ES2800475T3 - Method for extracting pea proteins - Google Patents

Abstract

A method of extracting pea proteins, comprising the steps of: (a) providing an aqueous composition comprising pea proteins; (b) isolating said pea proteins from said aqueous composition comprising pea proteins; (c) obtaining said isolated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8; and (d) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature in the range of 75 ° C to 210 ° C; wherein step (b) comprises adjusting the pH of said aqueous composition comprising pea proteins to a value in the range of 4.0 to 5.8, preferably in the range of 4.5 to 5.5; wherein step (d) comprises subjecting said precipitated pea proteins to a heat treatment at a temperature in the range of 115 ° C to 210 ° C for a time in the range of 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s.

Description

DESCRIPTION

Method for extracting pea proteins

Field of the invention

The present invention relates to methods for extracting and purifying proteins. In particular, the present invention relates to the extraction of pea protein. The invention further relates to pea proteins obtainable by the above methods, as well as to foods or food products containing such pea proteins. The invention also relates to the use of such pea proteins in the food or feed industry.

Background of the invention

Plant-based protein isolates represent a valuable alternative or supplement to animal protein in food or feed. For example in food, in addition to vegetable proteins, it can effectively replace animal proteins, often at lower cost. Additionally, many products that traditionally contain animal proteins, particularly dairy products, can be a major cause of food allergies.

Legumes are notable because most of them have symbiotic nitrogen-fixing bacteria in structures called root nodules. This arrangement means that root nodules are sources of nitrogen for legumes, making them relatively rich in plant protein. All proteins contain nitrogenous amino acids. Therefore, nitrogen is a necessary ingredient in the production of proteins. Therefore, legumes are among the best sources of plant protein. As legumes, such as peas ( Pisum sativum), in addition to being high in protein, are readily available and have a particularly well-balanced amino acid composition, they represent a protein source that is a valuable alternative to animal protein.

The main challenges in providing plant proteins revolve around the composition and purity of the proteins, and include aspects related to, for example, extraction, fractionation, and pre-isolation and post-isolation treatments. By the time the vegetable protein is isolated and available in a more or less pure form, all the previous manipulations have a great impact on the quality of the isolated vegetable protein. For example, the type and amount of impurities in protein isolates or extracts determine their final value. Such impurities include, for example, carbohydrates. While carbohydrates are generally unwanted impurities in the final protein isolate, some other impurities, such as vitamins or minerals may, by definition, not be undesirable, or may even be beneficial to the nutritional and / or physicochemical aspects of the isolate. protein. In addition to affecting the final composition of protein isolates or extracts, the extraction and / or purification process can drastically affect the physicochemical or functional properties of the protein isolate. In particular, protein solubility, viscosity, emulsifying capacity, color, taste or smell are strongly influenced by the techniques used.

As can be appreciated from the foregoing, obtaining a high quality protein isolate having specific desired properties can be cumbersome and often involves multiple costly and / or time-consuming manipulations. In view of this, there is still a need to improve the isolation of proteins from plants, in particular from legumes, such as pea.

FR2889416A1 refers to pea protein compositions characterized by a particular molecular weight distribution profile and water solubility, as well as methods for obtaining them.

FR2889417A1 relates to a granulated pea protein composition characterized by a mean particle diameter and a compressibility value, as well as methods for producing them.

WO 2010/022702 relates to a process for obtaining fat-containing legume protein.

Document US 2013/0017310 refers to a process for manufacturing soluble and functional plant proteins.

Khattab RY et al (Nutritional quality of legume seed as affected by some physical treatments, Part 1: Protein quality evaluation; Food Science and Technology, vol. 42, No. 6, 2009, p1107-1112;) describe the effects of different processing methods on the nutritional quality of cowpea, pea and bean seeds, such as the content of antinutritional factors, including tannins, phytic acid, trypsin inhibitors and oligosaccharides.

Camacho L et al (Nutritional improvement of legumes of habitual consumption fermented by cultures of the grain lactobacillus; Food, vol 16, 1991, p5-11) describe the effects of the fermentation of lactic acid in the content of oligosaccharides and phytates in the flour of lentils, chickpeas and peas.

Deshpande SS (Chapter 4: Fermentation of grain legumes, seeds and nuts in Latin America and the Caribbean, Fermented grain legumes, seeds and nuts: A global perspective (book series: FAO agricultural services bulletin), Food and Agriculture organization of the United Nations, vol. 142, 2000, p99-105, 107) describes fermented foods derived from cereal grains and tubers.

Schindler Sabrina et al (Improvement of the Aroma of Pea (Pisum sativum) Protein extracts by lactic acid fermented; Food biotechnology, vol. 26, 2012, No. 1) describe the formation of an unpleasant green or roasted taste during storage of extracts of legume proteins that limits its application in food. The pea protein extracts were subjected to lactic acid fermentation to improve flavor by reducing off-flavor formation or by masking undesirable green notes.

Accordingly, one of the objects of the present invention is to overcome or improve at least one of the disadvantages of the prior art, or to provide a useful alternative.

Summary of the invention

According to a first aspect of the present invention, a method for extracting pea proteins is provided. The method for extracting pea proteins comprises the steps of:

(a) providing an aqueous composition comprising pea proteins;

(b) isolating said pea proteins from said aqueous composition comprising pea proteins;

(c) obtaining said isolated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8;

(d) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature in the range of 75 ° C to 210 ° C,

wherein step (b) comprises adjusting the pH of said aqueous composition comprising pea proteins to a value in the range of 4.0 to 5.8, preferably in the range of 4.5 to 5.5;

wherein step (d) comprises subjecting said precipitated pea proteins to a heat treatment at a temperature in the range of 115 ° C to 210 ° C for a time in the range of 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s.

According to one embodiment of said method, the extraction of pea protein involves providing pea proteins which are subjected to isoelectric precipitation followed by heat treatment of the protein precipitate.

According to a second aspect of the present invention, there are provided pea protein extracts which can be obtained or are obtained by the method according to the first aspect of the invention.

According to a third aspect of the present invention, a pea protein composition is provided, comprising at least 60% by weight of protein based on the total dry matter of the composition, wherein said pea protein composition has an index nitrogen solubility at pH 7.0 of at most 15%, as measured in an aqueous composition comprising 3% by weight of said pea protein composition based on the total weight of the aqueous composition.

It is also disclosed herein that there is provided an edible composition, preferably a food or feed product, comprising the pea proteins according to the second aspect of the invention, or the pea protein composition according to the third aspect of the invention , or the pea proteins obtained by the method according to the first aspect of the invention.

In a fourth aspect, the present invention provides the use of pea protein extracts according to the second aspect of the invention, or pea protein composition according to the third aspect of the invention, or pea protein extracts obtained by the method according to the first aspect of the invention in food or feed products, preferably in bakery and confectionery food products.

In a fifth aspect, the present invention provides the use of pea proteins according to the second aspect of the invention, or pea protein composition according to the third aspect of the invention, or pea proteins obtained by the method according to the first aspect of the invention for clarifying beverages or beverages, preferably wine or fruit juices.

The present inventors have surprisingly discovered that pea proteins having particular functional, physicochemical and organoleptic characteristics when an aqueous composition comprising pea proteins is subjected to isolation steps such as protein precipitation, after which the proteins isolated are subjected to heat treatment.

In particular, it has been unexpectedly discovered that the methods according to the invention as described herein make it possible to obtain pea protein compositions, or pea protein extracts or concentrates having, inter alia, a lower ash content, higher density (both apparent and after settling), better flowability, better wettability, low solubility, lower viscosity, and lower gel strength, compared to pea protein extracts that are not obtained according to the methods of the invention such as described in this document.

The pea protein compositions of the invention have low affinity for water, which is of interest in applications with low water availability.

The specific characteristics of pea protein extracts and pea protein compositions according to the invention as described herein in particular make such pea protein extracts and pea protein compositions particularly suitable for use in the food or feed industry, in particular in bakery or confectionery food products, such as biscuits, breads, waffles, cakes, milk candies, extruded cereals and bars, etc. Surprisingly, it has been discovered that pea protein extracts and pea protein compositions according to the invention as described herein can be used in the above bakery and confectionery products and allow less water to be added during preparation. of these food products, while maintaining or even improving the quality (such as texture or taste) or shelf life of said food products and without compromising the workability of, for example, the dough used to prepare the bakery products. Another advantage of using less water in the preparation of bakery food products in particular is that the evaporation of water is facilitated during the cooking of such food products, which is not only more cost-effective, but also beneficially affects the overall quality. of food products (baked). The shelf life of food products containing pea protein extracts and pea protein compositions according to the invention can also be extended as described herein. Pea protein extracts and pea protein compositions as described herein according to the invention are also particularly suitable to replace, for example, animal proteins, such as milk proteins in food products, but also to replace other plant proteins, in particular allergenic plant proteins such as wheat protein, in food products. For example, pea protein extracts and pea protein compositions according to the invention as described herein can be used to partially or completely replace milk proteins in confectionery products, such as milk candies, or in dulce de leche bars, for which it has been surprisingly observed that a smoother texture can be obtained with the proteins according to the invention.

Furthermore, it has been unexpectedly discovered that pea protein extracts and pea protein compositions according to the invention as described herein are particularly suitable for use in rinsing or clarifying liquids, for example beverages or beverages. such as wine, beer, or fruit juices. Without wishing to be bound by any theory, it is hypothesized that the particularly low solubility of the pea protein extracts and the pea protein compositions according to the invention as described herein may be responsible for the clarification or clarification ability of proteins, in particular in relation to the reduction of the turbidity of liquids.

The independent and dependent claims establish particular and preferred characteristics of the invention. The features of the dependent claims may be combined with the features of the independent or other dependent claims as appropriate. The appended claims are also explicitly included by reference in the description.

Brief description of the figures

Figure 1 schematically represents an extraction process according to an embodiment of the invention.

Figure 2 represents a graph representing the gel strength at pH 6 of each extract (A) to (D).

Figure 3 represents a graph representing the profile of the nitrogen solubility index as a function of the pH for each extract (A) to (D).

Figure 4 represents a graph that represents the viscosity profile expressed as a function of pH for each extract (A) to (D).

Figure 5 represents a graph that represents the profile of the nitrogen solubility index expressed as a function of pH for each extract E to G.

Figure 6 represents a graph that represents the turbidity of different solutions comprising tannins after incubation for 96 h at 4 ° C.

Figure 7 represents a graph representing the turbidity of different solutions comprising tannins as a function of the incubation time at 4 ° C.

Figure 8 represents a graph representing the turbidity of different solutions comprising SiO2 after incubation for 96 h at 4 ° C.

Figure 9 represents a graph representing the turbidity of different solutions comprising SiO2 as a function of the incubation time at 4 ° C.

Figure 10 represents a graph representing the turbidity of different solutions after incubation for 96 h at 4 ° C.

Figure 11 represents a graph representing the turbidity of different pea protein solutions after incubation for 96 h at 4 ° C.

Figure 12 represents a graph representing the fermentation rate of different doughs prepared with pea protein extracts.

Figure 13 represents a graph representing the volume of the bread prepared in Example 5.

Figure 14 represents a graph that represents the hardness of the bread crumbs of the bread prepared in example 5. Figure 15 represents a graph that represents the hardness of the bars as a function of the useful life of the bars prepared in example 5 .

Figure 16 represents a graph that represents the profile of the nitrogen solubility index as a function of pH for each extract H and I.

Figure 17 represents a graph that represents the concentration of sugar / dry matter in% as a function of the fermentation time of the peas fermented with Lactobacillus fermentum LMG 6902, Lactobacillus fermentum LMG 18026, Lactobacillus Crispatus LMG 12005 or Lactobacillus Acidophilus LMG 8151.

Figure 18 represents a graph that represents the pH of shelled peas (7A) fermented with Lactobacillus fermentum LMG 6902, Lactobacillus fermentum LMG 18026, Lactobacillus Crispatus LMG 12005 or Lactobacillus Acidophilus LMG 8151 and the pH of the 7 aqueous solution (juice B) ) depending on the fermentation time.

Figure 19 represents a graph representing the acidity of shelled peas (8A) fermented with Lactobacillus fermentum LMG 6902, Lactobacillus fermentum LMG 18026, Lactobacillus Crispatus LMG 12005 or Lactobacillus Acidophilus LMG 8151 and the acidity of the solution (8Bose juice) ) depending on the fermentation time.

Figure 20 represents a graph that represents the lactic acid bacteria (Lactobacillus fermentum LMG 6902, Lactobacillus fermentum LMG 18026, Lactobacillus Crispatus LMG 12005 or Lactobacillus Acidophilus LMG 8151) concentration of the aqueous solution (juice) as a function of the fermentation time.

Detailed description of the invention

Before describing the present method of the invention, it should be understood that the present invention is not limited to the particular methods, components, products, or combinations described, as such methods, components, products, and combinations may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

As used herein, the singular forms "a", "an" and "the" include singular and plural references unless the context clearly indicates otherwise.

The terms "comprising", "comprises" and "composed of" as used herein are synonymous with "including", "including" or "containing", "contains", and are inclusive or open and they do not exclude additional, uncited members, elements, or method steps. It will be appreciated that the terms "comprising", "comprises" and "comprised of" as used herein comprise the terms "consisting of", "consists" and "consists of" as well as the terms "which consist essentially of ', "consists essentially" and "consists essentially of".

The enumeration of numerical ranges by extreme values includes all the numbers and fractions encompassed within the respective ranges, as well as the extreme values listed.

The term "about" or "about" as used herein refers to a measurable value such as a parameter, an amount, a time duration and the like, it refers to encompassing variations of / - 20% or less, preferably / - 10% or less, more preferably / - 5% or less, and still more preferably / - 1% or less of and from the specified value, insofar as such variations are appropriate for carry out the disclosed invention. It should be understood that the value referred to by the modifier "about" or "about" is also specifically and preferably disclosed.

While the terms "one or more" or "at least one", such as one or more or at least one or more members of a group of members, it is clear per se, by way of further examples, the term encompasses, between other things, a reference to any one of said members, or to two or more of said members, such as, for example, any of> 3,> 4,> 5,> 6 or> 7 etc. of said members, and even all said members.

Unless defined otherwise, all terms used to disclose the invention, including technical and scientific terms, have the meanings commonly understood by one of ordinary skill in the art to which the present invention belongs. For further guidance, definitions of terms are included to better appreciate the teaching of the present invention.

In the following passages, different aspects of the invention are defined in more detail. Each aspect thus defined may be combined with any other aspect or aspects unless clearly stated otherwise. In particular, any characteristic indicated as preferred or advantageous may be combined with any other characteristic or characteristics indicated as preferred or advantageous.

Reference throughout the present specification to "an embodiment" or "embodiment" means that a particular feature, structure, or particular characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Therefore, the occurrences of the phrases "in one embodiment" or "in the embodiment" in various places throughout this specification do not all necessarily refer to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner, as would be apparent to a person skilled in the art from the present disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the invention, and form different embodiments, as will be understood by those skilled in the art. The matter. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

In the following detailed description of the invention, reference is made to the accompanying drawings that form a part of the invention, and in which only specific embodiments in which the invention may be practiced are shown by way of illustration. The scope of the present invention is defined by the appended claims.

Claims numbered 1 through 68 are also described herein.

1. A method for extracting pea proteins, comprising the steps of:

(a) providing an aqueous composition comprising pea proteins;

(b) isolating said pea proteins from said aqueous composition comprising pea proteins, preferably using precipitation, flocculation, filtration and / or chromatography;

(c) obtaining said isolated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8;

(d) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature of at least 75 ° C.

Preferably, the aqueous suspension comprising pea proteins in step (c) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most, 30%, preferably at most 25%, and in one embodiment the dry matter can be adjusted to this extent by dilution with water.

2. The method according to claim 1, wherein step (d) comprises subjecting said aqueous suspension to a heat treatment at a temperature in the range of 75 ° C to 210 ° C, preferably in the range of 85 ° C to 160 ° C, for example 90 ° C to 150 ° C.

3. The method according to claim 1 or 2, wherein step (d) comprises subjecting said aqueous suspension to a heat treatment for at least 0.01 second, preferably for a time in the range of 0.01 second to 20 minutes, preferably, in the range of 10 seconds to 10 minutes.

4. The method according to any one of claims 1 to 3, wherein step (d) comprises subjecting said aqueous suspension to a heat treatment at a temperature in the range of 115 ° C to 210 ° C for a time in the range of range 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s.

5. The method according to any of the statements 1 to 4, wherein the time of subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature of at least 75 ° C in the step (d) decreases as the temperature increases.

6. The method according to any one of claims 1 to 5, wherein said step (b) comprises concentrating said pea proteins.

7. The method according to any of claims 1 to 6, wherein said step (b) comprises at least one precipitation, flocculation, filtration and / or chromatography step.

8. The method according to any of the statements 1 to 7, wherein step (b) comprises isoelectric precipitation.

9. The method according to any of the claims 1 to 8, wherein step (b) comprises adjusting the pH of said aqueous composition comprising pea proteins to a value in the range of 4.0 to 5.8, preferably, in the range 4.5 to 5.5.

