Field of the invention

The present invention relates to food grade soluble native rapeseed protein isolate, to a food grade soluble native rapeseed protein isolate that has a low microbe level, a process for obtaining food grade soluble native rapeseed protein isolate and use of the food grade soluble native rapeseed protein isolate in a food product.

Background of the invention

The use of vegetable based proteins in nutritional composition or human food applications is known, for example WO 2008/094434 discloses the use of wheat protein isolates as an alternative to the use of egg yolk protein in compositions. The use of soy based protein has also been described for example in WO 2014/018922. However, the use of wheat protein isolates may not be desirable for those with gluten allergies and there may also be intolerances to soy based proteins. 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 17 to 25% of seed dry weight. Processing rapeseed for oil for human consumption produces rapeseed meal (also referred to as cake, 60%) 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 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 <2% erucic acid and <30 mmol/g glucosinolates. The resultant rapeseed meal is currently used as a high-protein animal feed.

Protein are available as hydrolysates, concentrates and isolates. 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 it taste more bitter, but also allows it to be absorbed more rapidly during digestion than a 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 80% protein, which means that on a dry basis, 80% of the total weight is protein. Isolates are typically around 90% protein (dry basis). This is calculated using the Kjeldahl method. 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 6 subunits and has a total molecular weight of approximately 300 kDa. Napins are albumins and are low molecular weight storage proteins 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. EP 1389921 B1 discloses a process of forming a food composition, which comprises extracting rapeseed oil seed meal with an aqueous food-grade salt solution at a temperature of at least 5°C to cause solubilization of protein in the rapeseed oil seed meal and to form an aqueous protein solution having a protein content of 5 to 30 g/l and a pH of 5 to 6.8, and subsequently two protein fractions are separated out via micelles. This is done to improve solubility as the cruciferin fraction is usually considered as less soluble over a wide pH range when not in the presence of a salt. The resultant protein isolate is incorporated in said food composition in substitution for egg white, milk protein, whole egg, meat fibers, or gelatin. A similar micelle fractionation approach is disclosed in US 2010/041871 leading to separate fractions of cruciferin and napin. 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%.

Furthermore, it has been found that if during the preparation process, these vegetable proteins are heat treated, proteins denature and functional properties are lost. Still, to make rapeseed protein isolates safe for human and infant nutrition, a low microbe level is needed. This can be achieved by pasteurization, the drawback of which is that the proteins are denatured. Pasteurization denatures proteins in microbes and therefore also the protein in rapeseed protein isolate. This means that nutritional value is maintained but not the functionality. Alternatively, microfiltration can also be used, however this is not viable on a commercial scale.

There is therefore a need to find a suitable process to prepare rapeseed protein isolate with a low microbe number. A low microbe count is not only a requirement for the end-product but the microbial stability of the concentrate before drying is also important.

Detailed description of the invention

Traditionally, for materials having relatively high oil content (>35% on dry matter, rapeseed is approximately 40%) a combination of mechanical pressing and solvent extraction is used for an efficient extraction of the oil (Rosenthal ef a/. , Enzyme and Microbial Technology 19 (1996) 402-420). After the oil is extracted, the pressed material is heat treated to remove the solvent, resulting in a meal with an oil and protein content of 1-5% and 40-50% of the dry matter, respectively. Although the meal has a relative high protein content, the quality of the proteins is reduced significantly resulting from the harsh conditions (i.e., elevated temperature, solvents) employed during the oil extraction. The awareness that these oil extraction conditions are detrimental for the quality of the proteins is one of the factors bolstering the improvement of the cold pressing technology. During cold-pressing, no solvents (like e.g. hexane) are used and the oil is pressed out under mild conditions, resulting in better quality oil and an oilseed pressed meal of higher quality. This oilseed pressed meal has a relatively high oil content (typically >8%, for example >10%, on dry matter basis) and is an excellent source of proteins with preserved functionality. These proteins can be readily extracted from the meal by for instance an aqueous extraction (Rosenthal ei a/., Enzyme and Microbial Technology 19 (1996) 402-420, Rosenthal ei a/. , Trans iChemE, Part C, 76 (1998) 224-230 and Lawhon ei a/., J. Food Sci. 46 (1981 ) 912- 916). One of the biggest challenges of this type of processes is that during extraction proteins and oil are extracted concomitantly. This leads to an extract containing a significant amount of oil, present in most cases partly as a stable emulsion making its removal quite difficult. WO 2014/147068 discloses mild extraction of cold-pressed rapeseed meal to obtain protein-rich extracts that are practically fat-free.

