Production of soluble protein solutions from pulses

Abstract

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

Claims

1. A pulse protein product having a protein content of at least about 60 wt % (N6.25) d.b. and which is completely soluble at 1% w/v in water at acid pH values of less than about 4.4, is heat stable in aqueous media at acid pH values in the range of about 1.5 to about 4.4, such heat stability being determined by heating a 2% w/v aqueous protein solution of the pulse protein product at 95 C. for 30 seconds followed by cooling the heated solution to room temperature in an ice bath and measuring the clarity of the cooled solution in comparison to the clarity of the aqueous solution prior to heating, does not require stabilizers or other additives to maintain the protein product in solution, is low in phytic acid requires no enzymes in the production thereof.

2. The pulse protein product of claim 1 that has a clean flavor and no off odors.

3. The pulse protein product of claim 1 wherein the pulse protein has not been hydrolyzed.

4. The pulse protein product of claim 1 which is low in trypsin inhibitor activity.

5. The pulse protein product of claim 1 which has a protein content of at least about 90 wt % (N6.25) d.b.

6. The protein product of claim 1 which has a protein content of about 100 wt % (N6.25) d.b.

7. The pulse protein product of claim 1 which has a phytic acid content of less than about 1.5 wt %.

8. A pulse protein product having: 1. a molecular weight profile which is: 75 to 85%>100,000 Da 10 to 18%>15,000-100,000 Da 2 to 5%>5,000-15,000 Da 1 to 4%>1000-5000 Da, 2. a protein content of at least about 60 wt % (N6.25) d.b.; and 3. a solubility at 1% protein w/v in water at a pH of about 2 to about 4 of greater than about 90%.

9. The pulse protein product of claim 8 which is a yellow pea protein product.

10. A pulse protein product which is a lentil or dry pea protein product and which has a protein content of at least about 60 wt % (N6.25) d.b., which has a solubility at 1% protein w/v in water at a pH of about 2 to about 4 of greater than about 90%, and which has an absorbance of visible light at 600 nm (A600) for a 1% protein w/v aqueous solution at a pH of over the range of 2 to 4 of less than 0.150.

11. The pulse protein product of claim 10 which is a yellow pea protein product.

12. A pulse protein product which is a lentil or dry pea protein product and having a protein content of at least about 60 wt % (N6.25) d.b. which has an absorbance of visible light at 600 nm (A600) for an unheated 1% w/v protein aqueous solution at a pH in the range of 2 to 4 of less than 0.150.

13. The pulse protein product of claim 12 which is a yellow pea protein product.

14. A pulse protein product having a protein content of at least about 60 wt % (N6.25) d.b. which has a haze reading for an unheated 1% w/v protein aqueous solution at a pH over the range of 2 to 4, of less than 15%.

15. The pulse protein product of claim 14 which is a yellow pea protein product.

16. A pulse protein product having a protein content of at least about 60 wt % (N6.25) d.b. which has a haze reading for a 2% protein w/v solution in water at a pH over the range of 2 to 4, after heat treatment at 95 C. for 30 seconds, of less than 15%.

17. The pulse protein product of claim 16 which is a yellow pea protein product.

18. The pulse protein product of any one of claim 10 or 12 or 14 or 16 which has a protein content of at least about 90 wt % (N6.25) d.b.

19. A pulse protein product having a protein content of at least about 60 wt % (N6.25) d.b. which has colorimeter readings of L*=about 92 to about 100, a*=about 1 to about 1 and b*=0 to about 14, for a solution thereof in water prepared by dissolving sufficient pulse protein product to supply 3.2 g of protein per 100 ml of water, and having a pH of less than about 4.4 without the subsequent addition of a pH adjusting agent.

20. The pulse protein product of claim 19 which is a yellow pea protein product.

21. The pulse protein product of claim 19 which has a protein content of at least about 90 wt % (N6.25) d.b.

22. The pulse protein product of claim 7 which has a phytic acid content of less than about 0.5 wt %.

23. The pulse protein product of claim 12 wherein the A600 value is less than about 0.100.

24. The pulse protein product of claim 23 wherein the A600 value is less than 0.050.

25. The pulse protein product of claim 14 wherein the haze reading is less than about 10%.

26. The pulse protein product of claim 25 wherein the haze reading is less than about 5%.

27. The pulse protein product of claim 16 wherein the haze reading is less than about 10%.

28. The pulse protein product of claim 27 wherein the haze reading is less than about 5%.

29. The pulse protein product claimed in claim 21 wherein the protein content is about 100 wt % (N6.25).

Description

EXAMPLES

Example 1

(1) This Example evaluates the protein extractability of lentils, chickpeas and dry peas and the effect of acidification on the clarity of protein solutions resulting from the extraction step.

(2) Dry lentils, chickpeas, yellow split peas and green split peas were purchased in whole form and ground using a Bamix chopper until in the form of a relatively fine powder. The extent of grinding was not controlled by time or particle size. Ground material (10 g) was extracted with 0.15M CaCl.sub.2 (100 ml) for 30 minutes on a magnetic stirrer at room temperature. The extract was separated from the spent material by centrifugation at 10,200 g for 10 minutes and then further clarified by filtration with a 0.45 m pore size syringe filter. The ground starting material and the clarified extract were tested for protein content using a Loco FP 528 Nitrogen Determinator. The clarity of the extract at full strength and diluted with 1 volume of reverse osmosis purified (RO) water was determined by measuring the absorbance at 600 nm (A600). The full strength and diluted solutions were then adjusted to pH 3 with HCl and the A600 measured again. In this and other Examples where solution clarity was assessed by A600 measurement, water was used to blank the spectrophotometer.

(3) The protein contents and apparent extractabilities determined for each protein source are shown in Table 1.

(4) TABLE-US-00001 TABLE 1 Protein content and apparent extractability of protein sources protein source protein content (%) apparent extractability (%) lentil 24.20 47.5 chickpeas 18.97 52.2 yellow split peas 23.07 59.4 green split peas 22.38 64.3

(5) As may be seen from the results in Table 1, the apparent extractability of all the protein sources was quite good.

(6) Clarity of the full strength and diluted extract samples before and after acidification are shown in Table 2.

(7) TABLE-US-00002 TABLE 2 Effect of acidification on clarity of diluted and undiluted extract samples - calcium chloride extraction undiluted diluted initial initial final final initial initial final final sample pH A600 pH A600 pH A600 pH A600 lentils 5.22 0.093 3.04 0.253 5.30 1.196 2.96 0.037 chickpeas 5.15 0.189 3.07 0.228 5.25 2.714 2.79 0.099 yellow split 5.21 0.250 3.14 0.828 5.28 2.334 3.11 0.250 peas green split 5.23 0.288 3.18 0.577 5.31 2.248 2.97 0.161 peas

(8) As may be seen from the results of Table 2, full strength extract solutions from lentil, chickpea and split peas were clear to slightly hazy. Acidification without dilution increased the haze level in the samples. Dilution of the filtered extract with an equal volume of water resulted in notable precipitation and a corresponding increase in the A600 value. Acidification of the diluted solution largely re-solubilized the precipitate and resulted in a clear solution for lentils and chickpeas and a slightly hazy solution for the yellow and green split peas.

Example 2

(9) This Example contains an evaluation of the clarity of acidified, diluted or undiluted green split pea extracts with water and sodium chloride replacing the calcium chloride solution of Example 1 as the extraction solution.

