WO2024126712A1

WO2024126712A1 - Process for preparing hydrolyzed vegetable proteins

Info

Publication number: WO2024126712A1
Application number: PCT/EP2023/085875
Authority: WO
Inventors: Weixi QIU; Ying ZHA
Original assignee: Dsm Ip Assets B.V.
Priority date: 2022-12-14
Filing date: 2023-12-14
Publication date: 2024-06-20

Abstract

The present invention relates to a process for preparing hydrolyzed vegetable proteins comprising hydrolysis of vegetable proteins with an acid protease (such as an acid protease from Aspergillus niger) to prepare hydrolyzed vegetable proteins; a use of such certain protease for the preparation of a hydrolyzed vegetable protein; a hydrolyzed vegetable protein obtained or obtainable by such a process or such use; a use of such hydrolyzed vegetable protein as an ingredient in a food or beverages product; and a process for the preparation of a food or beverage comprising such a hydrolyzed vegetable protein.

Description

PROCESS FOR PREPARING HYDROLYZED VEGETABLE PROTEINS

Field

[001] The present invention relates to a process for preparing hydrolyzed vegetable proteins comprising hydrolysis of vegetable proteins with a certain protease to prepare hydrolyzed vegetable proteins; a use of such certain protease for the preparation of a hydrolyzed vegetable protein; a hydrolyzed vegetable protein obtained or obtainable by such a process or such use; a use of such hydrolyzed vegetable protein as an ingredient in a food or beverages product; and a process for the preparation of a food or beverage comprising such a hydrolyzed vegetable protein.

Background

[002] Proteins are a key element in animal and human nutrition. Nowadays proteins for nutrition are being sourced from animals (e.g. meat, fish, egg, dairy) or vegetables. There is an increasing desire to reduce the amount of animal based protein, for example because of customers wish to adhere to a specific diet out of religious restrictions/convictions, ethical or culinary preferences (such as, for example, a vegetarian or a vegan diet). Therefore, there is an increasing need for plant-based, i.e. vegetable-based, proteins that can be used in plant-based, i.e. vegetable-based, products.

[003] Soy protein is widely used, however in view of some intolerances to soy products there is also a need for alternative sources of vegetable proteins. Suitable alternatives include for example pea protein and rapeseed protein.

[004] A suitable source for vegetable based proteins are so-called vegetable meals, such as soy meal, peas meal (also called pea flour) or rapeseed meal.

[005] The composition of vegetable based proteins can be different from that of animal based proteins and vegetable based proteins can be more challenging to digest than animal based proteins.

[006] Proteases are enzymes that are known to be helpful in treatment of vegetable proteins.

[007] US2010/0081168A1 describes a method of treating vegetable proteins, comprising adding (i) an acid-stable protease and (ii) a phytase to vegetable proteins, wherein the protease comprises a certain amino acid sequence. It is indicated that such protease can be a microbial protease, wherein the term microorganism as used herein includes Archaea, bacteria, fungi and vira. A number of proteases were said to be analyzed for stability at pH 3, with the objective of identifying proteases that have the necessary stability to pass through the acidic stomach of mono-gastric animals. The key exemplified protease was a protease derived from the actinobacterium Nocardiopsis alba.

[008] EP4245149A1 describes a method for producing a processed plant protein-containing liquid composition, the method comprising a step of treating a plant protein-containing liquid composition with a protease and a protein deamidase. In passing it is stated that examples of the plant protein include proteins of plants (plant food ingredients) such as cereals such as oats, barley, wheat, rice, buckwheat, barnyard millet, millet, teff, and quinoa, pulses such as soybeans, peas, lupins, broad beans, and chickpeas, and nuts such as canary seeds, linseed, almonds, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, peanuts, coconuts, chestnuts, sesame, and pine nuts. Rapeseed is not mentioned.

[009] Improvements on the above are desired.

Summary

[010] The present inventors have surprisingly found several improvements overthe prior art. The process according to the invention can advantageously result in an increased protein content. In addition improvements in respect of functionality can be achieved, such as an improved solubility, bio-availability, dispersibility, emulsification, foamability, gelation, taste and/or texture, formability, and/or gelation.

[011] Accordingly the invention provides a process for preparing hydrolyzed vegetable proteins comprising hydrolysis of vegetable proteins with an acid protease to prepare hydrolyzed vegetable proteins, wherein the acid protease is preferably a, preferably fungal, acid endo-protease, most preferably an aspergillopepsin I protease.

[012] In addition, the invention provides a process for preparing hydrolyzed vegetable proteins, wherein the process comprises the steps of: a) preparing an aqueous mixture comprising water and the vegetable proteins; b) contacting the aqueous mixture, comprising the water and the vegetable proteins, with the acid protease, preferably at a pH in the range from 2 to 6, more preferably 3 to 5, preferably at a temperature in the range from 30°C to 60°C, more preferably 20°C to 50°C, most preferably 45°C to 55°C, preferably for a time period in the range from 10 minutes to 40 hours, more preferably 30 minutes to 30 hours, most preferably 1 hour to 20 hours, to hydrolyze the vegetable proteins; c) retrieving the hydrolyzed vegetable proteins from the aqueous mixture.

[013] The invention further provides a use of an acid protease, preferably a fungal acid endoprotease, more preferably an aspergillopepsin I protease, for the preparation of a hydrolyzed vegetable protein.

[014] Still further the invention provides hydrolyzed vegetable protein obtained or obtainable by a process as defined above or the use defined above.

[015] Finally the invention provides a use of a hydrolyzed vegetable protein, as defined above, as an ingredient in a food or beverages product and a process for the preparation of a food or beverage comprising a hydrolyzed vegetable protein as defined above.

[016] The present invention advantageously provides several improvements over the prior art. The process and use according to the invention can advantageously result in an increased protein content for food and beverages products. In addition improvements for selected raw materials in respect of functionality can be achieved, such as an improved solubility, bio-availability, dispersibility, emulsification, foamability, gelation, taste, such as a reduced bitterness, and/or texture. These advantages are illustrated in the description of the figures and in the examples. Further, the process advantageously allows for an enzymatic incubation and/or subsequent hydrolysis at a low pH, preferably a pH below 6.0, preferably below 5.0. More preferably a pH in the range from equal to or more than 2.0 to equal to or less than 6.0, more preferably in the range from equal to or more than 2.0 to equal to or less than 5.0, and most preferably in the range from equal to or more than 3.0 to equal to or less than 5.0. An additional advantage of operating the process at such a low pH, is that contamination by bacterial or other microbial growth is less likely to happen. Further, advantageously, the use of alkaline and/or alkaline earth salts for maintenance of a certain alkaline pH can be reduced or even eliminated, advantageously resulting in less salt being present in the final product.

Brief description of the drawings

[017] The invention is illustrated by the following figures:

[018] Figure 1 illustrates a process for enzymatic protein processing of gelatin powder. Gelatin powder was derived from an animal source.

[019] Figures 2a and 2b illustrates examples of processes for enzymatic protein processing of soy meal. This is an example of a process according to the invention. Soy meal is derived from a vegetable source (also referred herein as a plant source).

