Rapeseed protein hydrolysate

Abstract

The present invention relates to a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa.

Description

[0001] RAPESEED PROTEIN HYDROLYSATE

[0002] Field of the invention

[0003] The present invention relates a rapeseed protein hydrolysate. Further, the present invention relates to the process for preparing hydrolyzed rapeseed proteins. Finally, the present invention relates to the use of the rapeseed protein hydrolysate.

[0004] Background of the invention

[0005] Proteins are a crucial part of human diet, and this is for a major part met by proteins from animal origin, such as dairy proteins or protein from meat or fish. World population growth in combination with increasingly limited resources has resulted in the need for alternative protein sources to meet global protein demand. The production of plant-based foods requires less land and water and is associated with lower greenhouse gas emissions compared with animal-based foods.

[0006] Generally, the consumed proteins are intact proteins, however for specific reasons, hydrolysed proteins may have advantages. Nearly fully hydrolysed proteins lead to mostly free amino acids and some oligopeptides, for use in for instance flavours (savoury flavours from hydrolysed yeast proteins, rich in glutamate). Such hydrolysates can be made by proteolysis, using (combinations of) proteases, or in the case of several yeast extracts, autolysis. Alternatively, these can be made by chemical hydrolysis using highly concentrated acids.

[0007] In other cases, hydrolysis should not proceed to the individual free amino acids, but generate peptides of various length, depending on the application, which can be tuned by the strategy of hydrolysis, choice of proteases, sequence of addition of enzymes, environmental conditions (pH, temperature etcetera) and pretreatment (for instance heating). There are various reasons for such hydrolysates, mostly for nutritional or functional reasons.

[0008] Nutritional protein hydrolysates can be used for general improved protein absorption for sports recovery or medical nutrition, such as against sarcopenia. Bioactive peptides are claimed to present a greater potential as health-promoting ingredients, may be better absorbed in the intestine. As described by V.G. Tacias-Pascacio, et al. (Intern. J. Biol. Macromol. 2020 165 p2143-2196 Use ofAlcalase in the production of bioactive peptides: A review), "... bio-functionalities of peptides have been demonstrated on in vitro and in vivo studies, including positive impacts on cardiovascular, immune or nervous systems, such as inhibitors of the angiotensin-l-converting or dipeptidyl peptidase IV enzymes, antioxidant, antithrombotic, opioid, hypocholesterolemic or immunomodulating activities” [direct quote]

[0009] Moreover, by hydrolysis, potential allergenic peptides can be broken up, hence reducing or removing allergenicity.

[0010] Protein hydrolysates find their use also in feed for fermentation (such as for lactic acid bacteria or starters cultures and probiotics), biopharmaceuticals (to produce for instance toxins and vaccines), diagnostics (components of culture media), and cell cultures. For example, there is a need in the art for serum free media that can be used for the growth of cells such as cultured meat. Functional properties of protein hydrolysate may also improve compared to their unhydrolyzed origins, as is described by for instance Wouters et al. (Comp Rev Food Sci Safety 2016 15 p786 Relevance of functional properties of enzymatic plant protein hydrolysates in food systems)-. Depending on the strategy of hydrolysis, changes can be obtained that can include better solubility, better foaming, better emulsification, and may also impact gelation and water holding properties. Protein hydrolysates may for instance also act as plasticizer for use in films and casings (Zhang, C., et al. Food Chem. 2019 272, 694-701 The preparation and physiochemical characterization of rapeseed protein hydrolysate-chitosan composite films), and in more solid foods such as protein bars cheese or cheese alternatives or meat alternatives. Protein hydrolysates may also display anti-oxidative properties, by for instance improved chelating properties of specific protein hydrolysates, as has been mentioned particular for a hydrolysate of rapeseed protein isolate using a fungal protease, described by Durand et al. (Eur. J. Lipid Sci. Technol. 2021 , 123, 2000380 High Metal Chelating Properties from Rapeseed Meal Proteins to Counteract Lipid Oxidation in Foods: Controlled Proteolysis and Characterization and Kaugarenia_et al. Foods 2022, 11 , p2618 Optimization of Selective Hydrolysis of Cruciferins for Production of Potent Mineral Chelating Peptides and Napins Purification to Valorize Total Rapeseed Meal Proteins). In the latter it is even clear that using a fungal protease leads to hydrolysis of particularly cruciferin, and leaves napin mostly intact. Proteases are generally divided into two classes endoproteases (endopeptidase, endo proteinases) and exoproteases (exopeptidases). An endoprotease cleaves a peptide bond between amino acids within the protein chain, not at the termini. The exoproteases will cleave such peptide bond between amino acids at the termini of the protein molecule, either from the N-terminus or the C-terminus. Typical examples of endoproteases are those found in the digestive tract, such as pepsin, trypsin and chymotrypsin, as well as subtilisin.

[0011] Subtilisin is probably the most common protease used in food and non-food (such as laundry and dishwashing detergents), because of its aggressive character and its costeffectiveness. It originates from Bacillus subtilis, but industrially is mostly produced in Bacillus amyloliquefaciens. It is a serine protease with molecular weight of typically around 27kDa. See for instance Azrin et al Biotech and App Biochem - 2022 p1-18 Versatility of subtilisin: A review on structure characteristics and applications. Disadvantage of hydrolysates prepared by subtilisin is that the protein hydrolysates are perceived highly bitter.

[0012] Flavourzyme is also promoted as a good protease, it is a fungal protease complex produced by Aspergillus oryzae, and it contains both endoprotease and exopeptidase activities. At pH 7, when exo-protease activity prevails, a high degree of hydrolysis may be obtained, but that is mostly due to the generation of single amino acids, hydrolysed from the termini of the protein. At pH 5 the endoprotease activity is said to be more dominant. Often a combination of subtilisin (Alcalase) and Flavourzyme can be used to obtain functional hydrolysates, and it is also claimed to limit the bitterness level of the hydrolysates formed. It is claimed that the exo-activity of Flavourzyme leads to debittering of the peptides. US10619177 describes how common proteases such as Alcalase (tradename for subtilisin) and Flavourzyme (tradename for a fungal protease / peptidase complex produced by Aspergillus oryzae) are capable of hydrolysing soy protein and wheat gluten to a substantial extent at standard conditions (0.25% Alcalase on 12% soy bean meal, containing approximately 50% protein, reached a degree of hydrolysis of 12.4% in 2 hours at 70°; subsequent addition of 3% Flavourzyme at 50°C and 4 hours incubation led to a degree of hydrolysis of 22.4%).

[0013] Another strategy to reduce bitterness of protein hydrolysates is by treating a pre-hydrolysed protein further with a proline-specific endoprotease such as DelvoPlant PSP or DelvoPlant® PSP, as has been described by Edens et al. (J Agric Food Chem 2005 53 p7950-7957, Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates).

[0014] Rapeseed protein is abundantly available and is used more and more for the production of rapeseed protein products for the food and beverage industry.

[0015] Rapeseed seeds are rich in oil and contain considerable amounts of protein that accounts for 17 to 25% of seed dry weight. Processing rapeseed for oil for human consumption produces rapeseed meal as a by-product which contains about 30 to 40% protein. The rapeseed used for this purpose is usually of the varieties Brassica napus and Brassica juncea. These varieties contain only low levels of erucic acid and glucosinolate, and are also known as Canola. Canola is a contraction of Canada and ola, for "oil low acid“, but is now a generic term defined as rapeseed oil comprising <2% erucic acid and <30 mmol / g glucosinolate. The resultant rapeseed meal is currently used as a high-protein animal feed.