10. The method according to any one of claims 1 to 9, wherein step (c) comprises adjusting or maintaining the pH of the aqueous suspension to range from 4.0 to 5.8.

11. The method according to any one of claims 1 to 10, wherein said aqueous composition comprising pea proteins in step (a) has a pH of at least 6.0, preferably in the range of 6.0 to 9.0, preferably in the range of 6.5 to 8.5.

12. The method according to any of claims 1 to 11, wherein the pH of said aqueous composition comprising pea proteins in step (a) is adjusted to at least 6.0, preferably in the range of 6 0 to 9.0, preferably in the range 6.5 to 8.5.

13. The method according to any one of claims 1 to 12, wherein prior to step (a) an aqueous composition comprising peas, preferably shelled peas, is subjected to fermentation, preferably in the presence of lactic acid bacteria.

14. The method according to claim 13, wherein said fermentation is carried out in the presence of one or more Lactobacillus sp.

15. The method according to any one of claims 1 to 14, wherein before or during step (b) said aqueous composition comprising pea proteins, or said pea proteins are subjected to a heat treatment, preferably they are subjected to a temperature of at least 30 ° C, for example at least 40 ° C, for example at most 80 ° C, for example at least 50 ° C and at most 80 ° C, for example at least 53 ° C and a maximum of 78 ° C, for example at least 54 ° C and a maximum of 75 ° C.

16. The method according to any of the claims 1 to 15, wherein before or during step (b) said aqueous composition comprising pea proteins, or said pea proteins are subjected to pasteurization.

17. The method according to any of the claims 1 to 16, further comprising the step of drying said aqueous suspension after step (d), preferably with spray drying, preferably obtaining a pea protein composition having a pH in the range of 4.0 to 5.8, as measured at room temperature in 10 g of pea protein composition suspended in 90 g of water

18. Pea proteins that can be obtained by the method according to any of the statements 1 to 17.

19. Pea protein composition, comprising at least 60% protein by weight based on the total dry matter of the composition, wherein said pea protein composition has an index of nitrogen solubility (ISN) at pH 7.0 of at most 15%, as measured in an aqueous composition comprising 3% by weight of said pea protein composition based on the total weight of the aqueous composition, and preferably an ISN of at most 11%, preferably at most 10%, preferably at most 9%, preferably at most 8%. Preferably, the pea protein composition has a dry matter of at least 90% based on the total weight of the composition.

Nitrogen content (% atomic weight) of selected samples was determined using a LECO analyzer. The technique used was the classical Dumas method, which uses thermal conductivity detection (DCT): Heavy samples are burned in oxygen at 1200 ° C. Combustion products (including N2 and NOx) are swept with a helium carrier gas through combustion catalysts, scrubbers, and through a tube filled with reduced copper. Copper removes excess oxygen and reduces NOx to N2. The N2 is then measured with DCT.

20. Pea protein composition according to claim 19, wherein said composition has a pH in the range of 4.0 to 5.8, as measured at room temperature in 10 g of pea protein composition suspended at 90 g of water.

21. An edible composition, preferably a food or feed product, comprising the pea proteins according to claim 18, or the pea protein compositions according to any one of claims 19 or 20.

22. Use of the pea proteins according to claim 18, or the pea protein compositions according to any one of the claims 19 or 20, in food or feed products, preferably in bakery food products and in products confectionery foods, such as cookies, breads, waffles, cakes, milk candies, extruded cereals and bars, etc.

23. Use of pea proteins according to claim 18, or pea protein compositions according to any one of claims 19 or 20, to clarify beverages and / or concoctions, preferably wine, fruit juice, beer .

24. The method according to claim 13 or 14, wherein after said fermentation, the peas are ground.

25. The method according to any of the statements 1 to 17 and 24, wherein before step (a) said method comprises the steps of:

- provide peas, preferably shelled peas,

- optionally grinding said peas; and

- hydrating said peas, or said optionally ground peas;

26. The method according to any of the statements 1 to 17, 24 and 25, wherein before step (a) said method comprises the steps of:

(a1) fermenting an aqueous composition comprising peas, preferably in the presence of one or more lactic acid bacteria;

(b1) grinding said peas; thus obtaining ground peas;

(c1) fractionating said ground peas to obtain at least a fraction comprising proteins, also called aqueous composition comprising pea proteins.

27. The method according to any of claims 13, 14, 24 or 26, wherein said peas in step (a1) are subjected to fermentation until the pH in said peas is at most 5.5, preferably at most 5.0, more preferably in the range of pH 3.5 to pH 5.0, as measured at room temperature in 1 g of said peas that have been ground and then suspended in 9 g of water.

28. The method according to any one of claims 13, 14, 24 or 26 and 27, wherein said peas in step (a1) are subjected to fermentation until the pH in said peas is reduced by at least 1 pH unit, preferably in at least 1.5 pH units, as measured at room temperature in 1 g of said peas that have been ground and then suspended in 9 g of water.

29. The method according to any one of claims 13, 14, 24 or 26 to 28, wherein step (a1) comprises adding dried peas and / or shelled peas to an aqueous solution, preferably adding dried peas having a dry matter content in the range of 80% to 95% based on the total weight of the dry peas.

30. The method according to any one of statements 13, 14, 24 or 26 to 29, wherein said peas after stage (a1) and before stage (b1) have a dry matter content in the range 35% to 60% based on the total weight of the peas.

31. The method according to any one of claims 13, 14, 24 or 26 to 30, wherein step (a1) comprises fermenting said peas until they have a dry matter content in the range of 35% to 60% based on the total weight of the peas.

32. The method according to any one of claims 13, 14, 24 or 26 to 31, wherein said peas in step (a1) are subjected to fermentation for at least 3 h, preferably for at least 3 h and at most 24 h.

33. The method according to any one of claims 13, 14, 24 or 26 to 32, wherein said peas in step (a1) are subjected to fermentation at a temperature in the range of 30 ° C to 50 ° C preferably in the range of 35 ° C to 45 ° C.

34. The method according to any one of claims 13, 14, 24 or 26 to 33, wherein step (a1) comprises fermenting said peas in the presence of lactic acid bacteria, preferably in the presence of one or more Lactobacillus sp .

35. The method according to any one of claims 13, 14, 24 or 26 to 34, wherein said peas in step (a1) are subjected to fermentation in the presence of at least 102 cfu to, at most, 1010 cfu of lactic acid bacteria per ml of said aqueous composition comprising peas.

36. The method according to any one of claims 13, 14, 24 or 26 to 35, wherein fractionating said ground peas in step (c1) comprises separating at least part of the proteins comprised in the peas from the rest of the pea , preferably in a fraction comprising at least 50% by weight of protein based on the total dry matter of said fraction.

37. The method according to any one of claims 26 to 36, wherein fractionating said ground peas in step (c1) comprises adjusting the pH of the ground peas to a pH of at least 6, preferably at least 7, more preferably a pH of at least 8 and a maximum of 9. This pH adjustment can be carried out using any suitable base, such as sodium hydroxide, potassium hydroxide, calcium hydroxide. Preferably, this pH adjustment is carried out in an aqueous composition comprising ground peas having a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most 30%, preferably at most 25%. In one embodiment, the dry matter content of the ground peas is adjusted to the aforementioned dry matter content by adding water accordingly.

38. The method according to any one of claims 26 to 37, wherein fractionating said ground peas in step (c1) comprises subjecting said ground peas to one or more separation steps, preferably one or more decantation steps, preferably, one or more stages of centrifugal decantation.

39. The method according to any one of claims 13, 14, 24 or 26 to 38, wherein step (a1) comprises contacting shelled peas with an aqueous solution.

40. The method according to any one of claims 13, 14, 24 or 26 to 39, wherein step (a1) comprises contacting dry shelled peas with an aqueous solution, preferably dry shelled peas having a content of dry matter in the range of 80% to 95% based on the total weight of the dry shelled pea.

41. The method according to any one of claims 13, 14, 24 or 26 to 40, wherein step (a1) comprises fermenting said peas until they have a dry matter content in the range of 40% to 60% based on the total weight of the peas.

42. The method according to any one of statements 13, 14, 24 or 26 to 41, wherein said peas after stage (a1) and before stage (b1) have a dry matter content in the range 40% to 50% based on the total weight of the peas.

43. The method according to any one of claims 13, 14, 24 or 26 to 42, wherein before, during and / or after the milling step (b1) an aqueous solution is added, preferably water, preferably such as to obtain an aqueous composition comprising ground peas, said composition comprising 15% to 35% of dry matter based on the total weight of the composition, preferably comprising 15% to 35%, preferably 18% to 33% , for example 20% to 30%, such as at least 20%, for example at least 21%, for example at least 22%, for example at least 23%, for example For example at least 24%, for example at least 25%, 26%, 27%, 28%, 29%, for example at most 30%, for example at most 35%.

44. The method according to any one of claims 13, 14, 24 or 26 to 43, wherein said peas in step (a1) are fermented for a maximum of 24 h, for example, for a maximum of 20 h, for example, for a maximum of 18 h, for example, for a maximum of 12 h, for example, for a maximum of 10 h.

45. The method according to any one of statements 13, 14, 24 or 26 to 44, wherein at the end of stage (a1) said peas have an acidity in the range of 25 to 250 mEq OH- per g of green peas.

46. The method according to any one of claims 13, 14, 24 or 26 to 45, wherein fractionating said ground peas in step (c1) comprises adjusting the pH of the aqueous composition comprising the ground peas to a pH of at least 6, preferably at least 7, preferably at least 8, more preferably a pH of at least 7.5 and at most 9, preferably a pH of at least 7.5 and at most 8.5, and that separates a protein comprising a fraction of said ground peas. Preferably, this pH adjustment is made in the aqueous composition comprising ground peas having a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most 30%, preferably at most 25%. In one embodiment, the dry matter content of the ground peas is adjusted to the aforementioned dry matter content by adding water accordingly.

47. The method according to claim 46, wherein said at least one fraction comprising protein is subjected to a temperature of at least 30 ° C, for example at least 40 ° C, for example at least 50 ° C , for example at least 55 ° C, for example, at most 80 ° C, for example at least 50 ° C and at most 80 ° C, for example at least 53 ° C and at most 78 ° C, for example example of at least 54 ° C and a maximum of 75 ° C.

48. The method according to any one of claims 13, 14, 24 or 26 to 47, wherein said aqueous composition comprising peas in step (a1), comprises an aqueous solution, preferably water.

49. The method according to any one of claims 13, 14, 24 or 26 to 48, wherein the amount of peas in said aqueous composition comprising peas in step (a) preferably ranges from 150 to 500 kg of peas per m3 of aqueous composition comprising peas.

50. The method according to any one of claims 13, 14, 24 or 26 to 49, wherein said aqueous composition comprising peas before or at the beginning of the fermentation of step (a1) has a pH of at least 6 eg at least 6.2, eg at least 6.4, as measured in the aqueous composition comprising the peas, after said composition has been ground.

51. The method according to any one of claims 1 to 17, or 24 to 50, wherein before step (b) said aqueous composition comprising pea protein is subjected to a temperature of at least 30 ° C, for for example at least 55 ° C, for example at most 80 ° C, for example at least 50 ° C and at most 80 ° C, for example at least 55 ° C and at most 78 ° C.

52. The method according to any one of claims 13, 14, 24 or 26 to 51, wherein said lactic acid bacteria are selected from the group comprising Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus , Sporolactobacillus, Tetragenococcus, Vagococcus and Weisella and combinations thereof.

53. The method according to any one of claims 13, 14, 24 or 26 to 52, wherein the lactic acid bacteria are Lactobacillus sp, more preferably selected from the group comprising Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus panis, Lactobacillus mucosae, Lactobacillus pontis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus buchneri, Lactobacillus delbrueckii and Lactobacillus casei and mixtures thereof.

54. The method according to any one of claims 13, 14, 24 or 26 to 53, wherein the lactic acid bacteria are selected from the group comprising Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus panis, Lactobacillus mucosae, Lactobacillus pontis and mixtures thereof.

55. The method according to any one of claims 13, 14, 24 or 26 to 54, wherein the lactic acid bacteria are selected from the group comprising Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus panis, Lactobacillus mucosae, Lactobacillus pontis and mixtures thereof.

56. The method according to any one of claims 13, 14, 24 or 26 to 55, wherein said lactic acid bacteria is Lactobacillus fermentum, or Lactobacillus crispatus.

57. The method according to any one of claims 13, 14, 24 or 26 to 53, wherein said lactic acid bacteria is Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus acidophilus, or Lactobacillus plantarum.

58. The method according to any one of claims 13, 14, 24 or 26 to 53, wherein said lactic acid bacteria is Lactobacillus fermentum, Lactobacillus crispatus, or Lactobacillus acidophilus.

59. The method according to any one of claims 13, 14, 24 or 26 to 58, wherein the dried peas before the start of step (a1) have a pH of at least 6.0, preferably a pH of the range 6.0 to 7.0 (i.e. before fermentation), such as for example at least 6.0, for example at least 6.1, for example at least 6.2, for example , at least 6.3, for example, at most 6.9, for example, at most 7.0, preferably, in the range of 6.25 to 6.75, as measured at room temperature in 5 g of dried peas that have been ground with 95 g of water.

60. The method according to any one of claims 13, 14, 24 or 26 to 59, wherein said fermentation is anaerobic fermentation.

61. The method according to any one of claims 1 to 17, or 24 to 60, comprising the steps of: (i1) fermenting an aqueous composition comprising peas, preferably in the presence of one or more lactic acid bacteria ;

(ii1) grinding said peas in the presence of water; thus obtaining an aqueous composition comprising ground peas;

(iii1) fractionating said aqueous composition comprising ground peas to obtain at least one aqueous composition comprising pea proteins, preferably adjusting the pH of said aqueous composition to a pH of at least 6.

This pH adjustment can be done using any suitable base, such as sodium hydroxide, potassium hydroxide, calcium hydroxide. Preferably, this pH adjustment is carried out in an aqueous composition comprising ground peas having a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most 30%, preferably at most 25%. In one embodiment, the dry matter content of the ground peas is adjusted to the aforementioned dry matter content by adding water accordingly. 62. The method according to any one of claims 1 to 17, or 24 to 61, comprising the steps of: (i) grinding said peas; preferably dried peas;

(ii) fractionating said ground peas in the presence of an aqueous solution to obtain at least one aqueous composition comprising pea proteins;

(iii) isolating or concentrating said pea proteins from said aqueous composition comprising pea proteins;

(iv) obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8; and

(v) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature of at least 75 ° C.

Preferably, the aqueous suspension comprising pea proteins in step (iv) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most, 30%, preferably at most 25%, and in one embodiment the dry matter can be adjusted to this extent by dilution with water.

63. The method according to any of claims 1 to 17, or 24 to 62, comprising the steps of: (i) subjecting an aqueous composition comprising peas to fermentation, preferably in the presence of one or more lactic acid bacteria ;

(ii) grinding said peas;

(iii) fractionating said ground peas in the presence of an aqueous solution to obtain at least one aqueous composition comprising pea proteins;

(iv) isolating or concentrating said pea proteins from said aqueous composition comprising pea proteins;

(v) obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8; and

(vi) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature of at least 75 ° C.

Preferably, the aqueous suspension comprising pea proteins in step (v) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most, 30%, preferably at most 25%, and in one embodiment the dry matter can be adjusted to this extent by dilution with water.

64. The method according to any one of claims 26 to 63, wherein said fractionation step comprises fractionating said ground peas into a fraction comprising at least 50% by weight of protein based on the total dry matter of said fraction .

65. The method according to any of claims 26 to 64, wherein said fractionation step comprises separating at least part of the proteins comprised in the peas from the rest of the pea, preferably into a fraction that comprises at least 50% in protein weight based on the total dry matter of that fraction.

66. The method according to any one of claims 61 to 65, wherein obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8 comprises adjusting or maintaining the pH of the aqueous suspension to vary from 4.0 to 5.8.

67. Pea protein composition obtainable by the method according to any one of claims 1 to 17, or 24 to 66, comprising at least 60% by weight of protein based on the total dry matter of the composition , wherein said pea protein composition has a nitrogen solubility index at pH 7.0 of at most 15%, as measured in an aqueous composition comprising 3% by weight of said pea protein composition based on the total weight of the aqueous composition, and preferably said pea protein composition has an ISN of at most 11%, preferably at most 10%, preferably at most 9%, preferably, maximum 8%.

68. Pea protein composition according to any one of claims 19, 20 and 67, wherein said composition has a pH in the range of 4.0 to 5.8, as measured at room temperature in 10 g of pea protein composition suspended in 90 g of water.

In a first aspect, the invention relates to a method for extracting pea proteins, comprising the steps of:

(a) providing an aqueous composition comprising pea proteins;

(b) isolating or concentrating said pea proteins from said aqueous composition comprising pea proteins preferably using precipitation, flocculation, filtration and / or chromatography;

(c) obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8;

(d) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature of at least 75 ° C.

Preferably, the aqueous suspension comprising pea proteins in step (c) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most, 30%, preferably at most 25%, and in one embodiment can be adjusted to this extent by dilution with water.