[020] Figure 3 illustrates a process for enzymatic protein processing of pea flour. This is an example of a process according to the invention. Pea flour is derived from a vegetable source (also referred herein as a plant source).

[021] Figure 4 illustrates a process for enzymatic protein processing of rapeseed cake (also referred herein as rapeseed meal). This is an example of a process according to the invention. Rapeseed meal is derived from a vegetable source (also referred herein as a plant source).

Detailed description

Definitions

[022] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. [023] Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[024] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element. When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included. Thus, when referring to a specific moiety, e.g. "gene", this means "at least one" of that gene, e.g. "at least one gene", unless specified otherwise. [025] When referring to a compound of which several isomers exist (e.g. a D and an L enantiomer), the compound in principle includes all enantiomers, diastereomers and cis/trans isomers of that compound that may be used in the particular aspect of the invention; in particular when referring to such as compound, it includes the natural isomer(s).

[026] Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined.

[027] The terms "polypeptide", "peptide" and "protein" are preferably used herein to refer to a polymer of amino acid residues. The terms preferably apply to amino acid polymers in which one or more amino acid residue(s) is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide" and "protein" are preferably also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulphation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[028] The term “enzyme” preferably refers herein to a polypeptide (i.e. a protein) having a catalytic function.

The acid protease

[029] The invention advantageously provides processes for processing or treating of vegetable protein, also referred to as plant protein, with an acid protease, preferably an acid endo-protease. Such “acid protease”, respectively such “acid endo-protease” is herein also referred to as “acidic protease” respectively “acidic endo-protease”. Preferences for such processes are described herein above and herein below. The preferences for the acid protease, respectively acid endoprotease, are as described herein below.

[030] By a protease is herein suitably understood an enzyme having protease activity. By an acid protease is herein preferably understood a protease that is capable of hydrolyzing proteins at a pH below 6.0, preferably below 5.0. Preferably the acid protease is an acid protease that has an optimum activity and/or stability at a pH in the range from equal to or more than 2.0 to equal to or less than 6.0, more preferably in the range from equal to or more than 2.0 to equal to or less than 5.0, most preferably in the range from equal to or more than 3.0 to equal to or less than 5.0. Preferably the acid protease is an acid protease that has a reduced or no activity above a pH of 6.0.

[031] There are two types of proteases. Endo-proteases and exo-proteases. Preferably the protease is an endo-protease. An endo-protease is herein preferably understood to be a protease that hydrolyzes peptide bonds of non-terminal amino acids. That is, such an endo-protease can “cleave” peptide bonds in the inner regions of a protein to release for example oligopeptides. By an acid endo-protease is herein preferably understood an endo-protease that is capable of hydrolyzing peptide bonds of non-terminal amino acids in the protein at a pH below 6.0, preferably below 5.0. Preferably the acid endo-protease is an acid endo-protease that has an optimum activity and/or stability at a pH in the range from equal to or more than 2.0 to equal to or less than 6.0, more preferably in the range from equal to or more than 2.0 to equal to or less than 5.0, most preferably in the range from equal to or more than 3.0 to equal to or less than 5.0. Preferably the acid endoprotease is an acid endo-protease that has a reduced or no activity above a pH of 6.0.

[032] The acid protease, respectively acid endo-protease, is preferably a fungal acid protease, respectively a fungal acid endo-protease. By a fungal acid protease, respectively a fungal acid endo-protease, is herein suitably understood an acid protease, respectively an acid endo-protease, that is produced or producible in, and preferably obtained or obtainable from, a fungus. Preferably the fungal acid protease, respectively the fungal acid endo-protease, is produced or producible in, preferably obtained or obtainable from, an Aspergillus strain, more preferably an Aspergillus niger strain. In addition to the advantages from a functional perspective as described herein above and herein below, the use of a fungal acid protease, respectively a fungal acid endo-protease, has an advantage from a customer perspective. For products targeting customers with certain dietary requirements, such as vegan, kosher and/or halal customers, the use of certain animal- derived enzymes is not acceptable and the use of a fungal enzyme is preferred.

[033] Preferably the, preferably fungal, acid protease, more preferably the, preferably fungal, acid endo-protease is an aspergillopepsin I protease.

[034] Most preferably the, preferably fungal, acid protease, more preferably the, preferably fungal, acid endo-protease, is Maxipro® AFP, a fungal acid endo-protease commercially obtainable from DSM Food & Beverages, Delft, the Netherlands. Maxipro® AFP is classified as an aspergillopepsin I protease and possesses broad peptide bond specificity, it is an acid protease with an optimum pH in the range from 2 to 6. Up to now, Maxipro AFP was used in wine and fruit juice processing to clarify the wine and juice, as for example described by Marangona, et.al. [5], The advantages when using Maxipro® AFP for the different purpose of hydrolyzing vegetable proteins were unexpected.

[035] The above preferred acid protease, respectively acid endo-protease, can advantageously be used for the preparation of hydrolyzed vegetable proteins. That is, the above preferred acid protease, respectively acid endo-protease, can advantageously be used in a process for hydrolyzing vegetable proteins to produce hydrolyzed vegetable proteins.

[036] In addition to the above acid protease, respectively acid endo-protease, even more preferably fungal, acid endo-protease and most preferably aspergillopepsin I protease, other enzymes may be present, such as for example a protein deamidase such as described in EP4245149A1 , herein incorporated by reference. However, advantageously the acid protease, respectively acid endo-protease, even more preferably fungal, acid endo-protease and most preferably aspergillopepsin I protease can also be used as sole enzyme. That is, preferably the process, and more preferably step a) as described herein below, can be carried out in the absence of enzymes other than the acid protease, respectively acid endo-protease, even more preferably fungal, acid endo-protease and most preferably aspergillopepsin I protease. The vegetable proteins

[037] By a vegetable protein is herein understood a protein from a vegetable source. Such proteins are also referred to as plant-based proteins.

[038] Preferred vegetable sources for such vegetable proteins include rice, cereals, potato, seeds, such as oilseeds, nuts, beans and pulses. More preferred vegetable sources are soy beans, beans, peas, faba, wheat, maize, potato, rapeseed, sunflower seed, barley, rye and rice. Most preferred vegetable sources are soy, peas and rapeseed. However, also brewer’s spent grain can be a good source of vegetable proteins.

[039] Preferred vegetable proteins include rice protein, seed protein, nut protein, barley protein, bean protein and pulse protein. Also protein from brewer’s spent grain is preferred. Most preferred vegetable proteins are soy protein, pea protein and rapeseed protein.

The process

[040] The invention suitably provides a process for preparing hydrolyzed vegetable proteins comprising hydrolysis of vegetable proteins with an acid protease to prepare hydrolyzed vegetable proteins, wherein preferably the acid protease is an acid protease as described extensively above and wherein preferably the vegetable proteins are vegetable proteins as described extensively above.

