DE102011105909A1 - Process for the production of a rape protein isolate - Google Patents

Abstract

The present invention relates to a process for the preparation of a high-purity rape protein isolate and to a rape protein isolate prepared by the process according to the invention.

Description

The present invention relates to a process for the preparation of a high-purity rape protein isolate and to a rape protein isolate prepared by the process according to the invention.

Plant proteins are currently gaining importance as a raw material for the production of food, feed, additives and components for biodegradable polymers for a number of reasons. Due to the rapidly growing population of the earth, the associated problems in food production and the debate on the sustainability of food, especially animal proteins, there is increasing interest in using plant proteins as components for the production of food on a large, additional scale. These can serve as a source of protein on the one hand, on the other hand, they can adjust functional properties and z. B. act as an emulsifier or foaming agent. Also due to the lactose intolerance common in large parts of the world population, vegetable protein based products are of interest as an alternative to dairy products.

With regard to the use of plant proteins for animal feed applications, there is also a rapidly increasing demand and a search for alternative protein sources with high-quality amino acid distribution. An example in this context is the use of plant protein in aquaculture, as the capacity of fishmeal production is already exhausted and the need for aquaculture fish and shrimps will steadily increase over the next few years. Finally, because of their amphiphilic properties, plant proteins can be used as foam formers and emulsifiers for industrial applications and have the advantage here of being readily biodegradable. Likewise, such proteins can be used as components for the production of biodegradable materials from renewable resources.

For all the previous applications, rape proteins would be of interest, as they are produced in the production of rapeseed oil together with fibers and other phytonutrients. There they remain as a main ingredient in the press cake.

Rapeseed contains around 200 g of crude protein per 1000 g of dry matter. If one takes into account that a harvest of 6,500,000 t of rape was expected in Germany alone in 2010, there is considerable potential for animal and human nutrition. In animal nutrition, this potential has already been exploited, but up to the present time interfering with rape seed interferes with its digestibility, structure (amino acid side chains) and sensory properties of rape protein. These are in particular polyphenols and phytic acid.

The polyphenols mentioned are reactive compounds that react with the proteins depending on their structure. The reactions are mainly carried out on free amino groups (ε-amino group of lysine), on the SH groups and on the tryptophan side groups, ie the proteins are derivatized. As a result of these reactions, changes in protein properties occur. The solubility profile of these biopolymers changes, as does the isoelectric point, as well as the ratio of hydrophilic-hydrophobic properties. Furthermore, there is an increase in the molecular weights. The derivatization of the proteins also alters their enzymatic degradation behavior as, on the other hand, the derivatization of the enzymes alters their activity.

Phytic acid, another secondary ingredient of rapeseed, also has the ability to interact with rape protein. These are obviously non-covalent interactions. These reactions cause the isoelectric point of the rapeseed globulins to be shifted to the acidic region. This also alters functional properties (solubility, foam properties and the like) of the resulting protein products.

As long as there is no technological possibility to separate these interfering substances in a simple and industrially applicable manner from the high-quality rape protein and also to obtain an undenatured protein product, this potential can not be used. This applies to both animal feed production and food production.

Although the proteins contained therein are very high quality, but the Rapspresskuchen can only be added to the cattle feed to small amounts, if it still contains the aforementioned components that make the food incompatible.

The prior art discloses a number of processes which can be used to prepare a purified rape protein as a so-called isolate or concentrate:
Following a procedure of the IVV Freising (Protein preparation produced from rape seeds WO 2010/096943 ) the rapeseed cake can be aqueous extracted after pre-oiling, and proteins are precipitated by alcohol addition. The secondary plant substances, such as polyphenols, phytic acid and glucosinolates, remain in solution. This method makes it possible to produce a fiber-containing protein concentrate in relatively simple steps. The disadvantage of the method, however, is that the proteins partially denature by the alcohol precipitation and the separation of protein and fibers only partially succeeds according to this method).

The company Burcon Nutra Science Corporation is already with rapeseed protein formulations on the market, which are marketed under the name Puratein ^®. The corresponding procedure is described in the patents WO 2010/020042 . WO 2010/020039 . WO 2010/020038 . WO 2010/003245 . WO 2009/152621 . WO 2009/152620 . US 2007/0244300 and US 20080319171 and is based on the use of black-skinned rape varieties and a conventional oil mill technology.

The extraction of the proteins from the press cake is carried out by means of a salt solution, and the proteins are separated by a so-called micelle precipitation. The proteins do not undergo denaturation. However, the protein thus produced still has a relatively high content of glucosinolates, polyphenols and phytic acid. For the characterization of the salinity of the extraction solution gives the WO 2010/020039 on page 4 an ionic strength of at least 0.05, preferably at least 0.1 an ( US 2006193965 (A1) , Oilseed processing, US 20060381765 US 20060505 ).

In this case, a partial denaturation takes place, the secondary plant substances are separated as much as possible. About the contents of the products of mustard oil and in particular phenolic compounds are given no information. For the concentrates, maximum contents of glucosinolates of 3.5 and 3.8 μmol / g are given. The process is in the patent WO 09137934 A1 described. The essence of the process seems to be that the press cake is mixed with the solvent, preferably water, and salts or polysaccharides are added to the solvent for the extraction of the proteins from the rapeseed cake.

Another method of the company BioExx Extraction Technologies is mainly worked with yellow-skinned rape varieties (Brassica juncea), and the proteins are extracted with special solvents (iodine-tri-fluoro-methane), which do not denature. Lt. However, according to the manufacturer, the protein concentrate still has a residual content of glucosinolates and phytic acid. The process is in the WO 09137934 and the WO 11000094 described.

Finally, that describes WO 2008/144939 a process for aqueous extraction of protein from rapeseed cake to obtain an albumin (napin) rich fraction and a globulin (cruciferin) rich fraction. The albumin (napin) rich fraction is extracted at pH values between 2.5 and 5 from the press cake.

For the reasons mentioned above, there is an increasing demand for purified rape protein and alternative methods of rape protein rape. In particular, it is necessary to work with environmentally friendly processes without solvents and without extensive addition of salts.

