Preparative and purification processes relating to whey protein concentrate, whey protein phospholipid concentrate and milk fat globule membrane and the products obtained thereof

Abstract

The present invention relates generally to preparative and purification processes relating to whey protein and particularly to the preparation and purification of Whey Protein Concentrate (WPC), also known as Whey Protein Phospholipid Concentrate (WPPC), Milk Fat Globule Membrane (MFGM), procream, Whey Fat Concentrate (WFC) and whey retentate. We describe processes for increasing a protein concentration of a composition containing milk protein and milk fat, and processes for separating milk protein and milk fats in a composition containing milk protein and milk fat. The process includes extracting the composition with a solvent selected from hexane, 2-methyloxolane, ethanol and carbon dioxide, and combinations thereof. The present invention also relates to whey protein compositions, milk fat compositions and phospholipid-rich compositions obtainable or obtained by the processes of the invention.

Description

[0001] PREPARATIVE AND PURIFICATION PROCESSES AND PRODUCTS THEREOF

[0002] The present invention relates generally to preparative and purification processes relating to whey protein and particularly, although not exclusively, to the preparation and purification of Whey Protein Concentrate (WPC), also known as Whey Protein Phospholipid Concentrate (WPPC), Milk Fat Globule Membrane (MFGM), procream, Whey Fat Concentrate (WFC) and whey retentate. The terms procream, pro-cream, Procream and Pro-cream are synonymous and are considered to be generic descriptions of WPPC.

[0003] Dairy ingredients are among the most multifunctional ingredients in the food industry. Their unique protein, carbohydrate, fat and mineral composition contributes functional, as well as nutritional, benefits to foods.

[0004] Whey is obtained after the precipitation of milk casein in the cheese-making process and contains whey protein. The skilled person will be familiar with processes for obtaining whey from milk. Equally, by acid precipitation from skim milk, for example, but not limited to, by addition of: hydrochloric acid, lactic acid (either direct or produced by fermentation) or sulphuric acid.

[0005] WPPC and procream are generic terms used by the whey industry to describe the product that is microfiltered from whey to make a whey protein isolate (WPI). It is a coproduct of the whey protein isolate (WPI) production process, and it is the retentate fraction of the microfiltration of whey. Beta serum and buttermilk are also suitable sources of milk proteins.

[0006] WPPC contains proteins (60% to 75% by weight) and milk fat globules as well as milk fat globule membrane (MFGM) components including bioactive proteins and lipids such as phospholipids (up to 20% by dry matter weight). Beta serum and buttermilk are equally suitable for our purposes and contain proteins in amounts from 30 to 78 wt.%. WPPC is rich in essential amino acids, which are the building blocks of protein, and has a high nutritional value. Some embodiments provide or relate to purification of protein by solvent extraction of a whey feedstock.

[0007] In certain aspects, the present invention provides methods to:

[0008] 1 ) isolate and / or purify WPC or WPPC, in terms of removal of polar and / or non-polar fat molecules to enable a high protein purity product with a protein dry matter content of 40% to 80% by weight, to increase protein content to 80 wt.% or more, optionally up to 95 wt.% or higher; or

[0009] 2) isolate and / or purify WPC or WPPC, in terms of removal of polar and / or non-polar fat molecules to enable a high protein purity product at >80% protein dry matter by weight; or

[0010] 3) isolate and / or purify WPC or WPPC, in terms of removal of polar and / or non-polar fat molecules to enable a high protein purity product at 80% protein dry matter or more and fat dry matter <7.5%, but optionally 0.5-2%, 2-5.5%, 5.5-7.5%, 7.5-9%, 9-11 % or 11 -20% by weight.

[0011] In some aspects, the whey feedstock is a WPPC component and the present invention provides methods for enhancing the protein content of a WPPC component or composition.

[0012] In some aspects, the whey feedstock is a whey protein concentrate (WPC) and the present invention provides methods for preparing WPPC from whey protein concentrate.

[0013] The present invention also provides a process for the separation of whey protein components and fat components from a mixture of whey protein components and fat components.

[0014] In certain embodiments, methods and processes of the present invention involve the use of a solvent to remove fat, resulting in a concentrated protein stream and a fat rich stream.

[0015] The solvent is preferably a solvent approved for use in food processing. More specifically, in one aspect, the present invention provides a process for increasing a protein concentration of a feedstock composition containing milk protein and milk fat, the process comprising extracting the composition with a solvent selected from hexane, 2-methyloxolane, ethanol and, carbon dioxide, and combinations thereof.

[0016] In a second aspect, the present invention also provides a process for separating milk protein and milk fats in a feedstock composition containing milk protein and milk fat, the process comprising extracting the composition with a solvent selected from hexane, 2-methyloxolane, ethanol and carbon dioxide, and combinations thereof.

[0017] In certain examples, the feedstock composition is a feedstock composition comprising milk protein in an amount of from about 40 to 80 wt.%, from about 50 to 80 wt.% or from about 60 to 80 wt.%.

