阅读说明：本技术 酸性β-乳球蛋白饮料制品 (Acidic beta-lactoglobulin beverage product ) 是由 S·B·尼尔森 K·B·劳里德森 T·C·雅格 K·桑德加 G·德·穆拉·马西尔 H·贝特 于 2019-06-26 设计创作，主要内容包括：本发明涉及一种新型包装的热处理的饮料制品,其具有在2-4.7范围内的pH。此外,本发明进一步涉及一种生产包装的热处理的饮料制品的方法,还涉及该包装的热处理的饮料制品的不同用途。(The present invention relates to a novel packaged heat-treated beverage product having a pH in the range of 2-4.7. Furthermore, the present invention further 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 2-4.7, the beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is beta-lactoglobulin (BLG), and

-optionally, sweeteners, sugar polymers and/or flavouring agents.

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 sterile.

4. The packaged heat-treated beverage product of any one of the preceding claims, wherein the protein fraction of the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.11.

5. The packaged heat-treated beverage product of any one of the preceding claims, wherein the protein fraction of the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of less than 1.11.

6. The packaged heat-treated beverage product according to any one of the preceding claims, wherein the protein fraction has a degree of protein denaturation of at most 10%.

7. The packaged heat-treated beverage product according to any preceding claim, wherein the beverage product has a degree of protein denaturation of at most 10%.

8. A packaged heat-treated beverage product according to any preceding claim, wherein the beverage product has a pH in the range of 3.0-4.3.

9. Packaged heat-treated beverage product according to any one of the preceding claims, wherein the protein fraction of 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}*。

10. Packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product has a colour value Δ b, measured at room temperature, in the range-0.10 to +0.51 of the CIELAB colour scale, wherein,

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

11. 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 750 mM.

12. The packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product has a turbidity of at most 200 NTU.

13. The packaged heat-treated beverage product according to any preceding claim, wherein the beverage product has a turbidity in excess of 200 NTU.

14. Packaged heat-treated beverage product according to any one of the preceding claims, wherein the protein fraction contains at most 15% insolubles after centrifugation at 3000g for 5 minutes.

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

16. The packaged heat-treated beverage product according to any preceding claim, wherein the beverage product does not comprise any anti-coagulant.

17. Packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product comprises 4.0 to 35% w/w of the total amount of protein, preferably 4.0 to 30% w/w of the total amount of protein, relative to the weight of the beverage.

18. A packaged heat-treated beverage product according to any one of the preceding claims, wherein the beverage product further comprises carbohydrate, which is 0 to 95% of the total energy content of the product.

19. Packaged heat-treated beverage product according to any one of the preceding claims, wherein the lipid content of the beverage product is between 0 and 60% of the total product energy content.

20. 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%, even more preferably at most 6%, most preferably at most 4% relative to the total amount of protein of a standard whey protein concentrate from sweet whey.

21. The packaged heat-treated beverage product of any preceding claim, wherein the beverage product comprises BLG isolate.

22. A method of producing a packaged heat-treated beverage product having a pH of 2 to 4.7, the method comprising the steps of:

a) Providing a liquid solution comprising:

-2 to 45 wt% of total protein, of which at least 85% of the protein is BLG,

-optionally, sweeteners, sugar polymers and/or flavouring agents,

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

heat treating the liquid solution of step a) and/or the packaged liquid solution of step b), said heat treatment comprising at least pasteurization.

23. Use of a protein solution for controlling the turbidity of a heat-treated acidic beverage product, wherein the protein solution comprises 2 to 45% w/w total protein amount relative to the solution weight, wherein at least 85% w/w of the protein is BLG, and the pH of the heat-treated acidic beverage product is in the range of 2.0-4.7.

24. Use of a protein solution for controlling the astringency of a heat-treated acidic beverage product, wherein the protein solution comprises 2 to 45% w/w total protein, wherein at least 85% w/w of the protein is BLG, relative to the weight of the solution, and the pH of the heat-treated acidic beverage product is in the range of 2.0-4.7.

25. A packaged heat-treated beverage product according to any one of claims 1 to 21, 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 1-21 as a dietary supplement.

27. Use of the packaged heat-treated beverage product of claim 26, 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 2-4.7. 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.

Background

Nutritional supplements containing whey protein are commonly used for muscle synthesis, weight control, and maintenance of muscle and body weight. Nutritional supplements are directed to different types of consumers, such as male/female sports enthusiasts, professional athletes, children, elderly people and patients who are malnourished or at risk of malnourishment and/or have an increased protein demand.

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.

Beverages comprising whey proteins are well known. For example, a heat-treated acidic beverage comprising whey protein.

Etzel 2004(Etzel, 2004, manufacture and use of a protein fraction of a dairy product. the American society for Nutrition science, pp. 996-1002) describes a beverage comprising 2.5 wt% WPI at a pH of 2-7. They found that only after the addition of an anticoagulant a heat-treated beverage could be obtained.

WO 2018/115,520a1 discloses a method based on crystallization of BLG in a salting-in mode (salting-in mode) to produce an edible isolated beta-lactoglobulin composition and/or a composition comprising crystallized beta-lactoglobulin. The BLG crystals may then be separated from the remaining mother liquor.

WO 2004/049,819a2 discloses a method for improving the functional properties of globular proteins comprising the steps of: providing a solution of one or more globular proteins, the proteins in the solution at least partially aggregating in fibrils; and then performing one or more of the following steps in a random order: the pH is increased; increasing the salt concentration; concentrating the solution; the mass of solvent in the solution was varied. Preferably, the solution of one or more globular proteins is provided by heating at low pH or by addition of a denaturant. Also disclosed are the protein additive thus obtained, its use in food and non-food applications and food or non-food products containing the protein additive.

WO 2014/055,830a1 discloses a shelf-stable transparent liquid nutritional composition having a pH of 2.5 to 4.6 and comprising: water; at least one source of EGCg sufficient to provide an amount of EGCg of 200-1700 mg/L; and at least one source of protein sufficient to provide a total amount of protein of 25-45 g/L. The shelf-stable, clear liquid nutritional composition loses no more than 20% by weight of the solids content of EGCg present in an initial preparation of the composition during heat sterilization due to epimerization, degradation, or both epimerization and degradation. In a particular embodiment, the loss of EGCg is characterized by the amount of epimer product GCg present in the shelf-stable, clear liquid nutritional composition after heat sterilization. Also disclosed are methods of making the shelf-stable, clear liquid nutritional compositions.

WO 2011/112,695a1 discloses nutritional compositions and methods of making and using the nutritional compositions. The nutritional composition comprises whey protein micelles and leucine and provides leucine in an amount sufficient to improve protein synthesis in the human body, while the fluid matrix maintains a low viscosity and acceptable organoleptic properties.

WO 2011/051,436a1 discloses an at least partially transparent composition for human or animal consumption and a package for the composition. One embodiment of the present invention relates to an at least partially transparent container containing an at least partially transparent aqueous non-alcoholic composition. The container includes at least one polarizer (polarizer) to make the liquid crystal visible in the composition.

WO 2010/037,736a1 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 present invention has high alpha-lactalbumin and immunoglobulin G in addition to low beta-lactoglobulin.

FR 2296428 discloses protein compositions for dietary and therapeutic use based on whey (lactoserum) 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 to improve resistance to intestinal infections and to treat certain metabolic diseases (such as hyperphenylalaninemia). They may also be used for dermatological or cosmetic purposes and may form part of a low protein diet.

Disclosure of Invention

The inventors have observed that organoleptic properties such as astringency and mouthfeel play an important role in the selection of liquid nutritional beverages by consumers.

Some of the challenges of incorporating whey protein in heat-treated acidic beverages are: the unstable precipitates formed settle in the beverage, high viscosity and even gel formation, and unpleasant taste due to high astringency and/or dry mouthfeel (dry mouthfeel).

It is an object of the present invention to provide a packaged, heat-treated, whey protein-containing acidic beverage product having improved organoleptic and/or visual properties.

It is another object of the present invention to provide a high protein beverage which has a low viscosity, a pleasant taste, optionally also a low degree of astringency, and which may be transparent or opaque.

The present inventors have now found that such packaged heat-treated beverages can provide a broad acidic pH range, up to and including 4.7, while still having a low viscosity and, optionally, a low degree of astringency and dry mouthfeel. The present invention provides two beverages, a clear beverage and an opaque but stable beverage.

Accordingly, one aspect of the present invention relates to a packaged heat-treated beverage product having a pH in the range of 2-4.7, the beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, and

-optionally, a sweetener, a sugar polymer 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 in the range of 2-4.7, comprising the steps of:

a) Providing a liquid solution comprising:

-2 to 45 wt% of total protein, of which at least 85% of the protein is BLG,

-optionally, sweeteners, sugar polymers and/or flavouring agents,

b) packaging the liquid solution:

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

Yet another aspect of the invention relates to the use of a protein solution comprising 2 to 45% w/w total protein, wherein at least 85% w/w of the protein is BLG, relative to the weight of the solution, for controlling the turbidity of a heat-treated acidic beverage product having a pH in the range of 2.0-4.7.

Yet another aspect of the invention relates to the use of a protein solution comprising 2 to 45% w/w of the total amount of protein, wherein at least 85% w/w of the protein is BLG, relative to the weight of the solution, for controlling the astringency of a heat-treated acidic beverage product having a pH in the range of 2.0-4.7.

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

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

Drawings

Figure 1 shows a graph of a BLG and WPI beverage having a pH of 3.7 and a protein content of 6% w/w, heat treated at 120 ℃ for 20 seconds, or at 75 ℃ for 15 seconds.

FIG. 2 shows images of WPI-B at pH 3.0-3.7, 120 deg.C/20 sec and BLG at pH3.7, 120 deg.C/20 sec.

FIG. 3 shows images of WPI-B at pH 3.0-3.7, 75 deg.C/15 sec and BLG at pH3.7, 75 deg.C/15 sec.

Figure 4 shows images of WPI-B at pH3.7 and BLG at pH 3.9 (75 ℃/15 seconds).

Fig. 5 shows the turbidity of a 6% UHT treated (120 ℃/20 sec) BLG beverage product.

Fig. 6 shows the turbidity of a 6% pasteurized (75 ℃/15 sec) BLG beverage product.

Figure 7 shows the viscosity of a 6% UHT treated (120 ℃/20 sec) BLG beverage product.

Figure 8 shows the yellowness (b) of 6% UHT-treated (120 ℃/20 sec) beverage compositions.

Figure 9 shows the yellowness (b) of a 6% pasteurized (75 ℃/15 sec) beverage composition.

Figure 10 shows images of 15% BLG beverage at ph3.7 (left) and 6% WPI-a at ph3.7 (right) (75 ℃/15 s).

Fig. 11 shows the results of sensory evaluation of high protein BLG beverage compositions and images of 6 w/w% and 15 w/w% BLG samples at ph 3.7.

Fig. 12 shows high protein beverage products of 30, 27.5, 25, 20% BLG beverage products prepared by heating at 75 ℃ for 5 minutes. The viscosity remains at a very low level even after heating.

Figure 13 shows images of different WPI and BLG beverage samples.

Figure 14 shows sensory evaluation of beverages (from 0 to 15 ratings). WPI pH was 3.0(120 ℃/20 sec) and BLG pH was 3.7(75 ℃/15 sec).

Figure 15 shows the effect of pH and temperature on the taste of protein beverages.

Fig. 16 shows sensory data for astringency of BLG beverages at pH 3.0(120 ℃/20 sec) and pH 3.7(75 ℃/15 sec).

Fig. 17 shows sensory data for dry mouthfeel of BLG beverages at pH 3.7 and heat treated at 120 ℃/20 seconds or 75 ℃/15 seconds.

Figure 18 shows sensory data recording the effect of heat treatment temperature on whey odor.

Figure 19 shows an image of a mineral enriched 6% BLG beverage heat treated at 95 ℃ for 5 minutes, pH 3.7.

Figure 20 shows an image of a mineral enriched 6% BLG beverage heat treated at 75 ℃ for 5 minutes, pH 3.7.

Figure 21 shows the stability of a milky BLG beverage heat treated at 93 ℃ for 4 minutes with and without sucrose addition, at a pH of 4.3.

Fig. 22 shows an image of an opaque 6% protein BLG beverage prepared by heating at pH 4.2 or 4-5 at 75 ℃ for 5 minutes.

Figure 23 shows images of BLG and SPI beverages heat treated at 75 ℃ for 5 minutes at pH 3.7.

Figure 24 shows images of BLG and SPI beverages at pH 3.7.

Figure 25 shows yellowness (b) of beverage compositions (pH 3.0 and 3.7) containing 6 w/w% BLG and UHT treated (120 ℃/20s) after storage in the dark (20 ℃) for up to 6 months.

Figure 26 shows yellowness (b) of beverage compositions (pH 3.0 and 3.7) containing 6 w/w% BLG and pasteurized (75 ℃/15s) after storage in the dark (20 ℃) for up to 6 months.

Detailed Description

Definition of

In the context of the present invention, the term "beta-lactoglobulin" or "BLG" relates to beta-lactoglobulin obtained from a mammalian species, which is present in, for example, native, expanded and/or glycosylated form and comprises naturally occurring gene variants. The term further includes aggregated BLG, precipitated BLG and crystallized BLG. When referring to the amount of BLG, it refers to the total amount of BLG, which comprises BLG aggregates. The total amount of BLG was determined according to example 1.31. The term "BLG aggregate" refers to BLG as follows: the BLG is at least partially unfolded and further aggregates with other denatured BLG molecules and/or other denatured whey proteins, typically by means of hydrophobic interactions and/or covalent bonds.

BLG is the most predominant protein in bovine whey and whey, and there are several gene variants, the predominant variants in cows being labeled a and B. BLG is a lipoprotein and binds to a variety of hydrophobic molecules, representing roles in their transport. BLG has also been shown to bind iron via siderophores and may play a role in combating pathogens. Human breast milk lacks homologs of BLG (homologue).

Bovine BLG is a relatively small protein comprising 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 at pH below about 3, retaining its native state, as measured using nmr spectroscopy. However, BLG can also exist as tetramers, octamers and other multimers 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 obtained from a mammalian species, which is present in, for example, native, expanded and/or glycosylated form and comprises naturally occurring gene 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}100%, to determine the percentage of non-aggregated BLG relative to the total BLG. m is_{Total BLG}Concentration or content of BLG determined according to example 1.31, m_{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" relates to a solid material whose constituents (for example atoms, molecules or ions) are arranged in a highly ordered microstructure, forming a lattice extending in all directions.

In the context of the present invention, the term "BLG crystals" relates to protein crystals, which mainly comprise non-aggregated BLG (preferably native BLG) arranged in a highly ordered microstructure, forming a lattice extending in all directions. The BLG crystal may be, for example, monolithic or polycrystalline, and may also be, for example, a whole crystal, a crystal fragment, or a combination thereof. The crystal fragments are formed, for example, when the intact crystals are subjected to mechanical shear during processing. The crystal fragments also have a highly ordered microstructure of crystals, but may lack uniform surfaces and/or edges or corners of intact crystals. Examples of large numbers of intact BLG crystals are found in, for example, fig. 18 of PCT application No. PCT/EP2017/084553, and examples of BLG crystal fragments are found in fig. 13 of PCT application No. PCT/EP 2017/084553. In both cases, the BLG crystal and crystal fragments can be visually identified using an optical microscope, which is well defined, compact, and closely ordered. 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 appear as open or porous masses with unclear boundaries, opacity, and irregular size.

In the context of the present invention, the term "crystallization" relates to the formation of protein crystals. Crystallization may be initiated, for example, by spontaneous occurrence or by addition of crystallization seeds.

In the context of the present invention, the term "edible composition" relates to compositions which are safe for human consumption and use as food ingredients and which do not contain problematic amounts of toxic components, such as toluene or other harmful 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 in native and/or glycosylated form, and encompasses naturally occurring gene variants. The term also encompasses aggregated ALA and precipitated BLG. When referring to ALA content, it refers to the total amount of ALA comprising e.g. ALA aggregates. The total ALA content was determined according to example 1.31. The term "aggregated ALA" relates to ALA as follows: the ALA is typically at least partially unfolded and it further typically aggregates with other denatured ALA molecules and/or other denatured whey proteins by means of 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, and β -1, 4-galactosyltransferase (β 4Gal-T1) forms the catalytic component. Together, these proteins make LS available for lactose production by transfer of galactose moieties to glucose. One of the main structural differences of β -lactoglobulin is that ALA does not have any free thiol groups that can serve as a starting point for the covalent aggregation reaction.

In the context of the present invention, the term "non-aggregated ALA" also relates to ALA obtained from a mammalian species, which is present in e.g. native, expanded and/or glycosylated form and comprises naturally occurring gene 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}100% to determine non-aggregated ALA relative to total ALAPercentage (D). m is_{Total ALA}Is the ALA concentration or content determined according to example 1.31, m_{Non-aggregating ALA}Is the non-aggregated ALA concentration or content determined according to example 1.6.

In the context of the present invention, the terms "caseinomacropeptide" or "CMP" relate to the hydrophilic peptide of residue 106-169, "kappa-CN" or "kappa-casein" derived from the hydrolysis of mammalian species by aspartic proteases (e.g.chymosin), e.g.in native and/or glycosylated form, including naturally occurring gene 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 the total amount of protein. The BLG isolate preferably has a total amount of protein of at least 30% w/w, and preferably at least 80% w/w, relative to the total amount of solids.

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

In the context of the present invention, "liquid BLG isolate" refers to a BLG isolate in liquid form and is preferably an aqueous solution.

The term "whey" relates to the liquid phase remaining after 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 based on rennet precipitation of casein, and "sour whey" which is a whey product produced based on acid precipitation of casein. Acid-based precipitation of casein may be achieved by means such as addition of food acids or bacterial culture.

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

The term "whey protein" or "serum protein" refers to the proteins present in whey.

In the context of the present invention, the term "whey protein" refers to the protein found in whey or milk serum. The 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 the protein species found in whey and/or 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:

18% w/w ALA relative to the total amount of protein,

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

4% w/w BSA relative to the total amount of protein,

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

6% w/w of immunoglobulin relative to the total amount of protein,

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

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

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

In the context of the present invention, the term "mother liquor" refers 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 contains small BLG crystals that escape separation.

In the context of the present invention, the term "casein" refers to casein found in milk, including two natural micellar caseins found in raw milk, a single casein species and caseinate.

In the context of the present invention, a liquid that is "supersaturated" or "supersaturated with BLG" means that the concentration of dissolved, unaggregated BLG 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 from solution in molecular systems: phase diagrams, supersaturation and other basic concepts"; review of the chemical society, p. 2286, 2300, p. 7, 2014). The supersaturation may be determined by a number of different measurement techniques (e.g., by spectroscopy or particle size analysis). In the context of the present invention, the supersaturation of BLG is determined by the following method.

Method for testing the supersaturation of BLG in a liquid under specific conditions:

a) 50ml of the liquid sample to be tested was transferred into a centrifuge tube (VWR catalog No. 525-. It should be noted that the sample and subsequent fractions should be kept under the original physical and chemical conditions of the liquid in steps a) -h).

b) The samples were immediately centrifuged at 3000g for 3 minutes, accelerated up to 30 seconds and decelerated up to 30 seconds.

c) Immediately after centrifugation as much supernatant as possible (without disturbing the pellet if it has formed) is transferred to a second centrifuge tube (same size as in step a).

d) 0.05ml of a subsample (subsample A) was extracted from the supernatant

e) 10mg of BLG crystals having a particle size of at most 200 μm (non-aggregated BLG having a purity of at least 98% relative to the total solids) are added to a second centrifuge tube and the mixture is stirred.

f) The second centrifuge tube was allowed to stand at the original temperature for 60 minutes.

g) Step f) was immediately followed by centrifuging the second centrifuge tube at 500g for 10 minutes and extracting another 0.05ml of the subsample (subsample B) from the supernatant.

h) If there is a centrifugal precipitate in step g), it is recovered, resuspended in milliQ water and the suspension is immediately observed for the presence of crystals observable by microscopy.

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% w/w of BLG relative to the total weight of the subsample. Concentration of non-aggregated BLG in subsample A as C_BLG，ADenotes, of subsample BConcentration of non-aggregated BLG as C_BLG，BAnd (4) showing.

j) If c is_BLG，BLower than c_BLG，AAnd if 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 compositions that are free of particulate matter, and also include compositions comprising a 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) refer to dry or aqueous compositions comprising a total amount of protein of 20-89% w/w 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.

Alternatively, 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 of total protein, relative to 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. WPC or SPC comprises

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

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

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

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

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

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

The WPI or SPI preferably comprises:

90-100% w/w of the total amount of protein, relative to the total amount of 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.

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

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

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

4-35% w/w ALA, relative to the total amount of 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 of the total amount of protein, relative to the total amount of solids,

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

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

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

SPI typically contains no CMP or only trace amounts of 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 include more than one non-BLG protein present in whey 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 terms "consisting essentially of …" and "consisting essentially of …" mean that the claims or features in question encompass the materials or steps specified, as well as those 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". With the same logic, the phrase "n₁、n₂、…、n_i-1And/or n_i"means" n₁"or" n₂"OR … or" n_i-1"or" n_i"or component n₁、n₂、…、n_i-1And n_iAny combination of (a): .

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 or more preferably even less.

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 number of viable microorganisms of the composition. The term does not include the addition of chemicals that kill microorganisms. Furthermore, the term does not include the thermal exposure of the atomized droplets of liquid during spray drying, but includes possible preheating prior to spray drying.

In the context of the present invention, the pH of the powder is the pH after 10g of powder have been mixed with 90g of demineralized water (demineralized water) and is measured according to example 1.16.

In the context of the present invention, unless another reference (e.g. total solids or total protein) is specifically mentioned, the weight percentage (% w/w) of a component of a certain composition, product or material refers to the weight percentage of that component relative to the weight of the particular composition, product or material.

In the context of the present invention, the process steps "concentration" and the verb "concentration" refer to the concentration of the protein and include protein concentration on the basis of the total amount of solids and protein concentration on the basis of the total weight. This means that e.g. concentration does not necessarily require an increase in the absolute protein concentration w/w of the composition, as long as the protein content is increased relative to the total solids.