[0016] The predominant storage proteins found in rapeseed are cruciferins and napins (S.P. Perera, T.C. McIntosh, J.P.D. Wanasundra, Plant 2016, 5, p36, Structural Properties of Cruciferin and Napin of Brassica napus (Canola) Show Distinct Responses to Changes in pH and Temperature). Cruciferins are globulins and are the major storage protein in the seed. It is composed of 6 subunits and has a total molecular weight of approximately 300 kDa. Napins are albumins and are a low molecular weight storage protein with a molecular weight of approximately 14 kDa. Napins are more easily solubilized and in for example EP 1715752B1 a process is disclosed to separate out the more soluble napin fraction, preferably to at least 85 wt.%. Napins are primarily proposed for use in applications where solubility is key. DE 10 2014 005466 A1 also describes a process for obtaining purified cruciferin and napin fractions. During the process, also a protein mixture of the two with 55-60% napins and 40-45% cruciferins is obtained. The solubility of this protein mixture is approximately 75%.

[0017] Rapeseed proteins can be also divided into various fractions according to the corresponding sedimentation coefficient in Svedberg units (S). This coefficient indicates the speed of sedimentation of a macromolecule in a centrifugal field. For rapeseed proteins, the main reported fractions are: 12S, 7S and 2S. Cruciferin and napin are the two major families of storage proteins found in canola / rapeseed. Napin is a 2S albumin, and cruciferin is a 12S globulin.

[0018] Hydrolysates from rapeseed protein isolate are expected to have a high solubility even after heating or pH shifts, it is expected to improve I speed up the digestion of rapeseed protein, and will change the taste, flavour and mouthfeel characteristics of the rapeseed protein. I

[0019] R. He et al. (Food Research International, 2012, vol 49, pages 432-438, Antioxidant activities of rapeseed peptides produced by solid state fermentation) describe the preparation of rapeseed peptides by solid state fermentation with Bacillus subtilis. Rapeseed peptides were prepared by mixing a quantity of the fermented matter with water, centrifugation and fractionating the supernatant through an ultrafiltration membrane with molecular weight cut off of 5000 Da. Hence, the molecular weight distribution data shown in Figure 1 B do not provide any data above 5 kDa.

[0020] Whilst subtilisin (Alcalase) would be the first protease of choice to hydrolyse rapeseed protein isolate, it appears poorly capable of hydrolysing rapeseed protein isolate, literature such as of Vioque et al (J Enzyme Inhib. 2001 , 16 p 81-87 Alcalase rapeseed inhibitors: purification and partial characterization) claim that rapeseed protein isolates inhibit subtilisin. Still many reports claim Alcalase to be efficient in hydrolysing rapeseed protein isolate (Tacias-Pascacio et al. Intern. J. Biol. Macromol. 2020 165 p2143-2196 Use of Alcalase in the production of bioactive peptides: A review), yet this is often done in relatively high enzyme to substrate ratios, such as 0.24 AU / g protein (Durand et al. Eur. J. Lipid Sci. Technol. 2021 , 123, 2000380 High Metal Chelating Properties from Rapeseed Meal Proteins to Counteract Lipid Oxidation in Foods Controlled Proteolysis and Characterization) or 0.3 AU / g protein (Vioque et al. J Enzyme Inhib 2001 16 p 81- 87 Alcalase rapeseed inhibitors: purification and partial characterization and also often in combination with another enzyme such as Flavourzyme to further breakdown the size of the peptides.

[0021] M. Schweizer et al (Food Chemistry, 2007, vol 105, pages 1606-1613, Prediction of short peptides composition by RP-HPLC coupled to ESI mass spectrometry) describe the combination of reversed-phase chromatography and electrospray mass spectrometry to predict the amino acid composition of low molecular weight peptides present in rapeseed protein hydrolysates. The objective was to develop a method to predict the composition of peptides of low molar weight (<1000 Da). Two different rapeseed protein hydrolysates were prepared by using two separate enzymes: Alcalase (subtilisin) and PTN (trypsin extracted from porcine pancreas). The starting material was a rapeseed protein concentrate prepared by alkaline extraction followed by isoelectric precipitation, from defatted rapeseed meal, commonly defatted by hexane extraction. This process leads to a rapeseed protein composition rich in cruciferin and low in napin - see for instance Perera, McIntosh, and Wanasundara, Plant 2016, 5 referred to above. Such a composition low in napin is easily hydrolysed by common proteases such as subtilisin (Alcalase). Hydrolysates of RPC (with more than 75% protein content) with Alcalase 2.4 L and PTN 3.0 resulted in mixtures of 97% and 90% short peptides (<10 kDa), respectively.

[0022] Under extreme conditions such as pH swing to pH <3 and back to 8, Alcalase can be used to hydrolyse rapeseed protein, leading moreoverto high salt load (not easily removed from a protein hydrolysate as ultra- or nano filtration (UF / NF) may not work) and still a poor taste is obtained. Particularly, the hydrolysis of napins is difficult. Usually, a substantial part of the napins in the hydrolysate is intact (see also Kaugarenia_et al. Foods 2022, 11 , p2618 Optimization of Selective Hydrolysis of Cruciferins for Production of Potent Mineral Chelating Peptides and Napins Purification to Valorize Total Rapeseed Meal Proteins), and therefore there is a need in the art for improved rapeseed protein hydrolysates and methods for the production thereof.

[0023] Surprisingly, the present inventors found that the hydrolysis of a rapeseed protein isolate (RPI) by a combination of an endoproteases such as aspergillopepsin 1 with a proline-specific endoprotease leads to a hydrolysate where most of the napin has been hydrolysed, and that provides good taste.

[0024] Detailed description of the invention

[0025] In a first aspect, the present invention relates to a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa.

[0026] Preferably, wherein the present rapeseed protein hydrolysate comprises at least 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or at least 70% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa.

[0027] In a preferred embodiment, the present protein hydrolysate comprises: a. 1 to 10% (w / w) of protein fragments having a molecular weight of more than 17 kDa; b. 10 to 30% (w / w) of protein fragments having a molecular weight of 3,5 to 17 kDa; and / or c. 60 to 89% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa, preferably wherein the sum of the protein fragments does not exceed 100% (w / w).

[0028] Preferably, the present protein hydrolysate comprises: a. 1 to 6% (w / w) of protein fragments having a molecular weight of more than 17 kDa; b. 15 to 30% (w / w) of protein fragments having a molecular weight of 3,5 to 17 kDa; and / or c. 65 to 84% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa, preferably wherein the sum of the protein fragments does not exceed 100% (w / w).

[0029] Preferably, the molecular weight as used herein is defined by high performance sizeexclusion chromatography, HP-SEC. Preferably, the HP-SEC comprises:

[0030] -mixing 100 pl of protein hydrolysate 5% solution 900 pl of a 0.5% sodium dodecyl sulphate (SDS) solution in purified water, to obtain a 10 times (v / v) dilution of protein hydrolysate;

[0031] -denature the diluted protein hydrolysate;

[0032] -1 pl was further used for further analysis.

