PRODUCTION OF SOLUBLE PROTEIN PRODUCTS FROM HEMP ("H701")

Abstract

A hemp protein product, which may be an isolate, produces solutions at low pH values and is useful for the fortification of soft drinks and sports drinks without precipitation of protein. The hemp protein product is obtained by extracting a hemp protein source material with an aqueous calcium salt solution to form an aqueous hemp protein solution, separating the aqueous hemp protein solution from residual hemp protein source, adjusting the pH of the aqueous hemp protein solution to a pH of about 1.5 to about 4.4 to produce an acidified hemp protein solution, which may be dried, following optional concentration and diafiltration, to provide the hemp protein product.

Claims

1. A method of producing a hemp protein product having a protein content of at least about 60 wt %, preferably at least about 90 wt %, (N?6.25) on a dry weight basis, which comprises: (a) extracting a hemp protein source with an aqueous calcium salt solution to cause solubilization of hemp protein from the protein source and to form an aqueous hemp protein solution, (b) at least partially separating the aqueous hemp protein solution from residual hemp protein source, (c) optionally diluting the aqueous hemp protein solution, (d) adjusting the pH of the aqueous hemp protein solution to a pH of about 1.5 to about 4.4 to produce an acidified aqueous hemp protein solution, (e) optionally clarifying the acidified hemp protein solution if it is not already clear, (f) alternatively from steps (b) to (e), optionally diluting and then adjusting the pH of the combined aqueous hemp protein solution and residual hemp protein source to a pH of about 1.5 to about 4.4 then separating the acidified aqueous hemp protein solution from residual hemp protein source, (g) optionally concentrating the aqueous hemp protein solution while maintaining the ionic strength substantially constant by a selective membrane technique, (h) optionally diafiltering the optionally concentrated hemp protein solution, and (i) optionally drying the optionally concentrated and optionally diafiltered hemp protein solution.

2. The method of claim 1 wherein said aqueous calcium salt solution is an aqueous calcium chloride solution.

3. The method of claim 2 wherein said aqueous calcium chloride solution has a concentration less than about 1.0 M.

4. The method of claim 3 wherein said concentration is about 0.10 to about 0.15 M.

5. The method of claim 1 wherein said extraction step (a) is effected at a temperature of about 1? to about 65? C., preferably about 15? to about 65? C., more preferably 20? to about 35? C.

6. The method of claim 1 wherein said extraction with aqueous calcium salt solution is conducted at a pH of about 4.5 to about 11.

7. The method of claim 6 wherein said pH is about 5 to about 7.

8. The method of claim 1 wherein said aqueous hemp protein solution has a protein concentration of about 5 to about 50 g/L.

9. The method of claim 8 wherein said protein concentration is about 10 to about 50 g/L.

10. The method of claim 1 wherein said aqueous calcium salt solution contains an antioxidant.

11. The method of claim 1 wherein, following said separation step (b) and prior to said optional dilution step (c) or in step (f) after said separation step, said aqueous hemp protein solution is treated with an adsorbent to remove colour and/or odour compounds from the aqueous hemp protein solution.

12. The method of claim 1 wherein said aqueous hemp protein solution is diluted in step (c) or (f) with about 0.1 to about 10 volumes of aqueous diluent to a conductivity of less than about 105 mS.

13. The method of claim 12 wherein said aqueous hemp protein solution is diluted in step (c) or (f) with about 0.5 to about 2 volumes of aqueous diluent to provide a conductivity of said hemp protein solution of about 4 to about 21 mS.

14. The method of claim 12 wherein said aqueous diluent has a temperature of about 1? to about 100? C.

15. The method of claim 14 wherein said temperature is about 15? to about 65? C.

16. The method of claim 15 wherein said temperature is about 20? to about 35? C.

17. The method of claim 1 wherein said acidified hemp protein solution has a conductivity of less than about 110 mS if diluted or less than 115 mS if not diluted.

