.

By controlling pH, mineral content (especially Ca)²⁺Content) and protein concentration of the protein solution, whether or not to form gels, large gel fragments, protein nanogels or soluble whey protein aggregates can be controlled. As is well known to the skilled person. Protein nanogels are typically formed by heating a whey protein solution having a pH of about 5.5 to about 6.5, preferably about 5.8 to 6.2, and are favored over conventional whey protein concentrates in whey protein solutions having a reduced mineral content. Soluble whey protein aggregates are generally formed by heating a whey protein solution having a pH of about 6.5 to 8.5, preferably about 6.6 to 7.5, and a higher level of monovalent cations (e.g., sodium) is preferred. Such monovalent cations are typically added when increasing the pH.

Thus, in some particularly preferred embodiments of the present invention, the packaged heat-treated beverage product comprises at least 50% w/w protein nanogel relative to the total amount of protein, preferably at least 60% w/w protein nanogel relative to the total amount of protein, more preferably at least 70% w/w, even more preferably at least 80% w/w, most preferably at least 90% w/w protein nanogel.

For example, a preferred packaged heat-treated beverage product comprises:

-the total amount of protein is 5-20% w/w, preferably 8-19% w/w, more preferably 9-18% w/w, even more preferably 10-17%, most preferably 11-16% w/w, relative to the weight of the beverage,

-the total amount of BLG is at least 85% w/w, preferably at least 88% w/w, more preferably at least 90% w/w, even more preferably at least 90% w/w, and most preferably at least 92% w/w, relative to the total amount of protein,

-the total amount of protein nanogel is at least 50% w/w, preferably at least 60% w/w, more preferably at least 70% w/w, even more preferably at least 80% w/w relative to the total amount of protein.

It is particularly preferred that the packaged heat-treated beverage product comprises:

-the total amount of protein is 10 to 20% w/w, preferably 11-19% w/w, more preferably 12-18% w/w, even more preferably 13-17% w/w, most preferably 14-16% w/w,

-the total amount of BLG is at least 90% w/w, preferably at least 92% w/w, more preferably at least 94% w/w, most preferably at least 96% w/w,

-the total amount of protein nanogel is at least 50% w/w relative to the total amount of protein, preferably it is at least 60% w/w relative to the total amount of protein, more preferably at least 70% w/w, even more preferably at least 80% w/w.

The inventors have also found that protein nanogels develop viscosity less readily than native whey protein, thus making it possible to produce a sterile, pH neutral beverage with a high protein content but with a viscosity low enough to make it easy to drink.

Protein nanogels made by thermal denaturation of whey protein containing at least 85% w/w BLG appear to be particularly advantageous for use in high protein beverages and, without being bound by theory, it is believed that high BLG protein nanogels can provide a denser protein nanogel structure than protein nanogels based on conventional WPI and that this difference allows more protein to be included in a beverage without affecting its drinkability.

In some preferred embodiments of the present invention, a packaged heat-treated beverage product comprises:

-at least 50% w/w protein nanogel relative to the total amount of protein,

-up to 30% w/w soluble whey protein aggregates relative to the total amount of protein,

-up to 5% w/w of insoluble proteinaceous matter.

In some more preferred embodiments of the present invention, a packaged heat-treated beverage product comprises:

-at least 60% w/w protein nanogel relative to the total amount of protein,

-up to 20% w/w soluble whey protein aggregates relative to the total amount of protein,

-up to 5% w/w of insoluble proteinaceous matter.

In some even more preferred embodiments of the present invention, the packaged heat-treated beverage product comprises:

-at least 70% w/w protein nanogel relative to the total amount of protein,

-up to 15% w/w soluble whey protein aggregates relative to the total amount of protein,

-up to 5% w/w of insoluble proteinaceous matter.

The inventors have observed that when a beverage with an increased amount of soluble whey protein aggregates relative to the total amount of protein reaches an environment similar to the stomach, it is more intense than a beverage containing less soluble whey protein aggregates (see example 9). The ability of food products to develop viscosity and/or texture in the stomach has previously been linked to satiety and beverages with a high content of soluble whey protein aggregates thus cause an increase in satiety upon ingestion. This is very advantageous for people who wish to lose weight, and is particularly useful for patients suffering from obesity.

Thus, in a further particularly preferred embodiment of the invention, the packaged heat-treated beverage product comprises at least 60% w/w soluble whey protein aggregates relative to the total amount of protein, preferably at least 70% w/w, more preferably at least 80%, even more preferably at least 90% w/w soluble whey protein aggregates relative to the total amount of protein.

Optionally but also preferably, the packaged heat-treated beverage product comprises:

-the total amount of protein is 5 to 12% w/w, preferably 6-11% w/w, more preferably 7-10% w/w, even more preferably 8-10% w/w, most preferably 11-16% w/w,

-a total amount of BLG of at least 94% w/w relative to the total amount of protein, preferably at least 96% w/w relative to the total amount of protein, even more preferably at least 98% w/w relative to the total amount of protein, and

-at least 60% w/w soluble whey protein aggregates relative to the total amount of protein, preferably at least 70% w/w, more preferably at least 80% w/w, even more preferably at least 90% w/w soluble whey protein aggregates relative to the total amount of protein,

the packaged heat-treated beverage product preferably has:

turbidity of at most 100NTU, preferably at most 40NTU, even more preferably at most 10NTU, and

at 22 ℃ and 100s^-1At a shear rate of at most 100cP, preferably at most 50cP, more preferably 20cP, and more preferably at most 10 cP.

For example, the packaged heat-treated beverage product preferably comprises:

-the total amount of protein is 5-20% w/w, preferably 8-19% w/w, more preferably 9-18% w/w, even more preferably 10-17% w/w, most preferably 11-16% w/w,

-total amount of BLG is at least 85% w/w relative to total amount of protein, preferably at least 90% w/w relative to total amount of protein, even more preferably at least 94% w/w relative to total amount of protein, most preferably at least 6% w/w relative to total amount of protein, and

-at least 60% w/w soluble whey protein aggregates relative to the total amount of protein, preferably at least 70% w/w, more preferably at least 80% w/w, even more preferably at least 90% w/w soluble whey protein aggregates relative to the total amount of protein.

Soluble whey protein aggregates made by heat denaturation of whey protein containing at least 85% w/w BLG appear to be particularly advantageous for use in protein beverages and, without being bound by theory, it is believed that high BLG soluble aggregates provide a stronger gel upon acidification than soluble aggregates based on normal WPI. This difference makes it possible to produce a beverage that forms more gel/higher viscosity in the stomach upon digestion, thereby promoting satiety.

In some preferred embodiments of the present invention, a packaged heat-treated beverage product comprises:

-at least 60% w/w soluble whey protein aggregates relative to the total amount of protein,

-up to 20% w/w protein nanogel relative to the total amount of protein,

-up to 2% w/w of insoluble proteinaceous matter.

In some more preferred embodiments of the present invention, a packaged heat-treated beverage product comprises:

-at least 80% w/w soluble whey protein aggregates relative to the total amount of protein,

-up to 5% w/w protein nanogel relative to total protein,

-up to 2% w/w of insoluble proteinaceous matter.

One aspect of the present invention relates to a method of producing a packaged heat-treated beverage product having a pH in the range of 5.5 to 8.0, the method comprising the steps of:

a) providing a liquid solution comprising:

-the total amount of protein is from 1 to 20 wt%, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG),

-optionally, sweeteners and/or flavouring agents,

b) the liquid solution is packed and then the liquid solution is packed,

wherein the liquid solution of step a) and/or the packaged liquid solution of step b) is subjected to a heat treatment comprising at least pasteurization.

Preferably, the method of producing a packaged heat-treated beverage product having a pH of 5.5-8.0 comprises the steps of:

a) providing a liquid solution comprising:

-the total amount of protein is from 1 to 20 wt%, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG),

Optionally, sweeteners, sugar polymers and/or flavors,

b) the liquid solution is packed and then the liquid solution is packed,

wherein the liquid solution of step a) and/or the packaged liquid solution of step b) is subjected to a heat treatment comprising at least pasteurization.

The liquid solution of step a) preferably has the same composition as the heat-treated beverage product, except for the changes that cause the heat treatment. Thus, the features mentioned in the context of heat-treated beverage products apply equally to liquid solutions, the main difference being that liquid solutions generally have a lower degree of protein denaturation than heat-treated beverage products.

In some preferred embodiments of the liquid solution of the invention, at least 85% w/w of the protein is BLG. Preferably, at least 88% w/w of the protein is BLG, more preferably at least 90% w/w, even more preferably at least 91% w/w, most preferably at least 92% w/w of the protein is BLG.

Even higher relative amounts of BLG are both feasible and desirable, so in some preferred embodiments of the invention at least 94% w/w of the protein in the liquid solution is BLG, more preferably at least 96% w/w of the protein is BLG, even more preferably at least 98% w/w of the protein is BLG, and most preferably about 100% w/w.

For example, the liquid solution preferably comprises at least 97.5% w/w of BLG relative to the total amount of protein, preferably at least 98.0% w/w, more preferably at least 98.5% w/w, even more preferably at least 99.0%, most preferably at least 99.5% w/w of BLG relative to the total amount of protein, e.g. about 100.0% w/w of BLG relative to the total amount of protein.

In some preferred embodiments of the invention the sum of alpha-lactalbumin (ALA) and Caseinmacropeptide (CMP) comprises at least 40% w/w of the non-BLG proteins in the liquid solution, preferably at least 60% w/w, even more preferably at least 70% w/w, most preferably at least 90% w/w of the non-BLG proteins in the liquid solution.

In some preferred embodiments of the invention ALA comprises at most 80% w/w of the non-BLG proteins in the liquid solution, preferably at most 60% w/w, even more preferably at most 40% w/w, and most preferably at most 30% w/w of the non-BLG proteins in the liquid solution.

Even lower levels of ALA may be preferred, and thus in some preferred embodiments of the invention ALA comprises at most 20% w/w of the non-BLG proteins in the liquid solution, preferably at most 15% w/w, even more preferably at most 10% w/w, most preferably at most 5% w/w of the non-BLG proteins in the liquid solution.

In a further preferred embodiment of the invention each major non-BLG whey protein is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Even lower concentrations of major non-BLG whey proteins may be desirable. Thus, in a further preferred embodiment of the invention, each major non-BLG whey protein is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

The inventors have found that a reduction of lactoferrin and/or lactoperoxidase is particularly advantageous for obtaining a color neutral whey protein product.

Thus, in some preferred embodiments of the invention, lactoferrin is present in a weight percentage relative to the total amount of protein which is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% relative to the weight percentage of total protein in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoferrin may be desirable. Thus, in a further preferred embodiment of the invention, lactoferrin is present in a weight percentage relative to the total amount of protein which is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Similarly, in some preferred embodiments of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% relative to the total amount of protein in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoperoxidase may be desirable. Thus, in a further preferred embodiment of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

The protein of the liquid solution is preferably made from mammalian milk, and preferably from ruminant milk (e.g., milk from cattle, sheep, goats, buffalo, camels, llamas, horses, and/or deer). Proteins derived from bovine milk are particularly preferred. Thus, the protein of the liquid solution is preferably a milk protein.

The protein of the liquid solution is preferably whey protein and/or milk albumin, even more preferably bovine whey protein and/or milk albumin.

In some preferred embodiments of the invention, the liquid solution has an inherent tryptophan fluorescence emission (I330/I350) of at least 1.11, more preferably at least 1.13, even more preferably at least 1.15, and most preferably at least 1.17.

In some preferred embodiments of the invention, the liquid solution has a degree of protein denaturation of at most 20%, more preferably at most 10%, even more preferably at most 5%, most preferably at most 1%.

Both low protein denaturation and fluorescence emission rates are characteristic of liquid solutions in which the protein is predominantly in its native conformation. The native protein conformation is particularly preferred for the production of clear beverages.

The packaging of step b) may be any suitable packaging technique and any suitable container may be used to package the liquid solution.

However, in a preferred embodiment of the invention, the packaging of step b) is aseptic packaged aged, i.e. the liquid solution is packaged under aseptic conditions. For example, aseptic packaging may be performed using an aseptic filling system, and it preferably involves filling a liquid solution into one or more aseptic containers.

Aseptic filling and sealing is particularly preferred if the liquid solution is already sterile or has a very low microbial content prior to filling.

Examples of useful containers are, for example, bottles, cartons, bricks and/or bags.

In some preferred embodiments of the invention, the vessel wall has a light transmission of at most 10%, preferably at most 1%, more preferably at most 0.1%, even more preferably at most 0.01%, most preferably at most 0.001% at any wavelength in the range of 250-500 nm.

In a further preferred embodiment of the present invention the average light transmission of the vessel wall in the range of 250-500nm is at most 10%, preferably at most 1%, more preferably at most 0.1%, even more preferably at most 0.01%, most preferably at most 0.001%.

The light transmittance of a container wall is measured by providing a flat container wall and measuring the light transmission through the container wall at any relevant wavelength. The measurement is carried out using a standard spectrophotometer and by inserting a piece of the vessel wall into the light path (e.g. using a cuvette or similar device) such that the plane of the piece of the vessel wall is perpendicular to the light pathAnd (4) setting. The transmission at wavelength i is calculated as T_i＝I_{i, thereafter}/I_{i, before}X 100% where I_{i, before}Is the light intensity at wavelength I before reaching the vessel wall, and I_{i, thereafter}Is the intensity at wavelength i after the beam of light of the light path has passed through the wall of the container of the block.

By calculating all transmission measurements T taken in a given wavelength range_iAnd dividing the sum by the number of transmission measurements in the given wavelength range to calculate the average transmission.

In some preferred embodiments of the present invention, the vessel wall has a light transmission of at most 10%, preferably at most 1%, more preferably at most 0.1%, even more preferably at most 0.01%, most preferably at most 0.001% at any wavelength in the range of 250-800 nm.

In a further preferred embodiment of the present invention the average light transmission of the vessel wall in the range of 250-800nm is at most 10%, preferably at most 1%, more preferably at most 0.1%, even more preferably at most 0.01%, most preferably at most 0.001%.

The container, which is opaque or low-transparent, can be prepared, for example, using the following: coloured, absorbent-containing polymers or coated polymers or coloured or coated glass, or alternatively a metal layer incorporated in the container wall, for example in the form of an aluminium foil. Such opaque or low light transmitting containers are known in the food and pharmaceutical industries.

Non-limiting examples of suitable polymeric materials are, for example, polyethylene terephthalate (PET) or PET-like polymers.

In other preferred embodiments of the invention, at least a portion of the container wall, and preferably the entire container, is transparent. In some preferred embodiments of the present invention, the average light transmission of at least a portion of the vessel wall, and preferably the entire vessel wall, is at least 11%, preferably at least 20%, more preferably at least 50%, even more preferably at least 60%, and most preferably at least 80% over the range of 400-700 nm.

In some preferred embodiments of the method of the present invention, the liquid solution of step a) is subjected to a heat treatment comprising at least pasteurization, and then packaged in step b).

In another embodiment of the method of the present invention, the packaged liquid solution of step b) is subjected to a heat treatment comprising at least pasteurization.

In a particular embodiment, the heat treatment involves heating the beverage product to a temperature in the range of 70-80 ℃.

In some preferred embodiments of the invention, the temperature of the heat treatment is in the range of 70-80 ℃, preferably in the range of 70-79 ℃, more preferably in the range of 71-78 ℃, even more preferably in the range of 72-77 ℃, most preferably 73-76 ℃, e.g. about 75 ℃.

Preferably, the duration of the heat treatment is 1 second to 60 minutes when performed in the temperature range of 70-80 ℃. The highest exposure time is most suitable for the lowest temperature in the temperature range and vice versa.

In other preferred embodiments, the temperature of the heat treatment is at least 60 minutes at 70 ℃, or preferably at least 45 minutes at 75 ℃, or preferably at least 30 minutes at 80 ℃, or preferably at least 22 minutes at 85 ℃ or preferably at least 10 minutes at 90 ℃.

In a particularly preferred embodiment of the invention, the heat treatment is carried out at 70-78 ℃ for 1 second to 30 minutes, more preferably at 71-77 ℃ for 1 minute to 25 minutes, even more preferably at 72-76 ℃ for 2 minutes to 20 minutes.

In some preferred embodiments of the invention, the heat treatment process comprises heating to a temperature of 85 ℃ to 95 ℃ for 1 to 3 minutes.

Higher temperatures may also be preferred in some embodiments, particularly if deployment and optional aggregation of the BLG is desired. For example, the temperature of the heat treatment may be at least 81 ℃, preferably at least 91 ℃, preferably at least 95 ℃, more preferably at least 100 ℃, even more preferably at least 120 ℃, and most preferably at least 140 ℃.

In some preferred embodiments of the invention, sterilization involves a temperature of 120 to 150 ℃ for 4 to 30 seconds.

The heat treatment may, for example, involve a temperature in the range of 90-130 ℃ and a duration in the range of 5 seconds-10 minutes. The heat treatment may, for example, involve heating to a temperature in the range of 90-95 ℃ for 1-10 minutes, for example about 120 ℃ for about 20 seconds. Alternatively, the heat treatment may involve heating to a temperature in the range of 115-125 ℃ for a duration of 5-30 seconds, for example, about 120 ℃ for about 20 seconds.

Alternatively, for example, the heat treatment may be a UHT type treatment, which typically involves a temperature in the range of 135-144 ℃ and a duration in the range of 2-10 seconds.

Alternatively, but also preferably, the heat treatment may involve a temperature in the range of 145-180 ℃ and a duration in the range of 0.01-2 seconds, and more preferably a temperature in the range of 150-180 ℃ and a duration in the range of 0.01-0.3 seconds.

