WO2013157004A1

WO2013157004A1 - Method of recovery of milk fat globule membrane

Info

Publication number: WO2013157004A1
Application number: PCT/IL2013/050334
Authority: WO
Inventors: Vitaly SPITSBERG; Adie MANNHEIM; Michael ZALTZMAN
Original assignee: BA'EMEK ADVANCED TECHNOLOGIES Ltd
Current assignee: BA'EMEK ADVANCED TECHNOLOGIES Ltd
Priority date: 2012-04-18
Filing date: 2013-04-18
Publication date: 2013-10-24
Anticipated expiration: 2014-10-18

Description

METHOD OF RECOVERY OF MILK FAT GLOBULE MEMBRANE

FIELD OF THE INVENTION

This invention relates to isolation of ingredients from milk, specifically to isolation of milk fat globule membrane from milk, buttermilk or other milk products.

BACKGROUND OF THE INVENTION

Milk and its products such as butter, cheese, whey and buttermilk account for a major industrial arena in the food industry. These contain lactose, minerals, caseins, and serum proteins which can be used in various food or non food applications.

Industrial Buttermilk is the liquid phase released during destabilization (churning) of cream in the butter making process. Thus, for many years buttermilk was considered an invaluable by-product. However, over the last two decades a valorization process has been established due to the specific composition of proteins (casein and whey proteins), glycoproteins, enzymes and polar or neutral lipids from the milk fat globule membrane (MFGM). Notably, the high content of membrane found in buttermilk is responsible for structural integrity, and stability of the milk fat in the aqueous phase (Vanderghem et al. 2010).

Studies are focused on the development of techniques for recovery of MFGM fragments from buttermilk. A commonly used technique involves tangential filtration. However, since casein micelles and MFGM fragments, both present in buttermilk, are roughly similar in size, it is difficult to separate the MFGM components from casein using filtration techniques (Rombaut et al; Morin et al.).

Thus, there is a need to develop techniques that allow the efficient recovery of casein-free MFGM fractions. SUMMARY OF THE DISCLOSURE

The present disclosure provides, in accordance with a first of its aspects, a method for recovery of milk fat globule membrane (MFGM) from buttermilk (BM), the method comprises treating BM with a phosphate buffer to obtain a buffer treated suspension and subjecting the buffer treated suspension to at least one separation step to produce an MFGM main fraction comprising in its majority free MFGM.

In accordance with a second aspect, the present disclosure provides a method for recovery of milk fat globule membrane (MFGM) from a protein skim milk product comprising, subjecting a protein skim milk product to at least one continuous flow centrifugation (CFC) to precipitate MFGM and collecting said MFGM.

The methods of the present disclosure are particularly suitable and applicable for large scale production of MFGM.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the present disclosure and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figure 1 is a schematic flow chart of a method for recovering milk fat globule membrane (MFGM) from buttermilk (BM), in accordance with an embodiment of the present disclosure.

Figure 2 is a flow chart representation of the steps for the recovery MFGM from industrial BM in accordance with an embodiment of the present disclosure.

Figure 3 is an image of a 10% SDS-PAGE showing level of protein characteristics of MFGM obtained from BM before (lanes 1 and 2) and after (lanes 3 and 4) addition of sodium phosphate buffer in accordance with an embodiment of the invention,, lane 1- 7C^g, lane 2- 5C^g, lane 3- 5C^g, lane 4- 9C^g, lane 5 is a molecular weight marker; breast cancer antigen 1 (BRCA1); xanthine oxidase (XO); CD36 - glycoprotein IV; FABP - fatty acid binding protein. Figure 4 is a flow chart representation of the steps for the recovery of MFGM from combined bovine raw milk in accordance with an embodiment of the present disclosure.

Figure 5 is an image of a 10% SDS-PAGE of showing level of protein characteristics of MFGM obtained in accordance with an embodiment of the invention, from raw bovine milk (lane 2); lane 1 is a molecular weight marker; breast cancer antigen 1 (BRCA1); xanthine oxidase (XO); CD36 - glycoprotein IV; FABP - fatty acid binding protein.

DETAILED DESCRIPTION OF EMBODIMENTS

The beneficial health properties of MFGM are described. The MFGM that surrounds fat globules in milk is a natural source of sphingolipids, phoshpholipids and proteins. These may be found valuable for example in cancer treatment or support neonatal gut maturation and myelination of the central nervous system. Being of natural source, MFGM components are safe for oral administration.