[041] The hydrolysis of the vegetable proteins is preferably carried out by contacting the vegetable proteins and the acid protease in an aqueous environment. More preferably an aqueous mixture, comprising water and the vegetable proteins, is contacted with the acid protease. Preferably the pH during such contacting, i.e. during the hydrolysis, is a pH in the range from equal to or more than 2.0 to equal to or less than 6.0, more preferably from equal to or more than 3.0 to equal to or less than 5.0.

[042] Preferably the temperature during such contacting, i.e. during the hydrolysis, is a temperature in the range from equal to or more than 30°C to equal to or less than 60°C, more preferably from equal to or more than 20°C to equal to or less than 50°C, most preferably from equal to or more than 45°C to equal to or less than 55°C.

[043] Preferably the time period during which the vegetable proteins and the acid protease are contacted, i.e. the hydrolysis time, is a time period in the range from equal to or more than 10 minutes to equal to or less than 40 hours, more preferably from equal to or more than 30 minutes to equal to or less than 30 hours, most preferably from equal to or more than 1 hour to equal to or less than 20 hours.

[044] The process advantageously allows for an enzymatic incubation and/or subsequent hydrolysis at a low pH, preferably a pH below 6.0, preferably below 5.0. More preferably a pH in the range from equal to or more than 2.0 to equal to or less than 6.0, more preferably in the range from equal to or more than 2.0 to equal to or less than 5.0, and most preferably in the range from equal to or more than 3.0 to equal to or less than 5.0. An additional advantage of operating the process such a low pH, is that contamination by bacterial or other microbial growth is less likely to happen. That is, contamination, for example by bacterial or other microbial growth, can advantageously be reduced. Further, advantageously, the use of alkaline or alkaline earth salts, such as caustic (NaOH) or other salts, can be reduced or even eliminated, advantageously resulting in less salt being present in the final product. Hence, preferably the process is carried out in the absence of any, preferably ex-situ added, alkaline or alkaline earth salts.

[045] The invention thus advantageously also provides a process for preparing hydrolyzed vegetable proteins, wherein the process comprises the steps of: a) preparing an aqueous mixture comprising water and the vegetable proteins; b) contacting the aqueous mixture, comprising the water and the vegetable proteins, with an acid protease, preferably at a pH in the range from 2 to 6, more preferably 3 to 5, preferably at a temperature in the range from 30°C to 60°C, more preferably 20°C to 50°C, most preferably 45°C to 55°C, preferably for a time period in the range from 10 minutes to 40 hours, more preferably 30 minutes to 30 hours, most preferably 1 hour to 20 hours, to hydrolyze the vegetable proteins; c) retrieving the hydrolyzed vegetable proteins from the aqueous mixture.

Preferably the acid protease is an acid protease as described extensively above and preferably the vegetable proteins are vegetable proteins as described extensively above.

[046] As will be clear from the above, the processes according to the invention are not suitable to be carried out “in vivo” and may suitably be classified as “ex-vivo” processes . That is, they are not suitable to be carried out in any whole, living organism, such as an animal or human being.

Preparing an aqueous mixture

[047] The processes according to the invention may comprise a step, further named step a), comprising preparing an aqueous mixture comprising water and the vegetable proteins.

[048] In a preferred embodiment, step a) is comprising a sequence of steps including:

- providing of a vegetable source, preferably seeds, comprising vegetable proteins and vegetable oil;

- optionally milling of the vegetable source;

- solvent extracting of the vegetable oil from the vegetable source to prepare a liquid comprising the vegetable oil and a solid residue comprising the vegetable proteins, wherein preferably the solvent is an alkane, more preferably hexane;

- separating of the liquid and the solid residue, preferably by means of a solid-liquid separation, optionally followed by stripping of residual solvent from the solid residue by further means;

- optionally milling of the solid residue;

- preparing an aqueous mixture comprising water and the, optionally milled, solid residue comprising the vegetable proteins; and wherein such aqueous mixture can suitably be used in a step b) as described above in the section on the process or in the claims. This embodiment for step a) is especially preferable where the vegetable protein is derived from a vegetable source that is a seed, preferably rapeseed. An example of such a step a) is provided by figure 4. Advantageously, the above process steps allow for the use of caustic (NaOH) or other salts to be reduced or even eliminated, advantageously resulting in less salt being present in the final product. As indicated above, preferably the vegetable proteins are proteins from oilseeds, more preferably rapeseed.

[049] In another preferred embodiment, step a) is comprising a sequence of steps including:

- optionally milling of the vegetable source;

- solvent extracting of the vegetable oil from the vegetable source to prepare a liquid comprising the vegetable oil and a first solid residue comprising the vegetable proteins, wherein preferably the solvent is an alkane, more preferably hexane;

- separating of the liquid and the first solid residue, preferably by means of a solid-liquid separation, optionally followed by stripping of residual solvent from the first solid residue by further means;

- optionally milling of the first solid residue;

- solvent extracting of the vegetable proteins from the first solid residue, wherein the solvent is water or an aqueous salt solution, to prepare a liquid comprising water and vegetable proteins and a second solid residue comprising the starch;

- separating of the liquid comprising the water and the vegetable proteins from the second solid residue comprising the starch, preferably by means of a solid-liquid separation;

- optionally washing the vegetable proteins by precipitating the vegetable proteins from the liquid comprising water and vegetable proteins obtained in the preceding step and resuspending the precipitated vegetable proteins in water to prepare a liquid comprising water and washed vegetable proteins; wherein the liquid comprising the water and the, optionally washed, vegetable proteins can suitably be used as the aqueous mixture in step b) as described above in the section on the process or in the claims. This embodiment for step a) is especially preferable where the vegetable protein is derived from a vegetable source that is a bean, preferably soy. Examples of such a step a) are provided by figures 2a and 2b. Advantageously, the above process steps allow for the use of caustic (NaOH) or other salts to be reduced or even eliminated, advantageously resulting in less salt being present in the final product.

[050] In yet another preferred embodiment, step a) is comprising a sequence of steps including:

- providing of a vegetable source, preferably beans and/or pulses, comprising vegetable proteins and starch;

- optionally milling of the vegetable source;

- solvent extracting of the vegetable proteins from the vegetable source, to prepare a liquid comprising the vegetable proteins and a solid residue comprising the starch, wherein preferably the solvent is an aqueous salt solution, preferably an aqueous solution of an alkaline or alkaline earth salt, most preferably an aqueous solution of sodium hydroxide; - separating of the liquid comprising the vegetable proteins from the solid residue comprising the starch, preferably by means of a solid-liquid separation.

In one alternative, enzyme may be added before a subsequent precipitation. That is, in one alternative, such step a) may suitably comprise:

- solvent extracting of the vegetable proteins from the vegetable source, to prepare a liquid comprising the vegetable proteins and a solid residue comprising the starch, wherein the solvent is an aqueous salt solution and the liquid comprises water and vegetable proteins;

- separating of the liquid comprising the water and the vegetable proteins from the solid residue comprising the starch, preferably by means of a solid-liquid separation; wherein the liquid comprising the water and the vegetable proteins may suitably be used as the aqueous mixture in step b) as described above in the section on the process or in the claims.

