PLANT PROTEIN AND ITS METHOD OF PREPARATION

Abstract

The invention relates to a plant protein isolate containing less than 10 microgram, preferably less than 5 microgram, of the sum of hexanal, 2-pentyl-furan, (E)-2,4,heptadienal and 1-octen-3-ol per gram of dry matter and its method of preparation. The plant protein is preferably obtained from leguminous plant, more preferably from pea or fava bean, most preferably from pea. The method for extracting the plant protein isolate consists of the steps: (a) providing a protein containing seed, (b) milling said seed, (c) suspending the milled seed in water, (d) extracting proteins from said milled suspension and (e) washing the extracted proteins with water at a temperature between 60° C. and 100° C. and at a pH in the range of 4 to 5.5.

Claims

1. A plant protein isolate containing less than 10 μg, preferably less than 5 μg of the sum of hexanal, 2-pentyl-furan, (E)-2,4, heptadienal and 1-octen-3-ol per gram of dry matter.

2. The plant protein isolate of claim 1, wherein the protein isolate is obtained from a leguminous plant, preferably from pea or fava bean, more preferably from pea.

3. The plant protein isolate of claim 1, containing less than 5 mg of total saponins per gram of dry matter.

4. A method for extracting a plant protein isolate according to claim 1, comprising the steps of: (a) providing a protein containing seed, preferably leguminous seed, more preferably pea seed; (b) milling said seed; (c) suspending the milled seed in water; (d) extracting proteins from said milled suspension; (e) washing the extracted proteins with water at temperature between 60° C. and 100° C., preferably between 75° c. and 95° c., and pH in the range of 4 and 5.5, preferably between 4.5 and 5; (f) optionally passing the washed proteins obtained at the end of step (e) through a shearing pump or a homogenizer to improve protein functionality; and (g) optionally drying the proteins obtained in step (e) or (f).

5. The method of claim 4, wherein the volume of water needed to wash the proteins in step (e) is between 1 and 5 times the quantity of protein suspension, but preferably less than 3 times.

6. The method of claim 4, wherein the milling of step (b) is carried out in the absence of oxygen.

7. The method of claim 4, wherein the milling of step (b) is carried out at residual concentration of dioxygen of less than 300 μg/l, more preferably less than 200 μg/l.

8. The method of claim 4, wherein the pH in step (e) is adjusted using food grade acids, in particular including hydrochloric acid, citric acid or sulfuric acid.

9. The method of claim 4, wherein the washed proteins obtained at the end of step (e) or (f) are dried using a spray-dyer, preferably a multistage spray-dryer.

10. The method of claim 4, wherein the milling of step (b) is a wet milling step.

11. The method of claim 4, wherein the milling of step (b) is a dry milling step.

Description

FIGURES

[0073] FIG. 1: Chemical structure of main volatile compounds linked to the “beany” or “vegetal” taste.

[0074] FIG. 2: Inventive process #1 according to Example 3—high temperature and acid wash.

[0075] FIG. 3: Inventive process #2 according to Example 4—low oxygen grinding and high temperature acid wash.

[0076] FIG. 4: Comparison of protein isolates from prior art and the invention.

EXAMPLES

Example 1: Prior Art Process #1, Involving Solvent Purification

[0077] This example demonstrates the reference protein in organoleptic point of view. It uses solvents that must be avoided from industrial point of view (explosion hazard, clean-label . . . ).

