DE102011012569A1 - Method of obtaining secondary plant ingredients using a membrane having cation-exchanging groups - Google Patents

Abstract

The present invention relates to a method for isolating positively charged, phenolic secondary plant constituents from plant material using a membrane with cation-exchanging groups and a food additive obtainable by this method.

Description

The present invention relates to a process for isolating phenolic secondary plant ingredients from plant material and to a food supplement obtainable by this process.

Herbal ingredients, especially the so-called secondary plant ingredients, which have no greater caloric value, are increasingly attracting the attention of science and food technology, where they are under the keyword "Functional Food" as dietary supplements or food additives of interest.

Of particular importance here are low molecular weight compounds which have antioxidant properties and thus the ubiquitously formed, so-called "reactive oxygen species", reactive oxygen-containing molecules such as hydroxyl radicals OH ·, the superoxide O ₂ ^- , hydrogen peroxide H ₂ O. ₂ , singlet oxygen, which can react with nitric oxide NO to peroxynitrite ONO ₂ , as well as inactivate hypobromite and hypochlorite. In the context of their antioxidant activity, further positive effects of these low molecular weight compounds, such as the protection of endothelial cells, the suppression of tumor growth and the protection of the cardiovascular system, are discussed intensively. For example, the cardioprotective effect associated with regular red wine consumption is caused, inter alia, by OH-substituted diphenols such as resveratrol. Resveratrol is a substance that, as an antioxidant, presumably reduces the sensitivity to oxidation of low density lipoprotein (LDL) in the blood, inhibits aggregation of platelets, and inhibits endogenous cholesterol biosynthesis by inhibiting squalene monooxygenase.

The technical production or isolation of secondary plant ingredients poses particular challenges to their processing, since the stability of many isolated substances above a certain pH decreases rapidly. In addition, since many plant extracts have a high content of fruit acids and ions, separation via conventional ion exchange chromatography is made difficult or even impossible. In addition, processes known in the prior art often require high volumes of plant extract to be used, require toxicologically hazardous or corrosive substances such as methanol or acetic acid to elute the secondary plant constituents, and are often very tedious. The limited storage stability of many secondary plant ingredients, however, requires rapid processes to be able to convert the sensitive substances as quickly as possible in a stabilizing environment can.

Known in the art, for example, a method of anthocyanin accumulation of blueberry juice concentrate by means of a two meter long, highly hydrophobic Amberlite ^® XAD-column having a diameter of 11 cm. A disadvantage of this method, however, are the required high volumes of juice concentrate to be used, the elution with toxicologically questionable or corrosive substances, such as methanol and acetic acid, and the high time required for the separation of the anthocyanins from the juice concentrate.

DE 10 2009 024 410 A1 discloses a method of isolating phenolic secondary plant ingredients from plant material, such as e.g. Plums, in which the plant ingredients are adsorbed on a microporous membrane with affinity ligands from the group of boronates or metal chelates and subsequently eluted from the membrane.

WO 2008/136741 A1 discloses a process for removing polyphenols from beverages, wherein the beverages are treated with a polymer matrix having ether ligands, preferably polyether ligands having CC multiple bonds, fixed thereto. The polymer matrix can be present as a particulate or membrane-shaped adsorbent. The polyphenols are not isolated as end products and target substances of this method.

WO 2008/097154 A1 discloses a method for desulphurizing beverages.

The polymer matrices based on crosslinked polysaccharides used have a polyether coating, which can be prepared by grafting, for example, polyethylene glycol or diethylene glycol vinyl ether. The polyether functionalization of the matrices allows efficient removal of unwanted polyphenols from beverages. The polyphenols are not isolated as end products and target substances of this method.

US 5,141,611 discloses a process for removing polyphenols from beverages using polyamide membranes or particles having glutaraldehyde / resorcinol-based or glutaraldehyde-based surface modification in combination with melamine, 1,6-hexamethylenediamine or various amino acids. Likewise, the polyphenols are not isolated as end products and target substances of this process.