In another alternative, enzyme may be added after a subsequent precipitation. That is, in another alternative such step a) may suitably comprise the additional subsequent step of:

- retrieving of the vegetable proteins from the liquid, preferably by means of precipitation and/or drying, to obtain a solid residue comprising the vegetable proteins; and

- optionally milling of the solid residue;

- preparing an aqueous mixture comprising water and the, optionally milled, solid residue comprising the vegetable proteins; and wherein such aqueous mixture may suitably be used in a step b) as described above in the section on the process or in the claims.

The above embodiment for step a) is especially preferable where the vegetable protein is derived from a vegetable source that is a pulse, preferably peas or pea flour. An example of such a step a) is provided by figure 3.

[051] In addition to the above acid protease, respectively acid endo-protease, even more preferably fungal, acid endo-protease and most preferably aspergillopepsin I protease, other enzymes may be present, such as for example a protein deamidase such as described in EP4245149A1 , herein incorporated by reference. However, advantageously the acid protease, respectively acid endo-protease, even more preferably fungal, acid endo-protease and most preferably aspergillopepsin I protease can be used as sole enzyme. That is, preferably step(s) a) as described above can be carried out in the absence of enzymes other than the acid protease, respectively acid endo-protease, even more preferably fungal, acid endo-protease and most preferably aspergillopepsin I protease.

Contacting the vegetable proteins with the acid protease

[052] The processes according to the invention may comprise a dedicated step, further named step b), comprising contacting the vegetable proteins, suitably as part of the above described aqueous mixture, with the acid protease. After the contacting, it is preferred for the acid protease to be inactivated.

[053] Preferably, step b) is therefore comprising a sequence of steps including: - contacting the aqueous mixture, comprising water and vegetable proteins, with an acid protease, preferably a fungal acid endo-protease, at a pH in the range from 2 to 6, preferably 3 to 5, preferably at a temperature in the range from 30°C to 60°C, more preferably 20°C to 50°C, most preferably 45°C - 55°C, preferably for a time period in the range from 10 minutes to 40 hours, more preferably 30 minutes to 30 hours, most preferably 1 hour to 20 hours, to hydrolyze the vegetable proteins;

- subsequently inactivating the acid protease, respectively the fungal acid endo-protease, preferably by heating the acid protease, respectively the fungal acid endo-protease, to a temperature above 75°C, more preferably a temperature in the range from 80°C to 95°C, preferably for a period of more than 5 minutes, preferably in the range from 5 to 60 minutes, more preferably in the range from 5 to 10 minutes.

[054] The above is especially preferable where the vegetable protein is derived from a vegetable source that is a seed, preferably rapeseed, where such has additional advantages. An example of such a step b) is provided by figure 4.

Retrieving the hydrolyzed vegetable proteins

[055] The processes according to the invention may comprise a step, further named step c), comprising retrieving the hydrolyzed vegetable proteins.

[056] In a preferred embodiment, such step c) is comprising:

- precipitating of the hydrolyzed vegetable proteins and/or

- drying, preferably spray drying.

Use

[057] The invention further provides the use of an acid protease, preferably a fungal acid endoprotease, more preferably a aspergillopepsin I protease, for the preparation of a hydrolyzed vegetable protein. Preferences for such use, including preferences for the acid protease and the vegetable proteins have already been provided above.

[058] In one preferred embodiment the invention further provides the use of an acid protease, preferably a fungal acid endo-protease, more preferably a aspergillopepsin I protease, for increasing the foamability of a hydrolyzed vegetable protein, preferably a hydrolyzed soy protein.

[059] The invention also provides a foam so produced. That is, the invention suitably also provides foam comprising hydrolyzed vegetable protein, preferably hydrolyzed soy protein, wherein such vegetable protein, respectively soy protein, was hydrolyzed with an acid protease, preferably a fungal acid endo-protease, more preferably a aspergillopepsin I protease. Preferences for how to carry out such hydrolysis are as described herein above and herein below. Further preferences include the aspects, each independently, as exemplified in Examples 2 and 5.

[060] In addition, the invention provides hydrolyzed vegetable protein obtained or obtainable by a process as described above. [061] Advantageously the invention further provides the use of such hydrolyzed vegetable protein as an ingredient in a food or beverages product. Thus, the invention also provides a process for the preparation of a food or beverage comprising such hydrolyzed vegetable proteins.

[062] The hydrolyzed may for example advantageously be used in bakery & cereal products, functional foods, meat alternatives, fish alternatives, snacks, animal food, diary alternatives (such as plant-based ice-cream, plant-based milk, plant-based yoghurt and/or plant-based cheese), clinical/medical nutrition, egg alternatives, plant-based beverages, such as plant-based energy drinks, and/or infant nutrition.

[063] Inventors further note that for both oilseeds protein meal further processing and pluses protein extraction, typically chemical and/or solvent usage is involved. The usage of enzymes can be beneficial in the processing of these protein streams for the purpose of further increasing extraction yield, improve protein solubility at desired pH, and change functionality to meet specific final application (e.g. beverage, baking, plant based meat) needs.

Application of the invention in respect of oilseeds

[064] The invention can be especially advantageous when applied to oilseeds. Oilseeds are therefore a preferred source of vegetable proteins. Sunflowerseed and rapeseed are most preferred. The use of oil seeds as a source for vegetable proteins was mentioned by Bernard [1 ]. [065] The invention therefore also provides a process for preparing hydrolyzed vegetable proteins comprising hydrolysis of vegetable proteins with an acid protease to prepare hydrolyzed vegetable proteins, wherein the acid protease is preferably a, preferably fungal, acid endo-protease; and wherein preferably the vegetable proteins are proteins from a source of vegetable proteins wherein the source of vegetable proteins is preferably oilseeds, more preferably rapeseed.

[066] Cake, meal or flour obtained from oil seeds, such as rapeseed, is preferably processed in a manner as illustrated in figure 4.

[067] Vegetable protein meal obtained from oillseeds after oil extraction using for instance hexane, such as soy meal and rapeseed meal, can be processed for different purposes, including increasing protein content, improving solubility, and changing functionality (such as formability, gelation and emulsification).

[068] The oilseed proteins, preferably rapeseed proteins, are therefore preferred vegetable proteins. A concentrate comprising hydrolyzed oilseed protein, respectively rapeseed protein, obtained or obtainable with the processes according to the invention, advantageously can have a higher amount of protein and an improved solubility. Protein content can be increased from a protein content in the range of 30%-35% wt protein to a protein content in the range of 45%-50% wt protein. Solubility can be improved from10 % to more than 90%. Without wishing to be bound by any kind of theory it is believed that the increased protein content is due to an improved solubility of the hydrolyzed vegetable protein, respectively hydrolyzed rapeseed protein, generated during the processes according to the invention. Application of the invention in respect of soy meal

[069] The invention can be especially advantageous when applied to soy beans and the soy meal obtained therefrom.