[0078] Cleaned and de-hulled dry yellow peas were grinded at 20° C., and then the pea flour was suspended in hexane-ethanol azeotropic mixture (82:18, v/v) at a ratio of 1:5 (w/v) at 4° C. to extract the lipids. The slurry was stirred at a low speed for 1.0 h and was then vacuum filtered. The filter cake was passed through a 20-mesh sifter. The procedure was repeated five times. The defatted pea flour was immersed in 95% (v/v) ethanol at 20° C. for 1.0 hour with a flour solvent ratio of 1:5 (w/v). After vacuum filtering, the cake was removed residual solvent by vacuum rotary evaporation at 60° C. The defatted pea flour was suspended in distilled water in a ratio of 1:9 (w/v) of flour to water, and the pH was adjusted to 7.0 with 2 mol L−1 NaOH. After stirring for 1.0 h at 20° C., the suspension was centrifuged at 3,000 g for 15 min to recover the supernatant (the protein fraction). The protein extracting solution was heated to 125-130° C. for 30 seconds directly through steam injection to inactivate the endogenous enzyme, and cooled to 50° C. using the plate heat exchanger, and then precipitated by adjusting the pH to 4.5 with 2 mol L−1 HCl and centrifuged at 3,000 g for 15 minutes. The protein curd was immersed in 85% (v/v) ethanol three times at 20° C. for 1.0 h at a ratio of 1:5 (w/v). After vacuum filtering, the cake removed residual solvent through vacuum rotary evaporation at 60° C. Then the alcohol washed protein flour was re-suspended in distilled water at a ratio of 1:9 (w/v) of flour to water, and neutralized the pH to 7.0 with 2 mol L−1 NaOH. The protein solution was freeze-dried to obtain pea protein isolates without any off-flavors. The sample “Prior art process #1-solvent” was obtained.

Example 2: Prior Art Process #2, Involving Soaking, Wet Milling and Isoelectric Precipitation

[0079] The dry yellow peas were blended in distilled water, with a ratio of 1:5 (w/v) of peas to water, at room temperature for 10 hours. The de-hulled and soaked peas were grinded in the presence of oxygen with a ratio of 1:4 (w/v) of wet peas to water. Once separated by a screw extruder, the water extract was centrifuged at 3,000 g for 15 minutes in order to remove starch and internal fiber, allowing a protein solution to be obtained. The protein solution was heated to 125-130° C. for 30 seconds, directly through steam injection in order to inactivate the endogenous enzyme, and then cooled to 50° C. with the plate heat exchanger, and then precipitated by adjusting the pH to 4.5 with 2 mol L.sup.−1 HCl and centrifuged at 3,000 g for 15 minutes. The protein curd was re-suspended in distilled water with a ratio of 1:1 (w/v) of curd to water, in order to obtain a solids content ranging from 10% to 12%, and neutralized to a pH of 7.0 with 2 mol L.sup.−1 NaOH. These steps of the process are followed by high pressure homogenization (20 MPa), heating treatment (120° C., 30 s), flash evaporation, and spray drying (180° C., 80° C.). The sample “Prior art process #2” was obtained.

Example 3: Inventive Process #1, Involving High Temperature Acid Wash of the Extracted Proteins

[0080] The dry yellow peas were de-hulled and blended in distilled water with a ratio of 1:5 (w/v) of peas to water at room temperature. The peas were then grinded at a pH of 8.5-9.0 in the presence of oxygen. Once separated by a screw extruder, the solution was centrifuged at 3,000 g for 15 minutes in order to remove insoluble substances (mostly starch and internal fibers), and a raw protein solution was obtained. The raw protein solution was then adjusted to a pH of 7.0-7.5 with 2 M HCl and heated to 125-130° C. for 30 seconds directly through steam injection in order to inactivate the endogenous enzyme, and then cooled to 50° C. using the plate heat exchanger. The proteins were then precipitated by adjusting the pH to 4.5 with 2 mol L−1 HCl and centrifuged at 3,000 g for 15 min. The protein curd was isolated by centrifugation and immersed with 2 parts weight of 90° C. distilled water, which was adjusted to a pH of 4.5.

[0081] After 30 minutes of contact time under gentle stirring, the protein solution was pumped into a plate filter in order to separate the protein from the water, and the obtained protein curd was suspended in distilled water in order to obtain a solids content ranging from 10% to 12%, and an adjusted pH of 7.0 with 2.0 M NaOH. Then, it was reheated to 125-130° C. for 30 seconds and spray dried (180° C., 80° C.). The sample “inventive process #1-HTAW alone with oxygen” was obtained.

