Patent Description:
Beta-casein is the major protein found in human milk. The protein binds to calcium at its phosphorylated regions. The protein, as a whole, is disordered and is characterized as a random coil protein.

Beta-casein is also found in bovine milk, however, in a lower concentration than in human milk. At room temperature, bovine beta-casein is bound to the casein micelles of bovine milk, but at lower temperatures, e.g. <NUM>-<NUM> degrees C, beta-casein is known to dissociate partly from the casein micelles to form free beta-casein, e.g. in the form of single free beta-casein molecules or small beta-casein aggregates.

Several approaches for isolating beta-casein from milk have been described in the prior art.

<CIT> discloses production of beta-casein by microfiltration of a cooled liquid feed containing calcium aggregated caseinate.

<CIT> describes a process for producing a beta-casein enriched milk protein product by subjecting a cooled skimmed milk to microfiltration. The used MF filter has a pore size of <NUM>-<NUM> micrometer.

<CIT> discloses a method of producing beta-casein: The method is based on initial cold microfiltration of cooled skimmed milk to obtain a partly beta-casein depleted retentate and a permeate containing milk serum protein and a significant amount of beta-casein. The beta-casein containing permeate may be subjected to further purification.

<CIT> discloses a method of preparing milk protein fraction, including beta-casein, using a first microfiltration step and subjecting the retentate of the first microfiltration step to a second microfiltration. The first microfiltration step may be performed at a warm temperature and the second microfiltration step may be performed at a cold temperature.

The invention pertains to a method of producing a beta-casein-containing composition containing at least <NUM>% (w/w) beta-casein relative to the total amount of casein and at least <NUM>% (w/w) beta-casein relative to the total amount of proteins, the method comprising the steps of:.

thereby providing the beta-casein-containing composition.

The second permeate of step e) may for example be used as the beta-casein-containing composition. Alternatively, a combination of the second permeate of step e) and subsequent permeates of the MF-diafiltration of step f) may be used as the beta-casein-containing composition. Alternatively, the product resulting from step g) may be used as the beta-casein-containing composition.

As steps f) and g) are deemed optional, there are several variants to the method. Alternatively, the method may comprise the steps a), b), c), d), and e). For example the method may comprise the steps a), b), c), d), e), and f). In other embodiments, the method comprises the steps a), b), c), d), e), and g). The method may e.g. comprise the steps a), b), c), d), e), f), and g).

In some embodiments of the invention, the method may consist of the steps a), b), c), d), and e). For example the method may consist of the steps a), b), c), d), e), and f). In other embodiments, the method consists of the steps a), b), c), d), e), and g). The method may e.g. consist of the steps a), b), c), d), e), f), and g).

An exemplary embodiment of the invention is illustrated in <FIG>. Here the milk (<NUM>) contacts a heating unit (<NUM>) which adjusts the temperature of the milk to the pretreatment temperature (Tpre). The milk is held at Tpre for a duration, tpre, sufficient to allow at least a substantial part of the free beta-casein to bind to the casein micelles.

The warm milk is then transferred to a first microfiltration unit (<NUM>) and is separated into a first permeate (<NUM>) and a first retentate (<NUM>). This embodiment involves subsequent diafiltration of the first retentate (<NUM>), which is mixed with a diluent (<NUM>), and the resulting mixture is then transferred to yet a microfiltration unit (<NUM>'), resulting in a first MF-diafiltration retentate (<NUM>') and a first MF-diafiltration permeate (<NUM>'). Yet a step of MF-diafiltration is performed on the first MF-diafiltration retentate (<NUM>'). The temperature during the warm microfiltration, TwMF, may be the same as or different from Tpre, though is it preferred that TwMF is at least <NUM> degrees C.

Subsequently, the resulting retentate (<NUM>") is diluted and transferred to a cooling unit where the diluted retentate is cooled to the temperature Tcold. The diluted retentate is kept at a cold temperature for a duration, tcold, sufficient to dissociate a significant part of the casein-micelle-bound beta-casein, thereby providing a substantial part of free beta-casein in the cooled, diluted retentate. The free beta-casein is separated from the cooled, diluted retentate by cold MF (<NUM>) and cold MF-diafiltration (<NUM>' and <NUM>"). The free beta-casein moves through the MF filter and into the permeate streams (<NUM>, <NUM>', <NUM>") which are combined and transferred to a spray-drying system (<NUM>), which converts the combined permeates to a beta-casein-containing powder.

The permeates of the warm MF/MF-diafiltration (<NUM>, <NUM>', <NUM>") contain high quality serum protein and may be converted into powder or liquid form serum protein concentrates.

The retentate stream (<NUM>") leaving the cold MF/MF-diafiltration is a partly beta-casein depleted isolate of micellar casein and may e.g. be used as a food additive or in the production of cheese.

As said, the invention pertains to a method of producing a beta-casein-containing composition containing at least <NUM>% (w/w) beta-casein relative to the total amount of casein and at least <NUM>% (w/w) beta-casein relative to the total amount of proteins the method comprising the steps of:.

The beta-casein-containing composition obtainable by the method of the invention contains at least <NUM>% (w/w) beta-casein relative to the total amount of protein. For example, the beta-casein-containing composition may contain at least <NUM>% (w/w) beta-casein relative to the total amount of protein. The beta-casein-containing composition may contain at least <NUM>% (w/w) beta-casein relative to the total amount of protein. Alternatively, the beta-casein-containing composition may contain at least <NUM>% (w/w) beta-casein relative to the total amount of protein, such as e.g. at least <NUM>% (w/w) beta-casein.

The beta-casein-containing composition may contain an amount of beta-casein in the range of <NUM> - <NUM>% (w/w) relative to the total amount of protein. For example, the beta-casein-containing composition may contain an amount of beta-casein in the range of <NUM> - <NUM>% (w/w) relative to the total amount of protein. The beta-casein-containing composition may e.g. contain an amount of beta-casein in the range of <NUM> - <NUM>% (w/w) relative to the total amount of protein. Alternatively, the beta-casein-containing composition may contain an amount of beta-casein in the range of <NUM> - <NUM>% (w/w) relative to the total amount of protein.

The beta-casein containing composition obtainable by the method of the invention contains at least <NUM>% (w/w) beta-casein relative to the total amount of casein. For example, the beta-casein-containing composition may contain at least <NUM>% (w/w) beta-casein relative to the total amount of casein. The beta-casein-containing composition may contain at least <NUM>% (w/w) beta-casein relative to the total amount of casein. Alternatively, the beta-casein-containing composition may contain at least <NUM>% (w/w) beta-casein relative to the total amount of casein. For example, the beta-casein-containing composition may contain at least <NUM>% (w/w) beta-casein relative to the total amount of casein, such as e.g. at least <NUM>% (w/w) beta-casein.