The heat treatment may be carried out using equipment such as plate or tube heat exchangers, scraped surface heat exchangers or retort systems (retort systems). Alternatively, and particularly preferably, the heat treatment above 95 ℃, direct steam-based heating may be employed, for example, using direct steam injection, direct steam infusion or spray cooking. In addition, such direct steam-based heating is preferably used in combination with flash cooling. Suitable examples of carrying out spray cooking are found in WO2009113858a1, which is incorporated herein for all purposes. Suitable examples of implementing direct steam injection and direct steam infusion are found in WO2009113858a1 and WO 2010/085957 A3, which are incorporated herein for all purposes. The general aspects of high temperature treatment, such as may be found in "Thermal technologies in food processing" ISBN 185573558X, which is incorporated herein by reference in its entirety for all purposes.

In some preferred embodiments of the invention, pasteurization is combined with physical microbial reduction.

Useful examples of physical microbial reduction include one or more of bacterial filtration, ultraviolet radiation, high pressure treatment, pulsed electric field treatment, and ultrasound.

In some preferred embodiments of the present invention, the heat treatment is sterilization, thereby forming a sterilized liquid beverage product. Such sterilization may preferably be obtained by a combination of bacterial filtration and pasteurization.

In the context of the present invention, the term "bacterial filtration" relates to filtration performed at a pore size sufficient to retain microorganisms such as bacteria and spores, but which does not retain native BLG. Bacterial filtration, sometimes also referred to as sterile filtration, involves microfiltration of the liquid involved. Bacterial filtration is typically performed with membranes having a pore size of at most 1 micron, preferably at most 0.8 micron, more preferably at most 0.6 micron, even more preferably at most 0.4 micron, most preferably at most 0.2 micron.

Bacterial filtration, for example, may involve membranes having a pore size of 0.02-1 micron, preferably 0.03-0.8 micron, more preferably 0.04-0.6 micron, even more preferably 0.05-0.4 micron, most preferably 0.1-0.2 micron.

In some preferred embodiments of the invention, the liquid solution is subjected to bacterial filtration and then heat treatment using a temperature of at most 80 ℃, preferably at most 75 ℃. The combination of temperature and duration of the heat treatment is preferably selected to provide a sterile beverage product.

In other preferred embodiments of the invention, the liquid solution is subjected to bacterial filtration and then to a heat treatment using a temperature of at least 150 ℃ for at most 0.2 seconds, preferably at most 0.1 seconds. The combination of temperature and duration of the heat treatment is preferably selected to provide a sterile beverage product.

Depending on the heat treatment temperature used, it may be beneficial to cool the beverage product. According to a preferred aspect of the process of the present invention, after the heat treatment, the heat-treated beverage product is cooled in an optional step to preferably 0 to 50 ℃, preferably 0 to 25 ℃ or preferably 0 to 20 ℃, or preferably 0 to 15 ℃, preferably 0 to 10 ℃ or preferably 4 to 8 ℃ or preferably 2 to 5 ℃ or preferably 1 to 5 ℃.

If the beverage product has been pasteurized, it is preferably cooled to 0 to 15℃, more preferably 1 to 5℃, after the heat treatment.

According to one embodiment of the method, any acid or base may generally be used to adjust the pH of the liquid solution. One skilled in the art will recognize suitable means for adjusting the pH. Such as sodium or potassium carbonate, sodium or potassium bicarbonate or ammonium hydroxide. Preferably, a base such as KOH or NaOH is used to adjust the pH, although other bases including NaOH may be used to adjust the pH.

Those skilled in the art will recognize other means suitable for adjusting the pH. Suitable acids include, for example, citric, hydrochloric, malic or tartaric acid or phosphoric acid, most preferably citric and/or phosphoric acid.

In some preferred embodiments of the invention, the pH of the liquid solution is in the range of 6.5-7.5. Most preferably, the pH used is a pH of 6.5 to 7.0 or a pH of 6.8 to 7.2.

The pH of the liquid solution is preferably in the range of 5.5 to 6.2, or the pH of the liquid solution is in the range of 6.2-8.0.

Alternatively, the pH of the liquid solution may be in the range of 6.8 to 8.0, more preferably the pH of the liquid solution is in the range of 6.2-8.0.

The liquid solutions of the invention are found to be preferably clear and transparent, having a low viscosity in the pH range 6.2-8.0, preferably pH 6.3-7.6, more preferably pH 6.5-7.2.

The liquid solutions of the invention are found to have a low viscosity and a milky appearance, preferably in the pH range 5.5-8.0, preferably in the pH range 5.7-6.8, more preferably in the pH range 5.8-6.0.

In some preferred embodiments of the present invention, it was found that the liquid solution of the present invention is preferably heat treated at a pH in the range of 5.6-6.2, preferably at a pH in the range of 5.6-8.0, optionally mixed with a source of carbohydrates, fats, minerals and vitamins, adjusted to a preferred pH of 6.2-8.0, and subjected to a second heat treatment (UHT).

In some preferred embodiments of the invention, the liquid solution comprises a total amount of protein of 4.0 to 20% w/w by weight of the beverage.

In some embodiments of the invention, it is advantageous for the liquid solution to have a total amount of protein of 2.0 to 10.0% w/w of the weight of the beverage.

Thus, in some embodiments of the invention, the liquid solution preferably comprises 2.0 to 10% w/w of the total amount of protein relative to the weight of the liquid solution, preferably 3.0 to 10% w/w of the total amount of protein relative to the weight of the liquid solution, preferably 5.0 to 9.0% w/w of the total amount of protein relative to the weight of the liquid solution, preferably 6.0 to 8.0% w/w of the total amount of protein relative to the weight of the liquid solution.

In some embodiments of the invention, it is advantageous that the protein content of the liquid solution is relatively high, for example 10.0 to 20% w/w relative to the weight of the liquid solution.

Thus, in some embodiments of the invention, the liquid solution preferably comprises 10.0 to 20% w/w total protein by weight of the liquid solution, more preferably 12 to 19% w/w total protein by weight of the liquid solution, even more preferably 15-18% w/w total protein by weight of the liquid solution, most preferably 16 to 17% w/w total protein by weight of the liquid solution.

It is particularly preferred that the liquid solution comprises BLG isolate, e.g. when used in combination with other protein sources, preferably as the main protein source, possibly even as the sole protein source.

The BLG isolate is preferably a BLG isolate powder or a liquid BLG isolate comprises water and the amount of solids in the BLG isolate powder is 1-50% w/w.

A powder of a beta-lactoglobulin (BLG) isolate, preferably prepared by spray drying, having a pH in the range i)2-4.9, ii)6.1-8.5 or iii)5.0-6.0 and comprising:

-the total amount of protein is at least 30% w/w,

-the amount of BLG is at least 85% w/w relative to the total amount of protein, and

-the amount of water is at most 10% w/w.

The BLG isolate powder preferably has one or more of the following:

-bulk density of at least 0.2g/cm³，

An intrinsic tryptophan fluorescence emissivity (I330/I350) of at least 1.11,

-a degree of protein denaturation of at most 10%,

a thermostability at pH 3.9 of at most 200NTU, and

-up to 1000 colony forming units/g.

The BLG isolate powder is preferably an edible composition.

In some preferred embodiments of the invention, the pH of the BLG isolate powder is in the range of 2-4.9. Such powders are particularly useful for acidic foods, particularly acidic beverages.

In other preferred embodiments of the present invention, the pH of the BLG isolate powder is in the range of 6.1-8.5.

In some preferred embodiments of the invention the BLG isolate powder comprises a total amount of protein of at least 40% w/w, preferably at least 50% w/w, at least 60% w/w, more preferably at least 70% w/w, even more preferably at least 80% w/w.

Even higher protein contents may be desired and in some preferred embodiments of the invention the BLG isolate powder comprises a total amount of protein of at least 85% w/w, preferably at least 90% w/w, at least 92% w/w, more preferably at least 94% w/w, even more preferably at least 95% w/w.

The total amount of protein was measured according to example 1.5.

In some preferred embodiments of the invention the BLG isolate powder comprises at least 92% w/w BLG relative to the total amount of protein, preferably at least 95% w/w BLG relative to the total amount of protein, more preferably at least 97% w/w, even more preferably at least 98%, most preferably at least 99.5% w/w BLG.

In some preferred embodiments of the invention the sum of alpha-lactalbumin (ALA) and Caseinomacropeptide (CMP) comprises at least 40% w/w of the non-BLG proteins in the powder, preferably at least 60% w/w, even more preferably at least 70% w/w, most preferably at least 90% w/w of the non-BLG proteins in the powder.

In a further preferred embodiment of the invention each major non-BLG whey protein is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Even lower concentrations of major non-BLG whey proteins may be desirable. Thus, in a further preferred embodiment of the invention, each major non-BLG whey protein is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

The inventors have seen evidence that the reduction of lactoferrin and/or lactoperoxidase is particularly advantageous for obtaining a color neutral whey protein product.

Thus, in some preferred embodiments of the invention, lactoferrin is present in a weight percentage relative to the total amount of protein which is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoferrin may be desirable. Thus, in a further preferred embodiment of the invention, lactoferrin is present in a weight percentage relative to the total amount of protein which is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Similarly, in some preferred embodiments of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6% relative to the total amount of protein in a standard whey protein concentrate from sweet whey. Even lower concentrations of lactoperoxidase may be desirable. Thus, in a further preferred embodiment of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1% by weight relative to the total amount of protein in a standard whey protein concentrate from sweet whey.

Lactoferrin and lactoperoxidase were quantified according to example 1.29.

In some preferred embodiments of the invention the water content of the BLG isolate powder is at most 10% w/w, preferably at most 7% w/w, more preferably at most 6% w/w, even more preferably at most 4% w/w, most preferably at most 2% w/w.

In some preferred embodiments of the invention the BLG isolate powder comprises carbohydrate in an amount of at most 60% w/w, preferably at most 50% w/w, more preferably at most 20% w/w, even more preferably at most 10% w/w, even more preferably at most 1% w/w, most preferably at most 0.1%. The BLG isolate powder, for example, may comprise carbohydrates such as lactose, oligosaccharides and/or hydrolysates of lactose (i.e. glucose and galactose), sucrose and/or maltodextrin.

In some preferred embodiments of the invention the BLG isolate powder comprises lipids in an amount of at most 10% w/w, preferably at most 5% w/w, more preferably at most 2% w/w, even more preferably at most 0.1% w/w.

The inventors have found that it may be advantageous to control the mineral content to achieve certain desired properties of the BLG isolate powder.

In some preferred embodiments of the invention, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 10mmol/g protein. Preferably, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 6mmol/g protein, more preferably at most 4mmol/g protein, even more preferably at most 2mmol/g protein.

In other preferred embodiments of the invention, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 1mmol/g protein. Preferably, the total amount of Na, K, Mg and Ca in the BLG isolate powder is at most 0.6mmol/g protein, more preferably at most 0.4mmol/g protein, even more preferably at most 0.2mmol/g protein, most preferably at most 0.1mmol/g protein.

In other preferred embodiments of the invention, the total amount of Mg and Ca in the BLG isolate powder is at most 5mmol/g protein. Preferably, the total amount of Mg and Ca in the BLG isolate powder is at most 3mmol/g protein, more preferably at most 1.0mmol/g protein, even more preferably at most 0.5mmol/g protein.

In other preferred embodiments of the invention, the total amount of Mg and Ca in the BLG isolate powder is at most 0.3mmol/g protein. Preferably, the total amount of Mg and Ca in the BLG isolate powder is at most 0.2mmol/g protein, more preferably at most 0.1mmol/g protein, even more preferably at most 0.03mmol/g protein, most preferably at most 0.01mmol/g protein.

The inventors have found that a low phosphorous/low potassium variant of BLG isolate powder can be used, which is particularly useful for patients with renal disease. To produce such a product, the BLG isolate powder must have the same low levels of phosphorus and potassium.

Thus, in some preferred embodiments of the invention, the total content of phosphorus in the BLG isolate powder is at most 100mg phosphorus per 100g protein. Preferably, the BLG isolate powder has a total content of up to 80mg of phosphorus per 100g of protein. More preferably, the BLG isolate powder has a total content of up to 50mg of phosphorus per 100g of protein. Even more preferably, the BLG isolate powder has a total content of phosphorus of at most 20mg of phosphorus per 100g of protein. The BLG isolate powder has a total phosphorus content of up to 5mg phosphorus per 100 grams of protein.

In some preferred embodiments of the invention, the BLG isolate powder comprises up to 600mg potassium per 100g protein. More preferably, the BLG isolate powder comprises at most 500mg potassium per 100g protein. More preferably, the BLG isolate powder comprises at most 400mg potassium per 100g protein. More preferably, the BLG isolate powder comprises at most 300mg potassium per 100g protein. Even more preferably, the BLG isolate powder comprises at most 200mg potassium per 100g protein. Even more preferably, the BLG isolate powder comprises at most 100mg potassium per 100g protein. Even more preferably, the BLG isolate powder comprises at most 50mg potassium per 100g protein, and even more preferably, the BLG isolate powder comprises at most 10mg potassium per 100g protein.

The phosphorus content is related to the total amount of elemental phosphorus in the composition in question and is determined according to example 1.19. Similarly, the amount of potassium is related to the total amount of elemental potassium in the composition and is determined according to example 1.19.

In some preferred embodiments of the invention, the BLG isolate powder comprises at most 100mg phosphorus/100 g protein and at most 700mg potassium/100 g protein, preferably at most 80mg phosphorus/100 g protein and at most 600mg potassium/100 g protein, more preferably at most 60mg phosphorus/100 g protein and at most 500mg potassium/100 g protein, more preferably at most 50mg phosphorus/100 g protein and at most 400mg potassium/100 g protein, or more preferably at most 20mg phosphorus/100 g protein and at most 200mg potassium/100 g protein, or even more preferably at most 10mg phosphorus/100 g protein and at most 50mg potassium/100 g protein. In some preferred embodiments of the invention, the BLG isolate powder comprises at most 100mg phosphorus per 100g protein and at most 340mg potassium per 100g protein.

The low phosphorus and/or low potassium composition according to the invention can be used as a food ingredient for the production of a food product suitable for a group of patients with reduced kidney function.

In the context of the present invention, the turbidity of the clear liquid measured according to example 1.7 is at most 200 NTU.

Accordingly, in some preferred embodiments of the present invention, the pH of the BLG isolate powder is in the range of 2-4.9. Preferably, the pH of the BLG isolate powder is in the range of 2.5 to 4.7, more preferably 2.8 to 4.3, even more preferably 3.2 to 4.0, and most preferably 3.4 to 3.9. Alternatively, but also preferably, the pH of the BLG isolate powder may be in the range of 3.6-4.3.

The present inventors have found that for certain applications, for example, pH neutral foods, particularly pH neutral beverages, it is particularly advantageous to have a pH neutral BLG isolate powder. This is especially true for high protein, clear or opaque pH neutral beverages.

Accordingly, in some preferred embodiments of the present invention, the pH of the BLG isolate powder is in the range of 6.1-8.5. Preferably, the pH of the powder is in the range of 6.1-8.5, more preferably 6.2-8.0, even more preferably 6.3-7.7, most preferably 6.5-7.5.

In other preferred embodiments of the present invention, the pH of the BLG isolate powder is in the range of 5.0-6.0. Preferably, the pH of the powder is in the range of 5.1-5.9, more preferably in the range of 5.2-5.8, even more preferably in the range of 5.3-5.7, most preferably in the range of 5.4-5.6.

Advantageously, the bulk density of the BLG isolate powder for use in the present invention may be at least 0.20g/cm ³Preferably at least 0.30g/cm³More preferably at least 0.40g/cm³And even more preferably at least 0.45g/cm³And even more preferably at least 0.50g/cm³Most preferably at least 0.6g/cm³。

Low density powders (e.g., lyophilized BLG isolate) are fluffy and are easily inhaled into the air at the manufacturing site during use. This is problematic because it increases the risk of cross-contamination of the freeze-dried powder with other food products, and dusty environments are known to be responsible for hygiene problems. In extreme cases, dusty environments also increase the risk of dust explosions.

The high density version of the invention is easier to handle and less prone to flow into the surrounding air.

Another advantage of the high density variant of the present invention is that it takes up less space during transportation, thereby increasing the weight of BLG isolate powder that can be transported in one volume unit.

Another advantage of the high density variant of the invention is that it is not easily separated when used with powdered mixtures of other powdered food ingredients, such as powdered sugar (bulk density about 0.56 g/cm)³) Granulated sugar (bulk density about 0.71 g/cm)³) Citric acid powder (bulk density about 0.77 g/cm)³)。

The bulk density of the BLG isolate powder of the present invention may be in the range of 0.2 to 1.0g/cm ³In the range of 0.30 to 0.9g/cm, preferably³More preferably in the range of 0.40 to 0.8g/cm³In the range of from 0.45 to 0.75g/cm, even more preferably³In the range of from 0.50 to 0.75g/cm, even more preferably³And most preferably in the range of 0.6 to 0.75g/cm³Within the range of (1).

The bulk density of the powder was measured according to example 1.17.

The present inventors found that it is advantageous to maintain the natural texture of BLG, and have seen that when BLG is used in an acidic beverage, increased development of BLG leads to signs of increased dry mouthfeel.

Intrinsic tryptophan fluorescence emission (I330/I350) is a measure of the degree of expansion of the BLG, and the inventors have found that at higher intrinsic tryptophan fluorescence emission, less dry mouthfeel is observed in association with low or no expansion of the BLG. The intrinsic tryptophan fluorescence emission (I330/I350) was measured according to example 1.1.