(10) Dry green split peas were purchased in whole form and ground to a fine powder using a KitchenAid mixer grinder attachment. The extent of grinding was not controlled by time or particle size. Ground material (10 g) was extracted with 0.15M NaCl (100 ml) or RO water (100 ml) for 30 minutes on a magnetic stirrer at room temperature. The extract was separated from the spent material by centrifugation at 10,200 g for 10 minutes and then further clarified by filtration with a 0.45 m pore size syringe filter. The clarity of the filtrates at full strength and diluted with 1 volume of RO water was determined by measuring the absorbance at 600 nm. The full strength and diluted solutions were then adjusted to pH 3 with HCl and the A600 measured again.

(11) Clarity of the full strength and diluted extract samples before and after acidification are shown in Table 3.

(12) TABLE-US-00003 TABLE 3 Effect of acidification on clarity of diluted and undiluted extract samples - water and sodium chloride extractions undiluted diluted extraction initial initial final final initial initial final final solution pH A600 pH A600 pH A600 pH A600 water 6.56 0.113 3.14 >3.0 6.62 0.050 3.00 2.647 0.15M NaCl 6.19 0.021 2.96 >3.0 6.28 0.870 2.87 2.851

(13) As may be seen from the results in Table 3, extracts prepared with water or sodium chloride solution were very cloudy when acidified regardless of whether a dilution step was employed.

Example 3

(14) This Example evaluates the protein extractability of several types of dry beans and the effect of acidification on the clarity of protein solutions resulting from the extraction step.

(15) Pinto beans, small white beans, small red beans, romano beans, great northern beans and lima beans were purchased in whole, dry form and ground using a Bamix chopper until in the form of a relatively fine powder. The extent of grinding was not controlled by time or particle size. Black bean flour was also purchased. Ground material or flour (10 g) was extracted with 0.15M CaCl.sub.2 (100 ml) for 30 minutes on a magnetic stirrer at room temperature. The extract was separated from the spent material by centrifugation at 10,200 g for 10 minutes and then further clarified by filtration with a 0.45 m pore size syringe filter. The ground starting material or flour and the clarified extract were tested for protein content using a Leco FP 528 Nitrogen Determinator. The clarity of the extract at full strength and diluted with 1 volume of RO water was determined by measuring the absorbance at 600 nm. The full strength and diluted solutions were then adjusted to pH 3 with HCl and the A600 measured again.

(16) The protein contents and apparent extractabilities determined for each type of dry bean are shown in Table 4.

(17) TABLE-US-00004 TABLE 4 Protein content and apparent extractability of various dry beans type of bean protein content (%) apparent extractability (%) black bean 24.00 77.9 pinto bean 21.45 66.2 small white bean 24.41 63.5 small red bean 20.18 76.8 romano bean 18.07 86.9 great northern bean 21.77 85.9 lima bean 21.43 71.9

(18) As may be seen from the results in Table 4, the protein in all of the types of beans was readily extracted.

(19) Clarity of the full strength and diluted extract samples before and after acidification are shown in Table 5.

(20) TABLE-US-00005 TABLE 5 Effect of acidification on clarity of diluted and undiluted extract samples - calcium chloride extraction undiluted diluted 1 + 1 initial initial final final initial initial final final sample pH A600 pH A600 pH A600 pH A600 black bean 4.69 0.100 2.99 0.154 4.76 0.025 3.15 0.031 pinto bean 5.08 0.014 3.02 0.072 5.34 0.003 3.00 0.017 small white 5.08 0.026 3.03 0.092 5.23 0.022 3.03 0.019 bean small red bean 5.06 0.028 3.07 0.093 5.33 0.014 2.97 0.021 romano bean 4.96 n.d. 3.07 0.023 5.21 0.005 2.86 0.008 gr. northern 4.93 0.026 3.10 0.045 5.16 0.008 3.11 0.013 bean lima bean 5.13 n.d. 3.07 0.089 5.37 0.020 3.04 0.013 n.d. = not determined

(21) As may be seen from the results of Table 5, full strength extract solutions from all of the beans were quite clear. Acidification without dilution slightly increased the haze level in the samples but they remained quite clear. Dilution of the filtered extract with an equal volume of water did not result in the formation of any precipitate. This is in contrast to the precipitation seen upon dilution for the pulses tested in Example 1. The diluted bean protein solutions stayed clear when acidified.

Example 4

(22) This Example contains an evaluation of the clarity of acidified, diluted or undiluted small white bean extracts with water and sodium chloride replacing the calcium chloride solution of Example 3 as the extraction solution.

(23) Dry small white beans were purchased in whole form and ground to a fine powder using a Bamix chopper. The extent of grinding was not controlled by time or particle size. Ground material (10 g) was extracted with 0.15M NaCl (100 ml) or RO water (100 ml) for 30 minutes on a magnetic stirrer at room temperature. The extract was separated from the spent material by centrifugation at 10,200 g for 10 minutes and then further clarified by filtration with a 0.45 m pore size syringe filter. The protein content of the filtrates was determined using a Leco FP528 Nitrogen Determinator. The clarity of the extracts at full strength and diluted with 1 volume of RO water was determined by measuring the absorbance at 600 nm. The full strength and diluted solutions were then adjusted to pH 3 with HCl and the A600 measured again.

(24) Extraction with water and sodium chloride solution provided apparent extractabilities of 45.9% and 61.5% respectively. Clarity of the full strength and diluted extract samples before and after acidification are shown in Table 6.

(25) TABLE-US-00006 TABLE 6 Effect of acidification on clarity of diluted and undiluted extract samples - water and sodium chloride extractions undiluted diluted extraction initial initial final final initial initial final final solution pH A600 pH A600 pH A600 pH A600 water 6.48 0.079 2.95 >3.0 6.51 0.051 3.03 2.771 0.15M NaCl 6.13 0.116 3.01 >3.0 6.22 0.212 3.02 >3.0

(26) As may be seen from the results in Table 6, extracts prepared with water or sodium chloride solution were very cloudy when acidified regardless of whether a dilution step was employed.

Example 5

(27) This Example illustrates the production of green pea protein isolate at benchtop scale.

(28) 180 g of dry green split peas were finely ground using a KitchenAid mixer grinder attachment. 150 g of finely ground green split pea flour was combined with 1,000 ml of 0.15 M CaCl.sub.2 solution at ambient temperature and agitated for 30 minutes to provide an aqueous protein solution. The residual solids were removed and the resulting protein solution was clarified by centrifugation and filtration to produce a filtered protein solution having a protein content of 1.83% by weight. 655 ml of the filtered protein solution was added to 655 ml of RO water and the pH of the sample lowered to 3.03 with HCl solution.

(29) The diluted and acidified protein extract solution was reduced in volume from 1250 ml to 99 ml by concentration on a PES membrane having a molecular weight cutoff of 10,000 Daltons. An aliquot of 96 ml of concentrated protein solution was then diafiltered on the same membrane with 480 ml of RO water. The resulting acidified, diafiltered, concentrated protein solution had a protein content of 7.97% by weight and represented a yield of 65.5 wt % of the initial filtered protein solution that was further processed. The acidified, diafiltered, concentrated protein solution was dried to yield a product found to have a protein content of 95.69% (N6.25) d.b. The product was termed GP701-01 protein isolate.