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 terms "at least pasteurized" and "at least pasteurized" refer to a heat treatment with a microbicidal effect at a temperature equal to or higher than 70 ℃ for 10 seconds. References for determining the bacterial killing effect are escherichia coli O157: H7.

in the context of the present invention, the term "whey protein material (feed)" refers to a whey protein source of liquid BLG isolate. The whey protein material, typically WPC, WPI, SPC or SPI, has a lower content of BLG relative to the total amount of protein than 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 material. BLG-enriched compositions typically comprise the same whey protein as the whey protein material, but the BLG is present in the composition in a significantly higher concentration (relative to the total amount of protein) than the whey protein material. BLG-enriched compositions may be prepared from whey protein materials by chromatography, protein crystallization and/or membrane-based protein fractionation (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 whose BLG is supersaturated in the salt-soluble mode 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.

When a liquid, such as a beverage product, is sterilized and aseptically packaged in sterile containers, it typically has a shelf life of at least 6 months at room temperature. The sterilization process may kill spores and microorganisms that cause spoilage of the liquid.

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

The total energy content of a food product comprises the energy contributions of all the macronutrients present in the food product, such as energy from proteins, lipids and carbohydrates. The energy distribution from 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, a beverage comprises 20E% protein, 50E% carbohydrate and 30E% lipid, which 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 beverage has nutritional ingredients that are matched to a complete and healthy diet.

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

The term "Food for Special Medical Purposes (FSMP)" or "medical food" is a food for oral or tube feeding, which is used for a specific medical disorder, disease or condition having special nutritional requirements and used under medical supervision. The medical food may be a nutritionally complete or an nutritionally incomplete supplement/beverage.

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 such as vitamins, minerals and trace elements.

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 such as vitamins, minerals and trace elements.

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

In the context of the present invention, the terms "beverage", "beverage product" and "product" used as noun-tial wording refer to any water-based liquid that can be ingested as a beverage, for example. By pouring, sip or tube feeding.

In the context of the present invention, the term "protein fraction" relates to the protein of the composition in question, for example of 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 salivary secretion. Therefore, astringency is not a taste per se, but a physical mouth feel in the mouth and a time-dependent feeling.

In the context of the present invention, the term "dry mouthfeel" relates to the perception in the oral cavity that feels like dryness of the oral cavity and teeth and results in a minimization of salivary secretions.

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

In the context of the present invention, the term "mineral" as used herein, unless otherwise indicated, refers to any of a macro mineral, a trace or micro mineral, other minerals, and combinations thereof. The macrominerals include: calcium, phosphorus, potassium, sulfur, sodium, chlorine, magnesium. Trace or trace minerals include: iron, cobalt, copper, zinc, molybdenum, iodine, selenium and manganese. Other minerals include: 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 or obtained by processing a plant or animal. These terms also include synthetic lipid materials, so long as the synthetic material is suitable for human consumption.

In the context of the present invention, the term "transparent" includes beverage products that have a visually clear appearance and allow light to pass through and through to appear as distinct images. The turbidity of the clear beverage was at most 200 NTU.

In the context of the present invention, the term "opaque" includes beverage products having a visually non-clear appearance and a turbidity of greater than 200 NTU.

One aspect of the present invention relates to a packaged heat-treated beverage product having a pH in the range of 2.0 to 4.7, said beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, and

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

Packaged heat-treated beverage products comprising at least 85% w/w protein are highly beneficial for a variety of reasons. High BLG content in acidic beverages can also increase the pH range and lower the heating temperature while still maintaining clarity and being colorless, which can be achieved even at high protein concentrations. This means that the clear, colourless, high protein beverage can be produced at a much weaker acidic pH level than is produced using conventional WPI.

It was surprisingly found that the astringency, dry mouthfeel, sourness, whey smell and citric acidity of BLG beverages were lower compared to the traditional acidic WPI beverage.

Another advantage and extended pH range of the present invention is that milk beverages can be produced at high turbidity, low viscosity while still being white, not yellowing and still stable.

In some preferred embodiments of the packaged heat-treated beverage product of the present 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.

It is also possible and desirable that the relative content of BLG is even higher, so 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 has a BLG content of at least 97.5% w/w, preferably at least 98% 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, such as about 100.0% w/w, relative to the total amount of protein.

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 sterilized and is therefore aseptic.

In some preferred embodiments of the invention, the native configuration of the protein is maintained. Preferably, the protein is maintained in its native configuration by using a heat treatment that does not cause at least irreversible changes in the protein configuration of the BLG (and preferably does not cause all proteins).

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

Intrinsic tryptophan fluorescence emission ratio (intrinsic tryptophan fluorescence emission ratio), R-I330/I350, is a measure of protein naturalness. When R is at least 1.11, the natural configuration predominates, whereas when R is less than 1.11, at least partial unfolding and aggregation predominates. The method of analyzing intrinsic tryptophan fluorescence is described in example 1.1.

The inventors have found that heat-treated high protein beverages having an intrinsic tryptophan fluorescence emission ratio R ═ I330/I350 of at least 1.11 can be obtained while still having a low viscosity and being transparent. This is possible even when the protein fraction and/or the beverage product is subjected to a heat treatment corresponding to pasteurization, for example to a temperature below 90 ℃.

Thus, in some preferred embodiments of the invention, the protein fraction of the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.11, thus indicating that the protein is in a native state.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product 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.

It is particularly preferred that the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product or of the beverage product itself is at least 1.15, more preferably at least 1.16, even more preferably at least 1.17.

In some preferred embodiments of the invention, the packaged heat-treated beverage product comprising the protein fraction and optionally other ingredients (e.g., lipids, carbohydrates, vitamins, minerals, food acids, or emulsifiers) has a tryptophan fluorescence emission ratio of at least 1.11.

Thus, in some preferred embodiments of the present invention, the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.11.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the heat-treated beverage product 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.

In some preferred embodiments of the invention, the protein is denatured or at least partially denatured.

Thus, in some preferred embodiments of the present invention, the protein fraction of the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of less than 1.11, thus indicating that the protein is at least partially unfolded and predominantly aggregated.

In some embodiments of the present invention, the heat-treated beverage product has an intrinsic tryptophan fluorescence emission (I330nm/I350nm) of less than 1.10, more preferably less than 1.08, even more preferably less than 1.05, and most preferably less than 1.00.

However, in other preferred embodiments of the present invention, the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the heat-treated beverage product is less than 1.15, more preferably less than 1.14, even more preferably less than 1.13, and most preferably less than 1.12.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the heat-treated beverage product is less than 1.15, more preferably less than 1.14, even more preferably less than 1.13, and most preferably less than 1.12.

The beverage product may optionally contain other food additives in addition to the protein fraction, such as lipids, carbohydrates, vitamins, minerals, food acids or emulsifiers and the like. In some preferred embodiments of the invention, the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of less than 1.11, thus indicating that the protein is at least partially unfolded and predominantly aggregated.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the heat-treated beverage product is less than 1.10, more preferably less than 1.08, even more preferably less than 1.05, and most preferably less than 1.00.

Protein denaturation can also be described by another assay method than tryptophan fluorescence. This process is described in example 1.3.

In some preferred embodiments of the invention, the protein denaturation degree of the protein fraction of the packaged heat-treated beverage product is at most 10%. Preferably the degree of protein denaturation is at most 8%, more preferably at most 5%, even more preferably at most 3%, even more preferably at most 1%, most preferably at most 0.5%.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product has a protein denaturation degree of at most 10%. Preferably the degree of protein denaturation is at most 8%, more preferably at most 5%, even more preferably at most 3%, even more preferably at most 1%, most preferably at most 0.5%.

In some embodiments of the invention, when the protein fraction and/or beverage product has been subjected to e.g. a high temperature heat treatment, then the protein denatures more than 10%, preferably more than 20%, preferably more than 30%, preferably more than 40%, or preferably more than 50%, or preferably more than 70% or more, or preferably more than 80%, or preferably more than 90%, or preferably more than 95%, or preferably more than 99%.

For example, the protein denaturation degree of the protein fraction of the beverage product may be greater than 10%, preferably greater than 20%, more preferably greater than 30%, even more preferably greater than 40%, most preferably greater than 50%. Even higher degrees of denaturation may also be preferred, and thus the protein denaturation degree of the protein fraction of the beverage product may be more than 70%, preferably more than 80%, more preferably more than 90%, even more preferably more than 95%, most preferably more than 99%.

In some preferred embodiments of the present invention, the pH of the packaged heat-treated beverage product is in the range of 3.0 to 4.3. These pH ranges are particularly preferred for producing clear beverages with low viscosity and improved taste.

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 ensured that the pH could be raised during heat treatment, which improved the visual effect (colour and turbidity) and viscosity compared to heat treated WPI beverages. The present invention thus raises the pH range, in particular the upper pH limit, making it possible to heat treat acidic whey protein beverages at this upper pH limit and produce heat-treated acidic beverages of low viscosity and preferably also transparent.

It was surprisingly found that there is a significant difference between the sensory parameters of the BLG beverages of the present invention compared to beverages produced with WPI. It has been found that, surprisingly and advantageously, BLG beverages have a lower degree of astringency, dry mouthfeel, tartness, whey odor and tartness compared to WPI beverages. It has further been found that when the pH of an acidic beverage is raised, less sweetener is required to balance the tartness of the beverage and therefore a lower concentration of sweetener is required for such a beverage.

In some preferred embodiments of the invention, the pH of the packaged heat-treated beverage product is from 3.0 to 4.1, or preferably from 3.1 to 4.0, or preferably from 3.2 to 3.9, or preferably from 3.7 to 3.9, more preferably from 3.4 to 3.9, even more preferably from 3.5 to 3.9.

The pH of the packaged heat-treated beverage product may preferably be 3.7-4.3, more preferably 3.9-4.3, even more preferably 4.1-4.3.

Alternatively, but also preferably, the pH of the packaged heat-treated beverage product may be from 3.7 to 4.1, more preferably from 3.9 to 4.1.

These pH ranges are particularly important when the beverage product is to be pasteurized.

In some preferred embodiments of the invention, the pH of the packaged heat-treated beverage product is preferably from 3.0 to 3.9, or preferably from 3.2 to 3.7, or preferably from 3.4 to 3.6, or preferably from 3.5 to 3.7, or preferably from 3.4 to 3.6.

These pH ranges, in combination with high temperature treatments such as sterilization, are particularly suitable for producing clear beverages with low viscosity and improved taste.

In some preferred embodiments of the present invention, the pH of the packaged heat-treated beverage product is in the range of 4.1 to 4.7, which is particularly important for producing stable beverages having a milky appearance and high turbidity while still having a low viscosity. In some embodiments of the invention, the pH ranges from 4.2 to 4.6. In some other embodiments of the invention, the pH ranges from 4.2 to 4.5.

In some preferred embodiments of the invention the pH of the packaged heat-treated beverage product is in the range of 3.0 to 3.9 and the total amount of protein of the beverage product is 10 to 34% w/w, more preferably 12 to 30% w/w, even more preferably 15 to 25% w/w, relative to the weight of the beverage product.

In a further preferred embodiment of the invention the pH of the packaged heat-treated beverage product is in the range of 3.7-3.9 and the total amount of protein of the beverage product is 10-34% w/w, more preferably 12-30% w/w, even more preferably 15-25% w/w, relative to the weight of the beverage product.

In a further preferred embodiment of the present invention, the packaged heat-treated beverage product has the following characteristics:

-a pH in the range of 3.0 to 3.9, preferably 3.7 to 3.9,

-the total amount of protein is 10-34% w/w, more preferably 12-30% w/w, even more preferably 15-25% w/w, relative to the weight of the beverage product, and

intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

In a still further preferred embodiment of the present invention, the packaged heat-treated beverage product has the following characteristics:

-a pH in the range of 3.0 to 3.9, preferably 3.7 to 3.9,

-the total amount of protein is 10-34% w/w, more preferably 12-30% w/w, even more preferably 15-25% w/w, relative to the weight of the beverage product, and

-a degree of protein denaturation of at most 10%, preferably at most 5%, even more preferably at most 1%.

For both clear and opaque beverages, the visual appearance of the beverage product is important to the consumer. In particular for clear aqueous beverages or white creamy beverages, the inventors have found that it is advantageous to be able to control the color of the beverage (rather than to control the beverage to be non-colored).

However, even if a dedicated colouring agent is added during the production of the beverage, the inventors have found that it is advantageous to be able to avoid an additional colour source to avoid causing an undesirable change in the visual appearance of the beverage. The inventors found that the high BLG protein case described herein is more neutral/colorless in color and has less color change than conventional WPI. Conventional WPI has a yellowish appearance, which can be somewhat attenuated by the addition of oxidizing agents (e.g., bleach). However, the addition of an oxidizing agent is generally not desirable, and for the present invention, an oxidizing agent is not even required.

The CIELAB color scale was used to determine the color of the beverage as described in example 1.9. As an example, a positive Δ (delta) b value indicates that the color is more yellow than demineralized water, while a negative Δ b value indicates that the beverage is more blue than demineralized water. Therefore, consumers generally want the Δ b value to be close to 0 so that the beverage is neither yellow nor blue.

In some preferred embodiments of the invention, the packaged heat-treated beverage product has a color value Δ b on the CIELAB scale in the range of-0.10 to +0.51, in particular 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 on the CIELAB scale in the range of 0.0 to 0.40, preferably 0.10 to 0.25.

For opaque beverage products, for example, a packaged heat-treated beverage product having a turbidity of greater than 200NTU and preferably greater than 1000NTU preferably has a color value Δ b at the CIELAB scale in the range of-6 to-1.7, preferably in the range of-5.0 to-2.0.

In a further preferred embodiment of the invention the turbidity of the beverage product is higher than 200NTU, preferably higher than 1000NTU and the color value ab of the beverage product on the CIELAB scale is-10 to-0.5, more preferably-9 to-1.0.

In some preferred embodiments of the invention, the color value ab of the protein fraction of the packaged heat-treated beverage product is in the range of-0.10 to +0.51, 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 containing WPI with higher Δ b values and more yellow colour.

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

The a-value indicates the green-red component, green in the negative direction and red in the positive direction. In order to obtain a beverage that is neither red nor green, it is generally preferred that the color value Δ a is around zero.

It is generally preferred that the protein fraction of the packaged heat-treated beverage product has a Δ a on the CIELAB scale in the range of-0.2 to 0.2, particularly if the product has a turbidity of at most 200NTU, and more preferably at most 40 NTU. Preferably, the packaged heat-treated beverage product has a color value Δ a on the CIELAB color scale in the range of-0.15 to 0.15, preferably in the range of-0.10 to 0.10.

The present inventors have found that it is advantageous to control the mineral content of packaged heat-treated beverage products to achieve certain desired characteristics.

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.

The present inventors have surprisingly found that when using a BLG isolate as defined herein and in example 2, heat treated beverage products with high mineral concentrations can be produced without compromising viscosity. This offers the following possibilities: packaged heat-treated beverage products having high mineral content can be produced, and nutritionally complete or nutritionally incomplete supplement beverages can be produced.

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 750mM, preferably in the range of 100-600mM, or preferably in the range of 200-500 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 750 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 600mM, preferably at most 500mM, or preferably at most 400mM, or preferably at most 300mM, or preferably at most 200mM, preferably at most 170mM, most preferably at most 150mM, or preferably at most 130mM, 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 another exemplary embodiment, 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 some preferred embodiments of the invention, the packaged heat-treated beverage product comprises at most 150mM KCl and at most 150mM CaCl₂Or the packaged heat-treated beverage product comprises at most 130mM KCl and at most 130mM CaCl₂Or the packaged heat-treated beverage product comprises at most 110mM KCl and at most 110mM CaCl₂Or the packaged heat-treated beverage product comprises at most 100mM KCl and at most 100mM CaCl₂Or preferably the packaged heat-treated beverage product comprises at most 80mM KCl and at most 80mM CaCl₂Or, preferably, the packaged heat-treated beverage product comprises at most 50mM KCl and at most 50mM CaCl₂Or preferably, the packaged heat-treated beverage product comprises at most 40mM KCl and at most 40mM CaCl₂。

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 compositions, such as liquids, beverages, powders or other food products, having at least one, preferably two, even more preferably all of the following:

-at most 1.2% w/w ash relative to the total solids,

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

-a total content of sodium and potassium of at most 0.1% w/w relative to the total solids,

-a total phosphorus amount 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:

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

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

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

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

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

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

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

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

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

Particularly preferably, the low mineral composition has the following:

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

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

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

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

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 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 products are preferably low phosphorous and low potassium beverage products.

In the context of the present invention, the term "low phosphorus" refers to a composition, such as a liquid, powder or other food product, having a total content of phosphorus of at most 100mg phosphorus per 100g protein. Preferably, the total phosphorus content of the low phosphorus composition is at most 80mg phosphorus per 100g protein. More preferably, the total phosphorus content of the low phosphorus composition is at most 50mg phosphorus per 100g protein. Even more preferably, the total content of phosphorus in the low-phosphorus composition is at most 20mg of phosphorus per 100g of protein. Even more preferably, the total content of phosphorus in the low-phosphorus composition is 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 phosphorus per 100g protein. Preferably, the packaged heat-treated beverage product comprises at most 30mg phosphorus per 100g protein. More preferably, the packaged heat-treated beverage product comprises at most 20mg phosphorus per 100g protein. Even more preferably, the packaged heat-treated beverage product comprises at most 10mg phosphorus per 100g protein. Most preferably, the packaged heat-treated beverage product comprises at most 5mg phosphorus 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.

In the context of the present invention, the term "low potassium" refers to a composition, such as a liquid, powder or other food product, having a total content of potassium of at most 700mg potassium per 100g protein. Preferably, the total potassium content of the low-phosphorous composition is at most 600mg potassium per 100g protein. More preferably, the total potassium content of the low potassium composition is at most 500mg potassium per 100g protein. More preferably, the total potassium content of the low potassium composition is at most 400mg potassium per 100g protein. More preferably, the total potassium content of the low potassium composition is at most 300mg potassium per 100g protein. Even more preferably, the total potassium content in the low potassium composition is at most 200mg potassium per 100g protein. Even more preferably, the total potassium content in the low potassium composition is at most 100mg potassium per 100g protein. Even more preferably, the total potassium content of the low potassium composition is at most 50mg potassium per 100g protein, even more preferably, the total potassium content of the low potassium composition is at most 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, 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 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.

Heat-treated beverage products comprising low amounts of phosphorus and potassium may advantageously be supplemented with carbohydrates and lipids, the heat-treated beverage products preferably further comprising carbohydrates and lipids, the total amount of said carbohydrates being 30-60%, preferably 35-50E% of the total energy content of the beverage; the total amount of the lipid is 20-60%, preferably 30-50E% of the total energy content.

In one embodiment of the present invention, the packaged heat-treated beverage product comprises a multivitamin. In an exemplary embodiment, the packaged heat-treated beverage product comprises at least ten vitamins. In an exemplary embodiment, the packaged heat-treated beverage product comprises a multivitamin 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 preferred embodiments of the present invention, the packaged heat-treated beverage product comprises one or more edible 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 present invention, the packaged heat-treated beverage product further comprises a flavoring agent selected from the group consisting of salt, flavoring agent, odorant, and/or spice. In a preferred embodiment of the invention, the flavoring agent comprises chocolate, cocoa, lemon, orange, lime, strawberry, banana, tropical fruit flavors or combinations thereof. The choice of flavoring agent may depend on the beverage to be produced.

Transparency is a parameter used by consumers to evaluate products. One way to determine the transparency of a liquid food product is to measure the turbidity of the product as described in example 1.7.

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

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a turbidity of at most 200NTU, and the beverage is clear.

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

This is found when the applied heat treatment 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 preferably 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, and the beverage is opaque.

In some embodiments of packaged heat-treated beverage products, opaque beverage products are advantageous. This is advantageous, for example, when the beverage resembles milk and has a milky appearance. Nutritionally complete nutritional supplements are generally opaque in appearance.

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, even more preferably, a turbidity of greater than 2000 NTU.

The content of insolubles in the heat treated beverage product is a measure for the instability of the beverage and the extent to which the sediment settles 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 is precipitated after heating the sample after centrifugation at 3000x g for 5 minutes. See the analytical method in example 1.10.

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

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

In some preferred embodiments of the invention, the packaged heat-treated beverage product contains 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 often 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, most preferably no detectable insolubles at all.

Consumers prefer that the heat-treated beverages be liquid, easy to drink, and not gel.

One method of determining the viscosity of a beverage product is to measure 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 very low viscosity. This is advantageous when the beverage is used as a sports drink or in some embodiments of a nutritionally complete or an under-nourished supplement.

The inventors have surprisingly found that beverage products having an acidic pH and which have been subjected to a heat treatment such as pasteurization or even sterilization have a viscosity of at most 200 centipoise (cP) when measured at 22 ℃ at a shear rate of 100/s.

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 often 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, most preferably at most 1 cP.

It has previously been found that in order to produce an acidic clear heat-treated beverage comprising WPI, wherein the pH of the beverage is above pH 3.0, an anti-coagulant must be added to the beverage, see, for example, Etzel 2004(Etzel, m.r., 2004, dairy protein fraction manufacture and use, american society for nutrition science, p.996-1002).

The inventors have surprisingly found that transparent heat-treated beverages comprising at least 85% w/w BLG can be prepared even at a pH above pH 3.0 without the addition of an anticoagulant.

Thus, in some preferred embodiments of the present invention, the packaged heat-treated beverage product does not comprise any anticoagulant or alternatively comprises only trace amounts of anticoagulant.

In the context of the present invention, the term "anticoagulant" relates to food grade non-protein surfactants such as lauryl sulphate, polysorbates, mono-and/or diglycerides.

In some embodiments of the invention, the packaged heat-treated beverage product comprises at most 0.1% w/w anticoagulant, preferably at most 0.03% w/w anticoagulant, most preferably no anticoagulant. This embodiment is particularly preferred in the case of clear, low-fat beverages.

In some embodiments of the present invention, the packaged heat-treated beverage product does not comprise polyphenols.