[0018] In certain embodiments, the solvent is or comprises ethanol.

[0019] Suitably, extraction with a solvent comprising or consisting essentially of ethanol is carried out at atmospheric pressure or at a reduced pressure, optionally from about 1 bar to about 20 bar, further optionally about 5 bar to about 20 bar.

[0020] In certain embodiments, the carbon dioxide solvent is supercritical carbon dioxide or sub-critical carbon dioxide. Advantageously, the solvent is supercritical carbon dioxide (SCCO2).

[0021] In some examples, supercritical carbon dioxide extraction is carried out at a pressure of from about 100 bar to about 1000 bar; about 150 bar to about 600 bar, about 200 bar to about 500 bar or about 250 bar to about 400 bar; optionally at a pressure of about 300 bar, about 350 bar, about 410 bar or about 460 bar.

[0022] In some examples, supercritical carbon dioxide extraction is carried out at a temperature of about 20°C to about 80°C; about 40°C to about 70°C or about 50°C to about 60°C; optionally at a temperature of about 60°C. In certain examples supercritical carbon dioxide extraction is carried out at a scCCh flow rate of 1 to 70% by weight of the batch weight, or 20 to 60% or 30 to 50% or about 40%.

[0023] In certain embodiments, the extraction is carried out at a scCCh flow rate of about 1 kg / hour / kg of feedstock to about 50 kg / hour / kg of feedstock, optionally about 10 kg / hour / kg of feedstock, about 12 kg / hour / kg of feedstock, about 20 kg / hour / kg of feedstock, about 25 kg / hour / kg of feedstock, about 28 kg / hour / kg of feedstock, about 30 kg / hour / kg of feedstock or about 35 kg / hour / kg of feedstock.

[0024] Optionally, the extraction is carried out at a ratio of scCO2 to whey feedstock of 2:1 to 100: 1 , 10:1 to 70: 1 , 15: 1 to 50: 1 or 20: 1 to 30: 1 ; or about 22: 1 to about 25: 1 .

[0025] In certain embodiments, the feedstock composition is a whey protein concentrate (WPC) or a beta serum or buttermilk.

[0026] In some embodiments, the process is carried out with stepwise removal of solvent or is a continuous process.

[0027] In certain embodiments, the solvent is a co-solvent of supercritical carbon dioxide and at least one solvent selected from hexane, 2-methyloxolane and ethanol. Advantageously, the solvent is a co-solvent of supercritical carbon dioxide and ethanol.

[0028] In certain embodiments, the process comprises a first extraction of the feedstock composition with a first solvent, wherein the first solvent is supercritical carbon dioxide; and a second extraction with a second solvent.

[0029] In some examples, the second solvent is ethanol, hexane or 2-methyloxolane, or combinations thereof or is a solvent comprising supercritical carbon dioxide and ethanol, hexane or 2-methyloxolane or combinations thereof. Optionally the second solvent is a solvent comprising supercritical carbon dioxide and ethanol. In some examples, the process further comprises a third extraction with a third solvent; optionally wherein the third solvent is or comprises ethanol. Further optionally, the process further comprises a fourth extraction with a fourth solvent, wherein the fourth solvent is supercritical carbon dioxide.

[0030] In another embodiment, the process comprises a first extraction of the feedstock composition with a solvent comprising ethanol, hexane or 2-methyloxolane, or combinations thereof; and a second extraction with supercritical carbon dioxide.

[0031] In some embodiments, the process further comprises a step of isolating whey protein from the extracted feedstock composition; and / or a step of isolating a milk fat composition from the extracted feedstock composition; and / or a step of isolating a phospholipid-rich composition from the extracted feedstock composition.

[0032] In a further aspect, the present invention also provides a whey protein composition obtainable or obtained by a process as defined above.

[0033] In preferred embodiments, the whey protein composition has a protein content of 80 wt.% or more, 85 wt.% or more, 90 wt.% or more, 95 wt.% or more, or 99 wt.% or more.

[0034] In a yet further aspect, the present invention also provides a milk fat composition obtainable or obtained by a process as defined above.

[0035] In further aspect, the present invention also provides a phospholipid-rich composition and / or an isolated or substantially isolated phospholipid obtainable or obtained by a process as defined above.

[0036] It will be appreciated that the rate of extraction of material reduces over time. In other words, a larger proportion of material is extracted in the early periods of an extraction compared with later periods. The skilled person will adapt the duration of an extraction having regard, for example, to target yields. In certain examples, the extraction is conducted over a period of up to about 12 hours. In some examples, the extraction is conducted over a period of about 10 minutes to about 2 hours or about 3 hours. In certain examples, the processes of the present invention provide WPPC products having a protein dry matter content of 80 to 99.9 wt.%. Protein content was measured using the Dumas or Kjeldahl method, with a nitrogen factor of 6.38 (consistent with the dairy industry).

[0037] In certain examples, the process also provides a milk fat composition comprising 80 to 99.9 wt.% milk fat solids as measured using the Rose Gottlieb or Soxhlet method.