[0033] Preferably, the further analysis comprises: 1 pl of the clear solution was injected in a Waters ACQUITY HCIass-Bio UPLC system, equipped with a pump (Waters 186015041), autosampler (Waters 186015040), column heater (Waters 186015011) and photodiode array detector (Waters 186015032). The running buffer was 0.5% SDS solution in purified water. Separation was performed on a Phenomenex BioZen SEC-3, 1.8 pm, 4.6 mm X 150 mm column (Phenomenex 00F-4772-E0) at 50°C and 0.35 ml / min. Size exclusion chromatography (SEC) profiles were recorded using ultraviolet (UV) detection at 214 nm. The following standards were used: bovine serum albumin (Sigma P0914), chicken ovalbumin (Bio-Rad 1511901), equine myoglobin (Bio-Rad 1511901), lysozyme (Sigma, L6876), insulin (Sigma, I5500), glucagon (Sigma, G2044), [Glu1 ] Fibrinopeptide B (Sigma, F3261), glutathione oxidized (Sigma G4376), and glutathione reduced (G4251). These were dissolved in 0.5% SDS solution, denatured according to sample protocol, and injected. Their corresponding monomeric molecular weights and elution times were used to assign molecular weight fractions of >66 kDa, 66-44 kDa, 44-17 kDa, 17-14.4 kDa, 14.4-5.7 kDa, 5.7-3.5 kDa, 3.5-1 .6 kDa, 1 .6-0.6 kDa, 0.6-0.3 kDa and <0.3 kDa.

[0034] In a preferred embodiment, the invention provides a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa, wherein 40 to 65% (w / w) of the protein fragments is derived from cruciferins and 35 to 60% (w / w) of the protein fragments is derived from napins.

[0035] In yet another preferred embodiment, the invention provides a rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa, wherein 40 to 65% (w / w) of the protein fragments is derived from cruciferins and 35 to 60% (w / w) of the protein fragments is derived from napins, wherein the protein hydrolysate comprises a. 1 to 10% (w / w) of protein fragments having a molecular weight of more than 17 kDa; b. 10 to 30% (w / w) of protein fragments having a molecular weight of 3,5 to 17 kDa; and / or c. 60 to 89% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa, preferably wherein the sum of the protein fragments does not exceed 100% (w / w).

[0036] In a preferred embodiment, the present rapeseed protein hydrolysate has a degree of hydrolysis of between 1 and 30%, preferably between 2 and 20%, more preferably between 5 and 10%. Preferably the degree of hydrolysis is determined using the method described below.

[0037] Preferably, the present rapeseed protein hydrolysate has a degree of hydrolysis of 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%.

[0038] Alternatively, the present rapeseed protein hydrolysate has a degree of hydrolysis of 10 to 90%, 15 to 85%, 20 to 80%, 25 to 75%, 30 to 70%, 35 to 65%, 40 to 60% or 45 to 55%, or any combination of the lower and upper limits of these ranges.

[0039] In a preferred embodiment, the present rapeseed protein hydrolysate has a protein content of 40 to 100% (w / w). Such as 50 to 99%, 55 to 95%, 60 to 90%, 65 to 85% or 70 to 80%. Preferably the protein content is or comprises the present protein fragments. In a preferred embodiment, the present rapeseed protein hydrolysate is in the form of a concentrated liquid, a paste, a powder or a granulate.

[0040] Preferably, the present rapeseed protein hydrolysate is packed in individual packages of 1 to 50 gram, such as 2 to 40 gram, 3 to 35 gram, 4 to 30 gram, 5 to 25 gram or 6 to 15 gram.

[0041] Alternatively, the present rapeseed protein hydrolysate is packed in individual packages of 1 to 50 kilogram, such as 2 to 40 kilogram, 3 to 35 kilogram, 4 to 30 kilogram, 5 to 25 kilogram or 6 to 15 kilogram.

[0042] As described in the experimental part herein, the hydrolysate of a rapeseed protein isolate (RPI) as claimed and described herein is a hydrolysate where most of the napin has been hydrolysed, and that provides good taste. Moreover, the herein claimed and described rapeseed protein hydrolysate is a vegan hydrolysate.

[0043] According to another aspect, the present invention relates to a process for preparing (the present) hydrolyzed rapeseed proteins comprising the steps of: a) preparing an aqueous mixture comprising water and rapeseed proteins; b) contacting the aqueous mixture, comprising the water and the rapeseed proteins, with an aspergillopepsin 1 and / or a proline-specific endoprotease, 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 65°C, more preferably 40°C to 60°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 rapeseed proteins; and / or c) retrieving the hydrolyzed rapeseed proteins from the aqueous mixture.

[0044] This combination of proteases can be a sequential addition (such as first aspergillopepsin 1 , and then proline-specific endoprotease, or another protease under its optimum condition and then adapting the pH and temperature for the proline-specific endoprotease), or a combination of both enzymes, preferably in an environment (temperature, pH) where both enzymes show high activity.

[0045] In a preferred embodiment, the present method comprises first incubation of the rapeseed protein with aspergillopepsin 1 and thereafter incubation of the rapeseed protein with the prolinespecific endoprotease.

[0046] The inventors found that this method hydrolysis both the cruciferins and napins. The potential advantage of hydrolysis of napin is that this leads to peptides rich in cysteine, that may have advantages in for instance dough relaxation, de-aggregating highly aggregated proteins during for instance heat set gelation or extrusion, leading to other material properties. By acting partly as plasticizer, and more specifically by breaking open S-S bonds of the bulk protein. Hence, a napin protein hydrolysate thus may act as a natural glutathione replacer.

[0047] In another preferred embodiment, the present method comprises combined incubation of the rapeseed protein with aspergillopepsin 1 and proline-specific endoprotease. The present aspergillopepsin 1 (herein also referred to as fungal endoprotease aspergillopepsin 1) is also known as EC 3.4.23.18.

[0048] A "proline-specific endoprotease" is a protease that hydrolyses a protein or peptide at a position where the protein or peptide contains a proline-residue. A proline-specific endoprotease may have proline-specific endoprotease and / or proline-specific oligopeptidase activity (EC3.4.21 .26). A proline-specific endoprotease is preferably an enzyme that hydrolyses a peptide bond at the carboxy-terminal end of proline residues, resulting in a peptide and / or polypeptide fragment with a C-terminal proline.

[0049] A proline-specific endoprotease may for instance be derived from Aspergillus niger or Penicillium chrysogenum, such as disclosed in W02002 / 046381 and W02009 / 144269 respectively.

[0050] Other proline-specific endoproteases are known from WO2012 / 174127. WO2012 / 174127 discloses proline-specific protease from Botryotinia, fuckeliana, Aspergillus clavatus, Sclerotinia sclerotiotum, Mycosphaerelly graminicola, Neuropspora crasse, Talaromyces stipitatus and Gibberella zeae.

[0051] The skilled person is very well capable of determining suitable conditions for step (b) of the above-described method. As shown herein within the experimental part a preferred temperature is a temperature of at least 50 degrees Celsius, preferably a temperature in the range of 50 to 65 degrees Celsius. The experimental part also shows that a preferred starting pH is pH 3. It is further shown within the experimental part that an incubation time of at least 6 hours is preferred. The experimental part also provides guidance for suitable enzyme concentrations, which can range from 1 % to 11 % of both enzymes (dosage of the formulated enzymes is based on the protein powder), or with an activity (MaxiPro AFP) of 1000-1100 SAPU / g, this is 10-11 SAPU / g protein up to 11 Q- 121 SAPU / g protein, or for MaxiPro PSP with 5-9 PPU / g this is 0.05-0.09 PPU / g protein, up to 0.6 - 1 .0 PPU / g protein.