Furthermore, the rape protein must be isolated in undenatured form and the content of unwanted phytochemicals should be minimized as much as possible. In addition, it is of course also of interest to deplete the secondary plant substances separately and to regain them for other applications. However, according to the method described above, in order to prepare a rape protein isolate, it is always necessary to either work with a solvent for extraction or to precipitate the protein with a high salinity system.

The inventors of the present application have therefore set themselves the task of developing a method that allows the purification and isolation of high purity rape protein in non-denatured form and which is largely free of interfering ingredients such as phytochemicals, especially polyphenols and phytic acid. Furthermore, a process should be provided in which the ingredients which interfere with the isolation of proteins can be separately depleted and processed further. Finally, it was an object of the present inventors to develop that Procedure without the addition of toxic or in the production of food and animal feed unauthorized ingredients / additives.

• i) in Kontaktbringen des Rapspresskuchens mit einem Anionenaustauscher;
• ii) in Kontaktbringen des Rapspresskuchens mit einem Kationenaustauscher und/oder einem hydrophoben Adsorbens.

This object has been achieved by a process for obtaining a high purity rapeseed protein isolate from rapeseed cake comprising the steps

• i) contacting the rapeseed cake with an anion exchanger;
• ii) contacting the rapeseed cake with a cation exchanger and / or a hydrophobic adsorbent.

By means of this two-stage separation of the undesired ingredients in the rape protein isolate, these substances can be separated to a particularly high degree, i. H. a particularly pure rape protein isolate can be achieved. In addition, a two-stage separation offers the advantage that each of the separated substances are also present in a particularly pure form and can also be used.

For the purposes of the present application, the term "rapeseed cake" means any composition comprising more than 80% by weight of rapeseed, preferably more than 90% by weight, more preferably more than 95% by weight, and most preferably more as 99% by weight. It is preferred in this case if the rapeseed of the rapeseed presscake has already been subjected to a pressing process, that is to say the rapeseed has been at least mechanically pretreated. In a particularly preferred embodiment, the rapeseed in this mechanical pretreatment, a proportion of oil and / or fat of preferably at least 20 wt .-% (based on the original oil or fat content of rapeseed), preferably 40 wt .-%, in particular preferably at least 60% by weight, more preferably at least 70% by weight, more preferably at least 80% by weight, also preferably at least 90% by weight, also more preferably at least 95% by weight and most preferably at least 99% Weight% withdrawn. In addition, it is particularly preferred if, during the mechanical pretreatment process, the oil or fat contained in the rapeseed has been completely removed so that the rapeseed cake underlying the process according to the invention is free of oils and fats. In a particularly preferred embodiment of the method according to the invention, the rapeseed cake is de-oiled separately before step i) and / or ii) separately. The de-oiling of the rapeseed press cake before the steps i) and / or ii) can be carried out in any manner known to those skilled in the art as suitable for the purpose according to the invention. In a conventional Naxchentölung the press cake with z. For example, in the desolventation section, for example, hexane, care must be taken that it is performed under conditions that do not cause protein denaturation (eg, "flash desolventation").

The contacting of the rapeseed press cake with an anion exchanger according to step i) as well as bringing the rapeseed cake into contact with a cation exchanger and / or a hydrophobic adsorbent according to step ii) of the process according to the invention is preferably carried out by adding and mixing the anion exchanger or Cation exchanger and / or the hydrophobic adsorbent with the rapeseed cake. It is particularly preferred if the rapeseed cake in the bringing into contact according to step i) and / or ii) of the inventive method in an aqueous solution, d. H. in a mixture having at least 60% by weight, preferably at least 70% by weight, more preferably at least 75% by weight, especially preferably at least 80% by weight and most preferably at least 90% by weight (based on the Total mixture) is present in water.

The bringing into contact according to step i) and / or ii) of the rapeseed cake in aqueous solution with the anion exchanger or cation exchanger (and / or hydrophobic adsorbent) is thus preferably carried out by adding it to the aqueous mixture. In a particularly preferred embodiment, stirring is effected during or after the addition.

The bringing into contact is preferably carried out at a temperature of 5 to 90 ° C, preferably 10 to 80 ° C, more preferably 15 to 70 ° C, more preferably 17 to 60 ° C, particularly preferably 18 to 50 ° C, on preferably 20 to 40 ° C and most preferably 21 to 35 ° C.

The bringing into contact according to step i) and / or ii) is preferably carried out under atmospheric pressure.

In a preferred embodiment, the rapeseed cake prior to contacting in accordance with step i) and / or ii) is subjected to an aqueous extraction. The aqueous extraction can be carried out by adding to the rapeseed cake an amount of at least 60% by weight of water (as the main constituent of the extraction medium), preferably at least 65% by weight, more preferably at least 70% by weight, especially preferably at least 75% by weight .-%, also more preferably at least 80 wt .-%, more preferably at least 85% by weight and most preferably at least 90% by weight (based on the total mixture of water and rapeseed cake) is added. In a particular embodiment, stirring is effected during and / or after the addition. During the addition or after the addition of the water, a large part of the rape proteins contained in the rapeseed cake are dissolved. The extraction of the rape proteins from the rapeseed cake can be improved if a buffer is added to the water.

The pH is preferably adjusted to a value of 2 to 7, preferably 3 to 6 and most preferably 3.5 to 4.5 during the aqueous extraction.

The temperature during the aqueous extraction is preferably in a range of greater than 0 to 40 ° C, more preferably 1 to 25 ° C, more preferably 2 to 15 ° C, and most preferably 5 to 10 ° C. At temperatures below 10 ° C, the solubility of the rape proteins is highest.

In a particularly preferred embodiment, the extraction medium is free of salts. This allows the protein to be extracted very gently and without the risk of denaturation.

If the rapeseed cake before being brought into contact with step i) and / or ii) of the process according to the invention is subjected to an aqueous extraction, in a further preferred embodiment the solids and fibrous materials contained in the rapeseed cake will be brought into contact removed according to the steps i) and / or ii). This can be done for example by filtration or sedimentation. It is also possible to separate the extract from the solids and fibers by centrifugation.

The aqueous extraction can also be repeated, d. H. preferably 2 to 20 times, more preferably 3 to 10 times and most preferably 5 to 8 times.

