阅读说明：本技术 植物蛋白及其制备方法 (Vegetable protein and preparation method thereof ) 是由 周六明 M·伊贝尔 Y·华 C·张 X·孔 Y·陈 X·李 于 2019-09-25 设计创作，主要内容包括：本发明涉及一种植物蛋白分离物及其制备方法,该植物蛋白分离物含有小于10微克、优选地小于5微克的己醛、2-戊基-呋喃、(E)-2,4,庚二烯醛和l-辛烯-3-醇的总和/克干物质。该植物蛋白优选地获得自豆科植物,更优选地获得自豌豆或蚕豆,最优选地获得自豌豆。用于提取该植物蛋白分离物的方法由以下步骤组成：(a)提供含蛋白的种子,(b)研磨所述种子,(c)将研磨的种子悬浮于水中,(d)从所述研磨的悬浮液中提取蛋白,以及(e)在60℃至100℃的温度下并且在4至5.5的范围的pH下,用水洗涤提取的蛋白。(The present invention relates to a plant protein isolate containing less than 10 micrograms, preferably less than 5 micrograms of hexanal, 2-pentyl-furan, (E) -2,4, heptadienal and l-octen-3-ol sum per gram dry matter, and a process for its preparation. The plant protein is preferably obtained from a leguminous plant, more preferably from pea or broad bean, most preferably from pea. The process for extracting the plant protein isolate consists of the following steps: (a) providing a seed comprising protein, (b) grinding the seed, (c) suspending the ground seed in water, (d) extracting protein from the ground suspension, and (e) washing the extracted protein with water at a temperature of 60 ℃ to 100 ℃ and at a pH in the range of 4 to 5.5.)

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

2. The plant protein isolate as claimed in claim 1, wherein the protein isolate is obtained from a leguminous plant, preferably from pea or broad bean, more preferably from pea.

3. A plant protein isolate as claimed in claim 1 or 2, which contains less than 5mg total saponins per gram dry matter.

4. A process for extracting the plant protein isolate of any one of claims 1 to 3, consisting of the steps of:

(a) providing a protein-containing seed, preferably a leguminous seed, more preferably a pea seed;

(b) grinding the seed;

(c) suspending the ground seeds in water;

(d) extracting protein from the milled suspension;

(e) washing the extracted protein with water at a temperature of 60 ℃ to 100 ℃, preferably 75 ℃ to 95 ℃, and at a pH in the range of 4 to 5, preferably 4,5 to 5;

(f) optionally passing the washed protein obtained at the end of step (e) through a shear pump or homogenizer to improve protein functionality;

(g) optionally drying the protein obtained in step (e) or (f).

5. The process of claim 4 wherein the volume of water required to wash the protein in step (e) is 1 to 5 times the amount of protein suspension, but preferably less than 3 times.

6. The process of claim 4 or 5, wherein the milling of step (b) is carried out in the absence of oxygen.

7. The process according to claim 4 or 5, wherein the grinding of step (b) is carried out at a residual concentration of dioxygen of less than 300 μ g/l, more preferably less than 200 μ g/l.

8. The process of any one of claims 4 to 7, wherein the pH in step (e) is adjusted using a food-grade acid, in particular comprising hydrochloric acid, citric acid or sulfuric acid.

9. The process of any one of claims 4 to 8, wherein the washed protein obtained at the end of step (e) or (f) is dried using a spray dryer, preferably a multi-stage spray dryer.

10. A process according to any one of claims 4 to 9 wherein the grinding of step (b) is a wet grinding step.

11. The process of any one of claims 4 to 9, wherein the grinding of step (b) is a dry grinding step.

Technical Field

The present invention relates to plant proteins (including isolates and concentrates), preferably leguminous protein isolates, more preferably pea protein isolates, containing less than 10 μ g total volatile compounds per gram dry matter. Such vegetable proteins (including isolates and concentrates), preferably leguminous protein isolates, more preferably pea protein isolates, have a significantly lower off-flavour taste upon consumption than prior art vegetable protein isolates. The invention also relates to the extraction and purification process of the plant proteins (including isolates and concentrates), preferably leguminous protein isolates, more preferably pea protein isolates, of the invention. Finally, the invention also relates to the use of the plant proteins (including isolates and concentrates), preferably leguminous protein isolates, more preferably pea protein isolates, of the invention in the food, feed and pharmaceutical industries.

Background

Proteins, together with carbohydrates and lipids, constitute an important part of our diet. The protein demand is generally considered to be 12% to 20% of our daily food intake.

The protein consumed is typically of animal origin (e.g. meat, fish, egg and dairy products) or of vegetable origin (including cereals, oleaginous plants and leguminous plants).

In industrialized countries, protein intake is primarily from proteins of animal origin. Notably, many studies have shown that excessive consumption of animal-derived proteins and significantly less consumption of plant proteins is one of the causes of increased incidence of cancer and cardiovascular disease.

In addition, animal proteins have a number of disadvantages, including being allergenic (particularly for proteins from milk and eggs), and environmental degradation due to intensive agriculture necessary for animal protein production.

In view of this, manufacturers are gradually turning to vegetable proteins as substitutes for animal proteins. Indeed, it is known practice to use vegetable proteins instead of all or part of the animal proteins in food products.