The method may also be used for producing a serum protein concentrate, e.g. by collecting the first permeate and/or additional permeates from MF-diafiltration of the first retentate. Additionally, the method may be used for producing a beta-casein depleted micellar casein isolate, e.g. by collecting the second retentate and/or a subsequent retentate obtained by subjecting the second retentate to cold MF-diafiltration. For example, the method may be used for producing a beta-casein-containing composition, a serum protein concentrate, and a beta-casein reduced micellar casein isolate.

In the context of the present invention, the phrase "Y and/or X" means "Y" or "X" or "Y and X". Along the same line of logic, the phrase "n<NUM>, n<NUM>,. , ni-<NUM>, and/or ni" means " n<NUM>" or " n<NUM>" or. or "ni-<NUM>" or "ni" or any combination of the components n<NUM>, n<NUM>,. ni-<NUM>, and ni.

In the context of the present invention, the term "casein micelle" pertains to a spherical aggregate of casein species, such as alpha-s1-casein, alpha-s2-casein, beta-casein and kappa-casein. The casein species of the micelle are typically held together by calcium ions and hydrophobic interactions. Most of the casein of native milk is present in the form of casein micelles.

The human version of beta-casein is the major protein found in human milk. In bovine milk, however, the bovine version of beta-casein only constitutes approx. <NUM>-<NUM>% (w/w) of the total amount of protein. The beta-casein molecule binds to calcium at its phosphorylated regions, which are highly conserved. Beta-casein may e.g. be present in the form of casein-micelle-bound beta-casein or in the form of free beta-casein. The term "free beta-casein" refers to casein which is not bound to the casein micelles. Free beta-casein may for example be free molecules of beta-casein or so-called sub-micelles, which primarily contains a number of associated beta-caseins.

As said, step a) of the method involves adjusting the temperature of a milk to a pre-heating temperature (Tpre) of at least <NUM> degrees C, thereby providing a warm milk. Step a) may be perceived as a step of pre-heating the milk before the milk contacts the micro filtration filter.

The milk provided in step a) is preferably a liquid milk obtained from a mammal. As used herein the term "milk" includes raw milk, whole milk, skim milk, fat-free milk, low fat milk, and full fat milk. The term milk furthermore includes fresh milk or milk based on milk powder resuspended in water.

The solid contents of the milk may e.g. have been modified by dilution or concentration, i.e. the milk may e.g. be a concentrated milk or a diluted milk.

Fat-free milk is a non-fat or skim milk product. Low-fat milk is typically defined as milk that contains from about <NUM>% to about <NUM>% fat. Full fat milk often contains about <NUM>% fat.

Sources of milk include, but are not limited to, cow, sheep, goat, buffalo, camel, llama, mare and deer.

In some preferred embodiments of the invention, the milk comprises, or even consists of, bovine milk.

In some embodiments of the invention, the milk has been subjected to pasteurisation and/or bactofugation to eliminate, or at least reduce, the microbial load of the milk.

In some preferred embodiments of the invention, the milk of a) comprises <NUM>-<NUM>% w/w casein, <NUM>-<NUM>% w/w milk serum protein, and <NUM>-<NUM>% w/w milk fat. In even more preferred embodiments of the invention, the milk of step a) comprises <NUM>-<NUM>% w/w casein, <NUM>-<NUM>% w/w milk serum protein, and <NUM>-<NUM>% w/w milk fat.

While in theory all types of mammal milk may be used, it is particularly preferred that the milk has recently been milked from the source of the milk, e.g. from cows. For example, the milk may be at most <NUM> days old, i.e. at most <NUM> days since milking. Preferably, the milk is at most <NUM> day old. For example, the milk may be at most <NUM> days old. Even more preferably, the milk is at most <NUM> days old. For example, the milk may be at most <NUM> day old.

The use of newly milked milk for the present method is advantageous as it results in less degradation of beta-casein and therefore a better beta-casein yield than older milk.

The pre-heating temperature, Tpre, is at least <NUM> degrees C. For example, Tpre may be at least <NUM> degrees C. Alternatively, Tpre may be at least <NUM> degrees C. Tpre may e.g. be at least <NUM> degrees C.

Even higher pre-heating temperatures may be desired, thus, Tpre may be at least <NUM> degrees C. For example, Tpre may be at least <NUM> degrees C. Alternatively, the Tpre may be at least <NUM> degrees C. Tpre may e.g. be at least <NUM> degrees C.

In some embodiments of the invention, Tpre is in the range of <NUM>-<NUM> degrees C. For example, Tpre may be in the range of <NUM>-<NUM> degrees C. Alternatively, Tpre may be in the range of <NUM>-<NUM> degrees C. In some embodiments Tpre is in the range of <NUM>-<NUM> degrees C.

The duration of the pre-heating, tpre, may be varied depending on the pre-heating temperature, Tpre, used in the process. It is, however, preferred that the milk is sufficiently pre-heated to allow for association of free beta-casein to the casein micelles.

The present inventors have seen indications that a too short heat pre-treatment time and/or a too low pre-treatment temperature lead to a reduced yield of beta-casein. The inventors have discovered that it is possible to increase the yield of beta-casein by controlling tpre and Tpre.

The higher temperatures used during the pre-heating, the shorter heating times are required to provide an efficient re-association of free beta-casein to the casein micelles. If Tpre is in the range of <NUM>-<NUM> degrees C, the holding time may e.g. be in the range of <NUM> minute - <NUM> hour. If Tpre is in the range of <NUM>-<NUM> degrees C, the holding time may e.g. be in the range of <NUM> second - <NUM> minutes. If Tpre is in the range of <NUM>-<NUM> degrees C, the holding time may e.g. be in the range of <NUM> second - <NUM> seconds.

In some embodiments of the invention, Tpre is in the range of <NUM>-<NUM> degrees C, the holding time may e.g. be in the range of <NUM> minute - <NUM> hour.

The warm milk may furthermore contain the usual carbohydrates, fat and minerals found in mammal milk.

In the context of the present invention, the terms "method" and "process" are used interchangeably.

According to the invention the temperature of the milk is kept within the pre-heating temperature range for a duration, tpre, of most <NUM> hours. Alternatively, tpre may be at most <NUM> hours. tpre may for example be at most <NUM> hour. For example, tpre may be at most <NUM> hour.