It is possible to carry out the contacting according to step i) and / or ii) of the method according to the invention in the case of additionally performed aqueous extraction before or after the solids and fibers have been separated from the extraction medium or extract. In addition to an aqueous pre-extraction prior to bringing into contact according to i) and / or ii) of the process according to the invention, an acidic aqueous pre-extraction can still be carried out in a particular embodiment. Further acidic aqueous pre-extraction can further reduce the level of phytochemicals in the final rape protein isolate. By a second sour pre-extraction, the Phytinsäureabreicherung is preferably increased to at least 70 wt .-% (based on the Phytinsäureanteil in the starting material of the extraction). In the case of phenolic acids, the depletion preferably increases to at least 70% by weight, preferably to at least 95% by weight (based on the proportion of phenolic acid in the starting material of the extraction).

The anion exchanger in a preferred embodiment may be both organic and inorganic in nature. The anion exchanger is preferably an organic or inorganic material having a charge neutral point at a pH which is higher than the pH for the extraction of the rape protein. Preferred anion exchangers for the process of the present invention are aluminum oxides or hydrotalcites, with particular preference being given to those which have a cationic surface charge at pH values of 4.5. These are particularly preferred, since they can adsorb anionic compounds particularly well and bind particularly well in rapeseed isolate undesirable substances such as phytic acid. Likewise preferred anion scavengers may also contain a boehmite phase in addition to a hydrotalcite phase.

Particularly preferred are organic anion exchange resins having quaternary amino groups. An example of a very suitable, commercially available anion exchange resin is the product Purolite ^® A200 (Purolite THE COMPANY, 150 Monument Road, Bala Cynwyd, PA 19004, USA). Preferred anion exchangers, as described above, allow separation of the phytic acid to a high level and at the same time to minimize protein loss as the rape proteins bind to the anion exchanger more poorly.

The hydrophobic adsorbent is preferably a hydrophobic polymer resin. Examples of particularly suitable hydrophobic adsorbents are resins containing polystyrene, acrylate acid, methacrylic acid. Crosslinking with, for example, divivenbenzene is particularly preferred.

The cation exchanger is preferably a natural sheet silicate, which is particularly preferably selected from the group consisting of vermiculite, smectic sheet silicate such as Bentonite, minerals of talc such as talc, chlorite, kerolite or a mixture of two or more phyllosilicates.

The preferred cation exchangers according to the invention thus include charged clay minerals of the 2: 1 layer type with a negative charge of 0.2 to 0.6 per formula unit. Examples of smectites include bentonite, montmorillonite, beidellite, nontronite, hectorite and saponite. Another suitable smectite is stevensite, the structure of which can be derived from that of talc by vacancies of Mg 2+ ions.

Preferred bentonites typically have a cation exchange capacity of between 50 and 120 meq / 100 g and exhibit a high swelling capacity in water.

In a further embodiment according to the invention, the cation exchanger contains stevensite or a stevensite phase.

What is meant by Stevensit is familiar to the person skilled in the art. A closer characterization of stevensite can be found for example in JL Martin de Vidales et al., Clay Minerals, 1991, 26, 329-342 , and in GB Brindley et al., Mineralogical Magazine, 1977, 41, 443-452 to which express reference can be made.

In a further preferred embodiment, the cation exchanger contains a saponite. The definition of the typical mineral saponite may include in "Developments in Clay Science 1: Handbook of Clay Science", F. Bergaya, BKG Theng, G. Lagaly (Eds.), Elsevier, Amsterdam 2006 , and in particular herein Chapter 1, "General Introduction: Clays, Clay Minerals and Clay Science", F. Bergaya and G. Lagaly, p. 1 et seq be read. Saponite is a trioctahedral smectite with magnesium ions in the octahedral layer.

In a further preferred embodiment, the cation exchanger contains a cerolite. What is meant by kerolith is known to those skilled in the art. For example, here too GB Brindley et al., Mineralogical Magazine, 1977, 41, 443-452 to get expelled. The determination of kerolith can be carried out as described there.

The bringing into contact according to step i) and / or ii) of the process according to the invention is preferably carried out at a temperature of 2 to 90 ° C, preferably 5 to 80 ° C, more preferably 6 to 70 ° C, also preferably 7 to 60 ° C, more preferably 8 to 50 ° C, more preferably 9 to 40 ° C, even more preferably 10 to 30 ° C, and most preferably 15 to 25 ° C.

The bringing into contact according to step i) and / or ii) of the process according to the invention is preferably carried out at a pH of 3 to 6, more preferably 4 to 5, particularly preferably 4.

The proportion of the anion exchanger, cation exchanger and / or of the hydrophobic adsorbent is preferably in a range from 0.1 to 15% by weight (based on the total weight of rapeseed cake and optionally extractant), more preferably 0.5 to 10% by weight. , More preferably 1 to 5 wt .-% and most preferably 1.5 to 3.5 wt .-%.

In a preferred embodiment, the method of the present invention further comprises the step

iii) separating the purified rape protein isolate.

The separation can be carried out in any way that is known to those skilled than suitable for the purpose of the invention. The separation preferably takes place by filtration, sedimentation or centrifugation.

In a further particularly preferred embodiment, the method of the present invention further comprises the step

iv) ultrafiltration of the purified rape protein isolate according to step iii).

The ultrafiltration can be carried out in any manner which is known to those skilled in the art as suitable for the purpose of the invention. Particularly preferred is a "cut-off" of ultrafiltration such as ultrafiltration at a cut-off of 5 to 100 kDa, preferably 5 to 50 kDa, more preferably 10 to 30 kDa. Furthermore, a polysulfone membrane, a ceramic membrane or cellulose acetate is preferably used, with a polysulfone membrane being particularly preferred.