[070] Soy meal, suitably obtainable from soy beans, is preferably processed in a manner as illustrated in figure 2a and figure 2b. The process according to the invention and the processes illustrated in figure 2a and figure 2b can advantageously provide a soy protein concentrate having improved solubility. This is illustrated in the examples, where the hydrolyzed vegetable proteins, i.e. the hydrolyzed soy proteins, in a soy protein concentrate that was generated after hydrolysis with an acid protease in a process according to the invention, had an improved protein solubility, especially at a lower pH in the range from 3 to 5. Such an improved protein solubility at such lower pH is advantageous for application in acidic plant-based beverages. For example, plant-based energy drinks are typically acidic, having a pH in the range from 3 to 5. An improved solubility at such a pH allows one to increase the protein content in the plant-based energy drink

[071] The soy proteins are therefore preferred vegetable proteins. The hydrolyzed soy protein according to the invention, preferably obtained or obtainable with the process according to the invention, can therefore advantageously be used as an ingredient in a food or beverages product. Preferably such food or beverages product is a food or beverages product having a pH in the range from 3 to 5. More preferably such food or beverages product is a plant-based beverage, most preferably a plant-based energy drink, which plant-based energy drink preferably has a pH in the range from 3 to 5. The invention further provides a process for the preparation of a plant-based energy drink comprising a hydrolyzed soy protein, wherein such hydrolyzed soy protein was obtained in a process comprising hydrolyzing of a soy protein with an acid protease. Preferences for such acid protease and the process conditions are as described herein above.

[072] In addition to the above, the present invention thus preferably provides a plant-based beverage, having a pH in the range from equal to or more than 3 to equal to or less than 5, wherein the plant based beverage comprises a hydrolyzed vegetable protein, preferably a hydrolyzed soy protein, wherein such hydrolyzed vegetable protein, respectively hydrolyzed soy protein, was prepared by hydrolyzing a vegetable protein, respectively a soy protein, with an acid protease. Further the invention provides a process for preparing a plant-based beverage, comprising:

(i) preparing hydrolyzed vegetable protein, preferably hydrolyzed soy protein, by means of hydrolysis of vegetable protein, respectively soy protein, with an acid protease to prepare hydrolyzed vegetable proteins, wherein the acid protease is preferably a, preferably fungal, acid endo-protease; and

(ii) including the hydrolyzed vegetable protein, respectively hydrolyzed soy protein, in a beverage which is arranged to have a pH in the range from equal to or more than 3 to equal to or less than 5. Preferences are as described already herein above. Application of the invention in respect of pulses

[073] The invention can be especially advantageous when applied to pulses such as peas meal or pea flour. Production of pulse protein ingredients and their application in plant-based milk alternatives was described by Vogelsang-O’Dwyer et.al. [2],

5 [074] Vegetable protein from pulsus, such as pea and faba, can be extracted from the raw material to obtain high protein content products, such as protein concentrates and isolates. The extraction process allows for protein content increase but also for protein functionality improvement. [075] Pulse meal or pulse flour, such as peas meal or pea flour, is preferably processed in a manner as illustrated in figure 3. w [076] The pulse proteins, preferably pea proteins, are preferred vegetable proteins. The hydrolyzed pulse protein, respectively pea protein, obtained or obtainable with the processes according to the invention, has an advantageously improved emulsification capability and foamability. Advantageously the improved emulsification capabilities can be maintained for a prolonged time, even up to 7 days.

15

[077] Herein below the invention will be illustrated by a number of non-limiting examples.

Examples

Methods and materials

[078] In the examples the following enzymes were used:

Table 1 : Enzymes used in the experiments

[079] The enzymes Maxipro® BAP, Maxipro® NP and Maxipro® AFP are all commercially obtainable from DSM Food & Beverages, Delft, the Netherlands.

[080] As mentioned before, Maxipro® AFP (Acid Fungal Protease) is classified as an aspergillopepsin I protease, it possesses broad peptide bond specificity, it is an acid-protease and has its optimum pH in the acidic range, i.e. in the range from 2 to 6.

Explanation of the Examples

[081] As illustrated in example 1 below, the hydrolysis activity of Maxipro® AFP, when applied on animal- based protein (i.e. proteins from an animal source), such as gelatin, is very limited. Even when an overdosage of Maxipro® AFP of 10 times is applied, the hydrolysis results are not getting close to those of conventional product Maxipro® BAP.

[082] However, in contrast, when applied in the hydrolysis of vegetable proteins (i.e. plant-based proteins), such as soy meal, pea flour and rapeseed cake, as illustrated in respectively examples 2, 3 and 4, a more than substantial hydrolysis action was observed. In some cases the degree of hydrolysis even outperformed that of the conventional product Maxipro® BAP. That is, in some cases the hydrolysis with Maxipro® AFP resulted in an even higher degree of hydrolysis than was observed for conventional product Maxipro® BAP.

[083] The results are even more surprising as the hydrolysis action of Maxipro AFP is conducted at an acidic pH, in the examples being between 3 and 4. This is a pH that is unlikely to result in protein hydrolysis, as this range is close to the pl (isoelectric point) of the vegetable protein.

[084] As mentioned before, an additional advantage of operating the process such a low pH, is that contamination by bacterial or other microbial growth is less likely to happen. Further, advantageously, the use of alkaline or alkaline earth salts, such as caustic (NaOH) or other salts, can be reduced or even eliminated, advantageously resulting in less salt being present in the final product.

[085] Besides the surprising hydrolysis action, the treatment of vegetable proteins with Maxipro® AFP advantageously results in improved solubility and provides other functionality improvements. The actions and effects of Maxipro® AFP on different protein types are illustrated in the examples as follows:

- Soy meal: a high degree of hydrolysis, and significant solubility increase across the entire pH range of pH 3 to pH 8 can be obtained, as illustrated in example 2.

- Pea flour: a high degree of hydrolysis similar to neutral protease as illustrated in example 3. In addition improvements in emulsification and foamability can be obtained.

- Rapeseed cake: Maxipro® AFP was able to hydrolyze protein in rapeseed cake, enabling them to be extracted by solubilizing into the liquid phase, increasing the protein content from 30% wt to 40-45% wt, as illustrated in example 4.

Example 1 (comparative)

Pork gelatin hydrolysis with Maxipro® BAP and Maxipro® AFP

[086] The hydrolysis of pork gelatin using Maxipro® AFP or Maxipro® BAP is shown in this example. The process and analytical method are also described.

[087] The experiment was conducted at 30 g scale with pork gelatin powder. A 20% w/w gelatin suspension was made by adding tap water. The suspension was heated up to 50°C after adjusting its pH. Then, enzyme was added to start incubation. The time sample were taken after 2, 4, and 20h incubation. The samples were inactivated at 95°C for 10min. Solid-liquid separation was done by centrifugation at 4500 rpm for 20 min. The liquid phase, which contains the hydrolyzed gelatin, was collected and used for free amino nitrogen (FAN) and molecular weight distribution of peptides analysis.