(30) 8.30 g of GP701-01 was produced. A solution of GP701-01 was prepared by dissolving sufficient protein powder to provide 0.48 g protein in 15 ml RO water and the pH measured with a pH meter and the color and clarity assessed using a HunterLab Color Quest XE instrument operated in transmission mode. The results are shown in the following Table 7.

(31) TABLE-US-00007 TABLE 7 pH and HunterLab scores for solution of GP701-01 sample pH L* a* b* haze GP701-01 3.17 89.46 1.10 14.98 63.3

(32) As may be seen from the results in Table 7, the solution of GP701-01 was translucent and had a light color.

(33) The solution of GP701-01 was heated to 95 C., held at this temperature for 30 seconds and then immediately cooled to room temperature in an ice bath. The clarity was re-measured with the HunterLab instrument and the results are shown in Table 8.

(34) TABLE-US-00008 TABLE 8 HunterLab scores for solution of GP701-01 after heat treatment sample L* a* b* haze GP701-01 95.56 0.06 9.65 47.0

(35) As may be seen from the results in Table 8, heat treatment was found to improve the lightness and reduce the haze level of the solution while making it greener and less yellow. Although the level of haze in the solution was reduced, the protein solution was still translucent rather than transparent.

Example 6

(36) This Example illustrates the production of green pea protein isolate at benchtop scale but with the filtration step moved to after dilution and acidification of the extract.

(37) 180 g of dry green split peas were finely ground using a KitchenAid mixer grinder attachment. 150 g of finely ground green split pea flour was combined with 1,000 ml of 0.15 M CaCl.sub.2 solution at ambient temperature and agitated for 30 minutes to provide an aqueous protein solution. The residual solids were removed by centrifugation to produce a centrate having a protein content of 2.49% by weight. 800 ml of centrate was added to 800 ml of water and the pH of the sample lowered to 3.00 with diluted HCl. The diluted and acidified centrate was further clarified by filtration to provide a clear protein solution with a protein content of 1.26% by weight. By filtering the solution after dilution and acidification, the A600 of the solution before membrane processing in this trial was 0.012, compared to 0.093 for the diluted and acidified filtrate in Example 5.

(38) The filtered protein solution was reduced in volume from 1292 ml to 157 ml by concentration on a PES membrane having a molecular weight cutoff of 10,000 Daltons. An aliquot of 120 ml of concentrated protein solution was then diafiltered on the same membrane with 600 ml of RO water. The resulting acidified, diafiltered, concentrated protein solution had a protein content of 7.70% by weight and represented a yield of 42.5 wt % of the initial centrate that was further processed. The acidified, diafiltered, concentrated protein solution was dried to yield a product found to have a protein content of 94.23% (N6.25) d.b. The product was termed GP701-02 protein isolate.

(39) 8.55 g of GP701-02 was produced. A solution of GP701-02 was prepared by dissolving sufficient protein powder to provide 0.48 g protein in 15 ml of RO water and the pH measured with a pH meter and the color and clarity assessed using a HunterLab Color Quest XE instrument operated in transmission mode. The results are shown in the following Table 9.

(40) TABLE-US-00009 TABLE 9 pH and HunterLab scores for solution of GP701-02 sample pH L* a* b* haze GP701-02 3.23 90.78 0.77 14.00 47.2

(41) As may be seen from the results in Table 9, the GP701-02 solution was translucent and had a light color. The level of haze was lower than that determined for the solution of GP701-01 in Example 5.

(42) The solution of GP701-02 was heated to 95 C., held at this temperature for seconds and then immediately cooled to room temperature in an ice bath. The clarity was then re-measured with the HunterLab and the result is shown in Table 10 below.

(43) TABLE-US-00010 TABLE 10 HunterLab scores for solution of GP701-02 after heat treatment sample L* a* b* haze GP701-02 96.24 0.48 9.74 2.2

(44) As may be seen from the results in Table 10, heat treatment of the GP701-02 solution resulted in an extremely clear solution.

Example 7

(45) This Example illustrates the production of small white bean protein isolate at benchtop scale.

(46) About 150 g of small white beans were finely ground using a KitchenAid mixer grinder attachment. 120 g of finely ground small white bean flour was combined with 1,000 ml of 0.15 M CaCl.sub.2 solution at ambient temperature and agitated for 30 minutes to provide an aqueous protein solution. The residual solids were removed and the resulting protein solution was clarified by centrifugation and filtration to produce a filtered protein solution having a protein content of 2.02% by weight. 600 ml of the filtered protein solution was added to 600 ml of RO water and the pH of the sample lowered to 3.01 with diluted HCl. Some wispy particulates were visible in the sample after the pH adjustment and these were removed by passing the sample through 25 m pore size filter paper.

(47) A sample of the diluted and acidified protein extract solution was then reduced in volume from 1110 ml to 82 ml by concentration on a PES membrane having a molecular weight cutoff of 10,000 Daltons. An aliquot of 79 ml of the retentate was then diafiltered on the same membrane with 395 ml of RO water. The resulting acidified, diafiltered, concentrated protein solution had a protein content of 10.37% by weight and represented a yield of 67.6 wt % of the initial filtered protein solution that was further processed. The acidified, diafiltered, concentrated protein solution was dried to yield a product found to have a protein content of 93.75% (N6.25) d.b. The product was termed SWB701 protein isolate.

(48) 8.26 g of SWB701 was produced. A solution of SWB701 was prepared by dissolving sufficient protein powder to provide 0.48 g protein in 15 ml RO water and the pH measured with a pH meter and the color and clarity assessed using a HunterLab Color Quest XE instrument operated in transmission mode. The results are shown in the following Table 11.

(49) TABLE-US-00011 TABLE 11 pH and HunterLab scores for solution of SWB701 sample pH L* a* b* haze SWB701 3.09 97.42 0.22 5.29 73.2

(50) As may be seen from the results in Table 11, the solution of SWB701 was translucent and had a light color.

(51) The solution of SWB701 was heated to 95 C., held at this temperature for 30 seconds and then immediately cooled to room temperature in an ice bath. The clarity was re-measured with the HunterLab instrument and the results are shown in Table 12.

(52) TABLE-US-00012 TABLE 12 HunterLab scores for solution of SWB701 after heat treatment sample L* a* b* haze SWB701 98.57 0.17 4.05 50.0

(53) As may be seen from the results in Table 12, heat treatment was found to improve the lightness and reduce the haze level of the solution while making it greener and less yellow. Although the level of haze in the solution was reduced, the protein solution was still translucent rather than transparent.

Example 8

(54) This Example contains an evaluation of the solubility in water of the GP701-02 produced by the method of Example 6 and the SWB701 produced by the method of Example 7. Solubility was tested using a modified version of the procedure of Morr et al., J. Food Sci. 50:1715-1718.

(55) Sufficient protein powder to supply 0.5 g of protein was weighed into a beaker and then approximately 45 ml of reverse osmosis (RO) purified water was added. The contents of the beaker were 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 using a Leco FP528 Nitrogen Determinator. Aliquots of the dispersions were then centrifuged at 7,800 g for 10 minutes, which sedimented insoluble material. The protein content of the supernatant was then determined by Leco analysis.