Such replacement is not always easy because the functional properties of plant proteins differ from those of animal proteins. In this case, functional properties refer to physical or physicochemical properties that have an effect on the organoleptic quality of the food system produced during technical conversion, storage or home cooking preparation.

Among the vegetable proteins, the use of leguminous vegetable proteins is well known. Although milk proteins have powerful nutritional advantages, the high production costs limit their use in large-scale food processing. Alternatively, leguminous plant proteins may be substituted for milk proteins. Pea proteins in particular are now considered to be breakthrough proteins in the art. Pea protein isolate was obtained from seeds of non-GMO origin, not soy protein isolate.

One disadvantage of certain vegetable proteins, in particular leguminous vegetable proteins and pea proteins, is the fact that they are not odourless. This means that they may cause off-flavours even in products containing them. Consumers often describe off-flavors as "pea flavor", "beany flavor", "green flavor" or "botanical flavor".

A well-known and simple solution is to mask off-flavours during the formulation process by introducing chemical compounds into the solution. This may be made from off-flavour masking agents, flavouring agents and/or off-flavour modulating agents. Unfortunately, this type of solutions are often not fully functional, which means that they do not mask the off-flavours, but merely reduce them. Another disadvantage is that formulators must purchase additional compounds, thereby increasing formulation costs. Regulations, primarily food and drug regulations, may also be barriers to the use of such compounds. Another important factor is that today's consumers want "clean label" products and including these types of compounds on the product label can keep away some potential consumers.

A more preferred solution is to use the protein isolate directly with low off-flavours and some proposals have been made in some solutions for plant protein isolate producers.

For example, WO 2015/071498 explains how to use a wet milling extraction process in combination with lactic acid fermentation to extract purified pea protein isolate. This method is capable of producing pea protein isolates having a moderate variety of "good tastes", but unfortunately it does not produce a tasteless pea protein isolate. Referring to table 10 of the present patent application, each pea protein sample continues to be described as having a "beany" or "pea-like" taste.

In another example, WO 2017/120597 explains how to use salting-out precipitation in combination with high volume protein washing, in particular with high volume of water at neutral pH and average temperature. The process involves large amounts of salt and high volumes of neutral pH tap water (15 to 30 pea volumes). However, the "beany" and "bitter" tastes were still detected in pea protein isolates and were at the same level as in common commercial pea protein isolates (see fig. 18A, 18B and 18C).

Unfortunately, current commercial pea protein isolates still develop off-flavors when consumed, which are often described as "beany" or "vegetable-like" off-flavors. There remains a need for a tasteless leguminous protein isolate, preferably peas, which is free of any off-flavours.

In view of the above, it is an object of the present invention to overcome or reduce at least one of the disadvantages of the prior art and/or to provide a useful alternative.

Disclosure of Invention

A first aspect of the invention is a plant protein (including isolates or concentrates) containing less than 10 μ g of total volatile compounds per gram of dry matter, preferably less than 5 μ g of total volatile compounds per gram of dry matter.

In a preferred embodiment, the total volatile compounds are understood to be the sum of the contents of hexanal, 2-pentyl-furan, (E) -2,4, heptadienal and 1-octen-3-ol. Thus, in this example, the vegetable protein isolate according to the invention contains less than 10 μ g, preferably less than 5 μ g, of the sum of hexanal, 2-pentyl-furan, (E) -2,4, heptadienal and 1-octen-3-ol per gram dry matter. In a more preferred embodiment, total volatile compounds are understood to be the sum of all volatile compounds detected and analyzed by the method of the invention, which is described below.

In a more preferred embodiment, the vegetable protein further contains less than 5mg total saponins per gram dry matter.

In an even more preferred embodiment, the plant protein is obtained from a leguminous plant, preferably from pea or broad bean, wherein pea protein is most preferred.

All protein embodiments of the invention are characterized by a distinctly neutral taste and lack of "beany or" vegetable-like "off-flavors. These examples are further encompassed in the detailed description of the invention, as well as in the non-exhaustive list of examples.

The protein isolate according to the invention is also characterized by an improved solubility in water compared to proteins from the prior art. In particular, the protein isolate according to the invention has a solubility in water at 20 ℃ and pH 6 higher than 30%, preferably higher than 40%, more preferably about 50%, and a solubility at 20 ℃ and pH7 higher than 40%, preferably higher than 60%, more preferably higher than 70%, as determined according to the test described below.

A second aspect of the invention is a process for obtaining a plant protein (including an isolate or concentrate), preferably a leguminous protein isolate, more preferably a pea protein isolate. The method comprises the following steps:

(a) providing a protein-containing plant seed, preferably a leguminous seed, more preferably a pea seed;

(b) grinding the seed;

(c) suspending the ground seeds in water;

(d) extracting protein from the milled suspension, preferably by thermal coagulation at isoelectric pH;

(e) washing the protein with water at a temperature of 60 ℃ to 100 ℃, more preferably 75 ℃ to 95 ℃, and at a pH in the range of 4 to 5, more preferably 4.5-5;

(f) optionally passing the washed protein obtained at the end of step (e) through a shear pump or homogenizer to improve protein functionality;

(g) optionally drying the protein obtained in step (e) or (f).