[0038] A further embodiment comprises extraction on agglomerated microparticulated protein and milk fat globule membrane (MFGM) trial protein powder (dried WPPC).

[0039] In further aspects, the present invention provides processes of solvent extraction to: i) increase protein content to 80 wt.% protein dry matter or more and particularly to 80-95 wt.% or more; and / or ii) increase polar lipids / phospholipids content, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS) , and / or phosphatidylinositol (PI) content; for example a sphingomyelin content, based on total phospholipids, is of at least 2 wt.%, at least 6 wt.% or at least 10 wt.%, optionally 15-20%, 20-30%, or 20-35 wt.%; preferably at least 15 wt.% of total phospholipids or at least 20 wt.%, optionally in the range of 20-35 wt% or 20 to 30 wt.% and / or iii) alter phospholipids ratio and / or composition; and / or iv) increase a level of bioactive protein content, such as (but not limited to) Immunoglobulin G, Lactoferrin, Lactoperoxidase, Lysosomal alpha- mannosidase, Ribonuclease 4, Angiogenin 1 , Quiescin sulfhydryl oxidase, and / or Jacalin-like protein; and / or v) increase a level of bioactive MFGM proteins content, such as (but not limited to) butyrophilin, lactadherin, xanthine oxidase, mucins, and / or osteopontin; and / or vi) remove or isolate yellow / orange / red colour from WPPC powder; and / or vii) inactivate microbial and enzyme activity in the WPPC powder.

[0040] In some embodiments a desolventisation solution may be provided.

[0041] The present invention also provides or relates to whey protein concentrates; (WPC), beta serums and buttermilks consisting of, comprising or including product purified in accordance with a process of the present invention.

[0042] The present invention also provides a WPC80 product obtained or obtainable from WPPC.

[0043] The present invention also provides a WPI product obtained or obtainable from WPPC.

[0044] Uses of purified product may, for example, include the nutrition market, for example for WPC80 (from WPPC), phospholipids-enriched WPC (MFGM product), whey cream liquid, anhydrous milk fat.

[0045] The above and other aspects of the present invention will now be described in further detail, by way of example only, with reference to the following examples and the accompanying figures, in which:

[0046] Figure 1 is a schematic representation of a process apparatus used in an embodiment of the process of the present invention;

[0047] Figure 2 shows photographs of results obtained with hexane extraction in an embodiment of the process of the present invention; Figure 3 shows photographs of results obtained with 96% ethanol extraction in an embodiment of the process of the present invention;

[0048] Figure 4 is a schematic representation of a process apparatus used in an embodiment of a scCCh extraction process in accordance with the present invention;

[0049] Figure 5 shows photographs of results obtained with scCCh extraction in an embodiment of the process of the present invention;

[0050] Figure 6 is a photograph showing the results of a Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the results of pre- and post- extracted powders obtained with scCCh extraction (Example 3);

[0051] Figure 7 is a series of plots illustrating the analysis of sphingosine-based lipid using extraction from (i) scCCh, (ii) scCChand 5% (wt / wt) co-solvent extraction, (iii) scCCh and 10% (wt / wt) co-solvent extraction, (iv) scCCh and 20% (wt / wt) co-solvent extraction, (v) 100% ethanol extraction, and (vi) scCCh (from top to bottom, respectively);

[0052] Figure 8 is a series of plots illustrating the analysis of phosphatidylcholine based lipid using extraction from (i) scCCh, (ii) scCChand 5% (wt / wt) co-solvent extraction, (iii) scCCh and 10% (wt / wt) co-solvent extraction, (iv) scCCh and 20% (wt / wt) co-solvent extraction, (v) 100% ethanol extraction, and (vi) scCCh (from top to bottom, respectively); and

[0053] Figure 9 shows photographs of extracted material from the examples described below.

[0054] EXAMPLES

[0055] Unless otherwise defined, all terms (including technical and scientific terms) used herein are to be interpreted as is customary in the art. It will be further understood that terms in common usage should also be interpreted as is customary in the relevant art and not in an idealised or overly formal sense unless expressly so defined herein. Percentages are given as percentages by weight except where otherwise stated or apparent from the context.

[0056] Example 1 - hexane extraction

[0057] A sample of Volactive UltraWhey Velicious (Volac Whey Nutrition Ltd) whey protein concentrate powder was agglomerated with water to a moisture content of 4 wt.%. A sample of Volactive UltraWhey Velicious was also used without agglomeration. A sample of Milk Fat Globule Membrane (MFGM) was also obtained, from Volac Whey Nutrition Ltd.

[0058] Initial batch settling trials on a test-tube scale with hexane as the solvent at room temperature showed that agglomerated Volactive UltraWhey Velicious settled well at the bottom of the test-tube, with few particles remaining on the surface of the solvent or suspended in the body of the solvent. The non agglomerated Volactive UltraWhey Velicious showed powder on the surface of the solvent which did not settle with additional agitation. The MFGM material took longer to settle than either of the Volactive UltraWhey Velicious samples and some particles remained in suspension. In all examples, most of the whey sample settled quickly through the hexane, forming a layer on the bottom of the test-tube.