We have found that in our process, based on cold-pressed rapeseed meal, a rapeseed protein isolate is obtained with a high level of cruciferins while simultaneously displaying an unprecedented high solubility. There is no need to separate out the protein constituents and yet an unprecedented solubility across a broader pH range can be achieved and maintained. The hypothesis that solubility of rapeseed protein isolate can only be improved by reducing the amount of proteins with lower solubility, such as cruciferins, appears therefore not exclusive.

It has been found that there is no need to apply detrimental prior art heat treatment steps such as pasteurization to nevertheless obtain microbe levels as low as less than 1 ,000 CFU/g. The soluble native rapeseed protein isolate comprising both cruciferins and napins, obtained according to the present invention after mild extraction of rapeseed oil meal obtained using the cold-press method mentioned above, gave surprisingly low microbe levels.

In a first aspect of the invention, there is provided a native rapeseed protein isolate comprising 40 to 65 wt.% cruciferins and 35 to 60 wt.% napins and having a solubility of at least 88% over a pH range from 3 to 10 at a temperature of 23±2°C with a microbe level of less than 1 ,000 CFU/g. Preferably the microbe level is less than 500 CFU/g, more preferably from 200 CFU/g to 1 ,000 CFU/g. Generally, a level of around 1 ,000 CFU/g, such as from 800 to 1 ,200 CFU/g would be suitable for application in adults whereas a level of around 500 CFU/g, such as from 250 to 600 CFU/g would be suitable for application in children.

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

For use in human food consumption the native rapeseed protein isolate preferably comprises a low level of salt. This is established by measuring the conductivity. Preferably the conductivity of the native rapeseed protein isolate in a 2 wt.% aqueous solution is less than 9,000 μ3 αη over a pH range of 2 to 12. More preferably the conductivity of the native rapeseed protein isolate in a 2 wt.% aqueous solution is less than 4,000 μ3/αη over a pH range of 2.5 to 1 1 .5. For comparison, the conductivity of an aqueous 5 g/l sodium chloride solution is around 9,400 μ3/αη.

In another embodiment, the native rapeseed protein isolate has a phytate level of less than 0.4 wt.%, preferably of less than 0.25 wt.% and more preferably of less than 0.15 wt.%.

In still another embodiment, the native rapeseed protein isolate has a protein content of at least 90 wt.% (calculated as Kjeldahl N x 6.25) on a dry weight basis, more preferably at least 94 wt.%, most preferably at least 96 wt.% and especially at least 98 wt.%.

Preferably the native rapeseed protein isolate is substantially unhydrolyzed. By substantially unhydrolyzed is meant that the protein is not deliberately hydrolyzed.

In a second aspect of the invention, there is provided a process for obtaining a native rapeseed protein isolate according to the first aspect of the invention comprising the steps of: i) mixing cold-pressed rapeseed oil meal with an aqueous liquid at a temperature of from 45 to 65°C;

ii) separation of the aqueous liquid from the mixture obtained in step i);

iii) decreaming of the aqueous liquid obtained in step ii);

iv) adjusting the pH of the decreamed aqueous liquid obtained in step iii) to neutral by adding acid or base, and mixing with a precipitant to obtain a precipitate; v) removing the precipitate obtained in step iv) to obtain an aqueous liquid;

vi) concentrating and washing the aqueous liquid obtained in step v);

vii) isolating native rapeseed protein isolate from the concentrated and washed aqueous liquid obtained in step vi) by means of drying.

As outlined above, the rapeseed protein isolate is produced from cold pressed rapeseed press meal, the by-product of rapeseed oil production.

The process starts with an extraction step i), in which rapeseed meal is combined with an aqueous salt solution, for example 0 to 5% sodium chloride, at a temperature between 4 to 75°C, more preferably 20 to 75°C and most preferably 45 to 65°C. Preferably, in step i) said mixing is carried out such that the ratio between said cold-pressed rapeseed oil meal and said aqueous liquid is from 1 :2 to 1 :30 (w/w). Preferably the meal to water ratio is in the range of from 1 :5 to 1 :40, more preferably 1 :5 to 1 :20. After a period in the range of from 5 min to 2 hours the protein rich solution is separated from the insoluble material in the separation step ii). The protein rich solution is hereafter referred to as the extract.