However, in other preferred embodiments of the present invention, the packaged heat-treated beverage product comprises polyphenols. Polyphenols such as epigallocatechin-3-gallate (EGCG) have been shown to limit the aggregation of whey proteins upon heat treatment. Although they are not necessary for preparing the high protein beverage products of the present invention, they may be used as ingredients.

Thus, the packaged heat-treated beverage product may preferably comprise a total amount of polyphenols of 0.01-1% w/w, more preferably 0.02-0.6% w/w, even more preferably 0.03-0.4% w/w, more preferably 0.04-0.2% w/w.

In some preferred embodiments of the invention, the polyphenol comprises, or even consists essentially of, EGCG.

In some preferred embodiments of the invention, the packaged heat-treated beverage product comprises a total amount of protein of 4.0 to 35% w/w, preferably 4.0 to 30% w/w, relative to the weight of the beverage.

In a further preferred embodiment of the invention the packaged heat-treated beverage product comprises a total amount of protein of 5.0 to 45% w/w, more preferably 5.0 to 35% w/w, even more preferably 5.0 to 34% w/w, most preferably 5.0 to 32% w/w, relative to the weight of the beverage.

In some preferred embodiments of the present invention the packaged heat-treated beverage product comprises a protein content of at least 3-45% w/w, more preferably 11-40% w/w, even more preferably 15-38% w/w, most preferably 20-36% w/w.

In some embodiments of the invention, it is advantageous that the protein content of the packaged heat-treated beverage product is from 2.0 to 10.0% w/w relative to the weight of the beverage.

Thus, in some embodiments of the invention, the packaged heat-treated beverage product preferably comprises 2.0 to 10% w/w total protein by weight of the beverage, preferably 3.0 to 10% w/w total protein by weight of the beverage, preferably 5.0 to 9.0% w/w total protein by weight of the beverage, preferably 6.0 to 8.0% w/w total protein by weight of the beverage.

In some embodiments of the invention, it is advantageous that the protein content of the beverage is high (e.g. 10.0 to 45.0% w/w relative to the weight of the beverage).

Thus, in some embodiments of the invention, the packaged heat-treated beverage product preferably comprises 10.0 to 45.0% w/w total protein by weight of the beverage, preferably 10.0 to 20% w/w total protein by weight of the beverage, preferably 12 to 30% w/w total protein by weight of the beverage, preferably 15 to 25% w/w total protein by weight of the beverage, preferably 18 to 20% w/w total protein by weight of the beverage.

In a further preferred embodiment of the invention, the protein content of the packaged heat-treated beverage product is advantageously between 5.0 and 45.0% w/w, preferably between 6.0 and 35.0% w/w, more preferably between 7.0 and 34% w/w, even more preferably between 8.0 and 32% w/w, most preferably between 10 and 30% w/w, relative to the weight of the packaged heat-treated beverage product.

The present invention surprisingly makes it possible to provide packaged heat-treated beverage products having a protein content of more than 15% w/w, even more than 20% w/w. Thus, in some preferred embodiments of the present invention, the packaged heat-treated beverage product preferably comprises from 15 to 45.0% w/w of total protein by weight of the beverage product, preferably from 20 to 35% w/w of total protein by weight of the beverage product, more preferably from 21 to 34% w/w of total protein by weight of the beverage product, even more preferably from 25 to 32% w/w of total protein by weight of the beverage product.

In a further preferred embodiment of the present invention the packaged heat-treated beverage product preferably comprises 21 to 35% w/w of total protein by weight of the beverage product, preferably 25 to 35% w/w of total protein by weight of the beverage product, more preferably 28 to 35% w/w of total protein by weight of the beverage product, even more preferably 30 to 35% w/w of total protein by weight of the beverage product.

In a further preferred embodiment of the present invention the packaged heat-treated beverage product preferably comprises 21 to 33% w/w of the total amount of protein relative to the weight of the beverage product, preferably 25 to 33% w/w of the total amount of protein relative to the weight of the beverage product, more preferably 28 to 33% w/w of the total amount of protein relative to the weight of the beverage product.

The protein of the liquid solution is preferably prepared from the milk of a mammal, preferably from the milk of a ruminant, such as the milk of a cow, sheep, goat, buffalo, camel, llama, horse and/or deer. Proteins derived from bovine milk are particularly preferred. Thus, the protein of the liquid solution is preferably a bovine 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.

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 amount of lipids and/or optionally also a limited amount of carbohydrates.

In some preferred embodiments of the invention the preparation is in particular used as a sports drink and comprises a total amount of protein, e.g. 2-45% w/w relative to the weight of the drink, preferably 2-20% w/w relative to the weight of the drink, or preferably 2-10% w/w relative to the weight of the drink, most preferably 2-6% w/w relative to the weight of the drink.

In some preferred embodiments of the invention, the packaged heat-treated beverage product is particularly useful as a nutritional supplement for undernutrition and comprises a total amount of protein, e.g. in the range of 2-45% w/w relative to the weight of the beverage, preferably in the range of 2-20% w/w relative to the weight of the beverage, or preferably in the range of 3-10% w/w relative to the weight 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 a total amount of protein of, for example, 4-45% w/w relative to the weight of the beverage, or preferably 5-20% w/w relative to the 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 reduced kidney function.

In some preferred embodiments of the invention the packaged heat-treated beverage product comprises a total amount of protein, e.g. 2-45% w/w relative to the weight of the beverage, preferably 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.

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.

The packaged heat-treated beverage product 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 preferred 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 (sucralose), maltodextrin, corn syrup solids, sucrose (saccharose), maltose, sucrose ketose (sucralose), maltitol powder, glycerol, 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, mayonnaise (Fibersol), and combinations thereof.

In some preferred embodiments, the packaged heat-treated beverage product comprises a carbohydrate polymer, i.e., an oligosaccharide and/or polysaccharide.

In some preferred embodiments, the packaged heat-treated beverage product further comprises carbohydrate in the range of 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 in the range of 20 to 75% of the total energy content of the product, or preferably in the range of 30 to 60% of the total energy content of the product.

Even lower carbohydrate content is often preferred, and therefore in some preferred embodiments of the invention, the carbohydrate content of the preferably packaged heat-treated beverage product is from 0 to 30% of the total energy content of the product, more preferably from 0 to 20% of the total energy content of the product, even more preferably from 0 to 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 is 5% of the total energy content of the product, more preferably 1% of the total energy content of the product, even more preferably 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 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 invention, the packaged heat-treated beverage product is particularly useful as a nutritional supplement for undernutrition and comprises a total amount of carbohydrates in the range of 70-95%, preferably 80-90E% of the total energy content (E) 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 a total amount of carbohydrates in the range of 30-60%, preferably 35-50E%, of the total energy content 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 reduced kidney function.

In some preferred embodiments of the present invention, the packaged heat-treated beverage product comprises a total amount of carbohydrates in the range of 30-60%, preferably 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.

In one embodiment of the present invention, the beverage product 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, neotame, saccharin, stevia extract, a steviol glycoside, such as rebaudioside a, or a combination thereof. In some embodiments of the present invention, it is particularly preferred that the sweetener comprises or even consists of more than one High Intensity Sweetener (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, high intensity sugar sweeteners (e.g., aspartame, acesulfame potassium or sucralose) may be used in beverages that do not require the sweetener to provide energy, while natural sweeteners (e.g., steviol glycosides, sorbitol or sucrose) may be used for beverages with natural characteristics.

It may furthermore be preferred that the sweetener comprises or even consists of more than one polyol sweetener. 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 amount of lipids 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 60% of the total energy content of the product, or preferably from 0 to 50% of the total energy content of the product, or preferably from 0 to 45% of the total energy content of the product, or preferably from 0 to 30% of the total energy content of the product, or preferably from 0 to 20% of the total energy content of the product, or preferably from 0 to 10% of the total energy content of the product, or preferably from 0 to 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 is 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 useful as a nutritional supplement for undernutrition and comprises a total amount of lipids of, for example, 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 a total amount of lipids, for example, in the range of 20-50%, preferably 30-40E% 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, the packaged heat-treated beverage product comprises a total amount of lipids, for example, in the range of 20-60%, preferably 30-50E% of the total energy content.

The total amount of water in the beverage product is typically 50-98% w/w, preferably 45-97% w/w, more preferably 40-95% w/w, even more preferably 35-90% w/w, most preferably 30-85% w/w.

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

In some preferred embodiments of the invention, the total amount of water in the beverage product is 90-98% w/w, preferably 92-97.5% w/w, more preferably 94-97% w/w, even more preferably 95-97% w/w, most preferably 96-97% w/w. These embodiments are useful for clear aqueous beverages.

In some preferred embodiments of the invention, the beverage product is alcohol-free, 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 total solids of the beverage product is typically 1-45% w/w, preferably 5-40% w/w, more preferably 10-35% w/w, even more preferably 12-30% w/w, most preferably 16-25% w/w.

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

In some preferred embodiments of the invention the total amount of solids of the liquid solution is 1-10% w/w, preferably 1.5-8% w/w, more preferably 2-6% w/w, even more preferably 2-5% w/w, most preferably 2-4% w/w. These embodiments are useful for clear aqueous beverages

The non-solid portion of the beverage product is preferably water.

In some preferred embodiments of the invention the total amount of alpha-lactalbumin (ALA) and Caseinomacropeptide (CMP) comprises at least 40% w/w, 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 of the beverage product.

In a further preferred embodiment of the invention each major non-BLG whey protein (main 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% by weight relative to the total amount of protein of a standard whey protein concentrate from sweet whey.

Even lower concentrations of major non-BLG whey proteins are required. 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, expressed as a weight percentage relative to the total amount of protein of a standard whey protein concentrate from sweet whey, of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%.

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

Even lower levels of ALA are preferred, and therefore, in some preferred embodiments of the invention, ALA comprises at most 20% w/w, 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 beverage product.

The inventors have observed 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, the weight percentage of lactoferrin, expressed as a weight percentage relative to the total amount of proteins, relative to the total amount of proteins of a standard whey protein concentrate from sweet whey, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%. Even lower concentrations of lactoferrin may be desirable. Thus, in a further preferred embodiment of the invention, the weight percentage of lactoferrin, expressed as a weight percentage relative to the total amount of proteins, relative to the total amount of proteins of a standard whey protein concentrate from sweet whey, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%.

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

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

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

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 optionally 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:

-a total amount of protein of 2-45% w/w relative to the weight of the beverage, preferably 2-20% w/w relative to the weight of the beverage, or preferably 2-10% w/w relative to the weight of the beverage, most preferably 2-6% w/w relative to the weight of the beverage

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

-a total amount of lipids of 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 an incomplete nutritional supplement and comprises, for example:

-a total amount of protein of 2-45% w/w relative to the weight of the beverage, preferably 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 is 70-95%, preferably 80-90E%, of the total energy content (E) of the beverage, and

-the total amount of lipids is 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 comprises, for example:

-the total amount of protein is 4-45% w/w relative to the weight of the beverage, preferably 5-20% w/w relative to the weight of the beverage

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

the total amount of lipids is 20-50%, preferably 30-40E% 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. The content of phosphorus and other minerals such as potassium in the beverage product is very low.

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

-the total amount of protein is 2-45% w/w relative to the weight of the beverage, preferably 2-20% w/w relative to the weight of the beverage or preferably 3-12% w/w relative to the weight of the beverage, preferably 3-10% w/w relative to the weight of the beverage

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

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

The heat-treated beverage product is preferably in a suitable container as described herein, such as a bottle, box, brick, and/or bag.

The present inventors have surprisingly found that beverage products exposed to heat treatment in which at least some of the protein is denatured tend to develop color when stored under certain conditions. The inventors have subsequently found that the color generation is at least partly due to exposure to light (electromagnetic radiation) and that this phenomenon increases with increasing BLG concentration. The inventors have further found that this problem can be reduced or even avoided by selecting a container that blocks at least a portion of the ambient light.

Thus, in some preferred embodiments of the invention, the container 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% for any wavelength in the range of 250-500 nm.

In other preferred embodiments of the invention the container wall has an average 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% for any wavelength in the range of 250-500 nm.

The light transmission of the 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 performed using a standard spectrophotometer and a piece of the vessel wall is inserted into the light path (e.g. using a cuvette or similar arrangement) whereby the plane of the piece of the vessel wall is arranged perpendicular to the light path. Transmittance at wavelength i using T_i＝I_{i, after}/I_{i, front}100% of calculation, wherein I_{i, front}To achieve a light intensity at a wavelength I before reaching the vessel wall, I_{i, after}The intensity at wavelength i of the light beam in the light path after passing through the wall of the sheet of container.

By using all the transmission T measured in a given wavelength range _iThe sum is divided by the sum of the number of transmission measurements over a given wavelength range to calculate the average light transmission.

In some preferred embodiments of the invention the container 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% for any wavelength in the range of 250-800 nm.

In other preferred embodiments of the invention the container wall has an average 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% for any wavelength in the range of 250-800 nm.

Such a container with low light transmission can be produced, for example, as follows: colored absorbent-containing or coated polymers or colored or coated glass are used, or alternatively a metal layer is incorporated in the container wall, for example in the form of an aluminium foil. Such containers having no or low light transmission are well known in the food and pharmaceutical industries.

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

The inventors have further found that colour generation can be reduced or even avoided if the beverage product comprises mainly BLG in its natural configuration. Thus, it was found that the color problem can be mitigated by reducing the degree of protein denaturation, for example, by using a method that results in less denaturation.

Thus, in some preferred embodiments of the invention, at least a portion of the walls of the container are transparent, preferably the entire container is transparent. In some preferred embodiments of the invention, at least a portion of the wall of the container, preferably the entire wall of the container, has an average light transmission in the range of 400-700nm of at least 11%, preferably at least 20%, more preferably at least 50%, even more preferably at least 60%, most preferably at least 80%.

One aspect of the present invention relates to a method of producing a packaged heat-treated beverage product having a pH of 2-4.7, said method comprising the steps of:

a) providing a liquid solution comprising:

-2 to 45 wt% of total protein, of which at least 85% of the protein is BLG,

-optionally, sweeteners, sugar polymers 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, said heat treatment comprising at least pasteurization.

The liquid solution of step a) preferably has the same chemical composition as the heat treated beverage product described, except that the liquid solution has not undergone a final heat treatment. Thus the embodiments and preferred embodiments of the heat-treated beverage product referred to above and below apply equally to liquid solutions.

In some preferred embodiments, at least 85% w/w of the protein in the liquid solution of the invention 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, most preferably about 100% w/w.

For example, the liquid solution preferably comprises at least 97.5% w/w 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% w/w, most preferably at least 99.5% w/w BLG relative to the total amount of protein, e.g. about 100% w/w relative to the total amount of protein.

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 packaging, i.e. the liquid solution is packaged under aseptic conditions. For example, aseptic packaging can be performed by using an aseptic filling system, and it preferably involves filling the 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 low microbial content prior to filling.

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

In some preferred embodiments of the method, the packaged liquid solution of step b) is subjected to a heat treatment, said heat treatment comprising at least pasteurization. This embodiment is commonly referred to as a heat treatment or retort (retort) process within the container and involves heating the entire container and its contents to achieve pasteurization or even sterility. When heat treatment in a container is used, it is particularly preferred to maintain the temperature in the range of 70-82 ℃, more preferably in the range of 70-80 ℃ and most preferably in the range of 70-78 ℃. In this way, the level of protein spreading can be kept at a minimum.

In a further preferred embodiment of the method according to the invention, the liquid solution of step a) is subjected to a heat treatment, which heat treatment comprises at least pasteurization, and then packaged in step b).

In a particularly preferred embodiment, the heat treatment involves heating the beverage product to a temperature in the range of 70-82 ℃.

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 in the range of 73-76 ℃, e.g. about 75 ℃.

Preferably, when the heat treatment is performed at a temperature in the range of 70-82 ℃, the duration of the heat treatment is 1 second to 30 minutes. The highest treatment time is most suitable for the lowest temperature in this temperature range and vice versa. The lower the pH of the liquid solution, the higher the temperature that can be withstood without spreading.

In a particularly preferred embodiment of the invention, the heat treatment provides 70-80 ℃ for 1 second to 30 minutes, more preferably 71-77 ℃ for 1 minute to 25 minutes, even more preferably 72-76 ℃ for 2 minutes to 20 minutes.

In some preferred embodiments of the invention, the heat treatment 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 optionally BLG aggregation 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 in the range 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 of 5 seconds-10 minutes. The heat treatment may, for example, comprise 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 5-30 seconds, for example about 120 ℃ for about 20 seconds.

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

Alternatively, but also preferably, the heat treatment may comprise a temperature in the range of 145-180 ℃ and a duration of 0.01-2 seconds, and more preferably a temperature in the range of 150-180 ℃ and a duration 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. Alternatively, for heat treatments above 95 ℃, particularly preferably, 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 rapid cooling. Suitable examples of carrying out spray cooking can be found in WO2009113858a1, which is incorporated herein for all purposes. Suitable examples of implementing direct steam injection and direct steam injection can be found in WO2009113858a1 and WO2010/085957A3, which are incorporated herein for all purposes. General aspects of high temperature treatment can be found, for example, in "thermal technology in food processing" ISBN 185573558X, which is incorporated herein by reference for all purposes.

In some preferred embodiments of the invention, pasteurization is combined with another physical sterilization method.

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

In some particularly preferred embodiments of the invention, the heat treatment is selected so that it provides a degree of protein denaturation of at most 50%, preferably at most 20%, even more preferably at most 10%, most preferably at most 5%.

It is further preferred that the heat treatment is selected such that it provides an intrinsic tryptophan fluorescence ratio (I330/I350) of at least 1.11, preferably at least 1.13, more preferably at least 1.15, even more preferably at least 1.17.

In some preferred embodiments of the present invention, the heat treatment is sterilization, thereby resulting in a sterilized liquid beverage product. Such sterilisation may be obtained by a combination of bacterial filtration and heat treatment, such as pasteurisation. Sterilization may, for example, comprise heat treatment followed by bacterial filtration, or even more preferably, bacterial filtration followed by heat treatment.

In the context of the present invention, the term "bacterial filtration" relates to filtration performed with a pore size sufficient to retain microorganisms such as bacteria and spores but not native BLG. Bacterial filtration is sometimes also referred to as sterile filtration and involves microfiltration of the liquid involved. Bacterial filtration is typically performed using membranes with pore sizes 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 may for example use membranes with pore sizes 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 followed by a heat treatment at a temperature of at most 80 ℃, preferably at most 78 ℃. The duration of the heat treatment is preferably selected to be long enough to prepare a sterile beverage product.

In a further preferred embodiment of the invention, the liquid solution is subjected to bacterial filtration followed by a heat treatment at a temperature of at most 81-160 ℃, more preferably 100-155 ℃. The combination of temperature and preferably the selected duration of the heat treatment provides a sterile beverage product.

Depending on the heat treatment temperature used, it may be advantageous to cool the beverage product. According to a preferred aspect of the method 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 has been pasteurized, it is preferably cooled to 0 to 15 ℃, preferably to 1 to 10 ℃, and more preferably to 1 to 6 ℃ after the heat treatment.

Generally any acid or base may be used to adjust the pH according to one embodiment of the process. One skilled in the art will find suitable means to adjust the pH. Suitable acids include, for example, citric, hydrochloric, malic or tartaric acid or phosphoric acid, most preferably citric and/or phosphoric acid.

Useful examples of useful bases are salts of hydroxides, such as sodium or potassium hydroxide, carbonates or bicarbonates, carboxylates, such as citrates or lactates, and combinations thereof. Preferably, the pH is adjusted using a base such as KOH or NaOH.

In some preferred embodiments of the invention, the liquid solution has a pH in the range of 3.0 to 4.3. These pH ranges are particularly preferred for producing clear beverages with low viscosity and improved taste.

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, ensures an increase in pH during heat treatment, improving visual impact (colour and turbidity) and viscosity compared to heat treated WPI beverages. The invention thus increases the pH range in which acidic whey protein-containing beverages of low viscosity and preferably also clear can be prepared.

It was surprisingly found that there is a significant difference between the sensory parameters of the BLG beverages of the present invention compared to beverages produced with WPI. It was surprisingly and advantageously found that BLG beverages had a lower degree of astringency, dry mouthfeel, tartness, whey smell and tartness compared to WPI beverages. It has further been found that when the pH of an acidic beverage is raised, less sweetener is required to balance the tartness of the beverage and therefore a lower concentration of sweetener is required for such a beverage.

In some preferred embodiments of the invention, the pH of the packaged heat-treated beverage product is from 3.0 to 4.1, or preferably from 3.1 to 4.0, or preferably from 3.2 to 3.9, or preferably from 3.7 to 3.9, more preferably from 3.4 to 3.9, even more preferably from 3.5 to 3.9.

Thus, it is preferred that the pH of the liquid solution is from 3.0 to 4.1, or preferably from 3.1 to 4.0, or preferably from 3.2 to 3.9, or preferably from 3.7 to 3.9, more preferably from 3.4 to 3.9, even more preferably from 3.5 to 3.9.

The inventors have found that it is very difficult to prepare acidic protein beverages with a pH above 3.6, especially if the beverage should be transparent. However, the present invention makes this possible.

Therefore, the pH of the liquid solution is preferably 3.7 to 4.3, more preferably 3.9 to 4.3, even more preferably 4.1 to 4.3.

Alternatively, and also preferably, the pH of the packaged heat-treated beverage product is from 3.7 to 4.1, more preferably from 3.9 to 4.1.

These pH ranges are particularly relevant when the beverage product is a pasteurized beverage product.

In some preferred embodiments of the invention, the pH of the liquid solution is preferably 3.0 to 3.9, or preferably 3.2 to 3.7, or preferably 3.4 to 3.6, or preferably 3.5 to 3.7, or preferably 3.4 to 3.6.

These pH ranges, in combination with high temperature treatments such as sterilization, are particularly suitable for producing clear beverages with low viscosity and improved taste.

In some preferred embodiments of the invention, the pH of the liquid solution is in the range of 4.1-4.7, which is particularly important for producing stable beverages having a milky appearance and high turbidity while still having a low viscosity. In some embodiments of the invention, the pH ranges from 4.2 to 4.6. In some other embodiments of the invention, the pH ranges from 4.2 to 4.5.