[0059] For the purposes of the further trials, it was decided to proceed with agglomerated Volactive UltraWhey Velicious and MFGM.

[0060] Volac, Volactive, UltraWhey and Velicious are trade marks or registered trademarks of Volac Whey Nutrition Limited or Volac International Limited.

[0061] The pilot vessel included a conveyor for transporting the material to be extracted between an inlet and an outlet, defining a material flow path. Hexane solvent is introduced to the vessel over the material to affect extraction and is collected. The hexane was maintained at a temperature of 40-60°C throughout the trials. The material flow path included a series of extraction points at which the solvent, having passed over the material can be sampled (see Figure 1 , which shows the multistage configuration schematically).

[0062] A feedstock of 10 kg of whey material was added to the system at 5-minute intervals at a rate of 1 .7 to 1 .9 kg per hour. Hexane was flowed into the vessel at a rate of 70 millilitres per minute, with a planned residence time for any particular portion of the whey material of about 100 minutes.

[0063] Solids were recovered from the conveyor and vacuum dried at 60°C overnight. The results are shown in Figure 2, in which the reduced coloration of the extracted feedstocks is apparent.

[0064] Figure 2 shows the appearance of the powder and the oil after hexane extraction and vacuum drying:

[0065] A. (left) starting material for agglomerated Velicious, (right) post extraction agglomerated Velicious;

[0066] B. (left) starting MFGM trial material, (right) post extraction MFGM trial material;

[0067] C. dried oil as by-product from hexane extraction for agglomerated Velicious (right) and MFGM trial material (left); and

[0068] D. Picture showing milk fat.

[0069] The hexane used in the extraction was collected and steam distillation used to remove the hexane and thereby concentrate the materials extracted from the feedstock. Other methods of desolventising are equally suitable, such as flash desolventising. The resultant mixture had an oil content of 92.5 wt.% (agglomerated Volactive UltraWhey Velicious) and 92.8 wt.% (MFGM), compared with oil contents for the respective feedstocks of 12 and 15.1 wt.% respectively.

[0070] Initial trials indicated that both n-hexane and iso-hexane are equally suitable for our purposes.

[0071] The resultant samples were analysed for protein content. The agglomerated Volactive UltraWhey Velicious sample showed a protein dry matter content of 81.9 wt.%, compared with a protein dry matter value of 75.0 wt.% for the feedstock material. The MFGM material had a dry weight protein content of 85.0 wt.% compared with 73.3 wt.% for the MFGM feedstock.

[0072] The fat content of the extracted powders was also analysed and was seen to have reduced from around 12-15 wt.% in the feedstock materials, to around 2.2-3.3 wt.% in the extracted powders. The total phospholipid content of the product powders was found to be broadly unchanged between the feedstock and the extracted products.

[0073] Phospholipids analysis was performed on the starting and extracted materials in order to test if hexane extraction is able to selectively remove non-polar lipids (or butter oil or triacylglycerides), to yield a higher concentration of phospholipids in the extracted whey powder.

[0074] Total phospholipids in agglomerated Velicious powder were measured at 5.37% compared to post-hexane extraction of 5.11 %. Individual phospholipid analysis is set out in Table 1 (All values are mg / g. 10 mg / g corresponds with a content of 1 %):

[0075] TABLE 1

[0076] Only 0.11 % of the total phospholipids of the feedstocks was detected in the extracted oil.

[0077] The overall phospholipids composition remains substantially unchanged in the Volactive UltraWhey Velicious powder pre- and post- extraction, with phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) being the major species.

[0078] On the other hand, total phospholipids in MFGM trial powder increased from 4.59% to 6.08% post hexane extraction (Table 2).

[0079] TABLE 2

[0080] The overall phospholipids composition increased but the proportions remained substantially unchanged pre- and post- extraction, with phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) being the major species. About 0.7% of total phospholipids were detected in the extracted oil.

[0081] Protein quality (furosine) analysis

[0082] The effects of thermal treatments / processes on milk can be determined by using indicators such as furosine. The determination of furosine enables to assess the intensity of the initial phase of the Maillard reaction that causes heated milk to develop a brownish colour eventually. Furosine can be considered to be a proxy for thermal history and protein quality.

[0083] The level of furosine was analysed for the feedstock materials as well as for posthexane extracted samples after 4 and 8 hours of drying at 60°C set point. Hexane extraction and subsequent oven drying did not result in significant increase of furosine in the product powder.

[0084] Example 2 - ethanol extraction Using the same apparatus and procedure as described in Example 1 , ethanol (96%) was used as the extraction solvent using the same feedstocks. The ethanol was maintained at a temperature of about 60°C throughout. The results are shown in Figure 3.