EP 0 806 474 A1 discloses a method in which chromatography gels based on Sepharose ^®, which cation exchanging ligands have (sulfopropyl or carboxymethyl groups), used for beverage clarification or stabilization. In this process, the polyphenols are simultaneously removed with accompanying proteins as undesirable contaminants from beer. It is further disclosed how the cation exchangers can be regenerated by reuse of water, caustic soda or saline for reuse. The end EP 0 806 474 A1 Known methods do not contain any steps that permit isolation of the polyphenols as target substances, ie, no steps that permit separation of the polyphenols from the accompanying proteins or other contaminants from beer production.

Z. Borneman et al. in Journal of Membrane Science 134 (1997), 191-197 , a process for the removal of polyphenols and of a brown coloration from apple juice associated with the presence of these polyphenols. With respect to the removal of these polyphenols, polyethersulfone membranes (PES) modified with polyvinylpyrrolidone are superior to regenerated cellulose membranes to which no ligands are fixed. "Fouling" phenomena occurring during the process on the PES membrane can be reversed by regenerating the membrane with sodium hydroxide solution. Like the documents cited above, this document does not disclose any method for isolating polyphenols as target substances from fluids.

The object of the present invention is therefore to provide a process for the isolation of secondary plant constituents from plant material, which can be carried out in a simple manner, with little expenditure of time and at low pH values. This method should also avoid the use of toxicologically objectionable solvents as well as the use of large volumes of solvents to isolate the phytochemicals.

This object is achieved by the embodiments of the present invention characterized in the claims. In particular, according to the invention, there is provided a method of isolating phytochemicals from plant material that meets the above requirements. It was surprisingly found that by the use of microporous membranes carrying cation-exchanging groups, an extraction of positively charged secondary plant ingredients from plant material is possible.

• (a) das Bereitstellen eines Extraktes aus pflanzlichem Material, welches positiv geladene, phenolische sekundäre Pflanzeninhaltsstoffe enthält,
• (b) das Kontaktieren des Extraktes mit einer mikroporösen Membran, welche kationenaustauschende Gruppen zur Wechselwirkung mit den positiv geladenen, phenolischen sekundären Pflanzeninhaltsstoffe aufweist, wodurch die positiv geladenen, phenolischen sekundären Pflanzeninhaltsstoffe an der Membran adsorbiert werden, und
• (c) das Eluieren der positiv geladenen, phenolischen sekundären Pflanzeninhaltsstoffe von der Membran, wodurch eine die positiv geladenen, phenolischen sekundären Pflanzeninhaltsstoffe enthaltende Lösung erhalten wird.

Accordingly, an object of the present invention relates to a method for isolating positively charged phenolic plant secondary plant constituents, comprising the steps of:

• (a) providing an extract of plant material containing positively charged phenolic phytochemicals,
• (b) contacting the extract with a microporous membrane having cation exchange groups for interaction with the positively charged phenolic phytochemicals, thereby adsorbing the positively charged phenolic phytochemicals to the membrane, and
• (c) eluting the positively charged phenolic phytochemicals from the membrane to yield a solution containing the positively charged phenolic phytochemicals.

The term "isolate" also includes obtaining a solution containing the positively charged phenolic phytochemicals of the present invention. Optionally, the positively charged phenolic phytochemicals may be further purified from this solution and / or the positively charged phenolic phytochemicals may be obtained as such by removal of the solvent.

The term "vegetable material" in the present invention includes any vegetable material containing positively charged phenolic phytochemicals. In a preferred embodiment, the plant material is selected from the group consisting of fruits, in particular berries, vegetables, legumes, Tubers, onions, beets, tea, cocoa, coffee, wood, flowers, seeds, leaves and cones selected from conifers. In a preferred embodiment, the vegetable material is fruit skin, fruit juice, fruit pulp, fruit pulp, or the entire fruit, which is brought by suitable process steps into a state which permits subsequent efficient extraction of their positively charged secondary ingredients. In a particularly preferred embodiment, an extract of plums is provided in step a) of the method according to the invention.

