Patent Description:
Protein is a main feature of human nutrition. This may be sourced from animals (e.g. meat, fish, egg, dairy) or vegetables. There is a general desire to reduce the amount of animal-based protein. The use of egg protein is often undesirable. For example, due to problems with egg allergies, medical problems associated with cholesterol levels in eggs, religious restrictions/convictions, culinary preferences (such as, for example, a vegetarian or a vegan diet), cost fluctuations in the price of eggs, use of antibiotics and hormones in poultry production, and diseases associated with poultry (such as, for example, bird flu), the use of alternative proteins may be desired. The use of vegetable-based protein in human nutrition is known, for example <CIT> discloses the use of wheat protein isolates as an alternative to the use of egg yolk protein in compositions. However, the use of wheat protein isolates may not be desirable for those with gluten allergies. The use of soy-based protein instead of whey protein has also been described for example in <CIT>. Soy protein is widely used, however in view of some intolerances to soy products there is a need to find other sources of vegetable proteins.

Suitable alternatives include pea protein and rapeseed protein. Rapeseed seeds are rich in oil and contain considerable amounts of protein that accounts for <NUM> to <NUM>% of seed dry weight. Processing rapeseed for oil for human consumption produces rapeseed meal (<NUM>%) as a byproduct, which contains about <NUM> to <NUM>% protein. The rapeseed used for this purpose is usually of the varieties Brassica napus and Brassica juncea. These varieties contain low levels of erucic acid and glucosinolates 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 <<NUM>% erucic acid and <<NUM> mmol/g glucosinolates.

The predominant storage proteins found in rapeseed are cruciferins and napins. Cruciferins are globulins and are the major storage protein in the seed. A cruciferin is composed of <NUM> subunits and has a total molecular weight of approximately <NUM> kDa. Napins are albumins and are low molecular weight storage proteins with a molecular weight of approximately <NUM> kDa. Napins are more easily solubilized and in for example <CIT> a process is disclosed to separate out the more soluble napin fraction, preferably to at least <NUM> wt. Napins are primarily proposed for use in applications where solubility is key.

Rapeseed proteins can also be 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 <NUM>, <NUM> and <NUM>. Cruciferin and napin are the two major families of storage proteins found in rapeseed. Napin is a <NUM> albumin, and cruciferin is a <NUM> globulin. Furthermore, Schwenke and Linow (Nahrung (<NUM>) <NUM>, K5-K6) state that the cruciferin complex is present as a <NUM> kDa <NUM> hexamer when exposed to higher ionic strength (µ ≥ <NUM>/cm), and reversibly dissociates into <NUM> trimeric molecules of <NUM> kDa when exposed to low ionic strength conditions.

It has been found that high purity rapeseed protein isolate can have a broadly-based functionality in food products, unique among proteinaceous materials. The ability to utilize a protein that is vegetable in origin in food products enables truly vegetarian food products to be provided in instances where egg white and/or animal-derived protein have been used in the absence of any available substitute.

However, the use of plant proteins like rapeseed protein in food is still limited because of poor digestibility, taste, and nutritional value.

Poor digestibility of proteins in diets containing less refined cereals and grain legumes is due to the presence of less digestible protein fractions, high levels of insoluble fibers and/or high concentrations of anti-nutritional factors present or formed during processing. Dietary anti nutritional factors (ANFs) are known to negatively affect the protein digestibility, bioavailability of amino acids and protein quality of foods. The presence of high levels of trypsin inhibitors in soybean and other grain legumes have been shown to cause significant reduction in protein digestibility (up to <NUM>%) in rats and pigs (<NPL>). Based on the residues at their inhibitory sites, these inhibitors are either lysine- or arginine-type inhibitors. There are two major types of trypsin inhibitors, the Kunitz soybean trypsin inhibitor (KSTI) with an arginine at the reactive site and the Bowman-Birk inhibitor (BBI) with a lysine at the inhibitory site.

Another important factor in making plant-based protein food products is their taste and mouth feel, which notably is relevant in beverages. Vegetable proteins suffer from impaired taste such as bitterness and increased mouth feel even at low protein content. Also, many plant protein beverages contain very low amounts of proteins making them less nutritious and tasty foods.