In a preferred embodiment, the seed grinding in step (b) is carried out directly in water and in the absence of oxygen, preferably at a residual concentration of dioxygen of less than 300. mu.g/liter, preferably less than 200. mu.g/liter. The residual oxygen concentration may be determined according to the further described protocol.

In a more preferred embodiment, the milling is performed in the absence of additional water, and the milled suspension of step (c) is obtained by mixing the dried powder with water. This is preferably carried out at a residual concentration of dioxygen of less than 300. mu.g/l, preferably at a measurement of less than 200. mu.g/l. The residual oxygen concentration may be determined according to the further described protocol.

A third aspect of the invention is that it relates to the use of the plant protein (including isolates or concentrates), preferably leguminous protein isolates, more preferably pea protein isolates of the invention in food applications, feed applications, cosmetic applications and pharmaceutical applications.

The invention will be better understood from the following detailed description.

Drawings

FIG. 1: the chemical structure of the main volatile compounds is associated with a "beany" or "botanical" flavour.

FIG. 2: inventive Process #1 according to example 3-high temperature and acid wash.

FIG. 3: inventive method #2 according to example 4-low oxygen milling and high temperature acid washing.

FIG. 4: comparison of protein isolates from the prior art and the present invention.

Examples of the invention

Example 1: prior art method #1, involving solvent purification

This example shows the reference protein from an organoleptic point of view. It uses solvents that must be avoided from an industrial point of view (explosion hazard, cleaning of the label … …).

Clean and dehulled dry yellow peas were ground at 20 ℃ and then pea powder was suspended in hexane-ethanol azeotrope (82:18, v/v) at 4 ℃ in a ratio of 1:5(w/v) to extract lipids. The slurry was stirred at low speed for 1.0h and then vacuum filtered. The filter cake was passed through a 20 mesh screen. This procedure was repeated five times. Defatted pea flour was immersed in 95% (v/v) ethanol at 20 ℃ for 1.0 hour at a powder solvent ratio of 1:5 (w/v). After vacuum filtration, the residual solvent in the filter cake was removed by rotary evaporation under vacuum at 60 ℃. Defatted pea flour was suspended in distilled water at a 1:9(w/v) powder to water ratio and the pH was adjusted to 7.0 with 2mol L-1 NaOH. After stirring at 20 ℃ for 1.0h, the suspension was centrifuged at 3,000g for 15min to recover the supernatant (protein fraction). The protein extraction solution was heated directly to 125-130 ℃ for 30 seconds by steam injection to inactivate endogenous enzymes and cooled to 50 ℃ using a plate heat exchanger and then precipitated by adjusting the pH to 4.5 with 2mol L-1HCl and centrifuged at 3,000g for 15 minutes. The protein curd was immersed in 85% (v/v) ethanol three times at 20 ℃ for 1.0h at a ratio of 1:5 (w/v). After vacuum filtration, the filter cake was freed of residual solvent by rotary evaporation under vacuum at 60 ℃. The alcohol washed protein powder was then resuspended in distilled water at a 1:9(w/v) powder to water ratio and the pH was neutralized to 7.0 with 2mol L-1 NaOH. The protein solution was freeze dried to obtain pea protein isolate without any off-flavour. Sample "prior art method # 1-solvent" was obtained.

Example 2: prior art method #2, involving soaking, Wet milling and isoelectric precipitation

Dry yellow peas were mixed in distilled water at a pea to water ratio of 1:5(w/v) for 10 hours at room temperature. The de-husked and soaked peas were ground in the presence of oxygen at a wet pea to water ratio of 1:4 (w/v). Once separated with a screw extruder, the aqueous extract was centrifuged at 3,000g for 15 minutes to remove starch and internal fibers, thereby obtaining a protein solution. The protein solution was heated directly to 125-130 ℃ for 30 seconds by steam injection to inactivate endogenous enzymes, and then cooled to 50 ℃ using a plate heat exchanger, and then precipitated by adjusting the pH to 4.5 with 2mol L-1HCl, and centrifuged at 3,000g for 15 minutes. Resuspending the protein curd in distilled water at a ratio of curd to water of 1:1(w/v) to obtain a solids content ranging from 10% to 12%, and using 2mol L^-1NaOH neutralized to pH 7.0. These steps of the process are followed by high pressure homogenization (20MPa), heat treatment (120 ℃, 30s), flash evaporation, and spray drying (180 ℃, 80 ℃). Sample "prior art method # 2" was obtained.

Example 3: inventive method #1, high temperature acid wash of extracted proteins

Dried yellow peas were hulled and mixed in distilled water at a pea to water ratio of 1:5(w/v) at room temperature. The peas are then milled in the presence of oxygen at a pH of 8.5-9.0. Once separated with the screw extruder, the solution was centrifuged at 3,000g for 15 minutes to remove insoluble materials (mainly starch and internal fiber), and a crude protein solution was obtained. The crude protein solution was then adjusted to ph7.0-7.5 with 2M HCl and heated directly to 125-130 ℃ by steam injection for 30 seconds to inactivate endogenous enzymes, and then cooled to 50 ℃ using a plate heat exchanger. The protein was then precipitated by adjusting the pH to 4.5 with 2mol L-1HCl and centrifuged at 3,000g for 15 min. The protein curd was separated by centrifugation and immersed in 2 parts by weight of 90 ℃ distilled water (adjusted to pH 4.5).