In some preferred embodiments of the invention, the BLG isolate powder has an intrinsic tryptophan fluorescence emissivity (I330/I350) of at least 1.11.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission (I330/I350) of the BLG isolate powder is at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

If the BLG isolate powder contains a large amount of non-protein material, the protein component is preferably separated prior to measuring the intrinsic tryptophan fluorescence emission. Thus, in some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission rate of the protein component of the BLG isolate powder is at least 1.11.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emissivity (I330/I350) of the protein component of the BLG isolate powder is at least 1.12, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

The protein component may be separated from the BLG isolate powder, for example, by dissolving the BLG isolate powder in demineralized water and subjecting the solution to dialysis or ultrafiltration-based diafiltration using a filter that retains the protein. If the BLG isolate powder contains interfering levels of lipids, the lipids can be removed, for example, by microfiltration. Microfiltration and ultrafiltration/diafiltration steps may be combined together to remove lipids and small molecules from the protein component.

It is generally preferred that the bulk of the BLG in the BLG isolate powder is non-aggregated BLG. Preferably, at least 50% of the BLGs are non-aggregated BLGs. More preferably, at least 80% of the BLGs are non-aggregated BLGs. Even more preferably at least 90% of the BLG is non-aggregated BLG. Most preferably, at least 95% of the BLGs are non-aggregated BLGs. Even more preferably, about 100% of the BLG in the BLG isolate powder is non-aggregated BLG.

In some preferred embodiments of the invention, the protein denaturation degree of the BLG isolate powder is at most 10%, preferably at most 8%, more preferably at most 6%, even more preferably at most 3%, even more preferably at most 1%, most preferably at most 0.2%.

However, it may also be preferred that the BLG isolate powder has a significant level of protein denaturation, for example, if an opaque beverage is desired. Thus, in other preferred embodiments of the invention, the protein denaturation degree of the BLG isolate powder is at least 11%, preferably at least 20%, more preferably at least 40%, even more preferably at least 50%, even more preferably at least 75%, most preferably at least 90%.

If the BLG isolate powder has a significant level of protein denaturation, it is generally preferred to maintain a low level of insoluble protein material, i.e. precipitated protein material, which will precipitate in the beverage during storage. The content of insoluble material was determined according to example 1.10.

In some preferred embodiments of the invention the BLG isolate powder comprises at most 20% w/w insoluble proteinaceous material, preferably at most 10% w/w insoluble proteinaceous material, more preferably at most 5% w/w insoluble proteinaceous material, even more preferably at most 3% w/w insoluble proteinaceous material, most preferably at most 1% w/w insoluble proteinaceous material. It is even preferred that the BLG isolate powder does not contain any insoluble proteinaceous material at all.

The present inventors have found that the thermal stability of BLG isolate powder at pH 3.9 is a good indicator of its usefulness for clear high protein beverages. The thermal stability at pH 3.9 was measured according to example 1.2.

It is particularly preferred that the thermal stability of the BLG isolate powder at pH 3.9 is at most 200NTU, preferably at most 100NTU, more preferably at most 60NTU, even more preferably at most 40NTU, most preferably at most 20 NTU. Even better thermal stability is possible and the thermal stability of the BLG isolate powder at pH 3.9 is preferably at most 10NTU, preferably at most 8NTU, more preferably at most 4NTU, even more preferably at most 2 NTU.

The microbial content of the BLG isolate powder is preferably kept to a minimum. However, it is a challenge to obtain both high protein naturalness and low levels of microorganisms, as the microbial reduction process often results in protein unfolding and denaturation. The invention makes it possible to obtain very low contents of microorganisms while maintaining a high level of BLG naturalness.

Thus, in some preferred embodiments of the invention, the BLG isolate powder contains at most 15000 Colony Forming Units (CFU)/g. Preferably, the BLG isolate powder contains at most 10000 CFU/g. More preferably, the BLG isolate powder comprises at most 5000 CFU/g. Even more preferably, the BLG isolate powder comprises at most 1000 CFU/g. Even more preferably, the BLG isolate powder comprises at most 300 CFU/g. Most preferably, the BLG isolate powder comprises at most 100CFU/g, e.g., at most 10 CFU/g. In a particularly preferred embodiment, the powder is sterile. Sterile BLG isolate powders may be prepared, for example, by combining several physical microorganism reduction processes during the production of the BLG isolate powder, such as microfiltration and heat treatment at acidic pH.

In some preferred embodiments of the invention, the pH of the BLG isolate powder is in the range i)2 to 4.9, ii)6.1 to 8.5 or iii)5.0 to 6.0, and comprises:

-the total amount of protein is at least 30% w/w, preferably at least 80% w/w, even more preferably at least 90% w/w;

-the amount of beta-lactoglobulin (BLG) is at least 85% w/w, preferably at least 90% w/w, relative to the total amount of protein;

-the amount of water is at most 6% w/w;

-the amount of lipid is at most 2% w/w, preferably at most 0.5% w/w.

The BLG isolate powder has:

-an intrinsic tryptophan fluorescence emissivity (I330/I350) of at least 1.11;

-a degree of protein denaturation of at most 10%, and

-a thermostability at pH 3.9 of at most 200 NTU.

In some preferred embodiments of the invention, the pH of the BLG isolate powder is in the range of i)2 to 4.9 or ii)6.1 to 8.5, and comprises:

-the total amount of protein is at least 30% w/w, preferably at least 80% w/w, even more preferably at least 90% w/w;

-the amount of beta-lactoglobulin (BLG) is at least 85% w/w relative to the total amount of protein, preferably the amount of beta-lactoglobulin (BLG) is at least 90% w/w, and more preferably at least 94% w/w relative to the total amount of protein;

-the amount of water is at most 6% w/w;

-the amount of lipid is at most 2% w/w, preferably at most 0.5% w/w.

The BLG isolate powder has:

-an intrinsic tryptophan fluorescence emissivity (I330/I350) of at least 1.11;

a degree of protein denaturation of at most 10%, preferably at most 5%, and

-a thermostability at pH 3.9 of at most 70NTU, preferably at most 50NTU, even more preferably at most 40 NTU.

In some preferred embodiments of the invention, the pH of the BLG isolate powder is in the range of i)2 to 4.9 or ii)6.1 to 8.5, and comprises:

-the total amount of protein is at least 30% w/w;

-the content of beta-lactoglobulin (BLG) is at least 85% w/w, preferably at least 90% w/w, relative to the total amount of protein;

-the amount of water is at most 6% w/w.

The BLG isolate powder has:

-bulk density of at least 0.2g/cm³；

-an intrinsic tryptophan fluorescence emissivity (I330/I350) of at least 1.11;

-a degree of protein denaturation of at most 10%, and

-a thermostability at pH 3.9 of at most 200 NTU.

In other preferred embodiments of the present invention, the BLG isolate powder has a pH of 2 to 4.9 and comprises:

-the total amount of protein is at least 80% w/w, preferably at least 90% w/w, even more preferably at least 94% w/w;

-the amount of beta-lactoglobulin (BLG) is at least 85% w/w relative to the total amount of protein, preferably the amount of beta-lactoglobulin (BLG) is at least 90% w/w, even more preferably at least 94% w/w relative to the total amount of protein;

-the amount of water is at most 6% w/w;

-the amount of lipid is at most 2% w/w, preferably at most 0.5% w/w.

The BLG isolate powder has:

-bulk density of at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm³，

An intrinsic tryptophan fluorescence emissivity (I330/I350) of at least 1.11,

-a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%, and

-a thermostability at pH 3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10 NTU.

In other preferred embodiments of the present invention, the BLG isolate powder has a pH in the range of 6.1-8.5 and comprises:

-the total amount of protein is at least 80% w/w, preferably at least 90% w/w, even more preferably at least 94% w/w;

-the amount of beta-lactoglobulin (BLG) is at least 85% w/w relative to the total amount of protein, preferably the amount of beta-lactoglobulin (BLG) is at least 90% w/w, even more preferably at least 94% w/w relative to the total amount of protein;

-the amount of water is at most 6% w/w;

-the amount of lipid is at most 2% w/w, preferably at most 0.5% w/w.

The BLG isolate powder has:

-bulk density of at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm ³，

-a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%, and

-a thermostability at pH 3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10 NTU.

In other preferred embodiments of the present invention, the BLG isolate powder has a pH in the range of 6.1-8.5 and comprises:

-the total amount of protein is at least 80% w/w, preferably at least 90% w/w, even more preferably at least 94% w/w;

-the amount of beta-lactoglobulin (BLG) is at least 85% w/w relative to the total amount of protein, preferably the amount of beta-lactoglobulin (BLG) is at least 90% w/w, even more preferably at least 94% w/w relative to the total amount of protein;

-the amount of water is at most 6% w/w;

-the amount of lipid is at most 2% w/w, preferably at most 0.5% w/w.

The BLG isolate powder has:

-bulk density of at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm³，

-a degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%, and

-a thermostability at pH 3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10 NTU.

In other preferred embodiments of the present invention, the BLG isolate powder has a pH of 5.0-6.0 and comprises:

-the total amount of protein is at least 80% w/w, preferably at least 90% w/w, even more preferably at least 94% w/w,

-the amount of beta-lactoglobulin (BLG) is at least 85% w/w relative to the total amount of protein, preferably the amount of beta-lactoglobulin (BLG) is at least 90% w/w, even more preferably at least 94% w/w relative to the total amount of protein,

-the amount of water is at most 6% w/w,

-the amount of lipid is at most 2% w/w, preferably at most 0.5% w/w.

The BLG isolate powder has:

-bulk density of at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm³，

A degree of protein denaturation of at most 10%, preferably at most 5%, more preferably at most 2%,

-a thermostability at pH 3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10NTU, and

preferably, the BLG crystallinity is less than 10%.

A BLG isolate powder comprising at least 85% w/w BLG relative to the total amount of protein is typically provided by a process comprising the steps of:

a) providing a liquid BLG isolate having the following characteristics;

i) the pH value is within the range of 2-4.9,

ii) a pH value in the range of 6.1 to 8.5, or

iii) the pH value is in the range of 5.0 to 6.0,

said liquid BLG isolate containing at least 85% w/w BLG relative to the total amount of protein,

b) Optionally, performing physical microbial reduction on the liquid BLG isolate;

c) the liquid BLG isolate is dried, preferably by spray drying.

The BLG isolate is preferably prepared from mammalian milk, and preferably from ruminant milk, e.g. milk from cattle, sheep, goats, buffalo, camels, llamas, mares and/or deer. Proteins derived from bovine milk are particularly preferred. Thus, the BLG is preferably bovine BLG.

The liquid BLG isolate can be provided in a number of different ways.

Typically, providing a liquid BLG isolate involves or consists of isolating BLG from whey protein feed by one or more of the following methods to provide a BLG-enriched composition:

-crystallizing or precipitating BLG by salting-in (by salting-in),

crystallizing or precipitating the BLG in the BLG by salting out (by salting out),

-ion exchange chromatography, and

-fractionating the whey protein by ultrafiltration.

A particularly preferred way of providing a BLG-enriched composition is by crystallization of BLG, preferably by salting-in (by salting-out) or alternatively by salting-out (by salting-out).

The whey protein feed is preferably WPC, WPI, SPC, SPI or a combination thereof.

The term "whey protein feed" relates to a composition from which the BLG-enriched composition and subsequently the liquid BLG isolate are derived.

In some embodiments of the invention, a preparation of a BLG-enriched composition according to US 2,790,790a1 comprises, or even consists of, high salt BLG crystals in the pH range of 3.6-4.0.

In other embodiments of the invention, preparations of BLG-enriched compositions include or even consist of de Jongh et al (Mild Isolation Procedure reveals novel Protein Structural Properties of beta and Lactoglobulin (Milld Isolation products New Protein Structural Properties of beta-Lactoglobulin), J Dairy Sci., Vol.84 (3),2001, 562-pp.571) or the methods described by Vyas et al (Scale-Up of Native beta-Lactoglobulin Affinity Separation Processes), J.Dairy Sci.85: 1639-1645, 2002).

However, in a particularly preferred embodiment of the invention, BLG-enriched compositions are prepared by crystallization at pH 5-6 under salting-out conditions as described in PCT application PCT/EP2017/084553, which is incorporated herein by reference for all purposes.

In some preferred embodiments of the invention, the BLG enriched composition is an edible BLG composition according to PCT/EP2017/084553 comprising at least 90% BLG relative to the total amount of protein, and preferably comprising BLG crystals.

If the desired characteristics for use as a liquid BLG isolate are not already available, the BLG-enriched composition isolated from the whey protein feed may be subjected to one or more steps selected from the group consisting of:

-demineralising substances;

-adding minerals;

-dilution;

-concentration;

reduction of physical microorganisms, and

-pH adjustment.

Non-limiting examples of demineralization include, for example, dialysis, gel filtration, ultrafiltration UF/diafiltration, nanofiltration NF/diafiltration, and ion exchange chromatography.

Non-limiting examples of adding minerals include adding soluble, food acceptable salts, for example, salts of Na, K, Ca, and/or Mg. Such salts may be, for example, phosphate salts, chloride salts or salts of edible acids, such as citrate or lactate. The minerals may be added in solid, suspended or dissolved form.

Non-limiting examples of dilution include, for example, the addition of a liquid diluent, such as water, demineralized water, or an aqueous solution of a mineral, acid, or base.

Non-limiting examples of concentration include, for example, evaporation, reverse osmosis, nanofiltration, ultrafiltration, and combinations thereof.

If concentration has to increase the concentration of protein relative to the total amount of solids, it is preferred to use a concentration step such as ultrafiltration or optionally dialysis. If the concentration does not have to increase the concentration of protein relative to the total amount of solids, methods such as evaporation, nanofiltration and/or reverse osmosis may be employed.

Non-limiting examples of physical microbial reduction include, for example, heat treatment, bacterial filtration, ultraviolet radiation, high pressure treatment, pulsed electric field treatment, and ultrasound. These methods are well known to those skilled in the art.

Non-limiting examples of pH adjustment include, for example, the addition of a base and/or acid, preferably a food acceptable base and/or acid. Particular preference is given to using acids and/or bases which are capable of chelating divalent metal cations. Examples of such acids and/or bases are citric acid, citrate, EDTA, lactic acid, lactate, phosphoric acid, phosphate, and combinations thereof.

In some preferred embodiments of the invention, the color value ab of the liquid solution is in the range of-0.10 to +0.51 of the CIELAB color scale, in particular if the turbidity of the preparation is at most 200NTU, more preferably at most 40 NTU.

In a further preferred embodiment of the invention, the color value Δ b of the liquid solution is in the range of 0.0 to 0.40, preferably in the range of +0.10 to +0.25 of the CIELAB color scale.

The liquid solution of the invention may comprise other macronutrients than proteins.

In some embodiments of the invention, the liquid solution further comprises a carbohydrate. The total carbohydrate content of the liquid solution of the present invention depends on the intended use of the final heat-treated beverage product.

In some embodiments of the present invention, the packaged heat-treated beverage product further comprises at least one source of carbohydrates. In an exemplary embodiment, the at least one carbohydrate source is selected from the group consisting of: sucrose (sucrose), beet sugar (saccharose), maltose, dextrose, galactose, maltodextrin, corn syrup solids, sucralose, glucose polymers, corn syrup, modified starch, resistant starch, rice-derived carbohydrates, isomaltulose, white sugar, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohols, fructooligosaccharides, soy fiber, corn fiber, guar gum, konjac flour, polydextrose, fiber sol (Fibersol), and combinations thereof. In some embodiments of the invention, the packaged heat-treated beverage product comprises non-digestible saccharides, such as fructan, which comprises inulin or fructooligosaccharides.

In some preferred embodiments, the liquid solution further comprises a carbohydrate in an amount between 0 and 95% of the total energy content of the liquid solution, preferably between 10 and 85% of the total energy content of the liquid solution, preferably between 20 and 75% of the total energy content of the liquid solution, or preferably between 30 and 60% of the total energy content of the liquid solution.

Even lower carbohydrate contents are generally preferred, and therefore in some preferred embodiments of the invention, the carbohydrate content preferably constitutes between 0 and 30% of the total energy content of the preparation, more preferably between 0 and 20% of the total energy content of the preparation, even more preferably between 0-10% of the total energy content of the preparation.

In some preferred embodiments of the present invention, the carbohydrate content of the liquid solution is at most 3% of the total energy content of the liquid solution, more preferably at most 1% of the total energy content of the liquid solution, even more preferably at most 0.1% of the total energy content of the liquid solution.

In one embodiment of the invention, the liquid solution further comprises at least one additional ingredient selected from the group consisting of: vitamins, flavors, minerals, sweeteners, antioxidants, food acids, lipids, carbohydrates, prebiotics, probiotics, and non-whey proteins.

The further ingredients ensure that the final packaged heat-treated beverage product contains the required nutrients, i.e. nutrients that are particularly suitable for patients suffering from protein deficiency or athletes wishing to exercise muscles.

In one embodiment of the invention, the liquid solution further comprises at least one high intensity sweetener. In one embodiment, the at least one high intensity sweetener is selected from the group consisting of: aspartame, cyclamate, sucralose, acesulfame salts, neotame, saccharin, stevia extracts, steviol glycosides, e.g., rebaudioside a, or combinations thereof. In some embodiments of the present invention, it is particularly preferred that the sweetener comprises or even consists of one or more High Intensity Sweeteners (HIS).

HIS is present in both natural and artificial sweeteners, and typically has a sweetness intensity that is at least 10 times that of sucrose.

If used, the total amount of HIS is generally in the range of 0.01-2% w/w. For example, the total amount of HIS may be in the range of 0.05-1.5% w/w. Alternatively, the total amount of HIS may be in the range of 0.1-1.0% w/w.

The choice of sweetener may depend on the beverage to be produced, for example, a high intensity sweetener (e.g., aspartame, acesulfame potassium or sucralose) may be used in beverages that do not require the sweetener to provide energy, while for beverages with natural characteristics, a natural sweetener (e.g., steviol glycosides, sorbitol or sucrose) may be used.