[088] Free Amino Nitrogen (FAN) analysis asses the level of free amino acids and the level of terminal a-amino nitrogen groups in protein samples, which is an indirect indication of degree of hydrolysis of the protein. The FAN results of Maxipro® BAP, bacterial alkaline protease, and Maxipro® AFP acid protease - Aspergillopepsin I - incubated gelatin are given in Table 2, which shows that Maxipro® AFP was not able to match the hydrolysis action of Maxipro® BAP with a 10 times overdosage.

Table 2. Free Amino Nitrogen (FAN) results in gelatin samples after enzymatic treatment

[089] Measuring the peptide molecular weight (MW) distribution of the enzymatic treated gelatin is a direct indication of the action of the enzyme treatment. It shows what type of peptides are derived after hydrolyzing the protein. The peptide MW distribution of the hydrolyzed gelatin samples are given in Table 3, which shows that Maxipro® AFP was not able to match the action of Maxipro® BAP, even not so with 10 times overdosage.

Table 3. Peptide MW distribution of gelatin samples after enzymatic treatment

Method for Free amino nitrogen (FAN) analysis

[090] Ninhydrin color reagent reacts with terminal a-amino nitrogen groups to form a purple complex. The absorbance produced is proportional to the number of terminal a-amino nitrogen groups are reacting and therefore to the free amino nitrogen content present in the samples.

[091] The Ninhydrin color reagent A was prepared by dissolving 12.4 g Na2HPO4.2H2O, 15.0 g KH2PO4, 1 .25 g ninhydrin and 0.75 g fructose in a total of 250 ml of MilliQ-water. The pH of solution was adjusted to between 6.6 and 6.8.

[092] The dilution reagent B was prepared by dissolving 1 g KIO3 in 300 ml MilliQ-water and add 200 ml 96% ethanol

[093] The analysis was start with diluting the collected liquid fraction to a concentration of approximately 10ug glycine per ml. Then, 1 .0ml of diluted sample was mixed with 0.5ml of Ninhydrin color reagent A, and then heated at 100°C for 16min. After cooling the solution to 20°C for 20min, 2.5ml of dilution reagent B was added and mixed well. The solution was transferred into 1 ml cuvette and measure its absorption at a wavelength of 570nm. [094] The calibration line was made using glycine solution with concentrations between 0 and 21.4 pg/ml. The FAN value of sample was calculated as:

FAN = X * DF [pg FAN as glycine/mL]

Where X is the concentration of glycine derived from the calibration curve [pg/mL]; and Df is the dilution factor of the sample

Method for determining Molecular Weight (MW) distribution of peptides

[095] Analysis is done using Waters HCIass-Bio UPLC system (LC905) with a ACQUITY UPLC Protein BEH SEC Column (125A, 1 .7 pm, 4.6 mm X 150 mm). The chromatograms were generated on a UV detector at 214nm with a workflow of 0.3ml/min of 10OmM NaPhosphate buffered solution and 400mM NaCI (pH 6.4-6.5).

[096] The protein with known molecular weight were used as standards to assign the peak retention time. The standards used in the analysis are Bovine Serum Albumin (66 kDa), Ovalbumin (44 kDa), Eguine Myoglobin (17 kDa), [Glu]-Fibrinopeptide B (1.57 kDa), Glutathione oxidized (0.612 kDa), Glutathione reduced (0.307 kDa). Based on the retention time of peptides, the molecular weight of was assigned as shown in Table 4. The chromatogram was then divided into 5 sections with assigned MW. The molecular weight distribution of peptides is calculated as the percentage of the peak area of each section in the total peak area.

Table 4: Assignment of peptide molecular weight in KDa based on the retention time of the peak

Example 2 (according to the invention)

Soy meal hydrolysis with Maxipro® BAP and Maxipro® AFP

[097] The effect of Maxipro® AFP and Maxipro® BAP on soy protein solubility is shown in this example. The process and analytical method used are also described.

[098] The experiment was conducted at 2.5 kg scale with defatted soy flour. 500 g of soy flour was used to make 20% DM suspension. The pH was adjusted to 4.5. After 40 min agitation at 45°C, the suspension was centrifuged at 4000 g for 10 min. The pellet was collected and re-suspended in water at 20% DM solution. Enzyme was added after adjusting the pH of solution. The incubation was carried out for 2 h at 55°C. The solution was freeze dried to obtain soy protein concentrate powder. [099] The solubility of obtained soy protein concentrate was tested by suspending the protein concentrate at 2% protein content in pH environment from 3 to 8. After agitation for 30min, the protein suspension is centrifuged for 10min at 15000g. The protein content in liquid fraction was determined by the Kjeldahl assay. The solubility is calculated as a percentage of protein dissolved in aqueous phase, and the results are given in Table 5. It can be seen that when Maxipro® AFP is used, the solubility of the soy protein increased significantly at low pH (i.e. pH 3-5), which is a higher solubility compared to Maxipro® BAP treated protein.

Table 5. Protein solubility of soy protein concentrate with and without enzyme treatment

As illustrated by the results in Table 5, the hydrolyzed vegetable proteins in the soy protein concentrate that was generated after hydrolysis with the acid protease Maxipro® AFP have an improved protein solubility, especially at a lower pH in the range from 3 to 5.

Kjeldahl assay for determining protein content

[100] The analysis of the determination of Kjeldahl nitrogen involves two steps. The sample is first digested in sulfuric acid (95-97%) after addition of a catalyst (1 .5 g K2SO4 and 7.5 mg Se) during 90 minutes at 360°C. Then, sample is injected in San⁺⁺ Continuous Flow Analyzer System. It means the sample containing ammonium is mixed with a continuously streaming flow of a buffer solution.

NH4+ + OH- ->• NH3f + H2O

[101] The ammonia formed is separated in a diffusion cell from the solution over a hydrophobic semipermeable membrane and taken up by a streaming recipient flow containing an indicator. Due to the resulting pH shift, the buffer solution will change its color which is measured continuously in the flow photometer. The absorbance at 660nm is recorded continuously as Nitrogen peak. The Total Kjeldahl Nitrogen is defined as sum of present organic and ammonium Nitrogen. And the protein content is calculated as 6.25 times Kjeldahl Nitrogen value. Example 3 (according to the invention)

Pea flour hydrolysis with Maxipro® TNP and Maxipro® AFP

[102] The test and results of hydrolyzing pea flour using Maxipro® AFP or Maxipro® TNP are described.

[103] The experiment was conducted at 3.3 kg scale with pea flour. 500 g of pea flour was used to make 15% DM suspension. The pH was adjusted to 9. After 30 min agitation, the suspension was centrifuged at 3000 g for 5 min. The supernatant was then collected, and the pH was adjusted to 5.5. Enzyme was added in the supernatant after increasing the temperature to 50 °C. The incubation was carried out for 30min. The pH of the supernatant was lowered to 4.5, and then centrifuged at 500 g for 5 min. The pellet was collected, and re-suspended in water, the pH was then adjusted to 7. After which, the suspension was heated up to 90 °C for 10 min before been freeze dried to become pea protein isolate powder.