After a contact time of 30 minutes with gentle stirring, 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 to obtain a solids content ranging from 10% to 12% and adjusted to a pH of 7.0 with 2.0M NaOH. Then, it was heated again to 125 ℃ -130 ℃ for 30 seconds and spray dried (180 ℃, 80 ℃). Samples "inventive method #1-HTAW with oxygen" were obtained.

To demonstrate the synergy between high temperature and acidic pH washes, inventive method #1 was also repeated three times, with slight modifications:

low temperature washing (50 ℃) and at acid wash pH (4.5)

High temperature at high pH (7.0) and acidic wash pH (4.5)

Low temperature washing (50 ℃) and at neutral washing pH (7)

By comparing the results of example 3, this helps to explain how the innovative protein isolate can be obtained by the combination of the two parameters.

Example 4: inventive method #2, involving high temperature acid wash and low oxygen milling

Dried yellow peas were hulled and mixed in distilled water at a pea to water ratio of 1:5(w/v) at room temperature. Peas were then ground in oxygen free water at a ratio of 1:4(w/v) below 200. mu.g/l and then separated with a screw extruder. After standing for 1 hour under a nitrogen atmosphere, the protein solution was centrifuged at 3,000g for 15 minutes to remove insoluble materials (mainly starch and internal fiber), and then a crude protein solution was obtained. The crude protein solution was adjusted to ph7.0-7.5 while still under nitrogen atmosphere and then heated directly to 125-130 ℃ by steam injection for 30 seconds and finally cooled to 30-40 ℃ with a plate heat exchanger. The protein was then precipitated by adjusting to pH4.5 with 2mol L-1HCl and centrifuged at 3,000g for 15 minutes.

The protein curd was separated by centrifugation and immersed in 2 parts by weight of 90 ℃ distilled water (adjusted to pH 4.5). After a contact time of 30 minutes with gentle stirring, the protein solution was pumped into a plate filter to separate the protein from the water. After repeating this step twice with 90 ℃ water at pH4.5, 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 to obtain a solids content ranging from 10% to 12% and adjusted to a pH of 7.0 with 2.0M NaOH. Then heated to 125-130 ℃ for 30 seconds and spray dried (180 ℃, 80 ℃). Samples "inventive method #2-HTAW in combination with low oxygen milling" were obtained.

To show the synergistic effect of anaerobic milling and high temperature and acidic pH washing, inventive process #2 was also reproduced without high temperature and acidic pH washing.

Example 5: sensory test method

Sample preparation: 4% protein powder was dissolved in deionized water at room temperature (about 23 ℃ C.)

The panelists: 10 trained persons

Sensory evaluation was based on 3 descriptors: beany flavor, bitter and astringent. The scale of each descriptor is between 1 and 10, with 10 being the best score and 1 being the worst. The final sensory score was taken as the average of the total scores in all panelists and all 3 categories.

Example 6: comparison of the protein isolates of the invention produced in the prior art and examples 1 to 4

Table 1 below compares all protein isolates produced in examples 1 to 4. Reference commercial protein isolates are also included in the comparison.

The results clearly show that:

as predicted, better samples were obtained by the solvent reference method.

Only the processes involving high temperature acid washing will yield isolates with a sensory score above 7 and total volatile compounds below 10 μ g/g, which means that they have the closest figures to the solvent reference method.

High temperature acid washing combined with anaerobic grinding results in even better quality being obtained, which means that the total volatile compounds is below 5 μ g/g. Only anaerobic milling produced a product of moderate quality.

In summary, figure 4 clearly shows that the method of the invention results in protein levels never before achieved. Their sensory scores and volatile content are closer to the solvent reference than the commercial protein. No other solvent is so useful from an industrial point of view.

Example 7: comparison of solubility in Water of Prior Art isolates and inventive isolates

The following protocol will be used to measure solubility:

2.0g of the sample and 100g of distilled water were placed in a 400mL beaker at-20 ℃.

The pH is adjusted to 6 or 7 with 1N HCl and/or 1N NaOH and the mixture is made up to exactly 200.0g with distilled water.

This mixture was stirred for 30 minutes and subsequently centrifuged at 3000 Xg for 15 minutes.

After centrifugation, exactly 25.0g of supernatant was drawn into the crystallization dish (m 1). The petri dish was placed in an oven at 103 ℃ until it reached a constant mass (m 2).

-solubility ═ ((m2-m1)/25) × 100, expressed in g per 100g of dry matter in the solution

For comparability, the degree of hydrolysis of all protein samples was measured using the OPA method as described below.

The principle is as follows:

the "amino nitrogen" group of the free amino acids of the sample is reacted with N-acetyl-L-cysteine and phthaloyl dialdehyde (OPA) to form the isoindole derivative.

The amount of isoindole derivative formed during the reaction is stoichiometric with the amount of free amino nitrogen. Measured by the increase in absorbance at 340nm is the isoindole derivative.