• i) in-Kontakt-bringen des Rapspresskuchens mit einem Anionenaustauscher;
• ii) in-Kontakt-bringen des Rapspresskuchens mit einem Kationenaustauscher und/oder einem hydrophoben Adsorbens

wobei der Rapspresskuchen vor dem in-Kontakt-bringen gemäß Schritt i) und ii) einer mehrstufigen (2 bis 8 stufigen) wässrigen Extraktion unterzogen wurde und die Fest- und Faserstoffe ebenfalls vor dem in-Kontakt-bringen gemäß Schritt i) und ii) abgetrennt wurden. Besonders bevorzugt wird der Rapspresskuchen bei den einzelnen Stufen der wässrigen Extraktion bei jeder Stufe mit neuem wässrigen Extraktionsmittel in-Kontakt-gebracht.Particularly preferred embodiments of the process of the present invention is a process for obtaining a high purity rapeseed protein isolate from rapeseed cake comprising the steps

• i) contacting the rapeseed cake with an anion exchanger;
• ii) contacting the rapeseed cake with a cation exchanger and / or a hydrophobic adsorbent

wherein the rapeseed cake has been subjected to a multi-stage (2 to 8-stage) aqueous extraction prior to contacting in step i) and ii) and also the solids and fibers before contacting in step i) and ii) were separated. Most preferably, the rapeseed cake is contacted at each stage of the aqueous extraction at each stage with new aqueous extractant.

In other preferred embodiments, the rapeseed cake is de-oiled prior to aqueous extraction.

In addition, it is particularly preferred if an acid extraction of the rapeseed cake is carried out before or after the aqueous extraction.

The steps i) and ii) of the method according to the invention can also be carried out in the reverse order.

Measuring methods and procedures

Investigation methods for the characterization of the rapeseed cake

water content

Water content and volatile substance content Water and volatile components were determined according to the DGF unit method B-II 3 (87). The drying was carried out at 103 ± 2 ° C. The analyzer system TGA 601 from Leco was used as the destination device.

Mineral content

The determination was carried out by drying the samples at 105 ° C and subsequent ashing at 950 ° C under an oxygen atmosphere to constant weight. Weight constancy was given when the weight variation between two measurements at 9 minute intervals was less than 0.1%. For the determination, the analysis system TGA 601 (Leco, Mönchengladbach) was used.

crude protein

The crude protein content of the samples was determined according to LMBG §35 on the determination of nitrogen according to the Dumas method. To convert the nitrogen content into the crude protein content of the samples, the factor 6.25 was used. The determination of nitrogen was carried out with the system FP-528 (Leco, Mönchengladbach).

fat content

The fat content of the samples was determined according to Caviezel ^® with the DGF standard method KI 2c (00). The fat determination was carried out with the extraction unit B-815 and the fat determination device B-820 (Büchi, Flawil, Switzerland).

Investigation methods for the characterization of the aqueous rapeseed cake extract

Dry matter content (TS)

The dry substance is determined with the thermogravimetric analysis system TGA 601 from Leco. By heating to 105 ° C +/- 2 ° C the sample is withdrawn from the raw water to a constant weight setting (FHG-IVV, 2000a).

Total protein content

The total protein content is determined indirectly by the method of Dumas via the nitrogen determination. For the implementation, the analyzer Leco FP 528 is used. The degassed samples are completely burned under oxygen atmosphere at a temperature of 950 ° C. The resulting combustion gases collected, filtered and cooled. For reduction, they are passed over a copper reactor, where nitrogen oxides are reduced to elemental nitrogen and oxygen is removed. By adsorption, the by-products water and carbon dioxide are separated, so that the remaining nitrogen is detected by a Wärmeleitfähigkeitszelle cell. The protein content of the starting product can then be determined by multiplication by a factor of 6.25 (FHG-IVV, 2004).

phytic acid

Photometric determination method

The content of phytic acid is determined by the reaction of iron (III) chloride and sulfosalicylic acid. The reaction of iron (III) chloride and sulfosalicylic acid (called Wade reagent) gives a pink color, the adsorption maximum at 500 nm and is measured photometrically. In the presence of phytate, the iron complex is bound and is therefore no longer available for reaction with the sulfosalicylic acid, resulting in a weakening of the pink coloration.

The sample to be determined is extracted with 0.29 M HCl. Subsequently, 500 μl of sample are mixed with 800 μl of Wade reagent, centrifuged and measured photometrically at the wavelength of 500 nm. Using a calibration curve, the concentration of phytic acid in the sample is determined on the basis of the measured adsorption (FHG-IVV, 2009).

Determination by HPLC

In an external laboratory, the content of phytic acid (IP6) was determined by means of an HPLC method. Due to the precision of the method, other inositol phosphates (IP3-IP5) can also be determined. The hydrochloric acid-purified sample was separated by means of a Mono-Q column (HR 5/5 (50 × 5 mm).

phenolic acids

Photometric method

The determination of the phenolic compounds is carried out by a photometric method, which is based on the detection of sinapine and sinapinic acid by means of HPLC.

For the analysis, the extract to be examined is diluted with 100% methanol to give a 70% methanol solution. This is centrifuged in Eppendorf caps and diluted sufficiently with 70% methanol before the measurement to be in the range of the calibration line. The measurement takes place at a wavelength of 330 nm. The phenolic acids are quantified by calculating the calibration line.

Solid samples are mixed with 70% methanol and extracted three times in total. The supernatant is poured through a pleated filter and collected in a sealable flask. If necessary, a suitable dilution is performed before the photometric measurement (FHG-IVV, 2010).

HPLC method

The determination of the phenolic acids is carried out with a HPLC (high performance liquid chromatography) system from Dionex (HPLC pump P680, Automated Sample Injector ASI-100, column oven ICS-3000 DC, UV / VUS detector UVD170U) and a C18 column with polar endcapping (Synergi 4u Hydro-RP 80A).

For sample preparation, 1.5 g of solid sample are stirred for 30 minutes after addition of 13,500 μl of 70% methanol and a stir bar using a magnetic stirrer plate. After 30 minutes, the entire sample liquid is filtered off via a paper filter (602) into a 25 ml flask. The beaker is rinsed with 3,500 .mu.l of 70% methanol and this solution is also filtered off into the flask. After filtering off the filtrate, the filter is rinsed twice with 3,500 μl of 70% methanol each time. The filtrate is then made up to 25 ml with 70% methanol and shaken (about 30 sec). 300 μl of the aqueous sample are mixed with 700 μl of pure methanol by vortexing in an Eppendorf vial. After filtration through a 0.22 μm filter, the clear methanolic solutions are filled into HPLC vials and sealed.