[0052] Within this document Aspergillopepsin 1 of different batches with different names were used (MaxiPro AFP and Maxamyl AFP), as well proline-specific endoprotease (DelvoPlant PSP and MaxiPro PSP).

[0053] Commonly rapeseed protein isolate is obtained by aqueous extraction of rapeseed meal or rapeseed ‘cake’ obtained after the oil was removed (partially) by pressing and or optionally extraction by an organic solvent, which results in the rapeseed protein isolate and a (wet) cake residue. While rapeseed protein isolate has a protein content of higher than 80%, findings of this invention may also apply to a rapeseed protein concentrate with less than 80% protein, or even a rapeseed preparation with even lower protein concentration, such as rapeseed meal, or pressed rapeseed meal (pressed filter cake) or from the depleted wet cake residue that may contain substantial amount of protein.

[0054] In an alternative embodiment, the proteolysis from such rapeseed meal, press cake or depleted wet cake (residue) may occur in the presence of other enzymes, such as enzymes that further breakdown the cellular matrix and hence improve the accessibility for enzymes and release of the protein (hydrolysate) from the matrix, such as carbohydrases (cellulases, hemicellulases, pectinase) or phytase.

[0055] Preferably, the present rapeseed protein is rapeseed protein isolate.

[0056] Preferably, the present rapeseed protein comprises napins and cruciferins. Preferably the size of the relevant rapeseed proteins is determined by Blue Native SDS-PAGE and the amount of cruciferins and napins is determined by HP-SEC or by sedimentation velocity (SV- AUC). Preferably, the amounts of cruciferins and napins are calculated based on the total amount of rapeseed protein. Or alternatively, the amounts of cruciferins and napins are calcuated based on the sum of cruciferins and napins present in the rapeseed protein. Preferably, the amounts of cruciferins and napins are determined by size exclusion chromatography (SEC). Preferably, the amounts of cruciderins and napins are determined by size exclusion chromatography (SEC) using the following test: samples of protein isolate are dissolved in a 500 mM NaCI saline solution and analyzed by High Performance SEC using the same solution as the mobile phase, followed by detection using measuring UV absorbance at 220 nm, wherein the relative contribution of cruciferin and napin (wt. %) was calculated as the ratio of the peak area of each protein with respect to the sum of both peak areas.

[0057] Rapeseed protein may be in the form of an isolate or a concentrate. Rapeseed protein isolate may be prepared from cold-pressed rapeseed oil seed meal as described in WO 2018 / 007492 resulting in a product with a protein content of from 50-98% (w / w), or from 7095% (w / w) or of 90±5% (w / w). The rapeseed protein isolate may comprise of from 40-65% (w / w) cruciferins and of from 25-60% (w / w) napins wherein the size of the relevant proteins was verified by Blue Native PAGE and the ratio between the relevant proteins was determined by SEC-UV (Size Exclusion Chromatography, with UV detection), for example as described in WO 2018 / 007492. Alternatively, the rapeseed protein isolate may comprise at least 50, 60, 70, 80% (w / w), preferably at least 85% (w / w), preferably at least 90% (w / w), more preferably at least 95% (w / w) cruciferins as verified by Blue Native PAGE and / or SEC-UV, for example as described in WO 2018 / 007492. Alternatively, the rapeseed protein isolate may comprise at least 50, 60, 70, 80% (w / w), preferably at least 85% (w / w), preferably at least 90% (w / w), more preferably at least 95% (w / w) napins as verified by Blue Native PAGE and / or SEC-UV, for example as described in WO 2018 / 007492. More preferably the rapeseed protein isolate comprises 10-40% (w / w) napins and 40-60% (w / w) cruciferins, preferably as verified by Blue Native PAGE and / or SEC-UV, for example as described in WO 2018 / 007492. More preferably the rapeseed protein isolate comprises 25-35% (w / w) napins and 40-55% (w / w) cruciferins, preferably as verified by Blue Native PAGE and / or SEC-UV, for example as described in WO 2018 / 007492. Most preferably, the rapeseed protein isolate comprises 35-60% (w / w) napins and 40-65% (w / w) cruciferins, preferably as verified by Blue Native PAGE and / or SEC-UV, for example as described in WO 2018 / 007492.

[0058] Preferably, the present rapeseed protein comprises 40 to 65 wt. % 12S and 35 to 60 wt. % 2S. Preferably, the present rapeseed protein comprises 40 to 55 wt. % 12S and 45 to 60 wt. % 2S. In a preferred embodiment, the present rapeseed protein comprises 60 to 80 wt. % 12S and 20 to 40 wt. % 2S. Preferably, the present rapeseed protein comprises 65 to 75 wt. % 12S and 25 to 35 wt. % 2S.

[0059] In a preferred embodiment, the present rapeseed protein comprises 0 to 20 wt. % 12S and 80 to 100 wt. % 2S. Preferably, the present rapeseed protein comprises 0 to 10 wt. % 12S and 90 to 100 wt. % 2S. Preferably, the present rapeseed protein comprises 1 to 5 wt. % 12S and 95 to 100 wt. % 2S. Preferably, the present rapeseed protein comprises around 15 wt. % 12S and around 85 wt. % 2S. An example of such a rapeseed protein is Puratein® HS.

[0060] Preferably, the amounts of 12S and 2S is determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis. Preferably, the amounts of 12S and 2S is determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis using the following test: samples of protein isolate are dissolved in a 3.0% (or 500 mM) NaCI saline solution and amounts determined using interference optics.

[0061] In a preferred embodiment, the present rapeseed protein comprises 0 to 20 wt. % cruciferins and 80 to 100 wt. % napins. Preferably, the present rapeseed protein comprises 0 to 10 wt. % cruciferins and 90 to 100 wt. % napins. Preferably, the present rapeseed protein comprises 1 to 5 wt. % cruciferins and 95 to 100 wt. % napins. Preferably, the present rapeseed protein comprises around 15 wt. % cruciferins and around 85 wt. % napins. An example of such a rapeseed protein is Puratein® HS.

[0062] In a preferred embodiment, the present rapeseed protein (does not) comprises 70 to 95 wt. % cruciferins and 5 to 30 wt. % napins. Preferably, the present rapeseed protein (does not) comprises 80 to 90 wt. % cruciferins and 10 to 20 wt. % napins, such as around 90% cruciferins and 10% napins.

[0063] In an embodiment the rapeseed protein isolate is low in anti-nutritional factors and contains less than 1 .5% (w / w) phytate, preferably less than 0.5% (w / w) phytate and is low in glucosinolates (<5 pmol / g) and low in phenolics (<10 mg / g).

[0064] In one embodiment, the used rapeseed protein has a solubility (in water) of at least 88%, preferably at least 90%, more preferably at least 94% and most preferably at least 96% when measured over a pH range from 3 to 10 at a temperature of 23±2°C. This is also known as the soluble solids index (SSI).