According to the invention, steps (a) to (d) of the method according to the invention or as specified above can be performed, and preferably are performed in the following order, that is, step (a) precedes stage (b), which in turn precedes stage (c), which in turn precedes stage (d). However, it should be understood that the heat treatment in step (d) is in any case carried out after isolation of the proteins precipitated in step (c).

As used herein, the term "pea" refers to the round seeds contained in the pod of Pisum sativum and its subspecies, varieties, or cultivars. Preferably, the peas are yellow peas, preferably dried yellow peas, ie yellow peas that have been harvested in the dry state. Therefore, "pea proteins" as used herein refers to the proteins contained in pea seeds.

As used herein, "pea protein extraction" refers to the release and separation of pea proteins from other pea constituents. Pea protein extraction in accordance with certain embodiments of the invention may encompass isolation or purification of pea proteins. The person skilled in the art will understand that pea protein extracts do not consist entirely of proteins, and that a certain amount of additional components (impurities) may be present in pea protein extracts, such as lipids, carbohydrates, minerals. , etc.

In some embodiments of the invention, pea proteins, pea protein compositions, and pea protein extracts comprise, based on dry matter, at least 50% protein by weight (i.e., 50 g protein per 100 g of total dry matter), preferably at least 75% by weight of protein. In some embodiments, the pea protein extracts comprise, based on dry matter, at least 50% by weight to at most 95% by weight or 99% by weight of proteins, such as at least 75% by weight. % by weight to at most 99% by weight protein. Crude extracts typically comprise a smaller fraction of protein than refined or purified extracts.

As used herein, the term "aqueous composition comprising pea proteins" or "aqueous solution comprising pea proteins" refers to a composition or solution comprising water and pea proteins. In some embodiments, said solution may comprise additional constituents.

In one embodiment, the aqueous composition comprising pea proteins in step (a) of the method according to the invention or as described above comprises at least 1.0% dry matter based on the total weight of the composition , preferably, at least 2.0% dry matter, more preferably, at least 3.0% dry matter, such as, for example, at least 4.0% dry matter, such as, for example , at least 5.0% dry matter.

In another embodiment, the aqueous composition comprising pea proteins in step (a) of the method according to the invention or, as described above, comprises from 1.0% to 40% of dry matter, preferably from 2 0% to 30% dry matter, more preferably 3.0% to 20% dry matter, more preferably 3.0% to 15% dry matter, such as 3.0% to 10%

In one embodiment, the dry matter of the fraction comprising the protein comprises at least 50% by weight of pea proteins, preferably, at least 60% by weight of pea proteins, more preferably, at least 65% in weight of pea proteins, such as, for example, at least 70% by weight, such as at least 55% by weight and at most 80% by weight, for example, 60% by weight at 80% by weight, for example 60% by weight to 78% by weight.

In one embodiment, the pH of the aqueous composition comprising pea proteins has a pH or is adjusted to a pH of at least 6.0, preferably, the pH is or is adjusted to a pH of at least 6.5, preferably , in the range of pH 6.0 to 8.5, preferably in the range of pH 6.5 to 8.5, preferably in the range of pH 7.0 to 8.5, preferably pH 7, 3 to 8.0, such as, for example, at least pH 7.2, for example, at least 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7, 9, 8.0. For this purpose, for example, sodium hydroxide or any suitable base can be used to adjust the pH to the desired level.

In some embodiments, the aqueous composition comprising pea proteins in step (a) of the method according to the invention or, as described above, is an aqueous composition comprising ground peas.

As used herein, the term "milling" has its usual meaning in the art. By further guidance, milling, as used herein, may refer to the process of milling solid materials, i.e. peas, under exposure to mechanical forces that rip through the structure by overcoming internal bonding forces. Grinding can disintegrate the natural structure of peas. In a preferred embodiment, the ground particle size of a ground pea comprising at least 25% by weight of dry matter has a D50 of at most 300 pm, preferably at most 250 pm, for example at most, 200 pm, with D50 defined as the particle size for which fifty percent by volume of the particles are smaller than D50; and D50 measured by laser diffraction analysis on a Malvern-type analyzer.

For example, D50 can be measured by sieving or laser diffraction analysis. For example, Malvern Instruments laser diffraction systems can be used to advantage. Particle size can be measured by laser diffraction analysis on a Malvern-type analyzer. Particle size can be measured by analysis laser diffraction on a Malvern-type analyzer after the peas have been ground and are in a suspension of water with 25% dry matter. Suitable Malvern systems include Malvern 2000, Malvern MasterSizer 2000 (such as Mastersizer S), Malvern 2600 and Malvern 3600 series. Such equipment, together with their operation manual, meets or even exceeds the requirements set forth in ISO 13320. Malvern MasterSizer (like the Mastersizer S) can also be useful, as it can more accurately measure D50 towards the lower end of the range, for example for average particle sizes less than 8 | jm, applying Mie's theory, using appropriate optical media.

In certain embodiments, the ground peas are ground shelled peas, that is, peas removed from the pod. Shelled peas are peas from which the outer coating of the seeds is removed. The extraction of the sheath can be carried out by techniques known in the art, such as, for example, mechanically with descadores. It should be understood that when referring to shelled peas herein, in some embodiments, not all, but nevertheless, the vast majority of individual peas are shelled, such as preferably more than 90% of the peas are shelled.

In one embodiment, before, during, or after grinding the peas, an aqueous solution, preferably water, such as tap water or treated well water, preferably potable water, that is, water fit for human consumption, is added to the peas. . In a further embodiment, an amount of aqueous solution is added to the peas, such that said composition comprises 15% to 35% dry matter based on the total weight of the composition, preferably comprising 15% to 35% , preferably 20% to 30%, such as at least 19%, such as at least 20%, such as at least 21%, such as at least 22%, for example at least 23%, for for example at least 24%, for example at least 25%, for example at least 26%, for example at least 27%, for example at least 28%, for example at least 29%, for example, maximum 30%, for example maximum 35% dry matter based on the total weight of the composition. In a preferred embodiment, the milling process is a wet milling process, such that an aqueous solution is added to the peas before or during milling.

One skilled in the art will understand that if the aqueous composition comprising pea proteins is an aqueous composition comprising ground peas, all or substantially all of the pea constituents are comprised in the aqueous composition.

In a preferred embodiment, the aqueous composition comprising pea proteins in step (a) of the method according to the invention as described herein refers to a fraction comprising pea proteins, preferably obtained after grinding peas, and more preferably obtained after fractionating said ground peas. In one embodiment, step (c1) of the method according to the invention or, as described above, comprises fractionating said ground peas into a fraction comprising at least 50% by weight of protein based on the total dry matter of said fraction. As used herein, the term "fractionation" refers to a process by which at least part of the proteins comprised in the peas are separated from the rest of the pea. It should be understood that when referring to the fractionation step, in some embodiments, not all, but nevertheless, most of the individual proteins are separated, such as preferably at least 50% by weight, preferably at least 50% by weight. less 60% by weight of the proteins, based on the total protein content of the ground peas, are removed.

Providing an aqueous composition comprising pea proteins in step (a) can be achieved by any means known in the art, such as fractionating ground peas into a protein fraction. Fractionation of the ground peas into a fraction comprising proteins can be accomplished by any means known in the art, such as the addition of a suitable base or a salt.

Preferably, the ground peas are fractionated by adjusting the pH of the ground peas. Preferably, the ground peas are fractionated by increasing the pH of an aqueous composition comprising ground peas. Preferably, the fractionation step (c1) comprises adjusting the pH of the ground peas to a pH of at least 6, preferably at least 7, more preferably a pH of at least 8 and at most 9. Preferably, the fractionation step ( c1) comprises increasing the pH of an aqueous composition comprising ground peas. In a preferred embodiment, the pH of the composition is adjusted to a pH of at least 6, more preferably at least 7. In another preferred embodiment, the pH of the composition is adjusted to a value in the range of pH 6 to pH 9 , more preferably pH 7 to pH 9, such as at least 7.0, eg at least 7.1, eg at least 7.2, eg at least 7.3, eg at minus 7.4, for example, at least 7.5, for example, at least 7.6, for example, at least 7.7, for example, at least 7.8, for example, at least 7.9, for example, at least 8.0, for example, at least 8.1, for example, at least 8.2, for example, at least 8.3, for example, at least 8.4, for example, maximum 8.5, for example, maximum 8.6, for example, maximum 8.7, for example, maximum 8.8, for example, maximum 8.9, for example, maximum 9.0, whatever more preferably in the range of pH 7.5 to pH 8.5, most preferably pH 8 or about pH 8. Preferably, this pH adjustment is carried out in u An aqueous composition comprising ground peas having a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most 30%, preferably at most , 25%. In one embodiment, the dry matter content of the peas ground is adjusted to the dry matter content mentioned above by adding water accordingly. This pH adjustment can be made using any suitable base, such as sodium hydroxide, calcium hydroxide, potassium hydroxide, and the like. In a preferred embodiment, the pH of the compositions containing ground peas is adjusted by the addition of sodium hydroxide.

In a preferred embodiment, after pH adjustment, the fraction comprising proteins is separated from the aqueous composition comprising ground peas, by decantation or by the use of a hydrocyclone, preferably by decantation, preferably, centrifugal decantation (i.e., by middle of a decanting centrifuge), where the fraction that comprises the protein is the supernatant, and the sediment is a fraction that comprises, among others, the rest of the content of the ground peas and some residual proteins. In one embodiment, more than one fractionation step can be performed sequentially. For example, after decantation, the sediment can be suspended in an aqueous solution (preferably in an aqueous solution, which preferably has a similar or higher pH (preferably pH 8.5 or about pH 8.5) than in the first fractionation step) and is subjected to a decantation step, to recover additional proteins in the supernatant.

It should be understood that the grinding process of the peas can be carried out simultaneously with the fractionation of the ground peas, or alternatively, the grinding process of the peas can be carried out prior to the fractionation step.

It is to be understood that the fraction comprising proteins may also comprise additional constituents, notably those that are rendered soluble by or remain soluble by the fractionation step. In a preferred embodiment, the concentration (based on dry weight) of proteins in the fraction comprising the protein is at least 50% by weight, preferably, at least 60% by weight, such as at least 55% by weight and at most 80% by weight, for example, from 60% by weight to 80% by weight, for example, from 60% by weight to 78% by weight.

In one embodiment, the fraction comprising proteins comprises at least 1.0% dry matter based on the total weight of the composition, preferably, at least 2.0% dry matter, more preferably at least 3, 0% dry matter, such as, for example, at least 4.0% dry matter, such as, for example, at least 5.0% dry matter.

In another embodiment, the fraction comprising proteins comprises 1.0% to 40% dry matter, preferably 2.0% to 30% dry matter, more preferably 3.0% to 20% dry matter. more preferably 3.0% to 15% dry matter, such as 3.0% to 10%.

In one embodiment, the dry matter of the fraction comprising the protein comprises at least 50% by weight of pea proteins, preferably, at least 60% by weight of pea proteins, more preferably, at least 65% in weight of pea proteins, such as, for example, at least 70% by weight, such as at least 55% by weight and at most 80% by weight, or between 60% by weight and 80% by weight, or between 60% by weight and 78% by weight.

In some embodiments, in a further step, the fraction comprising the protein, also referred to herein as the aqueous composition comprising pea proteins is subjected to at least one heat treatment, preferably said fraction comprising protein is subjected to a temperature of at least 30 ° C, for example, at least 40 ° C, for example, at least 50 ° C, for example, said fraction comprising protein is subjected to a temperature in the range of 30 ° C to 90 ° C , more preferably, in the range of 50 ° C to 80 ° C, even more preferably, in the range of 55 ° C to 75 ° C, such as, for example, 55 ° C, 60 ° C, 65 ° C, 70 ° C or 75 ° C. In one embodiment, the heat treatment is 50 ° C to 60 ° C, eg 55 ° C to 65 ° C, eg 60 ° C to 70 ° C, eg 65 ° C to 75 ° C, for example, 70 ° C to 80 ° C. The skilled person will understand that said heat treatment can be pasteurization. Pasteurization is well known in the art and may comprise heat treatment at a specific temperature or temperature range for a specific time or time interval. The person skilled in the art will understand that, in general, when the temperature of the heat treatment increases, the duration of the heat treatment decreases.

Step (b) of the present process comprises isolating said pea proteins from said aqueous composition comprising pea proteins (ie, from said protein-comprising fraction). As used herein, the term "isolated" or "isolation" can refer to a process that separates proteins from said proteins that comprise the fraction. The term "concentration" can also be used interchangeably with "isolation". Accordingly, in one embodiment, in step (b) of the method according to the invention or as described above, the pea proteins are concentrated from said aqueous composition comprising pea proteins. Preferably, said isolation or concentration step can be carried out using precipitation, flocculation, filtration and / or chromatography, or a combination thereof.

Also described herein is a method for extracting pea proteins, comprising the steps of:

(a) providing an aqueous composition comprising pea proteins, wherein said composition is obtained by a method comprising the steps of:

(a1) grinding peas, preferably shelled peas;

(b1) fractionating said ground peas to obtain at least one fraction comprising proteins thereby forming an aqueous composition comprising pea proteins;

(b) isolating or concentrating said pea proteins from said aqueous composition comprising pea proteins preferably using precipitation, flocculation, filtration and / or chromatography;

(c) obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8;

(d) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature of at least 75 ° C.

Preferably, the aqueous suspension comprising pea proteins in step (c) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most, 30%, preferably at most 25%, and in one embodiment can be adjusted to this extent by dilution with water.

In one embodiment, the isolation or concentration of said pea proteins from said aqueous composition comprising pea proteins can be carried out using precipitation, flocculation, filtration and / or chromatography.

Preferably, the proteins are isolated or concentrated by isoelectric precipitation or by ultrafiltration. In a preferred embodiment, the isolation or concentration of pea proteins from said composition comprises at least one step of isoelectric precipitation of said proteins. Preferably, the pH of the composition comprising the pea proteins is adjusted to the isoelectric point of the proteins. As used herein, the term "isoelectric point" refers to the pH at which proteins have a net ionic charge of 0, or substantially 0 (that is, the sum of the positive and negative charges is 0, or substantially 0). While it is appreciated that the isoelectric point of individual proteins can vary, as used herein, the isoelectric pH of protein compositions as used herein refers to the pH of the composition at which the Overall charge of the proteins in the composition is 0, or substantially 0. The isoelectric pH of proteins and protein compositions can be determined by techniques known in the art. Herein, isoelectric pH is determined as the pH at which the solubility index of nitrogen is the lowest. In a preferred embodiment, the pH of the composition comprising the proteins is adjusted in the range of 4.0 to 5.8, preferably 4.5 to 5.5, such as, for example, 4.0, 4, 1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. The pH adjustment can be carried out by adding an acid, such as sulfuric acid or hydrochloric acid. At the isoelectric point, most proteins precipitate or aggregate.

In certain embodiments, isolation of precipitated or aggregated proteins is accomplished by separating a liquid fraction from an insoluble fraction, the latter comprising precipitated or aggregated pea proteins. Separation of precipitated or aggregated proteins can be effected by decantation, preferably centrifugal decantation. In a preferred embodiment, the dry matter content (by weight) after separation of the precipitated or aggregated proteins ranges from 20% to 40%, such as, for example, at least 25%, for example at least 26 %, for example at least 27%, for example at least 28%, for example at least 29%, 30%, 31%, 32%, 33%, 34% or 35%, preferably at least 27 % and maximum 38% based on the total weight of the precipitated or aggregated proteins. The dry matter content can be further adjusted, for example, by adding an aqueous solution to the precipitated or added proteins, thus obtaining a composition of precipitated proteins, preferably water, preferably potable water, that is, water suitable for human consumption. Preferably, the dry matter content can be adjusted to range from 10% to 25%, preferably 15% to 20%, such as, for example, at least 15%, for example, at least 16%, preferably, at least 17%, 18%, 19%, 20%, based on the total weight of the precipitated protein composition. Optionally, the protein isolation process can be repeated at least one more time. Preferably, the step of concentrating the proteins is performed only once.

In a preferred embodiment, the precipitated or aggregated proteins are preferably resuspended in an aqueous solution, preferably water, preferably potable water, that is, water suitable for human consumption. The dry matter content ranges preferably from 10% to 25%, preferably from 15% to 20%, such as, for example, at least 15%, for example at least 16%, 17%, 18%, 19% 20% of the protein composition resuspended.

According to the invention, the pH of the composition comprising the reconstituted proteins is adjusted (if necessary) or maintained in a range from 4.0 to 5.8, preferably from pH 4.5 to 5.5, such such as pH 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5. For this purpose, for example, sodium hydroxide or sulfuric acid can be used to adjust the pH to the desired level. Accordingly, obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8 in step (c) of the method described above comprises adjusting or maintaining the pH of the aqueous suspension. to range from 4.0 to 5.8, preferably from pH 4.5 to 5.5, such as, for example, pH 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5. Preferably, this pH adjustment is carried out in an aqueous composition comprising the reconstituted proteins having a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most 30%, preferably at most 25%. In one embodiment, the dry matter content of the composition is adjusted to the aforementioned dry matter content by adding water accordingly.

