WHEY PROTEIN BEVERAGES HAVING REDUCED ASTRINGENCY

Technical field of the invention The present invention relates generally to the field of whey proteins beverages and methods for the manufacture of such beverages. In particular, the present invention relates to stable and sterilized whey protein beverages with reduced astringency. The invention further relates to a method for manufacturing of whey protein isolate and use thereof in whey protein beverages.

Background of the invention

Whey protein is a protein fraction obtained from the milk of domesticated ruminants, such as cows, sheep and goats. Cow's milk contains two major protein fractions, viz., casein, which provides about 80 % by weight of the total protein, and whey protein, making up about 20% by weight of the total protein.

Whey protein is a source rich in essential branched chain amino acids (BCAAs), containing the highest known levels of any natural protein resource. BCAAs are important for athletes, because they are - unlike other essential amino acids - are metabolised in the muscle tissue.

Furthermore, because whey protein is commercially available in products containing small amounts of fat and carbohydrates, i.e. whey protein isolate, it may be a particularly valuable source for individuals with special nutritional needs e.g. for lactose intolerant individuals. In this way whey protein can be a valuable component of a diet program.

Given the advantages of whey protein, it has become a popular ingredient in the form of whey protein supplemented candy bars, whey beverages, and in whey protein powders. Ready-to-drink whey protein beverages have been on the marked for many years. However, astringency has been the major obstacle for the manufacture of acid whey protein beverages resembling convenient carbonated beverages that have granted shelf life of at least six month at ambient

temperature. Astringency implies a puckering or mouth drying sensation, which appears after a while in the mouth cavity following the intake of such a beverage.

Due to the prerequisite of at least six months shelf life, whey protein beverages need to undergo a sterilization treatment to remove spores and microorganisms causing spoilage of the milk products preferably by using heat or by using germ filtration techniques, such as ultrafiltration, diafiltration or nanofiltration. Preferred treatments include heat treatments at high temperatures for a relatively short range of time, such as using L)HT heat treatment (Ultra High Temperature). Employing L)HT unfolds the whey proteins and part of their core structure

(hydrophobic) will be exposed to the watery environment. At pH above 3.0 it is likely, due to hydrophobic interaction, that proteins will agglomerate and be building huge structures, which are recognized as haze or turbidity. Ultimately agglomerates will sediment and the beverage becomes cloudy and unacceptable to the average consumer.

In order to avoid the agglomeration and sedimentation of whey proteins and thus to obtain a palatable and transparent beverage, the pH of traditional whey protein beverages should under normal circumstances be no higher than pH 3.

It is assumed that astringency shows an optimum around pH 3.0 (Schneider and Foegeding, 2006; Beecher and Foegeding, 2006). The exact mechanism of astringency with respect to whey proteins is not fully understood. Salivary flow rate, viscosity and pH are a few variables that have been reported to be able to alter the perceived astringency of red wine, tannic acid, alum and cranberry juice.

Whey protein beverages become at a very low pH tart; consequently, a large quantity of sugar is needed to cover the tartness and sweeten the drink. Therefore, in order to achieve an acceptable drink at a low pH, if it has a high protein content, addition of sugar in high concentrations is needed.

Thus, it is desirable to be able to manufacture less sour beverages in combination with being able to add less sugar, but also in order to widen the range of possible additions of suitable flavours, colours and other pH-sensitive compounds such as pharmaceuticals.

Whey protein foods and beverages are increasing steadily in the nutritional market segment. It is therefore important to be able to control protein sedimentation, which may influence the overall flavour, palatability and appearance of the food or beverage. In the prior art there are suggestions for the improvement of transparency and palatability of whey protein beverages having a pH above 3.0. Some authors suggest solving the problem with transparency and palatability by avoiding sedimentation of the whey proteins after heat treatment during the manufacturing of the beverage.

For example, WO 2005/110112 discloses a method for the preparation of whey protein where the environment in which whey proteins are desorbed from the cation exchange column is controlled whereby a decreased denaturation of the protein is obtained, resulting in whey proteins which are soluble across a broad pH range. Moreover, Etzel (2004) suggests addition of food grade lauryl sulphate (16 g/l) to the beverages having a pH between 3.0 and 4.5 and a protein content of 25g/l in order to prevent aggregation of the denatured protein formed during thermal processing.

Etzel discloses protein solutions that are pictured transparent, but there is no discussion or mention of astringency. Schneider and Foegeding (2006) suggest to minimize astringency by increasing the pH of the beverage or to use a less heat sensitive protein source and/or to use molecular aggregation blockers such as Thioflavin-T and Quercetin. In Bleecher and Foegeding (2006) it is concluded that astringency decreases as pH is lowered due to the decrease in attractive charge between the whey proteins and saliva proteins and thus it is suggested controlling the net charge on whey proteins via modification with a reducing sugar or altering the ionic environment.

However, none of the methods of the prior art succeeds in providing a solution to the astringency problem in whey protein beverages having a pH above 3.0. Accordingly, methods for overcoming the problem of astringency, agglomeration and turbidity in whey protein beverages would be desirable.

In more detail, a more efficient and/or reliable method for preparing whey protein beverages in order to be able to produce stable, non-astringent and sterilized whey protein beverage with a wide range of different colours and flavours would be advantageous.

Summary of the invention

Apparently, an object of the present invention relates to ways of avoiding or controlling astringency in sterilized whey protein beverages, such as heat-treated whey protein beverages. In particular, it is an object of the present invention to provide a whey protein beverage with reduced astringency and in which at the same time it is possible to increase pH above 3.0.

Thus, one aspect of the invention relates to a whey protein beverage having a pH between 1.0 and 5.0, comprising 0.5 % - 15 % by weight whey protein and a shielding agent.

In a second aspect there is provided a whey protein beverage having a pH between 1.0 and 5.0, comprising 0.5 % - 15 % by weight whey protein and a shielding agent, said beverage has an astringency value which is 4 or less.