Alternatively or additionally, carbohydrate sweeteners may be used.

It may furthermore be preferred that the sweetener comprises or even consists of one or more polyol sweeteners. Non-limiting examples of useful polyol sweeteners are maltitol, mannitol, lactitol, sorbitol, inositol, xylitol, threitol, galactitol, or combinations thereof. If used, the total amount of polyol sweetener is typically in the range of 1-20% w/w. For example, the total amount of polyol sweetener may be in the range of 2-15% w/w. Alternatively, the total amount of polyol sweetener may be in the range of 4-10% w/w.

The liquid solution of the invention may comprise other macronutrients than proteins. In some embodiments of the invention, the liquid solution further comprises a lipid. The total lipid content in the final heat-treated beverage product of the present invention depends on the intended use of the heat-treated beverage product.

In some preferred embodiments of the invention, the lipid content of the liquid solution is between 0 and 50% of the total energy content of the liquid solution, or preferably between 0 and 45% of the total energy content of the liquid solution, or preferably between 0 and 30% of the total energy content of the liquid solution, or preferably between 0 and 20% of the total energy content of the liquid solution, or preferably between 0 and 10% of the total energy content of the liquid solution, or preferably between 0 and 5% of the total energy content of the liquid solution.

The content of lipids is according to ISO 1211: 2010 (determination of fat content)Gravimetric).

In some preferred embodiments of the invention, the lipid content of the liquid solution is at most 3% of the total energy content of the liquid solution, more preferably at most 1% of the total energy content of the liquid solution, even more preferably at most 0.1% of the total energy content of the liquid solution.

The liquid solution typically comprises a total amount of water in the range of 50-99% w/w, preferably in the range of 45-97% w/w, more preferably in the range of 40-95% w/w, even more preferably in the range of 35-90% w/w, most preferably in the range of 30-85% w/w.

In some preferred embodiments of the invention, the total amount of water comprised in the liquid solution is in the range of 55-90% w/w, preferably in the range of 57-85% w/w, more preferably in the range of 60-80% w/w, even more preferably in the range of 62-75% w/w, most preferably in the range of 65-70% w/w.

In some preferred embodiments of the invention, the liquid solution comprises a total amount of water in the range of 90-99% w/w, preferably 92-98.5% w/w, more preferably 94-98% w/w, even more preferably in the range of 95-98% w/w, most preferably in the range of 96-98% w/w.

In some preferred embodiments of the invention, the liquid solution is non-alcoholic, meaning that it comprises at most 1.0% w/w ethanol, more preferably at most 0.5% w/w, even more preferably at most 0.1% w/w, most preferably no detectable ethanol.

The liquid solution typically comprises a total amount of solids in the range of 1-45% w/w, preferably 5-40% w/w, more preferably 10-35% w/w, even more preferably in the range of 12-30% w/w, most preferably in the range of 16-25% w/w.

In some preferred embodiments of the invention the liquid solution comprises a total amount of solids in the range of 10-45% w/w, preferably 15-43% w/w, more preferably 20-40% w/w, even more preferably in the range of 25-38% w/w, most preferably in the range of 30-35% w/w.

In some preferred embodiments of the invention the liquid solution comprises a total amount of solids in the range of 1-10% w/w, preferably 1.5-8% w/w, more preferably 2-6% w/w, even more preferably in the range of 2-5% w/w, most preferably in the range of 2-4% w/w.

The part of the liquid solution that is not a solid is preferably water.

The present inventors have found that controlling the mineral content may be advantageous in achieving certain desired characteristics of the packaged heat-treated beverage product.

The present inventors have surprisingly found that when using a BLG isolate as defined herein, in examples 2 and 3, a heat-treated beverage product can be produced without reducing the viscosity and avoiding gelling. This provides the possibility that a packaged heat-treated beverage product with a high mineral content can be produced and a beverage of a nutritionally complete nutritional supplement or a nutritionally incomplete supplement can be produced.

In some embodiments of the invention, the liquid solution comprises a plurality of minerals. In one exemplary embodiment, the liquid solution comprises at least four minerals. In one embodiment, the four minerals are sodium, potassium, magnesium and calcium.

In some preferred embodiments of the invention the total amount of Na, K, Mg and Ca in the liquid solution is in the range of 0 to 400mM, preferably in the range of 10-200mM or preferably in the range of 20-100 mM.

In some preferred embodiments of the invention, the total amount of Na, K, Mg and Ca in the liquid solution is at most 400 mM.

In a further preferred embodiment of the invention the total amount of Na, K, Mg and Ca in the liquid solution is at most 300mM, preferably at most 200mM, or preferably at most 100mM, or preferably at most 80mM or preferably at most 60mM or preferably at most 40mM or preferably at most 30mM or preferably at most 20mM or preferably at most 10mM or preferably at most 5mM or preferably at most 1 mM.

In some preferred embodiments of the invention, the total amount of Mg and Ca is at most 75mM in the liquid solution, more preferably at most 40mM in the liquid solution, more preferably at most 20mM in the liquid solution.

In other preferred embodiments of the invention, the total amount of Mg and Ca is at most 10mM in the liquid solution, more preferably at most 8.0mM in the liquid solution, more preferably at most 6.0mM in the liquid solution, even more preferably at most 4.0mM in the liquid solution, most preferably at most 2.0mM in the liquid solution.

In another exemplary embodiment of the invention, the liquid solution comprises a plurality of minerals selected from the group consisting of: calcium, iodine, zinc, copper, chromium, iron, phosphorus, magnesium, selenium, manganese, molybdenum, sodium, potassium, and combinations thereof.

In other preferred embodiments of the invention, the liquid solution is a low mineral solution.

In the context of the present invention, the term "low mineral" relates to a composition, e.g. a liquid, a beverage, a powder or another food product, having at least one, preferably two, even more preferably all of the following:

-ash content of at most 1.2% w/w relative to total solids is transparent,

-the total content of calcium and magnesium is at most 0.3% w/w relative to the total amount of solids,

-the total content of sodium and potassium is at most 0.10% w/w relative to the total solids,

-the total content of phosphorus is at most 100 mg of phosphorus per 100 g of protein.

Preferably, the low mineral composition has at least one, preferably two or more, even more preferably all of the following:

-ash content of at most 0.7% w/w relative to total solids,

-the total content of calcium and magnesium is at most 0.2% w/w relative to the total amount of solids,

-the total content of sodium and potassium is at most 0.08% w/w relative to the total solids,

-a total phosphorus content of at most 80 mg phosphorus per 100 g protein.

Even more preferably, the low mineral composition has at least one, preferably two or more, even more preferably all of the following:

-ash content of at most 0.5% w/w relative to the total solids,

-the total content of calcium and magnesium is at most 0.15% w/w relative to the total amount of solids,

-the total content of sodium and potassium is at most 0.06% w/w relative to the total solids,

-a total phosphorus content of at most 50mg phosphorus per 100 g protein.

It is particularly preferred that the low mineral composition has the following characteristics:

-ash content of at most 0.5% w/w relative to the total solids,

-the total content of calcium and magnesium is at most 0.15% w/w relative to the total amount of solids,

-the total content of sodium and potassium is at most 0.06% w/w relative to the total solids,

-a total phosphorus content of at most 50mg phosphorus per 100 g protein.

In another exemplary embodiment of the invention, the liquid solution comprises a plurality of minerals selected from the group consisting of: calcium, iodine, zinc, copper, chromium, iron, phosphorus, magnesium, selenium, manganese, molybdenum, sodium, potassium, and combinations thereof.

The present inventors have found that the present invention allows the preparation of packaged heat-treated beverage products having very low levels of phosphorus and other minerals such as potassium, which is beneficial for patients suffering from kidney disease or other reduced kidney function disorders.

The liquid solution is preferably a low phosphorous beverage product.

The liquid solution is preferably a low potassium beverage product.

The liquid solution is preferably a low phosphorus and low potassium beverage product

In the context of the present invention, the term "low phosphorus" relates to compositions, such as liquids, powders or other food products, having a total content of phosphorus of at most 100mg phosphorus per 100g protein. Preferably, the low phosphorus composition has a total phosphorus content of at most 80mg phosphorus per 100g protein. More preferably, the low phosphorous composition may have a total phosphorous content of up to 50mg phosphorous per 100g protein. Even more preferably, the low phosphorus composition may have a total phosphorus content of up to 20mg phosphorus per 100g protein. Even more preferably, the total phosphorus content of the low-phosphorus composition may be up to 5mg phosphorus per 100g protein. The low phosphorous composition according to the invention may be used as a food ingredient for the production of a food product for a patient population with reduced renal function.

The phosphorus content is related to the total amount of elemental phosphorus in the composition in question and is determined according to example 1.19.

The amount of potassium is related to the total amount of elemental potassium in the composition in question and is determined according to example 1.19.

In some preferred embodiments of the invention, the liquid solution comprises at most 100mg phosphorus/100 g protein and at most 700mg potassium/100 g protein, preferably at most 80mg phosphorus/100 g protein and at most 600mg potassium/100 g protein, more preferably at most 60mg phosphorus/100 g protein and at most 500mg potassium/100 g protein, more preferably at most 50mg phosphorus/100 g protein and at most 400mg potassium/100 g protein, or more preferably at most 20mg phosphorus/100 g protein and at most 200mg potassium/100 g protein, or even more preferably at most 10mg phosphorus/100 g protein and at most 50mg potassium/100 g protein. In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises at most 100mg phosphorus per 100g protein and at most 340mg potassium per 100g protein.

The liquid solution comprising low amounts of phosphorus and potassium may advantageously be supplemented with carbohydrates and lipids, the heat-treated beverage product also preferably comprises a total amount of carbohydrates in the range of 30-60%, preferably in the range between 35-50E% of the total energy of the liquid solution, and a total amount of lipids in the range of 20-60%, preferably in the range between 30-50E% of the total energy content.

In one embodiment of the invention, the liquid solution comprises a plurality of vitamins. In an exemplary embodiment, the liquid solution comprises at least ten vitamins. In one exemplary embodiment, the liquid solution comprises a plurality of vitamins selected from the group consisting of: vitamin a, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin K, riboflavin, pantothenic acid, vitamin E, thiamin, niacin, folic acid, biotin, and combinations thereof.

In one embodiment of the invention, the liquid solution comprises a plurality of vitamins and a plurality of minerals.

In some preferred embodiments of the invention, the liquid solution comprises one or more food acids selected from the group consisting of: citric acid, malic acid, tartaric acid, acetic acid, benzoic acid, butyric acid, lactic acid, fumaric acid, succinic acid, ascorbic acid, adipic acid, phosphoric acid and mixtures thereof.

In one embodiment of the invention, the liquid solution further comprises a flavoring agent selected from the group consisting of salt, flavoring, odorant and/or fragrance. In a preferred embodiment of the invention, the flavoring agent comprises chocolate, cocoa, lemon, orange, lime, strawberry, banana, argan fruit flavors or combinations thereof. The choice of flavoring agent may depend on the beverage to be produced.

The inventors have found that the present invention, in particular the use of a protein component comprising at least 85% w/w BLG relative to the total amount of protein, allows the formation of protein nanogels at surprisingly high protein concentrations, which previously was thought to lead to uncontrolled gel formation. This is advantageous because it allows the direct production of high protein beverages without the need to concentrate the protein content after denaturing or concentrating the protein nanogel.

Thus, in some preferred embodiments of the invention, the liquid solution or an early solution (an early solution) for preparing the liquid solution comprises:

-the total amount of protein is in the range of 5-20% w/w, preferably in the range of 8-19% w/w, more preferably in the range of 10-18% w/w, most preferably in the range of 12-16% w/w,

the amount of BLG is at least 85% w/w relative to the total amount of protein, preferably at least 90% w/w relative to the total amount of protein, more preferably at least 92% w/w, most preferably at least 96%,

And having:

-a degree of protein denaturation of at most 30%, preferably at most 20%, even more preferably at most 10%, most preferably at most 5%, and

-a pH of 5.6-6.5, preferably 5.8-6.2, more preferably 5.9-6.1.

In the context of the present invention, the term "early solution" or "early solution for preparing a liquid solution" relates to an aqueous solution for preparing a liquid solution and typically has the same protein content as the liquid solution or a slightly higher protein content. For example, the use of an early solution is preferred when the protein, particularly native BLG, must be modified, for example by heat denaturation, prior to forming a liquid solution.

In some preferred embodiments of the invention, the liquid solution or the early solution (an early solution) used to prepare the liquid solution comprises a total amount of protein of 10-20% w/w, preferably 10-19% w/w, more preferably 10-18% w/w, most preferably 10-16% w/w.

In other preferred embodiments of the invention the liquid solution or the early solution (an early solution) used for preparing the liquid solution comprises a total amount of protein of 5-20% w/w, preferably 8-19% w/w, more preferably 10-18% w/w, most preferably 12-16% w/w.

-a pH of 5.6-6.5, preferably 5.8-6.2, more preferably 5.9-6.1.

In some preferred embodiments of the invention, the liquid solution or an early solution (an early solution) used to prepare the liquid solution has a degree of protein denaturation of at most 30%, preferably at most 20%, even more preferably at most 10%, most preferably at most 5%.

Even lower protein denaturation degrees may be preferred, and thus in some preferred embodiments of the invention, the liquid solution or the early solution (an early solution) used to prepare the liquid solution has a protein denaturation degree of at most 4%, preferably at most 2%, even more preferably at most 1%, most preferably at most 0.2%.

In some preferred embodiments of the invention, the lipid content of the liquid solution or the early solution (an early solvent) used for preparing the liquid solution is at most 5% w/w, more preferably at most 2% w/w, even more preferably at most 0.5% w/w, most preferably at most 0.1% w/w.

In some preferred embodiments of the invention, the carbohydrate content of the liquid solution or of the early solution (an early solution) used for preparing the liquid solution is at most 12% w/w, more preferably at most 6% w/w, even more preferably at most 2% w/w, most preferably at most 0.1% w/w.

However, in other preferred embodiments of the invention, the carbohydrate content of the liquid solution or the early solution (an earlier solution) used for preparing the liquid solution is 2-25% w/w, more preferably 4-20% w/w, even more preferably at most 5-18% w/w, and most preferably at most 6-15% w/w. These embodiments are useful, for example, if the final beverage must contain a large amount of carbohydrates. Alternatively, carbohydrates may be added to the heat-treated early solution (an earlier solution) after the protein nanogel is formed.

In some preferred embodiments of the invention, which may be used, for example, to form protein nanogels, the pH of the liquid solution or the early solution (an early solution) used to prepare the liquid solution is 5.6 to 6.5, preferably 5.8 to 6.2, more preferably 5.9 to 6.1.

The liquid solution or the earlier solution used for preparing the liquid solution (an early solution) is preferably subjected to a first heat treatment using a temperature of 70-145 deg.c, preferably 75-120 deg.c, more preferably 80-98 deg.c, even more preferably 82-96 deg.c, most preferably 85-95 deg.c.

Although the first heat treatment causing the protein nanogelling may be performed under high shear conditions, e.g. using a scraped surface heat exchanger or similar high shear device, surprisingly the protein nanogel may also be formed by heating under low shear or even no shear conditions, e.g. by immersing the packaged liquid solution in an oil bath or using a plate heat exchanger.

The first heat treatment preferably has a duration sufficient to provide a degree of protein denaturation of at least 40%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%. Even higher protein denaturation can be obtained by the first heat treatment, preferably at least 90%, and more preferably at least 95%.

The duration of the first heat treatment may be, for example, 0.1 second to 2 hours, preferably 0.5 minutes to 1 hour, more preferably 2 minutes to 30 minutes, most preferably 5 to 20 minutes.

In some preferred embodiments, the first heat treatment is the only heat treatment in the process of producing the packaged heat-treated beverage product. In this case, the liquid solution is subjected to a first heat treatment before or after packaging.

In other preferred embodiments, a first heat treatment is used to form the protein nanogel, followed by a second heat treatment, which is preferably used for final pasteurization or sterilization of the liquid solution. In this case, the first heat treatment is preferably applied to the early solution (an early solution) for preparing the liquid solution. This early solution (an earlier solution) may then be processed, for example, by mixing with other ingredients mentioned herein, such as carbohydrates, fats, minerals, and/or vitamins, to form a liquid solution. Such treatment may also involve other steps, such as pH adjustment, homogenization and/or emulsification, for example. Preferably, the liquid solution will comprise the early solution (an earlier solution), and optionally other ingredients mixed with the early solution (an earlier solution).

In some preferred embodiments of the invention:

applying a first heat treatment to the early solution (an early solution) used for preparing the liquid solution, thereby forming a protein nanogel,

optionally combining the heated early solution (an early solution) with other ingredients, e.g. by mixing,

-packaging the liquid solution in the form of the heated early solution (an early solution) itself or a combination of the heated early solution (an early solution) and other ingredients in a suitable container, and

-subjecting the packaged liquid solution to a second heat treatment comprising at least pasteurization and preferably sufficient to provide a sterile beverage product.

In some preferred embodiments of the invention, minerals, e.g., Ca, Mg, K and/or K, are added to the nanogel-containing, heat-treated early solution (an early solution). The inventors have observed that whey proteins are more resistant to pasteurization or even sterilization heat treatment in the presence of high mineral content when they have previously been converted to nanogels.

The term "beverage product" describes a liquid solution that has been subjected to a heat treatment that includes at least pasteurization.

The total amount of Ca and Mg of the liquid solution or the early solution (an early solution) used for preparing the liquid solution is preferably in the range of 0.001-0.1% w/w, more preferably in the range of 0.005-0.06% w/w, and most preferably in the range of 0.02-0.04% w/w.