In addition, the phospholipid fractions from MFGM can be used emulsions of liposome formulations for pharmaceutical or cosmetic applications. The high content of saturated phospholipids (about 50%) makes this fraction particularly resistant to oxidation.

In the health sector, sphingolipids derived from MFGM are gaining attention, in particular, sphingomyelins (SM).

Recovery of milk fat globule membrane (MFGM) from milk or a milk product is challenging especially in large scale production. The inventors have succeeded to recover MFGM for example as a precipitate from buttermilk, industrial grade, or from native non-thermally treated cream ("raw milk").

As shown in Example 1 herein, the inventors have developed a novel technique for recovering MFGM from buttermilk. The inventors have surprisingly found that use of phosphate buffer enables the release of MFGM from casein-MFGM complexes and thus enables the recovery of free MFGM without damaging the quality of the recovered MFGM. Further, as shown in Example 2 herein, the inventors have developed a novel technique to recover MFGM from raw milk. As detailed herein, the inventors have found that using a continuous flow centrifugation (CFC) enables large scale recovery of MFGM.

The term "buttermilk" is used herein at least with its conventional meaning, i.e. to refer to the liquid (aqueous) phase released from the butter mass during churning (destabilization) of cream in butter making processes. Like skimmed milk and whey, buttermilk contains lactose, minerals, caseins, and serum proteins.

Additionally, buttermilk has high content MFGM and in particular, phospholipids. In the context of the present disclosure, the term buttermilk also encompasses raw portions or fragments of the buttermilk, from which MFGM may also be recovered, including BM obtained from washed cream obtained from raw milk (at times referred to as native milk). This may include downstream products obtained during processing of buttermilk, e.g. for obtaining MFGM.

It is noted that BM obtained from washed cream contains low amounts of proteins, including low amount of casein, whey proteins and as such is referred to at times by the term "skim BM". "Skim milk" is also sometimes referred to the milk from which the cream has been removed.

Sources of BM may be, without being limited thereto from Tel Yosef Tnuva,

Israel.

The term "raw milk" used here denoted milk that has not been pasteurized or homogenized.

Pasteurization of milk typically uses temperatures below boiling, since at very high temperatures, casein micelles will irreversibly aggregate, or "curdle". The two main types of pasteurization used today are high-temperature, short-time (HTST) and extended shelf life (ESL). Ultra-high temperature (UHT or ultra-heat-treated) is also used for milk treatment.

Both buttermilk and MFGM are utilized in the food industry, particularly in the dairy sector, for emulsification, stabilizing, moisture retention, texture and flavor.

The "milk fat globule membrane" (MFGM) is used herein with its conventional meaning to refer to the membrane originating from buttermilk. MFGM is known to provide the milk fat in the liquid (aqueous) phase of the buttermilk with its structural integrity, protection and stability. Typically, MFGM is released into the aqueous phase during cream churning (destabilization of milk fat globules)and is known to contain specific proteins, glycoproteins, enzymes and lipids, including phospholipids among which phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomeylin (SM) are of particular interest. Additional MFGM components considered beneficial for their various food and health-related properties are the proteins and enzymes.

With respect to the lipid composition of MFGM, it has been described by Vanderghem et al. [ Vanderghem et al. ibid. , the content of which is incorporated herein by reference] to contain between 56%-80% neutral lipids out of the total lipids in MFGM. Also included are polar lipids, constituting 15-43% of total lipids in MFGM, and composed of phospholipids such as PC and PE (zwitterionic phospholipids constituting, respectively, about 35% and 30% of the MFGM isolate). Additionally, anionic phospholipids are present albeit, at lower amounts, for example, phosphatidylinositol (PI) and phosphatidylserine (PS) (at respectively 5% and 3%). Sphingomyelin (SM) was also found in a significant amount (about 22%).

MFGM also contains proteins, although at low abundance (constituting 1-4% of total milk proteins) [Vanderghem et al. ibid]. Examples of proteins include casein, and various enzymes.

When referring to "recovery" it is to be understood as relating to the obtaining of MFGM in a form and in an amount suitable for use in any industrial application, including, without being limited thereto, pharmaceutical, veterinary, cosmetic, food etc. Further, when referring to recovery is to be understood as meaning extraction of MFGM for example from BM in a free form, in an amount of at least 60%w/w, at times even 70%w/w or even 75%w/w out of the initial weight of BM.