Another aspect of the present invention relates to a method for the manufacture of a whey protein beverage having a pH between 1.0 and 5.0, said method comprising the steps of a) providing a liquid whey protein solution having a protein concentration of between 1 and 15 % by weight of the beverage; b) adjusting the pH of the liquid whey protein solution to between 1.0 and 5.0 by use of an acidulant or alkali; c) adding to the pH adjusted whey protein solution of step b) a shielding agent in an amount between 0.05 and 1% by weight of the beverage to obtain a whey protein composition; and d) subjecting the whey protein composition obtained in step c) to a sterilization treatment.

A further aspect relates to a method for the manufacture of a whey protein beverage having a astringency value is 4 or less at a pH between 1.0 and 5.0, said method comprising the steps of a) providing a liquid whey protein solution having a protein concentration of between 1 and 15 % by weight of the beverage;

b) adjusting the pH of the liquid whey protein solution to between 1.0 and 5.0 by use of an acidulant; c) adding to the pH adjusted whey protein solution of step b) a shielding agent in an amount within the range of from 0.05% to 1% by weight of the beverage to obtain a whey protein composition; and d) subjecting the whey protein composition obtained in step c) to a heat treatment resulting in the whey protein beverage.

Further aspects relates to whey protein beverages obtainable by the methods of the invention having an astringency value which is less than 4.

Yet another aspect of the present invention is to provide a method for the manufacturing of a whey protein isolate, the method comprising the steps of: i) microfiltration of liquid whey obtaining a permeate; ii) ultrafiltration of the permeate from step i) obtaining a concentrated retentate; iii) adjustment of the pH of the concentrated retentate from step ii) to a pH value below 3.8 using an acidulant; iv) ultrafiltration and diafiltration of the pH-adjusted retentate from step iii) at a pH value below 3.8 to obtain a whey protein isolate; and v) germ- filtration of the whey protein isolate obtained in step iv) on a ceramic membrane to obtain a whey protein isolate.

Further aspects relate to a whey protein isolate obtainable by the method according to the invention and a whey protein composition comprising the whey protein isolate according to the invention.

In even further aspects there is provided a method for obtaining an astringency value which is less than 4 in a whey protein beverage having a pH between 1 and 5, comprising the steps of adding a shielding agent in an amount within the range of from 0.05% to 1% by weight of the beverage to a whey protein solution having a protein concentration of between 1 and 15 % by weight of the beverage, and a whey protein beverage obtainable by the method of the present invention having an astringency value which is 4 or less.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions are defined as follows:

The expression "astringency" is often listed as a flavour defect or off flavour. But astringency is more a sensation in the mouth than a flavour. The term "astringency" is used in the present context for a puckering or mouth drying sensation, which appears after a while in the mouth cavity after the consumption of a food. This sensation "astringency" is also characterized by terms such as chalky, rough, dry, mouth coating, or filmy mouth-feel, suggesting finely divided insoluble particles in the mouth after consumption of a food. Thus, astringency is not a taste, but a physical mouth-feeling and time depended feeling in the mouth cavity. In the same sense the term "non-astringent" is used, i.e. when no puckering or mouth drying sensation can be observed in the mouth cavity when consuming a food product. The "astringency value" may, as described in the Examples below, be determined or measured by a trained tasting panel following conventional specific sensory methods or by the analytical method such as the "Saliva-Beverage Interaction test" as described in the below Examples.

In the present context, the term "whey protein isolate (WPI)" is used for the dry portion of whey obtained by the removal of non-protein constituents from whey so that the dry whey product contains more than 90% whey protein on DS% basis. The term "whey protein concentrate (WPC)" relates to the dry portion of whey obtained by the removal of sufficient non-protein constituents from whey so that the dry product contains between 34% and 85% protein. "Whey" is a collective term referring to the serum that remains after the manufacture of cheese or caseinates. The milk may be from one or more domesticated ruminants, such as cows, sheep, goats, yaks, water buffalo, horses, or camels. "Whey protein" is the term for a collection of globular proteins that can be isolated from liquid whey. For example, a cheese whey may typically contain a mixture of beta-lactoglobulin (~42%), alpha-lactalbumin (~22%), and GMP (glycomacropeptide) (~22%), which are soluble in their native forms, independent of pH.

In the present context, the expression "shielding agent" encompasses food grade organic compounds which may be amphiphilic surfactants, i.e. they posses both a hydrophobic group (their "tails") and hydrophilic groups (their "heads"). The shielding agents are capable of shielding the hydrophobic exposed parts (as a result from the heat treatment of the whey protein beverage) of the whey proteins and thus preventing hydrophobic interactions between whey proteins and proline rich proteins (PRP) naturally secreted in the mouth. Thus, the shielding agents according to the invention are soluble in both organic solvents and water. Accordingly, the shielding agents are not used as traditionally surfactants or emulsifiers, which could be saccharide-containing compounds (e.g. guar gum), for making it possible to mix oil or fat with water, but are acting with the protein and/or protecting the protein in the whey protein beverage of the invention.

"Transparency" is measured at 500 nm wavelengths in a cuvette with 1 cm path length utilizing standard spectrophotometer and/or alternative instrument, taking into consideration recommendations of a manufacturer for the optimum use of such instrument. 100% transparency is assigned to demineralised water.

Preferred embodiments

As shown in the below example, the use of an shielding agent resulted in an elimination or a significant reduction of astringency to an astringency value which is 4 or less in whey protein beverages having a pH above 3.0. Thus, it appeared, that the problems normally connected to whey protein beverages at pH between about 3.0 and 4.6, when sterilized, such as when heat treated, could be solved by the present invention.

Accordingly, in a first aspect there is provided a whey protein beverage having a pH between 1.0 and 5.0, comprising 0.5 % - 15 % by weight whey protein and a shielding agent.

In a second aspect there is provided a whey protein beverage having a pH between 1.0 and 5.0, comprising 0.5 % - 15 % by weight whey protein and a shielding agent, said beverage has an astringency value which is 4 or less.