The pre-heating temperature range is the temperature range in which the milk is pre-heated before it contacts the MF filter in step b).

Even shorter tpre may be used, for example if the pre-heating temperature is relatively high. Thus, in some embodiments of the invention, tpre is at most <NUM> minutes. Alternatively, tpre may by at most <NUM> minutes, or even shorter such as at most <NUM> minutes.

A very short tpre may be used, e.g. when Tpre exceeds <NUM> degrees C. Thus, in some embodiments of the invention, tpre is at most <NUM> minute. Alternatively, tpre may by at most <NUM> minute, or even shorter such as at most <NUM> minute.

For example, the milk maybe kept within the pre-heating temperature range for a duration, tpre, in the range of <NUM> second - <NUM> hours. Alternatively, tpre may be in the range of <NUM> second - <NUM> hours. tpre may for example be in the range of <NUM> seconds-<NUM> hour. For example, tpre may be in the range of <NUM> minute - <NUM> hour.

As said, relatively short tpre may be used, for example if the pre-heating temperature is relatively high. Thus, in some embodiments of the invention, tpre is in the range of <NUM> second -<NUM> minutes. Alternatively, tpre may be in the range of <NUM> seconds - <NUM> minutes. tpre may e.g. be in the range of <NUM> seconds - <NUM> minutes.

In some embodiments of the invention, the pre-heating temperature range is <NUM>-<NUM> degrees C and tpre is at most <NUM> hours. For example, the pre-heating temperature range may be <NUM>-<NUM> degrees C and tpre may be at most <NUM> hour. The pre-heating temperature range may e.g. be <NUM>-<NUM> degrees C and tpre may be at most <NUM> hour. Alternatively, the pre-heating temperature range may e.g. be <NUM>-<NUM> degrees C and tpre may be at most <NUM> hour.

In other embodiments of the invention, the pre-heating temperature range is <NUM>-<NUM> degrees C and tpre is at most <NUM> hours. For example, the pre-heating temperature range may be <NUM>-<NUM> degrees C and tpre may be at most <NUM> minutes. The pre-heating temperature range may e.g. be <NUM>-<NUM> degrees C and tpre may be at most <NUM> seconds. Alternatively, the pre-heating temperature range may e.g. be <NUM>-<NUM> degrees C and tpre may be at most <NUM> seconds.

In yet other embodiments of the invention, the pre-heating temperature range is <NUM>-<NUM> degrees C and tpre is at most <NUM> second. For example, the pre-heating temperature range may be <NUM>-<NUM> degrees C and tpre may be at most <NUM> seconds. The pre-heating temperature range may e.g. be <NUM>-<NUM> degrees C and tpre may be at most <NUM> seconds. Alternatively, the pre-heating temperature range may e.g. be <NUM>-<NUM> degrees C and tpre may be at most <NUM> seconds.

Step b) involves subjecting the warm milk to microfiltration, thereby providing a first MF permeate and a first MF retentate.

According to the invention the MF of step b) is performed using a filter that retains at least a substantial fraction of the casein micelles but allows for the passage of milk serum protein.

The pre-heating of the milk performed during step a) results in binding of a major part, and preferably substantially all, of the available beta-casein to the casein micelles.

In some preferred embodiments of the invention the filter for warm MF has a nominal pore size in the range of <NUM>-<NUM> micrometer. For example, the filter for warm MF may have a nominal pore size in the range of <NUM>-<NUM> micrometer. Alternatively, the filter for warm MF may have a nominal pore size in the range of <NUM>-<NUM> micrometer. The filter for warm MF may e.g. have a nominal pore size in the range of <NUM>-<NUM> micrometer.

In some preferred embodiments of the invention, the MF filter is used in crossflow mode.

A suitable microfiltration system can e.g. be found in<NPL>), which is incorporated herein by reference for all purposes.

More details regarding the implementation of microfiltration and MF-diafiltration can be found in the books "<NPL>) and "<NPL>, which are incorporated herein by reference for all purposes.

In some preferred embodiments of the invention the method of the present invention comprises step c), i.e. a step of subjecting the first retentate to MF-diafiltration.

The present inventors have found that the use of diafiltration in connection with the first microfiltration step is advantageous as it allows for washing away serum protein, which otherwise might show up as impurities in the final beta-casein product. While such impurities could be removed later in the process, the present inventors have found that it is both easy and convenient to do it before the second microfiltration step.

The MF-diafiltration of step c) may involve diluting the first retentate with a first diluent and subjecting the diluted first retentate to microfiltration to obtain a first diafiltration retentate and a first diafiltration permeate. The casein micelles are still retained by the MF filter while milk serum protein moves through the microfiltration filter and into the first diafiltration permeate. The dilution of retentate and subsequent microfiltration may be repeated several times, each time providing a retentate having a lower content of milk serum protein than in the previous cycle.

As will be understood, these filtration steps may be discrete steps performed one by one in a batch process or they may be performed simultaneously in a continuous process.

The use of MF-diafiltration is advantageous as it makes it possible to wash out most of the milk serum protein of the initial feed. MF-diafiltration is furthermore advantageous as it can be conducted at relatively low viscosity and therefore does not expose the casein micelles to excessive shear forces.

The MF and MF-diafiltration are typically conducted using low pressure, e.g. using a pressure of at most <NUM> bars, and preferably at most <NUM> bars. For example, the MF and MF-diafiltration may be conducted using a pressure of at most <NUM> bars. Alternatively, MF and MF-diafiltration may be conducted using a pressure of at most <NUM> bars. In preferred embodiments of the invention, the MF and MF-diafiltration are conducted using a pressure of at most <NUM> bar, such as e.g. at most <NUM> bar.

The filter for MF-diafiltration may be the same or similar to the one for the initial MF of the warm milk.

According to the invention the temperature of the feed and subsequent retentates of the MF-diafiltration is kept within the warm temperature range during at least part of the MF-diafiltration, and e.g. during the entire MF-diafiltration. This is to avoid washing out the beta-casein from the retentate during the diafiltration of step c).

In some preferred embodiments of the invention at least part of the MF-diafiltration involves the use of a first diluent having a concentration of Ca<NUM>+ of at least <NUM>/kg. For example, the first diluent may have a concentration of Ca<NUM>+ of at least <NUM>/kg. Alternatively, the first diluent may have a concentration of Ca<NUM>+ of at least <NUM> /kg. The first diluent may e.g. have a concentration of Ca<NUM>+ of at least <NUM>/kg.