In step (d) as described herein, said aqueous suspension having a pH in the range of 4.0 to 5.8 is subjected to heat treatment at a temperature of at least 75 ° C, preferably, the aqueous suspension is subjected to a temperature of at least 77 ° C, preferably, at least 78 ° C, preferably, at least 80 ° C, even more preferably, at least 85 ° C, even more preferably, at least 90 ° C, for example, at least 95 ° C, preferably at most 160 ° C, even more preferably, at most 210 ° C. Preferably, said proteins are subjected to a heat treatment at a temperature in the range of 75 ° C to 210 ° C, preferably, in the range of 85 ° C to 160 ° C, more preferably, in the range of 90 ° C to 150 ° C. The heat treatment can advantageously be carried out by means of one or more heat exchangers or by direct or indirect steam injection. In one embodiment, the duration of the heat treatment is at least 0.01 second, preferably in the range of 0.01 second to 20 min, preferably in the range of 10 seconds to 10 minutes. The person skilled in the art will appreciate that the higher the temperature, the shorter the duration of the heat treatment. For example, the heat treatment can be at a temperature in the range between 115 ° C and 210 ° C for a time in the range of 0.01 s to 15 s. Alternatively, for example, the heat treatment can be at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 15 s to 5 min. Alternatively, for example, the heat treatment can be at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 5 min to 15 min. In a preferred embodiment, the heat treatment is carried out at a temperature in the range of 75 ° C to 110 ° C, even more preferably at a temperature in the range of 80 ° C to 100 ° C, for a time in the range range from 2 min to 10 min, preferably, for a time in the range of 5 min to 8 min. In another preferred embodiment, the heat treatment is carried out at a temperature in the range between 130 ° C and 150 ° C for a time in the range of 1 s to 8 s. After heat treatment, the protein-containing compositions can be kept at a temperature in the range of 70 ° C to 90 ° C; preferably, in the range of 70 ° C to 85 ° C, before drying.

According to the invention, in step (d) of the method according to the invention as described herein, said aqueous suspension having a pH in the range of 4.0 to 5.8 is subjected to treatment thermal at a temperature in the range of 115 ° C to 210 ° C for a time in the range of 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s. Preferably, the heat treatment time decreases as the heat treatment temperature increases.

In one embodiment, said aqueous suspension having a pH in the range of 4.0 to 5.8 at a temperature in the range of 75 ° C to 210 ° C can be subjected to drying after step (d) of the method of in accordance with the invention as described herein. Drying can be accomplished by any means in the art, such as hot air application, evaporation, lyophilization, contact drying, steam drying, dielectric drying, roller drying, flash drying, etc. In a preferred embodiment, the aqueous suspension having a pH in the range of 4.0 to 5.8 at a temperature in the range of 75 ° C to 210 ° C is dried by spray drying. Optionally, the proteins can be subjected to granulation, by techniques known in the art.

In certain embodiments, the pea proteins in the aqueous composition comprising pea proteins in step (a) of the methods according to the invention as described herein can be derived from ground peas. In one embodiment, said ground peas are emitted from peas that have been hydrated, or said peas are dry ground and hydrated.

In another embodiment, said ground peas have been fermented before grinding. When whole peas are fermented prior to grinding, advantageously removal of fermenting microorganisms, as well as fermentation by-products, such as lactic acid, but also secreted compounds such as enzymes, which can affect downstream processing, they are easily separated and profitably from peas after fermentation. In addition, unexpectedly, when fermenting whole peas the content of monosaccharides, disaccharides and / or oligosaccharides of peas and, in particular, monomeric or dimeric sugars, such as glucose, fructose, sucrose, galactose and / or flatulent sugars, such as raffinose , stachyose, and verbascosa, all of which are within peas are drastically reduced, which is even more surprising when the limited duration of fermentation in some embodiments is taken into account.

As used herein, the term "sugar" or "free sugar" refers to monosaccharides, disaccharides, and / or oligosaccharides consisting of up to 10 monomer units. In some embodiments, when "total sugars" or "total free sugars" are referred to, these encompass the total of monosaccharides, disaccharides, and / or oligosaccharides consisting of up to 10 monomer units. In other embodiments, a specific subset of sugars is specified.

In one embodiment, an aqueous composition comprising peas is fermented, preferably in the presence of one or more lactic acid bacteria. Preferably, the peas that are fermented are unground peas (ie, whole peas). However, the peas can be, in one embodiment, split peas. In one embodiment, the peas are round when harvested and dried. After removing the pod, the natural division in the cotyledon of the seed can be separated manually or mechanically, resulting in "split peas".

Peas, as used herein, can be graded prior to fermentation. For example, stones or larger plant material, but also damaged peas, can be removed from the peas to be used according to the invention.

In such embodiments where the peas are fermented, the peas, preferably dry peas, preferably shelled peas, such as dried shelled peas are suspended in an aqueous solution. In a preferred embodiment, the aqueous solution is water. In one embodiment, the water can be potable water or well water that has been treated to make it potable. The water used is preferably potable water, that is, water suitable for human consumption.

In some embodiments, the amount of peas that is added to the aqueous solution to reconstitute the aqueous composition comprising peas preferably ranges from 150 to 500 kg of peas per m3 of aqueous composition comprising the peas, that is, per 150 to 500 kg of peas, an aqueous solution is added until a final volume of 1 m3 is reached.

In one embodiment, the aqueous composition comprising the peas at the beginning of fermentation has a pH of at least 6.0, preferably at least 6.2, for example at least 6.4, as measured in the composition. aqueous comprising peas, after said composition has been ground.

In a preferred embodiment, the peas that are contacted with the aqueous composition are naturally harvested dry, or in one embodiment the peas can be fresh peas. Preferably, the peas are dry peas, and have a dry matter content (by weight) of at least 80% (i.e., at least 80 g of dry matter per 100 g of total weight of the dry peas), more preferably , of at least 85%, for example, of at least 90%, for example, of at least 95%, such as in the range of 80% to 95%, for example 85% to 95 %, for example 90% to 95%.

As used herein, the term "fermentation" has its usual meaning in the art. By further guidance, fermentation is a microbiological metabolic process that involves the conversion of sugar to acids and / or gases using yeast and / or bacteria. Subjecting an aqueous composition comprising peas to fermentation as used herein, therefore, may refer to incubating the aqueous composition comprising peas with bacteria and / or yeast, preferably lactic acid bacteria, under suitable conditions so that bacteria and / or yeasts are metabolically active.

As used herein, "lactic acid bacteria" refers to a population of Gram-positive, low-GC, acid-tolerant, generally non-sporulating, non-breathing cocci or bacilli that are associated due to their common metabolic and physiological characteristics, and that they produce lactic acid as the main metabolic end product of carbohydrate fermentation. These bacteria can normally be found in lactic products and decaying plants. As used herein, lactic acid bacteria may be non-pathogenic in the sense that they do not cause harm or do not lead to harmful effects when ingested. Preferably, the lactic acid bacteria as used herein are one or more bacterial genera selected from Lactobacillus, Pediococcus, Lactococcus, Leuconostoc, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, and Weistragococus and combinations , and Vaistragococus. thereof. Most preferably, the lactic acid bacteria are Lactobacillus sp, most preferably selected from the group consisting of Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus panis, Lactobacillus mucosae, Lactobacillus pontis, Lactobacillus acidophilus, Lactobacillus plantarillus, Lactobacillus plantarii dellactobacillus, Lactobacillus plantarii. , and Lactobacillus casei, and mixtures thereof, for example from the group consisting of Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus panis, Lactobacillus mucosae, Lactobacillus pontis, Lactobacillus acidophilus and mixtures thereof, for example from the group consisting of Lactobacillus fermentum , Lactobacillus crispatus, Lactobacillus panis, Lactobacillus mucosae, Lactobacillus pontis and mixtures thereof, for example said bacterium is Lactobacillus fermentum, or Lactobacillus crispatus. In some embodiments, the fermentation may be spontaneous fermentation (i.e., in which fermenting microorganisms are not deliberately added, but the fermentation is carried out by microorganisms, preferably lactic acid bacteria, which occur in nature on / in peas and / or in the environment) or can be inoculated fermentations (i.e., in which fermenting microorganisms, preferably lactic acid bacteria, are deliberately added ). Fermentation can also be effected by transferring part or all of the aqueous fraction from one fermentation stage to the next fermentation to be started, for example by transferring at least 1/10 of the first fermentation volume to at least one second fermentation stage. In a preferred embodiment, the fermentation is anaerobic, (not strictly anaerobic) fermentation. In a preferred embodiment, said Lactobacillus fermentum is Lactobacillus fermentum LMG 6902 or LMG 18026. In a preferred embodiment, said Lactobacillus Crispatus is Lactobacillus Crispatus LMG 12005. In a preferred embodiment, said Lactobacillus acidophilus is Lactobacillus LMG 8151.

In one embodiment, the aqueous composition comprising peas is fermented until the pH in the peas is at most 5.5, preferably at most 5.0, more preferably, ranging from 3.5 to 5, preferably such as measured at room temperature in 1 g of said peas that have been ground and then suspended in 9 g of water, as described in the experimental section. In one embodiment, the aqueous composition comprising peas is fermented until the pH in the peas ranges from 3.5 to 4.5, for example 4.0 to 5.0, preferably 4.5 to 5 , 5, such as for example at least 3.5, for example at least 3.75, for example at least 4.0, for example at least 4.25, for example at least 4.50, for eg at least 4.75, eg at most 5.0, eg at most 5.25, eg at most 5.5, preferably, as measured at room temperature in 1g of said peas which have been ground and then suspended in 9 g of water, as described in the experimental section.

In one embodiment, the dried peas have a pH of at least 6.0, preferably in the range of 6.0 to 7.10 before fermentation, such as for example at least 6.0, for example at least 6.1, for example, at least 6.2, for example, at least 6.3, for example, 6.4, for example, 6.5, for example, 6.6, for example, 6.7, eg 6.8, eg 6.9, eg 7.10, preferably in the range 6.25 to 6.75, preferably as measured at room temperature in 1g of said peas which have been ground and then suspended in 9 g of water, as described in the experimental section.

In one embodiment, the aqueous composition comprising peas is fermented until the pH in the peas decreases by at least 1 pH unit, preferably by at least 1.5 pH units, such as for example at least 1, for example , at least 1.1, for example, at least 1.2, for example, at least 1.3, for example, at least 1.4, for example, at least 1.5, for example, at least 1, 6, for example, at least 1.7, for example, at least 1.8, for example, at least 1.9, for example, at least 2, for example, at least 2.1, for example, at least 2.2, for example, at least 2.3, for example, at least 2.4, for example, at least 2.5, for example, at least 2.6, for example, at least 2.7, by example, at least 2.8, for example, at least 2.9, for example, at least 3 pH units, preferably, as measured at room temperature in 1 g of said peas that have been ground and then suspended in 9 g of water, as described in the experimental section. In another embodiment, the aqueous composition comprising peas is fermented until the pH in the peas decreases by 1 pH unit to 3 pH units, preferably by 1.5 pH units to 3 pH units, such as , for example, in 1.5 pH units to 2.5 pH units, for example, in 2.0 pH units to 3.0 pH units, preferably, as measured at room temperature in 1 g of said peas that have been ground and then suspended in 9 g of water, as described in the experimental section. By way of example, and without limitation, at the beginning of fermentation, the pH in peas may be 6.5, and at the end of fermentation, the pH in peas may be 5.0, preferably as measured. at room temperature in 1 g of said peas that have been ground and then suspended in 9 g of water, as described in the experimental section.

In one embodiment, the aqueous composition comprising peas is fermented for at least 3 hours, preferably at least 4 hours, more preferably at least 6 hours. In another embodiment, the aqueous composition comprising peas is fermented for a time in the range of 3 h to 24 h, preferably in the range of 4 h to 24 h, more preferably, in the range of 4 h to 20 h, such such as, for example, at least 3 h, for example, at least 4 h, for example, at least 5 h, for example, at least 6 h, for example, at least 7 h, for example, at least 8 h, at least 9 h, around 10 h, around 11 h, around 12 h, around 13 h, around 14 h, for example, at most 15 h, for example, at most 16 h, for example, as Maximum 17 hours, for example, maximum 18 hours, for example, maximum 19 hours, for example, maximum 20 hours, for example, maximum 21 hours, for example, maximum 22 hours, for example, maximum 23 h, for example, maximum 24 h. The skilled person will appreciate that, for example, spontaneous fermentations may take longer than fermentations which are carried out by the addition of bacteria, in view of the different amounts of microorganisms at the beginning of the fermentation.

In one embodiment, the aqueous composition comprising peas is fermented at an optimum temperature for the fermenting microorganism, preferably, at a temperature that is at most 5 ° C higher or lower than the temperature that is optimal for the fermenting microorganism. fermentation. Optimal temperatures for bacteria and / or yeasts as defined herein are known in the art. By means of further guidance, and without limitation, an optimum temperature as defined herein refers to the temperature at which growth is maximized. In a further embodiment, the aqueous composition comprising peas is fermented at a temperature of at least 30 ° C, for example, in the range of 30 ° C to 50 ° C, preferably, in the range of 35 ° C to 45 ° C. In another embodiment, the aqueous composition comprising Peas are fermented at a temperature in the range of 30 ° C to 40 ° C, 35 ° C to 45 ° C, or 40 ° C to 50 ° C, preferably 40 ° C, or about 40 ° C.

In one embodiment, the aqueous composition comprising peas is fermented in the presence of fermenting microorganisms, such as bacteria and / or yeasts, preferably, comprising one or more lactic acid bacteria, more preferably, said lactic acid bacteria are selected from the group comprising one or more Lactobacillus sp .. In one embodiment, the fermentation is carried out in the presence of one or more of the microorganisms specified above, preferably lactic acid bacteria, at a concentration in the range of 102 cfu / ml to 1010 cfu / ml of said aqueous composition comprising peas, such as at least 104 cfu / ml, for example, at least 105 cfu / ml, for example, at least 106 cfu / ml, for example, at least 107 cfu / ml , for example, at least 108 cfu / ml, for example, at least 109 cfu / ml of said aqueous composition comprising peas. "Cfu" (colony forming units) are well known in the art and can be determined, for example, by plate counting. It is to be understood that "cfu / ml" refers to the amount of cfu per ml of the total aqueous composition comprising peas, ie including peas.

In another embodiment, the aqueous composition comprising the peas is subjected to fermentation in the presence of fermenting microorganisms, preferably comprising one or more lactic acid bacteria, preferably comprising one or more Lactobacillus sp., Wherein the microorganisms, preferably bacteria of lactic acid, are added at a concentration of at least 102 cfu / ml of aqueous composition comprising peas.

In one embodiment, the peas at the end of the fermentation and before the milling step have a dry matter content (by weight) in the range of 35% to 60%, preferably 35% to%, for example 40% to 50%, such as, for example, at least 40%, for example, at least 41%, at least 42%, for example, at least 43%, for example, at least 44% eg at least 45% eg at least 46% eg at least 47% approx 48% approx 49% eg at most 50% eg at most 55%, eg at most 60% based on the total weight of the peas at the end of the fermentation, ie after the peas have been isolated from the aqueous composition.

In a further embodiment, the peas are fermented until they have a dry matter content (by weight) in the range of 35% to 60%, preferably 35% to 55%, for example 40% to 50%, such as for example at least 40% for example at least 41% at least 42% for example at least 43% for example at least 44% for example at least 45 %, for example, at least 46%, for example, at least 47%, about 48%, about 49%, for example, at most, 50%, for example, at most, 55%, for example, maximum 60% based on the total weight of the peas at the end of the fermentation, that is, after the peas have been isolated from the aqueous composition. In this embodiment, the peas preferably have a dry matter content (by weight) before fermentation, or at the beginning of the fermentation of at least 80% (that is, at least 80 g of dry matter per 100 g of weight. total dried peas), more preferably at least 85%, for example, at least 90%, for example at least 95%, such as in the range of 80% to 95% %, for example 85% to 95%, for example 90% to 95%.

In one embodiment, peas that have undergone fermentation are ground. To this end, in one embodiment, the peas can be removed from the aqueous composition after fermentation and then subjected to milling. Preferably, the peas are washed or rinsed after fermentation and before grinding. Washing or rinsing can be carried out with an aqueous solution, preferably water, such as treated water or well water, preferably potable water, that is, water suitable for human consumption.

In a preferred embodiment, the method for extracting pea proteins from Pisum sativum ssp., Comprises the steps of:

(i) provide peas;

(ii) grinding said peas;

(iii) fractionating said ground peas in the presence of an aqueous solution to obtain at least one aqueous composition comprising pea proteins;

(iv) isolating or concentrating said pea proteins from said aqueous composition comprising pea proteins;

(v) obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8; and

(vi) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature in the range of 75 ° C to 210 ° C;

wherein step (b) comprises adjusting the pH of said aqueous composition comprising pea proteins to a value in the range of 4.0 to 5.8, preferably in the range of 4.5 to 5.5;

wherein step (d) comprises subjecting said precipitated pea proteins to a heat treatment at a temperature in the range of 115 ° C to 210 ° C for a time in the range of 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s.