18. The method of claim 17 wherein said conductivity is about 4 to about 26 mS.

19. The method of claim 1 wherein the pH of said aqueous hemp protein solution is adjusted in step (d) or (f) to about pH 2 to about 4.

20. The method of claim 1 wherein the acidified hemp protein solution is subjected to step (e).

21. The method of claim 1 wherein said acidified aqueous hemp protein solution is dried to provide a hemp protein product having a protein content of at least about 60 wt % (N?6.25) d.b.

22. The method of claim 1 wherein said acidified aqueous hemp protein solution is subjected to step (g) to produce a concentrated acidified hemp protein solution having a protein concentration of about 50 to about 300 g/L.

23. The method of claim 22 wherein said concentrated acidified hemp protein solution has a protein concentration of about 100 to about 200 g/L.

24. The method of claim 22 wherein said concentration step (g) is effected by ultrafiltration using a membrane having a molecular weight cut-off of about 1,000 to about 1,000,000 Daltons.

25. The method of claim 24 wherein said membrane has a molecular weight cut-off of about 1,000 to about 100,000 Daltons.

26. The method of claim 1 wherein said diafiltration step (h) is effected using water, acidified water, dilute saline or acidified dilute saline on the acidified hemp protein solution before or after partial or complete concentration thereof.

27. The method of claim 26 wherein said diafiltration step (h) is effected using about 1 to about 40 volumes of diafiltration solution.

28. The method of claim 27 wherein said diafiltration step (h) is effected using about 2 to about 25 volumes of diafiltration solution.

29. The method of claim 26 wherein said diafiltration step (h) is effected until no significant further quantities of contaminants or visible colour are present in the permeate.

30. The method of claim 26 wherein said diafiltration step (h) is effected until the retentate has been sufficiently purified so as, when dried, to provide a hemp protein isolate with a protein content of at least about 90 wt % (N?6.25) d.b.

31. The method of claim 26 wherein said diafiltration step (h) is effected using a membrane having a molecular weight cut-off of about 1,000 to about 1,000,000 Daltons.

32. The method of claim 31 wherein said membrane has a molecular weight cut-off of about 1,000 to about 100,000 Daltons.

33. The method of claim 26 wherein an antioxidant is present in the diafiltration medium during at least part of the diafiltration step (h).

34. The method of claim 22 or claim 26 wherein said concentration step (g) and optional diafiltration step (h) are carried out at a temperature of about 2? to about 65? C.

35. The method of claim 34 wherein said temperature is about 20? to about 35? C.

36. The method of claim 1 wherein said acidified aqueous hemp protein solution is subjected to steps (g) and (h) to produce a concentrated and/or diafiltered acidified hemp protein solution which, when dried, provides a hemp protein product having a protein concentration of at least about 60 wt % (N?6.25) d.b.

37. The method of claim 22 or claim 26 wherein said optionally concentrated and optionally diafiltered acidified hemp protein solution is treated with an adsorbent to remove colour and/or odour compounds.

38. The method of claim 22 or claim 26 wherein said optionally concentrated and optionally diafiltered acidified hemp protein solution is pasteurized prior to drying.

39. The method of claim 38 wherein said pasteurization step is effected at a temperature of about 55? to about 70? C. for about 30 seconds to about 60 minutes.

40. The method of claim 39 wherein said pasteurization step is effected at a temperature of about 60? to about 65? C. for about 10 to about 15 minutes.

41. The method of claim 30 wherein said optionally concentrated and diafiltered acidified hemp protein solution is subjected to step (i) to provide a hemp protein isolate having a protein content of at least about 90 wt % (N?6.25) d.b.

42. The method of claim 41 wherein said hemp protein isolate has a protein content of at least about 100 wt % (N?6.25) d.b.

43. A hemp protein product having a protein content of at least about 60 wt % (N?6.25) d.b. which is water soluble at acid pH values of less than about 4.4.