The use of diluents which contain a significant amount of calcium ions seems to reduce the wash-out of beta-casein during the warm MF/MF-diafiltration step and to improve the overall yield of beta-casein of the process.

According to the invention, the first diluent have a pH in the range of <NUM>-<NUM>. For example, the first diluent may have a pH of approx.

The first diluent preferably contain no or at least a very low content of protein.

In some embodiments of the invention, the first diluent comprises, or even consists of, ultrafiltration (UF) permeate of milk or whey.

Alternatively, the first diluent may be demineralised water or tap water.

The temperature used during the warm MF and MF-diafiltration, TwMF, is at least <NUM> degrees C. For example, TwMF may be at least <NUM> degrees C. Alternatively, TwMF may be at least <NUM> degrees C. TwMF may e.g. be at least <NUM> degrees C.

Even higher temperatures may be desired during the warm MF and MF-diafiltration, thus, TwMF may be at least <NUM> degrees C. For example, TwMF may be at least <NUM> degrees C. Alternatively, the TwMF may be at least <NUM> degrees C.

In some embodiments of the invention, TwMF is in the range of <NUM>-<NUM> degrees C. For example, TwMF may be in the range of <NUM>-<NUM> degrees C. Alternatively, TwMF may be in the range of <NUM>-<NUM> degrees C. In some embodiments TwMF is in the range of <NUM>-<NUM> degrees C.

The duration, twMF, of the warm MF and the optional warm MF-diafiltration, is preferably kept as short as possible. Thus, twMF, is preferably at most <NUM> hours. For example, twMF may be at most <NUM> hours. Alternatively, twMF may be at most <NUM> hours. twMF may be at most <NUM> hours. For example, twMF may be at most <NUM> hours. Alternatively, twMF may be at most <NUM> hours.

The retentate is preferably cooled to a temperature below <NUM> degrees C when it leaves the MF-diafiltration unit or, if no diafiltration is used, when it leaves the MF unit.

As said, step d) involves adjusting the temperature of a first composition derived from the first MF retentate to a cold temperature (Tcold) in the range of <NUM>-<NUM> degrees C and keeping the temperature of the first composition within that range for a duration (tcold) of at least <NUM> hour, thereby obtaining a cooled first composition.

The first composition is preferably a liquid aqueous composition. The first composition is derived from the first MF retentate in the sense that at least <NUM>% (w/w) of the casein micelles of the first composition originates from the first MF retentate and/or from a MF-diafiltration retentate thereof.

For example, at least <NUM>% (w/w) of the casein micelles of the first composition may originate from the first MF retentate and/or from a MF-diafiltration retentate thereof. Preferably, at least <NUM>% (w/w) of the casein micelles of the first composition originates from the first MF retentate and/or from a MF-diafiltration retentate thereof. Even more preferably, at least <NUM>% (w/w) of the casein micelles of the first composition originates from the first MF retentate and/or from a MF-diafiltration retentate thereof, such as e.g. substantially all the casein micelles.

In some preferred embodiments of the invention the first composition is the first MF retentate and/or a MF-diafiltration retentate thereof.

However, in other embodiments of the invention, the first MF retentate and/or a MF-diafiltration retentate thereof may be subjected to one or more additional process steps which lead to the formation of the first composition. Such additional process steps may e.g. involve temperature adjustment, concentration, dilution, demineralisation and/or pH adjustment.

In some embodiments of the invention the provision of the first composition involves concentrating the first MF retentate and/or a MF-diafiltration retentate thereof.

According to the invention the first composition comprises a total amount of casein of at least <NUM>% (w/w) relative to the total amount of protein of the first composition. For example, the first composition may comprise a total amount of casein of at least <NUM>% (w/w) relative to the total amount of protein of the first composition. Alternatively, the first composition may comprise a total amount of casein of at least <NUM>% (w/w) relative to the total amount of protein of the first composition. The first composition may e.g. comprise a total amount of casein of at least <NUM>% (w/w) relative to the total amount of protein of the first composition, such as approx. <NUM> (w/w).

The first composition typically comprises a total amount of milk serum protein of at most <NUM>% (w/w) relative to the total amount of protein of the first composition. For example, the first composition may comprise a total amount of milk serum protein of at most <NUM>% (w/w) relative to the total amount of protein of the first composition. Alternatively, the first composition may comprise a total amount of milk serum protein of at most <NUM>% (w/w) relative to the total amount of protein of the first composition. The first composition may e.g. comprise a total amount of milk serum protein of at most <NUM>% (w/w) relative to the total amount of protein of the first composition.

In some embodiments of the invention the first composition comprises a total amount of protein of at least <NUM>% (w/w) relative to the total weight of the first composition. For example, the first composition may comprise a total amount of protein of at least <NUM>% (w/w) relative to the total weight of the first composition. The first composition may e.g. comprise a total amount of protein of at least <NUM>% (w/w) relative to the total weight of the first composition. Alternatively, the first composition may comprise a total amount of protein of at least <NUM>% (w/w) relative to the total weight of the first composition.

In some embodiments of the invention the first composition comprises a total amount of protein in the range of <NUM>-<NUM>% (w/w) relative to the total weight of the first composition. For example, the first composition may comprise a total amount of protein in the range of <NUM>-<NUM>% (w/w) relative to the total weight of the first composition. The first composition may e.g. comprise a total amount of protein in the range of <NUM>-<NUM>% (w/w) relative to the total weight of the first composition. Alternatively, the first composition may comprise a total amount of protein in the range of <NUM>-<NUM>% (w/w) relative to the total weight of the first composition, such as e.g. in the range of <NUM>-<NUM>% (w/w).

In some embodiments of the invention the first composition comprises a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of protein. For example, the first composition may comprise a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of protein. The first composition may comprise a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of protein. Alternatively, the first composition may comprise a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of protein.

In some embodiments of the invention the first composition comprises a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of protein. For example, the first composition may comprise a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of protein.

The first composition may e.g. comprise a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of protein. Alternatively, the first composition may comprise a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of protein.

The total amount of beta-casein may be determined according to Bobe et al (<NPL>).

The total amount of casein may be determined according to ISO <NUM>-<NUM>:<NUM>, Milk - Determination of casein-nitrogen content - Part <NUM>: Indirect method (Reference method).

In some embodiments of the invention the first composition comprises a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of casein. For example, the first composition may comprise a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of casein. The first composition may e.g. comprise a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of casein. Alternatively, the first composition may comprise a total amount of beta-casein of at least <NUM>% (w/w) relative to the total amount of casein.