Preferably, the aqueous suspension comprising pea proteins in step (v) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most, 30%, preferably at most 25%, and in one embodiment can be adjusted to this extent by dilution with water.

In a preferred embodiment, the method for extracting pea proteins from Pisum sativum ssp, comprises the steps of:

(i) subjecting an aqueous composition comprising peas to fermentation, preferably in the presence of one or more lactic acid bacteria;

(ii) grinding said peas;

(iii) fractionating said ground peas in the presence of an aqueous solution to obtain at least one aqueous composition comprising pea proteins;

(iv) isolating or concentrating said pea proteins from said aqueous composition comprising pea proteins;

(v) obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8; and

(vi) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature in the range of 75 ° C to 210 ° C;

wherein step (b) comprises adjusting the pH of said aqueous composition comprising pea proteins to a value in the range of 4.0 to 5.8, preferably in the range of 4.5 to 5.5;

wherein step (d) comprises subjecting said precipitated pea proteins to a heat treatment at a temperature in the range of 115 ° C to 210 ° C for a time in the range of 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s.

Preferably, the aqueous suspension comprising pea proteins in step (v) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably at most, 30%, preferably at most 25%, and in one embodiment can be adjusted to this extent by dilution with water.

In a preferred embodiment, the method for extracting pea proteins from Pisum sativum ssp., Comprises the steps of:

(i1) providing an aqueous composition comprising pea proteins;

(ii1) isolating or concentrating said pea proteins from said aqueous composition comprising pea proteins, preferably using isoelectric precipitation, preferably adjusting the pH of said aqueous composition to a pH in the range 4.0 to 5.8;

(iii1) obtaining said isolated or concentrated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8;

(iv1) optionally adjust the dry matter content of the aqueous suspension to a value in the range of 10% to 25%, preferably 15% to 20%;

(v1) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature in the range of 75 ° C to 210 ° C;

wherein step (b) comprises adjusting the pH of said aqueous composition comprising pea proteins at a value in the range 4.0 to 5.8, preferably in the range 4.5 to 5.5;

wherein step (d) comprises subjecting said precipitated pea proteins to a heat treatment at a temperature in the range of 115 ° C to 210 ° C for a time in the range of 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s;

(vi1) drying said aqueous suspension having a pH in the range of 4.0 to 5.8, preferably obtaining a pea protein composition having a pH in the range of 4.0 to 5.8, as described measured at room temperature in 10 g of pea protein composition suspended in 90 g of water.

Preferably, the aqueous suspension comprising pea proteins in step (iii1) has a dry matter of at most 45%, preferably at most 40%, preferably at most 35%, preferably as maximum 30%, preferably maximum 25%, and in one embodiment can be adjusted to this extent by dilution with water.

The pea protein extracts obtained by the methods according to the present invention as described herein have different characteristics, such as different biochemical and / or organoleptic characteristics, as well as a difference in the values of the parameters associated with the quality compared to pea proteins known from the prior art.

Accordingly, in one aspect, the present invention encompasses pea protein extracts and pea protein compositions obtained by or obtainable by the methods according to the invention as described herein.

In a further aspect, the invention relates to a pea protein composition comprising at least 60% by weight of protein based on the total dry matter of the composition, wherein said pea protein composition has a solubility index nitrogen at pH 7.0 of at most 15%, as measured in an aqueous composition comprising 3% by weight of said pea protein composition based on the total weight of the aqueous composition, and preferably an ISN at pH 7.0 at most 11%, preferably at most 10%, preferably at most 9%, preferably at most 8%. In a preferred embodiment, said composition is obtained by the method of the invention.

In a further aspect, the invention relates to a pea protein composition having a gel strength at pH 6 of at most 100g, preferably at most 75g, even more preferably at most 50g. In another aspect, the invention relates to pea proteins having a gel strength at pH 6 in the range of 10 g to 100 g, preferably in the range of 10 g to 75 g, even more preferably in the range of 10 g. g to 50 g.

In a further aspect, the invention relates to a pea protein composition comprising at least 60% by weight of protein based on the total dry matter of the composition, wherein said pea protein composition has a solubility index nitrogen at pH 7.0 of at most 15%, as measured in an aqueous composition comprising 3% by weight of said pea protein composition based on the total weight of the aqueous composition, and preferably an ISN at pH 7.0 of at most 11%, preferably at most 10%, preferably at most 9%, preferably at most 8%, and a gel strength at pH 6 of, as maximum 100 g, preferably maximum 75 g, even more preferably maximum 50 g. In another aspect, the invention relates to pea proteins having a gel strength at pH 6 in the range of 10 g to 100 g, preferably in the range of 10 g to 75 g, even more preferably in the range of 10 g. g to 50 g.

In further aspects, the invention relates to pea proteins having one or more, preferably all of the A to H characteristics as indicated in Table 1.

Table 1

In preferred embodiments, pea proteins have any of the following combinations of characteristics from Table 1: A, B, C, D, E, F, G, H, A + B, A + C, A + D, A + E, A + F, A + G, A + H, B + C, B + D, B + E, B + F, B + G, B + H, C + D, C + E, C + F , C + G, C + H, D + E, D + F, D + G, D + H, E + F, E + G, E + H, F + G, F + H, G + H, A + B + C, A + B + D, A + B + E, A + B + F, A + B + G, A + B + H, A + C + D, A + C + E, A + C + F, A + C + G, A + C + H, A + D + E, A + D + F, A + D + G, A + D + H, A + E + F, A + E + G , A + E + H, A + F + G, A + F + H, A + G + H, B + C + D, B + C + E, B + C + F, B + C + G, B + C + H, B + D + E, B + D + F, B + D + G, B + D + H, B + E + F, B + E + G, B + E + H, B + F + G, B + F + H, B + G + H, C + D + E, C + D + F, C + D + G, C + D + H, C + E + F, C + E + G , C + E + H, C + F + G, C + F + H, C + G + H, D + E + F, D + E + G, D + E + H, D + F + G, D + F + H, D + G + H, E + F + G, E + F + H, E + G + H, F + G + H, A + B + C + D, A + B + C + E , A + B + C + F, A + B + C + G, A + B + C + H, A + B + D + E, A + B + D + F, A + B + D + G, A + B + D + H, A + B + E + F, A + B + E + G, A + B + E + H, A + B + F + G, A + B + F + H, A + B + G + H, A + C + D + E, A + C + D + F, A + C + D + G, A + C + D + H, A + C + E + F, A + C + E + G, A + C + E + H, A + C + F + G, A + C + F + H, A + C + G + H, A + D + E + F, A + D + E + G , A + D + E + H, A + D + F + G, A + D + F + H, A + D + G + H, A + E + F + G, A + E + F + H, A + E + G + H, A + F + G + H, B + C + D + E, B + C + D + F, B + C + D + G, B + C + D + H, B + C + E + F, B + C + E + G, B + C + E + H, B + C + F + G, B + C + F + H, B + C + G + H, B + D + E + F, B + D + E + G, B + D + E + H, B + D + F + G, B + D + F + H, B + D + G + H, B + E + F + G, B + E + F + H, B + E + G + H, B + F + G + H, C + D + E + F, C + D + E + G, C + D + E + H, C + D + F + G, C + D + F + H, C + D + G + H, C + E + F + G, C + E + F + H, C + E + G + H, C + F + G + H, D + E + F + G, D + E + F + H, D + E + G + H, D + F + G + H, E + F + G + H, A + B + C + D + E, A + B + C + D + F, A + B + C + D + G, A + B + C + D + H, A + B + C + E + F, A + B + C + E + G, A + B + C + E + H, A + B + C + F + G, A + B + C + F + H, A + B + C + G + H, A + B + D + E + F, A + B + D + E + G, A + B + D + E + H, A + B + D + F + G, A + B + D + F + H, A + B + D + G + H, A + B + E + F + G, A + B + E + F + H, A + B + E + G + H, A + B + F + G + H, A + C + D + E + F, A + C + D + E + G, A + C + D + E + H, A + C + D + F + G, A + C + D + F + H, A + C + D + G + H, A + C + E + F + G, A + C + E + F + H, A + C + E + G + H, A + C + F + G + H, A + D + E + F + G, A + D + E + F + H, A + D + E + G + H, A + D + F + G + H, A + E + F + G + H, B + C + D + E + F, B + C + D + E + G, B + C + D + E + H, B + C + D + F + G, B + C + D + F + H, B + C + D + G + H, B + C + E + F + G, B + C + E + F + H, B + C + E + G + H, B + C + F + G + H, B + D + E + F + G, B + D + E + F + H, B + D + E + G + H, B + D + F + G + H, B + E + F + G + H, C + D + E + F + G, C + D + E + F + H, C + D + E + G + H, C + D + F + G + H, C + E + F + G + H, D + E + F + G + H, A + B + C + D + E + F, A + B + C + D + E + G, A + B + C + D + E + H, A + B + C + D + F + G, A + B + C + D + F + H, A + B + C + D + G + H, A + B + C + E + F + G, A + B + C + E + F + H, A + B + C + E + G + H, A + B + C + F + G + H, A + B + D + E + F + G, A + B + D + E + F + H, A + B + D + E + G + H , A + B + D + F + G + H, A + B + E + F + G + H, A + C + D + E + F + G, A + C + D + E + F + H, A + C + D + E + G + H, A + C + D + F + G + H, A + C + E + F + G + H, A + D + E + F + G + H, B + C + D + E + F + G, B + C + D + E + F + H, B + C + D + E + G + H, B + C + D + F + G + H, B + C + E + F + G + H, B + D + E + F + G + H, C + D + E + F + G + H, A + B + C + D + E + F + G, A + B + C + D + E + F + H, A + B + C + D + E + G + H, A + B + C + D + F + G + H, A + B + C + E + F + G + H , A + B + D + E + F + G + H, A + C + D + E + F + G + H, B + C + D + E + F + G + H, A + B + C + D + E + F + G + H.

The skilled person will understand that when referring to "pea proteins" in some embodiments, in fact a composition is disclosed, which predominantly, but not exclusively, comprises pea proteins. Residual impurities can be present in such compositions. Said residual impurities can include, for example, minerals, sugars, etc. In a preferred embodiment, the term "pea proteins" preferably refers to a composition comprising (based on the total dry matter of the composition) at least 60% by weight of proteins, preferably, at least 75% by weight of proteins. , more preferably, at least 80% by weight. In another preferred embodiment, the term "pea proteins" refers to a pea protein extract or a pea protein composition comprising (based on the total dry matter of the composition) from 70% by weight to 98% by weight of proteins, preferably 75% by weight to 98% by weight protein, more preferably 80% by weight to 98% by weight protein, more preferably 85% by weight to 98% by weight, even more preferably , 88% by weight to 98% by weight.

Also described herein is a composition comprising pea proteins obtained by or obtainable by the methods according to the invention as described herein. In a preferred example, such a composition is an edible composition. As used herein, and as will be understood by one of ordinary skill in the art, an "edible" composition refers to a composition that is suitable for human or animal consumption. Preferably, said composition is a food or feed, more preferably a bakery food product, or a confectionery food product. In a preferred embodiment, said food product is a cookie, bread, cake, waffle, dulce de leche, extruded cereal or bar.

Accordingly, in a further aspect, the present invention relates to the use of the pea protein extract as described herein, in particular, the pea protein extract obtained or obtainable according to the methods described. in this document, in food or feed products. In a preferred embodiment, the food products are bakery food products or confectionery food products. Pea protein extract as described herein can, for example, partially or completely replace other proteins in foods or food products, such as, for example, proteins of animal origin, such as milk proteins. Particularly suitable applications of the pea protein extract as described herein can be used, for example, in processes for preparing bakery food products or confectionery products.

In a further aspect, the invention relates to the use of the pea protein extract as described herein, in particular, the pea protein extract obtained or obtainable according to the methods described herein. document for clarifying or clarifying liquids, such as beverages and beverages.

As used herein, the terms "clarify" and "clarify" have their usual meaning in the art. By way of further guidance, and without limitation, the term "rinse" refers to a process by which, for example, insoluble (suspended) elements are removed from a liquid by the addition of a rinse agent. The addition of a rinse agent can cause insoluble elements to aggregate, but also to form certain soluble elements of larger particles (eg, proteins), which can be easily removed, such as by filtration or centrifugation. In one embodiment, the rinsing or clarifying liquid is a beverage, that is, a liquid suitable for human or animal consumption. In a particularly preferred embodiment, said beverage is a fermented beverage, such as an alcoholic beverage, preferably wine (including, but not limited to, white wine, rosé wine, champagne, Port, Jerez, etc.). Also contemplated are the uses of pea proteins as described herein in accordance with the invention to clarify fermented beverages or beverages other than wine.

Aspects and embodiments of the invention are further supported by the following non-limiting examples.

Examples

Protocols

Unless otherwise specified, in the examples below, all parameters are measured as defined in this section. The measurement of the parameters as defined in this section also represents in preferred embodiments the method for measuring said parameters according to the invention as indicated in the respective aspects and embodiments of the detailed description above.

Unless otherwise specified, in the examples below, all parameters are measured as defined in this section. The measurement of the parameters as defined in this section also represents in preferred embodiments the method for measuring said parameters according to the invention as indicated in the respective aspects and the embodiments of the detailed description above.

PH measurement in dry peas or aqueous composition comprising peas or ground peas

The pH was measured with a WTW SERIES Inolab Termil 740 pH meter. The instrument was calibrated with pH 4.01 buffers (WTW pH 4.01 Technical Buffer, Model STP4, Order No. 108706) and pH 7 (WTW pH 7.00 Technical Buffer, Model STP7, Order No. 108708).

When the pH was measured in the aqueous composition, excluding the peas, a sample of the aqueous solution was taken directly from the fermentation vessel. The pH of the sample was measured once the value stabilized.

When the pH was measured in peas, the peas were taken from the fermentation vessel. The peas were drained in a strainer and then placed on absorbent paper for two minutes to remove excess juice. The peas were ground for one minute with a blender (Magic Bullet, Homeland Housewares). 1 g of ground peas was suspended in 9 g of deionized water (water conductivity <15 jS). The suspension was ground again with the blender. Finally, the pH of the suspension was measured (at room temperature) once the value stabilized.

When the pH was measured in dry peas, the peas were dry ground for one minute with a grinder (Kenwood). 5 g of ground dry peas were suspended in 95 g of deionized water (water conductivity <15 jS). The suspension was then homogenized on a stir plate for 1 minute. The pH of the suspension was measured once the value stabilized.

PH measurement in protein powder extract

The pH was measured with a WTW pH / Cond 340I / SET pH meter. The apparatus was calibrated with pH 4.01 buffers (WTW pH 4.01 Technical Buffer, Model STP4, Order No. 108706) and pH 7 (WTW pH 7.00 Technical Buffer, Model STP7, Order No. 108708). 5.0 g of protein powder extract was introduced into a 100 ml beaker and made up to 50 g (Ohaus ARC120 balance, sensitivity 0.01 g, capacity 3100 g) with demineralized water at room temperature. The suspension was stirred for 5 minutes on a stir plate (Stuart US151) at intensity 4. The pH of the suspension (at room temperature) was measured under stirring once the value stabilized.

PH measurement in protein suspension

The pH was measured with a WTW pH / Cond 340i / SET pH meter. The apparatus was calibrated with pH 4.01 buffers (WTW pH 4.01 Technical Buffer, Model STP4, Order No. 108706) and pH 7 (WTW pH 7.00 Technical Buffer, Model STP7, Order No. 108708). 50 ml of protein suspension was placed in a 100 ml beaker without further dilution. The pH of the sample was measured (at room temperature) once the value stabilized.

PH measurement of food products

The pH meter (Knick Portavo 902 PH) was calibrated with pH 4.01 buffers (WTW pH 4.01 Technical Buffer, Model STp4, Order No. 108706) and pH 7 (pH 7 Technical Buffer) .00 from WTW, Model STP7, Order No. 108708). The pH was measured by introducing the probe of the pH meter (Knick Portavo 902 PH) directly into the product (liquid food product, pasta, dough ...) at room temperature. In the case of solid food products, a 50% dilution was made in demineralized water and the solution was analyzed. After stabilization, the pH value was noted.

Enumeration of lactic acid bacteria

The dilutions of the sample were made with EPT Dilucups 9 ml Led techno.

The medium used was MRS agar (according to DE MAN, ROGOSA and SHARPE) from Merck Cat. No. 1.10661.0500. The peas or pea suspension were ground with a blender, Magic Bullet, Homeland Housewares. When a sample of the aqueous composition excluding peas was analyzed, a sample was taken directly from the fermentation vessel. 1 ml of sample was plated. If a dilution was required, 1 ml of sample was added to the dilucup dilution vessel and this step was repeated until the correct dilution was reached and then 1 ml of diluted sample was seeded. The Petri dishes were incubated 48 hours at 45 ° C.