The term "secondary plant ingredients" according to the invention comprises chemical compounds that are produced by plants neither in the energy metabolism nor anabolic or catabolic metabolism, and preferably the defense against pathogens and herbivores, the protection against environmental influences, such as UV radiation, or attracting of pollinators and seed wideners.

According to the present invention, the secondary plant ingredients are positively charged phenolic compounds. In the context of the present invention, the term "positively charged phenolic compounds" is understood to mean phenols and derivatives of phenols. Such compounds may comprise one or more aromatic rings, wherein the aromatic rings may be fused or may be bridged together by substituted alkyl groups and wherein the derivatives may have further OH groups in addition to the phenolic OH groups. In addition, the phenolic OH groups can be derivatized. For example, the phenolic OH groups and / or the other OH groups may be glycosylated. Furthermore, the secondary plant ingredients may be modified by methylation, acetylation or conversion to their aldehyde or acid function. The molecules of all these compounds have as a common feature a temporary, d. H. at least during the process according to the invention producible, or permanent cationic charge, which is compensated by a corresponding counter anion.

The present invention relates to the isolation of any suitable phenolic phytochemicals. Preferably, the secondary plant ingredients are selected from the group consisting of flavonoids, especially flavones, flavonols, flavanols, flavanones, flavanonols, anthocyanins, proanthocyanins, isoflavonoids and biflavonoids.

Preferred flavonoids are, for example, flavones, flavonols, flavanols, flavanones, flavanonols, proanthocyanins and anthocyanins. Preferred flavones are, for example, apigenin, luteolin, diosmetin, chrysoeriol, nobiletine, apirgenin, acetacetin, galangin, chrysin, tectochrysin, scutellarein, eupatorin, genkwanin, senensetin and their glycosides such as hyperoside, quercitrin and hesperidin. Preferred flavonols are, for example, kaempferol, quercetin, myricetin and their arabinosides, galactosides, glucosides, glycosides, rhamnosides and xylosides. Examples of preferred flavanones are isokuranetin, naringenin, hesperitin, eriodictyol and their glycosides, rutinosyl derivatives and neohesperidosyl derivatives. A preferred flavanonol is, for example, taxifolin. Preferred proanthocyanins are, for example, the glycosides of procyanidins and prodelphinidines, in particular their gallates. Preferred anthocyanins are, for example, the glycosides of pelargonidin, cyanidin, päonidin, delphinidin, petunidin and malvidin. Preferred isoflavonoids are, for example, the isoflavones, especially the soy isoflavones. Preferred isoflavones are, for example, genistein, daidzein, glycetein and their glycosides. Preferred lignans are, for example, the flax lignans, in particular matairesinol and Secoisolariciresinol-diglucosides.

Suitable positively charged phenolic phytochemicals are, for example, in K. Shetty, G. Paliyath, AL Pometto, RE Levin, "Functional Foods and Biotechnology", CRC Press LLC 2005, Taylor & Francis Group, pp. 152-159 , described.

Among the compounds mentioned, the anthocyanins, ie the dyes of various parts of plants, such as fruit skins, berries, or vacuole components, in particular the glycosides of pelargonidin, cyanidin, päonidin, delphinidin, petunidin and malvidin, are preferred according to the invention. Likewise, the proanthocyanins are particularly preferred on the structural basis of cathechin or epicathechin, also in the form of their gallates. The skilled person is aware of the diverse plant sources of anthocyanins and other phytochemicals. A list can be found for example in H.-D. Belitz and W. Grosch, "textbook of food chemistry", 4th edition, H.-Springer-Verlag Berlin, Heidelberg, New York, 1992, pp. 738-754, ISBN 3-540-55449-1 ,

Suitable processes for the preparation of plant extracts from vegetable material are known to the person skilled in the art, and include, for example, the homogenization of plant parts in a disperser or homogenizer.

• (a2) das Homogenisieren des Pflanzenextraktes derart, daß die im Pflanzenextrakt enthaltenen Partikel eine Größe von nicht mehr als 0,5 mm aufweisen.

