Method for preparing dairy product with reduced plasmin activity

Abstract

The invention relates to a method for preparing a dairy product with reduced plasmin activity. The present invention relates to a method for inactivating plasmin in a dairy product, comprising: separating casein and whey protein from a milk raw material by microfiltration, providing a casein concentrate and a whey protein concentrate; subjecting the casein concentrate to a heat treatment to provide a heat-treated casein concentrate; combining the heat treated casein concentrate having fully inactivated plasmin activity with a whey protein concentrate, lactose, milk minerals, and water to provide a dairy product having reduced plasmin activity; the dairy product having reduced plasmin activity is heat treated to provide a dairy product exhibiting fully inactivated plasmin activity. The dairy product prepared by the method maintains perfect sensory characteristics at different storage temperatures even if stored for a long time.

Description

[0001] This application is a divisional application of the original application with international application number PCT / FI2016 / 050603, Chinese national application number 201680049813.7, filing date 31 August 2016, and invention title "Method for the production of a milk product with reduced plasmin activity". TECHNICAL FIELD

[0002] The present invention relates to a method for the production of a milk product. The method provides a milk product with a long shelf life with maintained good sensory properties. BACKGROUND

[0003] Ultra-high temperature (UHT) treatment is a well-known process in the field of dairy to provide milk products with an extended shelf life at ambient temperature. UHT treatment can be carried out at a temperature of e.g. 135°C or higher for a time exceeding 1 second. UHT treatment destroys pathogenic and spoilage microorganisms and their spores in milk. However, UHT treatment does not necessarily inactivate enzymes contained in milk which can naturally occur in milk or originate from microorganisms. One example of such an enzyme is the indigenous milk protease, plasmin, which is bound to casein micelles in milk. Plasmin is characterized by its low activity at low temperatures but a strong increase at room temperature. Plasmin is heat stable and retains activity even under UHT treatment. The shelf life of a dairy product stored at room temperature is limited by the presence of plasmin which breaks down casein into smaller compounds resulting in a bitter taste or structural defects in milk.

[0004] The beta-lactoglobulin present in milk is a natural inhibitor of plasmin. Inactivation of plasmin typically requires a rigorous heat treatment, such as an extended heat treatment. In an extended heat treatment, the beta-lactoglobulin is extensively denatured and has an adverse effect on the sensory properties of the milk which typically has a strong cooked or even burnt taste. Without wishing to be bound by theory, it is believed that the beta-lactoglobulin reacts with the plasmin and inactivates it during the heat treatment. The presence of plasmin typically impairs the sensory properties of the dairy product, especially when stored at ambient temperature for a long period of time. In order to maintain good sensory properties of the milk throughout the sales period, the plasmin must be sufficiently inactivated.

[0005] WO2010 / 085957A1 discloses a method for the production of a long shelf life dairy product, wherein the milk is subjected to a physical separation of microorganisms and a high temperature treatment at 140-180°C for a time of at least 200 milliseconds.

[0006] WO2012 / 010699A1 discloses a method for the production of a long shelf life dairy product with reduced lactose content, wherein the lactose reduced milk is subjected to a high temperature treatment at 140-180°C for a time of at least 200 milliseconds.

[0007] WO 2009 / 000972 A1 discloses a method for producing well-preserved low-lactose or lactose-free dairy products. The proteins and sugars of milk are first separated into different fractions, which are then separately subjected to ultra-high temperature treatment. After the UHT treatment, the fractions are recombined. It is reported that the plasmin system can be inactivated and the Maillard browning reaction can be avoided, whereby taste, colour and texture defects of UHT-treated dairy products can be avoided.

[0008] There is a need for a simple, effective and economic method for preparing long shelf-life dairy products with perfect taste at different storage temperatures. SUMMARY

[0009] In the present invention,

[0010] The term "milk raw material" can be milk obtained from an animal such as a cow, a sheep, a goat, a camel, a mare or any other animal producing milk suitable for human consumption, or any liquid component derived therefrom;

[0011] The term "whey protein" has its well-defined meaning known to the person skilled in the art and refers to the protein fraction of milk that does not precipitate at pH 4.6. The term "whey" includes sweet whey and acid whey derived from cheese manufacture or casein manufacture, as well as ideal whey obtained by subjecting milk to various membrane filtrations (e.g. microfiltration, ultrafiltration, nanofiltration, reverse osmosis, dialysis), chromatography, crystallization, or combinations thereof;

[0012] The term "milk-derived sugar" mainly refers to lactose, but can include other sugars such as oligomeric galactose and lactose hydrolysis products, i.e. glucose and galactose;

[0013] The term "whey protein concentrate" is a fraction with a higher proportion of whey protein than the starting milk raw material used,

[0014] The term "casein fraction" includes casein concentrate and casein preparation.