The procedure is as follows:

test sample P for accurate weighing of a sample to be analyzed^*Introduced into a 100ml beaker. (depending on the amino nitrogen content of the sample, the test sample will be from 0.5 to 5.0 g).

Adding about 50ml of distilled water, homogenizing and transferring into a 100ml graduated cylinder, adding 5ml of 20% SDS and making up the volume with distilled water; stir 15 minutes at 1000rpm on a magnetic stirrer.

An amount of flask 1 of 1 tablet of the Megazyme kit was dissolved in 3ml of distilled water and stirred until complete dissolution. One tablet/test is provided.

This solution No. 1 was prepared just before use.

The reaction takes place directly in the spectrophotometer cuvette.

o blank:

3.00ml of solution No. 1 and 50. mu.l of distilled water were introduced.

o standard:

megazyme kit was introduced into 3.00ml of solution No. 1 and 50. mu.l of flask 3.

o sample:

3.00ml of solution No. 1 and 50. mu.l of the sample preparation were introduced.

The cuvettes were mixed and the absorbance measurements of the solution (a1) were read after about 2 minutes on a spectrophotometer at 340nm (the spectrophotometer was equipped with cuvettes having an optical path of 1.0cm, which can be measured at a wavelength of 340nm and verified according to the procedures described in the manufacturer's technical manual associated therewith).

The reaction was started immediately by adding 100. mu.l of the OPA solution of Megazyme kit to the spectrophotometer cuvette in the amount of flask 2.

The cuvettes were mixed and placed in the dark for about 20 minutes.

Next, absorbance measurements of the blank, standard and sample were read on a spectrophotometer at 340 nm.

The calculation method comprises the following steps:

the content of free amino nitrogen expressed as mass percentage of the product itself is given by the following formula:

wherein: Δ A ═ A2-A1

V is the volume of the flask

Mass of test sample in g

6803% extinction coefficient (in L.mol.) of isoindole derivative at 340nm^-1.cm^-1In units).

Molar mass of nitrogen (in g.mol.)^-1Is a unit)

3.15 final volume in cuvette (in ml)

Test samples in 0.05 ═ cuvettes (in ml)

The Degree of Hydrolysis (DH) is given by:

wherein the protein nitrogen is determined according to the DUMAS method of standard ISO 16634.

The following table summarizes all these analyses for the inventive process isolates as well as for the prior art isolates:

as is clear from the above table, inventive method samples #1 and #2 are the only samples having a solubility above 30%, preferably above 40%, more preferably about 50% at pH 6, and a solubility above 40%, preferably above 60%, more preferably above 70% at pH7.

This difference cannot be explained by the degree of hydrolysis in the same range: our inventive method also has an impact on the functional properties of the inventive isolate, especially improving its solubility at pH 6 and 7.

Detailed Description

The term "plant protein" is considered herein to be all types of proteins extracted from all types of plants. A plant must be understood as any of the various photosynthetic, eukaryotic, multicellular organisms of the kingdom plantae (which characteristically contain chloroplasts, have cell walls composed of cellulose, produce embryos, and lack the ability to move). Plants include trees, shrubs, herbs, ferns, mosses, and certain green algae. In particular, in the present application, the term plant applies to the family leguminosae, which includes peas and beans. Other preferred types of plants are flax, oat, rice and lentil.

"protein" in the present application is understood to mean a molecule consisting of one or more long-chain amino acid residues. In the present application, the protein may be native to the plant or modified, including hydrolyzed protein. These proteins may have different concentrations, including greater than 80% isolates or greater than 50% concentrates.

The term "leguminous" must be understood as meaning plants of the family Leguminosae (Leguminosae). These plants have seeds, unique flowers in the pods, and often nodules. These nodules contain symbiotic bacteria capable of fixing nitrogen.

The term "pea" is considered herein to be its broadest acceptable meaning. In particular, it includes all varieties of "photic peas" and "wrinkled peas", as well as all mutant varieties of "photic peas" and "wrinkled peas". These varieties are directed to uses (food for human consumption, animal feed, and/or other uses) typically for each pea type. In the present application, the term "pea" includes varieties of pea belonging to the genus pisum, and more specifically to the species sativum and aestivum. Said mutants are those specifically referred to as "r mutants", "rb mutants", "rug 3 mutants", "rug 4 mutants", "rug 5 mutants" and "lam mutants", as described in C-L HEYDLEY et al, entitled "Developing novel pea starch", Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society [ Proceedings of the seminar of the Industrial Biochemistry and Biotechnology Group of the Society ],1996, article pp.77-87.

The term "volatile" is used herein to refer to compounds that readily evaporate at ambient temperature and pressure. These compounds can be readily analyzed using chromatographic methods as described below.

The term "saponin" is considered herein to be any of a variety of plant glycosides that form soap bubbles when mixed and stirred with water. In particular, these saponins are amphiphilic glycosides that are chemically grouped by the phenomenon of soap-like foam produced when the saponin is shaken in an aqueous solution. They are structurally grouped on the basis of having one or more hydrophilic glycoside moieties in combination with lipophilic triterpene derivatives.

As described in the above summary of the invention, a first aspect of the invention is a plant protein isolate (including isolates or concentrates) containing less than 10 μ g of total volatile compounds per gram of dry matter, preferably containing less than 5 μ g of total volatile compounds per gram of dry matter.