<NPL>, discloses the kinetics of enzymolysis of rapeseed protein in relation to substrate concentration, initial enzyme concentration and hydrolysis time.

There is a need in the art to improve the properties of rapeseed protein, be it as concentrate, hydrolysate, isolate but certainly also as a food constituent. Hydrolysates are proteins that have been partially broken down by exposing the protein to heat, acid or enzymes that break apart the bonds linking amino acids. This makes hydrolysates taste more bitter, but also allows it to be absorbed more rapidly during digestion than native (non-hydrolyzed) protein. Isolates are purer than concentrates, meaning other non-protein components have been partially removed to "isolate" the protein. Many concentrates are around <NUM>% protein, which means that on a dry basis, <NUM>% of the total weight is protein. Isolates are typically around <NUM>% protein (dry basis). This is calculated using the Kjeldahl method.

Poor digestibility of vegetable proteins is partly due to the high levels of anti-nutritional factors (ANFs) such as trypsin inhibitors. The mouthfeel of a drink prepared from plant proteins is typically described as being not acceptable. Until the present invention, no acceptable solution is available to overcome these drawbacks of a plant protein drink.

Protein arginine deiminase (PAD) is an enzyme that deiminates arginine residues in proteins yielding citrulline as described in <CIT> or <CIT> by. For the trypsin inhibitors in which arginine occupies the reactive site, PAD action would render the inhibitor inactive towards trypsin. Chymotrypsin protease inhibitors and α-amylase inhibitors contribute as well to lowering food digestibility in a similar manner to trypsin inhibitors. Several of these inhibitors were shown to possess a crucial arginine residue important for the inhibitory function (<NPL>). About <NUM>% of all proteases inhibitors contain arginine at the reactive, P1 site (<NUM> sequences from <NUM> found in the MEROPS database for protease inhibitors). Trypsin inhibitors with arginine at P1 site can be found among all classes of vegetable proteins such as legumes, cereals, nuts, and seeds (soy, rapeseed, nuts, wheat, maize, barely, potatoes, rice, oats, tomatoes etc.), however not all inhibitory activities are found equally distributed within different species. For example, about <NUM>% of proteins in potato are protease inhibitors with many different classes of protease inhibitors present (more than <NUM> different proteases inhibitors identified) with distribution of these inhibitors dependent on the potato variety (<NPL>). Deactivation of Kunitz soybean trypsin inhibitor was shown by <NPL>) using mammalian PAD. However, deactivation of trypsin inhibitory activity in a complex protein matrix such as soy flour was never shown. For a benefit in a food product, a PAD activity in a complex food matrix is essential. In addition, mammalian PAD has a narrow selectivity for the arginine containing protein material; not all proteins containing arginine residues could be modified using the mammalian PAD2 enzyme. It was shown that chicken egg white ovomucoid, which contains an arginine residue at the trypsin inhibitory reactive site could not be modified by the mammalian PAD enzyme. <CIT> describes the use of PAD to improve a physical property such as gelling and foaming, or an emulsification property, or the solubility. However, the available prior art does not disclose or suggest the use of PAD to change sensory aspects, let alone describe how sensory aspects would be changed upon treatment by PAD, i.e. would a certain sensory aspect become stronger upon PAD treatment, or weaker?.

Surprisingly, the present invention demonstrates that different aspects of sensory and/or mouth feel and/or digestibility of a food comprising rapeseed protein can be improved using PAD.

Advantageously, the product and process of the invention relates to modifying sweetness, liquorice, astringency, powdery/chalk, fullness, thickness and/or digestibility of a food comprising rapeseed protein by incubating rapeseed protein isolate or a rapeseed protein solution with PAD and optionally processing said PAD-treated protein solution into a protein comprising food. Preferably, the product and/or process of the invention enables modification of at least one of the mentioned properties. For example, the product and/or process of the invention provides a method for modifying (preferably decreasing) sweetness of a food comprising rapeseed protein. Alternatively, the invention provides a process for modifying (preferably decreasing) liquorice of a food comprising rapeseed protein. The invention also provides a process for modifying (preferably decreasing) astringency of a food comprising rapeseed protein. The invention further provides a process for modifying (preferably decreasing) powdery/chalk of a food comprising rapeseed protein. Alternatively, the invention provides a process for modifying (preferably decreasing) fullness of a food comprising rapeseed protein. The invention also provides a process for modifying (preferably decreasing) thickness of a food comprising rapeseed protein. The invention further provides a process for modifying (preferably increasing) digestibility of a food comprising rapeseed protein. In another preferred embodiment, the process of the invention modifies at least <NUM>, <NUM> or <NUM> of the mentioned properties, such as a process for modifying powdery/chalk, fullness and thickness of a food comprising rapeseed protein.