As detailed herein, one challenge in the recovery of MFGM from buttermilk BM involves the separation between MFGM and casein micelles bound thereto. This is of particular importance in industrial, large scale manufacturing process. In raw milk (as well in BM), MFGM is associated, bound to or complexed with casein, casein micelles or any other form of casein. The desire is to obtain free MFGM.

As used herein the term "complexed" denoted an association between MFGM and casein possibly casein micelle. The association is not limited and may involve any chemical reaction namely, covalent or non-covalent interaction. Without being bound by theory, it was suggested by the inventors that calcium, present in casein micelle, may form a complex between MFGM and casein micelle through its binding to phospho- casein and phospholipids of MFGM.

In the context of the present disclosure large scale production of milk products, such as MFGM refers to recovery from several liters of a starting material, such as liters of raw milk. Thus, in the present disclosure, when referring to large scale production it is to be understood as encompassing amounts from at least 10 liters of a starting material.

Aiming at providing a solution for large scale recovery of free MFGM, a technique has been developed and is now disclosed herein that allows the separation of MFGM from bound casein by treating a starting material comprising MFGM-casein complexes with a phosphate buffer and applying centrifugation, particularly, continuous flow centrifugation (CFC) to the phosphate buffer treated material. It has been surprisingly found by the inventors that the use of the phosphate buffer prior to applying a separation stage resulted in the release of MFGM and enables the recovery of free MFGM (i.e. unbound or non-associated with casein) in large quantities and in highly purified grade.

The term "free MFGM" as used herein denotes the milk fat globule membrane when not associated, bound to or complexed in any way to casein, casein micelles or any other form of casein. In this context it is to be understood that casein refers to any form of casein, acid casein, salts of casein, phosphorous containing casein and rennet casein, casein subgroups (aSl, aS2, β and κ), casein fragments and any assembly of the above, such as micelles.

The MFGM fractions following the phosphate buffer treatment are not structurally or functionally damaged or otherwise defaulted during the procedure described herein and therefore are for use in pharmaceutical, nutritional as well as in other industrial applications. In other words, the procedure did not derogate in any way their applicability in pharmaceutical, nutritional and other industries.

In accordance with one aspect, there is thus provided by the present disclosure a method for recovery of MFGM from buttermilk (BM), the method comprises treating BM with a phosphate buffer to obtain a buffer treated suspension and subjecting the buffer treated suspension to at least one separation step to produce an MFGM main fraction comprising in its majority free MFGM (i.e. MFGM is the main constituent of this fraction).

As used herein the term "suspension" has its general meaning of a heterogeneous mixture containing particles that are sufficiently large for sedimentation but may also refer, in the context of the present invention to a solution that under suitable conditions may provide a precipitate and a supernatant.

In one embodiment, the method comprises (a) adjusting the pH of the BM to a level whereby a first precipitant (being a raw precipitant) and a first supernatant (being a raw supernatant) are formed, (b) suspending (treating) the raw precipitant with a phosphate buffer to form a buffer treated suspension; and (c) treating said buffer treated suspension to obtain a second precipitate and a second supernatant, the second supernatant comprises a main MFGM fraction ( "MFGM main fraction ").

The raw supernatant may also be subjected to treatment that provides an MFGM secondary fraction. Then, in order to increase MFGM recovery from BM, the main MFGM fraction and the MFGM secondary fraction may be combined to form the combined MFGM fraction.

The term "MFGM main fraction" is used to denote a fraction containing in its majority MFGM, and in some embodiments at least 60%, at times even at least 70%, 80% or even at least 90% of the free MFGM recovered in the present disclosure. In some embodiments, the MFGM main fraction comprises between 70% to 95% of the recovered MFGM.

Similarly, the term "MFGM secondary fraction" is used to denote a fraction containing free MFGM that constitutes at most 40%, at times no more than 30%, 20% or even less than 10% of the total free MFGM recovered in the method of the disclosure.

In line with the above, the term "combined MFGM fraction" is used to denote the combination of the MFGM main fraction with the MFGM secondary fraction.

When referring to "phosphate buffer" it is to be understood as encompassing any buffer prepared from organic or inorganic phosphate salt. Non-limiting examples of phosphate salts that may be used in accordance with the disclosure include sodium phosphate, potassium phosphate, rubidium phosphate, caesium phosphate and ammonium phosphate.