[104] Free Amino Nitrogen (FAN) analysis asses the level of free amino acids and the level of terminal a-amino nitrogen groups in protein samples, which is an indirect indication of degree of hydrolysis of the protein. The FAN results of Maxipro® TNP (neutral protease) and Maxipro® AFP incubated pea protein isolate are given in Table 6, which shows that Maxipro® AFP had a matching hydrolysis degree as Maxipro® TNP at the same dosage.

Table 6. Free Amino Nitrogen (FAN) results in pea protein isolate after enzymatic treatment

[105] Measuring the peptide molecular weight (MW) distribution of the enzymatic treated pea protein isolate is a direct indication of the action of the enzyme treatment. It shows what type of peptides are derived after hydrolyzing the protein. The peptide MW distribution of the hydrolyzed pea protein isolate samples are given in Table 7, which shows that Maxipro® AFP matched the action of Maxipro® TNP with the same dosage.

Table 7. Peptide MW distribution of pea protein isolate samples with and without enzymatic treatment

Example 4 (according to the invention)

Rapeseed cake hydrolysis with Maxipro® AFP

[106] The application process and effect of Maxipro® AFP on extraction yield of rapeseed protein is described.

[107] The experiment was conducted at 30 g scale with two types of rapeseed cake (A and B). A 20% w/w rapeseed cake suspension was made using tap water. The suspension was heated up to 50 °C after adjusting its pH to 4. Then, enzyme was added to start incubation. The time sample were taken after 2 and 24 h incubation. The samples were inactivated at 95 °C for 5 min. It was then centrifuged at 15000 g for 10 min. The liquid fraction was used for dry matter and protein content (kjeldahl) analysis

[108] The extraction yield on protein and the protein content in extracted products are given in Table 8. It can be seen that when Maxipro® AFP is used, significant increase of extraction yield and protein content in the extracted products can be achieved.

Table 8. Results of the rapeseed cake protein extraction test.

Dry matter

[109] The Halogen Moisture Analyzer is used for determining the dry matter of the liquid fraction. The instrument works on the thermogravimetric principle. At the start of the measurement the Moisture Analyzer determines the weight of the sample, the sample is then quickly heated by the integral halogen heating module and the moisture vaporizes. During the drying process the instrument continually measures the weight of the sample and displays the reduction in moisture. Once drying has been completed, the moisture or solids content of the sample is displayed as the final result.

[110] The dry matter of liquid sample is determined at 120°C as percentage of the final weight in the initial weight.

Example 5 (according to the invention)

Functionality test of soy protein product obtained from Example 2

[111] Dispersibility and foamability of the soy protein products obtained from the process as described in example 2, which will be named “SPP” in this example, were studied.

Dispersibility

[112] 1 g of SPP was added on top of 100g of water, the solutions were placed at lab room temperature (i.e. ~20 °C), and recorded the needed time for all SPP dispersed into water.

The time of addition is recorded as time 0, and then every minute, a snapshot is taken to determine if all SPP added is dispersed into water, and to estimate the amount of SPP that is dispersed into water. The results are shown in Table 9 below.

Table 9. Dispersibility test results of SPP processed with and without enzyme. +++ means all SPP is dispersed into water, ++ means 50% of SP is dispersed into water, + means 10% of SPP is dispersed into water, - means none of SPP is dispersed into water.

n.d. = not determined

Foamability

[113] The soy protein product (“SPP”) was gradually mixed into water at low agitation speed to a 10% protein (w/w) suspension. The pH of suspension was adjusted to 7 using 4M HCL or NaOH. After fully hydration for 1 h, 100g of protein suspension was transferred to a plastic beaker. It was whipped by a kitchen hand mixer for 4min with the highest mixing speed. Then 50g foam was gently transferred into a cylinder, The height of foam was recorded right away and after 1 h. The results are shown in Table 10 below

Table 10. Foamability test results of SPP process with and without enzyme, foam volume recorded immediately right after whipping (time 0), and after standing for 1 hour on the bench

Example 6 (according to the invention)

Pea flour processing with enzyme and effect on solubility of obtained pea protein concentrate

[114] In this example 960 grams of the pea flour was added into 4800 g tap water while agitating, temperature was adjusted to 45-50°C. The pH was adjusted to 3 or left as such depending on the enzyme used (see Table 11). The enzyme is then added, the dosage was 1% based on protein content of pea flour, and allow to incubate for 2 hours with agitation at 50 °C. After the incubation, pH was adjusted to 9 and the suspension was centrifuged at 4000G for 10 min. The supernatant was then collected, and its pH was adjusted to 7. The suspension was heated up to 90°C with a micro-pilot heat exchanger (OMVE) and held for 2 min to inactivate the enzyme.

[115] The obtained suspension was then freeze dried to powder from, which is the end product, pea protein concentrate.

[116] The solubility of obtained pea protein concentrate was tested by suspending the protein concentrate at 2% protein content in pH environment from 3 to 8. After agitation for 30min, the protein suspension is centrifuged for 10min at 15000g. The protein content in liquid fraction was determined by the Kjeldahl assay, as described in Example 2. The solubility is calculated as a percentage of protein dissolved in aqueous phase, and the results are given in Table 11 .

[117] It can be seen that when Maxipro AFP is used in combination with a phytase, Maxamyl P, the solubility of the obtained pea protein increased significantly, compared to both ‘no enzyme’ process and other enzyme processes, namely Maxipro BAP and TNP. Table 11. Protein solubility at different pH of pea protein concentrate processed with and without enzyme

Example 7 (according to the example)

Functionality test of pea protein product obtained from Example 6

[118] Dispersibility and texture of the pea protein products obtained from the process as described in example 6, which will be named PPP in this example, were studied.

Dispersibility

[119] 1 g of pea protein product (“PPP”) was added on top of 100g of water, the solutions were placed at lab room temperature (i.e. ~20 °C), and recorded the needed time for all PPP dispersed into water.

[120] The time of addition is recorded as time 0, and then every minute, a snapshot is taken to determine if all PPP added is dispersed into water, and to estimate the amount of PPP that is dispersed into water. The results are shown in Table 12 below.

Table 12. Dispersibility test results of PPP processed with and without enzyme. In the table “ +++” means all PPP is dispersed into water, “++” means 50% of PP is dispersed into water, “+ “means 10% of PPP is dispersed into water, “means none of PPP is dispersed into water.

n.d. = not determined

Texture

[121] For this test water and pea protein product (“PPP”) were weighted as described in Table

13. PPP was poured into water and mixed for 15min with 500rpm stirring. The solution was then poured on a smooth surface. The texture of solution was evaluated based whether clumps can be found. The findings are listed in Table 14.

Table 13 Ingredients of making PPP solution

Table 14 Texture of PPP solution. In the table “+” means no clumps can be found, means more than 5 clumps (>2mm) were found.

Reference

[1] Feed Ingredients | Feed Concentrates: Oilseed and Oilseed Meals; J.K. Bernard, Encyclopedia of Dairy Sciences (Second Edition), 2011 , Pages 349-355.

5 [2] Production of pulse protein ingredients and their application in plant-based milk alternatives;

Vogelsang-O’Dwyer et.al. Trends in Food Science & Technology, Volume 110, April 2021 , Pages 364-374.