As described in the example below, the astringency value may be determined or measured by a trained tasting panel following specific conventional sensory methods or by the herein described "Salvia-beverage interaction test". In the Example below the method of Sano et al. (2005) is used with minor modification, namely using black tea instead of tannic acid as a standard sample having an astringency value of 6-8. The method uses a scale from 1 to 10, where 10 is given for extreme astringency and 0 is given for no astringency. In accordance with the present invention, the astringency value is 4 or less. However, in preferred embodiments, the astringency value is 3 or less, such as 2 or less, including 1 or less or even an astringency value of 0.

Whey proteins useful in the present invention are whey proteins which show solubility over a wide pH-range, heat stability in solutions at low pH and which produce clear solutions with low viscosity. Furthermore, useful whey proteins must show a neutral taste. Presently preferred is whey protein isolate. Whey protein isolate may be produced by any known methods in the art. However, the manufacture of a useful, modified whey protein isolate is described below. One example of such a modified whey protein isolate is LACPRODAN ^® - DI9213, which is available from ArIa Foods Ingredients, Denmark.

However, soybean proteins may also be useful in the present invention. Similar to whey protein beverages, soybean-based protein beverages have increased in popularity as the availability of such products increases and improvements in organoleptic properties for such beverages occur. Currently, however, there are certain limitations present for widespread acceptance to consumers, primarily in terms of flavour and other aesthetic characteristics. Like in whey protein beverages, the main problem in soy beverages exits when the pH level is adjusted to a pH value of above 3 for enabling a successfully addition of organoleptic enhancers, such as flavourings and colorants. At such a specific pH level, the soy proteins are prone to thermal denaturation, leading to significant and highly undesirable aggregation or clustering of the protein molecules and resulting in the same undesirable sedimentation from solution as in whey protein beverages. As a result, an off-note, or bitter taste or astringency of soy protein beverages occurs. Thus, it is presently believed, that the use of a shielding agent in accordance with

the invention will result in elimination or a significant reduction of astringency or bitterness in soybean-protein beverages.

As described above, the exact mechanism of astringency with respect to whey proteins is not fully understood. However, the present inventors observed that when using a shielding agent as defined herein in whey protein beverages the astringency could be controlled or eliminated. Without being bound by theory, it is presently contemplated, that by shielding the hydrophobic exposed parts of the whey proteins after a sterilization treatment, such as a heat treatment, e.g. L)HT, using a shielding agent hydrophobic interactions between whey proteins and proline rich proteins (PRP) naturally secreted in the mouth will be obstructed or the interaction is weakened and allow for pH values above 3.0 of the whey protein final beverage without turbidity nor astringency. In addition the present inventors observed that the astringency may be reduced when sediments between PRP and whey protein quickly were solubilised in the mouth cavity, and thus controlling astringency in terms of time response.

The absence of astringency and raising the pH is desirable, because it provides the opportunity to manufacture less sour beverages with a widened range of suitable flavours, colours and/or pharmaceuticals. Implied in this possibility is that astringency may be reduced or eliminated at higher pH values.

Thus, in preferred embodiments, the whey protein beverage has a pH which is between 1.5 and 4.5, including between pH 2.0 and pH 4.0, such as between pH 3.0 and pH 4.0, e.g. between pH 3.0 and pH 3.5, including between pH 3.5 and pH 4.0, such as between pH 3.5 and pH 4.5. In presently preferred embodiment the pH is around 3.5, such as pH 3.5. In further embodiments, the whey protein beverage has a pH which is above 2.0, such as above pH 2.5, e.g. above pH 3.0, including above pH 3.2, such as above pH 3.5, e.g. above pH 3.7, including above pH 4.0, such as above pH 4.5.

It has been observed that whey protein beverages containing more than 1% protein will show astringency. In preferred embodiments, the whey protein beverage is one wherein the whey protein content is between 1.0% and 10.0% by weight of the beverage, such as between 1.5% and 9.0% by weight, including

between 2.0% and 8.0% by weight, e.g. between 2.5% and 7.0% by weight, such as between 3.0% and 6.0% by weight, including between 3.5% and 5.0% by weight of the beverage. In further preferred embodiments, the protein content is at the most 1%, such as at the most 2.5%, including at the most 3%, e.g. at the most 3.5%, such as at the most 4.0%, including at the most 5%, e.g. at the most 10%, such as the most 12.5%, including at the most 15% by weight of the beverage. In further embodiments, the whey protein beverage is one wherein the whey protein content is 1%, 2.5%, 3%, 3.5%, 4.0 %, 5%, 10%, 12.5%, or 15% by weight. In further useful embodiments, the protein content is at the at least 1%, such as at least 2.5%, including at least 3%, e.g. at least 3.5%, such as at least 4.0%, including at least 5%, e.g. at least 10%, such as at least 12.5%, including at least 15% by weight.

As shown in the below example, when adding a certain amount of a shielding agent to the whey protein beverage before the sterilization treatment, such as before the heat treatment, the interaction between the whey proteins and proline rich proteins (PRP) naturally secreted in the mouth will be obstructed or be weakened resulting in a reduction of astringency was observed. The concentration of the shielding agent useful for such a reduction may be between 0.05 and 1% by weight of the beverage, such as between 0.05 and 0.9%, including between 0.05 and 0.8%, e.g. between 0.05 and 0.7, such as between 0.04 and 0.6%, including between 0.03 and 0.5%, e.g. 0.04 and 0.4%, such as between 0.05% and 0.3%, such as between 0.05% and 0.25%, including between 0.07% and 0.2%, e.g. between 0.10% and 0.25%, such as between 0.15% and 0.20%, including between 0.07% and 0.15%, e.g. between 0.10% and 0.15% by weight of the beverage.

Any kind of food grade shielding agent may be used in the present invention, which is capable of shielding the hydrophobic exposed parts (as a result of the sterilization treatment, e.g. as a result from a heat treatment, of the whey protein beverage) of the whey proteins and thus preventing or weakened the hydrophobic interactions between whey proteins and proline rich proteins (PRP) naturally secreted in the mouth. Thus, in a preferred embodiment, the shielding agent is one which acts on and/or binds to the whey protein. Furthermore, the shielding agent is one which is capable of reducing astringency and/or where the presence

of the shielding agent results in an astringency value which is 4 or less, and should be used in amounts not influencing the taste of the beverage. Thus, shielding agents, such as e.g. lauryl sulphate, which need to be added to the beverage in such high amounts that the taste or taste sensation is affected are not desired in the present invention.