When a sample of peas was analyzed, whole peas were taken from the fermentation vessel. The peas were drained in a strainer and then placed on absorbent paper for two minutes to remove excess juice. The peas were ground for one minute. The ground peas were suspended (1 g of peas in 9 g of deionized water) in deionized water (conductivity <15 jS). The suspension was then ground with the blender. 1 ml of suspension was plated. If dilution was needed, 1 ml of the suspension was added to the dilucup dilution vessel and this step was repeated until the correct dilution was reached and then 1 ml of diluted sample was seeded. The Petri dishes were incubated 48 hours at 45 ° C.

Determination of dry matter

The total dry matter was determined gravimetrically as the residue remaining after drying. Moisture was evaporated from the sample by oven drying.

5 g of sample was weighed onto a preweighed dry aluminum dish (Ohaus precision balance, 410 g capacity, 0.001 g sensitivity). The sample was placed in an oven at 103 ° C until the residual weight remained constant (at least 24 h). The sample was cooled in a desiccator for 1 hour and then immediately weighed. The results are expressed in% (g of dry matter per 100 g of sample).

Dry matter (%) = (m3 - m1) / (m2 - ml) x 100

m1 = weight of the dry aluminum plate (in g)

m2 = weight of the aluminum plate with the sample before drying (in g)

m3 = weight of the aluminum plate with the sample after drying (in g)

Determination of dry matter of food products

The dry matter content of the food products was determined in duplicate after drying 5 g of sample at 104 ° C overnight.

Ash determination

The ash content was determined gravimetrically as the residual residue after heating in a muffle furnace at high temperature. Moisture was evaporated from the sample by oven drying.

2 g of sample was weighed into a pre-weighed dry porcelain crucible (Ohaus precision balance, 410 g capacity, 0.001 g sensitivity). The crucible was placed in a muffle oven at 550 ° C for 24 h. The crucible was placed for 1 h in an oven at 103 ° C and then in a desiccator for 1 h. After cooling, the crucible was weighed. The results are expressed in% (g of ash per 100 g of sample).

Ash (%) = (m3 - m1) / (m2-m1) x 100

m1 = weight of crucible (in g)

m2 = weight of crucible with sample (in g)

m3 = weight of crucible with ash (in g)

Determination of protein content by the Dumas method

The apparatus (Leco FP2000) was calibrated with EDTA sold by Leco under reference 502092. The amounts of EDTA weighed for the calibration ranged from 0.08 g to 0.50 g (0.08 g, 0.15 g, 0.25 g, 0.35 g, 0.40 g, 0.50 g). 0.3 g to 1 g of sample was weighed on a precision balance (Sartorius BP61S, capacity 61 g, sensitivity 0.1 mg) and placed in a ceramic pot. The ceramic pot was automatically placed in an oven at 1200 ° C where the sample was burned in a combustion tube by pyrolysis under controlled oxygen flow. Nitrogen compounds are converted to N2 and NOx, while other volatile decomposition compounds are retained through adsorbent filters and series of purification reagents. All nitrogen compounds are reduced to molecular N, which is determined quantitatively by a thermal conductivity detector. The nitrogen content was then calculated using a microprocessor.

The results are expressed as a percentage of protein (% N * 6.25):

% nitrogen = g of nitrogen per 100 g of sample

% protein =% nitrogen x 6.25

Determination of nitrogen content in ISN samples by the Dumas method

The apparatus (Leco FP2000) was calibrated with a 15 mg / ml glycine solution (glycine powder sold by Merck under the reference 1.04201.1000). The amounts of the 15 mg / ml glycine solution weighed for the calibration ranged from 0.1 g to 1.8 g (0.1 g, 0.4 g, 0.7 g, 1.1 g, 1.4 g, 1.8 g). 1 g to 1.8 g of sample was weighed on a precision balance (Sartorius BP61S, capacity 61 g, sensitivity 0.1 mg) and placed in a ceramic pot covered by a nickel insert. The ceramic pot was automatically placed in an oven at 1200 ° C where the sample was burned in a combustion tube by pyrolysis under controlled oxygen flow. Nitrogen compounds are converted to N2 and NOx, while other volatile decomposition compounds are retained through adsorbent filters and series of purification reagents. All nitrogen compounds are reduced to molecular N, which is determined quantitatively by a thermal conductivity detector. The nitrogen content was then calculated using a microprocessor.

The results were expressed as a percentage of nitrogen:

% nitrogen = g of nitrogen per 100 g of sample

Determination of the solubility index of nitrogen (ISN)

After dispersion of proteins in demineralized water, the nitrogen solubility index was determined by measuring the ratio between the percentage of nitrogen in the supernatant after centrifugation and the percentage of nitrogen in the starting suspension. The method was used in a powdered protein extract with a dry matter content of 90 to 99% (by weight) and was carried out in the month after the protein extract was dried. The measurement was carried out at room temperature.

9.0 g of sample was introduced into a 400 ml beaker and made up to 300 g (Ohaus ARC120 balance, sensitivity 0.01 g, capacity 3100 g) with demineralized water at room temperature. The suspension was homogenized with a spoon and then stirred for 5 minutes on a stirring plate (Stuart US151) at intensity 4. 10 ml of the starting suspension was collected and the nitrogen content was analyzed on an analyzer of Leco FP 2000 proteins. The suspension was divided into two 150 ml beakers, the pH was raised in one and decreased in the other. The pH of the suspension was adjusted to pH 3.5, 4.5, 5.5, 6.5, 7 and 8 with 1N HCl or 1N NaOH (pH meter WTW pH / Cond 340i / SET). For each pH adjustment, the pH value was recorded once stabilized and 10 ml of the suspension was collected in a 10 ml centrifuge tube. The aliquots of the suspension at different pH were centrifuged for 15 min at 6000 rpm (ALC 4239 R centrifuge). The different supernatants were collected and analyzed for nitrogen content on a Leco FP 2000 protein analyzer. For each pH tested, the nitrogen solubility index was calculated according to the following expression:

% nitrogen solubility index =% nitrogen in the supernatant /% nitrogen in the starting solution x 100

Determination of the isoelectric pH of the fraction comprising protein

300 g of fraction comprising protein having a protein content of 1% by weight based on the total weight of fraction comprising protein was introduced into a 400 ml beaker at room temperature. The suspension was stirred for 5 minutes on a stirring plate (Stuart US151) at intensity 4. 10 ml of the starting suspension was collected and the nitrogen content was analyzed on a Leco FP 2000 protein analyzer. The suspension was divided into two 150 ml beakers, the pH rose in one and decreased in the other. The pH of the suspension was adjusted to pH 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6 , 0, 6.25, 6.5, 6.75 and 7.0 with 1N HCl or 1N NaOH (pH meter WTW pH / Cond 340i / SET). For each pH adjustment, the pH value was recorded once stabilized and 10 ml of the suspension was collected in a 10 ml centrifuge tube. The aliquots of the suspension at different pH were centrifuged for 15 min at 6000 rpm (ALC 4239 R centrifuge). The different supernatants were collected and analyzed for nitrogen content on a Leco FP 2000 protein analyzer. For each pH tested, the nitrogen solubility index was calculated according to the following expression:

% nitrogen solubility index =% nitrogen in the supernatant /% nitrogen in the starting solution x 100

Isoelectric pH was determined as the pH at which the nitrogen solubility index was the lowest.

Determination of sugar

The sample was prepared with an eppendorf Centrifuge 5417R centrifuge and NANOSEP 100k OMEGA centrifuges.

The peas or pea suspension were ground with a blender, Magic Bullet, Homeland Housewares.

When a sample of the aqueous composition excluding peas was analyzed, a sample was taken directly from the fermentation vessel. The sample was diluted 20 times (1 g of pea juice in 19 g of deionized water) with deionized water (conductivity <15 jS). 0.5 ml of this dilution was placed in a filtration eppendorf and centrifuged at 14000 rpm for 10 minutes. The filtrate was then used for sugar analysis.

When a pea sample was prepared, whole peas were taken from the fermentation vessel. The peas were drained in a strainer and then placed on absorbent paper for two minutes to remove excess juice. The peas were ground for one minute. The ground peas were suspended (1 g of peas in 9 g of deionized water) in deionized water (conductivity <15 jS). The suspension was then ground with the blender.

The suspension was diluted 8 times (1 g of pea suspension in 8 g of deionized water) with deionized water (conductivity <15 µS). 0.5 ml of this dilution was placed in a filtration eppendorf and centrifuged at 14000 rpm for 10 minutes. The filtrate was then used for sugar analysis.

For the sugar analysis, a Thermo scientific Dionex ICS 5000 chromatographic system was used with the chromeleon 6.80 SR11 Build 3161 computer program. The separation was carried out using a 4 mm * 250 mm Carbopac PA100 (+ protector) at 40 ° C. Elution was carried out with 40 mM NaOH at a flow rate of 1 ml / min. The injection volume was 10 jl. Quadruple pulse detection was used for the detection of PAD. Calibration was performed with appropriate standard solutions that vary for each of the following sugars:

The concentration of the standard solution of sugars (Est1,2, 3 and 4) (mg / l) is given in the table below.

Acidity measurement

Acidity was measured with a SERIES WTW Inolab Termil 740 pH meter. The instrument was calibrated with buffers at pH 4.01 (WTW pH 4.01 Technical Buffer, Model STP4, Order No. 108706) and at pH 7 (WTW pH 7.00 Technical Buffer, Model STP7, Order No. 108708).

The peas or pea suspension were ground with a blender, Magic Bullet, Homeland Housewares.

When the acidity of the "pea juice" was measured, a sample (A) was taken directly from the fermentation vessel. Sample (A) was weighed. 1 mol / l sodium hydroxide solution (C) (# 1.09137.1000 TitriPURR; Density = d = 1.04 kg / I) was added slowly until the pH of the sample stabilized at pH 7 for at least two minutes. Then the mass of sodium hydroxide (B) was calculated.

Acidity (mEq / kg) = (B * (C / d) / A) * 1000

When the acidity of the peas was measured, whole peas were taken from the fermentation vessel. The peas were drained in a strainer and then placed on absorbent paper for two minutes to remove excess juice. The peas were ground for one minute. The ground peas were suspended (1 g of peas in 9 g of deionized water) in deionized water (conductivity <15 ^ S). The suspension was then ground with the blender. A suspension of peas was obtained.

An exact amount of the pea suspension (A ') was weighed. 1 mol / l sodium hydroxide solution (C) (# 1.09137.1000 TitriPURR; Density = d = 1.04 kg / I) was added slowly until the pH of the suspension stabilized at pH 7 for at least two minutes. Then the mass of sodium hydroxide (B ') was calculated.

Acidity (mEq / kg) = (B '* (C7d) / (A710)) * 1000

Determination of Viscosity with the Brookfield DVII Viscometer

Determining the viscosity of a protein suspension with a Brookfield DVII viscometer is the measure of its resistance to flow imposed by the rotation of a cylindrical probe. This resistance causes the rotation of a spring attached to the sensor of a drive system. The viscosity value, expressed in centiPoise (cP), is proportional to the percentage of torque indicated by the viscometer and to a multiplicative factor that depends on the probe used and its rotation speed. The method was used in a powdered protein extract with a dry matter content of 90 to 99% (by weight) and was carried out in the month after the protein extract was dried. The measurement was carried out at room temperature.

A 13.5% protein suspension (by weight) was prepared. 75 g of sample (Ohaus ARC120 balance, sensitivity 0.01 g, capacity 3100 g) were weighed into a 250 ml beaker and the required amount of demineralized water was weighed into a 1 l plastic beaker, both at room temperature. The powder was suspended in water with mechanical stirring (IKA, EURO-ST.P CV) at 700 rpm for 5 minutes with the use of an 80 cm diameter solvent (marketed by Roth under reference A322.1). The pH of the suspension was measured under stirring (pH meter WTW pH / Cond 340i / SET). Stirring was stopped for 3 minutes and the viscosity of the suspension was measured at three different locations with a Brookfield DVII + Pro viscometer at a speed of 50 rpm. The probe used for the measurement was chosen between SO1 and SO7, so that the percentage of twist was between 20% and 80%. The viscosity value was recorded after 4 seconds of rotation of the probe. The suspension was again placed under mechanical stirring for 5 minutes at 700 rpm during which the pH was adjusted to 6.4 with 3N HCl. Stirring was stopped for 3 minutes and the viscosity of the suspension was measured in the same way as before. Similarly, the viscosity of the suspension was measured at pH 6.2, 6.0 and 5.8 after 5 minutes of stirring at 700 rpm and 3 minutes of standing.

When the initial pH of the 13.5% protein suspension was 5.8 or less, the pH was raised to pH 7.5 with 3N NaOH, instead of being lowered with 3N HCl.

Determination of bulk density and density after settlement

The bulk density of a powder is the ratio of the mass of an uncompacted powder sample and its volume, including the contribution of the interparticulate void volume. Density after settling was obtained by mechanically compacting a graduated cylinder containing the powder sample. Bulk density and density after settling are expressed in grams per milliliter (g / ml). The method was used on protein powder extract and the measurement was carried out at room temperature.

60 g of powder were weighed into a 250 ml graduated cylinder (Ohaus ARC120 balance, sensitivity 0.01 g, capacity 3100 g). The volume of powder was measured before stirring. The apparent density was calculated as follows:

Apparent density = m / V1

m = mass of protein powder extract (expressed in g)

V1 = Volume of powder without settling (expressed in ml)

60 g of powder were weighed into a 250 ml graduated cylinder (Ohaus ARC120 balance, sensitivity 0.01 g, capacity 3100 g). The graduated cylinder was placed in the center of a settling apparatus (Retsch) and stirred for 12 minutes at intensity 60. The volume of powder after stirring was measured. The density after settlement was calculated as follows:

Density after settlement = m / V2

m = mass of protein powder extract (expressed in g)

V2 = Volume of settled powder (expressed in ml)

Determination of gel strength

The gel strength was determined by the maximum resistance of a gel to compression applied by a probe directed by a texture analyzer. Formation of a protein gel consisted of making a protein suspension that was subjected to heat treatment followed by cooling. The gel concentration was expressed in g or N. The method was used in a powdered protein extract with a dry matter content of 90 to 99% (by weight) and was carried out in the month after the protein extract was dried. The measurement was carried out at room temperature.

A 13.5% protein suspension (by weight) was prepared. 75 g of sample (Ohaus ARC120 balance, sensitivity 0.01 g, capacity 3100 g) were weighed into a 250 ml beaker and the required amount of demineralized water was weighed into a 1 l plastic beaker, both at room temperature. The powder was suspended in water with mechanical stirring (IKA, EURO-ST.P CV) at 700 rpm for exactly 10 minutes with the use of an 80 cm diameter solvent (sold by Roth under reference A322.1). On the other hand, the pH of the suspension was adjusted to 6.0 with 3N HCl or 3N NaOH according to the initial pH of the suspension (pH meter WTW pH / Cond 340i / s Et). The suspension was poured into two 220 ml glass flasks which were placed in a water bath at 80 ° C for 1 h. The glass jars were cooled for 10 minutes in a room temperature water bath and then placed for 16 hours in a cold room at 4 ° C. The glass jars were placed at room temperature for 15 minutes to bring them to room temperature. Gel strength was measured on a TA-XT2i texture analyzer (Stable Micro Systems, Ltd) with a 5 kg compression load cell and a conical probe (P45C Cone 45 ° Perspex). The gel force was the maximum force recorded at the end of penetration, expressed in g.

TA-XT2i Configuration:

◦ Measure the force in compression - hold until now

◦ Pre-test speed: 2 mm / s

◦ Test speed: 1mm / s

◦ Post-test velocity: 1mm / s

◦ Penetration distance: 35 mm

◦ Trigger type: Auto - 3 g

◦ Time 10 s

Powder flow measurement

The rheological behavior of a powder is characterized by the measurement of the resistance to flow. Fluency was measured with a Brabender microviscoamylograph (MVA). 20 g of sample (Ohaus ARC120 balance, sensitivity 0.01 g, capacity 3100 g) were weighed into the measuring container of the apparatus. The measurement ran for 5 minutes at 20 ° C using a cooling water bath. The rotation speed of the container was set at 75 rpm and the measuring range at 70 cmg. The apparatus measured the force required to maintain the rotation speed of the container at 75 rpm. The results correspond to the average of the measurements recorded during the last minute of the test and are expressed in the Brabender unit (UB). The lower the result, the higher the free-flowing powder.

Determination of wettability

Wettability is defined as the time in seconds required for all particles in a powder to become wet when placed on the surface of water. A dust sample is considered wet when it has sunk below the surface of the water or has a wet appearance. 1 g of powder sample was weighed into a glass beaker. 100 ml of demineralized water at room temperature was introduced into a 250 ml beaker (height 9 cm, diameter 7 cm). The test portion was poured from the glass beaker to the surface of the water. The time was recorded from the beginning of the transfer of the powder until all the particles were wetted. Results are expressed in seconds.