[0015] It is an object of the present invention to provide a method for inactivating plasmin in a dairy product, said method comprising the steps of:

[0016] - providing a milk raw material,

[0017] - separating casein and whey protein from said milk raw material by microfiltration, thereby providing a casein concentrate as microfiltration retentate and a whey protein concentrate as microfiltration permeate, the whey protein content of the casein concentrate being less than 20 wt%, in particular at most about 18 wt%, more particularly at most about 14 wt%, more particularly at most about 12 wt%, more particularly at most about 6 wt%, based on the total protein content of the casein concentrate,

[0018] - heat-treating the casein concentrate at a temperature of about 72°C to about 95°C, in particular about 80°C to about 95°C, thereby providing a heat-treated casein concentrate,

[0019] - providing a milk product with reduced plasmin activity comprising the heat-treated casein concentrate.

[0020] It is another object of the present application to provide a method for preparing a milk product with reduced plasmin activity, said method comprising the steps of:

[0021] - providing a casein product having a whey protein content of less than 20 wt%, in particular at most about 18 wt%, more in particular at most about 14 wt%, more in particular at most about 12 wt%, more in particular at most about 6 wt%, based on the total protein content of the casein product,

[0022] - heat-treating the casein product at a temperature of about 72°C to about 95°C, in particular about 80°C to about 95°C, thereby providing a heat-treated casein product,

[0023] - providing a milk product with reduced plasmin activity comprising the heat-treated casein product.

[0024] The present application provides a method for significantly reducing the plasmin activity of milk. The milk product prepared by the present method has perfect sensory properties at different storage temperatures.

[0025] There is a large amount of literature describing the separation of milk components, i.e. casein, whey protein, lactose and milk minerals, into different fractions by different separation techniques, such as chromatography and membrane filtration, including microfiltration, ultrafiltration, nanofiltration, diafiltration, reverse osmosis. The separated fractions can then be combined in appropriate ways to various milk products having different protein, lactose and mineral content, thereby providing the desired properties for the product in each case. Casein and whey protein can typically be separated into different fractions by microfiltration or chromatography. In microfiltration, plasmin is retained on the retentate side by the casein molecules, while the whey proteins pass the microfiltration membrane into the permeate.

[0026] It has surprisingly been found in the present invention that when the casein and whey proteins of milk are first separated and only the casein fraction with reduced whey protein content is subjected to a prolonged heat treatment based plasmin inactivation procedure, the defects in the organoleptic properties (e.g. taste) of prior art dairy products with prolonged shelf life can be avoided. After plasmin inactivation, the heat treated casein fraction can be recombined with natural ingredients such as whey proteins, lactose and milk minerals, and water, to provide a dairy product with various protein, lactose and mineral content and varying casein to whey protein ratios. By this method, heat sensitive whey proteins, in particular beta-lactoglobulin, can be kept denatured and the product does not develop a cooked taste.

[0027] The present invention also provides a method for preparing a dairy product showing reduced Maillard reaction. Maillard brown products generated in the Maillard reaction have an adverse effect on the organoleptic properties of dairy products, in particular ultra-high temperature (UHT) treated dairy products. Since in the present invention the casein fraction subjected to the plasmin inactivation procedure contains only a small amount of lactose, the Maillard reaction is weak and the formation of undesired Maillard products in the dairy product is avoided.

[0028] The method of the present invention provides a dairy product with a low degree of whey protein denaturation and plasmin activity, thus preserving the good organoleptic properties of the product.

[0029] The method of the present invention is simple, efficient and economical.

[0030] Another object of the present invention is a casein fraction having a whey protein content of at most about 18 wt%, in particular at most about 14 wt%, more in particular at most about 12 wt%, more in particular at most about 6 wt%, based on the total protein content of the casein fraction.

[0031] A further object of the present invention is the use of a casein fraction having a whey protein content of at most about 18 wt%, in particular at most about 14 wt%, more in particular at most about 12 wt%, more in particular at most about 6 wt%, based on the total protein content of the casein fraction, for the preparation of a dairy product with reduced plasmin activity. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The plasmin activity of the casein fraction before and after heat treatment is shown.