In a further embodiment, the shielding agent is a surfactant which is soluble in both organic solvents and water. In a preferred embodiment, the shielding agent is an amphiphilic surfactant, i.e. it posses both a hydrophobic group (its "tail") and hydrophilic groups (its "head"). The shielding agent may be a surfactant which is a monoglyceride.

In useful embodiments, the shielding agent is an amphiphilic surfactant which is non-ionic and hydrophilic. Furthermore, the shielding agent may be a polysorbat, including polysorbat 20, polysorbat 40, polysorbat 60, polysorbat 65, and polysorbat 80. The presently preferred shielding agent is polysorbate 80 (Tween 80).

Compounds which mimic the action of a shielding agent useful in the present invention include oleic acid such as oleic acid C18: l(9) cis.

The shielding agent may be pre-treated before the addition to the other ingredients of the whey protein beverage in order to activate the shielding agent. In useful embodiments, the shielding agent is preheated at a temperature between 50 and 80 ⁰ C, such as between 55 and 75°C, e.g. between 60 and 70 ⁰ C and preferably between 70 ⁰ C and 75°C.

In view of the present invention as such, it becomes possible to increase the pH to above 3.0 and thereby widen up the use of suitable pH-sensitive compounds. Thus, in preferred embodiments, the whey protein beverage according to the invention further comprises a compound selected from the group consisting of sugars, colours, flavours, minerals, vitamins and pharmaceuticals. In preferred embodiments the colours used are pH indicator.

In some embodiments, the whey protein beverage may be a carbonated beverage. The carbonation of the whey protein beverage may range from about 0.1 volumes of carbonation (per volume of liquid present in the beverage) to about 6 volumes of carbonation. More typically, the amount of carbonation present ranges from about 1.6 volumes to about 3.5 volumes, with the most typical concentration ranging from about 1.7 volumes to about 3.0 volumes. The carbonation may be added in the form of carbonated liquid, such as, for example, but not by way of limitation, carbonated water. The carbonation may be added by bubbling sterile carbon dioxide through the protein beverage until the desired amount of carbon dioxide is present or may be added by the addition of any edible carbonation source. It will be understood, that when carbonating the whey protein beverage according to the invention, the pH will de decreased and thus a reduced amount of the below mentioned acids are need to adjust the pH to the above defined levels.

A prerequisite for obtaining an appropriate shelf life for a dairy beverage for at least six month at ambient temperature is a sterilization treatment. The sterilization treatment may be a heat treatment, a germ filtration treatment such as diafiltration, ultrafiltration and nanofiltration, a pressure treatment, a treatment with a suitable compound capable of killing the spores or microorganisms in the beverage, or a combination hereof.

In a preferred embodiment, the sterilization treatment is a heat treatment. For whey protein beverages the temperature may preferable be chosen to be between 50 and 200 ⁰ C. However, under some circumstances the temperature/time may not be less than e.g. 120 ⁰ C for 20 seconds or 150 ⁰ C for 4 seconds. This heat treatment is also known as "Ultra High Temperature" (L)HT). It has been observed that if the temperature is less than 120 ⁰ C, increased gelling and agglomeration occur upon storage. Without being bound by theory, it is contemplated that the reason for the need of such an excessive heat/time is a) to inactivate proteases naturally present in the whey e.g. Cathepsin D., and b) to secure unfolding/denaturation of whey proteins to a certain degree, which will obstruct agglomeration at pH 3.0 in the final beverage upon storage at ambient temperature. It is to be noted that this criterion currently serves as definition for the molecular state.

Accordingly, the whey protein beverage according to the invention is one which may be heat-treated, such as heat-treated at a temperature within the range of from 50 to 200 ⁰ C, e.g. within the range of from 85 to 200 ⁰ C, such as within the range of from 120 - 200 ⁰ C, such as within the range of from 72 to 150 ⁰ C, including a temperature within the range of from 100 to 120 ⁰ C, e.g. a temperature within the range of from 110 to 120 ⁰ C, such as within the range of from 140 to 150 ⁰ C. However, under some circumstances the whey protein beverage may be heat treated at a temperature which is at the most 75°C, such as at the most 100 ⁰ C, including at the most 120 ⁰ C, e.g. at the most 150 ⁰ C, such as at the most 200 ⁰ C. In preferred embodiments, the whey protein beverage may be heat treated at a temperature which is at least 75°C, e.g. at least 85°C, such as at least 100 ⁰ C, including at least 120 ⁰ C, e.g. at least 150 ⁰ C, such as at least 200 ⁰ C.

In accordance with the invention, the whey protein beverage may be heat treated at the above temperatures for a specific period of time, such as within the range of from 2 to 30 seconds, including within the range of from 4 to 25 seconds, e.g. within the range of from 5 to 20 seconds, such as within the range of from 7 to 20 seconds, including within the range of from 10 to 25 seconds, e.g. within the range of from 10 to 20 seconds, such as within the range of from 15 to 30 seconds, including within the range of from 15 to 25 seconds, e.g. within the range of from 20 to 30 seconds.

Presently preferred embodiments are where the heat treatment is performed at a temperature of 72°C for 15 seconds, at a temperature of 160 ⁰ C for 20 seconds, at a temperature of 140 ⁰ C for 20 seconds, at a temperature of 15O ⁰ C for 20 seconds, and most preferable is a heat treatment at a temperature of 150 ⁰ C for 4 seconds.

Without being bound by theory, it is contemplated that the disappearance of the astringency is a result of the re-dissolution of the sediments in the mouth cavity within a short interval of time. Furthermore, it is presently believed that due to the high temperatures, the shielding agent, such as e.g. Tween 80, acts as a shielding agent and not as a traditional emulsifier or surfactant, i.e. the shielding

agent is not acting on the fat and/or oil part of the beverage but acts on the protein. Thus, for example the combination of increased heat treatment such as L)HT, e.g. 140 - 150 ⁰ C for 4 seconds, and the addition of a shielding agent, such as e.g. Tween 80, seem to shorten the perception of sediments and thus shorten or reduces astringency and/or increases the transparency significantly and considerably.