The term "sweetness" as used herein refers to the basic taste most commonly perceived when eating foods rich in sugar. The term "liquorice" as used herein refers to a taste sensation/component present in the sweet root of Glycyrrhiza glabra or in stevia plant species. The term "astringency" as used herein refers to the dry, puckering mouth feel similar to that caused by tannins that are for example found in many fruits or by proteins, especially in an acidic environment. The term "powdery/chalk" as used herein refers to the degree in which the product feels powdery or grainy in the mouth. The term "fulness" as used herein refers to the feeling of full, round body in the mouth. The term "thickness" as used herein refers to firmness in the mouth and represents the force needed to push the product in between tongue and palate. The term "digestibility" as used herein refers to the capability of being digested and relates to the quantity of food that is retained by the body versus the quantity of food that is consumed. Digestibility of a protein directly relates to the extent of hydrolysis that digestive proteases such as pepsin and pancreatic peptidases, obtained upon incubation with the said protein under physiological relevant conditions. The term "modifying" refers to either increasing or decreasing depending on the aimed goal.

The product and process of the invention relate to rapeseed protein isolate with improved sensory attributes. PAD-treated rapeseed protein isolate of the invention has significant reduction in eight sensory aspects when compared to untreated rapeseed protein isolate. Mouthfeel, flavor intensity, sweet flavor, bitter flavor, liquorice flavor, bitter aftertaste, length of aftertaste and astringency of aftertaste all are reduced by <NUM>% to <NUM>% compared to untreated rapeseed protein isolate depending on the specific sensory aspect and the amount of PAD used. Mouthfeel reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Flavor intensity reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Sweet flavor reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Bitter flavor reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Liquorice flavor reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Bitter aftertaste reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Length of aftertaste reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Astringency of aftertaste reduces from by about <NUM>% to <NUM>% or from about <NUM>% to <NUM>%. Hence the invention provides a novel rapeseed protein isolate with improved taste in multiple embodiments compared to existing rapeseed protein isolate.

Whether or not any of the mentioned properties is increased or decreased is determined by comparing to an otherwise identical prepared food except that the rapeseed protein solution is not incubated with PAD.

The step of incubating a rapeseed protein isolate or rapeseed protein solution with PAD can be performed at any suitable pH for any suitable time and with any suitable enzyme concentration. The skilled person can establish a suitable enzyme amount or a suitable incubation temperature or a suitable incubation pH or a suitable incubation time, for instance incubating protein with a PAD at a pH of between <NUM> and <NUM>, such as a pH of between <NUM> and <NUM>, such as a pH of between <NUM> and <NUM>, such as a pH between <NUM> and <NUM>, or a pH of between <NUM> and <NUM>, for instance at a pH of about <NUM>. A suitable temperature at which protein is incubated with PAD may be between <NUM> and <NUM>, such as a between <NUM> and <NUM>, or between <NUM> and <NUM>.

The PAD-treated rapeseed protein solution can for example be a final product, such as, but not limited to, a protein drink. Alternatively, the PAD-treated rapeseed protein solution is a food ingredient that can subsequently be further processed into a food comprising rapeseed protein.

A process of the invention comprises at least <NUM> step of incubating a rapeseed protein solution with a PAD. As described above and depending on whether the rapeseed protein solution is a food ingredient or a final protein comprising food, a process of the invention optionally comprises the step of processing said PAD-treated rapeseed protein solution into a rapeseed protein comprising food.