The method was carried out with a flow of 0.4 ml / min.
Eluent A: water-methanol, 90:10 with 2% acetic acid (100%)
Eluent B: methanol with 0.5% acetic acid

For sinapinic acid, the following formula of the calibration line with R ² = 0.9989 was determined: Peak area / 3.3215 = amount μg / ml sample

Due to a lack of standard, sinapine was analogously to the procedure of CHANDRA et al. ( CHANDRA, A .; RANA, J .; LI, Y., 2001: Separation, Identification, Quantification, and Method Validation of Anthocyanins in Botanical Supplement Raw Materials by HPLC and HPLC-MS, Journal of Agricultural and Food Chemistry, 49, pp. 3515-3521 ) is quantified by the molecular weight correction factor.

After quantification as sinapinic acid, the samples were multiply the correction factor, the quotient of the molar mass of sinapine and sinapinic acid: Correction factor = [M (sinapine) / M (sinapinic acid)] = (310.37 / 224.21) = 1.38

glucosinolates

In an external laboratory, the samples are concentrated, mixed with extractant and internal standard and stirred at 75 ° C. After subsequent centrifugation, the supernatant is placed on an ion exchanger, rinsed with buffer and treated with sulfatase. After the incubation period in the incubator, the samples are washed with HPLC water from the exchanger columns, filtered and analyzed by HPLC. Detection takes place at 229 nm.

Mustard oil Determination

The mustard oil content, calculated as allyl isothiocyanate, is determined in an external laboratory using the official 1975 method.

For glucosinolate determination, the sample is slurried in water. The mustard oils are split off under the action of ferments, distilled off after the addition of ethanol and taken up in dilute ammonia solution. The solution is heated with silver nitrate solution, cooled and filtered. The excess of silver nitrate is back titrated with an ammonium thiocyanate solution.

Using the same procedure, a blank solution is prepared without an analytical sample. The volume of silver nitrate solution consumed in the blank test is subtracted from the consumption of silver nitrate solution in the analytical sample solution. The value thus obtained indicates the silver nitrate solution in ml which has been consumed by the mustard oil of the analytical sample. 1 ml of 0.1 molar silver nitrate solution corresponds to 4.956 mg of allyl isothiocyanate ( GADAMER, 1975 ).

Adsorbents used

Inorganic Adsorbents

The following materials are γ-aluminas and mixed oxides of γ-alumina and γ-silica, which are normally used as catalysts.

EX M 1694 (mineralogical stevensite-kerolite)

This adsorber is a phyllosilicate with hydrophobic surface, which is present as a mixture of two types of talc. On the one hand as kerolite, a layered silicate with Mg ²⁺ instead of the Al ³⁺ octahedron and on the other hand as stevensite, a talc which has Mg ²⁺ gaps and therefore z. T. is negatively charged. A highly porous internal structure is characteristic of this adsorber. Tab. 1: Analytical values EX M 1694 analytical Data SiO ₂ 58.6% Al ₂ O ₃ 5.3% specific surface 230 m ² / g moisture content ~ 5% particle 25-100 μm 1.4%> 150 μm bulk weight ~ 580 g / l loss on ignition 8.5%

Organic adsorbent resins

Purolite A200 ^®

Purolite ^® A200 is a strongly basic polystyrene resin and has a high affinity for strong acid anions. The resin is spherical beads, which are characterized by a high chemical stability. It is particularly suitable for the treatment of aqueous media with a high content of free mineral acids and in addition has reversible sorptive capacities for complex organic materials, both in ionized and non-ionized form. Tab. 2: Product properties Purolite ^® A200 product features values structure Polystyrene gel crosslinked with divinylbenzene Functional groups Type 2 quaternary NH ₄ ⁺ groups Ionic form Cl Bulk density (Cl ^- form) 680-700 g / l Particle size range 0.3-1.2 mm max. 1% <0.3 mm, max. 5%> 1.2 mm water content 35-60% Specific weight (Cl ^- form, moist) 1.08 g / ml Moisture retention 45-51% pH stability 0-14

Purolite A500 ^®

In Purolite ^® A500 is a strong base, macroporous anion exchange resin. The porous structure of the opaque, spherical beads results in high binding capacities. The resin is therefore used for the deionization and adsorption of organic material. Tab. 3: Product properties Purolite ^® A500 product features values structure Polyacrylic acid cross-linked with divinylbenzene, Functional groups Type 1 quaternary NH ₄ ⁺ groups Ionic form Cl Bulk density (Cl ^- form) 670-700 g / l Particle size range 0.4-1.0 mm water content 43-75% Specific weight (Cl ^- form, moist) 1.08 g / ml Moisture retention 53-58% pH stability 0-14

Amberlite ^® XAD-4

Amberlite ^® XAD-4 is a hydrophobic, polyaromatic, strongly acidic cation exchange resin having a sulfonic acid group. Because it is modified with an acetyl group, it can be used for adsorption without a humidification process. The resin is whitish translucent, insoluble beads with high physical, chemical and thermal stability.

Due to its macro-crosslinked structure and large surface, it has high adsorptive properties and is particularly suitable for the binding of phenolic and aromatic compounds. Tab. 4: Product properties Amberlite ^® XAD-4 product features values structure Polysytrol-divinylbenzene Functional group Sulfonic acid group (SO ₃ ^- ) degree of crosslinking high Moisture retention 54-60% bulk density 680 g / l Spec. Weight 1.01-1.03 g / l Mean particle size 0,49-0,69 mm max. 5% <0.35 mm max. 5%> 1.18 mm surface ≥ 750 m ² / g porosity ≥ 0.50 ml / ml

Amberlite ^® IRP-69

Amberlite ^® IRP-69 is a strongly acidic cation exchange resin. The sulfonated copolymer of styrene and divinylbenzene has Na ⁺ as the exchangeable cation. It is therefore suitable for binding basic or cationic active ingredients. Amberlite ^® IRP-69 is a fine dry powder that is insoluble in water. Its ability to exchange ions is independent of pH. Tab. 5: Product properties Amberlite ^® IRP-69 product features values structure Polysytrol-divinylbenzene Functional group Sulfonic acid group (SO ₃ ^- ) Ionic form Na ⁺ water content Max. 10% particle size 10-25%> 75 μm max. 1%> 150 μm K exchange capacity 110-135 mg / g sodium content 9.4 to 11.5% Styrene content Max. 1 ppm