Color measurement

The L * a * b * coordinates were measured at 20 ° C using a CR5 chromium meter (Konica Minolta TA Sensing, Europe). L * denotes lightness on a scale of 0-100 from black to white; a *, (+) red or (-) green; and b *, (+) yellow or (-) blue.

Apparatus:

- CR5 chromameter (Konica Minolta TA Sensing Europe).

- Petri dish CR-A502

Procedure: Sample Preparation

- the Petri dish was filled with the sample for analysis on a uniform surface.

Method

- the Petri dish was placed in the apparatus in the specifically reserved place and the analysis of the Results began

- L * a * b * values are given by the chromameter (average of 3 measurements).

Water activity

Water activity is a measure of the energy state of the water in a system. It is defined as the pressure of water vapor in a substance divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one. Determination of water activity (aw) was performed using Rotronic Hygroskop DT, Krautli.

A cell was filled with the sample to be characterized and placed in the measurement chamber (Rotronic Hygroskop DT, Krautli). After stabilization, the water activity value was recorded.

Sensory analysis for proteins in solution

Sensory evaluation was performed by a trained 5-member panel. The training of the panelists was based on the recognition of 6 characteristics (sweetness, bitterness, metallic taste, salinity, acidity, umami and astringency). A descriptive analysis based on 4% dispersions was performed. After discussion to reach consensus, the descriptive terms that were most important to characterize the appearance, texture and taste of the solutions were selected.

Sensory analysis for baked goods

Sensory evaluation was performed by a trained 5-member panel. The training of the panelists was based on the recognition of 6 characteristics (sweetness, bitterness, metallic taste, salinity, acidity, umami and astringency). A descriptive analysis of the finished products was carried out. After discussion to reach consensus, the descriptive terms that were most important to characterize the appearance, texture and taste of the products were selected.

Expansion capacity in the furnace:

The expandability of the cookies was determined by measuring the length and width of 20 cookies with a gauge.

The average value was determined and expressed in mm.

Cookie hardness:

Cookie hardness is defined as the force required to break a cookie with a knife. The hardness of the cookie was evaluated by the Ta-XT2i texture analyzer.

Apparatus:

- TA-XT2i Texture Analyzer (Stable Micro Systems, Ltd)

- Compression load cell, 25 kg

- Set of blades with knife (HDP / BSK)

Process:

- Position the upper limit of the crosshead so that the Warner Bratzler blade is 1 mm above the surface of the sample

- TA-XT2i configuration:

◦ Measure the force in compression - back to top

◦ Pre-test speed: 3 mm / s

◦ Test speed: 2mm / s

◦ Post-test speed: 10mm / s

◦ Penetration distance: 5 mm

◦ Trigger type: Auto - 3 g

- The penetration test begins. The results were recorded by the texture analyzer and plotted on a graph.

Results

The hardness of the cookie was the maximum force recorded during the test (expressed as "maximum force"). The test results were obtained from 20 samples and the average value was calculated.

Cookie crunch:

Cookie crispiness is defined as the number of peaks recorded during compression of a cookie by a piston. The crispiness of the cookie was evaluated by the Ta-XT2i texture analyzer as described below.

Apparatus:

- TA-XT2i Texture Analyzer (Stable Micro Systems, Ltd)

- Compression load cell, 25 kg

- Piston T1 (9 cm length - 0.5 cm diameter)

Process:

- Position the upper limit of the crosshead so that the piston is 1 mm above the surface of the sample - TA-XT2i configuration:

◦ Measure the force in compression - back to top

◦ Pre-test speed: 2 mm / s

◦ Test speed: 0.1mm / s

◦ Post-test velocity: 5mm / s

◦ Penetration distance: 4 mm

◦ Trigger type: Auto - 3 g

- The penetration test begins. The results were recorded by Texture Analyzer and represented in a graph

Results

Cookie crispness was the number of peaks recorded during the test (expressed as "crisp"). The test results were obtained from 20 samples and the average value is calculated.

Yeast index for sourdough

This test assesses the ability of a bread dough to develop during fermentation:

After kneading, the dough was removed from the kneader and allowed to stand for 10 minutes. 7 small loaves (30 g each) were taken and rounded into small balls (with the hands). After shaping, they were placed in graduated glasses. Initial height (Vi) was observed. The graduated glass was placed in a fermentation cabinet at 32 ° C / 100% relative humidity for 35 minutes. At the end of the test, the vessels were removed from the cabinet and the final post-test height (Vf) was observed.

- The fermentation index (LI) was the difference in volume between the beginning and the end of the test:

LI = (Vf-Vi) x 3.14 x r2

(where r is the radius of the graduated glass)

Bread crumb hardness

The hardness of the bread crumbs was measured on a texture analyzer (TA XT2i, Stable Micro Systems, UK). Two 12.5 mm thick slices were compressed with a 36 mm diameter cylindrical stainless steel probe, penetration of up to 50% (distance = 6 mm) of their original height at a crosshead speed of 1 mm / s speed.

Apparatus:

- TA-XT2i Texture Analyzer (Stable Micro Systems, Ltd)

- Compression load cell, 5 kg

- AACC aluminum radius diameter P36R probe

Process:

- Position the upper limit of the crosshead so that the probe is 1 mm above the surface of the sample - TA-XT2i configuration:

◦ Measure the force in compression - back to top

◦ Pre-test speed: 1mm / s Test speed: 1.7mm / s

◦ Post-test speed: 10mm / s

◦ Penetration distance: 6 mm

◦ Trigger type: Auto - 5 g

- The penetration test begins. The results were recorded by the texture analyzer and plotted on a graph.

Results

- The hardness of the breadcrumb was defined as the force after 2.21 s (expressed as "hardness of the breadcrumb" in g)

- The test results were obtained from 20 samples and the average value was calculated.

Bread volume

The volume of bread was determined by the rapeseed displacement method (Standard AACC 10-05) in five repetitions. The average specific volume (volume / weight) was calculated.

Bar hardness

Rod hardness was defined as the maximum force recorded during compression of the rods by a piston. The hardness of the bars was evaluated using the Ta-XT2i texture analyzer as described below. Apparatus:

- TA-XT2i Texture Analyzer (Stable Micro Systems, Ltd)

- Compression load cell, 5 kg

- 52mm long piston - 5.7mm diameter)

Process:

- Position the upper limit of the crosshead so that the piston is 1 mm above the surface of the sample - TA-XT2i configuration:

Measure the force in compression - Back to top

Pre-test speed: 2mm / s

Test speed: 0.5mm / s

Post-test speed: 5mm / s

Penetration distance: 10mm

Trigger Type: Auto - 3g

- The penetration test begins. The results were recorded by Texture Analyzer and represented in a graph

Results

- The hardness of the bars was the maximum force recorded during the test (expressed as "hardness of the bars")

- The test results were obtained from 12 samples and the average value was calculated.

Example 1: Method for extracting pea proteins according to an embodiment of the present invention

This example was performed following the protocol as depicted schematically in Figure 1.

Step 1-Preparation of the pea protein concentrates:

Dry harvested peas, herein referred to as "dry peas" (having a dry matter content (based on weight) of approximately 87.7%) were screened and detuned by passing through a detuner. Subsequently, the peas were shelled in a sheller.

The peas were then subjected to fermentation with lactic acid bacteria (with Lactobacillus fermentum). So far, the peas have been soaked in drinking water in a batch manner. Drinking water was treated with well water to be safe for human consumption in accordance with European directive 98/83 / EC. In subsequent batches, part of the fermentation medium (aqueous phase excluding peas) from an earlier batch was used as an inoculum to effect subsequent fermentation. The peas were fermented in the presence of 10 8 cfu of lactic acid bacteria per ml of aqueous composition comprising peas. 400 kg of peas per m3 of the total volume of aqueous composition comprising peas were placed in a container. Fermentation was carried out anaerobically in a closed container without degassing at a temperature of 40 ° C, until a pH of 4.2 in peas was reached. During fermentation, the aqueous phase in the fermentation vessel was recirculated at approximately 20 m3 / hour. The peas were fermented for a duration of approximately 540 min. At the end of fermentation, the peas had absorbed water in an amount of approximately their initial mass before fermentation and had a dry matter content of approximately 45% (based on weight).

After fermentation, the peas were removed from the fermentation medium. The peas were then placed in a perforated rotary drum and washed to remove the remaining fermentation medium. After washing, the peas were subjected to wet milling. During milling, additional potable water was added so that the final composition had a dry matter content of approximately 25% (by weight). During the milling step, the pH was adjusted to about 8.0 by the addition of sodium hydroxide.

After grinding, the ground pea paste was subjected to centrifugal decantation. The supernatant containing soluble proteins and impurities (also referred to herein as an aqueous composition comprising pea proteins) had a dry matter content of about 4% (by weight).

The aqueous composition comprising pea proteins was subsequently subjected to heat treatment at 75 ° C for 15 seconds in a plate heat exchanger.

Subsequently, the pea proteins were concentrated by isoelectric precipitation. For this, the pH of the aqueous composition comprising pea proteins was adjusted to 4.7 with sulfuric acid. Separation of precipitated / aggregated proteins was carried out by centrifugal decantation. The resulting pea protein concentrate was obtained as an aqueous suspension having a dry matter content of approximately 25% (based on weight). Drinking water was added to reach a dry matter content of 13% (based on weight).

Step 2-Preparation of pea protein extracts from the concentrates of step 1:

The pH of the aqueous suspension was then adjusted to pH 5.3 with sodium hydroxide. The suspension was then subjected to heat treatment by heating it to approximately 90 ° C by means of a plate heat exchanger and keeping the aqueous suspension at a temperature of approximately 90 ° C for 7 min.

Finally, the aqueous suspension was spray dried. The inlet temperature of the spray dryer was approximately 190 ° C and the outlet temperature was approximately 72 ° C.

Example 2: The effect of acidification and heat treatment on the properties of pea protein extracts

Step 1-Preparation of the pea protein concentrates:

Dry harvested peas, herein referred to as "dry peas" (having a dry matter content (based on weight) of approximately 87%) were screened and detuned by passage through a detuner. Subsequently, the peas were shelled in a sheller.

The peas were then subjected to fermentation with lactic acid bacteria (with Lactobacillus fermentum). So far, the peas have been soaked in drinking water in a batch manner. In subsequent batches, part of the fermentation medium (aqueous phase excluding peas) from an earlier batch was used as an inoculum to effect subsequent fermentation. The peas were fermented in the presence of 10 8 cfu of lactic acid bacteria per ml of aqueous composition comprising peas. 400 kg of peas per m3 of the total volume of aqueous composition comprising peas were placed in a container. Fermentation was carried out anaerobically in a closed container without degassing at a temperature of 40 ° C, until a pH of 4.6 was reached in the peas. During fermentation, the aqueous phase in the fermentation vessel was recirculated at approximately 20 m3 / hour. The peas were fermented for a duration of approximately 480 min. At the end of fermentation, the peas had absorbed water in an amount of approximately their initial mass before fermentation and had a dry matter content of approximately 47% (based on weight).

After fermentation, the peas were removed from the fermentation medium. The peas were then placed in a perforated rotary drum and washed to remove the remaining fermentation medium. After washing, the peas were subjected to wet milling. During milling, additional potable water was added so that the final composition had a dry matter content of approximately 27% (by weight). During the milling step, the pH was adjusted to about 8 by the addition of sodium hydroxide.

After grinding, the ground pea paste was subjected to centrifugal decantation. The supernatant containing soluble proteins and impurities (also referred to herein as an aqueous composition comprising pea proteins) had a dry matter content of about 4.5% (by weight).

The aqueous composition comprising pea proteins was subsequently subjected to heat treatment at 75 ° C for 15 seconds in a plate heat exchanger.

Subsequently, the pea proteins were concentrated by isoelectric precipitation. For this, the pH of the aqueous composition comprising pea proteins was adjusted to 4.7 with sulfuric acid. Separation of precipitated / aggregated proteins was carried out by centrifugal decantation. The resulting pea protein concentrate was obtained as an aqueous suspension having a dry matter content of approximately 25% (based on weight).

Step 2-Preparation of pea protein extracts from the concentrates of step 1. Different pea protein extracts were prepared from the pea protein concentrate described above.

The extract (A) was obtained by adjusting the dry matter content of the aqueous suspension obtained in step 1 to approximately 15% (by weight) after the addition of water; followed by adjusting the pH of the suspension with sodium hydroxide until reaching a pH of approximately 7.5; and after heating to a temperature of about 98 ° C, the temperature was kept at about 95 ° C for about 6.5 minutes; and then the suspension was spray dried to obtain a powder having a dry matter content of about 95% (by weight).

The extract (B) was obtained by adjusting the dry matter content of an aqueous suspension prepared as described in step 1 to about 12.5% (by weight); and spray drying said aqueous suspension without pH adjustment (pH remained at 4.7) and without subsequent heat treatment.

The extract (C) was obtained by adjusting the dry matter content of an aqueous suspension prepared as described in step 1 to about 12.5% (by weight); followed by heat treatment of the suspension at a temperature of about 90 ° C for about 7 minutes without adjusting the pH (pH remained at 4.5), and then spray drying the aqueous suspension to obtain powder with a content of matter dry of about 95% (by weight).

The extract (D) was obtained by adjusting the dry matter content of an aqueous suspension prepared as described in step 1 to about 12.5% (by weight) after the addition of water; followed by adjusting the pH of the suspension with sodium hydroxide until reaching a pH of approximately 5.4; and post heat treatment at a temperature of about 90 ° C for about 7 minutes, and then spray drying the suspension to obtain a powder having a dry matter content of about 95% (by weight).

Gel strength, density after settling, and bulk density, flowability, and wettability of protein extracts (A) to (D) were measured. Results are shown in table 2.

Gel strength at pH 6 is depicted in Figure 2.

Table 2

The nitrogen solubility index (ISN) of each extract as a function of pH is given in Table 3 and is shown in Figure 3.

Table 3

The viscosity measured for each extract at different pH is given in Table 4 and the viscosity profile is shown in Figure 4.

Table 4

Example 3: Characterization of an extract according to an embodiment of the invention

Step 1-Preparation of the pea protein concentrates:

Dry harvested peas, herein referred to as "dry peas" (having a dry matter content (based on weight) of approximately 87%) were screened and detuned by passage through a detuner. Subsequently, the peas were shelled in a sheller.

The peas were then subjected to fermentation with lactic acid bacteria (with Lactobacillus fermentum). So far, the peas have been soaked in drinking water in a batch manner. In later batches, part of the medium Fermentation (aqueous phase excluding peas) from a previous batch was used as an inoculum to effect subsequent fermentation. The peas were fermented in the presence of 10 8 cfu of lactic acid bacteria per ml of aqueous composition comprising peas. 400 kg of peas per m3 of the total volume of aqueous composition comprising peas were placed in a container. Fermentation was carried out anaerobically in a closed container without degassing at a temperature of approximately 40 ° C, until a pH of 4.7 was reached in the peas. During fermentation, the aqueous phase in the fermentation vessel was recirculated at approximately 20 m3 / hour. The peas were fermented for a duration of approximately 430 min. At the end of fermentation, the peas had absorbed water in an amount of approximately their initial mass before fermentation and had a dry matter content of approximately 47% (based on weight).

After fermentation, the peas were removed from the fermentation medium. The peas were then placed in a perforated rotary drum and washed to remove the remaining fermentation medium. After washing, the peas were subjected to wet milling. During milling, additional potable water was added so that the final composition had a dry matter content of approximately 25% (by weight). During the milling step, the pH was adjusted to about 8 by the addition of sodium hydroxide.

After grinding, the ground pea paste was subjected to centrifugal decantation. The supernatant containing soluble proteins and impurities (also referred to herein as an aqueous composition comprising pea proteins) had a dry matter content of about 4% (by weight).

The aqueous composition comprising pea proteins was subsequently subjected to heat treatment at 75 ° C for 15 seconds in a plate heat exchanger.

Subsequently, the pea proteins were concentrated by isoelectric precipitation. For this, the pH of the aqueous composition comprising pea proteins was adjusted to 4.8 with sulfuric acid. Separation of precipitated / aggregated proteins was carried out by centrifugal decantation. The resulting pea protein concentrate was obtained as an aqueous suspension having a dry matter content of about 25% (based on weight).

Step 2-Preparation of pea protein extracts.

The extract (E) was obtained by adjusting the dry matter content of the aqueous suspension obtained in step 1 to approximately 16% (by weight) after the addition of water; followed by adjusting the pH of the suspension with sodium hydroxide until reaching a pH of approximately 7.4; and post heat treatment at a temperature of about 90 ° C for about 7 minutes; then by spray drying the suspension to obtain a powder having a dry matter content of about 95% (by weight).

Extract the pea protein concentrate (F) prepared according to the invention as described in example 1.

The extract (G) was obtained by adjusting the dry matter content of the aqueous suspension obtained in step 1 to approximately 16% (by weight) after the addition of water; followed by adjusting the pH of the suspension with sodium hydroxide until reaching a pH of approximately 6.1; and post heat treatment of the suspension at a temperature of about 98 ° C for about 7 minutes; and then by spray drying the suspension to obtain a powder having a dry matter content of about 95% (by weight).