The term protein arginine deiminase and peptidyl arginine deiminase (PAD) are used interchangeably herein. Protein or peptidyl arginine deiminases belong to a family of enzymes (EC <NUM>. <NUM>) that convert peptide or protein bound arginine into peptide or protein bound citrulline. This process is called deamination or citrullination. In the reaction from arginine to citrulline, one of the terminal nitrogen atoms of the arginine side chain is replaced by oxygen. The reaction uses one water molecule and yields ammonia as a side product. Whereas arginine is positively charged at a neutral pH, citrulline is uncharged. Surprisingly, it was found that a protein wherein at least part of the arginine has been converted into citrulline, and thereby resulting in protein with less charge, exhibited modified sweetness, liquorice, astringency, powdery/chalk, fulness, thickness and/or digestibility.

PAD may be derived from any suitable origin, for instance from mammalian or microbial origin. PAD's used in the present invention are advantageously derived from a microbial source, i.e. the PAD used in a process of the invention is a microbial PAD. For instance, PAD's may be derived from fungal origin such as from Fusarium sp. such as Fusarium graminearum, Chaetomium globosum, Phaesphaeria nodorum or from bacterial origin such as from the bacteria Streptomyces, e.g. Streptomyces scabies, Streptomyces clavuligeres. The wording "derived" or "derivable" from with respect to the origin of a polypeptide as disclosed herein, means that when carrying out a BLAST search with a polypeptide as disclosed herein, the polypeptide may be derivable from a natural source, such as a microbial cell, of which an endogenous polypeptide shows the highest percentage homology or identity with the polypeptide as disclosed herein.

PAD's are for instance known from <CIT>, disclosing a process for enzymatically treating a protein with a PAD, wherein at least <NUM>% of the arginine is transformed into citrulline.

A PAD may be a pure or purified PAD. A pure or purified PAD is an enzyme that may be at least <NUM>% pure, e.g., at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, at least <NUM>% pure, or at least <NUM>% pure, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% pure for instance as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.

Suitable amounts of PAD added to rapeseed protein isolate are from <NUM> to <NUM> U PAD/g of rapeseed protein isolate, or from <NUM> to <NUM> U PAD/g of rapeseed protein isolate, or from <NUM> to <NUM> U PAD/g of rapeseed protein isolate whereby the rapeseed protein isolate preferably is in an aqueous solution or suspension, such as for example a <NUM>% solution in water.

A PAD, or polypeptide having PAD activity may be produced in any suitable host organism by methods known in the art, for instance in fungi such as Aspergilli, e.g. Aspergillus niger or Aspergillus oryzae, Trichoderma, or the yeasts Saccharomyces, and Kluyveromyces or the bacteria of the genus Streptomyces or Bacilli. A suitable method to express a polypeptide having PAD activity in Aspergillus niger is for instance disclosed in Examples <NUM> and <NUM> in <CIT>.

The term "food comprising rapeseed protein" as used herein refers to any type of food as long as the food at least comprises rapeseed protein. The term food refers to solid foods as well as drinks. A rapeseed protein comprising drink prepared by a process of the invention can have a neutral or an acidic pH.

In a first aspect, the invention provides a rapeseed protein isolate comprising <NUM> to <NUM>% cruciferins and <NUM> to <NUM>% napins and having a solubility of at least <NUM>% when measured over a pH range from <NUM> to <NUM> at a temperature of <NUM>±<NUM>, wherein at least one of the amino acids in the cruciferins and/or napins is citrulline and wherein the rapeseed protein isolate comprises from <NUM>-<NUM>% of citrulline of the rapeseed protein.

In an embodiment, in the rapeseed protein isolate at least <NUM>%, or at least <NUM>%, or preferably at least <NUM>%, or at least <NUM>% or at least <NUM>% of the arginine residues that were originally present in the rapeseed protein are transformed into a citrulline residue. Arginine content in rapeseed protein is believed to be from <NUM>-<NUM>% (<NPL>). Accordingly, the rapeseed protein of the invention may contain from <NUM>-<NUM>% of citrulline, or from <NUM>-<NUM>% of citrulline.