The pH of the extract is preferably adjusted to neutral and the extract is further processed to clarify the material and remove non-protein substances. In the decreaming step iii), the residual fat and formed precipitates are removed via a solid/liquid separation step (e.g. filtration or centrifugation). Preferably, the decreaming in step iii) is carried out by means of centrifugation.

The extract is then concentrated and washed in an ultrafiltration/diafiltration (UF/DF) step vi). The UF/DF step has the purpose of concentrating the protein and removing anti-nutritional factors (e.g. polyphenols, residual phytate, glucosinolates). The concentrating and washing in step vi) is preferably carried out by means of ultrafiltration and diafiltration.

Finally, in step vii), the washed concentrate may be dried in a suitable dryer, such as a spray drier (single or multistage) with an inlet temperature in the range of from 150 to 200°C and an outlet temperature in the range of from 50 to 100°C resulting in the rapeseed protein isolate.

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

Preferably the rapeseed protein isolate is obtained in a process where the levels of napin and cruciferin are kept substantially constant (i.e. neither the napin or cruciferin levels are deliberately increased).

Surprisingly it was found that germ filtration of the extract before concentration was not required to obtain the low microbe concentrations of the first aspect of the invention. Furthermore, the concentrate showed good microbial stability. Although the microbial levels of the material before the removal of non-protein substance by centrifugation were above 1*10 ⁵ CFU/ml, after removal, preferably after precipitation and centrifugation to remove the precipitated material, the levels dropped below the 1*10 ² CFU/ml. This means that further processing to reduce the microbial count, for example by microfiltration is not required.

The process of the instant invention is characterized in that it is well-suited for large-scale application. Hence, in one embodiment the process is carried out at a scale of at least 500 kg, preferably of from 500 to 10,000 kg or from 1 ,000 to 5,000 kg in a period of from 2 to 10 hours.

In another embodiment of the invention there is provided a process for obtaining food grade soluble native rapeseed protein isolate and use of the food grade soluble native rapeseed protein isolate in a food product.

In a third aspect, the invention provides the use of the native protein isolate according to the first aspect of the invention in food products or pet food products.

In one embodiment, the invention provides the use of an emulsion in pet food products that comprise from 5% to 35% of native rapeseed protein isolate by weight of the pet food product, preferably from 25% to 30%. The native rapeseed protein isolate of the instant invention can be used as a germ-free ingredient in pet food, replacing e.g. wheat. The term "pet food" means any composition intended to be consumed by a pet. Meat or fish pet food can be a meat or fish emulsion product having a realistic meat- or fish-like image. The rapeseed protein isolate can be added to the meat or fish material before and/or after the meat or fish material is emulsified as described in e.g. WO 2015/1 14543. The pet can be any suitable animal, such as avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal.

Non-limiting Examples and comparative examples of the invention are described below.

EXAMPLES Test methods

Protein content

Protein content was determined by the Kjeldahl method according to AOAC Official Method 991.20 Nitrogen (Total) in Milk, using a conversion factor of 6.25 was used to determine the amount of protein (% (w/w)).

Conductivity

The conductivity of native rapeseed protein isolate in a 2 wt.% aqueous solution was measured using a conductivity meter: Hach senslON+ EC71. Solubility test

The below solubility test is adapted from Morr ef al. (J. Food Sci. (1985) 50, 1715-1718), the difference being the use of water instead of 0.1 M sodium chloride.