[0082] In order to demonstrate synergy between high temperature and acidic pH wash, Inventive process #1 was also reproduced three times with slight modifications: [0083] Low temperature wash (50° c.) and at acid wash pH (4.5) [0084] High temperature at high pH (7.0) and at acidic wash pH (4.5) [0085] Low temperature wash (50° c.) and at neutral wash pH (7)

[0086] By comparing the results of example 3, this helps explain how an innovative protein isolate is only reachable with a combination of both parameters.

Example 4: Inventive Process #2 Involving High Temperature Acid Wash and Low Oxygen Milling

[0087] The dry yellow peas were de-hulled and blended in distilled water with a ratio of 1:5 (w/v) of peas to water at room temperature. The peas were then grinded in oxygen-free water below 200 μg/I with a ratio of 1:4 (w/v), and then separated with a screw extruder. After standing for 1 hour under a nitrogen atmosphere, the protein solution was centrifuged at 3,000 g for 15 minutes to remove insoluble substances (mostly starch and internal fibers), and then a raw protein solution was obtained. The raw protein solution was adjusted to a pH of 7.0˜7.5 while still under nitrogen atmosphere, and then heated directly through the steam injection to 125˜130° C. for 30 seconds, and finally cooled to 30-40° C. using the plate heat exchanger. The protein was then precipitated by adjusting to a pH of 4.5 with 2 mol L−1 HCl and centrifuged at 3,000 g for 15 minutes.

[0088] The protein curd was isolated by centrifugation and immersed with 2 parts weight of 90° C. distilled water that was adjusted to a pH of 4.5. After 30 minutes of contact time under gentle stirring, the protein solution was pumped into a plate filter in order to separate the protein from the water. After repeating this step two times with 90° C. water at a pH of 4.5, the protein solution was pumped into a plate filter to separate the protein from the water., and the obtained protein curd was suspended in distilled water in order to obtain a solids content ranging from 10% to 12%, and an adjusted pH of 7.0 with 2.0 M NaOH. This was followed by reheating to 125-130° C. for 30 s and spray drying (180° C., 80° C.). The sample “inventive process #2—HTAW combing with low oxygen milling” was obtained.

[0089] In order to show synergy of oxygen-free milling and high temperature and acidic pH wash, Inventive process #2 was also reproduced without a high temperature and acidic pH wash.

Example 5: Organoleptic Testing Process

[0090] Sample preparation: 4% protein powder dissolved in deionized water at room temperature (around 23° c.)

[0091] Panelist: 10 trained people

[0092] Sensory evaluation is based on 3 descriptors: beany, bitter and astringent. The scale for each descriptor is between 1 and 10 with, with 10 being the best score and 1 being the worst. The final sensory score is taken from the average of the total amongst all panelists and in all 3 categories.

Example 6: Comparison of Prior Art and Inventive Protein Isolate Produced in Examples 1 Through 4

[0093] Table 1 below compares all protein isolates produced in examples 1 through 4. Reference commercial protein isolates are also included in the comparison.