In some embodiments of the invention the first composition comprises a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of casein. For example, the first composition may comprise a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of casein. The first composition may e.g. comprise a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of casein. Alternatively, the first composition may comprise a total amount of beta-casein in the range of <NUM>-<NUM>% (w/w) relative to the total amount of casein.

In some preferred embodiments of the invention, the method of the present invention comprises step c) and the first composition comprises, or even consists of, a diafiltration retentate or a protein concentrate thereof.

In the context of the present invention, a protein concentrate of a liquid contains a higher concentration of proteins than the liquid as such but substantially the same molar ratio between the individual proteins. A protein concentrate may e.g. be obtained by subjecting the liquid to ultrafiltration, reverse osmosis or solvent evaporation.

The temperature of the first composition is adjusted to a temperature, Tcold, in a cold temperature range of <NUM>-<NUM> degrees C to allow casein micelle-bound beta-casein to dissociate from the casein micelles. The first composition may either be cooled directly or it may be prepared from components that have already been cooled as prescribed herein.

The cold temperature range may e.g. be <NUM>-<NUM> degrees C. For example, the cold temperature range may be <NUM>-<NUM> degrees C. The cold temperature range may e.g. be <NUM>-<NUM> degrees C, such as about <NUM> degrees C.

The first composition is preferably kept within the cold temperature range for a duration, tcold, of at least <NUM> hour prior to step e).

In some preferred embodiments of the invention, the first composition is kept within the cold temperature range for a duration, tcold, of at least <NUM> hour prior to step e). For example, tcold may be at least <NUM> hours. Alternatively, tcold may be at least <NUM> hours, such as e.g. at least <NUM> hours. Even longer times may be used, thus, the first composition may e.g. be kept within the cold temperature range for a duration, tcold, of at least <NUM> hour prior to step e). For example, tcold may be at least <NUM> hours. Alternatively, tcold may be at least <NUM> hours, such as e.g. at least <NUM> hours.

The first composition may furthermore contain the usual small molecules, e.g. carbohydrates and minerals, found in mammal milk.

Step e) involves subjecting the cooled first composition to MF, thereby obtaining a second retentate and a second permeate, which second permeate is enriched with respect to beta-casein.

The second permeate is enriched with respect to beta-casein in the sense that it contains a higher weight percentage of beta-casein relative to the total amount of casein than the cooled first composition.

The microfiltration of step e) may e.g. make use of the same microfiltration system, including MF filter, which was used for the microfiltration of the warm milk.

It is preferred that the temperature of the cooled first composition and the resulting retentate is maintained within the cold temperature range during the cold MF of step e).

However, in some embodiments of the invention, the temperature of the cooled first composition is raised immediately before the second microfiltration. The present inventors have seen indications that increasing the temperature of the cooled first composition to a temperature in the range of <NUM>-<NUM> degrees C immediately before the second microfiltration step has the benefit of increasing the capacity of the microfiltration unit which reducing the energy consumption of the process and this without a significant loss in the beta-casein yield. Thus, in some preferred embodiments of the invention, Tcold is in the temperature range <NUM>-<NUM> degrees C and TcMF is in the range of <NUM>-<NUM> degrees C. For example, Tcold maybe in the temperature range <NUM>-<NUM> degrees C and TcMF may be in the range of <NUM>-<NUM> degrees C. Alternatively, Tcold maybe in the temperature range <NUM>-<NUM> degrees C and TcMF may be in the range of <NUM>-<NUM> degrees C.

In the context of the present invention, the term "immediately before the second microfiltration" means at most <NUM> minutes before the first composition contacts the membrane of the filtration unit performing the second microfiltration, preferably at most <NUM> minutes before, and even more preferred at most <NUM> minutes before, such as at most <NUM> minute before.

In some preferred embodiments of the invention the method comprises step f), i.e. a step of subjecting the second retentate to MF-diafiltration in order to wash out more beta-casein.

The present inventors have found that it is advantageous to perform diafiltration after the second microfiltration step as it allows for washing out more beta-casein from the second MF retentate, and thereby increasing the beta-casein yield per kg milk feed.

The MF-diafiltration of step f) may involve diluting the second retentate with a second diluent and subjecting the diluted second retentate to microfiltration to obtain a diafiltration retentate and a diafiltration permeate. The casein micelles are still retained by the MF filter while dissociated beta-casein moves through the microfiltration filter and into the diafiltration permeate. The dilution of diafiltration retentate and subsequent microfiltration may be repeated several times, each time providing a retentate having a lower content of beta-casein than in the previous cycle.

The second diluent typically has a pH in the range of <NUM>-<NUM>, and preferably in the range of <NUM>-<NUM>. For example, the second diluent may have a pH of approx.

The second diluent preferably contain no or only a very low content of protein.

In some embodiments of the invention, the second diluent comprises, or even consists of, ultrafiltration (UF) permeate of milk or whey.

Alternatively, the second diluent may be demineralised water or tap water. The present inventors have seen indications that the use of water as the second diluent in the second MF-diafiltration step increases the amount beta-casein that is released from the casein micelles during the second MF-diafiltration and thus seems to increase the over-all yield of beta-casein per kg milk feed.

The diafiltration permeate(s) of step f) contain free beta-casein and may be pooled with the second permeate.

The second permeate or the pooled second permeate and subsequent cold MF-diafiltration permeates may be used as the beta-casein composition of the invention.

It is preferred that the temperature of the retentates is maintained within the cold temperature range during the cold MF of step e) and also during the cold MF-diafiltration of step f) if the latter is included in the process.

The cold MF and cold MF-diafiltration are typically conducted using low pressure, e.g. using a pressure of at most <NUM> bars, and preferably at most <NUM> bars. For example, the MF and MF-diafiltration may be conducted using a pressure of at most <NUM> bars. Alternatively, MF and MF-diafiltration may be conducted using a pressure of at most <NUM> bars. The MF and MF-diafiltration may e.g. be conducted using a pressure of at most <NUM> bar, such as e.g. at most <NUM> bar.

The duration, tcMF, of the cold MF and the optional cold MF-diafiltration, is preferably kept as short as possible. Thus, tcMF, is preferably at most <NUM> hours. For example, tcMF may be at most <NUM> hours. Alternatively, tcMF may be at most <NUM> hours. tcMF may be at most <NUM> hours. For example, tcMF may be at most <NUM> hours. Alternatively, tcMF may be at most <NUM> hours.

In some preferred embodiments of the invention, the method contains a step g) of subjecting a second composition derived from the second permeate to one or more further processing steps, e.g. further purification and/or concentration steps.