In a preferred embodiment, the total volatile compound must be understood as the sum of the contents of hexanal, 2-pentyl-furan, (E) -2,4, heptadienal and 1-octen-3-ol (see formula in fig. 1). In a more preferred embodiment, total volatile compounds must be understood as the sum of all volatile compounds detected and analyzed by the present invention using the methods described below. These particular volatile compounds are associated with a "beany", "vegetable-like" or "pea-like" taste. The following examples show that the proteins of the invention contain less than 10. mu.g of these volatile compounds per gram of dry matter. Furthermore, in the examples section of the present application, it is shown that no commercial protein isolate or known extraction methods (not including the use of solvents) achieved this result.

In a more preferred embodiment, the plant protein isolate further contains less than 5mg total saponins per gram dry matter.

In an even more preferred embodiment, the plant protein isolate is obtained from a leguminous plant, preferably from pea or broad bean, most preferably from pea protein.

The link between off-flavours and vegetable protein compositions is well known to the person skilled in the art. Lead to thisCompounds that are odor-like can be divided into two classes. The person skilled in the art describes the first class as consisting of molecules with a typical molecular weight range of 30-300g.mol^-1Of (c) volatile compounds. Examples of such compounds are hexanal, 2-pentyl-furan, and the like. These volatile compounds often result in "beany", "vegetable-like" and/or "pea-like" tastes/off-notes. If the association between off-flavors such as "beany flavor" or "pea flavor" and volatile compounds is well known, it is known that no process or isolate is currently available at levels low enough to be barely noticeable to consumers. Only one method involves the use of solvents, which can be a serious disadvantage in industrial processes.

Such volatile compounds are present directly in leguminous plants, in particular peas, but they can also be synthesized during protein extraction by endogenous enzymes such as lipoxygenases, which oxidize residual lipids.

Total volatile compounds were evaluated by HS-SPME analysis program. This procedure was performed by examining the changes in Sorayya et al, volume flavor profile of select field flavors as the area infected by the crop and processing [ Volatile flavor characteristics of pea varieties of selected farmlands affected by crop year and processing ], Food Chemistry [ Food Chemistry ],124(2011), pages 326 to 335. A1 g pea protein sample was suspended in 100ml 15% (w/v) NaCl aqueous solution. After mixing 5ml of the solution, it was placed in a sample bottle. SPME fiber (50/30 μm, DVB/CAR/PDMS, Supelco Co., Shanghai, China) was used for flavor extraction. The fibers were conditioned at 250 ℃ for 1 hour prior to each use. A1 g sample of pea protein was suspended in 100mL of a 15% (w/v) NaCl (AR) aqueous solution at room temperature. After mixing, 5mL of the solution was placed in a 30mL clear glass vial (Supelco, shanghai, china) and then sealed with a cap containing a teflon-coated rubber septum and fitted with a small magnetic stir bar. An internal standard 2-methyl-3-heptanone (1mg/L solution) was added (Sigma Aldrich, Shanghai, China). The sample in the vial was heated in a water bath at 60 ℃ for 30 minutes and extracted using SPME fiber for 30min, and the magnetic stirring rate for the adsorption process was 500 rpm. The fibers were then injected into a GC-MS (SCION SQ-456-GC, Bruker, USA) equipped with a capillary column with a polar resin of DB-WAX (30m 0.25mm inner diameter, 0.25 μm film thickness; Agilent Technologies Inc., Guangzhou, Guangdong, China). Using no-split injection. The chromatograph temperature was set at 40 ℃ with an isotherm of 3 minutes, heated to 100 ℃ at a rate of 6 ℃ min-1, then heated to 230 ℃ at a rate of 10 ℃ min-1, and the final isotherm was 7 minutes. Mass spectrometry was run in an electron collision mode of 70 eV. The mass spectrometer scan mass was from m/z 33 to 350. The ionization source was set at 200 deg.C and the transmission line was set at 250 deg.C. Volatile compounds were identified by comparison to mass spectral libraries and by calculating and comparing GC retention indices of a series of alkanes (C8-C30). Retention indices are based on public data calculated under the same chromatographic conditions. Quantitative data were obtained by electronically integrating the area under the Total Ion Current (TIC) peak. The internal standard 2-methyl-3-cycloheptanone was then used to calculate the relative amounts and was normalized by considering the dry matter.

As regards the second category related to off-flavours, those skilled in the art are known to have 40-1,000g.mol^-1Typical molecular weight range of non-volatile compounds. For "bitter" off-flavors, it is saponin, oxidized phospholipids, etc. For "salty taste", they are sodium chloride, potassium chloride, and the like. For "sourness", they are butyric acid, acetic acid, etc. Saponins and their bitter off-taste are more challenging compounds in leguminous proteins, especially in peas.