44. The hemp protein product of claim 43 having a protein content of at least about 90 wt % (N?6.25) d.b.

45. The protein product of claim 43 having a protein content of at least about 100 wt % (N?6.25) d.b.

46. The hemp protein product of claim 43 which has a bland flavour.

47. The hemp protein product of claim 43 which is blended with water-soluble powdered materials for the production of aqueous solutions of the blend.

48. The blend of claim 47 which is a powdered beverage.

49. An aqueous solution of the hemp protein product of claim 43 having a pH of less than about 4.4.

50. The aqueous solution of claim 49 which is a beverage.

51. An aqueous solution of the hemp protein product of claim 43 having a pH of about 6 to about 8.

52. The aqueous solution of claim 51 which is a beverage.

53. The aqueous solution of claim 51 which is used in the production of a dairy analogue or dairy alternative product or a product that is a blend of plant and dairy ingredients.

Description

EXAMPLES

Example 1

[0058] This Example illustrates the production of the hemp protein isolate.

[0059] 22.5 kg of ground hemp press cake was combined with 150 L of 0.15 M CaCl.sub.2 solution at 25.8? C. and agitated for 30 minutes to provide an aqueous protein solution. The residual ground hemp press cake was removed and the resulting protein solution was clarified by centrifugation and filtration to produce a filtrate having a protein content of 1.31% by weight.

[0060] The filtrate was then diluted with reverse osmosis purified water and the pH of the sample lowered to 2.68 with HCl that had been diluted with an equal volume of water. The diluted and acidified protein solution had a protein content of 0.88 wt %.

[0061] The diluted and acidified protein solution was reduced in volume from 160 L to 7 L by concentration on a polyethersulfone (PES) membrane, having a molecular weight cut-off of 100,000 Daltons, operated at a temperature of approximately 30? C. The concentrated, acidified protein solution, with a protein content of 10.51 wt %, was diafiltered with 35 L of reverse osmosis purified water, with the diafiltration operation conducted at approximately 30? C. The resulting 7.38 kg of diafiltered protein solution had a protein content of 9.65 wt % and represented a yield of 50.4 wt % of the diluted and acidified protein solution that was further processed. The protein solution was then dried to yield a product found to have a protein content of 108.31 wt % (N?6.25) d.b. The product was given designation H001-H24-11A H701.

Example 2

[0062] This Example contains an evaluation of the phytic acid content of the hemp protein isolate produced by the method of Example 1 as well as the commercial hemp protein concentrate Hemp Pro 70 (Manitoba Harvest, Winnipeg, MB), the protein content of which was determined by combustion analysis using a Leco Nitrogen Determinator to be 65.76% d.b.

[0063] Phytic acid content was determined using the method of Latta and Eskin (J. Agric. Food Chem., 28: 1313-1315).

[0064] The phytic acid content of the H001-H24-11A H701 was 0.22% d.b. and that of Hemp Pro 70 was 1.43% d.b.

Example 3

[0065] This Example illustrates the colour of the hemp protein isolate prepared by the method of Example 1 and the commercial hemp protein concentrate Hemp Pro 70 in solution and in dry powder form.

[0066] Solutions of H001-H24-11A H701 and Hemp Pro 70 were prepared by dissolving sufficient protein powder to supply 0.48 g of protein in 15 ml of RO water. The pH of the solution was measured with a pH meter and the colour and clarity assessed using a HunterLab ColorQuest XE instrument operated in transmission mode. The results are shown in the following Table 1:

TABLE-US-00001 TABLE 1 pH and HunterLab readings for solutions of H001-H24-11A H701 and Hemp Pro 70 Sample pH L* a* b* Haze H001-H24-11A H701 3.24 97.69 ?0.21 10.54 67.5 Hemp Pro 70 7.45 1.53 3.64 2.42 96.5

[0067] As may be seen from the results in Table 1, the solution of H001-H24-11 A H701 was light in colour and translucent. The solution of Hemp Pro 70 was darker, more red, less yellow and had a higher haze level than the solution of H001-H24-11A H701.