The second composition is preferably a liquid aqueous composition. The second composition is derived from the second MF permeate in the sense that at least <NUM>% (w/w) of the beta-casein of the second composition originate from the second MF permeate and/or further permeate(s) obtained from step f).

For example, at least <NUM>% (w/w) of the beta-casein of the second composition may originate from the second MF permeate and/or further permeate(s) obtained from the MF-diafiltration of step f). Preferably, at least <NUM>% (w/w) of the beta-casein of the second composition originate from the second MF permeate and/or a further permeate(s) obtained from the MF-diafiltration of step f). Even more preferably, at least <NUM>% (w/w) of the beta-casein of the second composition originate from the second MF permeate and/or a further permeate(s) obtained from the MF-diafiltration of step f), such as e.g. substantially all the beta-casein.

In some preferred embodiments of the invention the second composition is the second MF permeate and/or a further permeate(s) obtained from the MF-diafiltration of step f). Alternatively, the second composition may be a protein concentrate of the second MF permeate and/or a further permeate(s) obtained from the MF-diafiltration of step f).

However, in other embodiments of the invention, the second MF permeate and/or a further permeate(s) obtained from the MF-diafiltration of step f) may be subjected to additional process steps which lead to the formation of the second composition. Such additional process steps may e.g. involve temperature adjustment, concentration, dilution, demineralisation, and/or pH adjustment.

In some embodiments of the invention the provision of the second composition involves concentrating the second MF permeate and/or a further permeate(s) obtained from the MF-diafiltration of step f).

In some embodiments of the invention the concentration of step g) involves heating the second composition to a temperature and for a duration sufficient for the formation of beta-casein sub-micelles and subsequently subjecting the second composition containing the beta-casein sub-micelles to ultrafiltration microfiltration under conditions which retain the beta-casein sub-micelles in the retentate and allows for the passage of serum proteins into the permeate.

The nominal molecular weight cut-off of the membrane used for the ultrafiltration may e.g. be in the range of <NUM>-<NUM> kDa, and preferably in the range of <NUM>-<NUM> kDa, such as e.g. in the range of <NUM>-<NUM> kDa.

In some embodiments of the invention the concentration of step g) increases the weight percentage of beta-casein of the second composition to at least <NUM>% (w/w) on a dry weight basis.

In some embodiments of the invention the concentration of step g) increases the weight percentage of beta-casein in the second composition to <NUM>-<NUM>% (w/w) on a dry weight basis.

In some embodiments of the invention the concentration of step g) increases the solids content of the second composition to at least <NUM>% (w/w). For example, the concentration of step g) may increase the solids content of the second composition to at least <NUM>% (w/w). Alternatively, the concentration of step g) may increases the solids content of the second composition to at least <NUM>% (w/w), such as at least <NUM>% (w/w).

The concentration of step g) may e.g. involve one or more processes selected from the group consisting of ultrafiltration, nanofiltration, reverse osmosis, evaporation, spray drying and freeze drying. For example, the concentration of step g) may e.g. involve two or more processes selected from the group consisting of ultrafiltration, nanofiltration, reverse osmosis, evaporation, spray drying, and freeze drying.

The present method may e.g. be implemented as a batch method or as a continuous method. Each step may be implemented as a discrete batch. Alternatively, groups of steps may be implemented as a continuous sub-process. For example,steps b) and c) may be implemented as a continuous sub-process. Alternatively, or additionally, steps e) and f) may be implemented as a continuous sub-process.

The MF systems used in the MF and/or MF-diafiltration steps are preferably systems that allow for controlling the temperature of the feed and retentate stream, e.g. by water-heating or water-cooling.

It is preferred to control both the temperature and the duration of various steps of the method. <FIG> and <FIG> illustrate two non-limiting examples the timing and temperature profile during such a method.

The symbols used in <FIG> and <FIG> have the following meaning:.

If the method is implemented as a continuous process, the durations related to the processing or specific conditions of the milk or the casein-micelle-containing retentates are the average time that a casein-micelle is subjected to the mentioned processing or specific condition.

<FIG> differs from <FIG> in that it has a Tpre which is significantly higher than the TwMF. In the method according to <FIG>, Tpre is approximately the same as TwMF.

The use of a short pre-heating at a relatively high pre-heating temperature followed by a warm MF/MF-diafiltration at lower TwMF represents an interesting embodiment as it seems to reduce the digestion of beta-casein caused by plasmin.

While it is preferred that the method of the present invention is implemented with tight control of temperature and timing, some fluctuation of the temperature may be acceptable, as long as Tpre, TwMF, Tcold stay within the intervals mentioned herein.

It is preferred that the time which the milk and the related casein-micelle-containing streams are held at high temperature is kept at a minimum. Thus, in some embodiments of the invention the average time, twarm, a casein-micelle is kept at a temperature of at least <NUM> degrees C is at most <NUM> hours.

For example, the average time, twarm, a casein-micelle is kept at a temperature of at least <NUM> degrees C may be at most <NUM> hours. The average time, twarm, a casein-micelle is kept at a temperature of at least <NUM> degrees C may e.g. be at most <NUM> hour. Alternatively, the average time, twarm, a casein-micelle is kept at a temperature of at least <NUM> degrees C may be at most <NUM> hours. Even faster processing is possible, thus, the average time, twarm, a casein-micelle is kept at a temperature of at least <NUM> degrees C may be at most <NUM> hour.

Reducing the average time at high temperature seems to reduce the level of plasmin digestion of beta-casein thereby improving the yield of beta-casein.

In some preferred embodiments of the invention, the retentate resulting from steps b) or c) is subjected to a plasmin inactivation step, such as e.g. a heat inactivation step. The heat inactivation step may for example involve adjusting the temperature of the retentate to a temperature in the range of <NUM>-<NUM> degrees C and keeping the temperature of the milk-related feed in that range for a period in the range of <NUM>-<NUM> seconds. The heat inactivation step may for example involve adjusting the temperature of the retentate to a temperature in the range of <NUM>-<NUM> degrees C and keeping the temperature of the milk-related feed in that range for a period in the range of <NUM>-<NUM> seconds.

It seems particularly advantageous to inactivate plasmin after milk serum proteins have been at least partly removed by the warm MF/MF-diafiltration, as the milk serum proteins are more prone to thermal denaturation than the caseins.

The inactivation of plasmin also results in reducing the level of plasmin digestion of beta-casein and thereby improves the yield of beta-casein.