[0033] Figure 2 The degree of proteolysis of a dairy product prepared by one embodiment of the method of the present invention during storage is shown. DETAILED DESCRIPTION

[0034] An object of the present invention is to provide a method for inactivating plasmin, said method comprising the steps of:

[0035] - providing a milk raw material,

[0036] - isolating casein and whey proteins from the milk raw material by microfiltration, thereby providing a casein concentrate and a whey protein concentrate as microfiltration permeate, the whey protein content of the casein concentrate being less than 20 wt%, in particular at most about 18 wt%, more particularly at most about 14 wt%, more particularly at most about 12 wt%, more particularly at most about 6 wt%, based on the total protein content of the casein concentrate,

[0037] - heat-treating the casein concentrate at a temperature of about 72°C to about 95°C, in particular about 80°C to about 95°C, thereby providing a heat-treated casein concentrate,

[0038] - providing a milk product having reduced plasmin activity, comprising the heat-treated casein concentrate.

[0039] If it is desired to adjust the protein, fat and / or lactose content to a desired level, the milk raw material can be pre-treated. For example, the milk raw material can be standardized for fat, and if desired, for protein content, in a manner generally known in the art. Furthermore, the milk raw material can be pre-treated by, for example, pasteurization, in order to reduce its microbial load in a manner generally known in the art. Removal of pathogenic and spoilage microorganisms is generally performed by physical separation, such as microfiltration, centrifugal bacteria removal or a combination thereof. In one embodiment of the present application, no physical separation of microorganisms of the milk raw material is performed.

[0040] Prior to isolating casein and whey, the milk raw material, optionally standardized (for fat, protein and / or lactose) and / or pre-treated for microbial removal (microfiltration, centrifugal bacteria removal), can be subjected to a heat treatment. The pore size of the microfiltration membrane in microbial removal is generally in the range of 0.8 to 1.4 pm. Examples of suitable heat treatments include, but are not limited to, to pasteurization, high pasteurization, or heating at a temperature lower than pasteurization temperature for a sufficient long time. In particular, UHT treatment at 138°C to about 180°C for at least 3 seconds (e.g. milk at least 138°C, 3 to 4 seconds), high pasteurization at 125°C to 138°C for 0.5 to 4 seconds (e.g. milk at 130°C, 1 to 2 seconds), pasteurization at 72°C to 75°C for 15 to 20 seconds (e.g. milk at 72°C, 15 seconds), heat retarding at 57°C to 68°C for up to 40 minutes (e.g. at 65°C, 2 seconds to 3 minutes), heat treatment at least 150°C for up to 0.3 seconds can be mentioned. The heat treatment can be direct (steam provided to milk, milk provided to steam) or indirect (tubular heat exchanger, plate heat exchanger, scraped surface heat exchanger).

[0041] If desired, the lactose content of the milk raw material can be reduced. In one embodiment, the lactose content is reduced enzymatically by adding lactase to the raw material. Lactases commonly used for lactose hydrolysis of milk can be used. The lactose content can also be reduced by other suitable means commonly known in the art, such as by membrane filtration, chromatography, electrodialysis, crystallization, centrifugation or precipitation. The various techniques can be combined in a suitable manner. The low lactose raw material can be further subjected to lactose hydrolysis to provide a lactose-free milk raw material.

[0042] In the present application, the milk raw material can thus be, for example, whole (full) milk, cream, low-fat milk, skim milk, buttermilk, colostrum, low-lactose milk, lactose-free milk, whey protein-depleted milk, calcium-depleted milk, milk reconstituted from milk powder, or a combination thereof (as such or a concentrate thereof), and pre-treated, such as heat-treated, as described above.

[0043] In one embodiment, the milk raw material is derived from cow's milk.

[0044] The milk raw material can contain fat and / or protein of plant origin.

[0045] In the method of the present application, casein is separated from the milk raw material, thereby providing a casein concentrate having a whey protein content of less than 20% by weight, based on the total protein content of the concentrate. In one embodiment, the whey protein content is at most about 18% by weight. In another embodiment, the whey protein content is at most about 14% by weight. In yet another embodiment, the whey protein content is at most about 12% by weight. In a particular embodiment, the whey protein content is at most about 6% by weight.

[0046] The total solids content of the casein concentrate can be adjusted to a desired level. For example, the obtained casein concentrate can be concentrated, e.g. by evaporation, into a powder.