(56) The solubility of the protein was then calculated using the following equation:
Solubility (%)=(% protein in supernatant/% protein in initial dispersion)100

(57) The natural pH values of the protein isolates produced in Examples 6 and 7 are shown in the following Table 13:

(58) TABLE-US-00013 TABLE 13 Natural pH of samples prepared in water at 1% w/v protein sample Natural pH GP701-02 3.23 SWB701 3.09

(59) The solubility results obtained are set forth in the following Table 14:

(60) TABLE-US-00014 TABLE 14 Solubility of products at different pH values Solubility (%) sample pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH GP701-02 100 100 100 31.1 35.7 37.8 100 SWB701 95.2 95.3 100 88.8 55.4 77.5 94.0

(61) As can be seen from the results of Table 14, both of the 701 products were extremely soluble over the pH range 2 to 4.

Example 9

(62) This Example contains an evaluation of the clarity in water of the GP701-02 produced by the method of Example 6 and the SWB701 produced by the method of Example 7.

(63) The clarity of the 1% w/v protein dispersions prepared as described in Example 8 was assessed by analyzing the samples on a HunterLab ColorQuest XE instrument operated in transmission mode to provide a percentage haze reading. A lower score indicated greater clarity.

(64) The clarity results are set forth in the following Table 15:

(65) TABLE-US-00015 TABLE 15 Clarity of solutions at different pH values as assessed by HunterLab analysis HunterLab haze reading (%) sample pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH GP701-02 11.9 16.3 17.4 91.8 92.1 92.0 14.0 SWB701 0.0 38.0 64.6 91.7 92.4 82.9 43.9

(66) As can be seen from the results of Table 15, the solutions of GP701-02 were substantially clear or slightly hazy in the pH range 2 to 4. The solutions of GP701-02 were cloudy at the higher pH values where the solubility was reduced. The solution of SWB701 had no detectable haze at pH 2, but was noticeably hazier as the pH increased. Note that the protein solubility was still very high in the pH range 3 to 4 even though the solutions were not clear.

Example 10

(67) This Example illustrates the production of black bean protein product at benchtop scale.

(68) 50 g of black bean flour was combined with 500 ml of 0.15 M CaCl.sub.2 solution at ambient temperature and agitated for 30 minutes to provide an aqueous protein solution. The residual solids were removed and the resulting protein solution was clarified by centrifugation and filtration to produce a filtered protein solution having a protein content of 1.18% by weight. 450 ml of the filtered protein solution was added to 450 ml of RO water and the pH of the sample lowered to 3.09 with diluted HCl.

(69) The diluted and acidified protein extract solution was then reduced in volume from 900 ml to 50 ml by concentration on a PES membrane having a molecular weight cutoff of 10,000 Daltons. An aliquot of 40 ml of the retentate was then diafiltered on the same membrane with 200 ml of RO water. The resulting acidified, diafiltered, concentrated protein solution had a protein content of 6.23% by weight and represented a yield of approximately 46.9 wt % of the initial filtered protein solution that was further processed. The acidified, diafiltered, concentrated protein solution was dried to yield a product found to have a protein content of 86.33% (N6.25) d.b. The product was termed BB701.

(70) 2.19 g of BB701 was produced. A solution of BB701 was prepared by dissolving sufficient protein powder to provide 0.48 g protein in 15 ml of RO water and the pH measured with a pH meter and the color and clarity assessed using a HunterLab Color Quest XE instrument operated in transmission mode. The results are shown in the following Table 16.

(71) TABLE-US-00016 TABLE 16 pH and HunterLab scores for solution of BB701 sample pH L* a* b* haze BB701 3.14 95.20 0.88 8.22 54.6

(72) As may be seen from the results in Table 16, the solution of BB701 was translucent and had a light color.

(73) The solution of BB701 was heated to 95 C., held at this temperature for 30 seconds and then immediately cooled to room temperature in an ice bath. The clarity was re-measured with the HunterLab instrument and the results are shown in Table 17

(74) TABLE-US-00017 TABLE 17 HunterLab scores for solution of BB701 after heat treatment sample L* a* b* haze BB701 95.89 0.54 7.81 25.2

(75) As may be seen from the results in Table 17, heat treatment was found to improve the lightness and reduce the haze level of the solution while making it less red and less yellow. Although the level of haze in the solution was reduced, the protein solution was still hazy rather than transparent.

Example 11

(76) This Example illustrates the production of yellow pea protein isolate at pilot scale.

(77) 20 kg of yellow split pea flour was combined with 200 L of 0.15 M CaCl.sub.2 solution at ambient temperature and agitated for 30 minutes to provide an aqueous protein solution. The residual solids were removed by centrifugation to produce a centrate having a protein content of 1.53% by weight. 180.4 L of centrate was added to 231.1 L of RO water and the pH of the sample lowered to about 3 with diluted HCl. The diluted and acidified centrate was further clarified by filtration to provide a clear protein solution with a protein content of 0.57% by weight and having a pH of 2.93.

(78) The filtered protein solution was reduced in volume from 431 L to 28 L by concentration on a PES membrane, having a molecular weight cutoff of 100,000 Daltons, operated at a temperature of about 30 C. At this point the acidified protein solution, with a protein content of 6.35% by weight, was diafiltered with 252 L of RO water, with the diafiltration operation conducted at about 30 C. The resulting diafiltered solution was then further concentrated to provide 21 kg of acidified, diafiltered, concentrated protein solution with a protein content of 7.62% by weight, which represented a yield of 58.0 wt % of the initial centrate that was further processed. The acidified, diafiltered, concentrated protein solution was dried to yield a product found to have a protein content of 103.27 wt % (N6.25) d.b. The product was termed YP01-D11-11A YP701 protein isolate.

Example 12

(79) This Example contains an evaluation of the protein and phytic acid content as well as the trypsin inhibitor activity of the yellow pea protein isolate produced by the method of Example 11 and a commercial yellow pea protein product called Propulse (Nutri-pea, Portage la Prairie, MB).

(80) Protein content was determined by a combustion method using a LecoTruSpec N Nitrogen Determinator. Phytic acid content was determined using the method of Latta and Eskin (J. Agric. Food Chem., 28: 1313-1315). Trypsin inhibitor activity (TIA) was determined using AOCS method Ba 12-75 for the commercial protein sample and a modified version of this method for the YP701 product, which has a lower pH when rehydrated.

(81) The results obtained are set forth in the following Table 18:

(82) TABLE-US-00018 TABLE 18 Protein content, phytic acid content and trypsin inhibitor activity of protein products % protein % phytic TIA (TIU/mg (N x 6.25) acid protein Batch Product d.b. d.b. (N x 6.25)) YP01-D11-11A YP701 103.27 0.27 4.6 Propulse 82.33 2.72 3.3

(83) As may be seen from the results presented in Table 18, the YP701 was very high in protein and low in phytic acid compared to the commercial product. The trypsin inhibitor activity in both products was very low.

Example 13

(84) This Example contains an evaluation of the dry color and color in solution of the yellow pea protein isolate produced by the method of Example 11 and a commercial yellow pea protein product called Propulse (Nutri-pea, Portage la Prairie, MB).

(85) The color of the dry powders was assessed using a HunterLab ColorQuest XE instrument in reflectance mode. The color values are set forth in the following Table 19:

(86) TABLE-US-00019 TABLE 19 HunterLab scores for dry protein products Sample L* a* b* YP01-D11-11A YP701 86.27 2.21 9.73 Propulse 82.39 3.29 20.94

(87) As may be seen from Table 19, the YP01-D11-11A YP701 powder was lighter, less red and less yellow in color compared to the commercial yellow pea protein product.