[0085] Figure 3 shows the appearance of the powder and of the oil before and after ethanol extraction and vacuum drying:

[0086] A. top left - starting MFGM trial material; top right - palletized MFGM trial material, (bottom left) post extraction MFGM trial material dried at temperature no greater than 60°C (run NHE 3075A); bottom right - post extraction MFGM trial material dried at temperature no greater than 65°C (run NHE 3075B); and

[0087] B&C. dried oil as by-product from ethanol extraction for (left) run 1 and (right) run 2.

[0088] With the MFGM feedstock, protein content increased, on a dry matter basis, from 72.4 wt.% in the feedstock to 81 to 85 wt.% in the extracted product.

[0089] In contrast with the hexane extraction, total phospholipids (in the MFGM trial material) were observed to drop from 5.37% in the feedstock to 1.23 wt.% in the post-ethanol extraction in the powder. The extracted oil contained 30 wt.% phospholipids, with PE, PC and SM being the major species.

[0090] The overall phospholipids composition variation in the product powder is set out in Table 3.

[0091] TABLE 3

[0092] Example 3 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.%

[0093] Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4, having the following numbered components:

[0094] 1 . CO2 Storage tank

[0095] 2. Heat exchanger

[0096] 3. Pump

[0097] 4. Flow meter

[0098] 5. Extraction Vessel

[0099] 6. Separator

[0100] 7. Valve

[0101] 8. Co-solvent pump (which may be omitted, or not used, as appropriate, for example in the apparatus used for Examples 3 to 7).

[0102] The construction of the apparatus will be familiar to the skilled reader and will not be described in further detail here, except where necessary for a fuller understanding of the disclosure.

[0103] A dried WPC70 powder, also known as a sample of Milk Fat Globule Membrane (MFGM), was obtained from Volac Whey Nutrition Ltd. 550g of this WPC70 powder having a protein dry matter content of 70 wt.% and fat dry matter content of 20.5 wt.%, the balance being other dry matter, was extracted in a 1 litre pressure vessel with supercritical carbon dioxide under conditions of 350 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 200g / min and a ratio of SCCO2 to whey feedstock of 22:1 by weight. The extraction was run under these conditions for approximately one hour. The carbon dioxide solvent was removed under standard conditions and recovered for re-use.

[0104] The process produced a WPC having a protein dry matter content of 86.8% by weight and a fat dry matter content of 3.5% by weight, the balance being non-protein / fat dry matter. The extracted material was determined to have a protein content of <0.5 wt.% (below the detection level), a fat content of around 97.5 wt.% and other dry matter of 2.5 wt.%.

[0105] The results are shown in Figure 5 and the analysis data set out in Tables 4 and 5.

[0106] Figure 5 shows the powder and oil appearance before and after supercritical CO2 extraction:

[0107] Left - starting MFGM trial material

[0108] Centre - extracted powder

[0109] Right - extracted oil / fat.

[0110] The processes of the present invention are particularly suitable for increasing a protein content of a whey protein-containing composition having a protein content of 60-80 % by weight to a protein content of 80 to 95 % by weight.

[0111] The processes of the present invention also provide a lipid component having a fat content of 80% or more, typically around 97% or greater. This provides a number of useful fat products, including a whey-derived butter-like product containing 80-95 wt.% milk fats, and a substantially anhydrous milk fat-like product having 95-99.9 wt.% milk fat. Such products, advantageously, exhibit substantially no bacteria or enzyme activity.

[0112] Table 4

[0113] The results are also illustrated graphically in Figure 6 which shows the results of a Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the pre- and post- extracted powders. Example 4 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.%

[0114] Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4.

[0115] A dried WPC70 powder (MFGM) was obtained from Volac Whey Nutrition Ltd. 1793g of this WPC70 powder, having a protein dry matter content of 69 wt.% and fat dry matter content of 24 wt.%, the balance being other dry matter, was extracted in a 5 litre pressure vessel with supercritical carbon dioxide under conditions of 460 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 28 kg / hour and a ratio of SCCO2 to whey feedstock of 15:1 by weight. The extraction was run under these conditions for approximately one hour.

[0116] The carbon dioxide solvent was removed under standard conditions and recovered for re-use.

[0117] The process produced a WPC having a protein dry matter content of 83% by weight and a fat dry matter content of 11 % by weight, the balance being non-protein / fat dry matter. The extracted material was determined to have a non-detectable protein content of <0.5 wt.%, a fat content of around 99.8 wt.% and moisture level of 0.14 wt.%.

[0118] The results of the analysis are set out in Table 6.

[0119] Table 6

[0120] Example 5 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.%

[0121] Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4.

[0122] A dried WPC70 powder (MFGM) was obtained from Volac Whey Nutrition Ltd. 2210g of this WPC70 powder having a protein dry matter content of 69 wt.% and fat dry matter content of 24 wt.%, the balance being other dry matter, was extracted in a 5 litre pressure vessel with supercritical carbon dioxide under conditions of 350 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 25 kg / hour and a ratio of SCCO2 to whey feedstock of 23:1 by weight. The extraction was run under these conditions for approximately two hours.