The total amount of Na and K of the liquid solution or of the early solution (an early solution) used for preparing the liquid solution is preferably in the range of 0.001-0.2% w/w, more preferably 0.01-0.1% w/w, and most preferably in the range of 0.04-0.06% w/w.

In some preferred embodiments of the invention, minerals, e.g., Ca, Mg, K and/or K, are added to the nanogel-containing, heat-treated early solution (an early solution). The inventors have observed that whey proteins are more resistant to pasteurization or even sterilization heat treatment in the presence of high mineral content when they have previously been converted to nanogels.

Thus, it is generally preferred to mix the nanogel-containing, heat-treated early solution (an early solution) with other ingredients comprising minerals in an amount sufficient to provide a liquid solution comprising a total amount of Ca and Mg of at least 0.1% w/w, preferably at least 0.3% w/w, more preferably at least 0.5% w/w.

The nanogel-containing, heat-treated early solution (an early solvent) is preferably mixed with other ingredients comprising minerals in an amount sufficient to provide a liquid solution comprising a total amount of Ca and Mg of 0.1-1.5% w/w, more preferably 0.3-1.2% w/w, even more preferably 0.5-1.0% w/w.

In addition, it is generally preferred that the nanogel-containing, heat-treated early solution (an early solution) is mixed with other ingredients comprising minerals in an amount sufficient to provide a liquid solution containing at least 0.2% w/w, preferably at least 0.5% w/w, more preferably at least 0.7% w/w of the total amount of Na and K.

The nanogel-containing, heat-treated early solution (an early solution) is preferably mixed with other ingredients including minerals in an amount sufficient to provide a liquid solution comprising a total amount of Na and K of 0.2-1.5% w/w, more preferably 0.5-1.2% w/w, even more preferably 0.7-1.0% w/w.

Particularly preferably, the liquid solution or the early solution (an early solution) for preparing the liquid solution is obtained by: the BLG isolate described herein (preferably obtained according to WO 2018/115520a 1) is mixed with water (preferably demineralized or pH-adjusted) and the pH is optionally adjusted to obtain the desired protein content, the pH being in the range of 5.6-6.4. Preferably the pH adjustment is stopped as soon as the liquid becomes transparent.

The soluble whey protein aggregates are preferably formed by heat treatment in a pH range of 6.6-8.0, more preferably 6.7-7.5, even more preferably 6.9-7.3. The heat treatment described in the context of protein nanogels is also useful for forming soluble whey protein aggregates. However, the protein content of the liquid solution is preferably in the range of 1-12% w/w, more preferably in the range of 3-11% w/w, even more preferably in the range of 5-10% w/w, most preferably in the range of 6-9% w/w.

For the preparation of soluble whey protein aggregates using a liquid solution of high protein concentration, the total amount of Ca and Mg of the liquid solution is preferably at most 0.01% w/w, more preferably at most 0.005% w/w, even more preferably at most 0.001% w/w.

For the preparation of soluble whey protein aggregates using a liquid solution of high protein concentration, the total amount of Na and K in the liquid solution is preferably at most 0.05% w/w, more preferably at most 0.01% w/w, most preferably at most 0.005% w/w.

One aspect of the invention relates to the use of a protein solution comprising a total amount of protein of 1 to 20% w/w relative to the weight of the solution, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG), for controlling the whiteness of a sterile beverage preparation having a pH in the range of 5.5-8.0.

Another aspect of the present invention relates to a packaged heat-treated beverage product as defined herein for use in a method of treating a disease associated with protein deficiency.

Another aspect of the present invention relates to the use of a packaged heat-treated beverage product as defined herein as a dietary supplement.

In a preferred embodiment of the invention, the packaged heat-treated beverage product as defined herein is used as a dietary supplement and is ingested before, during or after exercise.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, the beverage comprising:

-the total amount of protein is 1 to 20% w/w, relative to the weight of the beverage product, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG), preferably at least 90% w/w and

-optionally, sweeteners and/or flavouring agents,

wherein the protein component of the beverage product has a color value Δ b, measured at room temperature, in the range of-0.10 to +0.51 on the CIELAB color scale, wherein,

Δb*＝b_{samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}*。

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, the beverage comprising:

-the total amount of protein is 1 to 20% w/w, relative to the weight of the beverage product, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG), preferably at least 90% w/w and

-optionally, sweeteners and/or flavouring agents,

wherein the protein component of the beverage product has a color value Δ b, measured at room temperature, in the range of-0.10 to +0.51 on the CIELAB color scale, wherein,

Δb*＝b_{samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}*，

And a lipid content of at most 5% of the total energy content of the preparation.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, the beverage comprising:

-the total amount of protein is 1 to 20% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

wherein the protein component of the beverage product has a color value Δ b, measured at room temperature, in the range of-0.10 to +0.51 on the CIELAB color scale, wherein,

Δb*＝b_{samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}*，

And a lipid content greater than 5%, preferably greater than 20E% of the total energy content of the preparation.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH in the range of 5.8 to 8.00, said beverage comprising:

-the total amount of protein is 1 to 20% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

the turbidity of the beverage is greater than 200NTU, preferably greater than 40 NTU.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, the beverage comprising:

-the total amount of protein is 1 to 20% w/w, relative to the weight of the beverage product, wherein at least 85 w/w% of the protein is beta-lactoglobulin (BLG), preferably at least 90% w/w, and

-optionally, sweeteners and/or flavouring agents,

the beverage has a turbidity of at most 200NTU, preferably at most 40 NTU.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, the beverage comprising:

-the total amount of protein is from 3 to 20% w/w, more preferably from 3 to 18% w/w, even more preferably from 3 to 15% w/w, most preferably from 3 to 10% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

The turbidity of the beverage is greater than 200NTU, preferably greater than 40 NTU.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, the beverage comprising:

-the total amount of protein is from 3 to 20% w/w, more preferably from 3 to 18% w/w, even more preferably from 3 to 15% w/w, most preferably from 3 to 10% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

the beverage has a turbidity of at most 200NTU, preferably at most 40 NTU.

In some preferred embodiments of the invention, the packaged heat-treated beverage product has a pH in the range of 6.2 to 8.0, preferably 6.3 to 7.6, preferably 6.5 to 7.2, said beverage comprising:

-the total amount of protein is 1 to 20% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

the beverage has a turbidity of at most 200NTU, preferably at most 40 NTU.

More preferably, the packaged heat-treated beverage product has a pH in the range of 6.5 to 8.0, preferably 6.7 to 7.6, preferably 6.9 to 7.2, said beverage product comprising:

-the total amount of protein is 5 to 12% w/w, relative to the weight of the beverage product, wherein at least 90 w/w%, preferably at least 94% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

the beverage product has a turbidity of at most 40NTU, preferably at most 20NTU,

wherein the beverage product is preferably sterile.

In some preferred embodiments of the invention, the packaged heat-treated beverage product has a pH in the range of 6.2 to 8.0, preferably 6.3 to 7.6, preferably 6.5 to 7.2, said beverage comprising:

-the total amount of protein is from 3 to 20% w/w, more preferably from 3 to 18% w/w, even more preferably from 3 to 15% w/w, and most preferably from 3 to 10% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

the beverage has a turbidity of at most 200NTU, preferably at most 40 NTU.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH in the range of 6.5 to 8.0, preferably 6.7 to 7.6, preferably 6.9 to 7.2, and the beverage product comprises:

-the total amount of protein is from 3 to 20% w/w, more preferably from 3 to 18% w/w, even more preferably from 3 to 15% w/w, most preferably from 3 to 10% w/w, relative to the weight of the beverage product, wherein at least 90 w/w%, preferably at least 94% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

the beverage product has a turbidity of greater than 200NTU, preferably greater than 400 NTU.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH of 5.5 to 6.2, preferably 5.7 to 6.1, preferably 5.8 to 6.0, said beverage comprising:

-the total amount of protein is 1 to 20% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

the turbidity of the beverage is greater than 200NTU, preferably greater than 400 NTU.

In some preferred embodiments of the invention, the packaged heat-treated beverage product has a pH in the range of 5.8 to 8.0, preferably 6.3 to 7.6, preferably 6.5 to 7.2, said beverage comprising:

-the total amount of protein is 2-10.0% w/w relative to the weight of the beverage product, preferably the total amount of protein is 3.0-8.0% w/w relative to the weight of the beverage product, preferably the total amount of protein is 5.0-7.5% w/w, more preferably 4.0-6.0% w/w relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, a sweetener and/or a flavoring agent.

In some preferred embodiments of the invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, preferably 6.3 to 7.6, preferably 6.5 to 7.2, said beverage comprising:

-the total amount of protein is 2-10.0% w/w, preferably 3.0-8.0% w/w, preferably 5.0-7.5% w/w, more preferably 4.0-6.0% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners and/or flavouring agents,

wherein the total amount of magnesium and calcium is at most 10 mM.

In some preferred embodiments of the invention, the packaged heat-treated beverage product has a pH of 5.8 to 8.0, preferably 6.3 to 7.6, preferably 6.5 to 7.2, said beverage comprising:

-the total amount of protein is 2-10.0% w/w, preferably 3.0-8.0% w/w, preferably 5.0-7.5% w/w, more preferably 4.0-6.0% w/w, relative to the weight of the beverage product, wherein at least 85 w/w%, preferably at least 90% w/w of the protein is beta-lactoglobulin (BLG),

-optionally, sweeteners and/or flavouring agents,

the beverage comprises at most 100mg phosphorus per 100g protein and at most 700mg potassium per 100g protein, preferably at most 50mg phosphorus per 100g protein and at most 400mg potassium per 100g protein, or preferably at most 10mg phosphorus per 100g protein and at most 50mg potassium per 100g protein.

Preferred embodiments of the present invention relate to heat-treated beverage products obtainable by one or more of the methods described herein.

It should be noted that embodiments and features described in the context of one aspect of the invention are also applicable to other aspects of the invention.

All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

The invention will now be described in more detail in the following non-limiting examples.

Example 1: analytical method

Example 1.1: determination of protein naturalness by Tryptophan intrinsic fluorescence

Tryptophan (Trp) fluorescence spectroscopy is a well-described tool for monitoring protein folding and unfolding. Trp residues buried in native proteins typically show the highest fluorescence emission near 330nm than when present at more solvent-exposed positions (e.g., in unfolded proteins). In unfolded proteins, the wavelength used for Trp fluorescence emission is typically shifted to higher wavelengths, typically measured around 350 nm. We use this transition here to monitor the thermally induced development by calculating the ratio between the fluorescence emission at 330nm and 350nm to investigate the effect of heating temperature.

The analysis comprises the following steps:

diluting the beverage composition to 0.6mg/ml in MQ water;

transfer 300. mu.l of sample to a white 96-well plate, avoiding the formation of bubbles, or transfer 3mL of sample to a 10mm quartz cuvette;

tryptophan fluorescence emission intensity between 310nm and 400nm was recorded from the top by excitation at 295 using a 5nm gap;

samples were measured at 22 ℃ using a Cary Eclipse fluorescence spectrophotometer equipped with a plate reader attachment (G9810A) or a single cuvette holder;

the emission intensity ratio is calculated by dividing the fluorescence emission intensity measured at 330nm by the emission intensity at 350nm, R ═ I330/I350, and is used as a measure of protein naturalness.

o an R of at least 1.11 indicates the predominant native BLG conformation, and

an R of o less than 1.11 indicates at least partial unfolding and aggregation.

Example 1.2: thermal stability at pH 3.9

Thermal stability at pH 3.9:

the heat stability at pH 3.9 is a measure of the ability of the protein composition to remain clear when pasteurized at pH 3.9 for an extended period of time.

The thermal stability at a pH of 3.9 was determined by mixing the powder or liquid sample to be tested with water (or if it is a dilute liquid, concentrating it by low temperature evaporation) and adjusting the pH to 3.9 with a minimum of 0.1M NaOH or 0.1M HCl to form an aqueous solution having a pH of 3.9 and containing 6.0% w/w protein.

The pH-adjusted mixture was allowed to stand for 30 minutes, and then 25mL of the mixture was transferred to a 30mL thin-walled glass tube. It was heated to 75.0 degrees celsius for 300 seconds by immersion in a water bath at 75.0 degrees celsius. Immediately after heating, the glass test tube was transferred to an ice bath, cooled to 1-5 ℃, and then the turbidity of the heat-treated sample was measured according to example 1.7.

Example 1.3: determination of the degree of protein denaturation of whey protein compositions

It is known that the solubility of denatured whey protein at pH 4.6 is lower than at pH values below pH 4.6 or above pH 4.6, and therefore, the denaturation of whey protein compositions is determined by measuring the soluble protein content at pH 4.6 relative to the total protein content at a pH at which the proteins in the solution are stable.

More specifically, for whey proteins, the whey protein composition (e.g. powder or aqueous solution) to be analyzed is converted into:

-a first aqueous solution comprising 5.0% (w/w) total protein and having a pH of 7.0 or 3.0, and

-a second aqueous solution comprising 5.0% (w/w) total protein and having a pH of 4.6.

The pH adjustment was carried out using 3% (w/w) NaOH (aq) or 5% (w/w) HCl (aq).

The total protein content (P) of the first aqueous solution was determined according to example 1.5 _{pH 7.0 or 3.0})。

The second aqueous solution was stored at room temperature for 2h and then centrifuged at 3000g for 5 min. A sample of the supernatant was recovered and analyzed according to example 1.5 to obtain the protein concentration (S) in the supernatant_pH4.6)。

The protein denaturation degree D of the whey protein composition was calculated as follows:

D＝((P_{pH 7.0 or 3.0}-S_{pH 4.6})/P_{pH 7.0 or 3.0})×100％

Example 1.4: denaturation of protein was determined using reverse phase UPLC analysis (pH 4.6 acid precipitation)

BLG samples (e.g., unheated control and heated BLG beverage compositions) were diluted to 2% in MQ water. 5mL of the protein solution, 10mL of Milli-Q, 4mL of 10% acetic acid, and 6mL of 1.0M NaOAc were mixed and stirred for 20 minutes to allow the denatured protein to precipitate and aggregate at around pH 4.6. The solution was filtered through a 0.22 μm filter to remove aggregates and non-native proteins.

All samples were diluted to the same extent by the addition of polishing water.

For each sample, the same volume of sample was loaded onto a UPLC system with a UPLC chromatography column (Protein BEH C4;1.7 μm; 150X 2.1mm) and detected at 214 nm.

The samples were run under the following conditions:

and (3) buffer solution A: Milli-Q water, 0.1% w/w TFA

And (3) buffer solution B: HPLC grade acetonitrile, 0.1% w/w TFA

Flow rate: 0.4ml/min

Gradient: 24-45% B at 0-6.00 min; 45-90% B in 6.00-6.50 min; 90% B at 6.50-7.00 min; 90-24% B in 7.00-7.50 min and 24% B in 7.50-10.00 min

The peak area of BLG for protein standards (Sigma L0130) was used to determine the concentration of native BLG in the sample (5 th order calibration curve)

The sample was further diluted and re-injected if the linear range was exceeded.

Example 1.5: determination of the Total amount of protein

The total protein content (true protein) of the sample was determined by:

1) determination of the Milk-nitrogen content according to ISO 8968-1/2| IDF 020-1/2 (Milk-Determination of Nitrogen content) -part 1/2: Determination of the nitrogen content using the Kjeldahl method (Determination of Nitrogen content using the Kjeldahl method).

2) Determination of the Nitrogen content of Milk-4-IDF 020-4 (Milk-Determination of Nitrogen content) according to ISO 8968-4 (Milk-Determination of Nitrogen content) -part 4 Determination of the non-protein Nitrogen content (Determination of non-protein-Nitrogen content).

3) Calculating the total amount of protein (m)_{Total nitrogen}–m_{Non-protein nitrogen})×6.38。

Example 1.6: determination of non-aggregated BLG, ALA and CMP

The content of non-aggregated alpha-lactalbumin (ALA), beta-lactoglobulin (BLG) and Caseinomacropeptide (CMP) was analyzed by HPLC analysis at 0.4mL/min, respectively. mu.L of the filtered sample was injected onto 2 TSKgel3000PWxl (7.8mm 30cm, Tosohass, Japan) columns in tandem with a connected pre-column PWxl (6 mm. times.4 cm, Tosohass, Japan) equilibrated in the eluent (consisting of 465g Milli-Q water, 417.3g acetonitrile and 1mL trifluoroacetic acid) and using a 210nm UV detector.

Comparison of the peak area of the corresponding standard protein with the peak area of the sample for native alpha-lactalbumin (C)_α) Beta-lactoglobulin (C)_β) And caseinomacropeptide (C)_CMP) The content of (b) is quantitatively determined.

The total amount of additional protein (non-BLG protein) was determined by subtracting the amount of BLG (determined according to example 1.5) from the total amount of protein.

Example 1.7: determination of turbidity

Turbidity is the cloudiness or cloudiness of a fluid caused by a large number of particles, which are generally not visible to the naked eye, similar to smoke in air.

Turbidity is measured in Nephelometric Turbidity Units (NTU).

Add 20mL of beverage/sample to NTU glass and put in3000 IR turbidimeter. NTU values were measured after stabilization and repeated twice.

Example 1.8: measurement of viscosity

The viscosity of the beverage product was measured using a rheometer (Anton Paar, Physica MCR 301).

3.8mL of sample was added to cup DG 26.7. The sample was equilibrated to 22 ℃ and then at 50s^-1Pre-shear for 30 seconds, then equilibrate for 30 seconds at 1s^-1And 200s^-1In between, and for 1s^-1。

Unless otherwise stated, the viscosity is at 100s^-1Is presented in centipoise (cP) at shear rate of (a). The higher the measured cP value, the higher the viscosity.