In a preferred embodiment, the whey protein beverage is transparent. Such a transparent, non-astringent whey protein beverage is highly sought after because it has an appealing appearance and consistency as well as the desired palatability. As defined above, transparency relates to absorbance (A) measured at 500nm using a lcm cuvette. It is a standard American method. The EU uses 860nm, because 500nm is within the visual light wavelength thus affected by colour. Under the disclosed conditions of the measurement, 100% transparency was assigned to demineralised water. In accordance with the present invention the most preferred transparency of the whey protein beverage at 500 nm and 1 cm path length is A(500)< 0.05, such as A(500)< 0.04, including A(500)< 0.02, e.g. A(500)< 0.01.

As described above, it is desirable that a dairy beverage is stable, i.e. that it has a prolonged shelf life and/or storage stability, i.e. that there is substantially no change of the organoleptic properties of the beverage. A prolonged shelf life is to be seen as a prerequisite for a beverage according to the invention to be an attractive beverage in line with convenient carbonated beverages on the market. Thus, in a preferred embodiment, the whey protein beverage according to the invention has storage stability such that substantially no change of the organoleptic properties of said beverage occurs after storage for at least 6 months at a temperature of 20 ⁰ C, such as for at least 10 months or even at least 12 months.

In a further aspect of the present invention there is provided a method for the manufacture of a whey protein beverage having a pH between 1.0 and 5.0, said method comprising the steps of a) providing a liquid whey protein solution having a protein concentration of between 1 and 15 % by weight of the beverage; b) adjusting the pH of the liquid whey protein solution to between 1.0 and 5.0 by use

of an acidulant or an alkali; c) adding to the pH-adjusted whey protein solution of step b) an shielding agent in an amount within the range of from 0.05% and 1% by weight of the beverage to obtain a whey protein composition; and d) subjecting the whey protein composition obtained in step c) to a sterilization treatment resulting in the whey protein beverage.

In a yet further aspect there is provided a method for the manufacture of a whey protein beverage having an astringency value which is less than 4 at a pH between 1.0 and 5.0, said method comprising the steps of a) providing a liquid whey protein solution having a protein concentration of between 1 and 15 % by weight of the beverage; b) adjusting the pH of the liquid whey protein solution to between 1.0 and 5.0 by use of an acidulant or an alkali; c) adding to the pH adjusted whey protein solution of step b) an shielding agent in an amount within the range of from 0.05% to 1% by weight of the beverage to obtain a whey protein composition; and e) subjecting the whey protein composition obtained in step c) to a sterilization treatment resulting in the whey protein beverage.

In a preferred embodiment, the astringency value is determined by the method disclosed by Sano et al. 2005 with the modification as described above or by the "salvia-beverage interaction test" as described in the Examples. In accordance with the present invention, the astringency value is 4 or less. However, in preferred embodiments, the astringency value is 3 or less, such as 2 or less, including 1 or less or even an astringency value of 0.

Whey proteins useful in the liquid whey protein solution of the above to aspects are described above and may be selected from the group consisting of whey protein isolates, whey protein concentrate, intact whey protein, and combinations thereof. Presently preferred is whey protein isolate. One example of such a modified whey protein isolate is LACPRODAN ^® - DI9213, which is available from ArIa Foods Ingredients, Denmark. An example of the production of a specific useful whey protein isolate in the method for manufacture of a whey protein beverage is described below.

In preferred embodiments, the liquid whey protein solution has a protein concentration between 1.0 % and 10.0 % by weight of the beverage, such as

between 1.5% and 9.0% by weight, including between 2.0% and 8.0% by weight, e.g. between 2.5% and 7.0% by weight, such as between 3.0% and 6.0% by weight, including between 3.5% and 5.0% by weight of the beverage. In further preferred embodiments, the protein concentration is at the most 1%, such at the most 2.5%, including at the most 3%, e.g. at the most 3.5%, such as at the most 4.0%, including at the most 5%, e.g. at the most 10%, such as the most 12.5%, including at the most 15% by weight of the beverage. In further embodiments, the protein concentration in the liquid whey protein solution is 1%, 2.5%, 3%, 3.5%, 4.0 %, 5%, 10%, 12.5%, or 15% by weight of the beverage. In useful embodiments, the protein content in the liquid whey protein solution is at least 1%, such as at least 2.5%, including at least 3%, e.g. at least 3.5%, such as at least 4.0%, including at least 5%, e.g. at least 10%, such as at least 12.5%, including at least 15% by weight of the beverage.

An acidulant or an alkali is added in an amount effective to adjust the pH to the desired value of the liquid whey protein solution. In certain embodiments, the acidulant or the alkali is added in an amount effective to adjust the pH of the liquid whey protein solution to a value between 1.5 and 4.5, including between pH 2.0 and pH 4.0, such as between pH 3.0 and pH 4.0, e.g. between pH 3.0 and pH 3.5, including between pH 3.5 and pH 5.0, such as between pH 3.5 and pH 4.5. In a presently preferred embodiment the pH is adjusted to a pH value around 3.5, such as pH 3.5. In further embodiments, the pH is adjusted to a value above 2.0, such as above pH 2.5, e.g. above pH 3.0, including above pH 3.2, such as above pH 3.5, e.g. above pH 3.7, including above pH 4.0, such as above pH 4.5.

Various acidulants may be use in step b) of both methods for the adjustment of the pH of the liquid whey protein solution. In certain embodiments, the acidulant is an acid selected from the group consisting of phosphoric acid, citric acid, phosphoric-citric acid (e.g. a combination of phosphoric acid and citric acid), malic acid, glucono-delta-lactone acid, hydrochloric acid, lactic acid, fumaric acid, tartaric acid, acetic acid, adipic acid, carbonic acid, and combinations thereof. Alternatively, other acidulants may be used in step b), such as any food grade acidulants. As shown in the below example, an alkali, e.g. KOH, may be used in order to increase the pH to above 3.5. Additionally, stirring, agitation or other

methods of mixing may be utilized in step b) to promote blending of the liquid whey protein solution and the acidulant.