Amberlite ^® IRC-76

The exchange resin is a weakly acidic cation exchanger. IRC-76 is predominantly used to remove metals from aqueous solutions. However, the copolymer is sensitive to oxidation. Tab. 6: Product properties Amberlite ^® IRC-76 product features values matrix Acrylate copolymer Functional group Carboxyl group COO ^- Ionic form H ⁺ exchange capacity ≥ 3.90 eq / 1 (H ⁺ form) moisture content 52-58% (H ⁺ shape) density 1.14-1.18 g / cm ³ (H ⁺ form) grain size 500-750 μm max. 3% <0.300 mm max. 5%> 1,180 mm volume change H ⁺ → Na ⁺ : 60% bulk density 790 g / l

Amberlite ^® IRC-86

Amberlite ^® IRC-86 is a weakly acidic cation exchanger with high physical and chemical resistance. The appearance of the gel-like polyacrylic are yellow spheres. Tab. 7: Product properties Amberlite ^® IRC-86 product features values matrix Acrylate-divinylbenzene Functional group Carboxyl group COO ^- Ionic form H ⁺ exchange capacity ≥ 4.10 mol / l (H ⁺ form) moisture content 47-53% (H ⁺ form) density 1.17-1.195 g / cm ³ (H ⁺ form) grain size 580-780 μm max. 2.0% <0.300 mm max. 5.0%> 1.180 mm volume change H ⁺ → Na ⁺ : 100% bulk density 790 g / l

Lewatit CNP 105 ^®

Lewatit CNP 105 ^® is a macroporous methacrylic acid-divinylbenzene copolymer, and acts as a weak acidic cation exchange resin. Tab. 8: Product properties Lewatit ^® CNP 105 product features values matrix Methacrylic acid-divinylbenzene ionic form H ⁺ particle size 100-400 μm pore size ~ 27 nm exchange capacity 1.4 eq / l specific surface ~ 200 m ² / g

Examples and figures

The invention will be described and explained in more detail with reference to the following examples and figures. It should be understood that the examples and figures in no way limit the scope of the present invention, but are merely illustrative in nature, and more particularly illustrate preferred embodiments.

It shows

1 The adsorption of phytic acid on Purolite A 200 as a function of time at an adsorber concentration of 7% and at 24 ° C

2 The adsorption of phytic acid on Purolite A 500 as a function of time at an adsorber concentration of 7% and at 24 ° C

3 The adsorption of phytic acid on Purolite A 200 as a function of time and at adsorber concentrations of 3.5 and 7% in the extract under the defined environmental conditions of pH 4 and T = 24 ° C.

4 The adsorption of phenolic acids with Purolite ^® A 200 and Purolite ^® A 500 at a concentration of 7 wt .-% at a pH value of 4 and a temperature of 24 ° C

5 The adsorption of phenolic acids with Purolite ^® A 200 in a concentration of 7 wt .-% and 3.5 at a pH value of 4 and a temperature of 24 ° C

6 The adsorption of canola protein with Purolite ^® A 200 and Purolite ^® A 500 at a concentration of 7 wt .-% at a pH value of 4 and a temperature of 24 ° C

7 The adsorption of canola protein with Purolite ^® A 200 in a concentration of 7 wt .-% and 3.5 at a pH value of 4 and a temperature of 24 ° C

8th The adsorption of phenolic acids by various adsorbents at a pH of 4 and a temperature of 24 ° C over a period of 2 hours

9 The adsorption of phytic acid by various adsorbents at a pH of 4 and a temperature of 24 ° C over a period of 2 hours

Example 1: Acid aqueous extraction of de-oiled rapeseed cake

For the acidic aqueous extraction, a post-oiled rapeseed cake with the following composition was used: Table 9: Substance data of the presscake used for the extraction Product: Rapeseed (winter rape harvest 2008) de-oiled by Ölmühle Mara GmbH, after-tapped in the IVV Analysis values: TS protein ash fat [%] [%] [%] [%] 89.9 42.5 7.3 2.1 Analysis values: sinapin sinapinic mustard oils glucosinolates phytic acid [mg / 100g] [mg / 100g] [Mg / kg] [Mol / g] [%] 803 94 357 15.5 5.8

Depending on the required extract quantity, an 11 or 41 reactor is used for extract extraction. This is connected to a water bath and so tempered. An anchor stirrer mounted in a stirrer serves to mix the contents of the reactor. The optimized parameters for the extraction process, determined by Down (DOWY F., 2009: Separation of phytochemicals in the aqueous alkaline recovery of rape protein by adsorption, Diploma thesis, University of Applied Sciences Weihenstephan, Freising) are listed in Table 10 below. Tab. 10: Process parameters for the preparation of the acidic pre-extract parameter Time [min] Temperature [° C] PH value s: l ratio ionic strength 45 24 4 1:10 (1 + 10) 0

First, the desired amount of water is introduced into the reactor and their pH is adjusted to 4 with one molar hydrochloric acid. The water is tap water with an ionic strength of 0.0031 mol / l (HARTL, 2009).

Subsequently, within ten minutes alternately ground rapeseed pollen and one molar hydrochloric acid is added to the water introduced, the pH is kept as constant as possible to 4. After extraction for 45 minutes, centrifugation at 3300 g removes the residue from the extract. The extract obtained is used to carry out the adsorption experiments.

The concentrations of the phytic acid and phenolic compounds contained in the extract, as well as the proportion of protein and the dry matter are shown in Table 11. Tab. 11: Ingredients of the acidic extract product TS [%] Protein [%] Phytic acid [mg / ml] Phenolic acids [mg / ml] extract 3.28 38.03 5.26 1.60

Example 2 Purposeful removal of phytic acid from the aqueous rapeseed cake extract of Example 1

The aqueous extract from Example 1 was now treated with anion exchangers, with the aim of depleting the phytic acid. For the experiments, the commercial anion exchange resins Purolite ^® 200 and Purolite ^® 500 (150 Monument Road, Bala Cynwyd, PA 19004, USA THE COMPANY Purolite,) were purchased from Purolite used.