Alternatively, the viscosity was estimated using the Viscoman of Gilson (Gilson) and was measured at about 300s^-1At a shear rate of (c) was recorded.

Example 1.9: determination of color

The color was measured using a colorimeter (Konica Minolta, CR-400). 15g of sample was added to a small petri dish (55X 14.2mm, VWR Cat #391-0895) to avoid bubble formation. The protein content of the sample was normalized to 6.0 w/w% protein or less.

The colorimeter was calibrated to a white calibration plate (No. 19033177). The light source was set to D65 and the viewing angle was set to 2 degrees. The suspension was covered with a cover and the color (CIELAB color space, a-, b-, L-values) was measured as the average of three individual readings at different positions of the culture dish.

The demineralized water reference value had the following values:

L^*39.97±0.3

a*0.00±0.06

b*-0.22±0.09

based on the demineralized water measurement, the measurement is converted to a delta/difference value.

ΔL^*＝L_{Samples normalized to 6.0 w/w% protein}*-L_{Demineralized water}Measured at room temperature.

Δa*＝a_{Samples normalized to 6.0 w/w% protein}*-a_{Demineralized water}Measured at room temperature.

Δb*＝b_{Standard of meritSample reduced to 6.0 w/w% protein}*-b_{Demineralized water}Measured at room temperature.

The samples were normalized to 6.0 w/w% or less protein.

L a b color space (also known as CIELAB space) is a unified color space defined by the international commission on illumination (CIE) in 1976 for quantitative reporting of brightness and hue (ISO 11664-4: 2008(E)/CIE S014-4/E: 2007).

In this space, L denotes luminance (value from 0 to 100), and when L is 0, it is darkest black, and when L is 100, it is brightest white.

Color channels a and b represent true neutral gray values when a is 0 and b is 0. The a-axis represents the green-red component, green being the negative direction and red being the positive direction. b-axis indicates blue-yellow component, blue in negative direction and yellow in positive direction.

Example 1.10: beverage stability test/insoluble proteinaceous material

A whey protein beverage composition is considered stable if less than 15% of the total amount of protein in the heated sample precipitates after centrifugation at 3000g for 5 minutes:

approximately 20g of sample was added to the centrifuge tube and centrifuged at 3000g for 5 minutes

Kjeldahl analysis of proteins before centrifugation and supernatant after centrifugation for quantification of protein recovery, see example 1.5

The loss of protein was calculated as follows:

% of denaturation ((P)_{General assembly}-P_3000×g)/P_{General assembly})×100％

This parameter, sometimes referred to as the level of insoluble proteinaceous material, can be used to analyze liquid and powder samples. If the sample is a powder, 10g of the powder is suspended in 90g of demineralized water and hydrated at 22 ℃ for 1 hour with gentle stirring. Approximately 20 grams of sample (e.g., liquid sample or suspended powder sample) is placed in a centrifuge tube and centrifuged at 3000g for 5 minutes. According to example 1.5, the protein before centrifugation (P) was used _{General assembly}) And the supernatant after centrifugation (P)_3000×g) Kjeldahl analysis (Kjeldahl analysis) ofAnd (5) recovering the protein.

The amount of insoluble proteinaceous material was calculated as follows:

percent of insoluble proteinaceous material ((P)_{General assembly}-P_3000×g)/P_{General assembly})×100％。

Example 1.11: determination of gel Strength after acidification

To simulate the structural development of the beverage in the stomach during acidification, a rheometer (Anton Paar, Physica MCR301) was used. The beverage was diluted to a protein content of 2 w/w% and incubated at 42 ℃ for 30 minutes. Then 1 w/w% GDL (D-gluconic acid, Sigma Aldrich, 49-53 wt%) was added to the solution and stirred for 5 minutes. The solution (19.6mL) was added to the CC27-SS cup in the rheometer. The rheometer was equilibrated to 42 ℃ and the G' (storage modulus, Pa) was measured at 0.1Hz and 0.5% strain for 60 min. The pH was followed during acidification using a pH recorder (WTW, Multi 3410).

Example 1.12: determination of transparency by photography

A photograph of the beverage product was taken by placing the sample into a turbidity NTU measuring bottle that touched the paper with the "lorem ipsum" script. The measurement jar was photographed using a smartphone and the inventors evaluated whether the text could be clearly seen through the measurement jar.

Example 1.13: determination of the ash content

The ash content of the food product was according to NMKL 173: 2005 ash in food, gravimetric.

Example 1.14: determination of the conductivity

The "conductivity" (sometimes referred to as "specific conductance") of an aqueous solution is a measure of the conductive ability of the solution. The conductivity can be determined, for example, by measuring the alternating current resistance of the solution between the two electrodes, the results of which are usually given in milliSiemens per centimeter (mS/cm). Conductivity can be measured, for example, according to EPA (united states environmental protection agency) method 120.1.

Unless otherwise stated, the conductivity values referred to herein have been normalized to 25 ℃.

Conductivity was measured on a conductivity meter (WTW Cond 3210 with quadrupole 325 electrodes).

Before use, the system was calibrated as described in the manual. To avoid local dilution, the electrodes should be thoroughly rinsed in the same type of medium in which the measurement is performed. The electrodes are lowered into the medium so that the area where the measurement is made is completely submerged. The electrodes are then agitated to remove any air remaining on the electrodes. The electrodes are then held stationary until a stable value can be obtained and recorded by the display.

Example 1.15: determination of the Total solids of the solution

The Total solids of the solution can be determined according to NMKL 110, second edition, 2005 (Total solids (Water) -weight determination in milk and milk products). NMKL is "Nordisk MetodikkomLefor"abbreviation of.

The water content of the solution can be calculated as the relative content (% w/w) of 100% minus the total solids.

Example 1.16: determination of pH

All pH values were measured using a pH glass electrode and normalized to 25 ℃.

The pH glass electrode (with temperature compensation) was carefully rinsed and calibrated prior to use.

When the sample is in liquid form, the pH is measured directly in a liquid solution at 25 ℃.

When the sample was a powder, 10 grams of the powder was dissolved in 90mL of demineralized water at room temperature under vigorous stirring. The pH of the solution was then measured at 25 ℃.

Example 1.17: determination of bulk and bulk Density

The density of a dry powder is defined as the relationship between the weight and volume of the powder, which is analyzed under specified conditions using a special Stampf volumeter (i.e., graduated cylinder). The density is usually expressed in g/ml or kg/L.

In this method, a sample of dry powder is tamped into a measuring cylinder. After a specified number of taps, the volume of the product was read and the density calculated.

This method can define three types of densities:

pour density, i.e. mass divided by the volume of powder after transfer to the specified cylinder.

Loose bulk density, i.e. mass divided by the volume of powder after 100 taps according to the specified conditions of the present standard.

Bulk density, mass divided by the volume of powder after 625 taps according to the specified conditions of the present standard.

The method uses a special measuring cylinder 250ml, 0-250ml scale, a weight of 190 + -15 g (J.Engelsmann A.G.67059Ludwigshafen/Rh) and a Stampf measuring vessel, e.g.J.Engelsmann A.G.

The bulk and bulk densities of the dried product were determined by the following steps:

pre-treating;

the samples to be tested were stored at room temperature.

The sample is then thoroughly mixed (avoiding crushing the particles) by repeatedly rotating and turning the container. The container is filled to a level not exceeding 2/3.

And (3) treatment:

100.0. + -. 0.1 g of powder are weighed and transferred into a measuring cylinder. Volume V₀In milliliters.

If the cylinder does not hold 100g of powder, the amount of powder should be reduced to 50g or 25 g.

The cylinder was fixed to the Stampf volumetric apparatus and tapped 100 times. The surface is smoothed with a spatula and the volume V is read in ml₁₀₀。

The number of taps was changed to 625 ((including the 100 taps)). After tapping, the surface is flattened and the volume V is read in milliliters ₆₂₅。

And (3) calculating the density:

bulk density and bulk density in g/mL were calculated according to the following formulas:

bulk density of M/V

Where M represents the weighed sample in grams and V represents the 625 tapped volume in ml.

Example 1.18: determination of the moisture content in the powder

The moisture content of the food product is according to ISO 5537: 2004 (Determination of Dried milk-moisture content (Reference method))). NMKL is "Nordisk MetodikkomLefor"abbreviation of.

Example 1.19: determination of the content of calcium, magnesium, sodium, potassium, phosphorus (ICP-MS method)

The total amount of calcium, magnesium, sodium, potassium and phosphorus was determined using the following procedure: the samples were first decomposed using microwave digestion and then the total amount of minerals was determined using an ICP instrument.

The instrument comprises the following steps:

microwaves were from Anton Paar (Anton Paar) and ICP was Optima 2000DV from PerkinElmer Inc.

Materials:

1M HNO₃

2％HNO₃yttrium in (III)

Suitable for 5 percent of HNO₃Standard solution of medium calcium, magnesium, sodium, potassium and phosphorus

Pretreatment:

a quantity of powder was weighed out and then transferred to a microwave digestion tube. 5mL of 1M HNO was added₃. The samples were digested in the microwave as per the microwave specifications. The digestion tube was placed in a fume hood and the lid removed to allow the volatile fumes to evaporate.

A measurement step:

the pretreated samples were transferred to Digitube using known amounts of Milli-Q water. Adding 2% HNO into the digestion tube₃Yttrium solution in (about 0.25mL per 50mL diluted sample) and diluted to have been used with Milli-Q waterThe volume is known. Samples were analyzed on ICP using the procedure described by the manufacturer.

10mL of 1M HNO by using Milli-Q water₃And 0.5mL in 2% HNO₃The mixture of yttrium solutions in (1) was diluted to a final volume of 100mL to prepare blind samples.

At least 3 standard samples were prepared, the concentrations of which should include the expected sample concentrations.

The detection limit of the liquid sample is: 0.005g/100g sample for Ca, Na, K and P (phosphine) and 0.0005g/100g sample for Mg. The detection limits of the powder samples were 0.025g/100g of sample for Ca, Na, K and P (Pho) and 0.0005g/100g of sample for Mg.

When at or below the detection limit of P, the value of the detection limit is used in the example to account for the worst-case maximum amount of P present.

Example 1.20: determination of the Creatinine value

Creatinine values were determined as follows: "Evaluation of Maillard Reaction by creatinine De-determination During Infant Cereal Processing" Guerra-Hernandez et al, Journal of Cereal Science 29(1999) 171-. The creatinine values were calculated in units of mg creatinine/100 g protein.

Example 1.21: determination of the crystallinity of BLG in liquids

The following method was used to determine the crystallinity of BLG in liquids having a pH in the range of 5-6.

a) 10mL of the liquid sample concerned was transferred to a Maxi-Spin filter with a CA membrane with a pore size of 0.45 microns.

b) The filter was immediately spun at 1500g for 5 minutes, and the centrifuge was maintained at 2 ℃.

c) 2mL of cold Milli-Q water (2 ℃) was added to the retentate side of the spin filter (retenate side) and the filter was immediately spun at 1500g for 5 minutes while cooling the centrifuge to 2 ℃ and the permeate (permeate A) was collected, the volume was measured by HPLC and the BLG concentration determined using the method outlined in example 1.31.

d) 4mL of 2M NaCl was added to the retentate side of the filter, stirred rapidly and the mixture was allowed to stand at 25 ℃ for 15 minutes.

e) The filter was immediately spun at 1500g for 5 minutes and the permeate (permeate B) was collected.

f) The total weight of BLG in permeate a and permeate B was determined using the method outlined in example 1.31 and the results were converted to total weight of BLG rather than weight percent. The weight of BLG in permeate A is designated m_{Permeate A}The weight of BLG in permeate B is designated m_{Permeate B}。

g) The crystallinity of the liquid with respect to BLG is determined according to the following formula:

Degree of crystallinity ═ m_{Permeate B}/(m_{Permeate A}+m_{Permeate B})×100％

Example 1.22: determination of crystallinity of BLG in Dry powders

This method was used to determine the crystallinity of BLG in dry powders.

a) A5.0 gram sample of the powder was mixed with 20.0 grams of cold Milli-Q water (2 ℃) and allowed to stand at 2 ℃ for 5 minutes.

b) The liquid sample involved was transferred to a Maxi-Spin filter with a 0.45 micron CA membrane.

c) The filter was immediately spun at 1500g for 5 minutes, and the centrifuge was maintained at 2 ℃.

d) 2mL of cold Milli-Q water (2 ℃) was added to the retentate side of the spin filter, then immediately the permeate (permeate A) was collected at 1500g spin filter for 5 minutes, the volume was measured and the BLG concentration was determined by HPLC using the method outlined in example 1.31, converting the results to the total weight of BLG rather than weight percent. The weight of BLG in permeate A is designated m_{Permeate A}。

e) The crystallinity of BLG in the powder was then calculated using the following formula:

degree of crystallinity ((m)_{Total amount of BLG}-m_{Permeate A})/m_{Total amount of BLG})×100％

Wherein m is_{Total amount of BLG}Is the total amount of BLG in the powder sample of step a).

If the total amount of BLG in the powder sample is not known, it can be determined as follows: another 5g sample of the powder (from the same powder source) was suspended in 20.0 g Milli-Q water, then the pH was adjusted to 7.0 by adding aqueous NaOH, the mixture was allowed to stand at 25 ℃ for 1 hour with stirring, and finally the total amount of BLG in the powder sample was determined using example 1.31.

Example 1.23: determination of the conductivity of the Ultrafiltration (UF) permeate

15mL of the sample was transferred to an Amicon Ultra-15 centrifugal filter with a cut-off of 3kDa (3000NMWL) and centrifuged at 4000g for 20-30 minutes, or until a sufficient volume of ultrafiltration permeate had accumulated in the bottom of the filter to measure conductivity. Conductivity was measured immediately after centrifugation. Sample processing and centrifugation are performed at the temperature of the sample source.

Example 1.24: detection of dried BLG crystals in powders

The presence or absence of dried BLG crystals in the powder can be determined by the following method:

the powder sample to be analyzed was resuspended and gently mixed in demineralized water at a temperature of 4 ℃ in a weight ratio of 2 parts water to 1 part powder, and then rehydrated at 4 ℃ for 1 hour.

The rehydrated sample is examined microscopically to identify the presence of crystals, preferably using plane polarized light to detect birefringence.

The crystalline material is isolated and subjected to X-ray crystallographic analysis to verify the presence of the crystalline structure and preferably also to verify that the lattice (space group and unit cell size) corresponds to that of the BLG crystal.

The chemical composition of the isolated crystalline material was analyzed to verify that its solids consisted primarily of BLG.

Example 1.25: determination of the Total amount of lactose

Total amount of lactose determination of the dry milk, dry ice mix and processed cheese-lactose content according to ISO 5765-2:2002(IDF 79-2:2002) — part 2: enzymatic methods (Dried milk, Dried rice-mixes and processed cheese-Determination of lactose content-Part 2: Enzymatic method of the galactose mobility of the lactose) "Determination using the galactose moiety in lactose.

Example 1.26: determination of the Total amount of carbohydrates

The carbohydrate content was determined by using Sigma Aldrich carbohydrate total assay kit (Cat MAK104-1KT), where carbohydrates were hydrolyzed and converted to furfural and hydroxyfurfural, which were then converted to chromogens and monitored spectrophotometrically at 490 nm.

Example 1.27: determination of the Total amount of lipids

Lipid content according to ISO 1211:2010 (determination of fat content)Gravimetric method (Determination of Fat Content)Gravimetric Method)).

Example 1.28: measurement of Brix (Brix)

Brix measurements were performed using a PAL-alpha digital hand-held refractometer (Atago) calibrated on polished water (water filtered by reverse osmosis to obtain a conductivity of at most 0.05 mS/cm).

About 500. mu.l of the sample was transferred to the prism surface of the instrument and the measurement was started. The measured values are read and recorded.

The brix of the whey protein solution is proportional to the total solids content (TS), and TS (% w/w) is about brix 0.85.

Example 1.29: determination of lactoferrin and lactoperoxidase

The concentration of lactoferrin was determined by an ELISA immunoassay as outlined in Soyeur 2012(Soyeur et al; Mid-infrared prediction of lactoferrin content in cow's milk: potential indicator of mastitis (Mid-extracted prediction of lactoferrin content in bovine milk); Animal (2012),6:11, page 1830-1838).

The lactoperoxidase concentration was determined using a commercially available bovine lactoperoxidase kit.

Example 1.30: determination of the number of colony Forming units

The number of Colony forming units per gram of sample is determined at 30 ℃ according to ISO 4833-1:2013(E): Microbiology of food and animal feed-horizontal method of microbial count-Colony counting technique (Microbiology of food and animal feeding of microorganisms-Colony method for the efficiency of the environment of microorganisms-Colony-count technology).

Example 1.31: determination of the Total amount of BLG, ALA and CMP

The procedure is a liquid chromatography (HPLC) method for the quantitative analysis of proteins (e.g. ALA, BLG and CMP) and optionally other proteins in the composition. In contrast to the method of example 1.6, the present method also measures the protein present in aggregation, thus providing a measure of the total amount of protein species in the composition in question.

The separation format was Size Exclusion Chromatography (SEC) using 6M guanidine hydrochloride buffer as sample solvent and HPLC mobile phase. Mercaptoethanol is used as a reducing agent to reduce disulfides (S-S) in proteins or protein aggregates to produce unfolded monomeric structures.

Sample preparation can be easily achieved by dissolving 10mg protein equivalents in the mobile phase.

Two TSK-GEL G3000SWXL (7.7 mm. times.30.0 cm) columns (GPC columns) and a guard column were placed in series to achieve adequate separation of the major proteins in the starting material.

The eluted analytes were detected and quantified by UV detection (280 nm).