[0065] The above-described method results in a rapeseed protein hydrolysate. Such a hydrolysate comprises hydrolysed cruciferins and hydrolysed napins. Preferably, the vast majority (preferably at least 80% of the napins, more preferably at least 85, 90 or 95% of the napins) of the napins which were present in the starting material have been hydrolysed by the combination of endoproteases.

[0066] The presence of remaining (if any) intact napins may be observed by different techniques, for example by NMR, such as described by Rico et al. Biochemistry 1996 35 p15672-156821H NMR Assignment and Global Fold of Napin Bnlb, a Representative 2S Albumin Seed Protein, and Pantoja-Uceda et al. Biochemistry 2004, 43, p16036-16045 Solution Structure and Stability against Digestion of rproBnlb, a Recombinant 2S Albumin from Rapeseed: Relationship to Its Allergenic Properties. Such a method would enable the determination of effectiveness of napin hydrolysis.

[0067] Preferably, a process according to the invention does not comprise subtilisin.

[0068] According to another aspect, the present invention relates to the use of the present rapeseed protein hydrolysate in a growth medium for growing cells, or as growth factor for the culture of cells. Preferably the growth medium is a serum free medium. Preferably the rapeseed protein hydrolysate is as defined herein.

[0069] Further, the present invention relates to the use of the present rapeseed protein hydrolysate in a beverage. For example as nutrient or as antioxidant.

[0070] According to further aspect, the present invention relates to the use of aspergillopepsin 1 and proline specific endoprotease for hydrolysing napin. Preferably, the present invention relates to the use of aspergillopepsin 1 and proline specific endoprotease for hydrolysing the small napin subunit (of around 4.5 kDa which is visible on reducing SDS-PAGE at around the marker of 6 kDa). Napin comprises a large and a small subunit of respectively around 10 kDa and 4.5 kDa.

[0071] The features described for the different aspects such the rapeseed protein hydrolysate, the process and the use, are equally applicable to each of the aspects.

[0072] The invention is further illustrated in the examples below.

[0073] Description of figures

[0074] Figure 1. SDS electrophoresis gel from samples of exp 1.1 a, proteolysis of 5% RPI with resp. 0.1 % Alcalase; lane 1 : marker, lane 2, RPI before addition of Alcalase, lane 6, after 4 hours incubation.

[0075] Figure 2. SDS electrophoresis gel from samples of exp 1 .1 b / c, proteolysis of 5 or 20% RPI with 1 .0% Alcalase; lane 3, sample 1.1 b 5% RPI after 4 hr incubation with 1 .0% Alcalase; lane 5-7, 9 sample 1.1 C with 20% RPI after, 1.5, 3, 5 hours incubation with 1.0% Alcalase; Lane 10: marker.

[0076] Figure 3. SDS electrophoresis gel from samples of exp 1.4, proteolysis of 20% RPI with 1 .0% DelvoPlant PSP at pH 4; lane 1 marker, lane 2-5 after 0, 1 .5, 3, 4 hours incubation.

[0077] Figure 4. SDS-PAGE gel of samples A and B taken during incubation, lane 1 and 2 Marker M12, lanes 3 - 5, sample 2A taken at t=0, 3hr and 4hr respectively, lanes 6 - 8, sample 2B taken at t=0, 3hr and 4hr respectively.

[0078] Figure 5. SDS-PAGE gel of samples C taken during incubation; lanes 1 - 4, sample 2C taken at t=0, 3hr, 4hr of only MaxiPro AFP, and 4 hours MaxiPro AFP + 18 hours DelvoPlant PSP respectively. Righter lane Marker M12.

[0079] Figure 6. Growth curves of various strains. Bs: B. subtilis, Ec: E. coli, Pp: P. pastoris, and Sc: S. cerevisiae. X axis time in hours, Y axis Optical Density in Arbitrary Units. Markers for various combinations of growth Verduyn based media with the indicated nitrogen source, RPI: rapeseed protein isolate; 2A, 2B and 2C: hydrolysates described in example 2; urea: only urea; blank: no nitrogen source added.

[0080] Figure 7. A and B: SDS-PAGE results of hydrolysis reactions as described in Table 8 Figure 8. SDS-PAGE (reducing conditions) of larger batches of material: M: unstained

[0081] M12 marker; Lane 1 : Fresh 5% CanolaPRO solution (no pH changes, not heated); Lane 2: Sample 1 , 5% CanolaPRO, pH 3, 24h, 50°C; Lane 3: Sample 2, 5% CanolaPRO, pH 3, 1.5% MaxiPro AFP, 24h, 50°C; Lane 4: Samples, 5% CanolaPRO, pH 3, 1 .5% MaxiPro AFP, 1 .5% MaxiPro PSP, 24h, 50°C.

[0082] EXAMPLES

[0083] Materials and methods

[0084] Rapeseed protein isolate (RPI) was prepared from cold-pressed rapeseed oil seed meal as described in WO 2018 / 007492; the protein content was 90% (w / w). The resultant RPI comprised in the range of from 40 to 65% (w / w) cruciferins and 35 to 60% (w / w) napins, contained less than 0.26% (w / w) phytate and had a solubility of at least 88% when measured over a pH range from 3 to 10 at a temperature of 23±2°C. Batches GB20134-2 & GB20145-3 were used.

[0085] The following enzymes of table 1 were used.

[0086] Table 1

[0087] DelvoPlant PSP and MaxiPro PSP are the same enzymes, from different batches, and in the examples both names are used. The same holds for MaxiPro AFP and Maxamyl AFP

[0088] Analysis methods

[0089] SDS-PAGE electrophoresis - sample preparation

[0090] As such: 65 pl sample (protein solution) was added to 25 pl NuPAGE LDS sample buffer (4x) Invitrogen and 10 pl NuPAGE Sample Reducing Agent (10x) Invitrogen. Samples were heated for 10 min at 70°C. The samples were 5 times diluted in sample buffer (65 milliQ water : 25 LDS sample buffer : 10 Reducing agent).

[0091] SDS-PAGE Gel electrophoresis

[0092] SDS-PAGE was performed as follows. 10 pl of the sample solutions and 5-10 pl marker M12 (Invitrogen) were applied on NuPage 9-12% Bis Tris 10 wells gel. The gel was run at 200 V for 43 minutes, using the Xcell Surelock, with 600 ml 20x diluted MES SDS running buffer in the outer chamber and 200 ml 20x diluted MES SDS running buffer in the inner buffer chamber. After running, the gels were stained with 50 ml InstantBlue staining. And decolored for a duration of 1 to 3 nights.

[0093] Determination of primary amino groups by o-Phthaldialdehyde (OPA)

[0094] Primary amino groups were determined using what is commonly known as the “OPA” method, with the reagent o-Phthaldialdehyde. This is needed to calculate the degree of hydrolysis of a protein (see next). The degree of hydrolysis (DH (%)) is defined as the percentage of cleaved peptide bonds after hydrolysis. Analysis is always performed in combination with Kjeldahl Nitrogen; necessary for calculation “degree of hydrolysis”. o-Phthaldialdehyde (OPA) reacts in the presence of 1 ,4-Dithiothreitol (DTT) with primary amino groups of the cleaved protein fragments at ambient temperature. Color development of the reaction products is measured spectrophotometrically at 340 nm.

[0095] Degree of hydrolysis

[0096] The degree of hydrolysis was determined from the Kjeldahl Nitrogen (A10327) and amino groups (A2343).