TABLE-US-00001 TABLE 1 Sum of 4 volatiles listed in the 4 column Total 1-octen- 2-penty- (E)-2,4- on the volatiles Saponin DDMP Total Sensory Hexanal 3-ol furan Heptadienal Left compounds B saponin saponins score (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (μg/g) (mg/g) (mg/g) (mg/g) Prior art Prior art process 10 3.23 0.01 0.02 0.02 3.28 4.23 3.17 1.43 4.6 processes #1—solvent and Prior art process 2 17.47 0 0.28 0.28 18.03 40.82 6 2.17 8.17 products #2—soaking and wet milling Commercial product 3.4 8.8 1.82 4.11 4.11 18.84 32.05 1.88 0.99 2.87 #1—Propulse S Commercial product 2.4 16.37 0.67 2.66 2.66 22.36 36.89 1.52 1.58 3.1 #2—Purls 860 Commercial product 2.9 12.64 1.5 4.18 4.18 22.5 36.47 1.8 1.15 2.95 #3—Pisane F9 Commercial product 4 16.88 0.51 2.11 2.11 21.61 34.15 4.15 1.78 5.93 #4—Nutralys F85F Commercial product 3 20.91 0.61 2.28 2.28 26.08 39.97 3.64 1.5 5.14 #5—Nutralys S85F Inventive Inventive process 8 1.21 0.05 0.43 0.43 2.12 6.43 2.84 0.66 3.5 process #1—HTAW alone with oxygen Comparison with low 7 5.59 0.06 0.52 0.52 6.69 14.03 3.15 0.75 3.9 temperature wash (50° C.) Comparison with neutral 6.3 5.47 0.25 0.61 0.61 6.94 13.54 1.04 0.4 1.44 pH wash (at 90° C.) Comparison with neutral 5.6 7.57 0 1.39 1.39 10.35 18.63 1.28 0.56 1.84 pH wash (at 50° C.) Inventive process 9 0.8 0.03 0 0 0.83 4.84 3.87 0.45 4.32 #2—HTAW combined with low oxygen milling Oxygen free grinding 5 3.63 0.37 0.23 0.23 4.46 18.01 4.97 2.79 7.76 alone, without HTAW

[0094] The results clearly show that: [0095] The better samples were, as predicted, obtained through the solvent reference process [0096] Only processes involving high temperature acid wash lead to an isolate with a sensory score above 7 and total volatile compounds below 10 μg/g, which means that they had the closest numbers to the solvent reference process. [0097] The combination of high temperature acid wash with oxygen free grinding leads to an even better quality, meaning the total volatile compounds are below 5 μg/g. Using oxygen free grinding alone produces mid-range quality product

[0098] In conclusion, FIG. 4 clearly shows that the process of the invention leads to a level of protein quality that has never been reached before. Its sensory score and volatile contents are closer to the solvent reference than commercial protein. No other solvent used is as interesting from an industrial point of view.

Example 7: Comparison of Solubility in Water Between Prior Art Isolates and Inventive Isolates

[0099] Solubility will be measured using following protocol: [0100] 2.0 g of sample and 100 g of distilled water are placed in a 400 mL beaker at 20° C. [0101] pH is adjusted at 6 or 7, with 1N HCl and/or 1N NaOH and the mixture is made up to exactly 200.0 g with distilled water. [0102] This mixture is stirred for 30 minutes and then centrifuged for 15 minutes at 3000×g. [0103] After centrifugation, exactly 25.0 g of supernatant are withdrawn into a crystallizing dish (m1). The dish is placed in an oven at 103° C. until it reaches a constant mass (m2). [0104] Solubility=((m2−m1)/25)*100, expressed in g of dry matter per 100 g of solution

[0105] In order to be comparable, Hydrolysis Degree of all protein samples are measured using OPA method, which is described below.

[0106] Principle:

[0107] The “amino nitrogen” groups of the free amino acids of the sample react with N-acetyl-L-cysteine and o-phthalyldialdehyde (OPA) to form isoindole derivatives.

[0108] The amount of isoindole derivative formed during this reaction is stoichiometric with the amount of free amino nitrogen. It is the isoindole derivative that is measured by the increase in absorbance at 340 nm.

[0109] Procedure:

[0110] Introduce an accurately weighed test sample P* of the sample to be analyzed into a 100 ml beaker. (This test sample will be from 0.5 to 5.0 g as a function of the amino nitrogen content of the sample.)

[0111] Add about 50 ml of distilled water, homogenize and transfer into a 100 ml measuring cylinder, add 5 ml of 20% SDS and make up to the volume with distilled water; stir for 15 minutes on a magnetic stirrer at 1000 rpm.