[0047] The separation of casein and whey protein is performed by microfiltration. The pore size of the microfiltration membrane is typically about 0.08 pm.

[0048] In one embodiment, the microfiltration is performed at a temperature of 1 °C to 55 °C. In another embodiment, the microfiltration is performed at a temperature of 10 °C to 15 °C.

[0049] In one embodiment, the microfiltration is performed at a pressure of at most 3 bar. In another embodiment, the microfiltration is performed at a temperature of 10 °C to 15 °C and a pressure of less than 1.5 bar.

[0050] The isolated casein concentrate is heat treated at a temperature of from about 72°C to about 95°C to inactivate the plasmin in the concentrate. In one embodiment, the plasmin inactivation of the casein concentrate is at a temperature of from about 80°C to about 95°C. In one embodiment, the plasmin inactivation of the casein concentrate is for a time of from about 30 seconds to about 600 seconds. In another embodiment, the plasmin inactivation is for a time of from about 30 seconds to about 300 seconds. In another embodiment, the plasmin inactivation of the casein concentrate is at a temperature of from about 72°C to about 95°C for a time of from about 30 seconds to about 600 seconds. In yet another embodiment, the plasmin inactivation is at a temperature of from about 72°C to about 95°C for a time of from about 30 seconds to about 300 seconds. In yet another embodiment, the plasmin inactivation is at a temperature of from about 80°C to about 95°C for a time of from about 30 seconds to about 600 seconds. In yet another embodiment, the plasmin inactivation is at a temperature of from about 80°C to about 95°C for a time of from about 30 seconds to about 300 seconds. In a particular embodiment, the inactivation is at about 95°C for about 300 seconds.

[0051] The heat treated casein concentrate subjected to heat treatment has a reduced ratio of milk-derived sugar to casein as compared to normal milk. In one embodiment, the milk-derived sugar is lactose. In one embodiment, the ratio of milk-derived sugar to casein in the casein concentrate is at most about 1. In another embodiment, the ratio of milk-derived sugar to casein in the casein concentrate is at most about 0.5. In another embodiment, the ratio of milk-derived sugar to casein in the casein concentrate is at most about 0.2.

[0052] The heat treated casein concentrate with reduced plasmin activity can then be combined with natural milk ingredients such as whey protein, lactose, and milk minerals, as well as water, to provide various types of milk products. The protein, lactose, and mineral content of these products can be adjusted to desired levels. In addition, the ratio of casein to whey protein can be adjusted to that of normal milk (80 / 20), or higher or lower. The milk products can be flavored or unflavored.

[0053] In one embodiment, the milk product produced by the above-described methods of the present application comprises at least about 50% by weight of the heat treated casein concentrate based on the total protein content of the milk product. The milk ingredients suitable for use in the preparation of the milk product can be obtained as a commodity, or they can be prepared by various separation techniques including, but not limited to, membrane filtration, chromatography, precipitation, centrifugation, and evaporation. The separation of various milk components by membrane filtration, including microfiltration, ultrafiltration, nanofiltration, diafiltration, reverse osmosis, is widely described in the literature and is well known to those skilled in the art. The milk ingredients can be provided in liquid to powder form.

[0054] In another object, the present invention provides a method for the preparation of a milk product having reduced plasmin activity, said method comprising the steps of:

[0055] - providing a casein product having a whey protein content of less than 20 wt%, in particular at most about 18 wt%, more particularly at most about 14 wt%, more particularly at most about 12 wt%, more particularly at most about 6 wt%, based on the total protein content of the casein product,

[0056] - heat treating the casein product at a temperature of about 72°C to about 95°C, in particular about 80°C to about 95°C, thereby providing a heat treated casein product,

[0057] - providing a milk product having reduced plasmin activity comprising the heat treated casein product.

[0058] The casein product used in the method for the production of a milk product can be obtained in any suitable way. In one embodiment, the casein product is a casein concentrate obtained by microfiltration of a milk raw material to separate the casein and whey proteins into different fractions. The casein concentrate is obtained as a microfiltration retentate and the whey protein concentrate is obtained as a microfiltration permeate. The pore size of the microfiltration membrane is typically about 0.08 pm. In one embodiment, the microfiltration is performed at a temperature of 1 °C to 55 °C. In another embodiment, the microfiltration is performed at a temperature of 10 °C to 15 °C. In one embodiment, the microfiltration is performed at a pressure of at most 3 bar. In another embodiment, the microfiltration is performed at a temperature of 10 °C to 15 °C and a pressure of less than 1.5 bar.