In an embodiment, the rapeseed protein isolate has a solubility of at least <NUM>% when measured over a pH range from <NUM> to <NUM> at a temperature of <NUM>±<NUM>. Preferably, the rapeseed protein isolate has a solubility equal to or at least <NUM>% when measured in water over a pH range from <NUM> to <NUM> at a temperature of <NUM>±<NUM>. This is also known as the soluble solids index (SSI).

In an embodiment, the rapeseed protein isolate comprises at least <NUM>% (on dry matter) <NUM> rapeseed protein where the presence of <NUM> is verified by Blue Native PAGE.

For use in human food consumption the rapeseed protein isolate preferably comprises a low level of salt. This is measured by the conductivity. Preferably the conductivity of the rapeseed protein isolate in an aqueous <NUM>% solution is less than <NUM>,<NUM>/cm over a pH range of <NUM> to <NUM>. More preferably the conductivity of the rapeseed protein isolate in a <NUM>% aqueous solution is less than <NUM>,<NUM>/cm over a pH range of <NUM> to <NUM>. For comparison, the conductivity of a <NUM>/L sodium chloride aqueous solution is around <NUM>,<NUM>/cm.

In another embodiment, the rapeseed protein isolate has a phytate level less than <NUM> wt. %, more preferably less than <NUM> wt. % and most preferably less than <NUM> wt.

In another embodiment, the rapeseed protein isolate has a protein content of at least <NUM> wt. % (calculated as Kjeldahl N x <NUM>) on a dry weight basis, more preferably at least <NUM> wt. %, most preferably at least <NUM> wt. % and especially at least <NUM> wt.

In an embodiment the rapeseed protein isolate is un-hydrolyzed.

In a second aspect of the invention, there is provided a process for obtaining a rapeseed protein isolate comprising the steps of:.

wherein peptidyl arginine deiminase (PAD) is added during any or all of steps i), ii), iii), iv), v) and vi).

In an embodiment, PAD may be derived from any suitable origin, for instance from mammalian or microbial origin. PAD's used in the present invention are advantageously derived from a microbial source, i.e. the PAD used in a process of the invention is a microbial PAD. For instance, PAD's may be derived from fungal origin such as from Fusarium sp. such as Fusarium graminearum, Chaetomium globosum, Phaesphaeria nodorum or from bacterial origin such as from the bacteria Streptomyces, e.g. Streptomyces scabies, Streptomyces clavuligeres. In an embodiment the Fusarium-derived PAD disclosed in and prepared following Examples <NUM> and <NUM> of <CIT> is added during any or all of steps i), ii), iii), iv), v) and vi) above.

The rapeseed protein isolate is produced from rapeseed cold-pressed cake/meal, the byproduct of rapeseed oil production.

Steps i) to vii) are preferably carried out subsequently, i.e. in the order in which they are mentioned above.

In step i), preferably the aqueous liquid is an aqueous salt solution. More preferably the aqueous salt solution comprises sodium chloride. Most preferably the aqueous salt solution comprises <NUM> to <NUM>% sodium chloride (w/w). Preferably the extraction time is in the range of from <NUM> to <NUM> minutes, more preferably in the range from <NUM> to <NUM> minutes. Preferably during extraction, the protein:fat ratio in the protein rich liquid phase is maintained above <NUM>. Preferably the extracted rapeseed oil meal is washed with aqueous extraction liquid at a ratio of from <NUM>:<NUM> to <NUM>:<NUM>, more preferably of from <NUM>:<NUM> to <NUM>:<NUM>, still more preferably of from <NUM>:<NUM> to <NUM>:<NUM> (w/w).

In step ii), the separation may be carried out using any means known in the art including filtration and centrifugation. For example, standard filters, a filter press, a belt filter or other filters or centrifuges may be used, preferably at reduced pressure, i.e. of from -<NUM> to -<NUM> bar. Preferably the extraction temperature of from <NUM> to <NUM> is maintained.

In one embodiment, the mixing in step i) and the separating in step ii) may be carried out according to gravity-induced solid-liquid extraction as described in <CIT>.

In step iii), preferably the aqueous extraction liquid containing the proteins is decreamed using centrifugation. Alternatively, filtration may be used. Preferably the fat level is reduced by at least <NUM>% (w/v). The removal is measured with the decrease in concentration, while keeping the volume (reasonably) constant as about between <NUM> to <NUM>% of the volume is removed as "cream".