(88) Solutions of the yellow pea protein products were prepared by dissolving sufficient protein powder to supply 0.48 g of protein in 15 ml of RO water. The pH of the solutions was measured with a pH meter and the color and clarity assessed using a HunterLab Color Quest XE instrument operated in transmission mode. Hydrochloric acid solution was added to the Propulse sample to lower the pH to 3 and then the measurement repeated. The results are shown in the following Table 20.

(89) TABLE-US-00020 TABLE 20 pH and HunterLab scores for solutions of yellow pea protein products sample pH L* a* b* haze YP01-D11-11A YP701 3.45 93.97 0.54 12.70 5.0 Propulse 6.15 35.33 12.61 48.79 96.6 Propulse (pH adjusted) 3.00 37.83 11.55 47.87 96.9

(90) As may be seen from the results in Table 20, the YP01-D11-11A YP701 solution was transparent while the Propulse solution was very cloudy regardless of pH. The YP01-D11-11A YP701 solution was also much lighter, less red and less yellow than the Propulse solution regardless of its pH.

Example 14

(91) This Example contains an evaluation of the heat stability in water of the yellow pea protein isolate produced by the method of Example 11 and a commercial yellow pea protein product called Propulse (Nutri-pea, Portage la Prairie, MB).

(92) Solutions of the yellow pea protein products were prepared by dissolving sufficient protein powder to supply 1.6 g of protein in 80 ml of RO water. The natural pH of the solutions was determined with a pH meter. The samples were each split into two portions and the pH of one portion was lowered to 3.00 with HCl solution. The clarity of the control and pH adjusted solutions was assessed by haze measurement with the HunterLab Color Quest XE instrument operated in transmission mode. The solutions were then heated to 95 C., held at this temperature for 30 seconds and then immediately cooled to room temperature in an ice bath. The clarity of the heat treated solutions was then measured again.

(93) The clarity of the protein solutions before and after heating is set forth in the following Table 21:

(94) TABLE-US-00021 TABLE 21 Effect of heat treatment on clarity of 2% w/v protein solutions of yellow pea protein products haze before heat haze after heat sample pH treatment (%) treatment (%) YP01-D11-11A YP701 3.70 3.6 1.4 YP01-D11-11A YP701 3.00 2.8 1.3 (pH adjusted) Propulse 6.24 96.1 96.4 Propulse (pH adjusted) 3.00 96.6 96.6

(95) As can be seen from the results in Table 21, the solutions of YP01-D11-11A YP701 were transparent before and after heating at both pH levels. The solutions of Propulse were highly cloudy before and after heating at both pH levels.

Example 15

(96) This Example contains an evaluation of the solubility in water of the yellow pea protein isolate produced by the method of Example 11 and a commercial yellow pea protein product called Propulse (Nutri-pea, Portage la Prairie, MB). 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).

(97) 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 using a Leco TruSpec N 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 clear supernatant. The protein content of the supernatant was measured by Leco 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 ((100moisture 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)

(98) The natural pH values of the protein isolate produced in Example 11 and the commercial yellow pea protein product in water (1% protein) are shown in Table 22:

(99) TABLE-US-00022 TABLE 22 Natural pH of YP01-D11-11A YP701 and Propulse solutions prepared in water at 1% protein Batch Product Natural pH YP01-D11-11A YP701 3.56 Propulse 6.15

(100) The solubility results obtained are set forth in the following Tables 23 and 24:

(101) TABLE-US-00023 TABLE 23 Solubility of products at different pH values based on protein method Solubility (protein method) (%) Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH YP01- YP701 98.2 99.1 99.5 50.9 20.4 39.3 100 D11- Propulse 14.9 3.6 2.6 5.3 10.3 7.0 8.0 11A

(102) TABLE-US-00024 TABLE 24 Solubility of products at different pH values based on pellet method Solubility (pellet method) (%) Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH YP01- YP701 99.6 99.3 99.1 74.7 34.7 39.1 99.0 D11- Propulse 15.5 14.7 11.6 12.1 16.4 18.0 16.5 11A

(103) As can be seen from the results presented in Table 23 and 24, the YP01-D11-11A YP701 was highly soluble in the pH range of 2 to 4 and less soluble at higher pH values. The Propulse was very poorly soluble at all pH values tested.

Example 16

(104) This Example contains an evaluation of the clarity in water of the yellow pea protein isolate produced by the method of Example 11 and a commercial yellow pea protein product called Propulse (Nutri-pea, Portage la Prairie, MB).

(105) The clarity of the 1% w/v protein solutions prepared as described in Example 15 was assessed by measuring the absorbance at 600 nm, with a lower absorbance score indicating greater clarity. Analysis of the samples on a HunterLab ColorQuest XE instrument in transmission mode also provided a percentage haze reading, another measure of clarity.

(106) The clarity results are set forth in the following Tables 25 and 26:

(107) TABLE-US-00025 TABLE 25 Clarity of protein solutions at different pH values as assessed by A600 A600 Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH YP01- YP701 0.012 0.015 0.024 1.962 2.829 2.557 0.021 D11- Propulse 2.576 2.579 2.693 2.685 2.588 2.560 2.590 11A

(108) TABLE-US-00026 TABLE 26 Clarity of protein solutions at different pH values as assessed by HunterLab haze analysis HunterLab haze reading (%) Batch Product pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 Nat. pH YP01- YP701 0.0 0.1 1.1 95.9 96.7 96.4 0.7 D11- Propulse 96.2 96.3 96.7 96.7 96.2 96.4 96.4 11A

(109) As can be seen from the results of Tables 25 and 26, the solutions of YP01-D11-11A YP701 were transparent in the range of pH 2 to 4 but very cloudy at higher pH values. The solutions of Propulse were very cloudy regardless of pH.

Example 17

(110) This Example contains an evaluation of the solubility in a soft drink (Sprite) and sports drink (Orange Gatorade) of the yellow pea protein isolate produced by the method of Example 11 and a commercial yellow pea protein product called Propulse (Nutri-pea, Portage la Prairie, MB). 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.

(111) 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 analyzed using a Leco TruSpec N 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.

(112) When the solubility was assessed with pH correction, the pH of the soft drink (Sprite) (3.42) and sports drink (Orange Gatorade) (3.11) 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 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 analyzed using a Leco TruSpec N 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

(113) The results obtained are set forth in the following Table 27:

(114) TABLE-US-00027 TABLE 27 Solubility of yellow pea protein products in Sprite and Orange Gatorade no pH correction pH correction Solubility (%) Solubility (%) in Solubility (%) Solubility (%) in Batch Product in Sprite Orange Gatorade in Sprite Orange Gatorade YP01-D11-11A YP701 98.1 100 96.6 100 Propulse 3.2 4.6 5.6 7.4

(115) As can be seen from the results of Table 27, the YP01-D11-11A YP701 was highly soluble in the Sprite and the Orange Gatorade. As the YP701 is an acidified product, its addition did not significantly alter the pH of the beverages. The Propulse was very poorly soluble in the beverages tested. Addition of Propulse increased the pH of the drinks but the solubility of the protein was not improved by lowering the pH of the drink back to its original no-protein value.