The saponin extracts were analyzed according to a modified protocol inspired by: lynn Heng et al, Bitterness of saponin and the content of saponin in dry peas]Journal of the Science of Food and Agriculture]86(2006),1225-1231, and K.Decroos et al, Simultaneous quantification of differential glycosylation, acylated, and2, 3-dihydroxy-2, 5-dihydroxy-6-methyl-4H-pyran-4-one-linked so-formed phenol in an area using reversed-phase high-performance chromatography with improved diffraction detection [ using reversed-phase high-performance liquidSimultaneous quantification of soyasaponin with different glycosylation, acetylation and2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one conjugation by phase chromatography and evaporative light scattering detection]Journal of Chromatography A],1072(2005)185-193. Pea protein samples were defatted with hexane (AR, Sigma Aldrich, shanghai, china) and then refluxed for 6 hours and subsequently the pea proteins were air dried overnight in a fume hood. Defatted pea protein (1g) was extracted with 40ml of 60% (v/v) methanol (HPLC grade, Sigma Aldrich, shanghai, china) at 25 ℃ for 4 hours with constant shaking at 200rpm in an incubator shaker (SWB15, semer feishi (Thermo Fisher), shanghai, china). Before extraction, 100mg kg of the extract was added^-1Equilenin (an internal standard substance of (1) (estrogenic steroid, 3-hydroxyestra-1, 3,5,7, 9-pentaen-17-one)). The crude extract was filtered through an ashless filter paper (Whatman,110mm, national pharmaceutical group chemicals, Ltd., shanghai, china). The clear filtrate was freed of methanol by evaporation in vacuo at 40 ℃. This evaporation step was performed in less than 15 minutes using a 1L round bottom flask. The concentrate was made up to 5mL with distilled water and passed through a Sep-Pak C18 solid phase extraction column (Watts Plus tC18 cartridge, 37-55 μm, Suzhou, China) followed by a 15mL water rinse to remove unbound material. Bound compounds were eluted with 10ml of 100% (v/v) methanol (HPLC grade, Sigma Aldrich, shanghai, china) and analyzed by LC-MS. LC-MS chromatographic conditions follow. The capillary voltage was 4.4KV, the cone hole voltage was 40V, the ion source temperature was 100 deg.C, the solvent gas temperature was 250 deg.C, the photomultiplier voltage was 700V, and the flow rate was 4.2L/h. Liquid chromatography was performed on a Watts 2690 liquid chromatography system equipped with a Lichrospher C-18 (2.1X 250mm, Watts) column and detector Watts 996. The column temperature was 35 ℃ and the injection volume was 10uL, the flow rate was 0.3 mL/min. The gradient elution conditions for the LC-MS experiment were 0.5% formic acid (AR Sinopharm Chemical Reagent Co., Ltd.), Shanghai, China) for 30 minutes (0-30min), followed by acetonitrile (HPLC grade, Sigma Aldrich, Shanghai, China)) The ratio to 0.5% formic acid was 20:80 for 10min (30-40min) and the ratio was 40:60 for 1min (40-41min), and then adjusted to 0.5% formic acid. Molecular ion [ M + H ] in peak mass spectra of DDMP saponin and saponin B]⁺Are 1069 and 943, respectively. The relative amount of saponin was calculated using the internal standard equilenin and normalized by considering the dry matter.

The protein isolate according to the invention is also characterized by an improved solubility in water compared to proteins from the prior art. In particular, the protein isolate according to the invention has a solubility in water at 20 ℃ and pH 6 higher than 30%, preferably higher than 40%, more preferably about 50%, and a solubility at 20 ℃ and pH7 higher than 40%, preferably higher than 60%, more preferably higher than 70%.

Solubility can be measured by any method known in the art. Preferably, the following protocol will be used for the measurements:

2.0g of the sample and 100g of distilled water were placed in a 400mL beaker at-20 ℃.

The pH is adjusted to 6 or 7 with 1N HCl and/or 1N NaOH and the mixture is made up to exactly 200.0g with distilled water.

This mixture was stirred for 30 minutes and subsequently centrifuged at 3000 Xg for 15 minutes.

After centrifugation, exactly 25.0g of supernatant was drawn into the crystallization dish (m 1). The petri dish was placed in an oven at 103 ℃ until it reached a constant mass (m 2).

-solubility ((m2-m1)/25) × 100

A second aspect of the invention is a process for obtaining plant proteins (including isolates and concentrates), preferably leguminous protein isolates, more preferably pea protein isolates. The method involves the steps of: (a) providing a protein-containing plant seed, preferably a leguminous seed, more preferably a pea seed;

(b) grinding the seed;

(c) suspending the ground seeds in water;

(d) extracting protein from the milled suspension;

(e) washing the protein with water at a temperature of 60 ℃ to 100 ℃, more preferably 75 ℃ to 95 ℃, and at a pH in the range of 4 to 5, more preferably 4,5 to 5;

(f) optionally passing the washed protein obtained at the end of step (e) through a shear pump or homogenizer to improve protein functionality;

(g) optionally drying the protein obtained in step (e) or (f).

In step (a), plant seeds suitable for the present invention may be selected from the list of food compatible plant seeds, in particular pea, field bean, oat, lentil and flax … … pea seeds are indeed the best and most suitable seeds, followed by field bean.

Step (b) is intended to grind the seeds into a powder, which can be done by each method known to the person skilled in the art. It may include a prior soaking, bleaching or even a well-known baking step for the inhibition of endogenous enzymes such as lipoxygenases. The seeds may be ground to a powder prior to mixing the seeds into the water, a process known as "dry milling". However, it is also possible to carry out the grinding while suspending the seeds in water, also known as the "wet-grinding" process.