In some preferred embodiments of the invention, the total amount of protein in the liquid solution is 4.0 to 35% w/w, preferably 4.0 to 30% w/w, more preferably 5.0 to 30% w/w, relative to the weight of the beverage.

In a further preferred embodiment of the invention the total amount of protein of the liquid solution is 5.0 to 45% w/w, more preferably 5.0 to 35% w/w, even more preferably 5.0 to 34% w/w, most preferably 5.0 to 32% w/w, relative to the weight of the liquid solution.

In some preferred embodiments of the invention, the protein content of the liquid solution is at least 3-45% w/w, more preferably 11-40% w/w, even more preferably 15-38% w/w, most preferably 20-36% w/w.

In some embodiments of the invention, it is advantageous that the protein content of the liquid solution is between 2.0 and 10.0% w/w relative to the weight of the solution.

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 high (e.g., 10.0 to 45.0% 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 45.0% w/w total protein by weight of the liquid solution, preferably 10.0 to 20% w/w total protein by weight of the liquid solution, preferably 12 to 30% w/w total protein by weight of the liquid solution, preferably 15 to 25% w/w total protein by weight of the liquid solution, preferably 18 to 20% w/w total protein by weight of the liquid solution.

In a further preferred embodiment of the invention, the protein content of the liquid solution is advantageously between 5.0 and 45.0% w/w, preferably between 6.0 and 35% w/w, more preferably between 7.0 and 34% w/w, even more preferably between 8.0 and 32% w/w, most preferably between 10 and 30% w/w, relative to the weight of the liquid solution.

The present invention surprisingly makes it possible to provide packaged heat-treated beverage products having a protein content of more than 15% w/w, even more than 20% w/w. Thus, in some preferred embodiments of the invention, the liquid solution preferably comprises 15 to 45.0% w/w total protein by weight of the liquid solution, preferably 20 to 35% w/w total protein by weight of the liquid solution, more preferably 21 to 34% w/w total protein by weight of the liquid solution, even more preferably 25 to 32% w/w total protein by weight of the liquid solution.

In a further preferred embodiment of the invention the liquid solution preferably comprises 21 to 35% w/w of the total amount of protein relative to the weight of the liquid solution, preferably 25 to 35% w/w of the total amount of protein relative to the weight of the liquid solution, more preferably 28 to 35% w/w of the total amount of protein relative to the weight of the liquid solution, even more preferably 30 to 35% w/w of the total amount of protein relative to the weight of the liquid solution.

In a further preferred embodiment of the invention, the liquid solution preferably comprises 21 to 33% w/w of the total amount of protein relative to the weight of the beverage product, preferably 25 to 33% w/w of the total amount of protein relative to the weight of the liquid solution, more preferably 28 to 33% w/w of the total amount of protein relative to the weight of the liquid solution.

The protein of the liquid solution is preferably prepared from the milk of a mammal, preferably from the milk of a ruminant, such as the milk of a cow, sheep, goat, buffalo, camel, llama, horse and/or deer. Proteins derived from bovine milk are particularly preferred. Thus, the protein of the liquid solution is preferably a bovine 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 total amount of alpha-lactalbumin (ALA) and Caseinomacropeptide (CMP) is at least 40% w/w, 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 of the liquid solution.

In a further preferred embodiment of the invention, each major non-BLG whey protein is expressed in weight percentage relative to the total amount of protein, said weight percentage of each major non-BLG whey protein being 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 of a standard whey protein concentrate from sweet whey.

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

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

Even lower levels of ALA are preferred, and therefore, in some preferred embodiments of the invention, ALA comprises at most 20% w/w, 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 some preferred embodiments of the invention, the pH of the liquid solution is in the range of 3.0 to 3.9 and the total amount of protein in the liquid solution is 10 to 34% w/w, more preferably 12 to 30% w/w, even more preferably 15 to 25% w/w, relative to the weight of the liquid solution.

In a further preferred embodiment of the invention the pH of the liquid solution is in the range of 3.7 to 3.9 and the total amount of protein of the liquid solution is 10 to 34% w/w, more preferably 12 to 30% w/w, even more preferably 15 to 25% w/w, relative to the weight of the liquid solution.

In a further preferred embodiment of the invention, the liquid solution has the following properties:

-a pH in the range of 3.0 to 3.9, preferably 3.7 to 3.9,

-the total amount of protein is 10-34% w/w, more preferably 12-30% w/w, even more preferably 15-25% w/w, relative to the weight of the liquid solution, and

intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.13, more preferably at least 1.15, even more preferably at least 1.17, most preferably at least 1.19.

In a further preferred embodiment of the invention, the liquid solution has the following properties:

-a pH in the range of 3.0 to 3.9, preferably 3.7 to 3.9,

-the total amount of protein is 10-34% w/w, more preferably 12-30% w/w, even more preferably 15-25% w/w, relative to the weight of the liquid solution, and

-a degree of protein denaturation of at most 10%, preferably at most 5%, even more preferably at most 1%.

As mentioned above, the inventors have surprisingly found that transparent heat-treated beverages comprising at least 85% w/w BLG can be prepared even at a pH above pH 3.0 without the addition of an anticoagulant.

Thus, in some preferred embodiments of the invention, the liquid solution does not comprise any anticoagulant or alternatively only comprises trace amounts of anticoagulant.

In some embodiments of the invention, the liquid solution comprises at most 0.1% w/w anticoagulant, preferably at most 0.03% w/w anticoagulant, most preferably no anticoagulant. This embodiment is particularly preferred in the case of clear, low-fat beverages.

In some preferred embodiments of the invention, the liquid solution does not comprise polyphenols.

However, in other preferred embodiments of the invention, the liquid solution comprises polyphenols. Thus, the liquid solution may preferably comprise 0.01-1% w/w of the total amount of polyphenols, more preferably 0.02-0.6% w/w, even more preferably 0.03-0.4% w/w, more preferably 0.04-0.2% w/w.

In some preferred embodiments of the invention, the polyphenol comprises, or even consists essentially of, EGCG.

BLG isolates, for example in combination with other protein sources, preferably as the primary protein source, may even be the sole protein source.

The BLG isolate is preferably a BLG isolate powder or a liquid BLG isolate comprising water and a solid BLG isolate powder in an amount of 1-50% w/w.

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

-at least 30% w/w of total protein,

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

-up to 10% w/w water.

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

-bulk density of at least 0.2g/cm ³

Intrinsic tryptophan fluorescence emission ratio (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 BLG isolate powder has a pH in the range of 2-4.9. The powder is particularly useful as an acidic food product, particularly an acidic beverage.

In other preferred embodiments of the invention, the BLG isolate powder has a pH 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 where higher protein content is desired, 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 determined according to example 1.5.

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

In some preferred embodiments of the invention the total amount of alpha-lactalbumin (ALA) and Caseinomacropeptide (CMP) comprises at least 40% w/w, preferably at least 60% w/w, even more preferably at least 70% w/w of the non-BLG proteins in the powder, 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, expressed as a weight percentage relative to the total amount of protein of a standard whey protein concentrate from sweet whey, of at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%.

Even lower concentrations of major non-BLG whey proteins are required. 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, expressed as a weight percentage relative to the total amount of protein of a standard whey protein concentrate from sweet whey, of at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%.

The inventors have seen evidence 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, the weight percentage of lactoferrin, expressed as a weight percentage relative to the total amount of proteins, relative to the total amount of proteins of a standard whey protein concentrate from sweet whey, is at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%. Even lower concentrations of lactoferrin may be desirable. Thus, in a further preferred embodiment of the invention, the weight percentage of lactoferrin, expressed as a weight percentage relative to the total amount of proteins, relative to the total amount of proteins of a standard whey protein concentrate from sweet whey, is at most 4%, preferably at most 3%, more preferably at most 2%, even more preferably at most 1%.

Similarly, in some preferred embodiments of the invention, the lactoperoxidase is present in a weight percentage relative to the total amount of protein, said weight percentage of lactoperoxidase relative to the total amount of protein of a standard whey protein concentrate from sweet whey being at most 25%, preferably at most 20%, more preferably at most 15%, even more preferably at most 10%, most preferably at most 6%. Even lower concentrations of lactoperoxidase may be required. Thus, in a further preferred embodiment of the invention, the lactoperoxidase 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% relative to the total amount of protein of 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 amount of water 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 may, for example, 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 at most 10% w/w lipid, preferably at most 5% w/w, more preferably at most 2% w/w, even more preferably 0.1% w/w.

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

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 suffering from kidney disease. To make such a product, the BLG isolate powder must have the same low amounts of phosphorus and potassium.

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

In some preferred embodiments of the invention, the BLG isolate powder comprises at most 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. Even more preferably, the BLG isolate powder comprises at most 10mg potassium per 100g protein.

The content of phosphorus 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 in question 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/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.

The present inventors have found that for some applications, such as acidic food products, in particular acidic beverages, an acidic BLG isolate powder having a pH of at most 4.9, even more preferably at most 4.3, is particularly advantageous. This is especially true for high protein, clear acidic beverages.

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

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

The inventors have found that for some applications, such as pH neutral foods, in particular pH neutral beverages, it is particularly advantageous to have a BLG isolate powder that is pH neutral. This is especially true for high protein, clear or opaque pH neutral beverages.

Thus, in some preferred embodiments of the 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 to 6.0. Preferably, the pH of the powder is in the range of 5.1-5.9, more preferably 5.2-5.8, even more preferably 5.3-5.7, most preferably 5.4-5.6.

Advantageously, the bulk density of the BLG isolate powder used 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 such as lyophilized BLG isolate are fluffy and easily enter the air at the production site during use. This is problematic because it increases the risk of cross-contamination of the lyophilized 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 variants of the present invention is that they take up less space in transport, thereby increasing the weight of the transportable BLG isolate powder per unit volume.

Another advantage of the high density variants of the invention is that they are less prone to segregation (segregation) when used in powder mixtures with other powdered food ingredients, such as powdered sugar (bulk density about 0.56 g/cm)³) Granulated sugar (pile-up)Density of about 0.71g/cm³) 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 from 0.2 to 1.0g/cm ³Preferably 0.30 to 0.9g/cm³More preferably 0.40 to 0.8g/cm³And even more preferably 0.45 to 0.75g/cm³And even more preferably 0.50 to 0.75g/cm³Most preferably 0.6 to 0.75g/cm³。

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

The present inventors have found that when BLG is used in an acidic beverage, it is advantageous to maintain the natural configuration of the BLG, and it has been observed that increasing the development of BLG results in an increased degree of dry mouthfeel.

Intrinsic tryptophan fluorescence emissivity (I330/I350) is a measure of the extent of BLG expansion, and the inventors found that at high intrinsic tryptophan fluorescence emissivity, which correlates with low or no expansion of BLG, less dry mouthfeel was observed. Intrinsic tryptophan fluorescence emissivity (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, and most preferably at least 1.19.

If the BLG isolate powder contains a large amount of non-protein material, the protein fraction is preferably separated before measuring the intrinsic tryptophan fluorescence emissivity. Thus in some preferred embodiments of the invention, the protein fraction of the BLG isolate powder has an intrinsic tryptophan fluorescence emissivity of at least 1.11.

In some preferred embodiments of the invention, the intrinsic tryptophan fluorescence emissivity (I330/I350) of the protein fraction 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 fraction may be separated from the BLG isolate powder, for example, by dissolving the BLG isolate powder in demineralised water and subjecting the solution to dialysis or ultrafiltration-based diafiltration using a protein-retaining filter. If the BLG isolate powder contains interfering levels of lipids, the lipids can be removed by, for example, microfiltration. Microfiltration and ultrafiltration/diafiltration steps may be combined to remove lipids and small molecules from the protein fraction.

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 is also preferred that the BLG isolate powder has a significant level of protein denaturation if, for example, an opaque beverage is desired. Thus, in a further preferred embodiment 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 settles upon storage of the beverage. The level of insolubles was determined according to example 1.10.

In a preferred embodiment of the invention, the BLG isolate powder comprises at most 20% w/w insoluble protein material, preferably at most 10% w/w insoluble protein material, more preferably at most 5% w/w insoluble protein material, even more preferably at most 3% w/w insoluble protein material, most preferably at most 1% w/w insoluble protein material. Even more preferably, the BLG isolate powder may be free of any insoluble protein material.

The present inventors have found that the thermal stability of BLG isolate powder at pH3.9 is a good indicator of its usefulness for clear high protein beverages. The thermal stability at pH3.9 is determined according to example 1.2.

The thermostability of the BLG isolate powder at ph3.9 is particularly preferably 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, preferably the thermal stability of the BLG isolate powder at ph3.9 is 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, achieving both high protein naturalness and low levels of microorganisms is a challenge because the process of sterilization often results in unfolding and denaturation of the protein. 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 up to 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, such as at most 10 CFU/g. In a particularly preferred embodiment, the powder is sterile. The sterile BLG isolate powder may be prepared, for example, by combining several physical sterilization 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 of i)2 to 4.9, ii)6.1 to 8.5, or iii)5.0 to 6.0, and the BLG isolate powder comprises:

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

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

-at most 6% w/w water,

-up to 2% w/w, preferably up to 0.5% w/w of lipids,

the BLG isolate powder has:

an intrinsic tryptophan fluorescence emission ratio (I330/I350) of at least 1.11,

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

-thermal stability at pH3.9 of at most 200 NTU.

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

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

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

-at most 6% w/w water,

-up to 2% w/w, preferably up to 0.5% w/w of lipids,

The BLG isolate powder has:

an intrinsic tryptophan fluorescence emission ratio (I330/I350) of at least 1.11,

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

-a thermostability at pH3.9 of at most 70NTU, preferably at most 50NTU, even more preferably at most 40 NTU.

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

-at least 30% w/w of total protein,

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

-at most 6% w/w water,

the BLG isolate powder has:

-at least 0.2g/cm³The bulk density of the composite material is higher than that of the composite material,

an intrinsic tryptophan fluorescence emission ratio (I330/I350) of at least 1.11,

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

-thermal stability at pH3.9 of at most 200 NTU.

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

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

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

-at most 6% w/w water,

-up to 2% w/w, preferably up to 0.5% w/w of lipids,

the BLG isolate powder has:

-at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm³The bulk density of the composite material is higher than that of the composite material,

an intrinsic tryptophan fluorescence emission ratio (I330/I350) of at least 1.11,

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

-a thermostability at ph3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10 NTU.

In another preferred embodiment of the present invention, the BLG isolate powder has a pH ranging from 6.1 to 8.5, and comprises:

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

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

-at most 6% w/w water,

-at most 2% w/w lipid, preferably at most 0.5% w/w,

the BLG isolate powder has:

-at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm³The bulk density of the composite material is higher than that of the composite material,

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

-a thermostability at ph3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10 NTU.

In a further preferred embodiment of the present invention, the BLG isolate powder has a pH in the range of 6.1 to 8.5, and comprises:

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

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

-at most 6% w/w water,

-up to 2% w/w, preferably up to 0.5% w/w of lipids,

the BLG isolate powder has:

-at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm³The bulk density of the composite material is higher than that of the composite material,

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

-a thermostability at ph3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10 NTU.

In a further preferred embodiment of the present invention, the BLG isolate powder has a pH in the range of 5.0 to 6.0, and comprises:

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

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

-at most 6% w/w water,

-up to 2% w/w, preferably up to 0.5% w/w of lipids,

the BLG isolate powder has:

-at least 0.2g/cm³Preferably at least 0.3g/cm³More preferably at least 0.4g/cm³The bulk density of the composite material is higher than that of the composite material,

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

-a thermostability at pH3.9 of at most 50NTU, preferably at most 30NTU, even more preferably at most 10NTU, and

-preferably, BLG crystallinity below 10%.

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

a) providing a liquid BLG isolate having:

i) the pH range of 2 to 4.9,

ii) a pH range of 6.1 to 8.5,

iii) a pH range of 5.0 to 6.0,

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

b) optionally, the liquid BLG isolate is physically sterilized,

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

BLG isolates are preferably prepared from the milk of mammals and are preferably obtained from the milk of ruminants such as cows, sheep, goats, buffalos, camels, llamas, mares and or deer. Proteins obtained from milk are particularly preferred. The BLG is therefore preferably bovine BLG.

Liquid BLG isolates can be provided in a number of different ways.

Typically, providing a liquid BLG isolate involves separating BLG from a whey protein material to provide a BLG-enriched composition, or even consisting of, by one or more of the following methods:

-crystallizing or precipitating BLG by salting-in,

crystallization or precipitation of BLG by salting out (salting-out),

-ion exchange chromatography, and

-fractionation (fractionation) of whey proteins by ultrafiltration.

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

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

The term "whey protein material" refers to a composition from which a BLG-enriched composition and subsequent liquid BLG isolate can be obtained.

In some embodiments of the invention, the preparation of the BLG-enriched composition comprises or even consists of high salt BLG crystals according to US2790790a1 at ph 3.6-4.0.

In other embodiments of the invention, the preparation of a BLG-enriched composition comprises or even consists of the methods described by de Jongh et al (the anastomotic separation method reveals novel protein structural properties of beta-lactoglobulin, lacto-industrial science, Vol.84, No. 3, 2001, pp.562-571) or Vyas et al (a magnified study of the process of affinity separation of native beta-lactoglobulin, lacto-industrial science, Vol.85, pp.1639-1645, 2002).

However, in a particularly preferred embodiment of the present invention, the BLG-enriched composition is prepared by crystallization under salt-dissolving conditions at pH5-6, as described in PCT application PCT/EP2017/084553, herein incorporated 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 BLG crystals.

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

demineralization (demineralization),

-the addition of minerals,

-dilution of the aqueous phase,

-a concentration of the organic phase,

-physical attenuation, and

-pH adjustment.

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

Non-limiting examples of adding minerals include adding soluble, food acceptable salts, such as salts of Na, K, Ca, and/or Mg. Such a salt may be, for example, a phosphate salt, a chloride salt or a salt of a food acid, such as a citrate or lactate salt. The minerals may be added in solid, suspension 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 requires an increase in protein concentration relative to total solids, a concentration step such as ultrafiltration or dialysis is preferably used. If the concentration does not require an increase in protein concentration relative to total solids, methods such as evaporation, nanofiltration and/or reverse osmosis may be employed.

Non-limiting examples of physical sterilization 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 liquid solution has a color value Δ b on the CIELAB scale in the range of-0.10 to +0.51, 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 liquid solution has a color value Δ b on the CIELAB scale of 0.0 to 0.40, preferably +0.10 to + 0.25.

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 invention depends on the intended use of the final heat-treated beverage product.

In some preferred embodiments of the invention, the liquid solution 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, maltodextrin, corn syrup solids, sucrose, maltose, sucrose ketol, maltitol powder, glycerol, 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, yazaquin (Fibersol), and combinations thereof.

In some preferred embodiments of the invention, the liquid solution comprises a carbohydrate polymer, i.e. an oligosaccharide and/or a polysaccharide.

In some preferred embodiments, the liquid solution further comprises a carbohydrate that is 0 to 95% of the total energy content of the liquid solution, preferably 10 to 85% of the total energy content of the liquid solution, preferably 20 to 75% of the total energy content of the liquid solution, or preferably 30 to 60% of the total energy content of the liquid solution.

Even lower carbohydrate content is often preferred, and thus in some preferred embodiments of the invention, the carbohydrate content of the liquid solution is preferably from 0 to 30% of the total energy content of the preparation, more preferably from 0 to 20% of the total energy content of the preparation, and even more preferably from 0 to 10% of the total energy content of the preparation.

In some preferred embodiments of the invention, the carbohydrate content of the liquid solution is at most 5% of the total energy content of the liquid solution, more preferably at most 1% of the total energy content of the liquid solution, and 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.

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, neotame, saccharin, stevia extract, a steviol glycoside, such as rebaudioside a, or a combination thereof. In some embodiments of the present invention, it is particularly preferred that the sweetener comprises or even consists of more than one High Intensity Sweetener (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, high intensity sweeteners (e.g., aspartame, acesulfame potassium or sucralose) may be used in beverages that do not require the sweetener to provide energy, while natural sweeteners (e.g., steviol glycosides, sorbitol or sucrose) may be used for beverages with natural characteristics.

Alternatively or additionally, carbohydrate sweeteners may be used.

It may furthermore be preferred that the sweetener comprises or even consists of more than one polyol sweetener. 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 amount of lipids 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 60% of the total energy content of the liquid solution, or preferably 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 50-99% w/w of the total amount of water, preferably 45-97% w/w, more preferably 40-95% w/w, even more preferably 35-90% w/w, most preferably 30-85% w/w.

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

In some preferred embodiments of the invention the liquid solution comprises 90-99% w/w of the total amount of water, preferably 92-98.5% w/w, more preferably 94-98% w/w, even more preferably 95-98% w/w, most preferably 96-98% w/w. These embodiments are useful, for example, for clear aqueous beverages.

In some preferred embodiments of the invention, the liquid solution is alcohol-free, 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 1-45% w/w total solids, preferably 5-40% w/w, more preferably 10-35% w/w, even more preferably 12-30% w/w, most preferably 16-25% w/w.

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

In some preferred embodiments of the invention the liquid solution comprises 1-10% w/w total solids, preferably 1.5-8% w/w, more preferably 2-6% w/w, even more preferably 2-5% w/w, most preferably 2-4% w/w. The non-solid portion of the liquid solution in which these embodiments are useful, for example, for clear aqueous beverages, is preferably water.

The present inventors have found that it is advantageous to control the mineral content of packaged heat-treated beverage products to achieve certain desired characteristics.

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

The present inventors have surprisingly found that when using a BLG isolate as defined herein and in example 2, heat treated beverage products with high mineral concentrations can be produced without compromising viscosity. This offers the following possibilities: packaged heat-treated beverage products having high mineral content can be produced, and nutritionally complete or nutritionally incomplete supplement beverages can be produced.

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 750mM, preferably in the range of 100-600mM, or preferably in the range of 100mM to 600mM, or preferably in the range of 200-500 mM.