[0097] First, the primary NH2 per g protein (h) need to be calculated: h = ((106x F) / (NTKN x N factor) - p) / a

[0098] Where: h = mmol primary NH2 per g protein

[0099] F = mmol primary NH2 per g sample

[0100] NTKN = g total Kjeldahl nitrogen per g sample

[0101] 106= conversion factor NTKN mg / kg -> g / g

[0102] N factor= conversion factor N to protein (see table 2) a = slope of the hydrolysis curve (no dimension) (see table 2)

[0103] B = intercept of the hydrolysis curve (mmol primary NH2 per g protein)

[0104] The degree of hydrolysis can be calculated:

[0105] DH (%) = h / htot *1 00% where: h = mmol primary NH2 per g protein htot = mmol primary NH2 theoretically available per g protein (see table 2)

[0106] Table 2 Constants for some frequently used proteins

[0107] * Calculated based on amino acid composition of theoretical sequence of cruciferin and napin.

[0108] Alternative method of analysis of number of NH2 groups as indicator of the degree of hydrolysis (for example 5)

[0109] The OPA (o-Phthaldialdehyde) assay was performed to determine the increased amino groups of CanolaPRO hydrolysate. 52.5mg L-serine was dissolved in 50ml MilliQ to produce a 10mM stock solution. To get 8 calibration solutions, series dilution was performed as shown in

[0110] Table 3.

[0111] Table 3 Series dilution of serine stock solution

[0112] Samples were diluted 25 times by combining 40 .l sample and 960 .l MilliQ. 20pl of samples were added into a well of 96 wells microplate separately. After that, 200pl Fluoraldehyde™ o-Phthaldialdehyde Reagent Solution (OPA) was added to each well. Then the plate was covered with aluminium foil and kept at room temperature for 15 minutes and absorbance was read at 340nm in the plate reader.

[0113] Analysis of molecular weight distribution by HP SEC

[0114] Several samples were analysed on molecular weights by using high performance sizeexclusion chromatography, HP-SEC. After homogenization on a vortex stirrer (Scientific Industries G-560E), 100 pl of each sample (protein hydrolysates in approximately 5% solution) were mixed with 900 pl of a 0.5% sodium dodecyl sulphate (SDS) solution in purified water, to obtain a 10 times (v / v) dilution of each sample. The mixtures were denatured for 15 minutes at 70°C and 1000 rpm in a thermomixer (Eppendorf ThermoMixer C). For Example 2, 1 pl of the clear solution was injected in a Waters ACQUITY HCIass-Bio UPLC system, equipped with a pump (Waters 186015041), autosampler (Waters 186015040), column heater (Waters 186015011) and photodiode array detector (Waters 186015032). The running buffer was 0.5% SDS solution in purified water. Separation was performed on a Phenomenex BioZen SEC-3, 1.8 pm, 4.6 mm X 150 mm column (Phenomenex 00F-4772-E0) at 50°C and 0.35 ml / min. For Example 6, 1 pl of the clear solution was injected in a Waters ACQUITY Premier UPLC system, equipped with a pump (Waters 186018001), autosampler (Waters 186018002), column heater (Waters 186015011) and photodiode array detector (Waters 186018007). The running buffer was 0.5% SDS solution in purified water. Separation was performed on a Waters ACQUITY UPLC protein BEH SEC column, 200A, 1 .7 pm, 4.6 mm X 150 mm (Waters 186005225) at 50°C and 0.35 ml / min.

[0115] Size exclusion chromatography (SEC) profiles were recorded using ultraviolet (UV) detection at 214 nm. The following standards were used: bovine serum albumin (Sigma P0914), chicken ovalbumin (Bio-Rad 1511901), equine myoglobin (Bio-Rad 1511901), lysozyme (Sigma, L6876), insulin (Sigma, I5500), glucagon (Sigma, G2044), [Glu1] Fibrinopeptide B (Sigma, F3261), glutathione oxidized (Sigma G4376), and glutathione reduced (G4251). These were dissolved in 0.5% SDS solution, denatured according to sample protocol, and injected. Their corresponding monomeric molecular weights and elution times were used to assign molecular weight fractions of >66 kDa, 66-44 kDa, 44-17 kDa, 17-14.4 kDa, 14.4-5.7 kDa, 5.7-3.5 kDa, 3.5-1 .6 kDa, 1.6-0.6 kDa, 0.6-0.3 kDa and <0.3 kDa. The peak areas and peak percentages of each molecular weight fraction of the samples were exported and evaluated.

[0116] Example 1

[0117] Proteolysis of rapeseed protein isolate with standard proteases

[0118] Rapeseed protein isolate was incubated with standard proteases: Alcalase (subtilisin) and Flavourzyme (both Novozymes); and Delvoplant PSP, a proline-specific fungal endoprotease produced by dsm-firmenich. The rapeseed protein was dissolved at 5 or 20% concentration in water, and the pH was set and controlled (using 4N H3PO4 or 4N NaOH) and temperature were set as indicated in the table below. A sample was taken at t=0. Then enzyme solutions were added, see table below. Samples (40 ml) were taken every 60 min for a period of 4 hr. Proteolytic activity was inactivated by placing the tubes in a water bath at 90°C for 5 min. For the experiment with Flavourzyme, the pH was decreased to pH 5 after two hours, since Flavourzyme exhibits more endoprotease activity at this pH. At pH 7, exo-protease activity is more abundant.

[0119] The degree of hydrolysis was measured using the method above, figures are given in the table 4 below.

[0120] Table 4

[0121] Samples were heat treated to inactivate the protease, and subsequently analyzed using SDS-Page. The results for untreated and Alcalase-treated rapeseed protein isolate are shown in figure 1 , 2 and 3.

[0122] The figures 1-3 all show the intact bands of cruciferin (36, 31 and 21 kDa) and the napin bands (at 9 and 6 kDa, commonly poorly resolved in this SDS-PAGE set up). Only little or no indications of substantial hydrolysis appear from the analyses. The total amount of free amino acids was also measured and for none of the samples this exceeded 1 % free amino acids (on total protein), except for the treatment with Flavourzyme, showing more than 25% free amino acids (on total protein) - indicative of its endo-protease activity Conclusion: Despite high enzyme-to-substrate ratios and optimum conditions for the various enzymes, only limited hydrolysis of RPI was observed. Especially the low hydrolysis levels by Alcalase were surprising. Flavourzyme appeared to show good hydrolysis but that was due to its exo-protease preference at pH7, that led to high level of free amino acids, but not to substantial breakdown of the core of the rapeseed proteins.

[0123] Example 2

[0124] Hydrolysis of rapeseed protein isolate with aspergillopepsin 1 and proline-specific endoprotease

[0125] In this example hydrolysis of rapeseed protein hydrolysate is described using 5% RPI under the following conditions:

[0126] A. Pretreatment at pH 1 .8 using nitric acid followed by proteolysis with Alcalase at pH

[0127] 8

[0128] B. Proteolysis using fungal endoprotease aspergillopepsin 1 (MaxiPro AFP) at pH 3.5

[0129] C. Proteolysis using fungal endoprotease aspergillopepsin 1 (MaxiPro AFP) at pH 4 followed by second incubation with proline-specific endoprotease (DelvoPlant PSP) (pH 4)

[0130] Table 5 *Doses in reaction mixture, not on protein or dry matter

[0131] These runs were executed under food-grade conditions. 1 .5 L solution of rapeseed protein isolate (5% w / w) in water was made and the pH was adjusted (using 4N H3PO4 or 4N NaOH) and temperature were set. Sample 2A was first brought to pH2 using HCI solution and then brought back to 8 using 4N NaOH (effectively producing around 4g / L NaCI after pH swing). Samples at t=O were taken for analysis. The enzyme solutions were added - see table. Samples (40 ml) were taken every 60 min over a period of 4 hr. Samples were deactivated by placing the tubes in a water bath at 90°C for 5 min. At the end of the incubation time, the final mixtures were heated 90°C for 5 min to inactivate the enzyme. For sample C, after incubation with MaxiPro AFP, the temperature was reduced to 50°C and DelvoPlant PSP was added, the incubation was continued for 18 hr to obtain final the sample C, followed by heated 90°C for 5 min to inactivate the enzyme.