[0068] The colour of the dry powders was assessed using the HunterLab ColorQuest XE instrument operated in reflectance mode. The colour values are set forth in the following Table 2:

TABLE-US-00002 TABLE 2 HunterLab scores for H001-H24-11A H701 and Hemp Pro 70 dry powders Sample L* a* b* H001-H24-11A H701 85.45 0.90 9.58 Hemp Pro 70 52.29 2.78 24.44

[0069] As may be seen from the results presented in Table 2, the H001-H24-11A H701 powder was lighter, less red and less yellow than the Hemp Pro 70 powder.

Example 4

[0070] This Example contains an evaluation of the solubility in water of the hemp protein isolate produced by the method of Example 1 as well as the commercial hemp protein concentrate Hemp Pro 70, a product promoted as being water soluble. Solubility was tested based on protein solubility (termed protein method, a modified version of the procedure of Morr et al., J. Food Sci. 50:1715-1718) and total product solubility (termed pellet method).

[0071] Sufficient protein powder to supply 0.5 g of protein was weighed into a beaker and then a small amount of reverse osmosis (RO) purified water was added and the mixture stirred until a smooth paste formed. Additional water was then added to bring the volume to approximately 45 ml. The contents of the beaker were then slowly stirred for 60 minutes using a magnetic stirrer. The pH was determined immediately after dispersing the protein and was adjusted to the appropriate level (2, 3, 4, 5, 6 or 7) with diluted NaOH or HCl. A sample was also prepared at natural pH. For the pH adjusted samples, the pH was measured and corrected periodically during the 60 minutes stirring. After the 60 minutes of stirring, the samples were made up to 50 ml total volume with RO water, yielding a 1% w/v protein dispersion. The protein content of the dispersions was measured by combustion analysis using a Leco Nitrogen Determinator. Aliquots (20 ml) of the dispersions were then transferred to pre-weighed centrifuge tubes that had been dried overnight in a 100? C. oven then cooled in a desiccator and the tubes capped. The samples were centrifuged at 7,800 g for 10 minutes, which sedimented insoluble material and yielded a supernatant. The protein content of the supernatant was measured by combustion analysis and then the supernatant and the tube lids were discarded and the pellet material dried overnight in an oven set at 100? C. The next morning the tubes were transferred to a desiccator and allowed to cool. The weight of dry pellet material was recorded. The dry weight of the initial protein powder was calculated by multiplying the weight of powder used by a factor of ((100?moisture content of the powder (%))/100). Solubility of the product was then calculated two different ways:

Solubility(protein method)(%)=(% protein in supernatant/% protein in initial dispersion)?1001)

Solubility(pellet method)(%)=(1?(weight dry insoluble pellet material/((weight of 20 ml of dispersion/weight of 50 ml of dispersion)?initial weight dry protein powder)))?1002)

[0072] Values calculated to be greater than 100% were expressed as 100%.

[0073] The solubility results are set forth in the following Table 3. The natural pH for the sample of H001-H24-11A H701 was 3.31. The natural pH for the sample of Hemp Pro 70 was 7.69.

TABLE-US-00003 TABLE 3 Solubility of H001-H24-11A H701 and Hemp Pro 70 at different pH values as determined by protein method Solubility (%) product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH H001-H24-11A 94.4 100 99.1 81.7 67.0 63.7 99.1 H701 Hemp Pro 70 53.2 47.7 12.7 10.3 10.4 15.1 16.2

TABLE-US-00004 TABLE 4 Solubility of H001-H24-11A H701 and Hemp Pro 70 at different pH values as determined by pellet method Solubility (%) product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH H001-H24-11A 99.7 99.2 98.5 77.6 66.5 59.3 98.2 H701 Hemp Pro 70 50.0 46.0 27.2 24.3 25.6 26.9 30.3

[0074] As may be seen from the results presented in Tables 3 and 4, the H001-H24-11A H701 was highly soluble in the pH range 2 to 4. The Hemp Pro 70 was only partially soluble at all pH values tested.

Example 5

[0075] This Example contains an evaluation of the clarity in water of the hemp protein isolate produced by the method of Example 1 as well as the commercial hemp protein concentrate Hemp Pro 70.