[3] Whole soybean protein extraction processes: A review; Preecea et.al. Innovative Food w Science & Emerging Technologies, Volume 43, October 2017, Pages 163-172.

[4] Towards plant protein refinery: Review on protein extraction using alkali and potential enzymatic assistance; Sari et.al, Biotechnol. J. 2015, 10, Pages 1138-1157.

15 [5] Degradation of white wine haze proteins by Aspergillopepsin I and II during juice flash pasteurization; Marangona, et.al. Food Chemistry, Volume 135, Issue 3, 1 December 2012, Pages 1157-1165

Claims

Claims A process for preparing hydrolyzed vegetable proteins comprising hydrolysis of vegetable proteins with an acid protease to prepare hydrolyzed vegetable proteins, wherein the acid protease is preferably a, preferably fungal, acid endo-protease; and wherein preferably the vegetable proteins are proteins from a source of vegetable proteins; and wherein preferably the source of vegetable proteins is oilseeds, more preferably rapeseed. The process according to claim 1 , wherein the acid protease is produced in Aspergillus niger. The process according to any one of the preceding claims, wherein the acid protease is an aspergillopepsin I protease. The process according to any one of the preceding claims, wherein the acid protease is Maxipro® AFP, commercially obtainable from DSM Food & Beverages in the Netherlands. The process according to any one of the preceding claims, wherein the vegetable proteins comprise or consist of soy proteins, pea proteins, rapeseed proteins, barely proteins, rice proteins, and/or proteins from brewer’s spent grain. The process according to any one of the preceding claims, wherein the process comprises the steps of: a) preparing an aqueous mixture comprising water and the vegetable proteins; b) contacting the aqueous mixture, comprising the water and the vegetable proteins, with the acid protease, preferably at a pH in the range from 2.0 to 6.0, more preferably 3.0 to 5.0, preferably at a temperature in the range from 30°C to 60°C, more preferably 20°C to 50°C, most preferably 45°C to 55°C, preferably for a time period in the range from 10 minutes to 40 hours, more preferably 30 minutes to 30 hours, most preferably 1 hour to 20 hours, to hydrolyze the vegetable proteins; c) retrieving the hydrolyzed vegetable proteins from the aqueous mixture. The process according to claim 6, wherein step a) is comprising a sequence of steps including:

- optionally milling of the vegetable source; - solvent extracting of the vegetable oil from the vegetable source to prepare a liquid, comprising the vegetable oil, and a solid residue, comprising the vegetable proteins, wherein preferably the solvent is an alkane, more preferably hexane;

- optionally milling of the solid residue;

- preparing an aqueous mixture comprising water and the, optionally milled, solid residue comprising the vegetable proteins; and wherein such aqueous mixture is used in step b). The process according to claim 6, wherein step a) is comprising a sequence of steps including:

- optionally milling of the vegetable source;

- solvent extracting of the vegetable oil from the vegetable source to prepare a liquid, comprising the vegetable oil, and a first solid residue, comprising the vegetable proteins, wherein preferably the solvent is an alkane, more preferably hexane;

- optionally milling of the first solid residue;

- solvent extracting of the vegetable proteins from the first solid residue, wherein the solvent is water or an aqueous salt solution, to prepare a liquid, comprising water and vegetable proteins, and a second solid residue, comprising carbohydrates and/or fibers;

- separating of the liquid, comprising the water and the vegetable proteins, from the second solid residue, comprising the carbohydrates and/or fibers, preferably by means of a solidliquid separation;

- optionally washing the vegetable proteins by precipitating the vegetable proteins from the liquid, comprising water and vegetable proteins, obtained in the preceding step and resuspending the precipitated vegetable proteins in water to prepare a liquid comprising water and washed vegetable proteins; wherein the liquid comprising the water and the, optionally washed, vegetable proteins is used as the aqueous mixture in step b).

The process according claim 6, wherein step a) is comprising a sequence of steps including: - providing of a vegetable source, preferably beans and/or pulses, comprising vegetable proteins and starch;

- optionally milling of the vegetable source;

- solvent extracting of the vegetable proteins from the vegetable source, to prepare a liquid comprising the vegetable proteins and a solid residue comprising the starch, wherein preferably the solvent is an aqueous salt solution, preferably an aqueous solution of an alkaline or alkaline earth salt, most preferably an aqueous solution of sodium hydroxide;

- separating of the liquid comprising the vegetable proteins from the solid residue comprising the starch, preferably by means of a solid-liquid separation.

10. The process according to claim 9 wherein step a) is comprising:

- separating of the liquid comprising the water and the vegetable proteins from the solid residue comprising the starch, preferably by means of a solid-liquid separation; wherein the liquid comprising the water and the vegetable proteins is used as the aqueous mixture in step b).

11 . The process according to claim 9 wherein step a) is comprising the additional subsequent step of:

- optionally milling of the solid residue;

- preparing an aqueous mixture comprising water and the, optionally milled, solid residue comprising the vegetable proteins; and wherein such aqueous mixture is used in step b).

12. The process according to any one of claims 6 to 11 , wherein step b) is comprising a sequence of steps including:

- contacting the aqueous mixture, comprising water and vegetable proteins, with the acid protease at a pH in the range from 2 to 6, preferably 3 to 5, preferably at a temperature in the range from 30°C to 60°C, more preferably 20°C to 50°C, most preferably 45°C - 55°C, preferably for a time period in the range from 10 minutes to 40 hours, more preferably 30 minutes to 30 hours, most preferably 1 hour to 20 hours, to hydrolyze the vegetable proteins;

- subsequently inactivating the acid protease, preferably by heating the acid protease to a temperature above 75°C, more preferably a temperature in the range from 80°C to 95°C, preferably for a period of more than 5 minutes, preferably in the range from 5 to 60 minutes, more preferably in the range from 5 to 10 minutes.

13. The process according to any one of claims 6 to 12, wherein step c) is comprising:

- precipitating of the hydrolyzed vegetable proteins and/or

- drying, preferably spray drying.

14. Use of an acid protease, preferably a fungal acid endo-protease, more preferably a aspergillopepsin I protease, for the preparation of a hydrolyzed vegetable protein.

15. Hydrolyzed vegetable protein obtained or obtainable by a process as defined in any one of claims 1 to 13 or the use of claim 14.

16. Use of a hydrolyzed vegetable protein as defined in claim 15, as an ingredient in a food or beverages product.

17. Process for the preparation of a food or beverage comprising a hydrolyzed vegetable protein as defined in claim 15.

18. Process for the preparation of a beverage having a pH in the range from equal to or more than 3 and equal to or less than 5, comprising a hydrolyzed vegetable protein as defined in claim 15, wherein such hydrolyzed vegetable protein is a hydrolyzed soy protein.

19 Beverage having a pH in the range from equal to or more than 3 and equal to or less than 5, comprising a hydrolyzed vegetable protein as defined in claim 15, wherein such hydrolyzed vegetable protein is a hydrolyzed soy protein.