In accordance with the present method, a shielding agent is to be added to the pH-adjusted whey protein solution prior to the sterilization treatment.

Any kind of food grade shielding agent may be used in the present invention, which is capable of shielding the hydrophobic exposed parts (as a result from the heat treatment of the whey protein beverage) of the whey proteins and thus preventing or weakened the hydrophobic interactions between whey proteins and proline rich proteins (PRP) naturally secreted in the mouth. Thus, in a preferred embodiment, the shielding agent is one which acts on and/or binds to the whey protein. Furthermore, the shielding agent is one which is capable of reducing astringency and/or where the presence of the shielding agent results in an astringency value which is 4 or less, and should be used in amounts not influencing the taste of the beverage. Thus, shielding agents, such as e.g. lauryl sulphate, which need to be added to the beverage in such high amounts that the taste or taste sensation is affected are not desired in the present invention.

In a further embodiment, the shielding agent is a surfactant which is soluble in both organic solvents and water. In a preferred embodiment, the shielding agent is an amphiphilic surfactant. The shielding agent may be a surfactant which is a monoglyceride.

In useful embodiments, the shielding agent is an amphiphilic surfactant which is non-ionic and hydrophilic. Furthermore, the shielding agent may be a polysorbat, including polysorbat 20, polysorbat 40, polysorbat 60, polysorbat 65, and polysorbat 80. The presently preferred shielding agent is polysorbate 80 (Tween 80).

Compounds which mimic the action of shielding agent useful in the present invention include oleic acid such as oleic acid C18: l(9) cis.

The shielding agent may be preheated before the addition to the pH adjusted whey protein solution of step b) in order to activate the shielding agent. In useful

embodiments, the preheating step is carried out at a temperature between 50 and 80 ⁰ C, such as between 55 and 75°C, e.g. between 60 and 70 ⁰ C and preferably between 70 ⁰ C and 75°C.

In preferred embodiments, the shielding agent is added to the liquid whey protein solution in an amount within the range of from between 0.05 and 1% by weight of the beverage, such as between 0.05 and 0.9%, including between 0.05 and 0.8%, e.g. between 0.05 and 0.7, such as between 0.04 and 0.6%, including between 0.03 and 0.5%, e.g. 0.04 and 0.4%, such as between 0.05% and 0.3%, such as between 0.05% and 0.25%, including between 0.07% and 0.2%, e.g. between 0.10% and 0.25%, such as between 0.15% and 0.20%, including between 0.07% and 0.15%, e.g. between 0.10% and 0.15% by weight of the beverage.

In useful embodiments of the present invention, a further compound is added selected from the group consisting of sugars, colours, flavours, minerals, vitamins and pharmaceuticals and combinations thereof. Examples of specific compounds are described above.

As mentioned above, a sterilization treatment of the whey protein solution is necessary in order to observe the desired storage stability. The sterilization treatment may be a heat treatment, a germ filtration treatment such as diafiltration, ultrafiltration and nanofiltration, a pressure treatment, a treatment with a suitable compound capable of killing the spores or microorganisms in the beverage, or a combination hereof.

However, in preferred embodiments, the whey protein solution is heat-treated at a temperature within the range of from 50 to 200 ⁰ C, e.g. within the range of from 85 to 200 ⁰ C, such as within the range of 120 to 200 ⁰ C, such as within the range of from 72 to 150 ⁰ C, including a temperature within the range of from 100 to 120 ⁰ C, e.g. a temperature within the range of from 110 to 120 ⁰ C, such as within the range of from 140 to 150 ⁰ C. However, under some circumstances the whey protein solution is subjected to a heat treatment at a temperature which is at the most 75°C, such as at the most 100 ⁰ C, including at the most 120 ⁰ C, e.g. at the most 150 ⁰ C, such as at the most 200 ⁰ C. In preferred embodiments, the whey protein solution is heat-treated at a temperature which is at least 75°C, e.g. at

least 85°C, such as at least 100 ⁰ C, including at least 120 ⁰ C, e.g. at least 150 ⁰ C, such as at least 200 ⁰ C.

In accordance with the invention, the heat treatment may be performed at the above temperatures for a specific period of time, such as within the range of from 2 to 30 seconds, including within the range of from 4 to 25 seconds, e.g. within the range of from 5 to 20 seconds, such as within the range of from 7 to 20 seconds, including within the range of from 10 to 25 seconds, e.g. within the range of from 10 to 20 seconds, such as within the range of from 15 to 30 seconds, including within the range of from 15 to 25 seconds, e.g. within the range of from 20 to 30 seconds.

In presently preferred embodiments, the heat treatment is performed at a temperature of 72°C for 15 seconds, at a temperature of 160 ⁰ C for 20 seconds, at a temperature of 14O ⁰ C for 20 seconds, at a temperature of 15O ⁰ C for 20 seconds, and most preferable is a heat treatment at a temperature of 15O ⁰ C for 4 seconds.

The traditional method for the manufacturing of a whey protein isolate does not involve a pH-adjustment of the retentate obtained by the microfiltration and ultrafiltration of the liquid whey and a pasteurisation step prior to drying. However, the present inventors found, that the adjustment of the pH of the concentrated retentate did result in a whey protein isolate product, which is less salt and metallic, making it useful in a protein beverage. Thus, in a useful embodiment, the liquid whey protein solution is a whey protein isolate provided by the method comprising the steps of: i) microfiltration of liquid whey obtaining a permeate; ii) ultrafiltration of the permeate from step i) obtaining a concentrated retentate; iii) adjustment of the pH of the concentrated retentate from step ii) to a pH value below 3.8; iv) ultrafiltration and diafiltration of the pH-adjusted retentate from step iii) at a pH value below 3.8 to obtain a whey protein isolate; v) filtration of the whey protein isolate obtained in step iv) on a ceramic membrane; and vi) optionally, pasteurization and/or spray drying of the whey protein isolate obtained in step v). An example of a method for the provision of such a whey protein isolate is described in detail below.