In some embodiments, the phosphate salt used for preparation of the buffer solution is a sodium phosphate including any one or combination of sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, sodium trimetaphosphate, tetrasodium pyrophosphate and sodium hexametaphosphate. In one particular embodiment, the phosphate buffer is prepared with sodium hexametaphosphate.

In some embodiments, the phosphate buffer is used at a concentration of between 0.005M to 0.5M, at times between 0.01M to 0.3M, at times between 0.05-0.1 M. In some embodiments, the phosphate buffer is at a concentration of about 0.1M. In some other embodiments, the phosphate buffer is at pH above 6. In some other embodiments, the phosphate buffer is at pH of between 7 to 8, at times between 7.2-7.8. In some other embodiments, the phosphate buffer is at pH of about 7.2.

When referring to "separation" it is to be understood as any one or combination of filtration, settling, centrifugation and continuous flow centrifugation (CFC).

It has been found by the inventors that centrifugation improves MFGM recovery and thus a preferred embodiment, separation comprises at least one stage of centrifugation by CFC.

CFC includes delivery of liquid containing particles to the centrifugal rotor which is designed for sediment particles to stay inside the rotor but the supernatant flows out of the rotor. As such, CFC, enables on one hand, continuous processing of a large volumes of liquids such as milk and on the other hand, concentration to a small volume of the supernatant (pellet), such as MFGM.

Examples of a commercially available CFC are the continuous flow rotors from Sorvall, being capable of producing a centrifugal force of at least 50,000xg, and delivery of suspended matter at a flow rates 100-200 ml/min. As will be shown hereinbelow, such conditions allowed, at 2-4°C, precipitation of large amounts of desired intermediate products and of MFGM. It should be appreciated that by using higher centrifugal forces, the flow rate would increase accordingly. For example, when using 100,000xg, the flow rate would also increase above 100-200 ml/min. A person skilled in the art would be able to select the appropriate flow rate for other centrifugal forces by trial and error experiments.

In line with the above, reference is now made to Figure 1 providing a schematic illustration of a method for recovering MFGM from BM according to some embodiments of the invention.

Accordingly, BM (10) is subjected, in accordance with this embodiment, to pH adjustment to obtain a raw precipitant (12) and a raw supernatant (14). The raw precipitant (12) is then suspended with a phosphate buffer to obtain a buffer treated suspension (16). The buffer treated suspension (16) is then subjected to at least one separation stage where a second precipitant (18) and a second supernatant (20) are formed. The second supernatant comprises the free MFGM. The second supernatant is separated and concentrated to obtain the MFGM main fraction (22).

In accordance with some embodiments of the present disclosure, the adjusting of the pH of the BM (10) is to an acidic pH. The resulting raw precipitant (12) is rich in casein-bound MFGM.

In some embodiments, the pH of the BM is reduced to a pH value of below about 6, preferably between about 4 and about 6. In one embodiment, the pH is adjusted to be between about 4.4 and about 4.8.

In some embodiments, adjusting the pH is achieved by mixing the BM with HC1 (and verifying the pH with a pH meter or the like to be at the target pH or pH range), for a period of between several minutes to several hours, and at a temperature between 5°C to 50°C, at times between 20°C-25°C. In some embodiments, this pH adjusting step involves incubation of BM with diluted HC1, for example 0.1 N HC1 for between 0.5-1 hours, preferably for between 20-30 min at said temperatures.

In some embodiments, after adjusting the pH of the BM to an acidic pH, casein participates and the thus formed suspension or precipitant is cooled to a temperature below 20°C, at times below 10°C and even below 5°C. Alternatively, the raw precipitant is cooled to a temperature between 1°C to 20°C. The raw precipitant (12) is subjected to a separation stage, preferably at least one CFC, so as to remove the raw supernatant (14), which typically contains whey and lower amounts of MFGM.

Thus, in some embodiments, the acidic BM is allowed to cool and is then subjected to a separation stage after which the raw precipitant (12) is separated from the raw supernatant (14).