Example 18

(116) This Example contains an evaluation of the clarity in a soft drink and sports drink of the yellow pea protein isolate produced by the method of Example 11 and a commercial yellow pea protein product called Propulse (Nutri-pea, Portage la Prairie, MB).

(117) The clarity of the 2% w/v protein dispersions prepared in soft drink (Sprite) and sports drink (Orange Gatorade) in Example 17 were assessed using the A600 and HunterLab haze methods described in Example 16.

(118) The results obtained are set forth in the following Tables 28 and 29:

(119) TABLE-US-00028 TABLE 28 A600 readings for yellow pea protein products in Sprite and Orange Gatorade no pH correction pH correction A600 in A600 in A600 in Orange A600 in Orange Batch Product Sprite Gatorade Sprite Gatorade no protein 0.007 0.450 0.007 0.450 YP01-D11- YP701 0.048 0.338 0.043 0.345 11A Propulse 2.800 2.834 2.827 2.793

(120) TABLE-US-00029 TABLE 29 HunterLab haze readings for yellow pea protein products in Sprite and Orange Gatorade no pH correction pH correction Haze (%) Haze (%) Haze (%) Haze (%) in in Orange in in Orange Batch Product Sprite Gatorade Sprite Gatorade no protein 0.0 78.6 0.0 78.6 YP01-D11- YP701 5.7 56.7 4.9 57.7 11A Propulse 97.1 97.5 96.3 96.3

(121) As can be seen from the results of Tables 28 and 29, the addition of YP01-D11-11A YP701 to the soft drink and sports drink added little or no haziness, while the addition of the Propulse made the drinks very cloudy, even when the pH was corrected.

Example 19

(122) This Example illustrates the production of yellow pea protein isolate at pilot scale.

(123) 20 kg of yellow split pea flour was combined with 200 L of 0.15 M CaCl.sub.2 solution at 60 C. and agitated for 30 minutes to provide an aqueous protein solution. The residual solids were removed by centrifugation to produce a centrate having a protein content of 1.32% by weight 186.5 L of centrate was added to 225.8 L of RO water at 60 C. and the pH of the sample lowered to 3.34 with diluted HCl. The diluted and acidified centrate was further clarified by filtration to provide a clear protein solution with a protein content of 0.58% by weight.

(124) The filtered protein solution was reduced in volume from 400 L to 35 L by concentration on a polyethersulfone membrane, having a molecular weight cutoff of 100,000 Daltons, operated at a temperature of about 58 C. At this point the acidified protein solution, with a protein content of 4.94 wt %, was diafiltered with 350 L of RO water, with the diafiltration operation conducted at about 60 C. The resulting diafiltered solution was then further concentrated to provide 21.52 kg of acidified, diafiltered, concentrated protein solution with a protein content of 7.54% by weight, which represented a yield of 65.9 wt % of the initial centrate that was further processed. The acidified, diafiltered, concentrated protein solution was dried to yield a product found to have a protein content of 103.19 wt % (N6.25) d.b. The product was termed YP01-E19-11A YP701 protein isolate.

Example 20

(125) This Example illustrates a comparison of the flavor of the YP701, prepared as described in Example 19, with that of a commercial yellow pea protein product called Nutralys S85F (Roquette America, Inc. Keokuk, Iowa), with the evaluation done at low pH.

(126) 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 YP701 was determined to be 3.78. Food grade HCl was added to the solution of Nutralys S85F to lower the pH from 7.25 to 3.78. An informal panel of seven panelists was asked to blindly compare the samples and indicate which sample had a cleaner flavour, and of which sample they preferred the flavour.

(127) Seven out of seven panelists indicated that the YP701 had a cleaner flavour. Seven out of seven panelists preferred the flavour of the YP701.

Example 21

(128) This Example illustrates a comparison of the flavour of the YP701, prepared as described in Example 19, with that of the commercial yellow pea protein product Nutralys S85F, with the evaluation done at near neutral pH.

(129) 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 Nutralys S85F was determined to be 7.32. Food grade NaOH was added to the solution of YP701 to raise the pH from 3.67 to 732. An informal panel of eight panelists was asked to blindly compare the samples and indicate which sample had a cleaner flavour, and of which sample they preferred the flavour.

(130) Six out of eight panelists indicated that the YP701 had a cleaner flavour. Six out of eight panelists preferred the flavour of the YP701.

Example 22

(131) This Example illustrates a comparison of the flavour of the YP701, prepared as described in Example 19, with that a commercial yellow pea protein product called Propulse (Nutri-Pea, Portage la Prairie, MB), with the evaluation done at low pH.

(132) 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 YP701 was determined to be 3.78. Food grade HCl was added to the solution of Propulse to lower the pH from 6.17 to 3.78. An informal panel of seven panelists was asked to blindly compare the samples and indicate which sample had a cleaner flavour, and of which sample they preferred the flavour.

(133) Six out of seven panelists indicated that the YP701 had a cleaner flavour. Seven out of seven panelists preferred the flavour of the YP701.

Example 23

(134) This Example illustrates a comparison of the flavour of the YP701, prepared as described in Example 19, with that of the commercial yellow pea protein product called Propulse, with the evaluation done at near neutral pH.

(135) 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 Propulse was determined to be 6.18. Food grade NaOH was added to the solution of YP701 to raise the pH from 3.78 to 6.18. An informal panel of eight panelists was asked to blindly compare the samples and indicate which sample had a cleaner flavour, and of which sample they preferred the flavour.

(136) Seven out of eight panelists indicated that the YP701 had a cleaner flavour. Six out of eight panelists preferred the flavour of the YP701.

(137) In the Examples which follow, certain data pertaining to the YP01-D11-11A YP701 pea protein isolate and the commercial yellow pea protein product Propulse, already presented in Examples 11 to 16, is presented a second time for convenience of comparison with other pea protein isolates and commercial yellow pea protein products.

Example 24

(138) This Example illustrates the production of yellow pea protein isolates at pilot scale.

(139) a kg of b was combined with c L of d at e and agitated for f minutes. g kg of calcium chloride pellets (95.5%) dissolved in h L of RO water was then added and the mixture stirred for an additional i minutes. The residual solids were removed by centrifugation to produce a centrate having a protein content of j % by weight. k L of centrate was added to 1L of RO water at m and the pH of the sample lowered to n with diluted HCl. The diluted and acidified centrate was further clarified by filtration to provide a clear protein solution with a protein content of o % by weight.

(140) The filtered protein solution was reduced in volume from p L to q L by concentration on a polyethersulfone membrane, having a molecular weight cutoff of r Daltons, operated at a temperature of about s C. At this point the protein solution, with a protein content of t wt %, was diafilterd with u L of RO water, with the diafiltration operation conducted at about v C. The diafiltered protein solution was then concentrated to w, and then x L of the sample diafiltered with an additional y L of RO water, with the diafiltration operation conducted at approximately z C. The concentrated protein solution, having a protein content of aa wt % was further concentrated to a protein content of ab wt %, then diluted with RO water to a protein content of ac wt % to facilitate spray drying. The protein solution before spray drying, having a weight of ad kg was recovered in a yield of ac % of the initial centrate that was further processed. The concentrated and diafiltered protein solution was then dried to yield a product found to have a protein content of af wt % (N6.25) d.b. The product was given designation ag. The parameters a to ag are set forth in the following Table 30.