Equipment/materials:

1. HPLC Pump with Manual gasket Wash 515(Waters (Watts))

HPLC Pump controller Module II (Waters)

3. Auto sampler 717 (Waters)

4. Double absorbance detector 2487 (Waters)

5. Computer software capable of generating quantitative reports (Empower 3, Waters)

6. And (3) analyzing the column: two TSK-GEL G3000SWXL (7.8X 300mm, P/N:08541)

Protection of the column: TSK-guard column SW × L (6.0 × 40mm, P/N:08543)

7. Ultrasonic bath (Branson 5200)

8.25mm syringe filter with 0.2 μm cellulose acetate membrane. (514 0060, VWR)

And (3) treatment:

mobile phase:

A. storage buffer (Stock buffer solution)

1. 56.6g Na were weighed in a 1000mL beaker₂HPO₄、3.5g NaH₂PO₄And 2.9g EDTA.

Dissolved in 800mL of water.

2. The pH is measured and, if necessary, adjusted to 7.5. + -. 0.1 with HCl (lowering the pH) or NaOH (increasing the pH).

3. Transfer to 1000mL volumetric flask and dilute to the mark with water.

B.6M guanidine hydrochloride mobile phase

1. 1146g of guanidine hydrochloride are weighed into a 2000mL beaker, and 200mL of stock buffer (A) are added

2. The solution was diluted with water to about 1600mL while mixing with a magnetic stir bar (50 ℃ C.)

3. The pH is adjusted to 7.5. + -. 0.1 with NaOH

4. Transferred to a 2000mL volumetric flask, diluted to the mark with water

5. Filtration was carried out using a solvent filtration apparatus with a 0.22 μm membrane filter

Calibration standard

Calibration standards for each protein to be quantified were prepared by:

1. approximately 25mg of the protein reference standard was accurately weighed (to 0.01mg) into a 10mL volumetric flask and dissolved in 10mL of water.

This is a protein stock standard solution of the protein (S1).

2. Pipette 200. mu. l S1 into a 20ml volumetric flask and dilute to the mark with mobile phase.

This is the low working standard solution (low working standard solution) WS 1.

3. Pipette 500 μ L S1 into a 10mL volumetric flask and dilute to the mark with mobile phase.

This is standard solution WS 2.

4. Pipette 500 μ L S1 into a 5mL volumetric flask and dilute to the mark with mobile phase.

This is standard solution WS 3.

5. Pipette 750 μ L S1 into a 5mL volumetric flask and dilute to the mark with mobile phase.

This is standard solution WS 4.

6. Aspirate 1.0mL S1 into a 5mL volumetric flask and dilute to the mark with mobile phase.

This is the high working standard solution (high working standard solution) WS 5.

7. Using a calibrated disposable pipette, 1.5mL of WS1-5 was transferred to a separate vial.

Add 10. mu.L of 2-mercaptoethanol to each vial and cap. The solution was vortexed for 10 seconds.

The standards were allowed to stand at ambient temperature for about 1 hour.

8. The standards were filtered using a 0.22 μm cellulose acetate syringe filter.

The purity of the protein was measured using Kjeldahl (Kjeldahl) (N.times.6.38) and the area% of WS5, a standard solution, was determined using HPLC.

Protein (mg) ═ protein standard weight (mg) × P1 × P2

P1 ═ P% (Kjeldahl method)

Protein area% (HPLC) with P2 ═ protein

Sample preparation

1. The original sample, corresponding to 25mg of protein, was weighed into a 25ml volumetric flask.

2. About 20mL of mobile phase was added and the sample was dissolved for about 30 minutes.

3. The mobile phase was added to the scale (Add mobile phase to volume) and 167. mu.L of 2-mercaptoethanol was added to 25ml of sample solution.

4. Sonication is carried out for about 30 minutes, and the sample is then left at ambient temperature for about 1.5 hours.

5. The solution was mixed and filtered using a 0.22 μ l cellulose acetate syringe filter.

HPLC system/chromatographic column

Chromatographic column balance

1. The GPC protection column and two GPC analysis columns were connected in this order.

The new chromatography column is usually transported in phosphate buffered saline.

2. The water is gradually passed through the new column at a rate of 0.1 to 0.5mL/min over 30 to 60 minutes.

Rinsing was continued for about 1 hour.

3. The flow rate was gradually reduced from 0.5mL/min to 0.1mL/min and replaced with the mobile phase in the reservoir.

4. The pump flow rate was gradually increased from 0.1mL/min to 0.5mL/min over 30 to 60 minutes to avoid pressure shock and was maintained at 0.5 mL/min.

5. Ten samples were injected to saturate the column and then wait for the peak to elute.

This will facilitate the adjustment of the column.

This step is accomplished without waiting for each implant to be completed before the next implant.

6. Equilibrating with mobile phase for at least 1 hour.

Calculation results

The quantitative determination of the content of proteins to be quantified, such as alpha-lactalbumin, beta-lactoglobulin and caseinmacropeptide, is carried out by comparing the peak areas of the respective standard proteins with the peak areas of the samples. The results are reported as: g specific protein per 100g of original sample or weight percentage of specific protein relative to the weight of original sample.

Example 1.32: quantifying the amount of microgels, protein nanogels, soluble whey protein aggregates and native proteins

The amounts of insoluble proteinaceous matter, protein nanogels, soluble whey protein aggregates and native proteins in a beverage or powder sample were quantified using the following steps:

a) the sample to be tested (by mixing with demineralized water or by concentration) is converted into a solution containing 20g total protein/L (e.g. by dilution with demineralized water or by concentration), and the concentration of total protein is verified by measuring the total protein in an aliquot of the solution using example 1.5 and reported as c_a。

b) Centrifuging a first aliquot of the solution in a) at 3.000 Xg for 5 minutes to precipitate insoluble proteinaceous material, subsequently measuring the concentration of protein in the supernatant as described in step a), and reporting the total amount of protein in the supernatant as c _b. The content of insoluble protein aggregates (percentage relative to the total amount of protein) was calculated as (c)_a-c_b)/c_a×100。

c) Centrifuging a second aliquot of the solution in a) at 50.000 Xg for 1 hour to precipitate insoluble protein material and protein nanogels, and measuring the concentration of protein in the supernatant as described in step a) and noted c_c. The protein nanogel fraction was calculated as (c)_a-c_c)/c_a×100-(c_a-c_b)/c_a×100

d) The pH of the third aliquot was adjusted to 4.6 to precipitate all denatured and/or aggregated proteins in the sample. The sample was left at room temperature for 15 minutes and then centrifuged at 50.000 Xg for 1 hour to separate the precipitate. The total protein (mainly native protein) concentration of the resulting supernatant was measured as described in step a) and recorded as c_d。

The fraction of soluble whey protein aggregates in solution was calculated as

(c_a-c_d)/c_a×100-(c_a-c_c)/c_a×100。

The fraction of native protein in solution is calculated as c_d/c_a×100。

If the absolute concentration of insoluble proteinaceous matter, protein nanogels, soluble whey protein aggregates and/or native proteins of the original sample is desired, this can easily be calculated by using information about how much of the solution in the original sample generation step a).

All centrifugation steps were performed at 25 ℃ using a Beckmann Coulter Avanti JXN-30 centrifuge equipped with JA-30.50 rotors and using 50mL of sample in a 50mL Beckmann centrifuge tube (29X 103 mm).

Example 1.33: hydrodynamic diameter

The hydrodynamic diameter (mean intensity size (d.nm)) of the protein particles was determined by dynamic light scattering using a nanosizer (malvern). 800 μ L of demineralized water and 5 μ L of heat-treated protein beverage were mixed and then added to the UV cuvette. The dimensional measurements were carried out at room temperature (22 ℃).

Example 2: production of spray-dried acidic BLG isolate powder

Whey protein feed

The UF retentate from the sugar deficiency in the sweet whey from the standard cheese production process was filtered through a 1.2 micron filter and reduced in lipid via Synder FR membrane and then used as feed for the BLG crystallization process. The chemical composition of the feed is shown in table 1. It is noteworthy that all weight percentages of the specific proteins (e.g. BLG, ALA) mentioned in this example relate to the weight percentage of non-aggregated proteins relative to the total protein.

Regulation (Conditioning)

A Koch HFK-328 type membrane (70 m) was used²Membrane) the feed concentration of sweet whey feed was adjusted to a total solids of 21% (TS) ± 5 under ultrafiltration conditions at 20 ℃ at a feed pressure of 1.5-3.0bar in 46 cell spacers, and polished water (water having a conductivity of at most 0.05mS/cm after reverse osmosis filtration) was used as the diafiltration medium. The pH was then adjusted by adding HCl to make the pH about 5.5. Diafiltration was continued until the conductivity of the retentate dropped below 0.1mS/cm over a period of 20 minutes. The retentate was then concentrated until the permeate flow was below 1.43L/h/m ². The first sample concentrated retentate was removed and centrifuged at 3000g for 5 minutes. The supernatant of the first sample was used to determine BLG production.

Crystallization

The concentrated retentate was transferred to a 300L crystallization tank seeded with pure BLG crystal material made from rehydrated, spray-dried BLG crystals. Subsequently, the inoculated whey protein solution was cooled from 20 ℃ to about 6 ℃ within about 10 hours to allow BLG crystals to form and grow.

After cooling, a sample of the whey protein solution containing crystals (second sample) was taken and the BLG crystals were isolated by centrifugation at 3000g for 5 minutes. The supernatant and crystal pellets from the second sample were subjected to HPLC analysis as described below. The yield of the crystals was calculated as described below and determined to be 57%.

TABLE 1 chemical composition of the feed

BLG yield was determined using HPLC:

the supernatants of the first and second samples were diluted to the same extent by adding polishing water and the diluted supernatants were filtered through a 0.22 μm filter. For each filtered and diluted supernatant, the same volume of supernatant was loaded into an HPLC system with Phenomenex and detected at 214nm5μm C4LC column 250X 4.6mm, Ea.

The samples were run under the following conditions:

and (3) buffer solution A: MilliQ water, 0.1% w/w TFA

And (3) buffer solution B: HPLC grade acetonitrile, 0.085% w/w TFA

Flow rate: 1mL/min

Column temperature: 40 deg.C

Gradient: 82-55% A and 18-45% B in 0-30 min; 55-10% of A and 45-90% of B in 30-32 min; 32.5-37.5 minutes 10% A and 90% B; 10-82% A and 90-18% B for 38-48 min.

Data processing:

since both supernatants were treated in the same manner, the areas of the BLG peaks could be directly compared to calculate relative yield. Since the crystals contained only BLG and the samples had all been treated in the same way, the alpha-lactalbumin (ALA) concentration as well as the ALA area should be the same in all samples. Thus, the ALA area before and after crystallization was used as a correction factor (cf) when calculating the relative yield.

cf_α＝ALA_{Before crystallization}area/ALA_{After crystallization}Area)

The relative yield was calculated by the following formula:

yield of the product_BLG＝(1-(cf_α×BLG_{After crystallization}area)/BLG_{Before crystallization}Area) × 100

Acid dissolution of BLC crystals

The material remaining in the crystallization tank was separated using a decanter at 350g, 2750RPM, 150RPM Diff. Feed and polishing water were mixed in a 1: 2, and mixing. The BLG crystals/solid phase in the decanter (decanter) are then mixed with polishing water to make it a more dilute slurry, and then phosphoric acid is added to lower the pH to about 3.0 to rapidly dissolve the crystals.

After dissolution of the BLG crystals, the pure BLG protein liquid was concentrated to 15 brix at the same UF setting as the Ultrafiltration (UF) used to prepare the feed for crystallization, and the pH was adjusted to a final pH of about 3.8. The liquid BLG isolate was then heated to 75 ℃ for 5 minutes and then cooled to 10 ℃. The heat treatment reduced the microbial load from 137.000CFU/g before heat treatment to <1000CFU/g after heat treatment. The heat treatment did not cause any denaturation of the proteins, and the intrinsic tryptophan fluorescence ratio (I330nm/I350nm) was determined to be 1.20, indicating the natural confirmation (native confirmation) of the BLG molecule.

The BLG was dried on a pilot plant spray dryer at an inlet temperature of 180 ℃ and an outlet temperature of 75 ℃. The water content of the resulting powder sampled at the outlet was about 4% w/w and the chemical composition of the powder is shown in table 2. A sample of the dried powder was dissolved and the degree of protein denaturation was determined to be 1.5% and the intrinsic tryptophan fluorescence emission ratio (I330/I350) was measured to be 1.20.

Table 2 composition of BLG isolated powder (BDL ═ below detection limit)

The bulk density of the spray-dried powder (625 taps) was estimated to be 0.2-0.3g/cm³。

Example 3: production of spray-dried, pH neutral BLG isolation powder

The lactose reduced whey protein isolate shown in table 3 was condition optimized and used as feed for crystallization when using the same protocol and experimental set-up as example 2. The crystallization yield was calculated to be 68%.

We note that all weight percentages of the specific proteins mentioned in this example, such as BLG and ALA, relate to the weight percentage of non-aggregated protein relative to the total amount of protein.

TABLE 3 composition of the feed

The remaining material in the crystallizer tank was separated on a decanter at 350g, 2750RPM, 150RPM Diff. Prior to separation, the feed was mixed with polishing water at a ratio of 1: 2, and mixing. The BLG crystals/solid phase in the decanter (decanter) are then mixed with the polish to make it a more dilute slurry, and 0.1M potassium hydroxide is then added to adjust the pH to about 7 for rapid dissolution of the crystals.

After dissolution of the crystals, the pure BLG protein liquid was concentrated to 15 brix at the same UF setting as the Ultrafiltration (UF) used to prepare the whey protein solution for crystallization, adjusting the pH to a final pH of 7.0. The BLG was dried on a pilot plant spray dryer at an inlet temperature of 180 ℃ and an outlet temperature of 75 ℃. The water content of the resulting powder sampled at the outlet was about 4% w/w. The composition of the powder is shown in table 4. After drying, some of the powder was dissolved in demineralized water, and the degree of protein denaturation was determined to be 9.0% and the intrinsic tryptophan fluorescence ratio (330nm/350nm) was 1.16.

Table 4 chemical composition of BLG isolated powder. BDL ═ below the detection limit.

The bulk density of the spray-dried powder (625 taps) was estimated to be 0.2-0.3g/cm³

Example 4: preparation of universal whey protein beverage

The dried BLG protein isolate powder (containing > 85% BLG based on protein) was dispersed in approximately 95% demineralized water required to achieve the desired final protein concentration.

A pH neutral BLG isolate powder was prepared as described in example 3, whereas a BLG isolate powder of pH 5.5 was prepared as described in example 7 of PCT/EP 2017/084553.

Optionally, minerals, sweeteners, flavors, stabilizers, emulsifiers or other components may also be added, including sources of fat and carbohydrates.

The pH is adjusted to a final pH using 10% NaOH or 10% phosphoric acid (or other food grade acid).

The remaining water is added to achieve the desired protein concentration, and the composition is optionally homogenized.

For comparison, products greater than or equal to 85% BLG in the reference sample were prepared with whey protein isolate while retaining the remaining steps.

The samples were stored in a dark environment at 20 ℃.

Example 5: heat treatment of whey protein compositions

The heat treatment is carried out using a plate heat exchanger or a tube heat exchanger (manufacturer: OMVE HTST/UHT pilot plant HT320-20) heated at 143 ℃ for 2 to 6 seconds (high temperature, short time (HTST)).

The heat-treated beverage composition was poured into 100mL sterile bottles at 10 ℃ and immediately sealed.

In other experiments, heat treatment was performed by transferring the whey protein source into thin-walled glass vials containing 15-30mL samples. The vials were soaked in a pre-equilibrated water bath at a target temperature range of 86 ℃ to 95 ℃ for 1 to 18 minutes and then cooled on ice.

Example 6: production of heat-treated beverage products

In this example, BLG beverages and WPI beverages containing 6% protein and having a pH of 7.0 were prepared.

The BLG beverage is prepared by dissolving the percolated BLG-separated powder having pH of 7.0 in demineralized water at 10 deg.C.

For comparison, WPI samples were prepared using WPI-A. WPI-A was dissolved in demineralized water at 10 deg.C. 10% NaOH was slowly added to the solution. The final pH was adjusted to pH 7.0.

The solution was heat treated using a plate heat exchanger as described in example 5 at 143 ℃/4 seconds and tapped to provide a heat sterilized whey protein beverage composition.

The composition of the BLG powder used to prepare the beverage products is given in table 5 below, along with the composition of the WPI for comparison.

Table 5 composition of BLG powder (pH 7.0 powder) and WPI powder (BDL ═ below detection limit).

Description of the invention	Drying BLG	WPI-A
			ALA(w/w％)	0.2	8
BLG(w/w％)	88.6	61
			Ash content (Ash)	5.36	3.7
Ca	BDL	0.072％
			Lipid	0.33	＜0.1
K	2.36	1.14
			Mg	BDL	0.0075
Na	BDL	0.484
			P	0.63	0.214
Protein	88.31	89.5
			pH	7.0	6.7

Example 7: colorless transparent whey protein beverage containing BLG > 85%

A beverage product is prepared in which about 92% w/w of the protein is BLG, see example 4.

For comparison, a whey protein sample based on a WPI powder containing about 61% w/w BLG was prepared.

The protein content of the sample was 6% w/w. The pH was adjusted to pH 7.0 with NaOH.

The article was heat treated at 143 ℃ for 4 seconds.

The haze, viscosity, color and clarity of the articles were measured according to the procedures described in examples 1.7, 1.8, 1.9.

The results are shown in Table 6 below.

Table 6 properties of BLG and WPI beverages.

pH 7.0	WPI-A	BLG
			Turbidity NTU	26.17	17.38
L*	39.31	39.98
			a*	-0.02	0.03
b*	0.46	0.01
			Δ L (sample-water)	-0.66	0.01
Δ a (sample-water)	-0.02	0.03
			Δ b (sample-water)	0.68	0.23

To calculate Δ b, the following formula is used:

Δb*＝b_{samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}Measured at room temperature.