[0076] The clarity of the 1% w/v protein dispersions prepared as described in Example 4 was assessed by measuring the absorbance at 600 nm (water blank), with a lower absorbance score indicating greater clarity. Analysis of the samples on the HunterLab ColorQuest XE instrument in transmission mode also provided a percentage haze reading, another measure of clarity.

[0077] The clarity results are set forth in the following Tables 5 and 6.

TABLE-US-00005 TABLE 5 Clarity of H001-H24-11A H701 and Hemp Pro 70 solutions at different pH values as assessed by A600 A600 Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH H001-H24-11A 0.011 0.043 0.110 1.542 2.477 2.567 0.058 H701 Hemp Pro 70 2.644 2.744 3.153 3.063 3.031 3.023 3.130

TABLE-US-00006 TABLE 6 Clarity of H001-H24-11A H701 and Hemp Pro 70 solutions at different pH values as assessed by HunterLab analysis HunterLab haze reading (%) Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH H001-H24-11A 0.0 11.2 30.0 94.8 95.5 95.5 16.5 H701 Hemp Pro 70 96.2 96.0 97.2 99.0 97.7 96.7 96.3

[0078] As may be seen from the results in Tables 5 and 6, the greatest solution clarity for the H001-H24-11A H701 was observed at the lower pH values. The Hemp Pro 70 provided very cloudy solutions at all the pH values tested.

Example 6

[0079] This Example contains an evaluation of the protein solubility in a soft drink and sports drink of the hemp protein isolate produced by the method of Example 1 and the commercial hemp protein concentrate Hemp Pro 70. The solubility was determined with the protein added to the beverages with no pH correction and again with the pH of the protein fortified beverages adjusted to the level of the original beverages.

[0080] When the solubility was assessed with no pH correction, a sufficient amount of protein powder to supply 1 g of protein was weighed into a beaker and a small amount of beverage was added and stirred until a smooth paste formed. Additional beverage was added to bring the volume to 50 ml, and then the solutions were stirred slowly on a magnetic stirrer for 60 minutes to yield a 2% protein w/v dispersion. The protein content of the samples was determined by combustion analysis using a LECO Nitrogen Determinator then an aliquot of the protein containing beverages was centrifuged at 7,800 g for 10 minutes and the protein content of the supernatant measured.

Solubility (%)=(% protein in supernatant/% protein in initial dispersion)?100

Values calculated to be greater than 100% were expressed as 100%.

[0081] When the solubility was assessed with pH correction, the pH of the soft drink (Sprite) (3.59) and sports drink (Orange Gatorade) (3.29) without protein was measured. A sufficient amount of protein powder to supply 1 g of protein was weighed into a beaker and a small amount of beverage was added and stirred until a smooth paste formed. Additional beverage was added to bring the volume to approximately 45 ml, and then the solutions were stirred slowly on a magnetic stirrer for 60 minutes. The pH of the protein containing beverages was determined immediately after dispersing the protein and was adjusted to the original no-protein pH with HCl or NaOH solution as necessary. The pH was measured and corrected periodically during the 60 minutes stirring. After the 60 minutes of stirring, the total volume of each solution was brought to 50 ml with additional beverage, yielding a 2% protein w/v dispersion. The protein content of the samples was determined by combustion analysis using a Leco Nitrogen Determinator then an aliquot of the protein containing beverages was centrifuged at 7,800 g for 10 minutes and the protein content of the supernatant measured.

Solubility (%)=(% protein in supernatant/% protein in initial dispersion)?100

Values calculated to be greater than 100% were expressed as 100%.

[0082] The results obtained are set forth in the following Table 7.