[0132] Samples were characterized by SDS-PAGE as is shown in figure 4 and 5.

[0133] The SDS-PAGE gels all showed substantial hydrolysis, however a relatively pronounced band at around 6kDa remained visible in samples 2A and 2B which was due to one of the napin subunits. In the final sample 2C (lane 4 in figure 5) only a small trace of this band was still visible.

[0134] These samples at the end of full incubation were further analyzed with HP SEC. In this analysis also PeptoPro casein hydrolysate (DSM) was taken along as an example of a protein hydrolysate resulting from a process including proline-specific endoprotease. The samples were separated in various molecular weight classes, for convenience here grouped into three classes: >17 kDa: larger than napin; 17 - 3.5 kDa: fraction containing unhydrolyzed napin and its subunits; <3.5 kDa: oligopeptides and amino acids, hydrolyzed fragments. In the table it can be seen that in hydrolysate 2A and 2C a substantial part of the napins (having a molecular weight of 14 kDa) had been hydrolyzed, and that in 2B still a substantial portion of the napin was intact.

[0135] Table 6

[0136] Final products of the protein hydrolysate after dilution to a protein content of around 2% were tasted at room temperature without further adjustment of pH or sweetness, by an internal group of tasters. Overall, compared to standard rapeseed protein isolate in solution, the astringency was found substantially reduced or absent, with sample 2C as the least astringent, and the ‘typical hydrolysate’ tastes were also modest. The sample 2A (Alcalase after acidification and neutralization) led to a salty product.

[0137] Conclusion: Only after heavy pre-treatment by pH swing to very low pH and back to pH 8, subtilisin (Alcalase) was capable of hydrolysing rapeseed protein isolate. Aspergillopepsin I (MaxiPro AFP), however, could hydrolyse rapeseed protein isolate already directly at its optimum pH of 3.5 to 4. After subsequent hydrolysis with a proline-specific endoprotease (DelvoPlant PSP) napin was further broken down, resulting in a fairly well tasting hydrolysate.

[0138] Example 3 Use of rapeseed protein hydrolysate in a growth medium

[0139] The hydrolysates from example 2 were used for the growth of Bacillus subtilis (168 RUG2010A), E. coll (RV308), Pichia pastoris (SMD1168) and Saccharomyces cerevisiae CEN.PK113-7DAA) cells. A Verduyn growth medium was used without urea, with a pH of 6 adjusted with KOH. The Verduyn medium further contained KH2PO4, 0.3%, K2SO4, 0.66%, MgSO4.7H2O, 0.05%, urea (optional - only in one case) 0.23%, liquid vitamin mix, 0.1%, liquid spore elements mix 0.1 %, glucose 2%, the remaining part being demi water. The medium was filter sterilized and stored at 4°C.

[0140] The following experiments shown in table 7 were carried out.

[0141] Table 7

[0142] The growth media were prepared as follows. For experiment 1 : 10 ml growth medium, 5 ml demi water, 0.8 ml 50% glucose, 0.4 gram rapeseed protein isolate, 4.2 ml demi water to a final volume of 20 ml, and pH adjusted to pH 6.06. For experiments 2, 3 and 4: 10 ml growth medium, 0.8 ml 50% glucose, 8 ml 5% hydrolysate, and pH adjusted to 6, 1 .2 ml demi water was added to a final volume of 20 ml. For experiments 5 and 6, 10 ml growth medium, 0.8 ml 50% glucose, 0.092 gram urea (only experiment 5), pH adjusted to 6 and 9.2 ml demi water was added to a final volume of 20 ml.

[0143] To pre-culture the 4 strains, 75 pl of the cells were added to a 100 ml non-baffled shake flask containing 15 ml yeast extract pepton dextrose growth medium and incubated overnight at 30°C with 280 RPM. Thereafter, 4 ml of the cultures was centrifuged for 5 minutes at 4000g. Supernatant was discarded, and cells were resuspended in 8 ml demi water.

[0144] The growth media were inoculated with 0.05 ml of the resuspended cells in a Biolector using 48 plates containing 1 ml media. Growth conditions were 30°C, 900 RPM and every 10 minutes a measurement of the optical density [Arbitrary Units] was taken.

[0145] The results are shown in figure 6.

[0146] Conclusion: Taking Optical Density as measure for growth of microorganisms it is clear that for all species tested, growth is better on a hydrolysate compared to unhydrolyzed rapeseed protein isolate. Comparing the hydrolysates amongst each other, the hydrolysate obtained after hydrolysis using a general fungal endoprotease Aspergillopepsin 1 [MaxiPro AFP] + a prolinespecific endo protease [DelvoPlant PSP] performed best.

[0147] Example 4

[0148] Optimising the hydrolysis, other enzymes, temperature

[0149] In these trials, different enzymes, different temperatures (40, 50, 60°C), and different combinations of enzymes at their respective optimum pH (3, 4, 5, 7) were tried, as is indicated in table 8.

[0150] The enzyme incubation was done using 30 mL of 5%CanolaPRO solution (batch GB22312-17) for each sample, the pH was adjusted using phosphoric acid. After 4 hours of incubation, the enzyme was inactivated by heating the tube for 10 minutes in a water bath set at 90°C. The hydrolysis was assessed by SDS-PAGE under reducing conditions, as shown in figure 7.

[0151] Table 8 Set up of the enzyme incubation.

[0152] *The dosage of the enzyme was based on formulated enzyme on dry matter of protein. MaxiPro AFP is aspergillopepsin 1 and MaxiPro PSP is proline-specific endoprotease.

[0153] From Figure 7-A, it can be seen that only MaxiPro AFP could hydrolyse the rapeseed protein isolate substantially, only the 6 kDa band of the napin subunit is still present. Collupulin could hydrolyse the cruciferin substantially but left most of napin intact, pH 7 was better than pH 4. The other proteases left much of the rapeseed protein isolate intact, even at these relatively high dosages. MaxiPro BAP, a subtilisin-rich preparate, shows intact napin and one of the cruciferin subunits at ~20-22 kDa also is left untouched.

[0154] Figure 7-B shows that the combination of MaxiPro AFP and MaxiPro PSP was even better capable of hydrolysing napin, as can be seen by further decrease of the intensity of the bands representing napin under reducing conditions, at 9 and 6 kDa. The 9 kDA band was totally gone, the intensity of 6 kDa band was lowest at 50 and 60°C.