In some preferred embodiments of the invention, the total amount of Na, K, Mg and Ca in the liquid solution is at most 750 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 600mM, preferably at most 500mM, or preferably at most 400mM, or preferably at most 300mM, or preferably at most 200mM, preferably at most 170mM, most preferably at most 150mM, or preferably at most 130mM, 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 another exemplary embodiment, 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 some preferred embodiments of the invention, the liquid solution comprises at most 150mM KCl and at most 150mM CaCl ₂Or the liquid solution comprises KCl at most 130mM and CaCl at most 130mM₂Or the liquid solution comprises at most 110mM KCl and at most 110mM CaCl₂Or the liquid solution comprises KCl up to 100mM and CaCl up to 100mM₂Or, preferably, the liquid solution comprises at most 80mM KCl and at most 80mM CaCl₂Or preferably, the liquid solution comprises at most 50mM KCl and at most 50mM CaCl₂Or the liquid solution comprises at most 40mM KCl and at most 40mM CaCl₂。

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

In the context of the present invention, the term "low mineral" relates to compositions, such as liquids, beverages, powders or other food products, having at least one, preferably two, even more preferably all of the following:

-at most 1.2% w/w ash relative to the total solids,

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

-a total content of sodium and potassium of at most 0.1% w/w relative to the total solids,

-a total phosphorus amount 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:

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

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

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

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

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

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

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

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

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

Particularly preferably, the low mineral composition has the following:

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

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

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

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

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 reduced kidney function.

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 phosphorous and low potassium beverage product.

In the context of the present invention, the term "low phosphorus" refers to a composition, such as a liquid, powder or other food product, having a total content of phosphorus of at most 100mg phosphorus per 100g protein. Preferably, the total phosphorus content of the low phosphorus composition is at most 80mg phosphorus per 100g protein. More preferably, the total phosphorus content of the low phosphorus composition is at most 50mg phosphorus per 100g protein. Even more preferably, the total content of phosphorus in the low-phosphorus composition is at most 20mg of phosphorus per 100g of protein. Even more preferably, the total content of phosphorus in the low-phosphorus composition is 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 invention, the liquid solution comprises at most 80mg of phosphorus per 100g of protein. Preferably, the liquid solution comprises at most 30mg of phosphorus per 100g of protein. More preferably, the liquid solution comprises at most 20mg of phosphorus per 100g of protein. Even more preferably, the liquid solution comprises at most 10mg phosphorus per 100g protein. Most preferably, the liquid solution comprises at most 5mg phosphorus 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.

In the context of the present invention, the term "low potassium" refers to a composition, such as a liquid, powder or other food product, having a total content of potassium of at most 700mg potassium per 100g protein. Preferably, the total potassium content of the low-phosphorous composition is at most 600mg potassium per 100g protein. More preferably, the total potassium content of the low potassium composition is at most 500mg potassium per 100g protein. More preferably, the total potassium content of the low potassium composition is at most 400mg potassium per 100g protein. More preferably, the total potassium content of the low potassium composition is at most 300mg potassium per 100g protein. Even more preferably, the total potassium content in the low potassium composition is at most 200mg potassium per 100g protein. Even more preferably, the total potassium content in the low potassium composition is at most 100mg potassium per 100g protein. Even more preferably, the total potassium content of the low potassium composition is at most 50mg potassium per 100g protein, even more preferably, the total potassium content of the low potassium composition is at most 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 invention, the liquid solution comprises at most 600mg potassium per 100g protein. More preferably, the liquid solution comprises at most 500mg potassium per 100g protein. More preferably, the liquid solution comprises at most 400mg potassium per 100g protein. More preferably, the liquid solution comprises at most 300mg potassium per 100g protein. Even more preferably, the liquid solution comprises at most 200mg potassium per 100g protein. Even more preferably, the liquid solution comprises at most 100mg potassium per 100g protein. Even more preferably, the liquid solution comprises at most 50mg potassium per 100g protein, even more preferably, the liquid solution 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 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 preferably further comprising carbohydrates and lipids, the total amount of said carbohydrates being 30-60%, preferably 35-50E% of the total energy content of the beverage; the total amount of the lipid is 20-60%, preferably 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 an 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 edible 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 agent, odorant and/or spice. In a preferred embodiment of the invention, the flavoring agent comprises chocolate, cocoa, lemon, orange, lime, strawberry, banana, tropical fruit flavors or combinations thereof. The choice of flavoring agent may depend on the beverage to be produced.

One aspect of the present invention relates to the use of a protein solution comprising 2 to 45% w/w (preferably 3 to 35% w/w) total amount of protein, wherein at least 85% w/w (preferably at least 90% w/w) of the protein is BLG, and wherein the pH of the acidic beverage product is in the range of 2.0-4.7, relative to the weight of the solution, for controlling the turbidity of a heat-treated acidic beverage product.

Another aspect of the invention relates to the use of a protein solution comprising 2 to 45% w/w of the total amount of protein, wherein at least 85% w/w (preferably 90% w/w) of the protein is BLG, relative to the weight of the solution, for controlling the astringency of a heat-treated acidic beverage product having a pH in the range of 2.0-4.7.

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

Another aspect of the 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 a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the protein fraction of the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.11,

-the lipid content is at most 5% of the total energy content of the preparation.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

Optionally, a sweetener and/or a flavoring agent,

wherein:

-the protein fraction of the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.11,

-the lipid content is at most 5% of the total energy content of the preparation.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-10 to 45% w/w of total amount of protein, preferably 10-35% w/w, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, relative to the weight of the beverage, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the protein fraction of the beverage product has an intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.11,

-the lipid content is at most 5% of the total energy content of the preparation.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

-the turbidity of the packaged heat-treated beverage product is at most 200NTU, preferably at most 40 NTU.

In other preferred embodiments of the present invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

-the turbidity of the packaged heat-treated beverage product is at most 200NTU, preferably at most 40 NTU.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 3.5 to 4.7, preferably 3.7 to 4.3, even more preferably 3.7 to 4.1, the beverage product comprising:

-2 to 45% w/w of total amount of protein, preferably 5.0-35%, more preferably 6.0-32% w/w,

-at least 88% w/w of the protein is BLG, preferably at least 90% w/w, more preferably at least 92% w/w,

-a total amount of lipids of at most 5% w/w, preferably at most 1% w/w, even more preferably at most 0.2% w/w,

optionally, a sweetener and/or a flavoring agent,

the beverage product has:

-a viscosity of at most 100cP, preferably at most 20cP, more preferably at most 10cP,

An intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of at least 1.13, preferably at least 1.15, more preferably at least 1.16,

and

-optionally, a turbidity of at most 50NTU, preferably at most 20NTU, more preferably at most 10 NTU.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

the packaged heat-treated beverage product has a turbidity of at most 200NTU, preferably at most 40 NTU.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-10 to 45% w/w of total amount of protein, preferably 10-20% w/w, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, relative to the weight of the beverage, and

optionally, a sweetener and/or a flavoring agent,

the packaged heat-treated beverage product has a turbidity of at most 200NTU, preferably at most 40 NTU.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11,

-the protein fraction of the beverage product has:

a colour value Δ b of-0.10 to +0.51 on the CIELAB scale, in which,

Δb*＝b_{samples normalized to 6.0% w/w protein}-b_{Demineralized water}Measured at room temperature.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11,

-the protein fraction of the beverage product has:

a colour value Δ b in the range from-0.10 to +0.51 on the CIELAB colour scale, wherein

Δb*＝b_{Samples normalized to 6.0% w/w protein}-b_{Demineralized water}Measured at room temperature.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-10 to 45% w/w of total amount of protein, preferably 10-20% w/w, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, relative to the weight of the beverage, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11,

-the protein fraction of the beverage product has:

a colour value Δ b ranging from-0.10 to +0.51 on the CIELAB scale, wherein:

Δb*＝b_{samples normalized to 6.0% w/w protein}-b_{Demineralized water}Measured at room temperature.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

2 to 45% w/w of total amount of protein relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

Optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11,

the total amount of-Na, K, Mg and Ca is at most 750mM, preferably at most 400mM, preferably at most 200 mM.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-2 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11,

the total amount of-Na, K, Mg and Ca is at most 750mM, preferably at most 400mM, preferably at most 200 mM.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-10 to 45% w/w of total amount of protein, preferably 10-20% w/w, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, relative to the weight of the beverage, and

Optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11,

the total amount of-Na, K, Mg and Ca is at most 750mM, preferably at most 400mM, preferably at most 200 mM.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 3.0 to 4.7, preferably 3.9 to 4.6 or preferably 4.0 to 4.5, said beverage comprising:

2 to 45% w/w of total amount of protein relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11, and/or

-the protein denaturation degree of the protein fraction is at most 5%, and/or

-the lipid content is more than 5% of the total energy content of the preparation.

In a preferred embodiment of the present invention, the packaged heat-treated beverage product has a pH in the range of 3.0 to 4.7, preferably 3.9 to 4.6, more preferably 4.0 to 4.5, said beverage comprising:

-2 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

Optionally, a sweetener and/or a flavoring agent,

wherein:

turbidity in excess of 200NTU, preferably in excess of 1000NTU, and/or

Viscosity of at most 200 cP.

In a preferred embodiment of the present invention, the packaged heat-treated beverage product has a pH in the range of 3.0 to 4.7, preferably 3.9 to 4.6, more preferably 4.0 to 4.5, said beverage comprising:

-2 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11, and/or

-the protein denaturation degree of the protein fraction is at most 5%, and/or

-the lipid content is more than 5% of the total energy content of the preparation.

In a preferred embodiment of the present invention, the packaged heat-treated beverage product has a pH in the range of 3.0 to 4.7, preferably 3.9 to 4.6, more preferably 4.0 to 4.5, said beverage comprising:

-2 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

turbidity of more than 200NTU, preferably more than 1000NTU, and/or

-viscosity of at most 200 cP.

In a preferred embodiment of the present invention, the packaged heat-treated beverage product has a pH in the range of 3.0 to 4.7, preferably 3.9 to 4.6, more preferably 4.0 to 4.5, said beverage comprising:

-10 to 45% w/w of total amount of protein, preferably 10-20% w/w, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, relative to the weight of the beverage, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.11, and/or

-the protein denaturation degree of the protein fraction is at most 5%, and/or

-the lipid content is more than 5% of the total energy content of the preparation.

In a preferred embodiment of the present invention, the packaged heat-treated beverage product has a pH in the range of 3.0 to 4.7, preferably 3.9 to 4.6, more preferably 4.0 to 4.5, said beverage comprising:

-10 to 45% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

turbidity in excess of 200NTU, preferably in excess of 1000NTU, and/or

Viscosity of at most 200 cP.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.2, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-5 to 34% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.13,

-the lipid content is at most 5% of the total energy content of the preparation.

In a preferred embodiment of the present invention, the packaged heat-treated beverage product has a pH in the range of 2.0 to 4.7, preferably 3.0 to 3.9 or preferably 3.2 to 3.7, said beverage comprising:

-5 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.13,

-the lipid content is at most 5% of the total energy content of the preparation.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 3.7 to 4.3, preferably 3.9 to 4.3 or preferably 4.1 to 4.3, said beverage comprising:

-5 to 10% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

Optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.13,

-the lipid content is at most 5% of the total energy content of the preparation.

In a preferred embodiment of the invention, the packaged heat-treated beverage product has a pH in the range of 3.7 to 4.3, preferably 3.9 to 4.3 or preferably 4.1 to 4.3, said beverage comprising:

-10 to 35% w/w of total amount of protein, relative to the weight of the beverage, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, and

optionally, a sweetener and/or a flavoring agent,

wherein:

-the intrinsic tryptophan fluorescence emission ratio (I330nm/I350nm) of the protein fraction of the beverage product is at least 1.13,

-the lipid content is at most 5% of the total energy content of the preparation.

In some embodiments, the heat-treated beverage has a shelf life of at least 6 months at 25 ℃, the heat-treated beverage comprising:

-an edible BLG composition as defined in PCT/EP2017/084553 providing a total amount of BLG of at least 1% (w/w), preferably at least 5% (w/w),

sweeteners, for example sugar sweeteners and/or non-sugar sweeteners,

at least one food acid, such as citric acid or other suitable food acids,

-optionally, a flavoring agent, and

-up to 80mg phosphorus per 100g protein,

the pH of the heat-treated beverage is in the range of 2.5-4.0.

In a preferred embodiment the invention relates to the use of a protein solution comprising 3 to 30% 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 BLG, preferably at least 90 w/w%, for controlling the turbidity of a heat treated acidic beverage product having a pH in the range of 3.0-4.5.

In a preferred embodiment the invention relates to the use of a protein solution comprising 3 to 30% w/w of the total amount of protein, wherein at least 85% w/w of the protein is BLG, preferably at least 90% w/w, relative to the weight of the solution, said acidic beverage product having a pH in the range of 2.0-4.0, for controlling the astringency of a heat-treated acidic beverage product.

A preferred embodiment of the present invention relates to a heat-treated beverage product 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.

Examples

Example 1: analytical method

Example 1.1: determination of protein naturalness by intrinsic tryptophan fluorescence

Tryptophan (Trp) fluorescence spectroscopy is a well-known tool for monitoring protein folding and unfolding. Trp residues buried in native proteins generally show the highest fluorescence emission near 330nm compared to when Trp residues are 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 330nm and 350nm fluorescence emissions to investigate the effect of heating temperature.

The analysis comprises the following steps:

dilute the beverage composition to 0.6mg/ml with MQ water.

Transfer 300 μ Ι sample to a white 96-well plate, avoiding air bubbles, or transfer 3mL to a 10mm quartz cuvette.

Tryptophan fluorescence emission intensity between 310 and 400nm was recorded from the top by excitation at 295 using a 5nm gap.

The samples were measured at 22 ℃ using a Cary Eclipse fluorescence spectrophotometer equipped with a plate reader kit (G9810A) or a single cuvette holder.

The emission intensity ratio, calculated by dividing the fluorescence emission intensity measured at 330nm by the emission intensity measured at 350nm, R ═ I330/I350, was used as a measure of protein naturalness.

R of at least 1.11 describes the natural configuration of BLG as predominant, and

r less than 1.11 reports at least partial unfolding and aggregation.

Example 1.2: thermal stability at pH3.9

Thermal stability at pH 3.9:

the heat stability at pH3.9 is a measure of the ability of a protein composition to maintain clarity at pH3.9 after prolonged pasteurization.

The thermal stability at pH3.9 was determined as follows: an aqueous solution having a pH of 3.9 and containing 6.0% w/w protein (or if it is a dilution, it is concentrated by low temperature evaporation) is formed by mixing a powder sample to be tested or a liquid sample to be tested with water and the pH is adjusted to 3.9 by the minimum required amount of 0.1M NaOH or 0.1M HCL.

The pH-adjusted mixture was allowed to stand for 30 minutes, after which 25mL of the mixture was transferred to a 30mL thin-walled glass tube. It was then heated to 75.0 ℃ for 300 seconds by immersion in a 75 ℃ water bath. Immediately after heating, the glass tube was moved to an ice bath and cooled to 1-5 ℃ and the turbidity of the heated sample was determined according to example 1.7.

Example 1.3: determining the degree of protein denaturation of a whey protein composition

It is known that the solubility of denatured whey protein at pH4.6 is lower than at pH values below or above pH4.6, and therefore the degree of 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 a pH at which the protein is stable in solution.

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) of the total amount of protein and having a pH of 7.0 or 3.0, and

-a second aqueous solution comprising 5.0% (w/w) of the total amount of protein and having a pH of 4.6.

The pH was adjusted using either 3% (w/w) NaOH (aq) or 5% (w/w) HCL (aq).

Total protein content (P) of the first aqueous solution_{pH7.0 or 3.0}) As determined by example 1.5. The second aqueous solution was stored at room temperature for 2h and subsequently 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 is calculated by the following formula:

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

Example 1.4: protein denaturation was determined using reverse phase UPLC analysis (pH4.6 acidic precipitation)

BLG samples (e.g., unheated reference 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 aggregate and precipitate at about 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 purified water (polished water).

For each sample, the same volume was injected into a UPLC system with 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 against a protein standard (Sigma L0130) was used to determine the concentration of native BLG in the sample (5 th order calibration curve)

If the linear range is exceeded, the sample is further diluted and re-injected.

Example 1.5: determination of the Total amount of protein

The total amount of protein (true protein) in the sample was determined by:

1) determination of the IDF 020-1/2-milk-nitrogen content according to ISO 8968-1/2| -part 1/2: the nitrogen content was determined using Kjeldahl method (Kjeldahl) to determine the total nitrogen content of the sample.

2) According to ISO 8968-4| IDF 020-4-determination of milk-nitrogen content-part 4: and determining the non-protein nitrogen content of the sample.

3) Use (m)_{Total nitrogen}–m_{Non-protein nitrogen}) 6.38 calculate total protein.

Example 1.6: determination of non-aggregating BLG, ALA, 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 into 2 TSKgel3000PWxl (7.8mm30cm, Tosohass, Japan) columns in tandem with an attached pre-packed column PWxl (6 mm. times.4 cm, Tosohass, Japan) equilibrated with eluent (consisting of 465g Milli-Q water, 417.3g acetonitrile and 1mL trifluoroacetic acid) and using a UV detector at 210 nm.

Comparison of the peak area obtained for the sample with the peak area of the corresponding standard protein 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 other proteins (non-BLG proteins) was determined by subtracting the amount of BLG from the total amount of protein (determined according to example 1.5).

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 place in3000IR turbidimeter. NTU values were measured after stabilization and repeated twice.

Example 1.8: determination of viscosity

The viscosity of the beverage product was determined by 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-shearing at a speed of 30 seconds, then carrying out an equilibration time of 30 seconds and at a speed of 1s^-1To 200s^-1To 1s^-1Is scanned.

Unless otherwise stated, viscosity is in 100s^-1In centipoise (cP) at shear rate. The higher the cP value measured, the higher the viscosityLow.

Alternatively, the viscosity was estimated using Gilson's Viscoman and taken at about 300s^-1Shear rate report of.

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 (55X14.2mm, VWR Cat. No. 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 at D65 and the field angle (observer) was set at 2 degrees. The color (CIELAB color gamut, a, b, L values) was measured with the cover suspension as the average of three individual readings at different positions of the culture dish.

The demineralized water reference values were as follows:

L*39.97±0.3

a*0.00±0.06

b*-0.22±0.09

the measurement is converted to a delta/difference based on the demineralized water measurement.

ΔL*＝L_{Samples normalized to 6.0 w/w% protein}*-L_{Demineralized water}Assay at room temperature.

Δa*＝a_{Samples normalized to 6.0 w/w% protein}*-a_{Demineralized water}Assay at room temperature.

Δb*＝b_{Samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}Assay at room temperature.

Samples were normalized to 6.0 w/w% protein or less.

L a b color gamut (also known as CIELAB gamut) is a unified color gamut 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 field, L denotes luminance (value from 0 to 100), and L is 0 for the darkest black and 100 for the brightest white.

Color channels a and b represent true neutral gray values at a 0 and b 0. The a-axis represents the green-red component, where green is the negative direction and red is the positive direction. b-axis represents the blue-yellow component, blue in the negative direction and yellow in the positive direction.

Example 1.10 beverage stability test/protein insolubles

A whey protein beverage composition is considered stable if the total amount of protein precipitated after centrifugation of the heated sample at 3000g for 5 minutes is less than 15%:

add approximately 20g of sample to the centrifuge tube and centrifuge at 3000g for 5 minutes.

Kjeldahl analysis of the proteins before centrifugation and the supernatants after centrifugation was performed to quantify the protein recovery, see example 1.5.

Protein loss was calculated by the following formula:

this parameter, sometimes referred to as the level of insoluble protein, 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 20g of sample (e.g., liquid sample or suspended powder sample) is placed in a centrifuge tube and centrifuged at 3000g for 5 minutes. Protein (P) before centrifugation according to example 1.5_{General assembly}) And the supernatant after centrifugation (P)_3000xg) Kjeldahl analysis was performed to quantify the protein recovery.

The amount of protein insolubles was calculated by the following formula:

example 1.11: sensory evaluation

Descriptive sensory evaluations were performed on the heat-treated beverage products. The beverage product is heated using a plate heat exchanger.

A 1 volume sample was mixed with a 1 volume water and compared to the unheated whey protein isolate, lactic acid and citric acid were also used to form a profile prior to the final taste stage:

categories	The attributes are as follows:
		fragrance	Whey, acid (sour milk products)
Basic taste	Sour and bitter
		Flavor (I) and flavor (II)	Whey, citric acid, lactic acid
Taste of the product	Dryness and astringency

The mouth of the subject was cleaned between each sample using crackers, white tea, watermelon and water.

A small cup is used to provide 15mL of test sample at ambient temperature (20-25 ℃).

Each test sample was given three times to 10 persons in three different areas, respectively, in a random order.

Attributes (see table above) are scored on a scale of 15cm, where 0 is low intensity and 15 is high intensity.

Statistical analysis was performed by "Panelcheck" software using a three-way ANOVA test, repeated several times. The samples were fixed and the panels were set at random.

Significance differences between the assessed samples were corrected using Bonferroni, which implies the lowest significance difference (pair-wise comparison of the groups associated with the letters).

Example 1.12: determining transparency from images

Photographs of beverage products were taken by placing samples into turbidity NTU measuring vials that contacted paper written with "lorem ipsum". Using a smartphone to take a picture of the vial, the inventors evaluated whether the text could be clearly observed through the vial.

Example 1.13: determination of ash content

According to NMKL 173: 2005 ash, gravimetric in food "determination of ash of food products.

Example 1.14: determination of the conductivity

The "conductivity" (sometimes referred to as "specific conductance") of an aqueous solution is a measure of the ability of the solution to conduct electricity. The conductivity can be determined, for example, by measuring the alternating current resistance of the solution between the two motors, the results of which are typically expressed in units of millisiemens per centimeter (mS/cm). The conductivity can be measured, for example, according to EPA (united states environmental protection agency) method No. 120.1.

Unless otherwise stated, the conductivity values referred to herein have been normalized to 25 degrees celsius.