The plant extract can be pretreated before contacting with the membrane, for example by prefiltration. Such prefiltration is used to remove particles and trub substances from the plant extract. In a preferred embodiment of the present invention, however, the plant extract is not further pretreated after its preparation. In particular, the plant extract can be contacted with the membrane without prefiltration. In this case, the plant extract contains particles and Trubstoffe and may have a turbidity due to these ingredients. The size of the particles is not limited. In a preferred embodiment of the present invention, the particles contained in the plant extract have a size of not more than 0.5 mm. In order to achieve this, in a preferred embodiment, the method according to the invention may further comprise the step after step (a) and before step (b):

• (a2) homogenizing the plant extract such that the particles contained in the plant extract have a size of not more than 0.5 mm.

Suitable methods for homogenizing are known to the person skilled in the art. Likewise, methods for checking the particle size are known to the person skilled in the art, for example by microscopic inspection and computer-assisted image analysis.

The term "microporous membrane" in the context of the present invention refers to membranes having a pore size of 0.1 to 20 .mu.m, preferably 0.5 to 15 .mu.m, and more preferably 1 to 10 .mu.m. The determination of the pore size can be carried out with a so-called "Capillary Flow Porometry Test" (Capillary Flow Porometer 6.0, CAPWIN Software System, Porous Materials Inc.).

The microporous membrane may be in any form suitable for contacting the surfaces of the membrane with the plant extract. For example, the microporous membrane may be integrated into a membrane adsorber module. Suitable membrane adsorber modules are for example made DE 102 36 664 A1 known. Preferably, such a membrane adsorber module is particle-like, ie insensitive to clogging by particles and trub-containing media.

The integration of the microporous membrane into a particle-permeable membrane adsorber module is particularly advantageous when a particle-containing plant extract is used. The particles contained in the plant extract generally do not affect the adsorption of the phenolic secondary plant constituents to be isolated on the membrane. Pre-filtration is not necessary in this case, which simplifies the process according to the invention.

As the microporous membrane, all membranes capable of adsorbing the positively charged phenolic secondary plant ingredients can be used. For this purpose, the microporous membranes have suitable cation-exchanging groups.

As cation-exchanging ligands for positively charged phenolic phytochemicals, any suitable ligand capable of interacting with positively charged molecules can be used in the method of the invention. In a preferred embodiment of the present invention, the cation-exchanging groups are selected from the group of weak or strong cation-exchange ligands. Among the weak cation exchange ligands, particular preference is given to methacrylate, carboxymethyl or orthophosphate groups. Among the strong cation exchange ligands, preferred are sulfoalkyl (more preferably sulfopropyl or sulfoethyl), sulfonate and sulfate groups, as well as heparin and derivatives. Particularly preferred cation exchange ligands are carboxymethyl and sulfopropyl groups. Suitable methods for the derivatization of microporous membranes with cation-exchanging groups are known to the person skilled in the art and are described, for example, in US Pat EP 0 527 992 B1 described.

A microporous membrane suitable for use in the method of the present invention is, for example, a membrane of a cellulose hydrate matrix and pores extending from one major surface to the other major surface of the membrane, the membrane having cation exchange groups for adsorptive separation on its inner and outer surfaces.

As a starting material for such a microporous membrane is a cellulose ester membrane which is brought into contact with at least one solution under conditions which lead to a swelling of the cellulose ester matrix and on the other hand at the same time, d. H. in situ, hydrolysis (saponification) of the ester groups to hydroxyl groups, whereby a cellulose hydrate membrane is formed.

Cellulose ester membranes may be composed of cellulose monoacetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose butyrate and cellulose acetobutyrate or other suitable cellulose esters or cellulose nitrate, methylcellulose or ethylcellulose, as well as mixtures thereof.

Subsequent to the saponification, the resulting cellulose hydrate matrix is preferably crosslinked by reacting the hydroxyl groups with one or more at least bifunctional reagents. Thereafter, functional groups (cation exchange ligands) are introduced into the crosslinked matrix to enable adsorptive material separation.

In a further step, functional groups, for. B. to the hydroxyl groups, the crosslinked membrane. Suitable methods for binding functional groups are known to the person skilled in the art.