For each of these experiments, a portion of the extract prepared (250-400 ml) is placed in an 11-reactor and mixed with the desired amount of support material. The concentration of the adsorbent refers to the amount of extract used. In the case of a batch with 500 g of extract and a 2% adsorber feed, this means an adsorber amount of 10 g. Uniform mixing is ensured by an anchor stirrer at 180 rpm.

After sufficient contact time between extract and carrier, extract samples are taken. For the removal of the stirring process is interrupted, so that the adsorber can sink and is not in the sample removed. If necessary, the casting is additionally carried out through a fine-mesh sieve (≤ 0.2 mm). For powdered materials, the adsorbent-extract mixture is separated by centrifugation. The extracted extract sample is examined for the content of protein, phytic acid and phenolic acids.

The results show that Purolite ^® 200 depletes the phytic acid better than Purolite ^® 500 and that a contact time of 1 h with the adsorber is sufficient under the set stirring conditions. Furthermore, 3.5% by weight of adsorber is sufficient to deplete at least 80% of the phytic acid.

A strong adsorption with respect to the phytic acid and at the same time low binding to phenolic acids are optimal conditions for using the adsorber for selective phytic acid adsorption.

The adsorption of the rape protein should be as low as possible. On the one hand, therefore, the phytic acid can be present as a pure fraction on the adsorber and on the other hand, the protein is not lost for isolate recovery. Protein adsorption is shown in the following graph.

The adsorption of the protein moves in Purolite ^® A200 in the range of 5.5-7% and Purolite® A500 of 7-9% at an identical Adsorberkonzentration of 7% ( 6 ).

Example 3 Purified removal of phenolic acids from the aqueous rapeseed cake extract of Example 2

The adsorption of the phenolic acids takes place in the second adsorption stage. Since hardly any phenolic acids were adsorbed by the first adsorption stage and the phytic acid was removed to a large extent from the extract, an optimal condition for the targeted removal and the recovery of a phenolic acid fraction in pure form is given.

The following table lists the adsorbents used with concentrations and investigated contact times Tab. 12: Adsorbents for phenolic acid depletion adsorber Concentration [%] Time [h] EX M 1694 2.0 1, 2 Amberlite ^® XAD-4 3.5 1, 2 Amberlite ^® IRP-69 2.0 1, 2 Amberlite ^® IRC-76 3.5 1, 2 Amberlite ^® IRC-86 3.5 1, 2 Lewatit ^® 2.0 1, 2

The percent binding of the phenolic acids is based on the amount that is still in the extract after the first adsorption stage. Tab. 13: Adsorbed amount of phenolic acids in stage 2 Phenolic acids adsorbed / extract [mg / g] adsorber after 1 h after 2 h EX M 1694 0.81 0.81 Amberlite ^® XAD-4 0.27 0.42 Amberlite ^® IRP-69 0.66 0.66 Amberlite ^® IRC-76 0.41 0.48 Amberlite ^® IRC-86 0.37 0.47 Lewatit CNP 105 ^® 0.20 0.22

Inorganic Adsorber:

The inorganic adsorber EX M 1964 was used in a concentration of 2%. It shows the best adsorption performance in comparison to the investigated adsorbers ( 8th ). In addition, for the binding of about 68% of phenolic acids, which corresponds to an amount of 0.81 mg / g of extract, a one-hour contact time of the carrier material with the extract is sufficient. A longer process time does not increase the adsorption capacity.

Organic Adsorbers:

Amberlite ^®

The adsorbent resins Amberlite ^® XAD-4 and IRP-69 are strongly acidic cation exchangers. With an adsorption capacity of up to 40%, the adsorbent Amberlite ^® XAD-4 is in the middle performance range. For the adsorption of 0.42 mg of phenolic acids per gram of extract, however, a process time of two hours is necessary because after one hour of adsorber treatment only slightly more than half is bound to phenolic acids. Conveniently, the separation of the adsorber, which is possible due to its particle size with a fine-mesh sieve (0.2 mm). The external nature of the adsorber is similar to that of the Purolite A200, which is why a concentration of 3.5% was chosen.

The adsorbent Amberlite ^® IRP-69, however, is very finely powdery and drier than the XAD-4, which is why it was used in a lower concentration of 2%. Due to its nature, it can not be removed by sieving from the extract. A complicated separation by centrifugation is necessary. With an adsorption capacity of around 57%, which corresponds to the binding of 0.66 mg of phenolic acids per gram of extract, Amberlite ^® XAD-4 scores second best of the adsorbers tested and has already reached its adsorption maximum after the processing time of one hour.

The two resins Amberlite ^® IRP-76 and IRP-86 are weak cation exchanger and very similar in their structure. Their adsorption capacity with respect to the phenolic acids hardly differs. With an adsorption line of 40 and 41% (0.47 and 0.48 mg / g extract) their performance is comparable to the also beaded, but strongly acidic cation exchanger XAD-4 and is in the middle range. A process time of two hours is necessary to ensure the binding of the phenolic acids. The separation of the weak cation exchangers from the extract is easy, since both resins are beaded spheres, which are retained by a sieve with a mesh size of 0.2 mm.

Lewatit ^®

Lewatit ^® CNP 105 is also a weakly acidic cation exchanger whose structural structure is similar to that of the weakly acidic Amberlite ^® . With a phenol binding of 12%, or 0.22 mg / g extract, which were achieved after a two-hour adsorption, the cation exchanger shows a weaker performance compared.

Adsorption of phytic acid in stage 2

In addition to the binding of phenolic acids in the first stage, phytic acid adsorption in stage 2 is also of interest. This shows which adsorbers in addition to the binding of phenolic acids additionally show an affinity for phytic acid. In turn, the lowest possible phytic acid adsorption is desired for the recovery of a pure fraction. On the other hand, the greatest possible depletion of phytic acid with respect to the purity of the recovered protein is advantageous. The percentage bound phytic acid quantity refers to the residual content that remains after the first adsorption in the extract.

The Amberlite ^® XAD-4, IRC-76 and IRC-86 all show a similar level of binding of phytic acid remaining about 80% ( 9 ). The increase in phytic acid adsorption, which is due to an extension of the process time, can also be clearly recognized. All three Amberlite ^® adsorbers are spherical beads used with an adsorber concentration of 3.5%.