In further aspects, there is provided a whey protein beverage obtainable by the two methods according to the invention having an astringency value which is less than 4. However, in preferred embodiments, the astringency value is 3 or less, such as 2 or less, including 1 or less or even an astringency value of 0. The astringency value may be determined by the method of Sano et al (2005) or the methods described in the below Examples.

Furthermore, the whey protein beverage obtainable by the two methods according to the invention has in addition a transparency at 500 nm and 1 cm path length which is A(500)< 0.05, such as A(500)< 0.04, including A(500)< 0.02, e.g. A(500)< 0.01.

In a further aspect there is provided a method for the manufacturing of a whey protein isolate, the method comprising the steps of: i) microfiltration of liquid whey obtaining a permeate; ii) ultrafiltration of the permeate from step i) obtaining a concentrated retentate; iii) adjustment of the pH of the concentrated retentate from step ii) to a pH value below 3.8 using an acidulant; iv) ultrafiltration and diafiltration of the pH-adjusted retentate from step iii) at a pH value below 3.8 to obtain a whey protein isolate; and v) germ-filtration of the whey protein isolate obtained in step iv) on a ceramic membrane to obtain a whey protein isolate.

In preferred embodiments, the microfiltration in step i) is performed on a ceramic membrane having a pore size of between 0.05 μ to 0.20 μ or on an organic membrane having a pore size of between 0.40 μ to 0.80 μ.

In certain embodiments, the ultrafiltration in step ii) and iv) is performed on a standard L)F membrane having a pore size within the range of from 2 to 10 kD. The germ filtration may be performed on ceramic membrane having a pore size of between 0.5 and 1.4 μ.

Examples of acidulants useful in the step iii) of the method according to the invention may be an acid selected from the group consisting of phosphoric acid, citric acid, phosphoric-citric acid, malic acid, glucono-delta-lactone acid, hydrochloric acid, lactic acid, fumaric acid, tartaric acid, acetic acid, adipic acid,

carbonic acid, and combinations thereof. Presently preferred are the use of HCI and phosphoric acid.

Under some circumstances it may be useful to subject the whey protein isolate to a pasteurization step and/or a drying step, such as a spray-drying step.

The resulting whey protein isolate obtainable by the method according to the present invention has a reduced salted and metallic taste compared to a whey protein isolate manufactured by the traditional method for manufacturing a whey protein isolate, which takes place without pH adjustment and steril-filtration. Furthermore, the whey protein isolate obtainable by the present method does have a high solubility over a wide pH-range and a good heat stability in solutions with a low pH (< pH 3.5) and it leads to clear solutions with low viscosity in addition to having a neutral taste as well as a low fat and carbohydrate content. One example of such a whey protein isolate is LACPRODAN ^® - DI9213, which is available from ArIa Foods Ingredients, Denmark.

In a further aspect of the present invention there is provided a whey protein composition comprising the whey protein isolate according to the present invention and one further ingredient. Such a whey protein composition may be selected from the group consisting of a food, a feed, a beverage, a gel, a nutritional bar, a snack bar and a pharmaceutical preparation. Useful embodiments of this aspect are described above.

Such whey protein composition may be a particularly valuable source for individuals with special medical needs (e.g. lactose intolerant individuals), and a valuable component of a training or diet program.

In a yet further aspect there is provided a method for the manufacture of a whey protein beverage having an astringency value which is less than 4 at a pH between 1.0 and 5.0, said method comprising the steps of a) providing the whey protein isolate having a protein concentration of between 1 and 15 % by weight of the beverage, said whey protein isolate is manufactured by the method comprising the steps of: i) microfiltration of liquid whey obtaining a permeate; ii) ultrafiltration of the permeate from step i) obtaining a concentrated

retentate; iii) adjustment of the pH of the concentrated retentate from step ii) to a pH value below 3.8 using an acidulant; iv) ultrafiltration and diafiltration of the pH-adjusted retentate from step iii) at a pH value below 3.8 to obtain a whey protein isolate; and v) germ-filtration of the whey protein isolate obtained in step iv) on a ceramic membrane to obtain a whey protein isolate; b) adjusting the pH of the whey protein isolate to between 1.0 and 5.0 by use of an acidulant or an alkali; c) adding to the pH adjusted whey protein isolate of step b) an shielding agent in an amount within the range of from 0.05% to 1% by weight of the beverage to obtain a whey protein composition; and e) subjecting the whey protein composition obtained in step c) to a sterilization treatment resulting in the whey protein beverage.

Useful embodiments of this aspect are described above.

A still further aspect of the present invention provides a method for obtaining an astringency value which is 4 or less in a whey protein beverage having a pH between 1 and 5, comprising the steps of adding a shielding agent in an amount within the range of from 0.05% to 1% by weight to a whey protein solution having a protein concentration of between 1 and 15 % by weight of the beverage. Useful embodiments of this aspect are described above.

In a final aspect there is provided a whey protein beverage obtainable by the method of the invention having an astringency value of 4 or less. However, in preferred embodiments, the astringency value is 3 or less, such as 2 or less, including 1 or less or even an astringency value of 0. The astringency value may be determined by the method of Sano et al (2005) or the methods described in the below Examples. Useful embodiments of this aspect are described above.

It must be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

The following examples are included to demonstrate particular embodiments of the invention. However, those of skill in the art should, in view of the present

disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The following examples are offered by way of illustration and is not intended to limit the scope of the invention in any way.

The invention will now be described in further details in the following non-limiting examples.

Example 1

Preparation of whey protein beverages with different concentrations of a shielding agent

The purpose of this example is to prepare a whey protein beverage according to the invention. In particular, the influence of the concentration of a shielding agent upon the astringency is studied.