The casein-containing streams, e.g. the milk and the subsequent casein-containing retentates, typically have a pH in the range of <NUM>-<NUM>, and preferably a pH in the range of <NUM>-<NUM>. pH values mentioned herein are measured at <NUM> degrees C unless stated otherwise.

A beta-casein isolate was produced according to the method of the present invention.

Warm microfiltration/MF-diafiltration:
<NUM><NUM> of cooled non-pasteurised skimmed milk was pre-heated to <NUM> degrees C for <NUM> minutes in a heat-and-hold tank and subjected to continuous microfiltration using <NUM>" spiral wound membranes of the type FR6338 from Synder Filtration, Vacaville, California, US, with <NUM> mil spacer and a nominal cut-off value of <NUM>,<NUM> Daltons. The feed flow rate was <NUM>/h. Four loops were present in the continuous microfiltration equipment. The total membrane area was <NUM><NUM>. The filtration was carried out under the following conditions: The skimmed milk was concentrated with a concentration factor of <NUM>. The temperature was maintained at <NUM> degrees C, and the mean pressure was maintained at <NUM> bar across two filter elements with a feeding pressure of <NUM> bar. The permeate from the microfiltration was directed to a parallel ultrafiltration process, and the permeate from the ultrafiltration process was continuously directed back to the microfiltration retentate in order to carry out diafiltration of the microfiltration retentate. <NUM>% diafiltration was carried out, i.e. the volume of ultrafiltration permeate used for diafiltration was <NUM> times the volume of the skimmed milk supplied to the microfiltration process. The mean flux was approximately <NUM>/m<NUM>/h. The processed microfiltration retentate was continuously subjected to heat treatment at <NUM> degrees C for <NUM> seconds, cooled down to <NUM> degrees C and collected in a tank. A total of <NUM><NUM> of MCI (micellar casein isolate) solution was collected at the end of the microfiltration process. The protein content in the MCI solution was <NUM>% (gram protein per <NUM> gram solution).

Ultrafiltration of the permeate from the warm microfiltration:
The permeate from the microfiltration process was collected in a feed tank to the continuous ultrafiltration process. Simultaneously with the microfiltration process, ultrafiltration was carried out using <NUM>" spiral wound membranes of the type HFK-<NUM><NUM> from Koch Membrane Systems, Wilmington, Massachusetts, US, with <NUM> mil spacer and a nominal cut-off value of <NUM>,<NUM> Daltons. Two loops were present in the continuous ultrafiltration equipment. The total membrane area was <NUM><NUM>. The filtration was carried out under the following conditions: The temperature was maintained at <NUM> degrees C, and the mean pressure was maintained at <NUM> to <NUM> bars across three filter elements in order to supply ultrafiltration permeate to the microfiltration process with the same flow as microfiltration permeate was removed from the microfiltration process. The mean flux was approximately <NUM>/m<NUM>/h.

Storage of MCI solution:
The MCI solution was stored at <NUM> degrees C for a period of <NUM> hours.

Cold microfiltration of the MCI solution:
<NUM> litres of the stored MCI solution was subjected to microfiltration using <NUM>" spiral wound membranes of the type FR6338 from Synder Filtration, Vacaville, California, US, with <NUM> mil spacer and a nominal cut-off value of <NUM>,<NUM> Daltons. The total membrane area was <NUM><NUM>. The filtration was carried out under the following conditions: The temperature was maintained at approximately <NUM> degrees C, and the mean pressure was maintained at <NUM> bar across two filter elements with a feeding pressure of <NUM> bar. The permeate from the microfiltration was directed to a parallel ultrafiltration process, and the permeate from the ultrafiltration process was continuously directed back to the microfiltration retentate in order to carry out diafiltration of the microfiltration retentate. <NUM>% diafiltration was carried out, i.e. the volume of ultrafiltration permeate used for diafiltration was five times the volume of the MCI solution supplied to the microfiltration process. The mean flux was measured as <NUM>/m<NUM>/h.

Concentration of the permeate of the Cold MF by ultrafiltration:
The permeate from the cold microfiltration process was collected in a feed tank to the ultrafiltration process. Simultaneously with the cold microfiltration process, ultrafiltration was carried out using <NUM>" spiral wound membranes of the type HFK-<NUM><NUM> from Koch Membrane Systems, Wilmington, Massachusetts, US, with <NUM> mil spacer and a nominal cut-off value of <NUM>,<NUM> Daltons. The total membrane area was <NUM><NUM>. The filtration was carried out under the following conditions: The temperature was maintained at approximately <NUM> degrees C, and the mean pressure was maintained at <NUM> to <NUM> bars across two filter elements in order to supply ultrafiltration permeate to the microfiltration process with the same flow as microfiltration permeate was removed from the microfiltration process. The mean flux was approximately <NUM>/m<NUM>/h. When the filtration process was completed after <NUM> hours, approximately <NUM> litres of ultrafiltration retentate was collected. The retentate was subsequently subjected to diafiltration in which <NUM>,<NUM> litres of tap water was added with the same flow as filtrate was removed, in order to remove lactose. After the diafiltration the retentate was concentrated until the protein content in the retentate was <NUM>%. The final volume of the retentate was <NUM> litres. The filtration conditions were the same as above.

Pasteurisation and spray-drying of the beta-casein-containing UF-permeate: Approximately <NUM> litres of the final retentate from the cold ultrafiltration was subjected to pasteurisation at <NUM> degrees C for <NUM> seconds. After pasteurisation a one-stage spray drying of the protein solution was carried out using standard parameters including an air inlet temperature of <NUM> degrees C and an air outlet temperature of <NUM> degrees C. <NUM> of powder was obtained.

The content of protein in the powder was measured as <NUM>% (gram protein per <NUM> gram powder), and the content of dry matter in the powder was measured as <NUM>% (gram dry matter per <NUM> gram powder). The beta-casein content of the powder was analysed as described in Example <NUM> and determined to <NUM>% (gram beta-casein per <NUM> gram protein).

The content of the amino acid proline was analysed according to Example <NUM> and determined to <NUM> gram proline per <NUM> gram protein.

Another beta-casein isolate was produced according to the method of the present invention.