(141) TABLE-US-00030 TABLE 30 Parameters for the runs to produce YP 701 YP01-D11- YP01-E19- YP03-J05- YP05-A18- YP06-B06- YP06-B07- ag 11A YP701 11A YP701 11A YP701 12A YP701 12A YP701-01 12A YP701 a 20 20 30 70 70 70 b Yellow split Yellow split Yellow pea Yellow split Yellow split Yellow split pea flour pea flour protein pea flour pea flour pea flour concentrate c 200 200 300 300 300 300 d 0.15M CaCl.sub.2 0.15M CaCl.sub.2 0.15M CaCl.sub.2 RO water RO water RO water e Ambient 60 C. 60 C. 30 C. 30 C. 30 C. temperature f 30 30 30 60 60 60 g 0 0 0 4.52 4.53 4.53 h 0 0 0 10 10 10 i 0 0 0 30 15 15 j 1.53 1.32 3.50 2.92 3.37 2.86 k 180.4 386.5 254.9 223.3 210 220 l 231.1 225.8 346.2 223.0 137 143 m Ambient 60 C. 60 C. Ambient Ambient Ambient temperature temperature temperature temperature n 2.93 3.34 3.26 3.04 Approx. 3 3.03 o 0.63 0.58 1.62 1.25 1.45 1.37 p 431 400 548 550 385 405 q 28 35 51 101 77 72 r 100,000 100,000 10,000 10,000 10,000 10,000 s 30 58 56 53 48 51 t 6.35 4.94 10.03 4.05 4.82 5.29 u 252 350 510 202 154 144 v 30 60 58 53 57 58 w 21 kg 21.52 kg.sup. n/a 34.78 kg.sup. 30.75 L.sup. 36 L x n/a n/a n/a n/a 20.75 36 y n/a n/a n/a n/a 103.75 180 z n/a n/a n/a n/a 58 58 aa 7.62 7.54 9.85 10.02 8.82 9.97 ab n/a n/a n/a n/a 11.75 12.20 ac n/a n/a n/a 5.00 6.59 6.45 ad 21 21.52 52.98 57.9 33.8 54.66 ae 58.0 65.9 58.5 44.5 31.5 56.1 af 103.27 103.19 102.62 101.99 104.64 102.73

Example 25

(142) This Example illustrates the protein content of the commercial yellow pea protein products Propulse (Nutri-Pea, Portage la Prairie, MB), Nutralys S85F (Roquette America, Inc. Keokuk, Iowa) and Pisane C9 (Cosucra Groupe Warcoing S.A., Belgium). These protein products are among the most highly purified pea protein ingredients currently commercially available.

(143) The protein content of the commercial samples was determined and the values are shown in Table 31.

(144) TABLE-US-00031 TABLE 31 Protein content of commercial yellow pea products Product % protein ((N 6.25) d.b.) Propulse 82.33 Nutralys S85F 83.10 Pisane C9 86.87

(145) As may be seen from the values presented in Table 31, the protein content of the commercial yellow pea protein products was notably lower than the protein content of the yellow pea protein isolates prepared as described in Example 24.

Example 26

(146) This Example illustrates the molecular weight profile of the yellow pea protein isolates prepared as described in Example 24 as well as the commercial yellow pea protein products.

(147) Protein samples were analyzed by size exclusion chromatography using a Varian ProStar HPLC system equipped with a 3007.8 mm Phenomenex S-2000 series column. The column contained hydrophilic bonded silica rigid support media, 5 micron diameter, with 145 Angstrom pore size. 0.05M NaCl, pH 3.5 containing 0.02% sodium azide was used as the mobile phase and also to dissolve dry samples. The mobile phase flow rate was 1 mL/minute and components were detected based on absorbance at 280 nm. Protein samples were mixed with mobile phase solution to a concentration of 1% w/v for pea protein products, placed on a shaker for at least 1 hour then filtered using 0.45 m pore size filter discs. Sample injection size was 50 L. The HPLC ProStar system automatically calculated retention times and peak areas and printed out a summary report.

(148) Before the pea protein samples were analyzed, a standard curve was prepared using a Biorad protein standard (Biorad product #151-1901) containing proteins with known molecular weights between 17,000 Daltons (myoglobulin) and 670,000 Daltons (thyroglobulin) with Vitamin B12 added as a low molecular weight marker at 1,350 Daltons. A 0.9% w/v solution of the protein standard was prepared in the mobile phase and analyzed as described above. Based on the retention times of these molecules of known molecular weight, a regression formula was developed relating the log (MW) to the retention time in minutes.
Retention time(min)=2.353log(Molecular weight)+18.853(r.sup.2=0.99)

(149) This formula was used to calculate retention times that corresponded to molecular weights of 100,000 Da, 15,000 Da, 5,000 Da and 1,000 Da. When the pea protein samples were analyzed, the peak areas lying within these retention times were used to calculate the percentage of protein ((range peak area/total protein peak area)100) falling in a given molecular weight range. Note that the data was not corrected by protein response factor.

(150) The molecular weight profiles of the products prepared as described in Example 24 and the commercial products are shown in Table 32.

(151) TABLE-US-00032 TABLE 32 Molecular weight profile of pea protein products % % % % 15,000- 5,000- 1,000- >100,000 100,000 15,000 5,000 product Da Da Da Da YP01-D11-11A YP701 77 16 4 3 YP01-E19-11A YP701 89 10 1 0 YP03-J05-11A YP701 85 11 2 2 YP05-A18-12A YP701 80 13 3 3 YP06-B06-12A YP701-01 81 14 3 2 YP06-B07-12A YP701 84 13 2 1 Propulse 11 25 13 51 Nutralys S85F 4 25 7 64 Pisane C9 9 43 14 35

(152) As may be seen from the results presented in Table 32, the molecular weight profile of the yellow pea protein isolates prepared as described in Example 24 was different from the molecular weight profile of the commercial yellow pea protein products.

Example 27

(153) This Example contains an evaluation of the phytic acid content of the yellow pea protein isolates produced as described in Example 24 as well as the commercial yellow pea protein products. Phytic acid content was determined using the method of Latta and Eskin (J. Agric. Food Chem., 28: 1313-1315).

(154) The results obtained are set forth in the following Table 33.

(155) TABLE-US-00033 TABLE 33 Phytic acid content of protein products Product % phytic acid d.b. YP01-D11-11A YP701 0.27 YP01-E19-11A YP701 0.23 YP03-J05-11A YP701 0.15 YP05-A18-12A YP701 0.22 YP06-B06-12A YP701-01 0.04 YP06-B07-12A YP701 0.02 Propulse 2.72 Nutralys S85F 2.24 Pisane C9 1.94

(156) As may be seen from the results presented in Table 32, all of the pea protein isolates produced by the method of Example 24 were very low in phytic acid, having phytic acid contents much lower than the commercial yellow pea protein products.

Example 28

(157) This Example illustrates the protein solubility at pH 2 to 4 of the yellow pea protein isolates prepared as described in Example 24 as well as the commercial yellow pea protein products. Solubility was tested by a modified version of the procedure of Morr et al., J. Food Sci., 50: 1715-1718.