TABLE-US-00007 TABLE 7 Solubility of H001-H24-11A H701 in Sprite and Orange Gatorade No pH correction pH correction Solubility in Solubility in Orange Orange Solubility in Gatorade Solubility in Gatorade Product Sprite (%) (%) Sprite (%) (%) H001-H24-11A 96.6 100 100 98.1 H701 Hemp Pro 70 8.7 9.0 32.3 39.5

[0083] The natural pH of the H701 was similar to that of the beverages so protein addition had only a minor effect on beverage pH. As can be seen from the results of Table 7 the H001-H24-11A H701 was highly soluble in both the Sprite and the Orange Gatorade, with and without pH correction and was much more soluble than the Hemp Pro 70 protein.

Example 7

[0084] This Example contains an evaluation of the clarity in a soft drink and sports drink of the hemp protein isolate produced by the method of Example 1.

[0085] The clarity of the 2% w/v protein dispersions prepared in soft drink (Sprite) and sports drink (Orange Gatorade) in Example 6 were assessed using the spectrophotometric and HunterLab methods as described in Example 5. In this case however, the spectrophotometer was blanked with the appropriate beverage.

[0086] The results obtained are set forth in the following Tables 8 and 9.

TABLE-US-00008 TABLE 8 A600 readings for H001-H24-11A H701 in Sprite and Orange Gatorade No pH correction pH correction A600 in A600 in Orange Orange A600 in Gatorade A600 in Gatorade Product Sprite (%) (%) Sprite (%) (%) H001-H24-11A H701 0.189 0.352 0.530 0.444 Hemp Pro 70 nd 3.596 nd 3.157 nd = not determined

TABLE-US-00009 TABLE 9 HunterLab haze readings for H001-H24-11A H701 in Sprite and Orange Gatorade No pH correction pH correction Haze in Haze in Haze in Orange Haze in Orange Product Sprite (%) Gatorade (%) Sprite (%) Gatorade (%) no protein 0.0 50.9 H001-H24-11A 58.2 88.6 88.7 91.1 H701 Hemp Pro 70 98.4 99.3 96.4 95.9

[0087] As can be seen from the results in Tables 8 and 9, despite the excellent protein solubility, the H001-H24-11A H701 contributed haze to the Sprite and Orange Gatorade. However, samples prepared with Hemp Pro 70 were cloudier than the samples prepared with H001-H24-11A H701.

Example 8

[0088] This Example illustrates a comparison of the flavor of the H701, prepared as described in Example 1, with that of the commercial hemp protein concentrate Hemp Pro 70, with the evaluation done at low pH.

[0089] Samples were prepared for sensory evaluation by dissolving sufficient protein powder to supply 5 g of protein in 250 ml purified drinking water. The pH of the solution of H701 was determined to be 3.31. Food grade HCl was added to the solution of Hemp Pro 70 to lower the pH from 7.73 to 3.31. An informal panel of seven panelists was asked to blindly compare the samples and indicate which sample was more bland in flavour, and of which sample they preferred the flavour.

[0090] Six out of seven panelists found the flavour of the H701 to be more bland, while seven out of seven panelists preferred the flavour of the H701.

Example 9

[0091] This Example illustrates a comparison of the flavor of the H701, prepared as described in Example 1, with that of the commercial hemp protein concentrate Hemp Pro 70, with the evaluation done at near neutral pH.

[0092] Samples were prepared for sensory evaluation by dissolving sufficient protein powder to supply 5 g of protein in 250 ml purified drinking water. The pH of the solution of Hemp Pro 70 was determined to be 7.72. Food grade NaOH was added to the solution of H701 to raise the pH from 3.23 to 7.72. An informal panel of seven panelists was asked to blindly compare the samples and indicate which sample was more bland in flavour, and of which sample they preferred the flavour.

[0093] Four out of seven panelists found the flavour of the H701 to be more bland, while four out of seven panelists preferred the flavour of the H701.

SUMMARY OF THE DISCLOSURE

[0094] In summary of this disclosure, the present invention provides a novel hemp protein product, which may be in the form of an isolate, which is completely soluble at acid pH and is useful in the protein fortification of aqueous systems, including soft drinks and sports drinks, particularly powdered versions of these drinks, without leading to protein precipitation. Modifications are possible within the scope of this invention.