[0123] The carbon dioxide solvent was removed under standard conditions and recovered for re-use.

[0124] The process produced a WPC having a protein dry matter content of 83% by weight and a fat dry matter content of 11 % by weight, the balance being non-protein / fat dry matter. The extracted material was determined to have a non-detectable protein content of <0.5 wt.%, a fat content of around 99.9 wt.% and moisture level of 0.05 wt.%.

[0125] The results of the analysis are set out in Table 7. Table 7

[0126] Example 6 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.%

[0127] Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4.

[0128] A dried WPC70 powder (MFGM) was also obtained from Volac Whey Nutrition Ltd. 2300g of this WPC70 powder having a protein dry matter content of 69 wt.% and fat dry matter content of 24 wt.%, the balance being other dry matter, was extracted in a 5 litre pressure vessel with supercritical carbon dioxide under conditions of 410 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 25 kg / hour and a ratio of SCCO2 to whey feedstock of 24:1 by weight. The extraction was run under these conditions for approximately two hours.

[0129] The carbon dioxide solvent was removed under standard conditions and recovered for re-use.

[0130] The process produced a WPC having a protein dry matter content of 84% by weight and a fat dry matter content of 11 % by weight, the balance being non-protein / fat dry matter. The extracted material was determined to have a non-detectable protein content of <0.5 wt.%, a fat content of around 99.8 wt.% and moisture level of 0.08 wt.%.

[0131] The results of the analysis are set out in Table 8.

[0132] Table 8

[0133] Example 7 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.%

[0134] Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4.

[0135] A dried WPC70 powder (MFGM) was obtained from Volac Whey Nutrition Ltd. 2300g of this WPC70 powder having a protein dry matter content of 69 wt.% and fat dry matter content of 24 wt.%, the balance being other dry matter, was extracted in a 5 litre pressure vessel with supercritical carbon dioxide under conditions of 300 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 10 kg / hour and a ratio of scCO2to whey feedstock of 13:1 by weight. The extraction was run under these conditions for approximately three hours.

[0136] The carbon dioxide solvent was removed under standard conditions and recovered for re-use.

[0137] The process produced a WPC having a protein dry matter content of 82% by weight and a fat dry matter content of 13% by weight, the balance being non-protein / fat dry matter. The extracted material was determined to have a non-detectable protein content of <0.5 wt.%, a fat content of around 99.8 wt.% and moisture level of 0.05 wt.%.

[0138] The results of the analysis are set out in Table 9.

[0139] Table 9

[0140] Example 8 - carbon dioxide extraction, in combination with ethanol, of WPC having a dry protein content of 60-80 wt.% Super-critical CO2 extraction, with ethanol co-solvent., was performed using an apparatus as shown schematically in Figure 4.

[0141] A dried WPC70 powder (MFGM) was obtained, from Volac Whey Nutrition Ltd. 1771g of this WPC70 powder having a protein dry matter content of 69 wt.% and fat dry matter content of 24 wt.% the balance being other dry matter. The extraction was completed in the following variations:

[0142] (i) Extracted in a 5 litre pressure vessel with supercritical carbon dioxide under conditions of 350 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 12 kg / hour and a ratio of SCCO2 to whey feedstock of 18:1 by weight. The extraction was run under these conditions for approximately two and a half hours.

[0143] (ii) 5% ethanol was introduced as a co-solvent using a further ratio of total flow to whey feedstock of 15: 1 by weight.

[0144] (iii) 10% ethanol was introduced as a co-solvent using a further ratio of total flow to whey feedstock of 15: 1 by weight.

[0145] (iv) 20% ethanol was introduced as a co-solvent using a further ratio of total flow to whey feedstock of 9: 1 by weight.

[0146] (v) 100% ethanol was introduced and a total of 10 litres used for the extraction.

[0147] (vi) The 5 litre pressure vessel introduced supercritical carbon dioxide under conditions of 350 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 12 kg / hour and a ratio of SCCO2 to whey feedstock of 15:1 by weight. The extraction was run under these conditions for approximately two and a quarter hours.

[0148] The carbon dioxide solvent was removed under standard conditions and recovered for re-use. Ethanol was removed from the fractions. Figure 7 shows precursor triple quadrupole LC / MS scans for a 264.3 Da fragment (indicative of sphingosine based lipids) with extractions set out above, with the same reference numbering, with (from top to bottom): i. SCCO2 only; ii. 5% (wt / wt) ethanol as co-solvent,; iii. 10% (wt / wt) ethanol as co-solvent; iv. 20% (wt / wt) ethanol as co-solvent; v. 100% ethanol; and vi. SCCO2 only.

[0149] Figure 8 shows precursor triple quadrupole LC / MS scans for a 184.0 Da fragment (indicative of phosphatidylcholine based lipids) with extractions set out above, with the same reference numbering, with (from top to bottom): i. SCCO2 only; ii. 5% (wt / wt) ethanol as co-solvent,; iii. 10% (wt / wt) ethanol as co-solvent; iv. 20% (wt / wt) ethanol as co-solvent; v. 100% ethanol; and vi. SCCO2 only.