While the raw supernatant (14) comprises a minor amount (fraction) of MFGM, the abundant (large, major) amount of MFGM was found to be present in the raw precipitant (12), physically associated together with casein micelles by calcium phosphate bridges. The inventors have found that under appropriate conditions, most of the MFGM was co-precipitated with casein in the raw precipitate (12 in Figure 1) enabling recovery of the major fraction (main fraction) and only a small fraction of MFGM (about l%-2%, Table 1) (secondary fraction) was recovered in the first raw supernatant (14). Notably, attempts to recover MFGM with sodium chloride buffer and sodium citrate were not so successful resulting in either not effective release of MFGM from casein or with only small quantities of free MFGM.

Further, attempts to dissociate the casein micelles from MFGM using ultracentrifugation techniques following the addition of sodium citrate [Corredig M. & Dalgleish D.G., 1997. Isolates from industrial buttermilk: emulsifying properties of materials derived from the milk fat globule membrane. J. Agric. Food Chem., 1997, 45:4595-4600]. The MFGM isolate by this technique proved to have very poor emulsifying properties compared to the whole buttermilk isolate.

The inventors have now found that successful release of the MFGM from the casein is possible when the raw precipitant (12) is treated with a phosphate buffer, whereby a suspension (16) is formed (at times referred to as the "buffer treated suspension"). The suspension with the buffer solution ("buffer treated suspension") is then subjected to a second separation stage comprising one or more separation steps or to any other technique that provides an MFGM supernatant (20, the "second supernatant") and a second precipitate (18). It was surprisingly found that the second supernatant (20) is rich with free MFGM while the second precipitant (18) contains most of the casein and only minor amounts of MFGM. The second supernatant (20) is then, preferably subjected to a concentration stage to provide a MFGM main fraction (22). In one embodiment, the second supernatant is subject to at least CFC to obtain the main MFGM fraction.

The raw supernatant (14) also contains whey and MFGM, although lower amounts of MFGM as compared to the amount of MFGM recovered from the raw precipitant. This raw supernatant (14) may also be subjected to one or more separation stages to obtain a secondary MFGM fraction (24).

The MFGM main fraction (22) and the MFGM secondary fraction (24) may be combined to constitute a combined MFGM fraction (26). In the non-limiting exemplified procedure below it has been found that the main MFGM fraction comprise about 80% of MFGM out of the combined MFGM fraction, and only between 1% - 2% of the free MFGM was found in the secondary MFGM fraction.

In some embodiments, the main MFGM fraction (22), the secondary MFGM fraction (24) and the combined MFGM fraction (26) may be further processed, together or separately, to be in a form suitable for storage. Such processing may involve, without being limited thereto, freezing, freeze-drying and spray drying.

In some embodiments, casein may also be recovered from BM. Accordingly, and with further reference to Figure 1, the second precipitate (18) is processed to obtain casein (28). The casein processing may involve, for instance, washing.

In another aspect, the present disclosure provides a method for recovery of MFGM from protein-skim milk or any protein-skim product therefrom (e.g. cream, protein skim buttermilk, etc.) as the starting material. Accordingly, to obtain the protein skim starting material, raw milk or a product therefrom is washed at least once with water whereby protein content is removed and protein skim milk is obtained (i.e. milk that is low in protein content). The protein skim milk is then subjected to at least one CFC whereby MFGM precipitates. This MFGM is free of casein. This method is especially suitable for large scale recovery of MFGM from water-washed cream.

Thus, according to a second aspect, the present disclosure provides a method for large scale recovery of milk fat globule membrane (MFGM) from protein skim milk product, the method comprises subjecting the protein skim milk product to at least one CFC to obtain a precipitant comprising MFGM. In the context of this aspect of the present disclosure it is to be understood that the term "protein-skim milk product" encompass milk or any product therefrom which was subjected to at least one washing with water. Without being bound by theory, it is believed that washing of raw milk or milk cream etc. at least once with water results in the removal of large portions of milk proteins, including casein, from the washed product thus allowing precipitation of free (non casein bound) MFGM.

In one embodiment, the protein skim milk product is subjected to CFC at a centrifugal force of at least 50,000gx at a flow rate of 100-200ml/min.

The recovered MFGM obtained by the techniques disclosed herein may be used in variety of applications.

In some embodiments, MFGM may be used in a nutraceutical composition.

The term "nutraceutical composition" used herein to denote any composition comprising a substance that is a food, or part of a food. In some embodiments, both dietary supplements and functional foods are considered nutraceuticals.

In some other embodiments, MFGM may be used in a pharmaceutical composition. Such compositions are suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intra-adipose tissue and intradermal) administration or administration via an implant.