1.1. Materials and methods

4.15 kg whey protein isolate (WPI) from ArIa Foods (LACPRODAN ^® - DI9213) was dissolved in 86 kg water. The WPI contains: Protein 90 %

Lactose 0.2 %

Fat 0.2 %

Ash 5.0 %

Moisture 5.0 % pH 2.8 - 3.5

To the WPI-solution the following ingredients were added : Trinatriumcitrat 9 g

25% sucralose solution 54 g

Lime PB3002648 (Danisco) 90 g

Pineappel QA00180377 (Quest) 45 g pH Adjusted to pH 3.5 with 1 : 5 diluted KOH

The solution was left overnight at 5 ⁰ C. Subsequently, the following amounts of Tween 80 were added to 10 kg of the solution :

Trial No Tween 80, g

^" ϊ (Too

2 1.00

3 2.00

4 4.00

5 7.00

6 10.00

7 15.00

8 20.00

9 30.00

Each solution was subjected to a L)HT treatment using Polarterm with 10 mm filter at 15O ⁰ C for 4 seconds, cooled to 85 ⁰ C, followed by a two stage homogenization at 100 and 200 bar. Subsequently, the solutions were tapped at 85 ⁰ C into sterile bottles and cooled in ice water. Finally, the bottles were stored at 2O ⁰ C 3 days before astringency was measured by the following method:

Method for the evaluation of astringency (Sano et al. 2005, modified):

1. Before tasting : drink ¹ /2-l glass cold water; 2. A standard sample (see below for details) has to be consumed first. The standard sample can also be used during the analysis in order to compare with the test sample (after two minutes) 3. TASTING:

3.1. Take in 15-2OmL of the test sample in the oral cavity. 3.2. Keep the test sample in the oral cavity for 10-15sec.

3.3. Swallow the test sample.

3.4. Note/describe whether astringency develops and if, how fast and at which level after 2 minutes. Use the scale 0-10, where 10 = extreme astringency and O = no astringency. 4. Drink ¹ /2-l glass cold water and optionally, eat a cracker;

5. Keep a break for at least 10 minutes;

6. Max. 3-6 tests pr. session.

Standard sample As a standard sample black tea KENYA HIGHLAND (available from Vinspecialisten Søren østergaard, Herning Centret, Merkurvej 84,7400 Herning, DENMARK). The tea is prepared by the following method:

1. To 100OmL boiling water a tea bag with 2 teaspoon tea is added; 2. Let the tea draw for 5 minutes under stirring;

3. Remove the bag;

4. Keep the tea in the refrigerator until the temperature is about 8-1O ⁰ C.

5. The standard sample is ready for use and corresponds to a moderate astringency, i.e. 6-8 on the above astringency scale. Compare with tannic acid solutions standards having 0.00 - 0.38 - 0.60 - 0.93 - 1.45 - 2.26 mM corresponding to a scale value of 0 - 2 - 4 - 6 - 8 -10, respectively.

1.2 Results

The score of the astringency were given within the scores of 1-10 with the highest score as the biggest degree of astringency. The results are shown in table 1.1

1.3 Conclusion

It appears that a lower degree of astringency is achieved with concentrations of Tween 80 between 0.07 and 0.150% (w/w) in whey protein beverages having a protein concentration of 3.7%, corresponding to 0.02% to 0.004% Tween pr. % protein.

Table 1.1 Results

Tween 80 PH Astringency %(w/w) value

0 3.51 10

0.010 3.51 10

0.020 3.52 8

0.040 3.53 7

0.070 3.53 3

0.100 3.53 4

0.150 3.53 4

0.200 3.53 6

0.300 3.54 4

Example 2

Saliva-Beverage Interaction Test

The present salvia-beverage interaction test is based on the method disclosed by Beecher et al. (2008) and thus based on their observations that a change in optical density (turbidity) was linearly related to sensorially determined astringency.

The formation of aggregate, as indicated by an increased turbidity of whey protein-saliva mixtures, can be explained based on two possible mechanisms (Beecher et al, 2008). First, as proposed by Sano et. al. (2005), whey proteins at

acidic pH when mixed with neutral pH saliva could result in a beverage-saliva solution at a pH close to the pH to the pi of whey proteins, favouring aggregation as observed by increase in turbidity. In this case, the whey protein aggregates are the cause of astringency. Alternatively, it could be due to interactions between the positively charged whey proteins (pH<pI) and negatively charged saliva proteins. If salivary proteins have isoelectric points below the isoeletric points of whey proteins there will be a pH range that result in a net electrostatic attraction causing aggregation.

2.1 Material and method (Beecher et al., 2008, modified ^" )

Tannic acid is used to make a standard for astringency value.

Concentration of Tannic acid Astringency

Value

0,0OmM 0

0.38mM 2

0.6OmM 4

0.93mM 6

1.45mM 8

2.26mM 10

Sample preparation

• Natural saliva or synthetic saliva is centrifuged at 12,000xg at 23°C for 20min, the supernatant is used immediately;

• Samples of protein beverages or standard chemical (tannic acid) and saliva is combined in a 1 : 1 ratio;

• The mixed composition/sample is subsequently vortex briefly to ensure proper mixing; and

• The mixed composition is allowed to equilibrate at room temperature for 15min; • Turbidity (optical density) is measured at 400nm, before and after centrifugation at 13,500xg for 15min.

The samples different in turbidity (before and after centrifugation) is compared to the standard and is thereby given an astringency score.

References

Beecher, J. W., Drake, M. A., Luck, P. E. and Foegeding, E. A., 2008. Factors regulating astringency of whey protein-fortified beverages, J. Dairy Sci. 91 :2553- 10 2560.

Beecher, J. W. and Foegeding, E. A., 2006, Factors regulating astringency of whey protein-fortified beverages, North Carolina State University.

15 Etzel, M. R., 2004, Manufacture and use of dairy protein fraction. American Society for Nutritional Science, pp. 996-1002.

Sano, H., Egashira, T., Kinekawa, Y., Kitabatake, N., 2005, J. Dairy Sci. 88:2312- 2317. 20

Schneider, P. A. and Foegeding, E. A, 2006, Aggregation of α-Lactalbumin at pH 3.5-6.0, North Carolina State University.