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 to be measured is completely immersed. The electrodes are then agitated to remove any air remaining on the electrodes. The electrodes are then held stationary until a stable value is obtained and recorded from the display.

Example 1.15: determination of total solids of solution

The total solution solids can be determined according to NMKL110 second edition, 2005 (total solids (water) -gravimetric determination in milk and dairy products). NMKL is "Nordisk MetodikkomLefor"abbreviation of.

The water content of the solution can be calculated by subtracting the relative content of the total solids (% w/w) from 100%.

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 degrees celsius.

When the sample is a powder, 10g of the powder is dissolved in 90ml of demineralized water at room temperature with vigorous stirring. The pH of the solution was then measured at 25 ℃.

Example 1.17: determination of bulk and bulk Density

The density of the dry powder is defined as the relationship between the weight and volume of the powder and is analyzed under specific 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 dry powder sample is tamped in a measuring cylinder. After a specific number of taps, the volume of the product was read and the density calculated.

Three types of densities can be defined by this method:

Pour density, i.e. the mass of powder divided by the volume after transfer to a specified cylinder.

Loose density, i.e. the mass of the powder divided by the volume after 100 taps according to the conditions specified in the standard.

Bulk density, the powder mass divided by volume after 625 taps according to the conditions specified in the 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 volume meter, e.g.J.Engelsmann A.G..

The bulk and bulk densities of the dried products were determined by the following procedure.

Pretreatment:

the samples to be tested were stored at room temperature.

The sample is then thoroughly mixed by repeatedly rotating and inverting the container (to avoid breaking the particles). The container is not filled more than 2/3.

The process comprises the following steps:

100.0. + -. 0.1g of powder was weighed and transferred to a measuring cylinder. Reading volume V in ml₀。

If 100g of the powder could not be loaded into the cylinder, it was reduced to 50g or 25 g.

The cylinder was mounted on a Stampf volumeter and tapped 100 down. The surface was scraped off with a spatula and the volume V read in ml₁₀₀。

The number of taps was changed to 625 (including the 100 taps). After tapping, the surface was scraped off and the volume V read in ml ₆₂₅。

And (3) calculating the density:

bulk and bulk densities in g/ml were calculated according to the following formula:

bulk density of M/V

Where M is the sample weight in g and V is the volume in ml after 625 taps.

Example 1.18: determination of the Water content of the powder

The moisture content of the food product is according to ISO 5537: 2004 (determination of dried dry milk-moisture content (reference method)). NMKL is "Nordisk MetodikkomLefor"abbreviation of.

Example 1.19: determination of the amount of calcium, magnesium, sodium, potassium, phosphorus (ICP-MS method)

The total amount of calcium, magnesium, sodium, potassium and phosphorus was determined using the following scheme: the samples were first decomposed using microwave digestion and then the total amount of minerals was determined using an ICP instrument.

Equipment:

microwaves were from Anton Paar (Anton Paar) and ICP was Optima 2000DV from PerkinElmer Inc.

Materials:

1M HNO₃

2％HNO₃yttrium in (III)

At 5% HNO₃Suitable standard solutions of calcium, magnesium, sodium, potassium and phosphorus

Pretreatment:

0.2g of the powder sample or 1g of the liquid sample was weighed 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 instructions. The digestion tube after completion of digestion was placed in a fume hood, and the lid was removed to allow volatile fumes to escape.

Measurement process:

the pretreated samples were transferred to Digitube using known amounts of Milli-Q water. Adding yttrium into a digestion tube to dissolve the yttrium in 2 percent of HNO₃Is added (about 0.25mL per 50mL of diluted sample) and diluted with Milli-Q water to a known volume. Samples were analyzed on ICP according to the procedure described by the manufacturer.

10mL of 1M HNO3 and 0.5mL of yttrium in 2% HNO by using Milli-Q water₃The mixture of solutions in (1) was diluted to a final volume of 100mL to prepare blind samples.

At least 3 standard samples were prepared at the expected sample concentrations.

The detection limit of the liquid sample is: ca. Na, K and phosphorus were 0.005g/100g of sample, and Mg was 0.0005g/100g of sample. The detection limits of the powder samples were: ca. Na, K and phosphorus were 0.025g/100g of sample, and Mg was 0.0005g/100g of sample.

When phosphorus is at or below the detection limit, the values of the detection limit are used in the examples to illustrate the maximum amount of phosphorus present in the worst case.

Example 1.20: determination of the furosine value:

the determination of the furfuryl acid value and the determination of the total amount of protein are carried out as described in "evaluation of the Maillard reaction by furfuryl acid determination during infant cereal processing" in "Guarer-Hernandez et al, J.cereals 29 (1999) 171-176", and according to example 1.5. The furoic acid value is expressed in 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 in the pH range 5-6.

a) 10mL of the liquid sample to be tested was transferred to a Maxi-Spin filter with a 0.45 micron CA membrane.

b) The filter was immediately spun at 1500g for 5 minutes. The centrifuge was maintained at 2 deg.C

c) 2mL of cold Milli-Q water (2 ℃) was added to the retentate side of the spin filter and the filter was immediately spun at 1500g for 5 minutes while keeping the centrifuge cool at 2 ℃, the permeate (permeate A) was collected, the volume was measured and the BLG concentration determined by HPLC 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) Immediately spin the filter at 1500g for 5 minutes and collect the permeate (permeate B)

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 called m_{Permeate B}。

g) The crystallinity of BLG in a liquid is determined by:

Degree of crystallinity ═ m_{Permeate B}/(m_{Permeate A}+m_{Permeate B})*100％

Example 1.22: determination of the crystallinity of BLG in Dry powder

The method is used to determine the crystallinity of BLG in dry powders.

a) A5.0 g sample of the powder was mixed with 20.0g cold Milli-Q water (2 ℃) and allowed to stand at 2 ℃ for 5 minutes.

b) The liquid sample to be tested 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. The centrifuge was maintained at 2 deg.C

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, the BLG concentration was determined by HPLC using the method outlined in example 1.31, and the results were converted to the total weight of BLG rather than weight percent. The weight of BLG in permeate A is designated m_{Permeate A}。

f) The crystallinity of BLG in the powder was then calculated using the following formula:

degree of crystallinity (m)_{BLG assembly}-m_{Permeate A})/m_{BLG assembly}*100％

Wherein m is_{BLG assembly}The total amount of BLG of the powder sample in step a).

If the total amount of BLG in the powder sample is unknown, it can be determined as follows: another 5g sample of the powder (from the same powder source) was suspended in 20.0g Milli-Q water, the pH adjusted to 7.0 by the addition of aqueous NaOH, the mixture was allowed to stand at 25 ℃ for 1 hour with stirring, and finally the total amount of BLG was determined for the powder sample using example 1.31.

Example 1.23: determination of the osmotic conductivity of ultrafiltration

15mL of the sample was transferred to an Amicon Ultra-15 centrifugal filter unit that retained 3kDa (3000NMWL) and centrifuged at 4000g for 20-30 minutes until a sufficient amount of ultrafiltration permeate had accumulated at the bottom of the filter unit 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 powder

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 in demineralised water at a temperature of 4 ℃ in a weight ratio of 2 parts water to 1 part powder, gently mixed 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 crystal 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

The total amount of lactose is according to ISO 5765-2: 2002(IDF 79-2: 2002) "dried milk, dry ice mix and processed cheese-determination of lactose content-part 2: enzymatic methods that utilize the galactose moiety of lactose.

Example 1.26: determination of the total amount of carbohydrates:

the carbohydrate content was determined by using Sigma Aldrich total carbohydrate assay kit (cat 104-1KT), where carbohydrates were hydrolyzed and converted to furfural and hydroxyfurfural, which were then converted to chromogens and detected spectrophotometrically at 490 nm.

Example 1.27: determination of the Total amount of lipids

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

Example 1.28: determination of Brix (brix)

Brix was measured using a PAL-alpha digital hand-held refractometer (Atago) calibrated with purified water (poleshed water) (reverse osmosis filtered water, conductivity up to 0.05 mS/cm).

Approximately 500. mu.l of 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 (TS), which is about 0.85 brix (% w/w).

Example 1.29: determination of lactoferrin and lactoperoxidase

The concentration of lactoferrin was determined by an ELISA immunoassay outlined in Soyeurt 2012 (Soyeurt et al; mid-infrared prediction of lactoferrin content in cow's milk: potential indicator of mastitis; animal (2012), 6:11, pp 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

According to ISO 4833-1: 2013 (E): microbiology of food and animal feed-horizontal method of microbial enumeration-colony counting technique at 30 ℃ to determine the number of colony forming units per gram of sample.

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 such as ALA, BLG and CMP as well as other proteins in the composition. Unlike 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 515(Waters) with manual seal rinse

HPLC Pump controller Module II (Waters)

3. Automatic sample injector 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 bar SWxL (6.0x 40mm, P/N:08543)

7. Ultrasonic bath (Branson 5200)

8.25mm syringe filter with 0.2 μm cellulose acetate membrane. (514 0060, VWR)

The process comprises the following steps:

mobile phase:

A. stock buffer

1. 56.6g of Na were weighed₂HPO₄、3.5g NaH₂PO₄And 2.9g of EDTA were added to a 1000mL beaker. Dissolve 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, followed by 200mL of stock buffer (A)

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 protein (S1)

2. Pipette 200. mu. l S1 into a 20ml volumetric flask and dilute to the mark with mobile phase. This is a 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 a high working standard solution (high working standard solution) WS 5.

7. Using a calibrated disposable pipette, 1.5mL of WS1 to WS5 were transferred into individual vials. Add 10. mu.L of 2-mercaptoethanol to each vial and cap the vial. The solution was vortexed for 10 seconds. The standards were allowed to stand at ambient temperature for about 1 hour.

8. The standard solution was filtered using a 0.22 μm cellulose acetate syringe filter.

The purity of the protein was measured using kjeldahl method (N x 6.38.38) and the area% of WS5 as 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. An original sample of 25mg equivalents 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 a constant volume and 167. mu.L of 2-mercaptoethanol was added to 25ml of the 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 acetate syringe filter.

HPLC system/chromatographyColumn

Chromatographic column balance

1. The GPC protection column and two GPC analysis columns were connected in this order. The new column is usually loaded with phosphate buffer.

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 was gradually increased from 0.1 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 performed without waiting for each sample injection to be completed before the next sample injection.

6. Equilibrating with mobile phase for at least 1 hour.

Calculation results

The quantitative determination of the content of proteins to be quantified (for example. alpha. -lactalbumin,. beta. -lactoglobulin and caseinmacropeptide) is carried out by comparing the peak areas of the corresponding standard proteins with the peak areas of the samples. The results are expressed as g of the specific protein per 100g of the original sample, or as a weight percentage of the specific protein relative to the weight of the original sample.

Example 2: production of spray-dried acidic BLG isolate powder

Whey protein material

The UF retentate from the sugar deficiency in sweet whey from the standard cheese manufacturing process was filtered through a 1.2 μm filter and then subjected to lipid lowering using a Synder FR membrane and then used as a raw material for BLG crystallization process. The chemical composition of the starting material is shown in table a. It should be noted that all weight percentages of the specific proteins mentioned in this example (e.g. BLG, ALA) relate to the weight percentage of non-aggregated proteins relative to the total amount of protein.

Regulating

A46 mil spaced Koch HFK-328 type membrane (70 m) was used²Membrane), feed pressure of 1.5-3.0bar, adjusting the sweet whey feedstock under ultrafiltration conditions at 20 ℃ to a feedstock concentration of 21% Total Solids (TS) ± 5, and using purified water (water after reverse osmosis filtration to obtain a conductivity of at most 0.05 mS/cm) as 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/m². A first sample of the concentrated retentate was taken and centrifuged at 3000g for 5 minutes. The supernatant of the first sample was used to determine the BLG yield.

Crystallization

The concentrated retentate was transferred to a 300L crystallization tank where the concentrated retentate was seeded with pure BLG crystal material made from rehydrated spray-dried BLG crystals. Subsequently, the inoculated whey protein solution is cooled from 20 ℃ to about 6 ℃ in about 10 hours, allowing BLG crystals to form and grow.

After cooling, a sample of whey protein solution containing crystals (second sample) was taken and centrifuged at 3000g for 5 minutes to isolate BLG crystals. The supernatant and crystal particles from the second sample were subjected to HPLC analysis as described below. The crystallization yield was calculated as described below and determined to be 57%.

TABLE A chemical composition of the starting materials

BLG yield was determined using HPLC:

the supernatants of the first and second samples were diluted to the same extent by adding purified 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 injected into the HPLC system with Phenomenex5μm C4LC column 250x 4.6mm, Ea., and detection at 214 nm.

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, BLG peak areas could be directly compared to calculate relative yields. Since the crystals contained only BLG and the samples had all been treated in the same way, the concentration of alpha-lactalbumin (ALA) and thus the area of ALA in all samples should be the same. Thus, the ALA area before and after crystallization was used as a correction factor (cf) when calculating the relative yield.

The relative yield was calculated by the following formula:

acid dissolution of BLC crystals

The material remaining in the crystallization tank was separated using a decanter (decanter) (350g, 2750RPM, 150RPM differential, 64 intervals (spacer) and 75L/h feed flow) and the feedstock was mixed with refined water at a ratio of 1: 2, and mixing. The BLG crystals/solid phase in the decanter is then mixed with refined water to make it a more dilute slurry, and then phosphoric acid or HCl 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 Ultrafiltration (UF) setting as used to prepare the starting material 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 was found to reduce the microbial load from 137.000CFU/g before heat treatment to <1000CFU/g after heat treatment. Heat treatment did not cause any protein denaturation, and the intrinsic tryptophan fluorescence ratio (I330nm/I350nm) was determined to be 1.20, indicating the native configuration of the BLG molecule.

The BLG was dried on the spray dryer of the pilot plant 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 B. A sample of the dried powder was solubilized 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.

Composition of blg isolate 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: preparation of conventional whey protein beverage

A dry BLG protein isolate powder containing > 85% BLG (protein based) is dispersed in up to about 95% demineralized water required to achieve the desired final protein concentration.

An acidic BLG isolate powder was prepared as described in example 2, whereas a BLG isolate powder of pH 5.5 was prepared as described in example 7 of PCT/EP 2017/084553.

Dissolution of the BLG material may be accelerated by the addition of an acid (selected from more than one food grade acid, such as phosphoric acid, hydrochloric acid, citric acid, malic acid or salts thereof in dissolved or powdered form) as described in PCT/EP 2017/084553. If the pH is lowered after dissolution with the addition of acid, the pH preferably does not exceed the desired target pH (i.e., to avoid unnecessary acid and/or base titrations).

Optionally, minerals, sweeteners, flavors, stabilizers, emulsifiers or other components may also be added, including sources of fat and carbohydrates. The method used comprises the following steps:

if fat is contained, the oil is first heated to 70 ℃ in a water bath;

Mixing with emulsifiers (typically up to 0.2% w/w in the final formulation); for example, emulsifier Grindsted Citrem LR10, a citrate ester recommended for phosphate-free applications. Cooling to 60 ℃ (to avoid/reduce potential denaturation/aggregation upon mixing with protein);

slow mixing with preheated water (60 ℃);

add all pre-mixed powder raw materials (to avoid "fish eyes"); this includes premixing the carbohydrate and protein;

optionally, adding minerals (NaCl, KCl, CaCl)₂And MgCl₂) To achieve the required concentrations of Na, K, Ca and Mg (target values in the middle of the allowable range) for food products (FSMP) that meet specific medical objectives. Minerals are dissolved in demineralized water and added up to the desired concentrations of Na, K, Ca and Mg. Other food grade FMSP-compliant minerals may be further used and added in dissolved or powdered form;

if necessary, the pH is adjusted to the final pH using up to 10% NaOH or up to 10% phosphoric acid (or other food grade acid);

adding the remaining water to achieve the desired protein concentration; and

optionally, homogenizing the composition ('upstream homogenization');

heat treating the composition;

Optionally, homogenization ('downstream homogenization').

For comparison, the whey protein isolate replaced > 85% of the BLG product while the remaining steps were retained to prepare the reference sample.

The samples were stored in a dark environment at 20 ℃.

Example 4: heat treatment of whey protein compositions

Heat treatment of the beverage: the beverage was heated at 120 ℃ for 20 seconds (high temperature, short time (HTST) to obtain denatured BLG) or at 75 ℃ and held for 15 to 5 minutes (BLG left natural) using a plate heat exchanger (manufacturer: OMVE HTST/UHT pilot plant HT320-20) equipped with a 10 μm bonded ultra fine fiber (Microfiber) cartridge (code 12-57-60k) (flagship filter). Other heat treatment conditions may also be applied.

The heat-treated beverage composition was tapped into a 100mL sterile bottle at 75-85 ℃ and immediately sealed and placed on ice.

In other experiments, heat treatment was performed by transferring the whey protein source into thin-walled glass vials containing 15-30mL of the sample. The vials were soaked in a pre-equilibrated water bath at a target temperature range of 75 ℃ to 95 ℃ for 1 to 5 minutes and then cooled on ice.

Example 5: production of heat-treated beverage products

In this example, BLG beverages and WPI beverages containing 6% protein and having a pH of 3.7 were prepared.

BLG beverages were prepared as follows: BLG isolate powder (as described in example 7 of PCT/EP 2017/084553) at pH 5.5 was dissolved in demineralised water at 10 ℃. Slowly add 10% H to the solution₃PO₄. The final pH was adjusted to 3.7.

The solution was heat treated at 120 ℃ for 20 seconds or at 75 ℃ and held for 15 seconds to 5 minutes using a plate heat exchanger as described in example 4. The beverage was tapped to provide a heat sterilized whey protein beverage composition.

WPI beverages were prepared using the same procedure but from WPI powder.

The composition of the BLG powder used to prepare the beverage products is given in table 1 below, and the composition of the WPI is also listed for comparison.

Table 1. composition of BLG powder (pH 5.5 powder) and WPI powder.

A beverage product comprising BLG and WPI having a pH of 3.7 and a protein content of 6% w/w, wherein 95.9% w/w of the protein is BLG, is heat treated at 120 ℃ for 20 seconds and at 75 ℃ for 15 seconds. In the WPI beverage (WPI-B), 57 w/w% of the protein is BLG. Different samples were analysed for turbidity (example 1.7), viscosity (example 1.8) and colour (example 1.9).

The results are shown in figure 1 and table 2 below.

Table 2.

And (4) conclusion:

the turbidity of the BLG sample remained low at 75 ℃, while the WPI sample had high turbidity. The WPI samples were also opaque as shown in figure 1.

The sterilized BLG sample had a turbidity of 7.0NTU compared to WPI with a turbidity of 263 NTU.

The viscosity is also kept low.

It is therefore possible to produce a clear beverage with a BLG content of about 96 w/w% of the protein content at ph3.7, which cannot be achieved in the WPI sample because it becomes opaque under the same conditions.

Example 6 a: it was demonstrated that the pH range of clear whey protein beverages could be extended.

BLG samples were prepared in which about 92 w/w% of the 6 w/w% protein was BLG, and for comparison, two different WPI samples were prepared, which contained 60 w/w% (WPI-a) and 57 w/w% (WPI-B) BLG, respectively.

A 6 w/w% whey protein composition was prepared as described in example 3 (BLG isolate powder was prepared according to example 2) using 10% phosphoric acid to adjust the final pH to obtain a selected pH of 3.0 to 3.9, respectively. In one aspect of the experiment, samples adjusted to pH levels between 3.0 and 3.9 were UHT treated at 120 ℃ for 20 seconds, tapped, sealed, and cooled. In another aspect of this experiment, samples at pH3.0 and 3.9 were pasteurized at 75 ℃ for 15 seconds according to example 4. Different samples were analysed for turbidity (example 1.7), viscosity (example 1.8), colour (example 1.9) and visual appearance (example 1.12).

The results are shown in FIGS. 2-10.

As a result:

figure 2 shows an image of WPI-B with pH 3.0-3.7 heat treated at 120 ℃ for 20 seconds, and a BLG beverage with pH3.7 heat treated at 120 ℃ for 20 seconds. FIG. 3 shows images of WPI-B with pH 3.0-3.7 heat treated at 75 ℃ for 15 seconds and BLG with pH3.7 heat treated at 75 ℃ for 15 seconds. Figure 4 shows images of WPI-B at pH3.7 and BLG at pH3.9 heated at 75 ℃ for 15 seconds.

Surprisingly, the inventors have found that even when UHT sterilized at ph3.7 (fig. 2), or when pasteurized (fig. 3 and 4), can even exceed ph3.7(ph3.9-4.1), the BLG beverage product remains visually clear, while in this case the WPI is opaque. These findings are further supported by the turbidity measurements shown in fig. 5(UHT) and fig. 6 (pasteurization), which are below 40NTU at ph3.7 and 3.9, respectively, while WPI is well above 40 NTU.

The viscosity of the BLG beverage product remains low after UHT treatment. The low viscosity indicates that the beverage sample is ready to drink. The viscosity increased dramatically with WPI, especially at high pH (fig. 7).

The inventors further found that the yellowness (b-value) of heat-treated WPI beverages (UHT and pasteurised) containing low amounts of BLG far exceeded the BLG reaching at least ph3.7, see fig. 8(UHT) and fig. 9 (pasteurisation).

And (4) conclusion:

the use of whey protein beverage with at least 85% w/w protein to BLG ensures at least two significant opportunities to provide the consumer with a whey protein beverage of the desired attributes:

1. the pH is increased during heat treatment to provide improved visual effect (colour, haze) and viscosity compared to WPI.

2. Allowing pasteurization to maintain the advantages of 1) while further extending the achievable pH range.

Example 6 b: it was demonstrated that the pH range of clear whey protein beverages could be extended.

In this example, BLG beverages containing 6 wt% and 12 wt% protein at pH 2.7, 3.0, 3.3 and 3.7 were prepared according to example 3. Table 3 shows a BLG powder for preparing a beverage, which powder comprises 98.2 w/w% BLG protein. The contents of Na, K, Ca and Mg were below the detection limit.