[0112] Dissolve 1 tablet of flask 1 of the Megazyme kit in 3 ml of distilled water and stir until fully dissolved. Provide one tablet per test.

[0113] This solution No. 1 is to be prepared extemporaneously.

[0114] The reaction takes place directly in the spectrophotometer cuvettes. [0115] Blank: [0116] Introduce 3.00 ml of solution No. 1 and 50 μl of distilled water. [0117] Standard: [0118] Introduce 3.00 ml of solution No. 1 and 50 μl of flask 3 of the Megazyme kit. [0119] Sample:

[0120] Introduce 3.00 ml of solution No. 1 and 50 μl of the sample preparation.

[0121] Mix the cuvettes and read the absorbance measurements (A1) for the solutions after about 2 minutes on the spectrophotometer at 340 nm (spectrophotometer equipped with cuvettes with a 1.0 cm optical path, which can measure at a wavelength of 340 nm, and verified according to the procedure described in the manufacturer's technical manual related thereto).

[0122] Start the reactions immediately by adding 100 μl of the OPA solution flask 2 of the Megazyme kit to the spectrophotometer cuvettes.

[0123] Mix the cuvettes and place them in darkness for about 20 minutes.

[0124] Next, read the absorbance measurements for the blank, the standard and the samples on the spectrophotometer at 340 nm.

[0125] Calculation Method:

[0126] The content of free amino nitrogen, expressed as a mass percentage of product per se, is given by the following formula:

[00001]$\left[{\mathrm{NH}}_2\%\mathrm{crude}\right]=\frac{\left({\Delta A}_{\mathrm{sample}}={\Delta A}_{\mathrm{blank}}\right)\times 3.15\times 14.01\times V\times 100}{6803\times 0.05\times 1000\times m}=\frac{\left({\Delta A}_{\mathrm{sample}}-{\Delta A}_{\mathrm{blank}}\right)\times 12.974\times V}{m\times 1000}$

[0127] in which: ΔA=A2−A1

[0128] V=volume of the flask

[0129] m=mass of the test sample in g

[0130] 6803=extinction coefficient of the isoindole derivative at 340 nm (in L.Math.mol.sup.−1.Math.cm.sup.−1).

[0131] 14.01=molar mass of nitrogen (in g.Math.mol.sup.−1)

[0132] 3.15=final volume in the cuvette (in ml)

[0133] 0.05=test sample in the cuvette (in ml)

[0134] The degree of hydrolysis (DH) is given by the formula:

[00002]$\mathrm{DH}=\frac{\mathrm{Protein}\mathrm{nitrogen}\left(\%\right)}{\mathrm{Amino}\mathrm{nitrogen}\left(\%\right)\times 100}$

[0135] in which the protein nitrogen is determined according to the DUMAS method according to standard ISO 16634.

[0136] Table below resume all these analysis on Inventive Process isolates and also on Prior Art isolates:

TABLE-US-00002 Solubility Solubility @ pH 6 @ pH 7 (g dry (g dry matter/ matter/ Hydrolysis 100 g of 100 g of Degree solution) solution) Inventive Process #1 53.0 79.2 Inventive Process #2 52.8 74.2 Propulse S 4.6 20.7 36.1 Puris 860 3.7 14.4 16.8 Nutralys S85F 4.6 20.8 57.1 Nutralys F85F 4.5 23.4 57.4

[0137] From lecture of above Table, it is clear that inventive process samples #1 and #2 are the only samples that possess a solubility at pH 6 above 30%, preferably above 40%, more preferably around 50% and a solubility at pH 7 above 40%, preferably above 60%, more preferably above 70%.

[0138] This difference can't be explained by hydrolysis degrees which are in the same range: our inventive process has also an impact on the functional properties of inventive isolates, especially raising its solubility at pH 6 and 7.