The wettability, flowability, density after settling and apparent density were measured for each extract.

The results are given in Table 5.

Table 5

Pea protein extracts E, F and G were evaluated for color. The results are shown in Table 6.

Table 6

The pH and gel strength at pH 6 of each extract were measured. The results are shown in Table 7.

Table 7

The nitrogen solubility index (ISN) of each extract as a function of pH is given in Table 8 and is shown in Figure 5.

Table 8

The viscosity measured for each extract at different pH is given in Table 9.

Table 9

The sensory characteristic of each extract was determined after dispersion in tap water at 4% by weight and the results are given in Table 10.

Table 10

Example 4: Use of pea proteins according to the invention in the clarification of wines

The clarification of the wine is carried out to decrease the turbidity and improve the brightness and the flavor.

A positively charged protein solution (pH <pHi) is added to the wine (pH 2.8 to 4.0). In the presence of negatively charged wine particles, electrical charges are neutralized and stable hydrophilic colloids become unstable, resulting in their precipitation.

The clarifying agents commonly used in the presentation of wines are gelatin, egg albumin, casein or vegetable proteins. Finishing aids such as silica (silicon dioxide) or tannins are also often used.

Different types of pea proteins were evaluated at two different concentrations (5 g / hl and 10 g / hl) in alcoholic solutions of 12 °, at pH 3.2, 4 ° C, in the presence of tannins or SiO2. The absorbance of the solutions was measured after standing (from 1 h to 96 h).

The process was as follows: in a 2 liter beaker, 1750 g of water (= 1750 ml) were added. A volumetric flask was filled with 250 ml of 96 ° ethanol. The ethanol was added to the water and mixed thoroughly. The pH of the resulting solution was adjusted to 3.2 with 1M HCl (weight noted). The tannins or silicon dioxide were added to the solution, and mixed thoroughly. The solution was covered and allowed to stand for 1 hr. The pea proteins were added and this constituted the incubation start time (t = 0 h). The solution was stored at 4 ° C.

Sampling: it was carried out as follows; A plastic syringe was filled with 30 ml of the solution, taken in the center of the beaker without shaking the solution, a cell was filled to measure the absorbance with the solution. The absorbance at 420 nm was recorded using a spectrometer. The solution was then filtered using a 0.45 µm Whatman filter. The absorbance of the filtrate was measured at 420 nm.

Turbidity was measured using the following equation:

Turbidity = absorbance before filtration - absorbance after filtration

1. Protein concentrations of 5 and 10 g / hl with tannins as adjuvants

Various solutions were prepared. Tannins were used at a concentration of 0.3 ml / l.

Blank T (also called Control T) was the negative control with tannins (without any clarifying agent). The tannins were a ready-to-use solution: ST solution from the Institut Oenologique de Champagne consisting of a solution of tannic acid (CAS number: 1401-55-4) and copper sulfate.

Perle T was the positive control with tannins (also known as Perle), and also contained 0.115 g / hl of Colle Perle (hydrolyzed gelatin from the Institut Oenologique de Champagne) as a clarifying agent.

The extracts E and F prepared in Example 3 were used as pea proteins.

Solution A comprised the Blank T solution and the pea protein from extract E.

Solution B comprised the Blank T solution and the pea protein from extract F.

The turbidity of the solutions after different incubation times at 4 ° C was measured as described above. The results are shown in Table 11 and Figure 7. The turbidity of the solutions after 96 h of incubation is shown in Figure 6.

Table 11

2. Protein concentration of 5 g / hl with SiO? as an adjuvant

SiO2 was used at 0.6 ml / l.

Blank S (also known as Control S) was the negative control with SiO2 (no clarifying agents)

Perle S was the positive control with SIO2 and containing 0.115 g / hl of Colle Perle (hydrolyzed gelatin provided by the Institut Oenologique de Champagne)

The extracts E and F prepared in Example 3 were used as pea proteins.

Solution A1 comprised the White S solution and the pea protein from extract E.

Solution B1 comprised the Blank solution S and the pea protein from extract F. The turbidity of the solutions after an incubation time other than 4 ° C was measured as described above. The results are shown in Table 12 and Figure 9. The turbidity of the solutions after 96 h of incubation is shown in Figure 8.

Table 12

Effect of adjuvants at a protein concentration of 5 g / hl

Table 13 and Figure 10 show the effect of the different adjuvants tested at 1 and 2 after 96 h of incubation at 4 ° C, in comparison with the solutions containing only the proteins and without adjuvants (none). None: pea proteins at 5 g / hl in alcoholic solutions of 12 °, at pH 3.2, 4 ° C prepared as follows: in a 2-liter beaker, 1750 g of water were added (= 1750 ml). A volumetric flask was filled with 250 ml of 96 ° ethanol. The ethanol was added to the water and mixed thoroughly. The pH of the resulting solution was adjusted to 3.2 with 1M HCl (weight observed). Pea proteins were added. The solution was stored at 4 ° C

Table 13

4. Comparison with other pea proteins (without adjuvants)

Solution A2 comprised a 12 ° alcoholic solution and pea protein from extract E.

Solution B2 comprised a 12 ° alcoholic solution and pea protein from extract F.

Solution C1 to F1 comprised a 12 ° alcoholic solution and commercial pea proteins.

The different extracts are represented in Table 14. The turbidity of the solutions was measured after different incubation times. The effect on turbidity is indicated in Table 15 and Figure 11.

Table 14

Table 15

Example 5: Food products comprising pea proteins according to the invention

The inclusion of pea proteins in various food products was evaluated.

1. Cookies

Cookie doughs were prepared. Extract E (not according to the invention) and Extract F (according to the invention) as prepared in Example 3 were used as pea proteins. Tests were performed to evaluate the pH of the cookie dough prepared with Extract E, and / or to modify the pH of the dough prepared with Extract F and different acidity correcting agents and raising agents.

1a) The dough was prepared as shown in Table 16

Table 16

An analysis of the mass is given in Table 17.

Table 17

1b) The dough was prepared as shown in Table 18.

A dough (Table 18) was prepared in which the amount of water added in the dough prepared with extract F was reduced, an optimum water reduction of 3% was found, and a dough with excellent texture and sheability was obtained. For the dough made with extract E, it was found that it was not possible to reduce the water content as the dough becomes too hard.

Table 18

An analysis of the mass is given in Table 19

The cookies were made from the dough from trials 3 and 4. They were baked in an Eloma Backmaster oven at 150 ° C for 20 minutes and cooled to room temperature before packaging. Table 20 shows an analysis of cookies prepared with the respective masses from trial 3 (cookie A) and trial 4 (cookie B).

Table 20

2. Protein enriched bread

The breads were prepared using a pea protein extract G as prepared in Example 3, or a pea protein extract F. Bread recipes are provided in Table 21. In addition to identical doughs prepared with the pea proteins according to the invention (extract F) and pea proteins not according to the invention (G), several adaptations were made (reduction of water content by 5% for F with or without pH adjustment of the mass; as well as the decrease in water content for G).

Table 21

The mixer used was a spiral kneader (type Vema QR24). After kneading, the dough was removed from the kneader and allowed to stand for 10 minutes. The dough was divided into 500 g dough rolls, which were made in the first round and then spread by hands. The bread was placed in previously greased metal cans and stored for 1 hour and 15 minutes in a controlled fermentation cabinet (t ° 32 ° C).

Oven: the bread was baked at 200 ° C for 60 minutes in a deck oven (Salva Modular). 20 minutes before the end of cooking, the steam extraction was opened. The bread was then cooled to room temperature and packed in a suitable packaging.

The dough was prepared according to the above recipes in Table 21 and evaluated as indicated in Table 22.

Table 22

It was found that the dough prepared with pea protein extract F, when the water content was reduced, the dough was less sticky, had good elasticity and was easier to work with. For the dough made with extract G, it was not possible to reduce the water content by more than 2%, the dough becomes too hard otherwise.

The fermentation index of the dough was measured. Table 23 and Figure 12 show the fermentation index for the respective doughs.

Table 23

The quality of the prepared bread was evaluated. Results are shown in Table 24. Measurements of bread volume and bread crumb hardness are shown in Figures 13 and 14, respectively.

Table 24

3. Dulce de leche bars

The dulce de leche bars were prepared using Extract E and F as prepared in Example 3. The recipes for the dulce de leche bar are shown in Table 25.

Table 25

The process to prepare the bars was as follows

- Melt the fat at 45 ° C in a water bath

- Mix the syrups and add the fat

- Mix the powders in a Hobart

- Add the syrups and shake for a few minutes until obtaining a homogeneous paste

- the dough was placed in a plastic bag and spread, leaving it to rest overnight

- Cut the bars and cover them with chocolate

It has been observed that the mixture (B) containing the extract F according to the invention takes less time to homogenize than the mixture (A), approximately 2 minutes less.

The pH, Aw (water activity) and hardness of the respective dulce de leche bars over time (months) were measured and the results are illustrated respectively in Tables 26, 27 and 28. The hardness of the candy bar Dulce de leche was also measured and the results are shown in Figure 15.

Table 26

Table 27

Table 28

The sensory analysis of the respective dulce de leche bars is illustrated in Table 29.

Table 29

Example 6: Characterization of an extract according to an embodiment of the invention (without fermentation step)

Step 1-Preparation of the pea protein concentrates:

Dry harvested peas, herein referred to as "dry peas" (having a dry matter content (based on weight) of approximately 87%) were screened and detuned by passage through a detuner. Subsequently, the peas were shelled in a sheller.

The peas were dry ground to obtain a pea flour. Drinking water was added to the pea flour so that the final composition had a dry matter content of about 25% (by weight). Subsequently, the pH was adjusted to about 8 by the addition of sodium hydroxide.

After pH adjustment, the pea paste was subjected to centrifugal decantation. The supernatant containing soluble proteins and impurities (also referred to herein as an aqueous composition comprising pea proteins) had a dry matter content of about 4% (by weight).

The aqueous composition comprising pea proteins was subsequently subjected to heat treatment at 75 ° C for 15 seconds in a plate heat exchanger.

Subsequently, the pea proteins were concentrated by isoelectric precipitation. For this, the pH of the aqueous composition comprising pea proteins was adjusted to 4.8 with sulfuric acid. Separation of precipitated / aggregated proteins was carried out by centrifugal decantation. The resulting pea protein concentrate was obtained as an aqueous suspension having a dry matter content of approximately 25% (based on weight).

Step 2-Preparation of pea protein extracts from the concentrates of step 1:

The extract (H) was obtained by adjusting the dry matter content of the aqueous suspension obtained in step 1 to approximately 18% (by weight) after the addition of water; followed by an adjustment of the pH of the suspension with sodium hydroxide until reaching a pH of approximately 5.4; and post heat treatment at a temperature of about 85 ° C for about 7 minutes; then by spray drying the suspension to obtain a powder having a dry matter content of about 95% (by weight).

The extract (I) was obtained by adjusting the dry matter content of the aqueous suspension obtained in step 1 to approximately 17% (by weight) after the addition of water; followed by an adjustment of the pH of the suspension with sodium hydroxide until reaching a pH of approximately 5.4; and the subsequent heat treatment was carried out by direct steam injection at a temperature of about 140 ° C for about 4 seconds; and then spray drying the suspension at 90 ° C to obtain a powder having a dry matter content of about 96% (by weight).

Gel strength, density after settling, and bulk density, flowability, and wettability of protein extracts (H) and (I) were measured. The results are shown in Table 30.

Table 30

The nitrogen solubility index (ISN) of both extracts as a function of pH is shown in Table 31 and is shown in Figure 16.

Table 31

The viscosity measured for both extracts at different pH is given in Table 32.

Table 32

Example 7: Comparative studies of fermented peas with different strains of Lactobacillus (Lactobacillus fermentum LMG 6902, Lactobacillus fermentum LMG 18026, Lactobacillus Crispatus LMG 12005 or Lactobacillus Acidophilus LMG 8151)

Dry harvested peas, herein referred to as "dry peas" (having a dry matter content (based on total dry pea weight) of about 87%) were sieved and detuned by passing through a detonator. Subsequently, the peas were shelled in a sheller.

The peas were then subjected to fermentation with lactic acid bacteria (Lactobacillus fermentum LMG 6902, Lactobacillus fermentum LMG 18026, Lactobacillus Crispatus LMG 12005 or Lactobacillus Acidophilus LMG 8151). 2000 g of peas were soaked in 3663 g of demineralized water sterilized at a temperature of 40 ° C, in a container. The fermentation medium comprising the mentioned bacterial strains was added at the same time. The vessel was in a thermostatted bath and a pump was added to recycle the aqueous phase at approximately 250 ml / min.

For Lactobacillus fermentum LMG 6902 and Lactobacillus fermentum LMG 18026 (both were obtained from BCCM / lMG Laboratorium voor Microbiologie, Universiteit Gent (UGent) Belgium), the fermentation medium was prepared as described in the procedure provided by the BCCM (F109C Revival de lyophilized cultures; recommended medium 66). Then 37 ml of the fermentation medium was added to the vessel.

For Lactobacillus crispatus LMG 12005 (50 Bn) and Lactobacillus Acidophilus LMG 8151 (100 Bn) (both were obtained from THT s.a. Gembloux, Belgium), the fermentation medium was prepared by placing 37 g of Lactobacillus flakes directly into the container.

The peas were fermented in the presence of approximately 108 cfu of lactic acid bacteria per ml of aqueous composition comprising peas. Fermentation was carried out in a closed container without degassing at a temperature of approximately 40 ° C.

Figures 17-20 respectively illustrate the evolution of the sugar content, pH, acidity and concentration of lactic acid bacteria as a function of the fermentation time.

Claims (13)

1. A method for extracting pea proteins, comprising the steps of: (a) providing an aqueous composition comprising pea proteins; (b) isolating said pea proteins from said aqueous composition comprising pea proteins; (c) obtaining said isolated pea proteins as an aqueous suspension having a pH in the range of 4.0 to 5.8; and (d) subjecting said aqueous suspension having a pH in the range of 4.0 to 5.8 to a temperature in the range of 75 ° C to 210 ° C; wherein step (b) comprises adjusting the pH of said aqueous composition comprising pea proteins to a value in the range of 4.0 to 5.8, preferably in the range of 4.5 to 5.5; wherein step (d) comprises subjecting said precipitated pea proteins to a heat treatment at a temperature in the range of 115 ° C to 210 ° C for a time in the range of 15 s to 0.01 s; at a temperature in the range of 95 ° C to 115 ° C for a time in the range of 5 min to 15 s; at a temperature in the range of 75 ° C to 95 ° C for a time in the range of 15 min to 5 min; at a temperature in the range of 75 ° C to 110 ° C for a time in the range of 10 min to 2 min; at a temperature in the range of 80 ° C to 100 ° C for a time in the range of 8 min to 5 min; or at a temperature in the range of 130 ° C to 150 ° C for a time in the range of 8 s to 1 s. The method according to claim 1, wherein isolating pea proteins from said aqueous composition of step (b), comprises concentrating said pea proteins. 3. The method according to claims 1 or 2, wherein step (b) comprises at least one precipitation, flocculation, filtration and / or chromatography step. 4. The method according to any one of claims 1 to 3, wherein step (b) comprises isoelectric precipitation. 5. The method according to any one of claims 1 to 4, wherein step (c) comprises adjusting or maintaining the pH of the aqueous suspension at a value in the range of 4.0 to 5.8, preferably, in the range of 4.5 to 5.5. 6. The method according to any one of claims 1 to 5, wherein said aqueous composition comprising pea proteins in step (a) has a pH of at least 6, preferably in the range of 6.0 to 9.0, preferably in the range 6.5 to 8.5. 7. The method according to any one of claims 1 to 6, wherein before step (a) an aqueous composition comprising peas is subjected to fermentation in the presence of lactic acid bacteria. 8. The method according to claim 7, wherein said fermentation is carried out in the presence of one or more Lactobacillus sp. The method according to any one of claims 1 to 8, wherein before or during step (b) said aqueous composition comprising pea proteins, or said pea proteins are subjected to a heat treatment, preferably pasteurization; said fraction comprising protein is subjected to a temperature of at least 30 ° C, for example, at least 40 ° C, for example, at least 50 ° C, for example, said fraction comprising protein is subjected to a temperature in the range from 30 ° C to 90 ° C, more preferably, in the range of 50 ° C to 80 ° C, even more preferably, in the range of 55 ° C to 75 ° C. Pea protein extract obtainable by the method according to any one of claims 1 to 9. 11. Pea protein composition, comprising at least 60% by weight protein based on the total dry matter of the composition, wherein said pea protein composition has a nitrogen solubility index at pH 7.0 of at most 15%, as measured in an aqueous composition comprising 3% by weight of said pea protein composition based on the total weight of the aqueous composition. 12. Pea protein composition according to claim 11, wherein said composition has a pH in the range of 4.0 to 5.8, as measured at room temperature in 10 g of pea protein composition suspended at 90 g of water. Use of the pea protein extract according to claim 10 or the pea protein composition according to claim 11 or 12 in food or feed products, preferably in bakery and confectionery food products, or for clarifying liquids.