Table 3 composition of BLG isolate powder (BDL ═ below detection limit)

The final pH of the beverage was adjusted to pH 2.7, 3.0, 3.3 and 3.7 using 1M phosphoric acid. The beverage was heat treated using a water bath at 75 ℃ or 95 ℃ for 5 minutes as described in example 4.

The turbidity (example 1.7) and protein naturalness determined by intrinsic tryptophan fluorescence emission ratio R ═ I330/I350 (example 1.1) of the different samples were analyzed.

Table 4 shows the results.

TABLE 4 analytical data for high protein beverages (6 and 12 w/w%) prepared from BLG at pH 2.7 to 3.7 heated at 75 ℃ and 95 ℃ for 5 minutes.

As a result:

the results clearly show that clear BLG beverages containing 6 w/w% or 12 w/w% protein with turbidity below 16NTU can be produced. The beverage is heat treated at 75 deg.C or 95 deg.C for 5 min. The beverage showed no visible aggregation or sedimentation. This was compared to the WPI sample of example 6a, see figure 4, which shows that the WPI sample at pH 3.7 (6 w/w% protein) was cloudy after heat treatment at 75 ℃ for 15 seconds.

Furthermore, it was surprisingly found that all beverages comprising 6 w/w% or 12 w/w% protein, having a pH of 2.7 to 3.7, heat treated at 75 ℃ for 5 minutes, have a predominant native configuration, as indicated by a surprisingly high tryptophan fluorescence emission ratio of 1.17-1.18.

Example 6 c: color stability of the beverage after 6 months of storage at 20 ℃.

In this example, BLG beverages containing 6 wt% protein at pH 3.0 and 3.7 were prepared according to example 3. Table 3 shows BLG powders used for preparing beverages.

The final pH of the beverage was adjusted to pH 3.0 and 3.7 using 10% phosphoric acid. The beverage was heat treated using a plate heat exchanger at 75 ℃ for 15 seconds or UHT at 120 ℃ for 20 seconds as outlined in example 4.

After heat treatment, the beverage was stored at 20 ℃ in the dark for 6 months.

The different samples were analysed for colour after day 0, 6 weeks, 3 months and 6 months (example 1.9).

The results are shown in FIGS. 25 and 26.

As a result:

the results show that the BLG beverage has a slower rate of color change during storage in the pasteurized (75 ℃/15s) samples compared to the UHT treated (120 ℃/20s) BLG beverage. It was surprisingly found that the colour (yellowness) of the pasteurised and UHT treated BLG beverage after 6 months of storage was even lower than the yellowness of the freshly prepared WPI beverages (WPI-a and WPI-B). Figures 8(UHT) and 9 (pasteurisation) show that freshly prepared WPI beverages have b values of 0.33-0.43 at pH 3.0 and 0.7-1.15 at pH 3.7. This allows a beverage comprising at least 6 w/w% BLG to be used as a protein source for whey protein beverages, in particular for the production of colourless beverages.

Example 7: preparation of heat sterilized high protein beverage using BLG

BLG samples were prepared in which about 92% w/w of the protein was BLG (0.42 w/w% ALA) and for comparison WPI samples were prepared using about 60 w/w% WPI-A in which the protein was BLG (8 w/w% ALA) and the WPI powder had a pH of 3.3.

The BLG isolate powder product (from example 2, powder pH 3.9) was dispersed in tap water to produce beverages with protein concentrations of 6.0, 10, 15, 20, 25 and 32 w/w% and pH adjusted to 3.7 using 10% phosphoric acid.

A BLG isolate powder (see Table 3 above) containing 98.2 w/w% BLG protein was dispersed in demineralized water to prepare a beverage having a protein concentration of 12 w/w%. The pH was adjusted to pH3.7 using 1M HCl.

The solution was heat treated at 75-120 ℃ for an incubation time of 15 seconds to 5 minutes and immediately cooled on ice as described in example 4 and table 5.

Different samples were analysed for viscosity (example 1.8), protein naturalness as determined by intrinsic tryptophan fluorescence emission ratio R ═ I330/I350 (example 1.1), visual appearance (example 1.12) and turbidity (example 1.7).

TABLE 5 analytical data for high protein beverages prepared from BLG at pH3.7 under heating conditions of 75 deg.C, 90 deg.C and 120 deg.C.

As a result:

the results are shown in table 5 above and in figures 10 to 12.

Figure 10 shows an image of a 15 w/w% BLG beverage heated at 75 ℃/15 seconds at ph3.7, which is clear and transparent (left), while 6% WPI-a heated at 75 ℃/15 seconds at ph3.7 (right) is opaque.

Fig. 11 shows sensory evaluation of high protein BLG beverage compositions, and images at pH3.7 of 6 w/w% and 15 w/w% BLG samples, both samples being clear.

Fig. 12 shows a high protein beverage preparation prepared by heating a BLG beverage at 75 ℃ for 5 minutes, all samples having a low viscosity and being liquid, the protein content of the BLG beverage being 32 w/w%, 27.5 w/w%, 25 w/w%, 20 w/w% (left to right).

The inventors have surprisingly found that even with heating at 75 ℃ for up to 5 minutes, all solutions retain a low viscosity, indicating little or no denaturation.

The viscosity observed at the typical high protein of non-aggregated native protein (flow behavior described by Inthavong, Kharlamova, Nicolai, Chassenieux & Nicolai, 2016) was about 10cP at 200 g/l.

The fluorescence spectra of the amino acids confirmed that the BLG retained its native configuration, e.g., intrinsic tryptophan emissivity (I330/I350) of at least 1.11 when heated slowly (75 ℃), whereas more severe heating resulted in denaturation, e.g., intrinsic tryptophan emissivity (I330/I350) of less than 1.11.

RP-HPLC analysis confirmed the tryptophan fluorescence results, revealing denaturation of 6% BLG beverages by heating at 75 ℃ for 5 minutes, and at 95 ℃ for 5 minutes for 41%.

The results show that the viscosity remains low even after heating.

It has been found that BLG beverage products can be heated above the denaturation temperature. However, heating at 95 ℃/5 minutes did result in gelation of BLG beverages containing more than 16 w/w% protein, while BLG beverages at 12 w/w% (95 ℃/5 minutes), 10 w/w% (90 ℃/5 minutes) and 6 w/w% (120 ℃/15 seconds) all remained liquid. At least partial denaturation/aggregation occurs under these heating conditions as evidenced by a reduction in intrinsic tryptophan emissivity (I330/I350).

However, it was found that when a BLG beverage product containing 12 w/w% protein was heat treated at 75 deg.C/5 minutes, its BLG retained its native configuration, as evidenced by an intrinsic tryptophan emission ratio (I330/I350) of 1.17, the beverage was also clear and liquid.

To the great surprise of the inventors, no significant difference in dry mouthfeel was found between the 6% BLG beverage product and the 15% BLG beverage product heated to 75 ℃ in the sensory group (see example 1.11 and fig. 11 for analysis).

Example 8: whey protein beverage product with improved taste

BLG samples and WPI samples were prepared. The composition of the samples is shown below.

The BLG isolate powder used was prepared by example 2.

	BLG	WPI-A
			BLG content w/w% of the protein	92	60
The ALA content of the protein w/w%	0.42	8
			pH of the powder	3.9	3.0

The samples were analyzed by 10 human sensory groups (see example 1.11). The WPI samples were more yellow and had higher b values, and they had higher turbidity compared to BLG beverages, especially at higher pH values. The analytical data are shown in table 3.

TABLE 6 analytical data for whey protein beverages prepared from BLG at pH3.0 and pH3.7 under heating at 75 ℃ and 120 ℃.

Turbidity (NTU) at 100s^-1Viscosity and color values b, L and a of (cP)

The visual appearance of the samples in table 6 is shown in fig. 13.

Data for sensory evaluation are shown in fig. 14-18.

The following formula is used for calculating Δ b:

Δb*＝b_{samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}Assay at room temperature.

The following formula is used for calculating Δ a:

Δa*＝a_{samples normalized to 6.0 w/w% protein}*-a_{Demineralized water}Assay at room temperature.

The following formula was used for calculating Δ L:

ΔL*＝L_{samples normalized to 6.0 w/w% protein}*-L_{Demineralized water}Assay at room temperature.

The color values of the demineralized water were:

l39.97, a 0 and b-0.22.

As a result:

by taking advantage of the opportunity to increase the pH and decrease the heating temperature while maintaining clarity and colorless properties, significant differences in taste were observed between beverages produced with WPI-a and BLG. The astringency, dry mouthfeel, sour taste, whey smell and citric acid taste of the BLG beverage were lower than those of the WPI beverage, as shown in fig. 14.

Fig. 15 shows that by raising the pH to 3.7 before heat treatment, sourness in the BLG beverage was reduced at both 120 ℃ and 75 ℃, while maintaining product clarity and low color. This cannot be done using WPI because, as can be seen in table 2 and figure 1, a clear and clear beverage cannot be produced at ph 3.7.

FIG. 16 illustrates that the astringency is significantly reduced when the temperature and pH are changed from pH3.0, 120 ℃/20 seconds to pH3.7, 75 ℃/15 seconds.

Figure 17 illustrates that by lowering the heating temperature from 120 ℃/20 seconds to 75 ℃/15 seconds, the dry mouthfeel is significantly reduced (native at 75 ℃ and denatured protein at 120 ℃).

Figure 18 illustrates that whey odor is reduced when BLG is maintained in its native state at pH3.7 using 75 ℃/15 seconds heating, under which conditions a clear, colorless WPI beverage cannot be produced.

It can also be seen from figure 3 that a clear WPI beverage could not be produced at a pH of 3.7 and heat treatment at 75 ℃/15 seconds.

Example 9 a: low color sweet BLG beverage product

A6% w/w BLG beverage was prepared, see BLG powder composition below. The beverage was prepared as described in example 3.

	BLG
		BLG content w/w% of the protein	92
The ALA content of the protein w/w%	0.42
		pH of the powder	3.9

The prepared BLG beverage contained 6% protein and had a pH of 3.7.

As the carbohydrate sucrose, 8 w/w% sucrose was used. Again, the test was performed with the high intensity sweetener sucralose. The sample was heat treated in a water bath at 93 ℃ for 4 minutes, after which it was cooled in an ice bath.

The different samples were analyzed for visual appearance (example 1.12), color (example 1.9), haze (example 1.7) and viscosity (example 1.8).

The results are shown in Table 7 below.

TABLE 7 addition of sucrose to 6 w/w% protein BLG samples

Using Viscoman

As a result:

it was found that a sweet BLG beverage can be produced using 8 w/w% sucrose as a sweetener and heat-treating it at 93 c for 4 minutes. The addition of 8 w/w% sucrose had only a weak effect on viscosity, turbidity and clarity (see table 5), and the color was not affected by the sucrose addition.

A BLG beverage with additives normally present in commercial beverages, e.g. in sports nutrition, is prepared comprising 6% w/w protein BLG, pH 3.7, heat treated at 75 ℃ for 5 minutes. See table 8 below for the composition.

TABLE 8 examples of commercial products

Composition (I)	Content (wt.)	Unit of
			BLG	660	g
Trisodium citrate	1.0	g
			100% sucralose	1.17	g
10% phosphoric acid	47	G
			Adding water to constant volume of 10kg	9.3	kg

TABLE 9 results for both formulations

As a result:

as can be seen from table 9, both the BLG beverage with the additive and the BLG beverage without the additive maintained low viscosity, transparency, and substantially no color.

Example 9 b: sweet low color clear BLG beverage

In this example, a BLG beverage containing 2 w/w%, 6 w/w% and 10 wt% protein, pH 3.7 was prepared as described in example 3. The BLG powder used to prepare the beverage contained 98.2 w/w% protein as BLG, see table 3 (example 6 b). In addition, the BLG beverage is sweetened using sucrose as a carbohydrate source, at a final concentration of 5 or 18 w/w% sucrose. The beverage was heat treated in a water bath at 75 ℃ or 95 ℃ for 5 minutes and then cooled on ice.

Further, a beverage comprising 10 w/w% protein, 17 w/w% carbohydrate and having a pH of 3.7 was prepared using the BLG powder in table 3. These samples were heat treated according to example 4 using a plate heat exchanger at 120 ℃ for 20 seconds.

Different samples were analysed for viscosity (example 1.8), turbidity (example 1.7), colour (example 1.9), protein naturalness (example 1.1) and protein insolubles (example 1.10) determined as intrinsic tryptophan fluorescence emission ratio R ═ I330/I350.

The results are shown in Table 10.

TABLE 10 Heat-treated clear sweetened protein beverage comprising BLG and pH 3.7.

As a result:

the results show that a stable (< 15% protein insolubles) 2 w/w% beverage containing 18 w/w% of carbohydrates in the form of sucrose can be prepared by heat treatment at 75 ℃ and 95 ℃ for 5 minutes at pH 3.7.

The viscosity and turbidity of the 2 w/w% BLG beverage was surprisingly low after heating at 75 ℃ and 95 ℃ and the beverage was colorless with Δ b values of 0.29 and 0.31, respectively. Heating at 75 ℃ produced a beverage consisting mainly of native whey protein, as evidenced by the ratio I330/350 of 1.19. It was further unexpectedly found that a predominantly native protein was also obtained by heating at 95 ℃ for 5 minutes, as indicated by a tryptophan fluorescence emission ratio of 1.13.

Table 10 further demonstrates that stable colorless BLG beverages of low viscosity and turbidity can be prepared with 6 w/w% protein (containing 5 or 18 w/w% sucrose) or even with 10 w/w% protein (containing 5 w/w% sucrose).

Furthermore, it was also demonstrated that the BLG of these beverages remained in the native state after heat treatment at 75 ℃ for 5 minutes, as indicated by a tryptophan fluorescence emission ratio of above 1.11, and that the beverages at least partially unfolded/aggregated after heating at 95 ℃ for 5 minutes, as indicated by a tryptophan fluorescence emission ratio of below 1.11.

For a 6 w/w% BLG beverage containing 17% sucrose, which was heat treated at 120 ℃ for 20 seconds by sterilization, it had both low viscosity and turbidity and was colorless, and its Δ b value was 0.47.

All beverages retained surprising clarity and yellowness was kept to a minimum even when the concentration of protein and carbohydrate sources was increased, see table 10. This clearly demonstrates that it is feasible to make BLG beverages in which the energy distribution of the carbohydrate is at most 33E% to at least 90E%.

Example 10 a: exemplary methods for transparent BLG beverage products comprising added minerals

The BLG powder used in this example had a pH of 5.5 and contained about 96% w/w protein as BLG (and 0.4% w/w protein as ALA).

An acidic BLG isolate powder was prepared according to example 2 and a beverage product was prepared according to example 5.

High temperature heat treated beverage product:

a 6% BLG beverage product was prepared at ph 3.7. Addition of liquid KCl and CaCl from 1M stock solution₂. They were heat-treated at 95 ℃ for 5 minutes.

As a result:

the results are summarized in table 11 below and fig. 19.

Fig. 19 shows a 6% BLG beverage with added minerals, pH3.7, heat treated at 95 ℃ for 5 minutes.

A: adding 0mM mineral

B: adding 15mM CaCl₂

C: adding 20mM KCl

D: adding 10mM KCl and 15mM CaCl₂

Heating at 95 deg.C for 5 min at pH3.7, adding minerals (0-20mM KCl, 0-15mM CaCl)₂Or 10mM CaCl₂And 10mM) of the BLG beverage product the turbidity remained below 30 NTU.

Gelation (cloudy gel) was observed upon addition of 30mM KCl.

Adding 20mM CaCl₂Gelation (clear gel) was observed.

The results clearly show that the effect of protein content on WPI is greater than the difference in minerals, as the amount of added minerals in table 10 greatly exceeds the difference between BLG and WPI products.

The samples in table 11 below remained clear (see fig. 19) and the viscosity was within the limits:

TABLE 11 BLG beverage additive ore Substance (CaCl)₂And KCl), the pH of which was 3.7, was heat-treated at 95 ℃ for 5 minutes.

Adding CaCl2,mM	Adding KCl, mM	Turbidity NTU	Viscosity, cP
				0	0	13.8	0.77±0.1
15	0	25.7	1.37±0.2
				0	20	19.6	1.08±0.1
10	20	23.9	1.44±0.3

Using Viscoman

Low temperature heat treated beverage product:

a 6% BLG beverage product was prepared at ph 3.7. Addition of liquid KCl and CaCl from 1M stock solution₂. They were heat-treated at 75 ℃ for 5 minutes.

As a result:

the inventors have surprisingly found that exceptionally high mineral concentrations are allowed when pasteurisation temperatures (75 ℃, 5 minutes) are used, see table 12 below.

Fig. 20 shows a 6% BLG beverage with added minerals, pH3.7, heat treated at 75 ℃ for 5 minutes.

A: adding 0mM mineral

B: adding 100mM KCl

C: adding 100mM CaCl₂

D: adding 100mM KCl and 100mM CaCl₂

Even if 100mM KCl or 100mM CaCl is added before heating₂The beverage product also remained clear when added to the beverage composition, see fig. 20. Furthermore, even if 100mM KCl and 100mM CaCl were added simultaneously₂The viscosity is also surprisingly low.

TABLE 12 addition of minerals (CaCl)₂And KCl) the viscosity and turbidity of the BLG beverage, which had a pH of 3.7, was heat-treated at 75 ℃ for 5 minutes.

Using Viscoman

Example 10 b: BLG beverage containing minerals

The BLG powder used in this example had a pH of 3.79 and contained about 98.2% w/w protein as BLG (see Table 3 of example 6 b). The contents of minerals sodium, potassium, calcium, magnesium and phosphorus in the powder are all below the detection limit.

High temperature heat treated beverage product:

according to the embodiment3, BLG beverage products containing 6 wt% protein at pH 2.7, 3.0, 3.3 and 3.7 were prepared. The BLG beverage comprises minerals NaCl, KCl, CaCl dissolved in demineralized water to 5, 3 and 1M, respectively, added from stock solution₂And MgCl₂. The mineral concentrations are shown in table 13 below.

Table 13 minerals added to BLG beverages.

24kcal/100 ml: the calculations are based on the following assumptions: protein 4kcal/g, fat 9kcal/g, carbohydrate 4kcal/g beverages were heat treated at 95 ℃ for 5 minutes using a water bath as outlined in example 4.

The different samples were analysed for turbidity (example 1.7), protein naturalness (example 1.1) and protein insolubles (example 1.10) determined as intrinsic tryptophan fluorescence emission ratio R ═ I330/I350.

The results are shown in Table 14 below.

Table 14: adding mineral (NaCl, KCl, CaCl)₂And MgCl₂) The turbidity, naturalness and insoluble particles of the resulting BLG beverage are heated at 95 deg.C for 5 minutes at a pH of 2.7 to 3.7.

As a result:

the results show that after a heat treatment at 95 ℃ for 5 minutes, a stable and clear acidic heat treated beverage can be prepared. The heat-treated BLG beverage comprises 6 w/w% protein and minerals (Na, K, Ca and Mg) and has a turbidity below 13NTU at pH 2.7 to 3.7 and a content of insoluble particles below 3%. This is possible even if the total amount of Na, K, Ca and Mg in the final product is 27.9-28.2mM (see mineral concentrations in Table 13).

Low temperature heat treated beverage product (added minerals):

preparation according to example 3BLG beverages containing 6 wt% and 12 wt% protein at pH 2.7, 3.0, 3.3 and 3.7. The BLG beverage comprises minerals NaCl, KCl, CaCl dissolved in demineralized water to 5, 3 and 1M, respectively, added from stock solution₂And MgCl₂. The mineral concentrations are shown in table 15 below.

Table 15: minerals added to BLG beverages.

24kcal/100ml and 48kcal/100 ml: the calculations are based on the following assumptions: protein 4kcal/g, fat 9kcal/g, carbohydrate 4kcal/g

The beverage was heat treated at 75 ℃ for 5 minutes using a water bath as outlined in example 4.

The turbidity of the different samples was analyzed (example 1.7) and the protein naturalness determined as intrinsic tryptophan fluorescence emission ratio R ═ I330/I350 (example 1.1).

The results are shown in Table 16 below.

Table 16: adding mineral (NaCl, KCl, CaCl)₂And MgCl₂See table 14) turbidity and naturalness of 6 w/w% and 12 w/w% BLG beverages, heated at 75 ℃ for 5 minutes, pH 2.7 to 3.7.

As a result:

the results show that after a heat treatment at 75 ℃ for 5 minutes, a clear acidic heat-treated beverage can be prepared. The beverage showed no visible aggregation or sedimentation. The heat-treated BLG beverage comprises 6 w/w% protein and minerals (Na, K, Ca and Mg) and has a turbidity of 10NTU or less than 10NTU at each pH of 2.7 to 3.7. This is possible even if the total amount of Na, K, Ca and Mg in the final product is 27.9-28.9mM (see mineral concentrations in Table 15).

It was surprisingly found that even in the case where the total amount of Na, K, Ca and Mg was 27.9-28.9mM (6 w/w% protein) and 50-52.6mM (12 w/w% protein), a natural (tryptophan fluorescence emission ratio of 1.17-1.20) and clear (less than 10NTU) BLG beverage could be prepared by heat treatment at 75 ℃ for 5 minutes, see Table 16.

It was also surprisingly found that a 12 w/w% protein BLG beverage had a turbidity of 2.8NTU at pH 3.0, even with a higher total 50.6mM Na, K, Ca and Mg mineral concentration (sample J), which was lower than the turbidity of 8.63NTU measured in a 6% WPI beverage at pH 3.0, which 6% WPI beverage contained lower concentrations of minerals (Na, K, Ca and Mg) at or below the detection limit, see table 6 of example 8. The detection limit of different minerals is Na: 0.005g/100g ═ 2.2mM, K: 0.005g/100g ═ 1.3mM, Ca: 0.005/100g ═ 1.2mM and Mg: 0.0005/100 g-0.2 mM, the total amount of which was 4.9 mM.

The surprising result provides a number of new possibilities for producing clear beverages containing additives, which additives (e.g. minerals) have an influence on the protein stability. By using a low heat treatment temperature of 75 ℃, the protein structure remains native and thus mineral tolerance is improved, since minerals increase/induce protein aggregation only when the protein is unfolded.