[0059] The casein product used in the method for the production of a milk product can be in a form from liquid to powder. In one embodiment, the casein product is a casein concentrate obtained as a microfiltration retentate and is further concentrated, e.g. by evaporation, to have the desired total solids content.

[0060] Furthermore, the whey protein concentrate can conveniently be further concentrated by ultrafiltration to provide a whey protein concentrate as an ultrafiltration retentate. The whey protein concentrate can be in a form from liquid to powder.

[0061] The casein product used in the method of producing a dairy product is subjected to a heat treatment similar to that described above to inactivate the plasmin in the casein concentrate. Thus, the heat treatment is carried out at a temperature of from about 72°C to about 95°C so as to inactivate. In one embodiment, the plasmin inactivation of the casein product is carried out at a temperature of from about 80°C to about 95°C. In one embodiment, the plasmin inactivation of the casein product is carried out for a time of from about 30 seconds to about 600 seconds. In another embodiment, the plasmin inactivation is carried out for a time of from about 30 seconds to about 300 seconds. In another embodiment, the plasmin inactivation of the casein product is carried out at a temperature of from about 72°C to about 95°C for a time of from about 30 seconds to about 600 seconds. In yet another embodiment, the plasmin inactivation is carried out at a temperature of from about 72°C to about 95°C for a time of from about 30 seconds to about 300 seconds. In yet another embodiment, the plasmin inactivation is carried out at a temperature in the range of from about 80°C to about 95°C for a time of from about 30 seconds to about 600 seconds. In yet another embodiment, the plasmin inactivation is carried out at a temperature of from about 80°C to about 95°C for a time of from about 30 seconds to about 300 seconds. In a particular embodiment, the inactivation is carried out at about 95°C for about 300 seconds.

[0062] The casein product used in the method of producing a dairy product has a reduced ratio of milk-derived sugar to casein as compared to normal milk. In one embodiment, the milk-derived sugar is lactose. In one embodiment, the ratio of milk-derived sugar to casein in the casein product is at most about 1. In another embodiment, the ratio of milk-derived sugar to casein in the casein product is at most about 0.5. In another embodiment, the ratio of milk-derived sugar to casein in the casein product is at most about 0.2.

[0063] The heat-treated casein product having reduced plasmin activity is combined with other milk components such as whey proteins, lactose and milk minerals, and optionally water, in appropriate proportions to provide a dairy product. The dairy product produced by the method of the present application comprises the heat-treated casein product. In one embodiment, the dairy product produced by the method of the present application comprises at least about 50% by weight of the heat-treated casein product based on the total protein content of the dairy product. The milk components suitable for use in the preparation of the dairy product can be obtained as a commodity, or they can be prepared by various separation techniques including, but not limited to, membrane filtration, chromatography, precipitation, centrifugation and evaporation. The separation of various milk components by membrane filtration, including microfiltration, ultrafiltration, nanofiltration, diafiltration, reverse osmosis, is widely described in the literature and is well known to those skilled in the art. The milk components can be provided in the form of a liquid to a powder.

[0064] After the heat treated casein product is combined with other milk components and optionally water to provide a milk product having the desired protein, lactose, and fat content, the product is optionally subjected to a heat treatment which destroys spoilage microorganisms in the product. In one embodiment, a heat treatment is performed. Suitable heat treatments for use in the present application are similar to those performed on milk raw materials and described above. Examples of suitable heat treatments include, but are not limited to, pasteurization, high temperature pasteurization, or heating at a temperature lower than pasteurization temperature for a sufficient length of time. In particular, one can mention UHT treatment at 138°C to about 180°C for at least 3 seconds (e.g., milk at least 138°C, 3 to 4 seconds), high temperature pasteurization at 125°C to 138°C for 0.5 to 4 seconds (e.g., milk at 130°C, 1 to 2 seconds), pasteurization at 72°C to 75°C for 15 to 20 seconds (e.g., milk at 72°C, 15 seconds), heat retarding at 57°C to 68°C for up to 40 minutes (e.g., at 65°C, 2 seconds to 3 minutes), heat treatment at at least 150°C for up to 0.3 seconds. The heat treatment can be direct (steam provided to milk, milk provided to steam) or indirect (tubular heat exchanger, plate heat exchanger, scraped surface heat exchanger).