In some further embodiments, the MFGM is suitable for use as a dietary supplement, nutraceutical food and/or as a drug additive.

In accordance with the present invention, the MFGM of the invention is for use in other health care applications as known in the art.

DETAILED DESCRIPTION ON SOME NON-LIMITING EXAMPLES

EXAMPLE 1: PREPARATION OF MFGM FROM INDUSTRIAL BUTTERMILK (BM)

Buttermilk (BM) was obtained from Tel Yosef factory, a division of Tnuva (Afula, Israel). SDS-PAGE and western immunoblotting analysis of the BM, according to Spitsberg et al. [Spitsberg Vitaly et al. Eur J Biochem. 1995; 230(3): 872-8] showed the presence of MFGM proteins in BM as detailed below. MFGM was recovered from BM according to the procedure steps illustrated in Figure 2.

As shown in Figure 2, BM was diluted with water (at a 1 : 1 ratio) and treated to have a pH of 4.6 by an addition of diluted HC1. The acidic BM was maintained at this pH of 4.6 at 37°C for 30 minutes until a first precipitate was obtained. This first precipitate was found to contain casein.

The first precipitate was allowed to cool to 2°C-4°C in a refrigerated thermostat/chamber. Once cooled, the acidic cooled BM (comprising the precipitate) was centrifuged at 2,000-3, OOOxg or CFC (for large scale) with a rotor speed of 50,000xg at 100-200 ml/min and 2°C-12°C to obtain a first supernatant (raw supernatant) (14 in Figure 1) containing whey and minor portion of MFGM and a pellet containing casein (the casein containing pellet corresponds to the first precipitate (raw precipitant) (12 in Figure 1).

It was found by the inventors that most of the MFGM was co-precipitated with casein in the casein containing pellet (12 in Figure 1) and only a small fraction of MFGM (about l%-2%, Table 1) was recovered in the first raw supernatant (14).

The inventors have suggested that the yield of MFGM is low as it is bound to casein in the precipitate. After various attempts to separate between MFGM and casein in the casein pellet ( first raw precipitate), the inventors have found that suspending the pellet (raw precipitant) in a sodium phosphate solution (0.05-0.1 M at pH 7.2-7.8) for 20-30 minutes, and subjecting the suspension to centrifugation using CFC rotor at 2000-3000xg speed at 100-200 ml/min at 2-8°C for 20 minutes, provided a second precipitate and a second supernatant (18 and 20 in Figure 1 respectively).

Analysis showed that at this stage casein was removed. It was thus suggested by the inventors that calcium phosphate bridges are responsible for the association between phospholipids of MFGM and the phosphate groups of casein (kappa casein). Without being bound by theory, it was suggested by the inventors that MFGM was released from the MFGM-casein assembly by the addition of calcium phosphate that induces competition binding causing the release MFGM from the complex with casein.

After this step the second pellet containing casein (corresponds to (18) in Figure 1) is removed and the supernatant (second supernatant) (20 in Figure 1) was centrifuged using a CFC rotor at 32,000-50,000xg speed with a flow rate 100-200 ml/min at 2-8°C for 1 hour. The supernatant is removed and the pellet containing MFGM was suspended in water and freeze dried to obtain main fraction of MFGM {"MFGM main fraction") (22 in Figure 1).

The supernatant obtained from the centrifugation (of the first raw supernatant (14) in Figure 1), containing whey and MFGM, was centrifuged using a CFC rotor at 50,000xg at 100-200 ml/min at 4°C to remove the whey and obtain a MFGM secondary fraction comprising a minor MFGM fraction ((24) in Figure 1). The first and the second MFGM faction corresponding to MFGM main fraction and MFGM secondary fraction, obtained in this step were then combined to the combined MFGM fraction ((26) in Figure 1) and the combined MFGM fractions were then subjected to freeze drying to obtain lyophilized MFGM.

The successful separation between casein and MFGM is evident from Figure 3.

Figure 3 shows the levels of proteins, which are characteristic to MFGM. Lanes 1 and 2 correspond to the fraction before the addition of buffer, namely the fraction comprising low amount of MFGM, minor fraction, and lanes 3 and 4 correspond to the fraction after the addition of sodium phosphate, namely the fraction comprising the major fraction of MFGM.