Sufficient protein powder to supply 0.8 g of protein was weighed into a beaker. A small amount of demineralized water was added to the powder and the mixture was stirred until a smooth paste was formed. Additional demineralized water was then added to make a total weight of 40 g (yielding a 2% w/w protein dispersion). The dispersion was slowly stirred for at least 30 min using a magnetic stirrer. Afterwards the pH was determined and adjusted to the desired level (2, 3, 4, etc.) with sodium hydroxide or hydrochloric acid. The pH of the dispersion was measured and corrected periodically during 60 minutes stirring. After 60 minutes of stirring, an aliquot of the protein dispersion was reserved for protein content determination (Kjeldahl analysis). Another portion of the sample was centrifuged at 20,000 g for 2 min. The supernatant and pellet were separated after centrifugation. The protein content was also determined by Kjeldahl analysis. Protein solubility (%) = (protein in supernatant / protein in total dispersion) x 100. Alternative methods for determining solubility are available and in some case use buffers, like borate-phosphate buffer in WO 201 1/057408. However, such as values are incomparable with the ones obtained in the instant application that are determined in the absence of buffer. MW determination by Blue Native PAGE

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 500 mM sodium chloride. As high salt concentrations are incompatible with electrophoretic separation, the sample was diluted 10-fold with water (final salt concentration: 50 mM). Coomassie® G-250 (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 14 kDa indicate 2S, around 150 kDa indicate 7S and around 300 kDa indicate 12S proteins.

Cruciferin/napin (C/N) ratio

The C/N ratio was determined by Size Exclusion Chromatography (SEC) analysis. Samples were dissolved in a 500 mM 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 280 nm. 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.

Phvtate level

Phytates were measured at Eurofins using method QD495, based on Ellis ef al. (Anal. Biochem. (1977) 77, 536-539).

Example 1

Preparation of rapeseed protein isolate from cold-pressed rapeseed oil seed meal

The rapeseed protein isolate was produced from cold-pressed rapeseed oil seed meal having an oil content of less than 15% on dry matter basis, cleaned and processed below 75°C.

In the extraction step, the cold-pressed rapeseed oil seed meal was mixed with an aqueous salt solution (1 to 5% sodium chloride), at a temperature between 40 to 75°C. The meal to aqueous salt solution ratio was in the range of from 1 :5 to 1 :20. After about 30 minutes to 1 hour the protein rich solution (extract) was separated from the insoluble material. The pH of the extract was adjusted to neutral and the extract was further processed to clarify the material and remove non-protein substances. In the decreaming step, the residual fat was removed using centrifugation. Non-protein substances were removed by adjusting the pH of the material to neutral in the presence of a salt with which phytate precipitates (e.g. calcium chloride). The formed precipitate is removed via a solid/liquid separation step (e.g. a membrane filter press or centrifugation) in which the impurities are removed in a solid salt form (e.g. calcium phytate). The extract was then concentrated and washed in an ultrafiltration/diafiltration (UF/DF) step. Finally, the washed concentrate was dried in a spray drier with an inlet temperature in the range of from 150 to 200°C and an outlet temperature in the range of from 50 to 100°C resulting in the rapeseed protein isolate. Several batches were prepared and tested.

The microbial count was measured using the international FDA measuring standard after the separation step where it was found to be in the range of 10 ⁶ CFU/ml. After precipitation and removal of the precipitate by centrifugation the microbial count was below the limit of detection of 10 ² CFU/ml. The microbial removal is also important for the stability of the concentrate after the UF/DF step. Concentrate prepared according to the invention and without the use of a microbe filter, was stored at 4°C and analyzed each day for 7 days. The results from this analysis showed that the concentrate was stable at around 1.1*10 ² CFU/ml and no further growth was seen. This suggests that the concentrate can be stored at 4°C and is stable from a microbial point of view. The conductivity of the resultant native rapeseed protein isolates in a 2% solution was less than 4,000 \}S/cm over a pH range of 2.5 to 1 1.5.

Blue Native PAGE: Main bands were observed roughly around 300 kDa, between the 242 and 480 kDa MW markers. Some staining was visible as a smear as lower MW (150 kDa and below). No clear bands were observed at 150 kDa. Based on these results, the rapeseed product contains the 12S form of cruciferin.

The resultant native rapeseed protein isolate comprised in the range of from 40 to 65% cruciferins and 35 to 60% napins.

The resultant native rapeseed protein isolate contained less than 0.26 wt.% phytate.

The resultant native rapeseed protein isolates had a solubility of at least 88% when measured over a pH range from 3 to 10 at a temperature of 23±2°C as shown for two batches in the below table.

PH 3 4 5 6 7 8 9 10

Sample 1 98 96 89 95 95 97 97 98 Solubility (%)

Sample 2 102.5 97.5 94.3 93.9 97.0 93.0 94.0 99.8 Solubility (%)