[0150] The products of each extraction are also shown in the photographs of Figure 9.

[0151] The process produced a WPI having a protein dry matter content of 92% by weight and a fat dry matter content of 2 % by weight, the balance being non-protein / fat dry matter.

[0152] The results of the analysis are set out in Table 10.

[0153] Table 10

[0154] Other fractions produced are rich in phospholipid components, with fractionation of the specific phospholipids as shown in the plots of Figure 7 and Figure 8.

[0155] Example 9 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.%, followed by ethanol extraction of the resulting powder

[0156] Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4.

[0157] A dried WPC70 powder (MFGM) was also obtained, from Volac Whey Nutrition Ltd. 1771 g of this WPC70 powder having a protein dry matter content of 69 wt.% and fat dry matter content of 24 wt.% the balance being other dry matter. The extraction was completed in the following steps:

[0158] 1 . Extracted in a 5 litre pressure vessel with supercritical carbon dioxide under conditions of 350 bar and an operating temperature of 60°C, with a SCCO2 flow rate of 12 kg / hour and a ratio of SCCO2 to whey feedstock of 18:1 by weight. The extraction was run under these conditions for approximately two and a half hours. The carbon dioxide solvent was removed under standard conditions and recovered for re-use.

[0159] 2. Powder removed and three separate ethanol extractions completed at 70°C using: a. 70% aqueous ethanol b. 90% aqueous ethanol c. 100% ethanol absolute 3. Ethanol partially removed using a filter followed by oven removal at 60°C.

[0160] The process produced a WPI having a protein dry matter content of 91 -94% by weight.

[0161] Observations

[0162] The examples demonstrate that processes of the present invention are particularly suitable for increasing a protein content of a whey protein-containing composition having a protein content of 60-80 % by weight to a protein content of 80 to 95 % by weight. The processes of the present invention also provide a lipid component having a fat content of 80% or more, typically around 99+%. This provides a number of useful fat products, including a whey-derived butter-like product containing 80-95 wt.% milk fats, and a substantially anhydrous milk fat-like product having 99.8-99.9 wt.% milk fat. Such products, advantageously, exhibit substantially no bacteria or enzyme activity.

[0163] Through these examples, we have demonstrated processes capable of: i) increasing protein content to 80% protein dry matter and above; ii) increasing polar lipids / phospholipids content, for example phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS) and / or phosphatidylinositol (PI); iii) altering the phospholipids ratio and / or composition of the WPPC; iv) increasing the level of bioactive proteins in WPPC, including Immunoglobulin G, Lactoferrin, Lactoperoxidase, Lysosomal alpha mannosidase, Ribonuclease 4, Angiogenin 1 , Quiescin sulfhydryl oxidase and / or Jacalin-like protein; and v) removing and / or isolating coloration which may be undesirable (such as yellow / orange / red coloration) from WPPC powder

[0164] Figure 9 shows photographs of the products of the extractions of Example 8, showing the quality of the materials obtained. The example embodiments are described in sufficient detail to enable those of ordinary skill in the art to embody and implement the systems and processes herein described. It is important to understand that embodiments can be provided in many alternative forms and should not be construed as limited to the discussion set forth herein.

[0165] Accordingly, while embodiments can be modified in various ways and take on various alternative forms, specific embodiments thereof are shown in the drawings and described in detail above as examples. There is no intent to limit to the particular forms disclosed. On the contrary, all modifications, equivalents, and alternatives falling within the scope of the appended claims should be included.

[0166] Although embodiments of the invention have been disclosed herein, it is understood that the invention is not limited to the embodiments and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention.

Claims

CLAIMS1 . A process for increasing a protein concentration of a feedstock composition containing milk protein and milk fat, the process comprising extracting the composition with a solvent selected from hexane, 2-methyloxolane, ethanol and, carbon dioxide, and combinations thereof.

2. A process for separating milk protein and milk fats in a feedstock composition containing milk protein and milk fat, the process comprising extracting the composition with a solvent selected from hexane, 2-methyloxolane, ethanol and carbon dioxide, and combinations thereof.

3. A process as claimed in claim 1 or claim 2 wherein the feedstock composition comprises milk protein in an amount of from 40 to 80 wt.%, from 50 to 80 wt.% or from 60 to 80 wt.%.

4. A process as claimed in any preceding claim wherein the solvent is or comprises ethanol, hexane or 2-methyloxolane; optionally ethanol or hexane, further optionally ethanol.

5. A process as claimed in claim 4 wherein the extraction is carried out at atmospheric pressure or at a reduced pressure, optionally at a reduced pressure of from about 1 bar to about 20 bar, further optionally about 5 bar to about 20 bar.