In step iv) the precipitant may comprise several divalent ions including salts of magnesium, zinc, iron, and calcium. Preferably an aqueous calcium chloride solution is used, as advocated in the prior art, for example in <CIT> (albeit for precipitation in pulse pea protein, a different species). Preferably the final concentration of the calcium chloride solution in the extract is in the range of from <NUM> to <NUM>/L, more preferably in the range from <NUM> to <NUM>/L, especially in the range from <NUM> to <NUM>/L and most especially in the range from <NUM> to <NUM>/L. The mixing with the precipitant can take place before, during or after pH adjustment. pH adjustment is to neutral, preferably to a value between pH <NUM> and pH <NUM>, more preferably to a value between pH <NUM> and pH <NUM>.

In step v), the precipitate (e.g. the phytate salt of calcium, magnesium, iron, zinc, or the like) may be removed by any of several methods including filtration, centrifugation, or the addition of enzymes such as phytases.

In step vi), preferably concentration is carried out by ultrafiltration to reach a concentration of at least <NUM> x. Preferably washing is carried out by diafiltration water using approximately <NUM> times the volume of the concentrate. For example, if the initial volume is <NUM>, the liquid is concentrated to about <NUM> (6x concentration) and is then washed with <NUM> (10x) of water. Preferably the washing is carried out at a temperature in the range of from <NUM> to <NUM> using an <NUM> to <NUM> kDa polyether sulfone PES membrane. Alternatively, a regenerated cellulose may also work Cold ultrafiltration may also be used. Preferably the washing is carried out using a <NUM> to <NUM>% sodium chloride solution followed by a <NUM>% to <NUM>% sodium chloride solution. Alternatively, the second wash may be with distilled water.

In step vii) the drying is preferably carried out using standard drying techniques such as evaporation, lyophilization, spray-drying and the like, with or without the application of reduced pressure. Preferably spray drying is used.

In an embodiment the rapeseed protein isolate is obtained in a process without a fractionating step for separating out cruciferins and napins.

In an embodiment the rapeseed protein isolate is obtained in a process where the levels of napin and cruciferin are kept substantially constant within the claimed range (i.e. neither the napin (<NUM>) or cruciferin levels (<NUM>) are deliberately increased). Preferably the rapeseed protein isolate comprises at least <NUM>% (on dry matter) of <NUM> rapeseed protein wherein presence of <NUM> is verified by Blue Native PAGE. More preferably the rapeseed protein isolate comprises at least <NUM>%, most preferably at least <NUM>%, especially at least <NUM>% and most especially at least <NUM>% (on dry matter) of <NUM> rapeseed protein wherein presence of <NUM> is verified by Blue Native PAGE.

Disclosed herein is a rapeseed protein isolate obtainable by the process of the second aspect having a reduction in sensory aspect of from <NUM>% to <NUM>% compared to untreated rapeseed protein isolate wherein said sensory aspect is mouthfeel or flavor intensity or sweet flavor or bitter flavor or liquorice flavor or bitter aftertaste or length of aftertaste or astringency of aftertaste or combinations of any or all thereof. Alternatively, the reduction in sensory aspect compared to untreated rapeseed protein isolate is from <NUM>% to <NUM>% or from <NUM>% to <NUM>%.

Disclosed herein is the use of a rapeseed protein isolate according to the first aspect of the invention as a foaming agent or as an emulsifying agent for food products.

In a further aspect of the invention there is provided a food product comprising a rapeseed protein isolate according to the first aspect of the invention.

The invention will be explained in more detail in the following Examples, which are not limiting the invention.

Protein content was determined by Kjeldahl method AOAC Official Method <NUM> Nitrogen (Total) in milk, using a conversion factor of <NUM> used to determine the amount of protein (% (w/w)).

The below solubility test is adapted from <NPL>), the difference being the use of water instead of <NUM> sodium chloride.