[0065] The protein, lactose, fat, and mineral content of the milk product can be adjusted to the desired level. In addition, the ratio of casein to whey protein can be adjusted to that of normal milk (80 / 20), or higher or lower. The milk product can be flavored or unflavored. The fat content of the milk product produced by the method of the present application is typically 0.05% to 10%, particularly 1.0% to 3.0%. The protein content of the milk product is at least about 0.9%. In one embodiment, the protein content is about 0.9% to about 20%.

[0066] The milk product produced by the method of the present application can be dried to a powder.

[0067] In one embodiment, the method of the present application includes a lactose hydrolysis step. Lactose hydrolysis can be performed at any suitable step. In one embodiment, lactose hydrolysis of the milk raw material is performed prior to separation of the casein and whey proteins. In another embodiment, lactose hydrolysis is performed after the heat treated casein product is combined with other milk components, but prior to optional heat treatment. In one embodiment, lactose hydrolysis is performed after heat treatment of the milk product.

[0068] In one embodiment, the milk product is low lactose having a lactose content of at most 1%. In another embodiment, the milk product is lactose free, having a lactose content of at most 0.01%.

[0069] In one embodiment, the milk product produced by the method of the present application is packaged under aseptic conditions.

[0070] The process of the present application can be a continuous process or a batch process.

[0071] The dairy products prepared by the process of the present application include, but are not limited to, milk products having different fat, protein and lactose content, milk products having increased whey protein content, milk products having increased casein protein content or milk products having increased total protein content, and mixtures thereof, infant formula and baby food having reduced total protein content.

[0072] The dairy products prepared by the process of the present application can be dried into a powder or further processed into other dairy products including fermented acid dairy products such as yoghurt, fermented milk, viili, fermented cream, sour cream, quark, butter milk, kefir, dairy shot drinks and cream cheese, or ice cream.

[0073] Another object of the present application is a casein fraction having a whey protein content of at most about 18 wt.%, in particular at most about 14 wt.%, more particularly at most about 12 wt.%, even more particularly at most about 6 wt.%, based on the total protein content of the casein fraction. In one embodiment, the ratio of milk-derived sugars to casein in the casein fraction is at most about 1. In another embodiment, the ratio of milk-derived sugars to casein in the casein fraction is at most about 0.5. In another embodiment, the ratio of milk-derived sugars to casein in the casein fraction is at most about 0.2. The casein fraction of the present application can be used for the preparation of various dairy products which maintain perfect sensory properties even at different storage temperatures during long term storage.

[0074] Another object of the present application is the use of a casein fraction for the preparation of a dairy product having reduced plasmin activity, the casein fraction having a whey protein content of at most about 18 wt.%, in particular at most about 14 wt.%, more particularly at most about 12 wt.%, even more particularly at most about 6 wt.%, based on the total protein content of the casein fraction. In one embodiment, the ratio of milk-derived sugars to casein in the casein fraction is at most about 1. In another embodiment, the ratio of milk-derived sugars to casein in the casein fraction is at most about 0.5. In another embodiment, the ratio of milk-derived sugars to casein in the casein fraction is at most about 0.2.

[0075] The following examples are given to further illustrate the present application and are not intended to limit the application thereto. The percentages of the various components in the tables below are by weight. In these examples, the following analytical methods were used:

[0076] Plasmin activity: modified method of M. Korycka-Dahl et al. (M. Korycka-Dahl, B. Ribadeau Dumas, N. Chene, J. Martal: Plasmin activity in milk, Journal of Dairy Science, 66(4) (1983), pp. 704-711)

[0077] SDS-PAGE: according to Laemmli (1970) using 18% Criterion TGX precast gels (Bio-Rad, USA). Protein bands were stained with Coomassie Brilliant Blue R-250 (Bio-Rad, USA) and compared to molecular weight markers (PrecisionPlus Protein standards, Bio-Rad, USA) (Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685)

[0078] Native whey proteins: modified size exclusion chromatography (Syva oja, E.-L., Korhonen, H., Determination of colostral immunoglobulins by gel filtration chromatography, IDF Special issue 9404, International Dairy Federation, Bruessels (1994) 216-219)

[0079] Furosine: according to IDF standard (IDF 193:2004(E) / ISO 18329:2004(E). Milk and milk products - Determination of furosine content - Ion-pair reverse-phase high-performance liquid chromatography method, 11 p.). Furosine reacts to chemical changes caused by heat treatment of proteins. The higher the furosine content, the greater the changes that have occurred in the proteins.