Warm microfiltration/MF-diafiltration:
Pre-treatment and microfiltration of cooled pasteurised skimmed milk was carried out essentially as described in Example <NUM>, except for the following: <NUM><NUM> of skimmed milk was used, and the skimmed milk was pre-heated to <NUM> degrees C for <NUM> minutes in a heat-and-hold tank. Both <NUM>" and <NUM>" spiral wound membranes were used, and five loops were present in the continuous microfiltration equipment. The total membrane area was <NUM><NUM>. The feed flow rate was <NUM>/h. The mean pressure was maintained at <NUM> bar across two filter elements with a feeding pressure of <NUM> bar. <NUM>% diafiltration was carried out, i.e. the volume of ultrafiltration permeate used for diafiltration was <NUM> times the volume of the skimmed milk supplied to the microfiltration process. The mean flux was measured as <NUM>/m<NUM>/h. A total of <NUM><NUM> of MCI (micellar casein isolate) solution was collected at the end of the microfiltration process. The protein content in the MCI solution was <NUM>% (gram protein per <NUM> gram solution).

Ultrafiltration of the permeate from the warm microfiltration:
The ultrafiltration process was carried out essentially as described in Example <NUM> except for the following: Membranes with <NUM> mil, <NUM> mil and <NUM> mil spacer were used. Four loops were present in the continuous ultrafiltration equipment. The total membrane area was <NUM><NUM>. The mean flux was approximately <NUM>/m<NUM>/h.

Cold microfiltration of the micellar casein isolate:
The <NUM><NUM> of stored MCI solution was subjected to continuous microfiltration using <NUM>" and <NUM>" spiral wound membranes of the type FR6338 from Synder Filtration, Vacaville, California, US, with <NUM> mil spacer and a nominal cut-off value of <NUM>,<NUM> Daltons. Five loops were present in the continuous microfiltration equipment. The total membrane area was <NUM><NUM>. The filtration was carried out under the following conditions: The temperature was maintained at <NUM> degrees C, and the mean pressure was maintained at <NUM> bar across two filter elements with a feeding pressure of <NUM> bar. The permeate from the microfiltration was directed to a parallel ultrafiltration process, and the permeate from the ultrafiltration process was continuously directed back to the microfiltration retentate in order to carry out diafiltration of the microfiltration retentate. <NUM>% diafiltration was carried out, i.e. the volume of ultrafiltration permeate used for diafiltration was <NUM> times the volume of the MCI solution supplied to the microfiltration process. The mean flux was approximately <NUM>/m<NUM>/h. The processed microfiltration retentate was continuously collected in a tank.

Concentration of the permeate of the Cold MF by ultrafiltration:
The permeate from the microfiltration process was collected in a feed tank to the continuous ultrafiltration process. Simultaneously with the microfiltration process, ultrafiltration was carried out using <NUM>" spiral wound membranes of the type HFK-<NUM><NUM> from Koch Membrane Systems, Wilmington, Massachusetts, US, with <NUM>, <NUM> and <NUM> mil spacer and a nominal cut-off value of <NUM>,<NUM> Daltons. Four loops were present in the continuous ultrafiltration equipment. The total membrane area was <NUM><NUM>. The filtration was carried out under the following conditions: The temperature was maintained at <NUM> degrees C, and the mean pressure was maintained at <NUM> to <NUM> bars across three filter elements in order to supply ultrafiltration permeate to the microfiltration process with the same flow as microfiltration permeate was removed from the microfiltration process. Continuously the retentate from the ultrafiltration process was subjected to diafiltration using demineralised water. The mean flux was approximately <NUM>/m<NUM>/h. At the end of the filtration, <NUM> litres of ultrafiltration retentate was collected.

Pasteurisation and spray-drying of the beta-casein-containing UF-permeate:
The <NUM> litres of ultrafiltration retentate was concentrated by reverse osmosis (RO) using standard operating conditions. <NUM> litres of RO concentrate was obtained, and the protein content in the RO retentate was <NUM>%. The RO retentate was subjected to pasteurisation at <NUM> degrees C for <NUM> seconds. After pasteurisation, a one-stage spray drying of the protein solution was carried out using standard parameters including an air inlet temperature of <NUM> degrees C and an air outlet temperature of <NUM> degrees C. <NUM> of powder was obtained.

The content of protein in the powder was measured as <NUM>% (gram protein per <NUM> gram powder), and the content of dry matter in the powder was measured as <NUM>% (gram dry matter per <NUM> gram powder). The beta-casein content of the powder was analyse as described in Example <NUM> and determined to <NUM>% (gram beta-casein per <NUM> gram protein).

The purity of beta-casein in powdered products was determined by <NPL>, and a water/acetonitrile solvent system. Prior to the analysis the sample is dissolved in <NUM> urea and <NUM> dithiothreitol with the purpose of obtaining a denatured and reduced protein solution.

The amino acid profile of the powdered products was analysed by standard amino acid analysis.

The beta-casein enriched powder produced in Example <NUM> was compared to a commercially available beta-casein product. This comparison was carried out by means of analysing the two products by capillary electrophoresis.

The obtained electropherograms are shown in <FIG> (product of Example <NUM>) and <FIG> (prior art product), and the individual known peaks are denominated in Table <NUM>.

Claim 1:
A method of producing a beta-casein-containing composition containing at least <NUM>% (w/w) beta-casein relative to the total amount of casein and at least <NUM>% (w/w) beta-casein relative to the total amount of proteins, the method comprising the steps of:
a) pre-heating a milk by adjusting it to a pre-heating temperature (Tpre) of at least <NUM> degrees C, said milk is kept within the pre-heating temperature range for a duration tpre of at most <NUM> hours thereby providing a warm milk,
b) subjecting the warm milk to microfiltration (MF), wherein the temperature of the feed during MF of the warm milk is maintained at a temperature (TwMF) of at least <NUM> degrees C and using a filter that retains at least a substantial fraction of the casein micelles, but allows for the passage of milk serum protein thereby providing a first MF permeate and a first MF retentate,
c) subjecting the first MF retentate to MF-diafiltration, using a first diluent having a pH in the range of <NUM>-<NUM>, and wherein the temperature of the feed and retentate stream is kept within the warm MF temperature range during at least part of the MF-diafiltration,
d) adjusting the temperature of a first composition derived from the first MF retentate, said first composition comprises a total amount of casein of at least <NUM>% (w/w) relative to the total amount of protein of the first composition, to a cold temperature (Tcold) in the range of <NUM>-<NUM> degrees C and keeping the temperature of the first composition within that range for a duration (tcold) of at least <NUM> hour, thereby obtaining a cooled first composition,
e) subjecting the cooled first composition to MF, thereby obtaining a second retentate and a second permeate, which second permeate is enriched with respect to beta-casein, and
f) optionally, subjecting the second MF retentate to MF-diafiltration,
g) optionally, subjecting a second composition derived from the second permeate to one or more further processing steps, e.g. further purification and/or concentration steps,
thereby providing the beta-casein-containing composition.