Soluble rapeseed protein isolate

Abstract

The present invention discloses a native rapeseed protein isolate comprising 40 to 65% cruciferins and 35 to 60% napins and having a solubility of at least 88% when measured over a pH range from 3 to 10 at a temperature of 23±2° C.; wherein the native rapeseed protein isolate comprises from 5% to 65% on dry matter of 12S rapeseed protein where the presence of 12S is verified by Blue Native PAGE.

Claims

1. A native rapeseed protein isolate comprising 40 to 65 wt % cruciferins and 35 to 60 wt % napins and having a solubility in water of at least 88% when measured over a pH range from 3 to 10 at a temperature of 23±2° C.; wherein the native rapeseed protein isolate comprises from 25% to 65% on dry matter basis of 12S rapeseed protein where the presence of 12S is verified by Blue Native PAGE.

2. The native rapeseed protein isolate according to claim 1 having a conductivity in a 2 wt. % aqueous solution of less than9,000 μS/cm over a pH range of 2 to 12.

3. The native rapeseed protein isolate according to claim 1 comprising at least 35% on dry matter basis of 12S rapeseed protein.

4. The native rapeseed protein isolate according to claim 1 comprising less than 20% on dry matter basis of 7S rapeseed protein.

5. The native rapeseed protein isolate according to claim 1 with a cruciferin/napin ratio in the range of from 0.9 to 1.3.

6. The native rapeseed protein isolate according to claim 1 with a phytate level less than 0.4 wt. %.

7. The native rapeseed protein isolate according to claim 1 with a solubility of at least 94% when measured over a pH range from 3 to 10 at a temperature of 23±2° C.

8. A food product or a pet food product comprising the native rapeseed protein isolate according to claim 1.

9. A pet food product according to claim 8 that comprises from 5% to 35% wt % of the native rapeseed protein isolate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows Blue Native PAGE gels applied to rapeseed protein isolates from different batches. The gels were stained with Simply Blue and scanned with the Spot Cutter.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

(2) In a first aspect of the invention, there is provided a native rapeseed protein isolate comprising 40 to 65% cruciferins and 35 to 60% napins and having a solubility of at least 88% when measured over a pH range from 3 to 10 at a temperature of 23±2° C.; wherein the native rapeseed protein isolate comprises from 5% to 65% on dry matter of 12S rapeseed protein where the presence of 12S is verified by Blue Native PAGE. Preferably, the native rapeseed protein isolate comprises from 10% to 65%, most preferably from 15% to 65%, especially from 25% to 65% and most especially from 35% to 65% (on dry matter) of 12S rapeseed protein where the presence of 12S is verified by native PAGE. As outlined above, a certain content of 12S proteins is not necessarily the same as cruciferins as the cruciferin 300 kDa 12S hexamer can dissociate into 7S trimeric molecules of 150 kDa.

(3) In one embodiment, the native rapeseed protein isolate has a solubility 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).

(4) For use in human food consumption the native rapeseed protein isolate preferably comprises a low level of salt. This can be 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 μS/cm 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 μS/cm over a pH range of 2.5 to 11.5. For comparison, the conductivity of a 5 g/I aqueous sodium chloride solution is around 9,400 μS/cm.

(5) In another embodiment, the native rapeseed protein isolate has a phytate level less than 0.4 wt. %, more preferably less than 0.3 wt. % and most preferably less than 0.15 wt. %.

(6) In still another embodiment, the native rapeseed protein isolate has a protein content of at least 90 wt. % (calculated as Kjeldahl N×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. %.

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

(8) Preferably the rapeseed protein isolate is obtained in a process 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.

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

(10) 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.

(11) 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.

(12) 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.

(13) 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.

(14) 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.

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

(16) 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 (2S) or cruciferin (12S) levels are deliberately increased).

(17) 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.sup.5 CFU/ml, after removal, preferably after precipitation and centrifugation to remove the precipitated material, the levels dropped below the 1*10.sup.2 CFU/ml. This means that further processing to reduce the microbial count, for example by microfiltration is not required.

(18) 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.

(19) 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.

(20) In a second aspect, the native rapeseed protein isolate may be used in any human nutritional food applications including as a foaming agent to replace egg whites, as an emulsifying agent to replace for example egg yolk in mayonnaise and simply as a nutritional component providing an excellent amino acid profile. Hence, the invention provides the use of a native rapeseed protein isolate according to the first aspect of the invention as a foaming agent for food products or as an emulsifying agent for food products.

(21) In a third aspect, the invention provides a food product or a pet food product comprising a native rapeseed protein isolate according to the first aspect of the invention.

(22) 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%. Native rapeseed protein isolate can be used as a gluten-replacing ingredient in pet food, which is advantageous as some pets can be sensitive to gluten. 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 2015/114543. The pet can be any suitable animal, such as avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal.

EXAMPLES

Test Methods

(23) Protein Content

(24) 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)).

(25) Conductivity

(26) The conductivity of native rapeseed protein isolate in a 2 wt. % aqueous solution was measured using a conductivity meter: Hach sensION.sub.+ EC71.

(27) Solubility Test:

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

(29) 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)×100.

(30) Alternative methods for determining solubility are available and in some case use buffers, like borate-phosphate buffer in WO 2011/057408. However, such as values are incomparable with the ones obtained in the instant application that are determined in the absence of buffer.

(31) MW Determination by Blue Native PAGE

(32) 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.

(33) 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.

(34) Cruciferin/Napin (C/N) Ratio

(35) 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.

(36) Phytate Level

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

Example 1

Preparation of Rapeseed Protein Isolate from Cold-Pressed Rapeseed Oil Seed Meal

(38) 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.

(39) 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 via a liquid/liquid separation step 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.

(40) The conductivity of the resultant native rapeseed protein isolates in a 2% solution was less than 4,000 μS/cm over a pH range of 2.5 to 11.5.

(41) Blue Native PAGE: Main bands were observed roughly around 300 kDa, between the 242 and 480 kDa MW markers (FIG. 1). 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.

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

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

(44) 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.

(45) TABLE-US-00001 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 (%)