The Lewatit ^® CNP 105 showed the lowest phytic acid adsorption at 35%.

Total reduction of phytochemicals after two-stage adsorption

With regard to the total reduction of phytic acid and phenolic compounds, the following results could be obtained. Tab. 14: Total reduction of phytic acid after two-stage adsorption adsorber step 1 Level 2 after 1 h [%] after 1 h [%] after 2 h [%] Purolite A200 ^® 86.42 EX M 1694 > 98 > 98 Amberlite ^® XAD-4 92.53 97.32 Amberlite ^® IRP-69 > 98 > 98 Amberlite ^® IRC-76 94.00 98.29 Amberlite ^® IRC-86 93.81 97.33 Lewatit CNP 105 ^® 88.87 89.78 Tab. 15: Total reduction of phenolic acids after two-stage adsorption adsorber step 1 Level 2 after 1 h [%] after 1 h [%] after 2 h [%] Purolite A200 ^® 2.23 EX M 1694 68.54 68.96 Amberlite ^® XAD-4 29.13 41.74 Amberlite ^® IRP-69 57.52 57.77 Amberlite ^® IRC-76 36.39 41.91 Amberlite ^® IRC-86 35.63 43.26 Lewatit CNP 105 ^® 13.29 15.58

Table 14 shows that it is possible to remove the phytic acid almost completely from the extract. Primarily due to the powder adsorbents EX M 1694 and Amberlite ^® IRP-69 of the second adsorption stage, the phytic acid can be reduced to such an extent that its content in the extract is below the linear detection limit of 0.01 mg / ml.

The total reduction of phenolic acids in the aqueous extract shown in Table 15 is possible up to about 69% by EX M 1694. The second powdered adsorbent Amberlite ^® IRC-69 also performs best with a reduction to around 58% of the adsorbers tested.

When comparing the beaded weak acid (Amberlite ^® IRC-76 and IRC-86) and strong acid (Amberlite ^® XAD-4) cation exchange resins can be seen no significant difference in the phenolic acid binding. At best, the content of phenolic compounds can be reduced to around 43%. Lewatit ^® CNP 105 has the lowest reduction of phenolic compounds with a total decrease of just under 16%.

Performance of adsorbers based on their dry matter content

Since different concentrations of the respective support materials were used in the adsorption experiments, a final consideration of the bound amounts based on the dry adsorber used is made in Tables 16 and 17. These tables also list for economic aspects how much dry adsorbent material was needed for each adsorption performance. Tab. 16: Phytinsäureadsorption based on the TS of the adsorber adsorber c [%] TS [%] c (dry) [%] PS / adsorber [mg / g] Time [h] Purolite A200 ^® 3.5 58,65 2.05 183.5 1 Purolite A500 ^® 7.0 47.77 3.34 121.4 1 Tab. 17: Phenolic acid adsorption based on the TS of the adsorber Adsorber [mg / g] c [%] TS [%] c (dry) [%] Phe / Ads. Time [h] EX M 1694 2.0 91,50 1.83 37.1 1 Amberlite ^® XAD-4 3.5 47.14 1.65 16.3 1 25.7 2 Amberlite ^® IRP-69 2.0 95.81 1.92 34.3 1 Amberlite ^® IRC-76 3.5 46,40 1.62 25.2 1 29.3 2 Amberlite ^® IRC-86 3.5 54.87 1.92 19.5 1 24.4 2 Lewatit CNP 105 ^® 2.0 32.39 0.65 30.6 1 34.6 2

Table 16 shows the phytic acid adsorption data. Of interest here is the adsorber Purolite ^® A200, which was specified for the first adsorption stage. Percent, the use of 2.05% dry carrier is required to bind 183.5 mg of phytic acid (PS) based on one gram of dry adsorbent.

The resin Purolite ^® A500 has been studied only in a higher concentration of 3.34% of dry material, so that per gram of the carrier (121.4 mg) has taken place a lower binding to phytic acid.

Table 17 gives the phenolic acid adsorption data. Concerning the use according dry adsorber materials EX M cut 1694 (37.1 mg / g), Amberlite ^® IRP-69 (34.3 mg / g) and Lewatit ^® CNP 105 (34.6 mg / g) best from ,

With a phenolic acid binding of 29.3 mg / g Amberlite ^® IRC-76 is the next better adsorber. Due to its pearl shape, the resin can also be easily separated from the extract. Amberlite ^® XAD-4 and IRC-86 are the worst performers in this regard.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

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Claims (13)

A process for obtaining a high purity rapeseed protein isolate from rapeseed cake comprising the steps i) contacting the rapeseed cake with an anion exchanger; ii) contacting the rapeseed cake with a cation exchanger and / or a hydrophobic adsorbent. The method of claim 1, wherein the rapeseed cake is de-oiled prior to contacting in step i) or ii). The method of claim 1 or 2, wherein the rapeseed cake is subjected to aqueous extraction prior to contacting in step i) or ii). Method according to one of claims 1 to 3, further comprising the step iii) separating the purified rape protein isolate. The method of claim 4, further comprising the step iv) ultrafiltration of the purified rape protein isolate according to step iii). A process according to any one of claims 3 to 5, wherein the aqueous extraction is carried out at a pH of from 3 to 6. A method according to any one of claims 3 to 6, wherein the extraction medium is free of salts. A method according to any one of claims 1 to 7, wherein the rapeseed cake is subjected to an acidic aqueous extraction prior to contacting in step i) or ii). The method of any one of claims 1 to 8, wherein the hydrophobic adsorbent is a hydrophobic polymer resin. A process according to any one of claims 1 to 8, wherein the cation exchanger is a natural layered silicate. The method of claim 10, wherein the natural layered silicate is selected from the group consisting of vermiculite, smectite layered silicate such as bentonite, a mineral of the talc group such as talc, chlorite, cerolite or a mixture of the aforementioned phyllosilicates. A process according to any one of claims 1 to 8, wherein the anion exchanger is an anion exchange resin having quaternary amino groups or an alumina. A rape protein extract produced by a process as defined in any one of claims 1 to 12.

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