The aim of step (c) is to suspend the ground powder in water. In the case of wet milling, water is introduced prior to milling. During dry milling, the powder is introduced with water at a concentration of 20% to 30% dry weight, preferably 25% dry weight.

The object of step (d) is to extract proteins from the ground seeds. Wet extraction methods are particularly suitable for the present invention. A preferred process is described in US 7186807(B2) in its entirety, which is incorporated by reference into the present application.

In the first step of the patented process, the powder obtained by grinding peas, which have been previously washed, sorted and bleached, is suspended in water. When suspending the powder in water, it is most advantageous to select a powder with an average particle size equal to or less than 100 μm, in a concentration of 20% to 30% dry weight, preferably 25% dry weight. The pH of the solution is not a limiting factor but most advantageously the pH of the suspension is not corrected, which means working in the pH range between 6.2 and 7.

In the second step, it is most advantageous to expose this aqueous powder suspension directly to the course of a centrifugal decanter. This may prevent the pea fibre fraction from being removed by previous sieving. The applicant company observed that, according to the configuration used in potato starch plants, the use of a centrifugal decanter for this separation operation makes it possible to easily separate it into two distinct fractions, soluble and protein on the one hand, and fiber and starch on the other hand.

In the third step, the protein can be easily separated from the fraction containing the mixture of soluble and protein thus obtained. This is achieved by selecting one of several techniques for precipitating proteins at their isoelectric pH, and/or ultrafiltration-type membrane separations. The preferred method is to use a combination of isoelectric pH and thermal coagulation of proteins, known as "thermal coagulation".

These obtained proteins (mainly globulins in the case of peas) are the starting material for step (e). The protein solution (typically less than 20% dry matter at commercial weight) is adjusted to a pH of 4 to 5.5, preferably to a pH of 4.5-5, more preferably to a pH of 4.5.5, preferably to a pH of 4.5, preferably to a temperature of about 90 ℃, in a water bath at a temperature of 60 ℃ to 100 ℃, more preferably 75 ℃ to 95 ℃, even more preferably about 90 ℃. The protein solution may be pumped directly into a plate filter or centrifuge to separate the whey, or after a contact time which may be as long as 30 minutes. The protein curd can then be washed, preferably 2 times, with 1 to 5 volumes of water, at a pH adjusted to 4 to 5.5, preferably to a pH of 4.5-5, more preferably to pH4.5, at a temperature of 60 ℃ to 100 ℃, more preferably 75 ℃ to 95 ℃, even more preferably at about 90 ℃. The washed protein curd was then fixed and resuspended in distilled water to obtain a solids content ranging from 10% to 12%, and the pH was adjusted to 6.5 to 7.0 with 2.0M NaOH. One option for this step may be to end up with high pressure homogenization (20MPa) and spray drying. In an alternative embodiment, steps (d) and (e) may be performed simultaneously. In this case, after addition of 1 to 5 volumes of water, the protein is coagulated, preferably thermally coagulated, by heating at a temperature of 60 ℃ to 100 ℃, more preferably 75 ℃ to 95 ℃, even more preferably at about 90 ℃ and at a pH of 2.5. These three parameters are essential to obtain an isolate with sufficient organoleptic quality. The best results were obtained with 1 to 5 volumes of water by heating at about 90 ℃ and ph 4.5.

In a second preferred embodiment, the seed grinding in step (b) is performed in the absence of oxygen. By oxygen-free it must be understood that the residual content of oxygen is less than 300ug/l, preferably less than 200 ug/l. The residual oxygen content is measured with a device common and known in the art, such as an oxygen meter, preferably at 15 ℃. The preferred method is dry milling, but wet milling can also be used. In the case of dry milling, oxygen can be analyzed by a tunable diode laser gas analyzer (TDL, Mettlo Toledo, shanghai, china), while in the case of wet milling, a dissolved oxygen analyzer (M400, Mettlo Toledo, shanghai, china) can be used. The combination of the absence of oxygen during the grinding step and the high temperature acid wash of step (d) appears to produce a synergistic effect and to produce high quality levels of leguminous pea protein isolate, preferably pea protein isolate. Unlike the results of the present invention, low oxygen milling alone did not produce protein with good organoleptic qualities. Such low residual oxygen content can be obtained by methods well known in the industry, such as purging nitrogen in the vessel in which the seeds are ground. In a more preferred embodiment, steps (c) and (d) are also carried out in the absence of oxygen, preferably with a residual oxygen content of less than 300. mu.g/l, preferably less than 200. mu.g/l. The use of nitrogen and water without dissolved oxygen in the headspace of the treatment device is a common method of ensuring such an embodiment.

In both embodiments of step (d) as described above, optional homogenization of the resulting protein may be performed with a shear pump to enhance solubility, if desired. Common known methods like pasteurization or the introduction of food grade auxiliary compounds can also be added to the process. Finally, the protein obtained can be dried by common techniques such as spray-drier.

The invention will be better understood with reference to the following examples and the accompanying drawings. These examples are intended to represent specific embodiments of the present invention and are not intended to limit the scope of the present invention.