To calculate Δ a, the following formula is used:

Δa*＝a_{samples normalized to 6.0 w/w% protein}*-a_{Demineralized water}Measured at room temperature.

To calculate Δ L, the following formula is used:

ΔL*＝L_{samples normalized to 6.0 w/w% protein}*-L_{Demineralized water}Measured at room temperature.

The color value of demineralized water was:

L*＝39.97，a*＝0，b*＝-0.22。

as a result:

the results presented in table 6 demonstrate that when a protein component comprising at least 85 w/w% BLG is used, a clear, colorless and transparent beverage is produced at pH 7.0. BLG beverages also have low viscosity.

In contrast, samples containing WPI in which about 61 wt% of the protein was BLG yellow and had higher b values.

Example 8: whey protein beverage and high-temperature heat treatment

An opaque milky beverage containing BLG was produced. BLG powder (pH 5.5) was dissolved in tap water and adjusted to pH 6.0 using 3% NaOH, followed by heat treatment at 94 ℃ for 14 minutes. The BLG beverage contains about 96% w/w protein as BLG.

A10 w/w% BLG beverage with pH 6.0 was prepared.

See composition of BLG and WPI samples below:

turbidity, viscosity, color and clarity were measured according to the procedures described in examples 1.7, 1.8, 1.9 and beverage stability in example 1.12.

The results are shown in table 7 below and fig. 1.

Table 7: the properties of the milky BLG beverage at pH 6.0.

Sample (I)	BLG
		L*	94.1
a*	-0.69
		b*	0.91
Δ L (sample-water)	54.13
		Δ a (sample-water)	-0.69
Δ b (sample-water)	1.13
		Viscosity (Viscoman) cP	2.3cP
Turbidity NTU	>10.000

As a result:

the results presented in table 7 and figure 1 show that when 10 w/w% protein comprising at least 85 w/w% BLG is used and heat treated (corresponding to sterilization), a milky/opaque colorless beverage is produced at pH 6.0. In contrast, the samples containing WPI (WPI-A and WPI-B) had gelled, and therefore it was impossible to produce a beverage (see FIG. 2).

Example 9: digestion of an exemplary BLG beverage comprising predominantly protein nanogels or soluble whey protein aggregates.

The purpose of this example is to explore the structure formation during gastric digestion of different whey protein beverages by in vitro simulation of gastric digestion.

Three heat-treated nutritional compositions were prepared. Two of the compositions contained > 85% BLG (beverages a and B) and a traditional WPI beverage (beverage C).

A beverage was produced according to example 4 and heat treated according to example 5.

Table 8 lists the ingredients of the protein powder used to prepare the exemplary beverages.

Table 8: composition of protein component (BDL ═ below detection limit).

Beverage A: a 6 w/w% BLG solution was prepared by dissolving a protein powder containing 98.2% BLG (table 8). The pH was adjusted to pH 7.0 using 10% NaOH and sterilized by UHT treatment at 143 ℃ for 6 seconds to produce BLG beverage solution a.

Beverage B: a 6 w/w% BLG solution was prepared by dissolving a powder containing 95.9% BLG (table 8). The pH was adjusted to pH 6.0 using 10% NaOH and heat treated at 86 ℃ for 18 minutes to produce BLG beverage solution B.

Beverage C: by dissolving protein powder containing 61% BLGThe final "WPI" was used to prepare 6 w/w% WPI beverage (Table 8). Adjusting the pH to pH 7.0 with 10% NaOH and heat treating at 143 deg.C for 6 seconds; beverage C was used as reference.

Beverages a and C were transparent, while beverage B was opaque and milky.

The type and amount of insoluble protein material, soluble aggregates, protein nanogels and native whey proteins present in the beverage were determined as described in example 1.32 and the results are shown in figure 3.

Results-soluble aggregates, protein nanogels, insoluble proteinaceous material and native whey protein:

it was found that BLG beverage (a) and WPI beverage (C) produced clear beverages, contrary to the creamy appearance of BLG beverage (B). The composition of the aggregates was evaluated by the method described in example 1.32, as shown in fig. 3. Figure 3 shows that beverage a (BLG pH 7.0) and C (WPI reference, pH 7.0) contain mainly soluble whey protein aggregates (67% and 44%, respectively), whereas beverage B (BLG pH 6.0) contains mainly protein nanogels (74%). In all three beverages, the insoluble protein material content was less than 1%.

Residual native protein was found at about 28-33% in both blg (a) and wpi (c) beverages, probably due to an incomplete aggregation process, whereas 13% of native protein was retained in blg (b). Thus, the beverage composition contains different protein structures, which may lead to differences in its digestion.

A fourth beverage (beverage D) was prepared by mixing 1 volume of heat-treated solution a with 1 volume of heat-treated solution B. Beverage D was found to contain 45% protein nanogel, 19% soluble whey protein aggregates and 37% native protein.

Method of simulating gastric digestion:

to explore the structure formation of different whey protein beverages during gastric digestion, we performed in vitro simulations of gastric digestion.

The samples were subjected to simulated oral and gastric digestion according to the protocol previously described in Mulet-Cabero, a. -i., Mackie, a.r., Wilde, p.j., Fenelon, M.A. & Brodkorb, a. (2019). Process-induced changes in cow's milk affect the structural mechanism and kinetics of gastric digestion in vitro. The food hydrocolloid, pages 86, 172-183, is modified slightly below.

Before digestion, samples (30g) were mixed with a solution consisting of buffer salts at 37 ℃ in the same amounts and concentrations as were subsequently used in the actual simulated digestion. The mixture was titrated to pH 2.0 with 0.1M HCl using pH Stat (Metrohm 602pH Stat), which is necessary to determine the actual buffering capacity of the food product and requires programming of the titrator to perform the actual digestion.

For the actual digestion, the sample (30) g was mixed with human model saliva (containing no salivary amylase because no starch is present in the beverage) at 37 ℃ for 2 minutes, the amount of model saliva (1.65g) being determined from the solids content (6%) of the sample.

The sample was transferred to a thermostatted reaction vessel for the gastric digestion process, which initially contained 10% of the total acid and gastric juice salts and simulated the fasted state of the stomach. The rate of addition of the remaining 90% of the buffer salt, 0.1M HCl and water should be such that the addition is complete after 105 minutes, which is the calculated duration of the gastric digestion process. The pepsin solution (0.5ml, 254400U/ml) was added using a syringe pump at 4.762 μ l/min so that all the pepsin solution was added before the 105 min digestion was complete. The solution was added near the vessel wall to simulate the secretion of the gastric mucosal surface. The pH of the mixture was monitored using a Metrohm monotrode (6.0258.010) equipped with an integrated PT100 thermometer mounted vertically and centrally in a digestion vessel placed in a rotating oscillating mixer (15rpm, ± 5 relative to horizontal) allowing gentle mixing of the samples^O) Is located in the center of (1). During digestion (17.5, 35, 52.5, 70, 87.5 and 105 minutes, respectively), six samples were taken from the bottom of the digestion vessel with a 4mm bore sampling pipette.

The samples were photographed in glass petri dishes on a black background using a cell phone (Samsung Galaxy S8+) under ambient light at a resolution of 4032 x 3024 pixels at a height of about 15 centimeters.

SDS PAGE samples were analyzed on 4-12% gradient gels (Bolt Gel, Invitrogen) under reducing conditions using the manufacturer's protocol (constant voltage, 200V, 22 min). A sample lane (The sample lanes) was loaded with standard (Invitrogen Mark 12). After running the gel, the gel was fixed in an acidic solution (50% water, 40% methanol, 10% acetic acid) for 2 hours, washed with water (3 × 5 min, 100ml water each), and then stained overnight (50ml Simply Blue, Invitrogen).

The gels were imaged using a Chemi Doc XRS system (Bio-Rad).

The results are shown in fig. 4 and 5. Fig. 4 illustrates semi-dynamic in vitro digestion of beverages A, B and c (wpi), while fig. 5 illustrates at the top: SDS-PAGE analysis of protein aliquots extracted at selected time points (17.5-105 min) during semi-dynamic in vitro digestion of samples. The pH values at different time points of the digestion study are shown at the bottom.

Results-simulated gastric digestion:

unexpectedly, despite the high ALA and CMP content of the WPI beverages, very similar visual protein coagulation behaviour was observed throughout the digestion of beverage a (BLG at pH 7.0) and C (WPI at pH 7.0). For both beverages (a and C), a protein clot has been observed at 17.5 minutes, likely due to initial mixing with acid and gastric juice salt, and the amount of clot increased at 35-52.5 minutes to form an opaque liquid (see fig. 4). The latter time points correspond to a pH range of about 5.6 to 4.2, with visible protein clots/agglomerates found up to 70 minutes (see fig. 5). It was further found that coagulation/agglomeration was accompanied by an increase in viscosity. When more pepsin and gastric juice salts are added, thereby further effectively lowering the pH, the digestion becomes transparent, the viscosity decreases and the protein clot gradually disappears.

However, it was surprisingly found that beverage B (BLG at pH 6.0) remained opaque throughout the digestion and contained no visible protein clumps at 35-52.5 minutes, and that the viscosity of beverage B was only slightly higher during this stage of digestion (fig. 4).

Figure 5 shows that in the early stages of digestion (17.5 min), the protein band between the 14kDa and 21kDa markers (corresponding to BLG) did predominate in beverage a, whereas multiple bands were present in WPI (beverage C).

In addition to the main BLG band, drink B also contained a band near the 37kDa marker band, which likely corresponded to BLG dimer. Both beverages a and c (wpi) exhibited protein bands with molecular weights below that of BLG as digestion time increased (as well as the continuous addition of gastric juice salts and pepsin).

Unexpectedly, the intensity of these low molecular weight protein/polypeptide bands in beverage a and beverage c (wpi) was significantly reduced in beverage B. This is probably due to the enhanced resistance to pepsin hydrolysis of BLG, mainly present in the form of protein nanogels, compared to soluble aggregates. This discovery may allow beverage manufacturers to regulate the state of protein transfer from the gastrointestinal tract to the intestine in beverages by using beverages that contain predominantly soluble whey protein aggregates or protein nanogels or by using mixtures.

Gel strength after acidification:

the gel strength of beverages A, B and C was measured during the acidification process as described in example 1.11. The results are shown in FIG. 6.

Results-measurement of gel strength after acidification:

fig. 6 illustrates a simulation of gastric acidification. The gel strength was measured during acidification for three types of beverages, which mainly contained soluble aggregates (beverage a and c (wpi)) or protein nanogels (beverage B). Due to the lower BLG content and higher CMP and other protein content, the concentration of soluble whey protein aggregates in WPI is expected to decrease (table 8).

Although the digestion patterns of beverages a and C were similar (see fig. 4 and 5), it was surprisingly found that the viscosity of BLG beverage a increased significantly compared to the WPI sample when acidified. This is probably due to the high purity of the BLG incorporated in the aggregates and the much higher content in BLG compared to WPI where CMP and ALA are less prone to aggregation, see table 8.

Example 10: high protein beverage comprising BLG (protein nanogel)

A nutritional high protein beverage comprising 10 w/w%, 11 w/w%, 12 w/w%, 13 w/w%, 14 w/w%, 15 w/w% and 16 w/w% whey protein, wherein ≥ 95.9% w/w is BLG prepared from BLG powder A (shown in Table 9). The pH was adjusted to pH 6.0 with 3% w/w NaOH. The solution was heat treated in a water bath at 90 ℃ for 5 minutes as described in example 5. Thereafter, the sample was cooled in ice water and tempered to room temperature according to example 5.

Table 9: chemical composition of BLG isolate powder A

The different beverages were analyzed for viscosity (example 1.8), visual appearance of the image (example 1.9) and size (hydrodynamic diameter) and hydrodynamic diameter (example 1.33).

As a result:

the results are shown in FIGS. 7, 8 and 9. The chemical composition of the 16 w/w% BLG beverage heat-treated at 90 ℃ for 5 minutes is illustrated in table 10 below.

The inventors found that the viscosity of the BLG samples remained surprisingly very low even after heat treatment at 90 ℃ for 5 minutes at a protein concentration of 10% w/w, even higher up to at least 16% w/w protein, see fig. 7 and 8. This is completely unexpected. Comparable WPI samples will gel at 10 w/w% WPI pH 6.0 (see example 8 and figure 2).

Despite the high protein concentration during the heat aggregation, no precipitation or particles were observed in the beverage, which makes the nutritional composition particularly suitable for high protein beverage applications.

The hydrodynamic diameter of the protein particles was measured (example 1.33) as shown in figure 9. The measured hydrodynamic diameter of the protein particles was 185-323nm, indicating that the high protein beverage contained protein nanogel particles, which has been described by Phan-Xuan et al, 2014 (Phan-Xuan, t., Durand, d., Nicolai, t., Donato, L., Schmitt, c., & Bovetto, L. (2014)) as particles having a hydrodynamic radius of 100-300 nm. The thermally induced formation of the beta-lactoglobulin microgel is driven by the addition of calcium ions. Food hydrocolloids, 2012, 34, 227 and 235.

Table 10: chemical composition of 16 w/w% BLG beverage.

	w/w％
		Ca	0.025
K	0.008
		Mg	0.004
Na	0.038
		PH	6.04
PHO	BDL
		Protein	15.9

Example 11: high protein beverage at neutral pH

Nutritional high protein beverages containing 6 w/w%, 10 w/w% and 12 w/w% whey protein (of which 95.9% w/w is BLG) were prepared from BLG powder a (shown in table 9) to evaluate the stability and turbidity of the beverages. The pH was adjusted to pH 6.0 and 7.0 with 3% w/w NaOH or HCl. The solution was heat treated in a water bath at 90 ℃ for 5 minutes as described in example 5. Thereafter, the sample was cooled in ice water and tempered to room temperature according to example 5.

Different samples were analysed for viscosity (example 1.8), turbidity (example 1.7), colour (example 1.9) and amount of insoluble proteinaceous material (example 1.10).

The results are shown in table 10 below and fig. 10.

TABLE 10 Performance of high protein BLG beverages at pH 6.0 and pH 7.0. Gray cells are not defined because the sample gelled.

As a result:

clear beverage:

it was surprisingly found that when powder a (table 9) containing 0.1455 w/w% calcium was used, a stable colorless clear beverage could be produced at pH 7.0 even at high protein concentrations with a protein content of 10 w/w%. The inventors have carried out experiments to confirm that WPI based beverages will form gels under similar conditions.

Milky beverage:

it was found that a milky beverage (4.5cP) with very low viscosity could be produced even at a protein concentration of 12 w/w% protein at pH 6.0 after heat treatment at 90 ℃ for 5 minutes. There was no visible sign of particles or precipitation.

In contrast, we found that the corresponding WPI samples, having a protein content of 10 w/w%, contained only 57-61 w/w% BLG of the total protein gel, as shown in example 8.

Example 12: high protein beverage with lipid content at neutral pH of 25% and 50% of total energy content

The nutritional high protein beverage prepared from BLG powder A (as shown in Table 9) contained 3 w/w%, 6 w/w%, 10 w/w% and 12 w/w% whey protein, with 95.9% w/w being BLG. Lipids were added to make up 25% and 50% of the total energy content in order to assess the opportunity to prepare a nutritional beverage in the presence of fat.

The water and lipids were equilibrated in a water bath at 70 ℃. 0.2% Grindsted Citrem LR10 was dissolved in heated oil and then slowly mixed with preheated water. The solution was cooled to 60 ℃, the powder was added and stirred for 30-45 minutes to obtain a beverage composition.

The pH was adjusted to pH 6.0 or pH 7.0 with 3% NaOH or HCl. The solution was heat treated in a water bath at 90 ℃ for 5 minutes. Thereafter, the sample was cooled in ice water and tempered to room temperature according to example 5.

A homogenization step may also be included in the above steps if it is desired to obtain a homogeneous sample.

Different samples were analysed for viscosity (example 1.8), turbidity (example 1.7), colour (example 1.9) and amount of insoluble proteinaceous material (example 1.10).

The results are shown in Table 11 below.

Table 11. properties of the lipid-added milky BLG beverage.

As a result:

it has been found that stable high protein BLG beverages can be produced even with lipid content of 25% and 50% of the total energy content and still have very low viscosity.

When 50% of the total energy content is lipid, a 10 w/w% protein milk beverage (containing at least 85 w/w% BLG) with very low viscosity (5.5cP) can be produced at pH 7.0.

It was also found that at pH 6.0 a 12 w/w% beverage could be produced with a lipid content of 25% and 50% of the total energy content. They have a white milky appearance and a very low viscosity (7.8 cP). The turbidity was at least 11000 NTU. The beverage was stable and no insoluble protein material was observed after 5 minutes at 3000 g.

Example 13: high pH beverages

A nutritional beverage comprising BLG was produced at pH 8.0 to demonstrate its stability and appearance at high pH.

A nutritional high protein beverage containing 3 w/w% whey protein, at least 85 wt% of which is BLG, was prepared from BLG powders a and B (as shown in table 12).

Table 12: chemical composition of two BLG isolate powders.

The pH was adjusted to pH 8.0 with 3% NaOH. The solution was heat treated in a water bath at 90 ℃ for 5 min. Thereafter, the sample was cooled in ice water and tempered to room temperature according to example 5.

The results are shown in Table 13.

Table 13: pH 8.0 beverage properties.

As a result:

the results show that clear stable beverages with a pH of 8.0 can be prepared. These beverages are surprisingly colorless and transparent when powder a or powder B is used, and have very low viscosity and turbidity at pH 8.0.