6. A process as claimed in claim 4 or claim 5 wherein the extraction is carried out at a temperature of about 5°C to about 80°C; about 40°C to about 70°C or about 50°C to about 60°C; optionally at a temperature of about 60°C7. A process as claimed in any one of claims 4 to 6 wherein the solvent is absolute ethanol or an aqueous ethanol having a water content of up to about 40 wt.%, up to about 30 wt.%, up to about 20 wt.%, up to about 10 wt.% or up to about 5 wt.%.

8. A process as claimed in any one of claims 1 to 3 wherein the carbon dioxide solvent is supercritical carbon dioxide or sub-critical carbon dioxide.

9. A process as claimed in claim 8 wherein the solvent is supercritical carbon dioxide.

10. A process as claimed in claim 9 wherein the extraction is carried out at a pressure of from about 100 bar to about 1000 bar; about 150 bar to about 600 bar, about 200 bar to about 500 bar or about 250 bar to about 400 bar.

11. A process as claimed in claim 10 wherein the extraction is carried out at a pressure of about 300 bar, about 350 bar, about 410 bar or about 460 bar.

12. A process as claimed in any one of claim 8 to 11 wherein the extraction is carried out at a temperature of about 20°C to about 80°C; about 40°C to about 70°C or about 50°C to about 60°C; optionally at a temperature of about 60°C.

13. A process as claimed in any one of claims 8 to 12 wherein the extraction is carried out at a scCCh flow rate of about 1 to about 70% by weight of the batch weight, or about 20 to about 60% or about 30 to about 50% or about 40%.

14. A process as claimed in any one of claims 8 to 13 wherein the extraction is carried out at a scCCh flow rate of about 1 kg / hour / kg of feedstock to about 50 kg / hour / kg of feedstock, optionally about 10 kg / hour / kg of feedstock, about 12 kg / hour / kg of feedstock, about 20 kg / hour / kg of feedstock, about 25 kg / hour / kg of feedstock, about 28 kg / hour / kg of feedstock, about 30 kg / hour / kg of feedstock or about 35 kg / hour / kg of feedstock.

15. A process as claimed in any one of claims 8 to 14 wherein the extraction is carried out at a ratio of scCCh to whey feedstock of 2: 1 to 100: 1 , 10:1 to 70: 1 , 15:1 to 50: 1 or 20: 1 to 30: 1 ; or about 22: 1 to about 25: 1 .

16. A process as claimed in any preceding claim wherein the feedstock composition is a whey protein concentrate (WPC) or a beta serum or buttermilk.

17. A process as claimed in any one of claims 1 to 16 wherein the process is carried out with stepwise removal of solvent.

18. A process as claimed in any one of claims 1 to 16 wherein the process is a continuous process.

19. A process as claimed in any preceding claim wherein the solvent is a cosolvent of supercritical carbon dioxide and at least one solvent selected from hexane, 2-methyloxolane and ethanol.

20. A process as claimed in claim 19 wherein the solvent is a co-solvent of supercritical carbon dioxide and ethanol.

21. A process as claimed in any preceding claim wherein the process comprises a first extraction of the feedstock composition with a first solvent, wherein the first solvent is supercritical carbon dioxide; and a second extraction with a second solvent.

22. A process as claimed in claim 21 wherein the second solvent is ethanol, hexane or 2-methyloxolane, or combinations thereof or is a solvent comprising supercritical carbon dioxide and ethanol, hexane or 2-methyloxolane or combinations thereof.

23. A process as claimed in claim 22 wherein the second solvent is a solvent comprising supercritical carbon dioxide and ethanol.

24. A process as claimed in any of claims 21 to claim 23 further comprising a third extraction with a third solvent; optionally wherein the third solvent is or comprises ethanol.

25. A process as claimed in claim 24 further comprising a fourth extraction with a fourth solvent, wherein the fourth solvent is supercritical carbon dioxide.

26. A process as claimed in any one of claims 1 to 20 wherein the process comprises a first extraction of the feedstock composition with a solvent comprising ethanol, hexane or 2-methyloxolane, or combinations thereof; and a second extraction with supercritical carbon dioxide.

27. A process as claimed in any preceding claim further comprising a step of isolating whey protein.

28. A process as claimed in any preceding claim further comprising a step of isolating a milk fat composition.

29. A process as claimed in any preceding claim further comprising a step of isolating a phospholipid-rich composition.

30. A whey protein composition obtainable or obtained by a process as claimed in any one of claims 1 to 26.

31. A whey protein composition as claimed in claim 30 wherein the composition has a protein content of 80 wt.% or more, 85 wt.% or more, 90 wt.% or more, 95 wt.% or more, or 99 wt.% or more.

32. A whey protein composition having a protein content of 80 wt.% or more, 85 wt.% or more, 90 wt.% or more, 95 wt.% or more, or 99 wt.% or more.

33. A milk fat composition obtainable or obtained by a process as claimed in any one of claims 1 to 26.

34. A phospholipid-rich composition obtainable or obtained by a process as claimed in any one of claims 1 to 26.P11979PC0135. An isolated phospholipid or mixture of phospholipids obtainable or obtained by a process claimed in any one of claims 1 to 26.

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