[0080] Free tyrosine equivalent: As described by Matsubara et al. (Matsubara, H., Hagihara, B., Nakai, M., Komaki, T., Yonetani, T., Okunuki, K., Crystalline bacterial proteinase II. General properties of crystalline proteinase of Bacillus subtilis N', J. Biochem. 45 (4) (1958) 251-258). Free tyrosine equivalent can be used to study the level of proteolysis in the product.

[0081] Example 1. Isolation of milk by membrane filtration

[0082] Skim milk was ultrafiltered at a volume concentration ratio (VCR) of 3.7 at about 10°C. The membrane used for ultrafiltration was Koch HKF131 from Koch Membrane Systems. Table 1 shows the composition of the skim milk and the obtained milk protein concentrate (i.e. the ultrafiltration retentate).

[0083] Example 2. Isolation of milk by membrane filtration

[0084] The skim milk was microfiltered at a temperature of 10-15°C at a pressure of less than 1.5 bar, thereby concentrating the casein in the microfiltration retentate. The membrane used in the microfiltration was Synder FR from Synder Filtration, Inc. The skim milk was first microfiltered at a concentration factor of about 4. The microfiltration was then continued by diafiltration, wherein tap water was added to the resulting microfiltration retentate in an amount equal to the amount of retentate obtained. The microfiltration was continued until an amount of permeate was discharged that was equal to the amount of water added. The diafiltration step was repeated twice. The permeate obtained from the two diafiltration steps was combined and the combined mixture was ultrafiltered at a temperature of 10-15°C with a Koch HKF 131 membrane to concentrate the whey proteins in the ultrafiltration retentate. The ultrafiltration was continued until the protein content of the retentate reached 9%.

[0085] The permeate of the ultrafiltration was concentrated by nanofiltration (membrane Desal DK, filtration temperature 10°C), thereby concentrating the lactose in the nanofiltration retentate to a total solids content (TS) of the retentate of about 20%.

[0086] The obtained nanofiltration permeate was concentrated by reverse osmosis (membrane Filmtec RO, filtration temperature about 10°C), thereby concentrating the minerals in the retentate. The filtration was continued until the TS of the retentate was about 2.5%.

[0087] Table 1 shows the composition of the various fractions obtained in the microfiltration, ultrafiltration, nanofiltration and reverse osmosis in Examples 1 and 2 above. Table 1 further shows the composition of the cream separated from the whole milk. All the milk components and fractions given in Table 1 can be used for the preparation of dairy products.

[0088] Table 1.

[0089]

[0090] * Non-nitrogen proteins

[0091] Example 3. Preparation of a skimmed dairy product according to the method of the application

[0092] A skimmed dairy product was prepared with the method of the application. The casein concentrate obtained in Example 2 was heat pre-treated by indirect heating (95°C, 5 minutes) to inactivate the fibrinolysin. Samples were taken from the casein concentrate before and after the heat pre-treatment step. The results of the fibrinolytic activity in the casein concentrate before and after heat pre-treatment are shown in Figure 1 The results show that the fibrinolytic activity can be completely inactivated by heat pre-treatment of the casein concentrate.

[0093] The heat pre-treated casein concentrate was used together with the other membrane filtration fractions obtained in Example 2 for the preparation of a skimmed dairy product. The recipe and composition of the skimmed dairy product are listed in Table 2. The components were combined and mixed thoroughly. The mixture was heat treated at 157°C for 0.1 seconds by direct steam injection (steam injection equipment from SPX, Denmark) and aseptically packaged. The packages were stored at 20°C and 6°C.

[0094] Table 2.

[0095]

[0096] Fibrinolytic activity, furosine content and the amount of native beta-lactoglobulin and alpha-lactalbumin were determined from samples taken from the mixture before heat treatment and from the heat treated (157°C, 0.1 seconds) and aseptically packaged product. The results are listed in Table 3. The fibrinolytic activity in the mixture before heat treatment was already very low due to the heat pre-treatment step. No activity was detected in the heat treated product. In addition, the heat treated product had a very low furosine content and a high content of native beta-lactoglobulin and alpha-lactalbumin, indicating that minor chemical changes occurred during the heat treatment.

[0097] Table 3

[0098]

[0099] The degree of proteolysis during storage was evaluated by determining the tyrosine equivalent of the samples. The results are shown in Figure 2No significant proteolysis was detected in the samples during storage, indicating that plasmin was very effectively inactivated by the method of the present application.