As shown, during the separation of MFGM from BM, the level of free xanthine oxidase (XO), which is one of the proteins of MFGM, markedly increases after addition of sodium phosphate, namely in the major MFGM fraction (lanes 3 and 4) compared to the level before the addition of buffer, namely minor fraction (lanes 1 and 2). In addition, the level of butyrophilin (BTN) also increases after this treatment. These two findings indicate that protein bound to MFGM was released from MFGM after phosphate buffer treatment leading to an increased amount of MFGM._The obtained MFGM also contained varied amount of contaminants, casein (30-33 kDa) and thermally modified β-lactoglobulin (15 kDa).

The presence of fatty acid binding protein (FABP) in MFGM was confirmed by western blot analysis with antibody directed to bovine milk FABP [Spitsberg et al. 1995 ibid.]. Table 1 shows that the level of MFGM before the suspension in phosphate buffer, namely in the first supernatant (raw supernatant) is considerably low (1.9mg in 100ml BM).

In addition, as shown in Table 1, the degree of MFGM release depends on the selection of appropriate conditions including buffer and pH.

As also shown in Table 1, the high degree of MFGM recovery from casein was obtained by using sodium-hexametaphosphate buffer 0.1M at a pH of 7.1 or 7.2.

The release of MFGM was shown to be pH depend with hardly no MFGM released at pH values of 4.6-6, and showing an increase with increased pH values.

Suspending the casein pellet in other phosphate buffer was found to be less effective. For example suspending in sodium chloride buffer (0.1 M) or sodium sulfate (0.1 M) did not produce any MFGM indicating that no MFGM was released and suspending in sodium citrate or sodium oxalate provided a low amount of MFGM.

The inventors have suggested the following model to possibly support the results shown above:

Casein is present in milk and buttermilk as micelles which are colloidal particles formed by casein aggregates wrapped up in soluble κ-casein molecules. Calcium, present in casein micelle, may form a complex between MFGM and casein micelle through its binding to phospho-casein and phospholipids of MFGM.

In the presence of sodium phosphate buffer the assembly of MFGM - casein micelle can dissociate into its components, especially at pH between 7 and 8.

From the results it may be understood that at pH values of between 7 and 8, (HP0₄)^2" is a predominant phosphate ion in the solution. This ion thus appears to compete for the binding of Ca and thereby withdraw Ca from the assembly MFGM-Ca- casein: The interaction of (HPO₄)^2" with Ca in turn leads to formation of an immiscible salt CaHPC>4 which is easily removed from the solution.

In contrast, at pH values below 7, (H₂PO₄)^" seems to be a predominant phosphate ion which does not compete with the binding to Ca and the salt Ca(]¾P0₄)₂ remains soluble in water and the MFGM-Ca-casein thus remain intact. In Summary:

At pH 7.4 using 95% Na^PC^:

MFGM-phospholipids—Ca—phospho-k-casein-micelle pH 7.4 using 95% Na₂HPO_¾ j

MFGM-phospholipids phospho-k-casein-micelle CaHP0₄

MFGM-phospholipids— Ca— phospho-k-casein-micelle pH 4.5 using 99% Na H₂PO_¾ j

MFGM-phospholipids-phospho-k-casein-micelle-H₂P0₄ (No Dissociation)

It should be noted that further unsuccessful attempts to produce MFGM from BM were done by the inventors by using ultrafiltration and diofiltration techniques. As experienced by the inventors, diafiltration usually leads to equal distribution of MFGM proteins between permeate and retenate associated with a high content of casein.

Using this protocol enabled to obtain high yield of MFGM from a large volume of BM. In other words, the procedure provided herein and outlined in Figures 1 and 2 enables large scale recovery of MFGM from buttermilk.

Tablel. Recovery of MFGM from industrial BM

was obtained after the "second centrifugation" i.e. after treatment of casein pellet with sodium phosphate, or other reagents; ^c nd=not detected.

EXAMPLE 2: PREPARATION OF MFGM FROM BOVINE RAW MILK

An outline of the procedure is provided in Figure 4.

Combined bovine raw milk, including a mixture of milk from different farms, was obtained from Tnuva, Rehovot, Israel and was separated at a temperature of 40°C-50°C into cream (fat) and skim milk by a standard milk separator and the cream was washed with water so as to allow increase in fat content in the cream (to up to 64%) and to wash out proteins such as whey proteins from the cream.