Sufficient protein powder to supply <NUM> of protein was weighed into a beaker. Water was added to the powder and the mixture was stirred until a smooth paste was formed. Additional water was then added to make a total weight of <NUM> (<NUM>% w/w protein dispersion). The dispersion was slowly stirred for at least <NUM> using a magnetic stirrer. Afterwards the pH was adjusted to the desired level (<NUM>, <NUM>, <NUM>, etc.) with sodium hydroxide or hydrochloric acid. After stirring for <NUM> minutes, an aliquot of the dispersion was reserved for protein content determination (Kjeldahl analysis). Another portion of the sample was centrifuged at <NUM>,<NUM> for <NUM>. The supernatant and pellet were separated after centrifugation. The protein content was also determined by Kjeldahl analysis.

Alternative methods for determining solubility are available and in some case use buffers, like borate-phosphate buffer in <CIT>. However, such as values are incomparable with the ones obtained in the instant application that are determined in the absence of buffer.

In the case of Native PAGE, the protein charge has an impact on the electrophoretic mobility. In the case of Blue Native PAGE (and to the contrary of Clear Native PAGE), the Coomassie Brilliant Blue dye provides the necessary charges to the protein complexes for the electrophoretic separation. The proteins were dissolved in <NUM> sodium chloride. As high salt concentrations are incompatible with electrophoretic separation, the sample was diluted <NUM>-fold with water (final salt concentration: <NUM>). Coomassie® G-<NUM> (SimplyBlue™, ThermoFischer Scientific) was used and gels were scanned with an ExQuest™ Spot Cutter (BioRad). Resultant bands after carrying out Blue Native PAGE were observed. It would be expected that bands around <NUM> kDa indicate <NUM>, around <NUM> kDa indicate <NUM> and around <NUM> kDa indicate <NUM> proteins.

The C/N ratio was determined by Size Exclusion Chromatography (SEC) analysis. Samples were dissolved in a <NUM> sodium chloride saline solution and analyzed by HP-SEC using the same solution as the mobile phase. Detection was done by measuring UV absorbance at <NUM>. The relative contribution of cruciferin and napin (%) was calculated as the ratio of the peak area of each protein with respect to the sum of both peak areas.

Phytates were measured at Eurofins using method QD495, based on <NPL>).

Measurement of PAD activity was performed as described in <CIT> by measuring the formation of citrulline residues in α-N-benzoyl-L-arginine-ethyl ester (BAEE). The incubation mixture contained <NUM> tris-HCl buffer (pH <NUM>), <NUM> CaCl<NUM>, <NUM> DTT, <NUM> BAEE in a final volume of <NUM>µL. Incubation was performed at <NUM> for <NUM>, and the reaction was stopped by adding <NUM>µL <NUM> N HClO<NUM>. Citrulline was determined by colorimetry according to the method of <NPL>). One unit of PAD is expressed as <NUM>µmol of citrulline formed/min/mg of protein.

Rapeseed protein isolate was prepared from cold-pressed rapeseed oil seed meal as described in <CIT>. The conductivity of the resultant rapeseed protein isolate in a <NUM>% solution was less than <NUM>,<NUM>/cm over a pH range of <NUM> to <NUM>. Blue Native PAGE: Main bands were observed roughly around <NUM> kDa, between the <NUM> and <NUM> kDa MW markers. The resultant rapeseed protein isolate comprised in the range of from <NUM> to <NUM>% cruciferins and <NUM> to <NUM>% napins, contained less than <NUM> wt. % phytate and had a solubility of at least <NUM>% when measured over a pH range from <NUM> to <NUM> at a temperature of <NUM>±<NUM>.

Cloning and expression of the polypeptide having PAD activity was performed as disclosed in Examples <NUM> and <NUM> of <CIT>.

Rapeseed proteins containing TIA were incubated with PAD and the reduction in TIA was measured using a BAEE assay. The preparation of the proteins with and without PAD incubation and the assay used are described below.

Rapeseed cake sample preparation: To <NUM> of rapeseed cake <NUM>µL <NUM>% NaCl was added and <NUM> units of PAD. The rapeseed was solubilized for <NUM> minutes at <NUM> in a Thermomixer at <NUM> rpm. Hereafter the sample was centrifuged for <NUM> minutes at <NUM>. The control rapeseed cake sample was solubilized in the absence of PAD.