Preferably, functional groups are bonded to the cellulose membrane via epoxide groups or aldehyde groups. The introduction of the epoxide groups can take place already in the crosslinking step or subsequently.

Membranes of the company. Sartorius Stedim Biotech GmbH are particularly preferred in the present process "Sartobind® ^®" is used.

The step (b) of contacting the plant extract with the microporous membrane comprises all forms of contacting the plant extract with the microporous membrane. The contacting is done in such a way that the positively charged, phenolic secondary plant ingredients contact the cation-exchanging groups of the membrane to be adsorbed to them. Step (b) may be accomplished by, for example, passing the plant extract tangentially along at least one membrane surface. However, it is also possible that when using a membrane with pores extending from one major surface to the other major surface of the membrane, the plant extract is passed through the membrane, which is preferably convective permeable.

For the purposes of the present invention, the term "adsorption" means all possibilities of reversibly binding positively charged, phenolic secondary plant constituents to the cation exchanger ligands. This reversible bond can be chemical and / or physical in nature.

According to the present invention, in step (c), the elution of the positively charged phenolic secondary ingredients from the membrane occurs. In this way, a solution containing the phenolic secondary plant ingredients is obtained. The term "elution" in the context of the present invention, the desorption and the associated rinsing steps are summarized. The liquid used for elution is the "eluent".

As the eluent, in the process of the present invention, any solvent having a higher affinity for the cation-exchanging groups used than the positively-charged phenolic phytochemical ingredient to be isolated can be used. In a preferred embodiment of the process according to the invention, the eluent used in step c) is water or from the group of aqueous solutions of alkali metal or alkaline earth metal salts of inorganic or organic acids, preferably alkali metal and alkaline earth metal salts of mineral acids (preferably hydrochloric, sulfuric, Phosphoric or nitric acid). Further preferred salts are the alkali metal or alkaline earth metal salts of the halogens, particularly preferably NaCl.

Among the organic acids, the salts of di- and tricarboxylic acids are preferred, particularly preferred are citric and lactic acids.

Furthermore, the additional admixture of C1-C4 alcohols, such as ethanol or butanol, to eluent has proven to be advantageous.

In this way, the subsequent step of elution can be simplified. Preferred aqueous media for this washing step are those which do not elute the phenolic phytochemicals. In particular, water is preferred as the aqueous medium.

In a particularly preferred embodiment of the process according to the invention, the cation-exchanging groups are sulfonate groups and the microporous membrane consists of cellulose hydrate, wherein the eluent used in step c) is a solution of sodium chloride and potassium phosphate in a mixture of water, ethanol and 1- or isobutanol ,

• (b2) das Entfernen von auf der Membran verbliebenen Pflanzenresten aus dem Extrakt durch Waschen mit einem wäßrigen Medium.

In a further preferred embodiment, the method according to the invention further comprises the step after step (b) and before step (c):

• (b2) the removal of remaining on the membrane plant residues from the extract by washing with an aqueous medium.

The present invention further relates to a food additive which is obtainable by the process according to the invention.

The present invention will be further illustrated by the following non-limiting examples and figures.

Show

1 : the course of the adsorption and elution of anthocyanins when using a membrane overflowable on both sides
and

2 : the adsorption of anthocyanins on a strongly acidic membrane adsorber by bilateral overflow with circular delivery.

Example 1: Production of a plant extract

The following describes the preparation of an anthocyanin-containing plant extract.

The plant extract was prepared from commercially available prunes (Prunus domestica) (Unipack Fruits, South Africa). The fruits had a diameter of 7 to 8 cm, a fresh weight of about 150 to 200 g and were characterized by a very dark, purple-colored fruit skin (shell). The shell of a fruit has been completely removed and in 300 ml of a solution of 0.02 mol / l potassium orthophosphate, which was adjusted to pH 2.0 with phosphoric acid (here and hereinafter referred to as "buffer"), with an "Ultra-Turrax ^® T25 "disperser (Janke and Kunkel, Staufen im Breisgau) homogenized at an idling speed of 8000 rpm for 5 min. The result was a turbid suspension, with particles in a size range of a maximum of 0.5 mm. The particle size was determined with a transmitted light microscope "Axiovert 40" (Zeiss) with attached camera and the image analysis software "Axiovision" (Zeiss). No size distribution was determined, but only ensured that the particles do not exceed the size of 0.5 mm. The extract was passed through a pleated filter and made up to 500 ml with buffer and mixed thoroughly. The result was a cloudy, black-red liquid, hereinafter called "crude extract", which was used in the following examples.