[0155] Conclusion: based on the profiles in the SDS-PAGE gels, proved MaxiPro AFP effective in hydrolysis, but the combination of AFP and PSP was more effective in hydrolysis. Other enzymes were less capable of hydrolysing rapeseed protein isolate. A temperature of at least 50°C is more optimum than lower temperatures.

[0156] Example 5 Further Optimising the hydrolysis

[0157] To find more optimum hydrolysis conditions, a multifactor trial was performed in which incubation time, pH, enzyme concentration of both MaxiPro AFP and MaxiPro PSP were varied using a Design of Experiments (DoE) set up. The temperature was set at 50°C. 5% CanolaPRO (batch number DI00000089) solution was used. The DoE was blocked by days, 30 samples were run in four consecutive days. The setting of factors is shown in Table 9. After incubation, the pH (and hence the change in pH) was measured, and the level of free NH2 groups by the OPA method. These parameters were used to calculate optimum conditions using the Design Expert program. Based on these results, a selection of samples was characterised using SDS-PAGE.

[0158] Table 9 setting of factors

[0159] *Dosage of the enzyme is based on the protein powder but not the volume of the solution

[0160] Starting pH and concentration of MaxiPro AFP had the strongest effect on delta pH, at pH 3 and 10% MaxiPro AFP on protein, the increase in pH was largest. The effect of MaxiPro PSP on the change in pH was small.

[0161] In the model related to the increase of number of amine groups, the starting pH and concentration of MaxiPro PSP had little effect on the increase of the number of amine groups, concentration of MaxiPro AFP dominated this model.

[0162] Based on this experiment, the optimized parameters to break down cruciferin and most of the napin based on the SDS-PAGE gel were pH3, 10% MaxiPro AFP, and 10% MaxiPro PSP, for at least 6 hours. Furthermore, it can be seen that both proteases could be added at once, no need for sequential addition; MaxiPro AFP was needed to break down the majority of the protein, but breaking napin needed MaxiPro PSP. Longer incubation time results in lighter bands around 6kDa.

[0163] Example 6

[0164] Bigger batches of hydrolysate material have been made, used for tasting and for detailed analysis with HP SEC

[0165] Based on the conditions found in previous examples, larger batches of hydrolysates and control products were made. Materials: CanolaPRO, batch DI00000139; proteases: MaxiPro AFP, batch 4241561011 ; 1.5% formulated product on product = 0.075% on overall solution, MaxiPro PSP, batch FG23187; 1 .5% formulated product on product = 0.075% on overall solution. Food grade phosphoric acid was used to tune the pH

[0166] The production was performed using the Hettich water bath set up with overhead stirrers (Hettich Benelux BV Geldermalsen, the Netherlands).

[0167] Three batches each of 2L 5%CanolaPRO solution were prepared, by adding 100g CanolaPRO powder to 1900ml drinking water spoon by spoon with stirring at 199rpm. After the protein was dissolved, the pH was adjusted to 3 by phosphoric acid. The solutions were brought to 50°C and stirred. Enzymes were added and solutions were left to incubate at 50°C for 24 hours:

[0168] • Sample 1 (2L): without enzyme

[0169] • Sample 2 (2L): 1 .5% MaxiPro AFP

[0170] • Sample 3 (2L): 1 .5% MaxiPro AFP and 1 .5% MaxiPro PSP

[0171] The samples were pasteurized at 90°C for 10 minutes and cooled to room temperature in the Hettich bath and placed in the ice bath. Parts of the samples were freeze dried, other parts were kept frozen in separate bottles for further testing. The samples were characterised by increase in pH (Table 10), SDS-Page, see figure 8, and SEC UV, results are shown in table 11.

[0172] Table 10. Table 11

[0173] Both the SDS-PAGE as well as the SEC-UV showed that MaxiPro AFP could substantially hydrolyse the rapeseed protein isolate, and the combination of AFP and PSP was even better in breaking down the protein. From the SDS-PAGE it can be seen that especially for sample 3, the 6 kDa band of the napin subunit was very faint, indicative for substantial hydrolysis of napin as such and this 6 kDa subunit in particular.

[0174] The samples were tasted by a group of internal tasters, diluted 1 :1 with water to 2.5%, and compared to unformulated PeptoPro casein hydrolysate (see example 2). Rapeseed protein hydrolysates were considered less astringent, less sweet and less liquorice than the non treated acidified rapeseed protein isolate. Astringency was relatively high for all, but for drinks at low pH without sweetening, the low pH makes discriminating levels of astringency difficult. Both hydrolysates were considered better in taste than unformulated PeptoPro.

[0175] To give a first impression of what a formulated drink would look like, 4 parts of hydrolysate sample 3 was diluted with 1 part of commercial lemon flavoured syrup (Karvan Cevitam Zero Citroen, with artificial sweeteners and citric acid, from the supermarket) and 3 parts of water. Although the pH of the formulated product was reduced again to 3.4, the flavour and sweetener mellowed the taste and mouthfeel to an acceptable drink.

Claims

CLAIMS1. A rapeseed protein hydrolysate comprising protein fragments, wherein at least 50% (w / w) of the protein fragments has a molecular weight of less than 3.5 kDa.

2. A rapeseed protein hydrolysate according to claim 1 , having a degree of hydrolysis of between 1 and 30%, preferably between 2 and 20%, more preferably between 5 and 10%.

3. A rapeseed protein hydrolysate according to claim 1 or claim 2, having a protein content of 40 to 100% (w / w).

4. A rapeseed protein hydrolysate according to any of the claims 1 to 3, wherein the protein hydrolysate is in the form of a concentrated liquid, a paste, a powder or a granulate.

5. A rapeseed protein hydrolysate according to any of the claims 1 to 4, wherein the protein hydrolysate comprises: a. 1 to 10% (w / w) of protein fragments having a molecular weight of more than 17 kDa; b. 10 to 30% (w / w) of protein fragments having a molecular weight of 3,5 to 17 kDa; c. 60 to 89% (w / w) of protein fragments having a molecular weight of less than 3.5 kDa.

6. A process for preparing hydrolyzed rapeseed proteins comprising the steps of: a) preparing an aqueous mixture comprising water and rapeseed proteins; b) contacting the aqueous mixture, comprising the water and the rapeseed proteins, with an aspergillopepsin 1 and a proline specific endoprotease, 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 65°C, more preferably 40°C to 60°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 rapeseed proteins; c) retrieving the hydrolyzed rapeseed proteins from the aqueous mixture.

7. A process for preparing hydrolyzed rapeseed proteins according to claim 6, comprising first incubation of the rapeseed protein with aspergillopepsin 1 and thereafter incubation of the rapeseed protein with the proline specific protease.

8. A process according to claim 6 or 7, wherein said rapeseed proteins are a rapeseed protein isolate (RPI), preferably an RPI comprising 40 to 65% (w / w) cruciferins and 35 to 60% (w / w) napins.

9. Use of a rapeseed protein hydrolysate in a growth medium for growing cells.

10. Use according to claim 8, wherein the growth medium is a serum free medium.11 . Use of a rapeseed protein hydrolysate as growth factor, preferably as growth factor for the culture of cells.

12. Use of a rapeseed protein hydrolysate in a beverage.

13. Use according to claim 9 to 12, wherein the rapeseed protein hydrolysate is as defined in claim 1 to 5.

14. Use of aspergillopepsin 1 and proline specific endoprotease for hydrolysing napin, preferably for hydrolysing the small napin subunit.

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