[0100] It will be obvious to a person skilled in the art that, as technology advances, the inventive concept can be implemented in various ways. The application and its embodiments are not limited to the examples described above but can vary within the scope of the claims.

Claims

1. A method for inactivating plasmin in dairy products, the method comprising the following steps: - Provide dairy raw materials, - Casein and whey protein are separated from the milk raw material by microfiltration, thereby providing a casein concentrate as a microfiltration residue and a whey protein concentrate as a microfiltration extract, wherein the whey protein content of the casein concentrate is at most 6% by weight, based on the total protein content of the casein concentrate. - The casein concentrate is heat-treated at a temperature of 72°C to 95°C for 30 to 600 seconds to provide a heat-treated casein concentrate with completely inactivated plasmin activity. - A heat-treated casein concentrate with completely inactivated plasmin activity is combined with the whey protein concentrate, lactose, milk minerals, and water to provide a dairy product with reduced plasmin activity. - The dairy products with reduced plasmin activity are subjected to heat treatment at at least 150°C for up to 0.3 seconds to provide dairy products exhibiting completely inactivated plasmin activity.

2. A method for preparing a dairy product with completely inactivated plasmin activity, the method comprising the following steps: - Provide dairy raw materials, - Casein and whey protein are separated from the milk raw material by microfiltration, thereby providing a casein concentrate as a microfiltration residue and a whey protein concentrate as a microfiltration extract, wherein the whey protein content of the casein concentrate is at most 6% by weight, based on the total protein content of the casein concentrate. - The casein concentrate is heat-treated at a temperature of 72°C to 95°C for 30 to 600 seconds to provide a heat-treated casein product with completely inactivated plasmin activity. - A heat-treated casein product with completely inactivated plasmin activity is combined with said whey protein concentrate, lactose, milk minerals, and water to provide a dairy product with reduced plasmin activity, comprising a heat-treated casein product exhibiting completely inactivated plasmin activity, and also comprising whey protein, lactose, milk minerals, and optionally water. The dairy products with reduced plasmin activity are subjected to heat treatment at at least 150°C for up to 0.3 seconds to provide dairy products exhibiting completely inactivated plasmin activity.

3. The method as described in claim 1 or 2, wherein, The pore size of the microfiltration membrane is 0.08 µm.

4. The method as described in claim 1 or 2, wherein, The microfiltration is performed at temperatures ranging from 1°C to 55°C.

5. The method of claim 4, wherein, The microfiltration is performed at a temperature of 10°C to 15°C.

6. The method as described in claim 1 or 2, wherein, The microfiltration is performed at a pressure of up to 3 bar.

7. The method as described in claim 1 or 2, wherein, The microfiltration is performed at a temperature of 10°C to 15°C and a pressure of less than 1.5 bar.

8. The method as claimed in claim 1 or 2, wherein, The heat treatment of the casein concentrate is carried out at a temperature of 72°C to 95°C for 30 to 300 seconds.

9. The method as claimed in claim 1 or 2, wherein, The heat treatment of the casein concentrate is carried out at a temperature of 80°C to 95°C for 30 to 600 seconds.

10. The method as claimed in claim 1 or 2, wherein, The heat treatment of the casein concentrate is carried out at a temperature of 80°C to 95°C for 30 to 300 seconds.

11. The method as claimed in claim 1 or 2, wherein, The heat treatment of the casein concentrate was carried out at 95°C for 300 seconds.

12. The method as claimed in claim 1 or 2, wherein, The casein concentrate or the casein product is concentrated into a powder.

13. The method as claimed in claim 1 or 2, wherein, The ratio of milk-derived sugars to casein in the casein concentrate or casein product is at most 1.

14. The method of claim 13, wherein, The ratio of milk-derived sugars to casein in the casein concentrate or casein product is at most 0.

5.

15. The method of claim 13, wherein, The ratio of milk-derived sugars to casein in the casein concentrate or casein product is at most 0.

2.

16. The method as claimed in claim 1 or 2, wherein, No physical separation of microorganisms was performed.

17. The method as claimed in claim 1 or 2, wherein, The amount of the heat-treated casein concentrate or the heat-treated casein product in the dairy product is at least 50% by weight of the total protein content of the dairy product.

18. The method of claim 1 or 2, further comprising a lactose hydrolysis step.

19. The method as claimed in claim 1 or 2, wherein, The dairy products are aseptically packaged.

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