The extract was diluted 1:50 with the aforementioned buffer, and an adsorption spectrum in the wavelength range of 240-650 nm was recorded with a spectrophotometer of the type Spekord from Jenoptik, Jena, Germany. A maximum at 280 nm and 513 nm (broad) was found. The further determination of the concentration of the plant dye was carried out at 513 nm.

To determine the content of the anthocyanin in the individual fractions examined, the extinctions of the respective fraction can be converted into mg / l. A solution of 16 mg / l anthocyanin mixture at pH 2.5 produces an absorbance of 0.45 at the wavelength 520 nm ( L. Jurd, Food Sci. 29, 1964, p. 16-19 ).

Example 2 Production of a Two-Sided, Overflowable Module with Adsorptive Membrane for the Extraction of Anthocyanins from Plant Material

A device for receiving an adsorption membrane was created. Two rectangular plates (upper and lower part) with the dimensions 12 × 6 cm were at the end faces, ie at the sides facing each other in parallel arrangement of the plates, with one, to the narrower side of the plates in parallel in one Distance of 1 cm running inlet and outlet channel provided with a depth of 3 mm. The inlet or outlet channel was connected in each case via a feed bore with a hose nozzle.

From a 1 mm thick sheet of soft silicone, two frames were cut with 10 × 5 cm inside and 12 × 6 cm outside dimensions. Recesses were made to accommodate threaded rods, allowing both panels to be joined together to form a 2mm high channel between them. In each case, a rectangular piece measuring 10 × 5 cm was cut out of the membranes to be examined and inserted into the channel between two woven fabrics of the type XNP 4410 from Convet Plastics bv, Genk, Belgium, with the same dimension.

The result was a device with two parallel channels on opposite sides of a microporous membrane, which were overflowed by a suitable pump on both sides simultaneously with the fluid to be treated.

The elution solution consisted of a mixture of 10% by volume of EtOH, 10% by volume of isobutanol, filled up with 80% by volume of buffer to a final volume of 1 l, in which 1 mol / l of NaCl was dissolved.

There was a strongly acidic cation exchange membrane of the type ^® Sartobind S from Sartorius Stedim Biotech, designation 18842, lot 1000353. D: 22M (membrane made of reinforced, stabilized cellulose with sulfonic acid groups) with the dimensions 12 x 6 cm with buffer wetted and incorporated. Experiments were carried out with the 1:50 diluted extract in different operating modes and overflow velocities. In each experiment, 50 ml of starting solution were submitted.

The results are summarized in Tables 1 and 2.

It means:
E _tot : the total extinction determined in the respective fractions at 513 nm, expressed in extents units "EE".
Column 1, no .: running test number with the same membrane in the device,
Column 2, type: The abbreviation "lin" means that the solution was conveyed once through the adsorber, the abbreviation "zirk" means that the volume was circulated for 10 minutes through the adsorber.

The unit ml / min indicates the flow rate through the adsorber.

AL: means the number of extinction units "EE" in the template at the beginning of the experiment indicated by the respective experiment number.

DL: means the number of extinction units "EE" in the template, or in linear mode ("lin"), in the receiver at the end of the experiment.

Geb: means the absolute difference of the two values "AL" and "DL", "Geb%" the corresponding indication of this difference referred to "AL" in percent.

EL: means the total amount elutable by the adsorber, which can be determined from the extinction at 513 nm, "Rec%" is the proportion of "EL" relative to "Geb" in percent.