Rapeseed protein isolate sample preparation: Rapeseed protein isolate was prepared as described in <CIT>. To a <NUM>% (w/v) solution of this material, <NUM> U/ml PAD was added, and the solution was incubated for <NUM> hours at <NUM> (Thermomixer <NUM> rpm). The control sample was incubated with the same volume of water instead of PAD.

TIA measurement with BAEE assay: The assay used is essentially as described in the Sigma protocol "assay method for trypsin inhibitor activity". <NUM>µL <NUM>/mL Trypsin in <NUM> HCl (Sigma; Trypsin from bovine pancreas; <NUM> units/mg solid) was added to <NUM> <NUM> HCl. Subsequently <NUM>µL of the protein sample prepared as described above (with or without enzyme incubation) was added into the mixture. As a control, <NUM>µL <NUM> HCl was added instead of the inhibitor in the trypsin activity measurement. These solutions were incubated for <NUM> minutes. To <NUM> <NUM> sodium phosphate pH <NUM> (<NUM>) <NUM>µL BAEE (Sigma; Na-Benzoyl-L-Arginine ethyl ester hydrochloride <NUM>/ml in the same buffer) was added and <NUM>µL <NUM> HCl. Trypsin activity was determined after addition of <NUM>µL of the Trypsin solutions and the reaction was monitored at <NUM>. The spectrophotometer was calibrated with <NUM>µL <NUM> HCl instead of the trypsin solution. The reaction was followed for <NUM> minutes with <NUM> data points per minute. An increase in absorbance at <NUM> indicates cleavage of the BAEE by the trypsin. When trypsin was incubated with the protein sample prior the substrate BAEE addition, the absorbance at <NUM> decreased in time, indicating inhibition of enzyme activity. When protein sample was incubated with PAD prior addition to the trypsin, the absorbance at <NUM> is partially restored indicating that trypsin inhibition by the protein sample diminished. From the slopes with and without PAD treated samples a reduction of TIA levels by PAD can be calculated. In the below Table, the reduction of TIA measured by the BAEE for the rapeseed protein samples is presented.

To a <NUM>% solution of rapeseed protein isolate in water (<NUM>, see Materials), <NUM> units of PAD were added. The resulting suspension was incubated for <NUM> hours at <NUM>-<NUM>. Subsequently the sample was frozen and lyophilized.

In alternative approaches leading to similar modified protein isolate comprising citrulline, PAD was added at different steps of the process as described in <CIT>. Hence the process comprised the steps of:.

wherein PAD was added during any or all of steps i), ii), iii), iv), v) and vi).

Aqueous rapeseed protein powder suspensions were made (<NUM>, <NUM>% w/w) in tap water and the pH was adjusted to <NUM> with <NUM> H<NUM>SO<NUM>. Suspensions were incubated for <NUM> h at <NUM> with and without PAD addition at different enzyme dosage. The enzyme was further inactivated, by heating the materials at <NUM>, with a holding time of <NUM> minutes. The samples were evaluated by means of descriptive analysis (QDA) with the sensory panel (n=<NUM>) on attributes relevant for the product in the test. During the test the samples were offered according to an optimally balanced design and were scored on <NUM>-<NUM> unstructured line scales in EyeQuestion in duplicate. The data were analyzed with an ANOVA to find significant differences between the individual samples. Differences with p<<NUM> were considered as significant; the results are displayed in <FIG>.

The in vitro digestibility of the product obtained in Example <NUM> was measured at NIZO Food Research Center using the SYMPHYD platform as described in <NPL>). The results of the in vitro model for protein digestion of rapeseed protein isolate with and without treatment with PAD are shown in <FIG>.

Claim 1:
A rapeseed protein isolate comprising <NUM> to <NUM>% cruciferins and <NUM> to <NUM>% napins and having a solubility of at least <NUM>% when measured over a pH range from <NUM> to <NUM> at a temperature of <NUM>±<NUM>, wherein at least one of the amino acids in the cruciferins and/or napins is citrulline and wherein the rapeseed protein isolate comprises from <NUM>-<NUM>% of citrulline of the rapeseed protein.