Example 11 a: whey protein milk beverage, high temperature heat treatment

An exemplary method of producing an opaque milky beverage comprising BLG and optionally a carbohydrate source. The BLG powder was dissolved in tap water and pH adjusted according to example 3 and heat treated at 93 ℃ for 4 minutes. The BLG beverage, which contained about 92% w/w protein as BLG and about 0.42% w/w protein as ALA, was produced based on an acidic BLG isolate powder with a pH of 3.9 (example 2).

A 6% BLG beverage with pH 4.3 was prepared. Sucrose 8% was added as a carbohydrate source. Turbidity, viscosity, color, clarity and beverage stability were measured according to the procedures described in examples 1.7, 1.8, 1.9 and example 1.10.

The results are shown in tables 17 and 18 below and in fig. 21.

TABLE 17 stability of milk beverages comprising BLG, heat treatment 93 deg.C/4 min, 6% protein, pH 4.3

Using Viscoman

TABLE 18 stability of milky BLG beverage containing sucrose, Heat treatment 93 deg.C/4 min, 6% protein, pH 4.3

A WPI sample containing 6% protein and pH 4.3 was prepared. The WPI samples were heat treated at 94 ℃ for 5 minutes. 0% sucrose or 8% sucrose was added to the WPI-a sample, while 0% sucrose or 6% sucrose was added to the WPI-B sample.

	BLG	WPI-A	WPI-B
				BLG content w/w% of the protein	92	60	8
The ALA content of the protein w/w%	0.42	57	10
				pH of the powder	3.9	3.0	6.8

As a result:

figure 21 illustrates the stability of a milky BLG beverage at pH4.3 heat treated at 93 ℃ for 4 minutes in the presence and absence of sucrose. A: 0% sucrose (before centrifugation), B: 8% sucrose (before centrifugation), C: 0% sucrose (after centrifugation), D: 8% sucrose (after centrifugation).

The results presented in tables 17 and 18 and figure 21 show that a high end point pH, e.g. pH4.3, enables the manufacture of a milky beverage, which is preferred in some embodiments of the invention, e.g. when the consumer prefers a milky whey protein beverage. It was also found that in the preparation with or without sucrose, the viscosity was low even at a pH of 4.3.

The color also remains neutral. This is particularly preferred by consumers who prefer a non-yellowing milky beverage. When b is high and positive, a yellowish coloration is seen.

The beverage was also found to be stable as evidenced by a < 15% reduction in protein and high turbidity after 5 minutes centrifugation at 3000x g.

Milky WPI beverages at 6 w/w% protein could not be produced based on WPI-a or WPI-B at pH4.3 because they would gel and therefore have a high viscosity, which is true for both WPI samples with and without sucrose addition.

Example 11 b: whey protein milk beverage, high temperature heat treatment

Carbohydrate-free high temperature treated milk beverage

The BLG powder used in this example had a pH of 3.79 and contained about 98.2% w/w protein as BLG (see Table 3 of example 6 b).

BLG beverages containing 6 wt% protein at pH 4.2 and 4.4 were prepared according to example 3 using 0.75M NaOH.

The beverage was heat treated at 95 ℃ for 5 minutes using a water bath according to example 4.

The different samples were analysed for turbidity (example 1.7), viscosity (example 1.8), protein naturalness (example 1.1), colour (example 1.9) and protein insolubles (example 1.10) determined as intrinsic tryptophan fluorescence emission ratio R ═ I330/I350.

The results are shown in Table 19 below.

Table 19: 6 wt% BLG milky beverage, pH 4.2 and 4.4, heat treated at 95 deg.C for 5 minutes.

Δ b was calculated using the following formula:

Δb*＝b_{samples normalized to 6.0 w/w% protein}*-b_{Demineralized water}Assay at room temperature.

Δ a was calculated using the following formula:

Δa*＝a_{samples normalized to 6.0 w/w% protein}*-a_{Demineralized water}Assay at room temperature.

Δ L was calculated using the following formula:

ΔL*＝L_{samples normalized to 6.0 w/w% protein}*-L_{Demineralized water}Assay at room temperature.

The color values of the demineralized water were:

l39.97, a 0 and b-0.22.

As a result:

the results shown in table 19 demonstrate that at high end point pH (e.g. even pH 4.4) a milky beverage can be produced, which is preferred in some embodiments of the invention, for example when the consumer prefers a whey protein beverage of milky appearance.

It has surprisingly been found that stable (protein insolubles below 5%) low viscosity (below 2cP) milky BLG beverages (turbidity at least 11000NTU) can be successfully prepared even at pH values of 4.4 when the beverage is heat treated at 95 ℃ for 5 minutes.

When the beverage was heat treated at 95 ℃ for 5 minutes, at least partial denaturation/aggregation occurred at pH 4.2 and 4.4, as evidenced by the intrinsic tryptophan emission ratio (I330/I350) of 0.99 at pH 4.2 and 1.01 at pH 4.4.

In contrast, example 11a describes that it is not possible to prepare a 6 w/w% protein creamy WPI beverage based on WPI-A or WPI-B with a pH of 4.3, since they would gel and therefore have a high viscosity.

High temperature treated milk beverage with carbohydrate source:

the BLG powder used in this example had a pH of 3.79 and contained about 98.2% w/w protein as BLG (see Table 3 of example 6 b).

BLG beverages containing 2 wt% and 6 wt% protein were sweetened according to example 3 using sucrose as carbohydrate source until the final sucrose concentration was 5 or 18 w/w% sucrose and the pH was adjusted to 4.2.

The BLG beverage was heat treated at 95 ℃ for 5 minutes using a plate heat exchanger, as outlined in example 4.

The different samples were analysed for turbidity (example 1.7), viscosity (example 1.8), protein naturalness (example 1.1), colour (example 1.9) and protein insolubles (example 1.10) determined as intrinsic tryptophan fluorescence emission ratio R ═ I330/I350.

The results are shown in Table 20 below. The energy percentage (% E) of protein and carbohydrate was calculated.

Table 20: BLG milky beverage containing 2 wt% and 6 wt% of carbohydrate, pH 4.2, heat treated at 95 deg.C for 5 minutes.

As a result:

the results shown in table 20 indicate that a high end point pH (e.g., pH 4.2) and a heat treatment at 95 ℃ for 5 minutes ensured the production of a milk beverage comprising protein and carbohydrate, wherein the carbohydrate comprises 45.5%, 75% and 90% of the total energy of the beverage. All three beverages had high turbidity above 1276NTU and low viscosity below 10.3 cP. The beverage was stable with less than 1.6% protein insoluble. All three beverages containing protein and carbohydrate developed at least partial unfolding/aggregation as evidenced by intrinsic tryptophan emission ratios (I330/I350) of 1.03 or less.

All beverages had a milky and opaque appearance.

Example 12 a: whey protein milk beverage, long-term low-temperature heat treatment

Exemplary methods of producing a milky beverage comprising BLG at different pH. The BLG powder was dissolved in tap water and the pH was adjusted to pH 4.2-4.5 using 10% phosphoric acid according to example 3. The preparation was heat treated at 75 ℃ for 5 minutes with a protein content of 6% w/w. The BLG beverage contained about 92% w/w protein as BLG and 0.42% w/w protein as ALA and was produced on the basis of BLG powder at pH 3.9.

Turbidity, viscosity, color and visual clarity were measured according to the procedures described in examples 1.7, 1.8, 1.9 and 1.12.

The results are shown in table 21 below and fig. 22.

Fig. 22 shows an image of an opaque 6% protein BLG beverage made by heating at 75 ℃ for 5 minutes at pH 4.2-4.5.

TABLE 21 Properties of opaque BLG beverages prepared at pH 4.2-4.5 and heating at 75 deg.C for 5 minutes

	pH 4.2	pH 4.5
Turbidity (NTU)	2489.6	4282.9
			Viscosity (cP)	0.954	0.943
L*	32.33±0.02	40.14±0.06
			a*	-0.23±0.05	-0.67±0.02
b*	-1.34±0.01	-3.42±0.01

As a result:

it was found that beverages with a pH of 4.2 to 4.5 had a milky and opaque appearance and high turbidity, while still having a low viscosity.

Example 12 b: whey protein milk beverage, long-term low-temperature heat treatment

In this example, BLG beverages containing 6 wt% protein at pH4.2, 4.4 and 4.6 were prepared as described in example 3. The BLG powder used to prepare the beverage contained 98.2% w/w protein as BLG (see Table 3 of example 6 b).

The final pH was adjusted to 4.2, 4.4 and 4.6 using 0.75M NaOH. The beverage was heat treated at 75 ℃ for 5 minutes using a water bath as outlined in example 4.

The different samples were analysed for turbidity (example 1.7), viscosity (example 1.8), amount of insoluble particles (example 1.10) and colour (example 1.9).

The results are shown in Table 21.

TABLE 21 Properties of opaque BLG beverages at pH 4.2-4.6 after heating at 75 deg.C for 5 minutes.

As a result:

it was surprisingly found that stable (< 7% insoluble particles) low viscosity milky beverages could be produced even with heat treatment at 75 ℃ for at least 5 minutes at pH4.2, pH4.4 and pH4.6 and that the stable low viscosity milky beverages exhibited lower viscosity even than the WPI-a and WPI-B beverages of pH 3.7 in example 6 a.

Example 12 c: whey protein milk beverage with carbohydrate, long-time low-temperature heat treatment

The BLG powder used in this example had a pH of 3.79 and contained about 98.2% w/w protein as BLG (see Table 3 of example 6 b).

BLG beverages containing 6 wt% and 10 wt% protein were sweetened using sucrose as the carbohydrate source until the final sucrose concentration was 5 or 18 w/w% sucrose. The pH was adjusted to 4.2 using 0.75M NaOH and the beverage was prepared as described in example 3.

The BLG beverage was heat treated at 75 ℃ for 5 minutes using a water bath as outlined in example 4.

Different samples were analysed for turbidity (example 1.7), viscosity (example 1.8) and protein insolubles (example 1.10).

The results are shown in Table 22 below. The energy percentage (% E) of protein and carbohydrate was calculated.

Table 22 BLG milky beverage containing 6 wt% and 10 wt% carbohydrate at pH 4.2 heated at 75 ℃ for 5 minutes.

As a result:

the results in table 22 demonstrate that at a high end point pH (e.g., pH 4.2), a creamy beverage comprising protein and carbohydrate can be produced by heat treatment at 75 ℃ for 5 minutes.

It has surprisingly been found that milky beverages are stable with insoluble particles below 0.7%. The milky beverage had a low viscosity at pH 4.2 and carbohydrates accounted for 33.3%, 45.5% and 75% of the total energy.

Example 13: colourless whey protein beverage containing > 85% BLG

A beverage product was prepared in which about 92% w/w of the protein was BLG and about 0.42% w/w of the protein was ALA (BLG isolate powder pH 3.9), see example 3.

For comparison, a whey protein sample (pH of SPI powder 6.7) comprising about 80% w/w BLG and about 4% w/w ALA SPI (serum protein isolate) was prepared.

The protein content of the sample was 6% w/w.

The pH of the beverage was adjusted to pH 3.7.

The turbidity, viscosity, color, clarity and beverage stability of the product were measured according to the procedures described in examples 1.7, 1.8, 1.9 and example 1.10.

The results are given in table 23 below and in fig. 23 and 24.

TABLE 23 Properties of BLG and SPI beverages under different Heat treatments

pH 3.7	BLG, No Heat treatment	SPI, no heat treatment	SPI 5 min at 75 DEG C	SPI 95 ℃ for 5 minutes
					Turbidity NTU	1.47	21.82	52.64	74.21
Viscosity (cP)	1.31	0.764	1.07	1.44
					L*	39.86±0.03	39.41±0.05	39.36±0.07	39.36±0.08
a*	-0.03±0.03	-0.30±0.01	-0.29±0.02	-0.28±0.02
					b*	-0.08±0.04	1.53±0.01	1.43±0.02	1.52±0.01

Viscosity was determined by Viscoman (example 1.8)

As a result:

it was found that SPI (about 80% BLG, about 4% ALA) increased more in viscosity due to heat treatment compared to BLG preparations at pH 3.7.

In addition, the b-value of SPI beverage was higher and therefore more yellow than the color of BLG sample.

Example 14 a: a nutritional whey protein beverage comprises more than or equal to 85% BLG, a carbohydrate source and a lipid source.

Example 14a describes an exemplary method of preparing a heat sterilized beverage product in which at least 85% w/w of the protein is BLG.

The inventors have surprisingly found that the addition of 100mM KCl and 100mM CaCl results from ₂The 6% nutritional composition of (b) remains liquid (viscosity about 1cP) even after heating at 75 ℃ for 5 minutes, so that the BLG beverage (. gtoreq.85%) receives a surprisingly large mineral concentration during the heat treatment by pasteurization at 75 ℃ (holding time up to at least 5 minutes).

Since the heat stability of whey proteins is often compromised by high mineral doses, we further investigated the possibility of producing a nutritionally complete acidic BLG beverage to produce a sterile nutritional beverage containing > 85% BLG, carbohydrates, fats and minerals, and the combination of which meets current FSMP (special medical food) requirements.

Proteins were solubilized and mixed with lipids and carbohydrates in the exemplified ratios according to the energy profiles described in table 24.

The food grade acids and minerals are selected to suit the food requirements for a particular medical purpose (FSMP).

Vitamins may also be provided in the beverage to meet the FSMP requirements and produce a nutritionally complete nutritional supplement.

Table 24: composition of an exemplary nutritional composition comprising protein, carbohydrate, and a fat source.

A6 w/w% BLG nutritional beverage further comprising 13.5 w/w% sucrose and 4.7 w/w% rapeseed oil was mixed at 70 ℃. The ingredients of protein, fat and carbohydrate are selected to suit medical nutritional recommendations.

In certain aspects, as shown in Table 24, (1)40mM KCl and 14mM CaCl₂Or (2)80mM KCl and 28mM CaCl₂With other components or (3) without minerals.

The solution was homogenized at 200 bar.

The solution was heat treated by immersion in a water bath at 75 ℃ or 95 ℃ for 5 minutes and cooled on ice.

Table 25: nutritional composition comprising BLG, a carbohydrate source, a fat source and added minerals.

And (4) conclusion:

it was found that an opaque beverage can be produced using BLG in combination with a fat and carbohydrate source, heated at 75 ℃ and 95 ℃.

At 75 ℃ it remains in the native state (tryptophan fluorescence ratio of 1.18 despite its fat content), whereas at 95 ℃ denaturation is caused (tryptophan fluorescence). The viscosity remains low. Since it can maintain its natural configuration, it can manage minerals that are critical to medical nutrition (FSMP requirements). Furthermore, the ability of the nutritional composition to remain in a liquid state in the presence of selected minerals clearly demonstrates its feasibility for use in medical nutrition.

Example 14 b: a nutritional whey protein beverage comprising a carbohydrate source and a lipid source and added minerals.

Example 14b describes a method of producing a creamy beverage comprising BLG meeting FSMP requirements, a carbohydrate source, a fat source, and added minerals.

Since the heat stability of whey proteins is often affected by high doses of minerals, this example investigated the opportunity to prepare a nutritionally complete acidic BLG beverage meeting current FSMP (food for special medical purposes) requirements by sterilizing a nutritional beverage comprising 6-16.7% w/w BLG source (98.2% purity) and further comprising 2.7-7.3% w/w fat source (rapeseed oil) and 7.5-18% w/w sucrose (as described in table 26 below).

Table 26 also shows the pH and mineral composition using food grade acids and salts to reach pH 3.5 and the levels of Na, K, Ca and Mg in the final beverage. Vitamins may be further provided to ensure that the BLG beverage meets FSMP requirements to produce a nutritionally complete nutritional supplement.

The BLG powder used in this example had a pH of 3.79 and contained about 98.2% w/w protein as BLG (see Table 3 in example 6 b). The BLG powder contained less than detectable levels of the minerals sodium, potassium, calcium, magnesium and phosphorus.

Table 26: minerals added to BLG beverages.

The measured contents of Na, K, Mg and Ca in the terminally heat-sterilized nutritional compositions shown in Table 26 (ICP-MS, example 1.19) met the FSMP requirements for a total of 131-. These results clearly demonstrate the feasibility of using BLG in medical nutrition.

A nutritional beverage composition was prepared as described in example 3 and the vials were heat treated by immersion in a water bath at 75 ℃ for 5 minutes according to example 4.

Different samples were analysed for viscosity (example 1.8), turbidity (example 1.7), colour (example 1.9) and protein-insoluble (example 1.10) determined as intrinsic tryptophan fluorescence emission ratio R ═ I330/I350 protein naturalness (example 1.1) and protein insolubles.

The results are shown in Table 27 below.

TABLE 27 mineral addition (NaCl, KCl, CaCl)₂And MgCl₂) Thereafter, the viscosity, turbidity, color, naturalness and protein insolubles of the BLG beverage were heated at 75 ℃ for 5 minutes at pH 3.5.

As a result:

it was surprisingly found that a milk-like beverage can be produced using a BLG source comprising more than 98.2% protein as BLG, in combination with a fat and carbohydrate source, by heating at 75 ℃ for 5 minutes. This is possible even if large amounts of Na, K, Ca and Mg are present and the total concentration of these minerals is 130-167 mM.

At 75 ℃, BLG remains in its native state (tryptophan fluorescence emission ≧ 1.17) and, despite the addition of large amounts of minerals, stable milky (turbidity >5000NTU) beverages with surprisingly low levels of protein insolubles (< 1% protein insolubles after 3000x g centrifugation) and low viscosities were prepared. As the total dry matter amount increased (16.7% bLG, 3.34% fat, 10% sucrose), the viscosity increased slightly to 14.2 cP. The beverage is substantially colorless.

Example 15: low-phosphorus protein beverage

Four samples of low phosphorous beverage were prepared using the purified BLG product of example 3 (crystalline product obtained from PCTEP2017/084553, raw material 3). All the dried ingredients were mixed with demineralized water to obtain 10kg of each sample and hydrated at 10 ℃ for 1 hour.

The samples were placed at 90 ℃ for 180 seconds and aseptically filled into sterile containers.

The packaged beverage has a shelf life of at least 1 year at ambient temperature.

All ingredients used to prepare 5 beverages had a low phosphorus content, so the phosphorus content of the obtained beverages was well below 80mg/100g protein. Therefore, these four beverages are suitable for use as protein beverages for patients with kidney disease.

Low phosphorous BLG beverage comprising carbohydrate and fat:

we have also shown that beverages with very low phosphorous content (less than 1mg phosphorous/g protein) in the final beverage can be produced using BLG protein powder comprising at least 85 w/w% BLG (which is particularly low in phosphorous content, table 3) and a source of sucrose and fat (as shown in table 28) which also contains low levels of phosphorous. This is demonstrated over a large protein concentration range from 6% w/w to 20% w/w BLG.

TABLE 28 energy and phosphorus content in exemplary BLG beverages particularly suitable for patients with renal disease. The asterisks indicate the phosphorus content in the exemplary BLG beverage calculated based on the maximum possible phosphorus content in the used BLG powder.

These BLG beverages are particularly suitable for delivering protein, fat and carbohydrate nutrients to renal patients due to the low phosphorus content of all the raw materials used, and the relatively low phosphorus content of the final beverage, which is less than 80mg/100g protein (see table 28).

Example 16 a: milky whey protein beverage containing minerals and heat-treated at low temperature

The BLG powder used in this example had a pH of 3.79 and contained about 98.2% w/w protein as BLG (see Table 3 of example 6 b). The contents of minerals sodium, potassium, calcium, magnesium and phosphorus in the powder are all lower than the detection limit.

BLG beverages containing 6 wt% protein at pH 4.2, 4.4 and 4.6 were prepared as described in example 3. The BLG beverage contains minerals Na, K, Ca and Mg, and the mineral concentrations are shown in table 29 below.

Table 29: mineral concentration of BLG beverage

The beverage was heat treated at 75 ℃ for 5 minutes using a water bath as outlined in example 4.

Different samples were analysed for turbidity (example 1.7), viscosity (example 1.8), protein insolubles (example 1.10) and colour (example 1.9).

The results are shown in Table 30 below.

Table 30: turbidity, viscosity, protein insolubles and color of BLG beverages at pH 4.2, 4.4 and 4.6 after addition of minerals (Na, K, Ca and Mg) and heating at 75 ℃ for 5 minutes.

As a result:

it was found that by heat treatment at 75 ℃ for 5 minutes at a pH of 4.2 to 4.6, a stable (0-4% protein insoluble) and natural (tryptophan fluorescence emission ratio of 1.16 to 1.17) BLG beverage having a low viscosity (0.7-1.5cP) and a low negative yellowness (. beta.) could be prepared even at a higher mineral content, wherein the total amount of Na, K, Ca and Mg was 29.1-35.3mM (table 29).

Example 16 b: milky whey protein beverage containing minerals and carbohydrates and heat-treated at low temperature

The BLG powder used in this example had a pH of 3.79 and contained about 98.2% w/w protein as BLG (see Table 3 of example 6 b). The contents of minerals sodium, potassium, calcium, magnesium and phosphorus in the powder are all below the detection limit.

A BLG beverage containing 6 wt% and 10 wt% protein at pH 4.2 was prepared as described in example 3. The BLG beverage contains minerals Na, K, Ca and Mg, and the mineral concentrations are shown in table 31 below. The beverage further comprises 5 wt% or 18 wt% sucrose.

Table 31: mineral concentration of BLG beverage

The beverage was heat treated at 75 ℃ for 5 minutes using a water bath as outlined in example 4.

Different samples were analysed for turbidity (example 1.7), viscosity (example 1.8) and protein insolubles (example 1.10).

The results are shown in Table 32 below.

Table 32: turbidity, viscosity and insoluble particles of BLG beverage at pH 4.2 after addition of minerals (Na, K, Ca and Mg) and carbohydrates, heated at 75 ℃ for 5 minutes.

As a result:

it has surprisingly been found that a milky beverage containing sucrose and minerals (Na, K, Ca and Mg components in a total amount of 29.1-35.3mM) is produced. These beverages have low viscosity (below 3.6cP) and high stability (< 1.4% insoluble particles).