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

TABLE 3 Phospholipids Pre-extraction Post-extraction Phosphatidylethanolamine (PE) 14.9 2.7 Phosphatidyl choline (PC) 14.6 1.55 Sphingomyelin (SM) 14.7 1.7 Phosphatidyl serine (PS) 5.7 4.5 Phosphatidyl inositol (PI) 3.2 1.45 Other 0.7 0.3 Total (by sum) 53.7 12.3 Example 3 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.% Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4, having the following numbered components: 1. CO2 Storage tank 2. Heat exchanger 3. Pump 4. Flow meter 5. Extraction Vessel 6. Separator 7. Valve 8. Co-solvent pump (which may be omitted, or not used, as appropriate, for example in the apparatus used for Examples 3 to 7). 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. 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. 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.%. The results are shown in Figure 5 and the analysis data set out in Tables 4 and 5. Figure 5 shows the powder and oil appearance before and after supercritical CO2 extraction: Left - starting MFGM trial material Centre - extracted powder Right - extracted oil / fat. 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. 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.% milkfats, and a substantially anhydrous milkfat-like product having 95-99.9 wt.% milk fat. Such products, advantageously, exhibit substantially no bacteria or enzyme activity. Table 4 Starting material Extracted powder Extracted oil Protein Nx6.38 (dry matter) (%) 72.4 86.8 ND Fat (by Rose Gottlieb) (%) 16.25 3.53 92.13 Lactose (%) 0.3 0.3 ND E col a |6T9 a 8 a a a p-g ............ 8» ui a a 8 a 8 a 8 a 8 a Other 3 in a 8 00 a u? a k 00 <r-1 a § 3 g u R 5 s 5 3 i 8 ■r4 8 T"“i 3 5 8 s 9 3 £ a a a a s a S a 8 a 3 a s o s a E S a S a W a S o a W a a « a £ **4 co W S Remarks                       ) co c g o c o S3 I c Q 8 X 8 £ § ss o in c £ ■C 3 o © 3=5 1 1 .s CM 8 45 I w c '€ 1 a 8 ¢0 s c s I ? co 5 CM c a s I 1 co c a 45 1 1 8 ® s I fi c a 65 I ? i CO c g 1 & ? 03 s I 5 ? i c a a 8 S CM f a ? 2 CO C a s s Cl a a § a 8 s s r 1 1 8 t fx a z s 1 CO: U. co t 1 £ a z i 1 a a z 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 5 the pre- and post- extracted powders. Example 4 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.% Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4. 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. The carbon dioxide solvent was removed under standard conditions and recovered for re-use. 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.%. The results of the analysis are set out in Table 6. Table 6 Starting material Extracted powder Extracted oil Protein Nx6.38 (dry matter) (%) 68.9 83.3 ND Fat (by Rose Gottlieb) (%) 24.0 11.4 99.1 (Soxhlet method) Lactose (%) Not measured Phosphatidylcholine (%) 2.21 2.94 Phosphatidylethanolamine (%) 1.58 1.74 Phosphatidylinositol (%) 0.38 0.61 Phosphatidylserine (%) 0.67 0.88 Sphingomyelin (%) 1.28 1.57 Example 5 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.% Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4. 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. The carbon dioxide solvent was removed under standard conditions and recovered for re-use. 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.%. The results of the analysis are set out in Table 7. Table 7 Starting material Extracted powder Extracted oil Protein Nx6.38 (dry matter) (%) 68.9 83.1 ND Fat (by Rose Gottlieb) (%) 24.0 11.2 97.8 (Soxhlet method) Lactose (%) Not measured Phosphatidylcholine (%) 2.21 Phosphatidylethanolamine (%) 1.58 Phosphatidylinositol (%) 0.38 Phosphatidylserine (%) 0.67 Sphingomyelin (%) 1.28 Example 6 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.% Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4. 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. The carbon dioxide solvent was removed under standard conditions and recovered for re-use. 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.%. The results of the analysis are set out in Table 8. Table 8 Starting material Extracted powder Extracted oil Protein Nx6.38 (dry matter) (%) 68.9 83.7 ND Fat (by Rose Gottlieb) (%) 24.0 11.0 98.8 (Soxhlet method) Lactose (%) Not measured Phosphatidylcholine (%) 2.21 Phosphatidylethanolamine (%) 1.58 Phosphatidylinositol (%) 0.38 Phosphatidylserine (%) 0.67 Sphingomyelin (%) 1.28 Example 7 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.% Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4. 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 scC02 to whey feedstock of 13:1 by weight. The extraction was run under these conditions for approximately three hours. The carbon dioxide solvent was removed under standard conditions and recovered for re-use. 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.%. The results of the analysis are set out in Table 9. Table 9 Starting material Extracted powder Extracted oil Protein Nx6.38 (dry matter) (%) 68.9 82.0 ND Fat (by Rose Gottlieb) (%) 24.0 13.1 98.9 (Soxhlet method) Lactose (%) Not measured Phosphatidylcholine (%) 2.21 Phosphatidylethanolamine (%) 1.58 Phosphatidylinositol (%) 0.38 Phosphatidylserine (%) 0.67 Sphingomyelin (%) 1.28 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. 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: (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. (ii) 5% ethanol was introduced as a co-solvent using a further ratio of total flow to whey feedstock of 15:1 by weight. (iii) 10% ethanol was introduced as a co-solvent using a further ratio of total flow to whey feedstock of 15:1 by weight. (iv) 20% ethanol was introduced as a co-solvent using a further ratio of total flow to whey feedstock of 9:1 by weight. (v) 100% ethanol was introduced and a total of 10 litres used for the extraction. (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. 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. 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. The products of each extraction are also shown in the photographs of Figure 9. 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. The results of the analysis are set out in Table 10. Table 10 Starting material Extracted powder Protein Nx6.38 (dry matter) (%) 68.9 91.6 Fat (by Rose Gottlieb) (%) 24.0 2.1 Lactose (%) 0.8 0.2 Phosphatidylcholine (%) 2.21 0.17 Phosphatidylethanolamine (%) 1.58 0.38 Phosphatidylinositol (%) 0.38 0.78 Phosphatidylserine (%) 0.67 1.00 Sphingomyelin (%) 1.28 0.06 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. Example 9 - carbon dioxide extraction of WPC having a dry protein content of 60-80 wt.%, followed by ethanol extraction of the resulting powder Super-critical CO2 extraction was performed using an apparatus as shown schematically in Figure 4. A dried WPC70 powder (MFGM) was also 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 steps: 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. 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. The process produced a WPI having a protein dry matter content of 91 -94% by weight. Observations 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. 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 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 5 alternative forms and should not be construed as limited to the discussion set forth herein. Accordingly, while embodiments can be modified in various ways and take on various alternative forms, specific embodiments thereof are shown in the drawings and 10 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. Although embodiments of the invention have been disclosed herein, it is understood 15 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

1. 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 scCO2 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 SCCO2 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 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.

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 milkfat 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.

35. An isolated phospholipid or mixture of phospholipids obtainable or obtained by a process claimed in any one of claims 1 to 26.

Images