(158) 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 or 4) with diluted NaOH or HCl. 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 using a Leco TruSpec N Nitrogen Determinator. Aliquots of the dispersions were then centrifuged at 7,800 g for 10 minutes, which sedimented insoluble material and yielded a clear supernatant. The protein content of the supernatant was measured by Leco analysis and the solubility of the product calculated as follows:
Solubility (%)=(% protein in supernatant/% protein in initial dispersion)100

(159) The protein solubility results obtained are set forth in the following Table 34

(160) TABLE-US-00034 TABLE 34 Solubility of products at different pH values Solubility (%) Product pH 2 pH 3 pH 4 YP01-D11-11A YP701 98.2 99.1 99.5 YP01-E19-11A YP701 94.8 90.3 100 YP03-J05-11A YP701 100 98.2 93.3 YP05-A18-12A YP701 100 100 100 YP06-B06-12A YP701-01 100 100 100 YP06-B07-12A YP701 98.9 100 100 Propulse 14.9 3.6 2.6 Nutralys S85F 38.3 19.7 15.0 Pisane C9 20.8 14.0 12.9

(161) As may be seen from the results presented in Table 33, all of the pea protein isolates prepared as described in Example 24 were highly soluble in the pH range 2-4. The solubility of all of the commercial yellow pea protein products was low in the pH range 2-4.

Example 29

(162) This Example contains an evaluation of the clarity in water of the yellow pea protein isolates prepared as described in Example 24 as well as the commercial yea pea protein products.

(163) The clarity of the 1% w/v protein solutions prepared as described in Example 28 was assessed by measuring the absorbance at 600 nm, with a lower absorbance score indicating greater clarity. Analysis of the samples on a HunterLab ColorQuest XE instrument in transmission mode also provided a percentage haze reading, another measure of clarity.

(164) The clarity results are set forth in the following Tables 35 and 36.

(165) TABLE-US-00035 TABLE 35 Clarity of protein solutions at different pH values as assessed by A600 A600 Product pH 2 pH 3 pH 4 YP01-D11-11A YP701 0.012 0.015 0.024 YP01-E19-11A YP701 0.027 0.022 0.033 YP03-J05-11A YP701 0.026 0.027 0.034 YP05-A18-12A YP701 0.010 0.009 0.020 YP06-B06-12A YP701-01 0.011 0.012 0.020 YP06-B07-12A YP701 0.013 0.015 0.022 Propulse 2.576 2.579 2.693 Nutralys S85F 1.430 2.045 2.398 Pisane C9 2.031 2.368 2.516

(166) TABLE-US-00036 TABLE 36 Clarity of protein solutions at different pH values as assessed by HunterLab haze analysis HunterLab haze reading (%) Product pH 2 pH 3 pH 4 YP01-D11-11A YP701 0.0 0.1 1.1 YP01-E19-11A YP701 2.8 0.9 4.3 YP03-J05-11A YP701 0.3 0.5 1.6 YP05-A18-12A YP701 0.0 0.0 0.0 YP06-B06-12A YP701-01 0.0 0.0 0.0 YP06-B07-12A YP701 0.0 0.0 0.0 Propulse 96.2 96.3 96.7 Nutralys S85F 96.3 96.8 96.9 Pisane C9 97.5 97.6 97.8

(167) As may be seen from the results presented in Tables 35 and 36, all of the solutions prepared from the yellow pea protein isolates prepared as described in Example 24 were very clear. The solutions prepared from the commercial yellow pea protein products were cloudy.

Example 30

(168) This Example contains an evaluation of the heat stability in water of the yellow pea protein isolates prepared as described in Example 24 as well as the commercial yellow pea protein products.

(169) 2% w/v protein solutions were prepared in RO water. The natural pH of the solutions was determined with a pH meter. The solutions of the commercial yellow pea protein products were split into two portions and the pH of one portion was lowered to 3.00 with HCl solution. The clarity of the solutions was assessed by haze measurement with the HunterLab ColorQuest XE instrument operated in transmission mode. The solutions were then heated to 95 C., held at this temperature for 30 seconds and then immediately cooled to room temperature in an ice bath. The clarity of the heat treated solutions was then measured again.

(170) The clarity of the protein solutions before and after heating is set forth in the following Table 37.

(171) TABLE-US-00037 TABLE 37 Effect of heat treatment on clarity of 2% w/v protein solutions Haze before heat Haze after heat Product pH treatment (%) treatment (%) YP01-D11-11A YP701 3.89 2.6 0.9 YP01-E19-11A YP701 3.99 7.6 7.1 YP03-J05-11A YP701 3.73 5.4 3.0 YP05-A18-12A YP701 3.38 0.0 0.0 YP06-B06-12A YP701-01 3.59 0.0 0.0 YP06-B07-12A YP701 3.56 0.0 0.0 Propulse 6.24 96.1 96.4 Propulse (pH adjusted) 3.00 96.6 96.6 Nutralys S85F 7.44 97.2 97.1 Nutralys S85F (pH adjusted) 3.00 97.2 97.1 Pisane C9 7.76 97.6 97.5 Pisane C9 (pH adjusted) 3.00 97.4 97.6

(172) As may be seen from the results presented in Table 37, the yellow pea protein isolates prepared as described in Example 24 produced solutions which were very low in haze before and after heat treatment. The solutions of commercial yellow pea protein product were highly cloudy before and after heating at natural pH and pH 3.

Example 31

(173) This Example contains the solution color values determined for the yellow pea protein isolates prepared as described in Example 24 as well as the commercial yellow pea protein products.

(174) Protein solutions were prepared by dissolving sufficient protein product to supply 0.48 g of protein in 15 ml of RO water. The pH of the solutions was measured with a pH meter and the color and clarity assessed using a HunterLab ColorQuest XE instrument operated in transmission mode. Hydrochloric acid solution was added to the samples of commercial yellow pea protein product to lower the pH to 3 and then the measurement repeated. The results obtained are set forth in the following Table 37.

(175) TABLE-US-00038 TABLE 38 Color values for solutions of pulse product Product pH L* a* b* Haze (%) YP01-D11-11A YP701 3.45 93.97 0.54 12.70 5.0 YP01-E19-11A YP701 3.79 95.44 0.09 8.62 14.2 YP03-J05-11A YP701 3.62 93.64 0.52 10.97 6.0 YP05-A18-12A YP701 3.44 96.51 0.35 9.29 0.0 YP06-B06-12A YP701-01 3.57 96.77 0.39 8.74 1.5 YP06-B07-12A YP701 3.43 96.42 0.35 9.32 2.1 Propulse 6.15 35.33 12.61 48.79 96.6 Propulse (pH adjusted) 3.00 37.83 11.55 47.87 96.9 Nutralys S85F 7.32 53.48 6.20 34.01 97.5 Nutralys S85F (pH adjusted) 3.00 53.70 7.00 32.66 97.4 Pisane C9 7.68 45.04 8.57 47.57 98.8 Pisane C9 (pH adjusted) 3.00 46.62 8.30 45.88 98.3

(176) As may be seen from the results presented in Table 38, the yellow pea protein isolates prepared as described in Example 24 produced solutions that were low in haze and lighter, less red and less yellow than the solutions of commercial pea protein product.

SUMMARY OF THE DISCLOSURE

(177) In summary of this disclosure, the present invention provides novel pulse protein products which are completely soluble and form heat stable, preferably transparent, solutions at acid pH and are useful in the protein fortification of aqueous systems, including soft drinks and sport drinks, without leading to protein precipitation. Modifications are possible within the scope of this invention.