At this stage, the cream fat may be frozen -70°C and before use thawed. Upon use, the cream fat was melted, diluted with water in a ratio 1 : 1 and subjected to churning (destabilization) performed in a large Stefan blender at 2,000-3,000 rpm and at room temperature (around 25°C) for 20-30 min, to separate between buttermilk and butter by filtration.

As appreciated, the buttermilk obtained from churning of water washed cream (30-40% fat) and filtration is different than the industrial buttermilk described in Example 1 provided above, since it contains low amounts of protein such as casein, whey proteins and of calcium.

The butter was collected by filtration through three layers of gauze, suspended and melted with an equal volume of water (1 :1 ratio) at 48°C and then separated into butter and whey, by a standard milk separator.

The whey was combined with the buttermilk, obtained after churning of a cream, and 10 L of combined low protein content buttermilk was processed by CFC using a CFC rotor of Sorval (USA) centrifuge at 50,000xg at 4-12°C and at a flow rate of 100 - 200 ml/min, for about 1 or 2 hours to obtain a brown colored pellet (15g). The supernatant containing the whey was removed and the pellet was re-suspended in distilled water and freeze dried.

Analysis of the pellet by SDS-PAGE (Figure 5) showed that the pellet contains milk fat global membrane (MFGM).

Claims

CLAIMS:

1. A method for recovery of milk fat globule membrane (MFGM) from buttermilk (BM), the method comprises treating BM with a phosphate buffer to obtain a buffer treated suspension and subjecting the buffer treated suspension to at least one separation step to produce an MFGM main fraction comprising in its majority free MFGM.

2. The method of Claim 1, comprising pH adjustment of the BM before treatment with said phosphate buffer to a level whereby a raw precipitant and a raw supernatant are formed, the raw precipitant being then treated with said phosphate buffer to form the buffer treated suspension.

3. The method of Claim 2, wherein the pH is adjusted to an acidic pH.

4. The method of Claim 3, wherein the pH is adjusted to a pH between 4.0 and 6.0.

5. The method of Claim 4, wherein the pH is adjusted to a pH between 4.4 and 4.8.

6. The method of any one of Claims 2 to 5, comprising cooling the BM to a temperature below 5°C before treatment with said phosphate buffer.

7. The method of any one of Claims 2 to 6, comprising separating said raw precipitant, said raw precipitant comprising casein and MFGM.

8. The method of any one of Claims 1 to 7, wherein the phosphate buffer comprises a phosphate salt selected from the group consisting of sodium phosphate, potassium phosphate, rubidium phosphate, cesium phosphate and ammonium phosphates.

9. The method of Claim 8, wherein the phosphate buffer comprises sodium phosphate, sodium polyphosphate, sodium hexametaphosphate.

10. The method of any one of Claims 1 to 9, wherein the buffer treated suspension is subjected to one or more separation steps whereby said MFGM main fraction is recovered in a second supernatant.

11. The method of Claim 10, comprising precipitating MFGM from the second supernatant to obtain the MFGM main fraction.

12. The method of Claim 11, comprising treating the raw supernatant to obtain an MFGM secondary fraction.

13. The method of Claim 12, comprising combining said MFGM main fraction with the MFGM secondary fraction to obtain a combined MFGM fraction.

14. The method of Claim 13, wherein said MFGM main fraction constitutes at least 80% of the MFGM of the combined MFGM fraction.

15. The method of any one of Claims 1 to 14 comprising freezing or freeze drying the main MFGM fraction.

16. The method of Claim 13 or 14, comprising freezing or freeze drying the combined MFGM fraction.

17. The method of any one of Claims 1 to 15, wherein said one or more separation steps is selected from the group consisting of filtration, settling, centrifugation or continuous flow centrifugation (CFC).

18. The method of Claim 16, wherein separation is at least by CFC.

19. The method of any one of Claims 1 to 18, wherein at least said buffer treated suspension is subjected to CFC.

20. A method for recovery of milk fat globule membrane (MFGM) from a protein skim milk product comprising, subjecting a protein skim milk product to at least one continuous flow centrifugation (CFC) to precipitate MFGM and collecting said MFGM.

21. The method of Claim 20, wherein said CFC comprises centrifugation at a force of at least 50,000xg and at a flow of between 100-200ml/min.

22. The method of Claim 20 or 21, wherein said protein skim milk product is water washed cream.

23. The method of any one of Claims 20 to 22, for large scale production of MFGM.