An oblique arrow pointing from the upper right to the lower left in the table from a higher line to a lower line means that the depleted solution was recharged onto the adsorber after elution of the adsorber. Table 1: overview of the experiments with the Sartobind ^® S-type membrane of Example 2 under different operating conditions

After performing the five experiments in Table 1, a fresh membrane of the same type was incorporated into the device. Table 2: Further experiments with the extract and a fresh membrane

The gradual increase in pump performance in Runs 12 and 13 did not result in any acceleration in the decrease in concentration. The speed was increased from 6 to 75 ml / min or 12 to 72 ml / min in ten increments every three minutes.

1 shows the decrease in extinction units (EE) for AL or EL of the starting solution on repeated application of the anthocyanene-depleted solution to the same adsorber, followed by elution according to the results for experiments Nos. 8 to 11 according to Table 2.

The diamond-shaped symbols of row 1 correspond to the quantity of anthocyanins offered in each case, the symbols in square form of row 2 indicate the respectively eluted material. For each run, about 15 extinction units (RE) are depleted. This amount is found only in the first elution (Experiment 8), in the following three elutions less anthocyanin than the theoretical 15 EE is found. This indicates a non-elutable amount of anthocyanin remaining on the adsorber under these conditions.

2 shows the adsorption of anthocyanins on a strongly acidic membrane adsorber by bilateral overflow with circular promotion. The data are taken from Experiment No. 6 of Table 2. E 513 denotes the extinction of the solution studied at 513 nm as a measure of the anthocyanin content, and the x-axis indicates the circulation time in minutes.

The symbols in diamond form represent the concentration of anthocyanin in the template measured over the circulation time. The recirculation curve is exponentially decaying and approaching a limit corresponding to the capacity of the adsorbent for anthocyanins under the chosen experimental conditions.

Example 3 Production of a particle-permeable membrane adsorber module with cation-exchanging groups

Analogous to DE 102 36 664 A1 a particle-permeable membrane adsorber module was produced. To this was a rectangular piece that described in Example 2 strongly acidic cation exchange membrane Sartobind ^® S of Sartorius Stedim Biotech GmbH with a width of 12 cm and a length of 100 cm together with a fabric of the same size of the type XNP 4410 (Convet Plastics bv , Genk, Belgium) was wound onto a 1 cm diameter plastic rod and this package centered in a plastic tube 14 cm in length and 47 mm in diameter so that the outermost membrane coil of the inner tube wall was directly and liquid-tight. This resulted in a particle-permeable channel formed by the tissue and delimited on both sides by the wound-up membrane. Round blanks with a diameter of 47 mm were punched from the same fabric. Eight of these blanks were placed on the packing on the inflow and outflow side of the coil. The ends of the pipes were closed with centrally sized rubber stoppers of suitable size provided with hose nipples. The tube was inserted vertically into a holder and a hose pump with a silicone hose connected to the input olive. A suitable silicone hose was also connected to the drain hose olive. Both tubes were placed in a template vessel. The dead volume of the entire system from the adsorber module and the hoses was 200 ml. A total of three identical modules were created, which are referred to below as M1, M2 and M3.

Example 4: Adsorption of anthocyanins on a strongly acidic membrane adsorber M1 by controlled linear flow of M1

The receiver was filled with 500 ml of buffer and circulated through the adsorber module for 5 minutes at a flow rate of 300 ml / min. The buffer was discarded, 300 ml of fresh buffer filled and the process repeated.

50 ml of the crude extract was added to 450 ml of buffer and mixed by means of a magnetic stirrer. This slightly cloudy suspension was passed through the vertical adsorber from bottom to top by means of the peristaltic pump at a flow rate of 50 ml / min. A sample was taken every 50 ml volume at the outlet and the absorbance E _{513 of} the suspension in a spectrophotometer at 513 against the buffer as a blank (see Samples 1-9). The results obtained are shown in Table 3:
E _Frak represents the measured value in the respective fraction and EE the total extinction fraction in the fraction as product of E ₅₁₃ and the volume of the respective fraction, ie 50 ml. E ₅₁₃ (%) results from the extinction value of the respective sample E _Frak divided by the initial value E _Frak (AL) = 0.85 and from the conversion into the percentage value. The bound amount of anthocyanins results from the subtraction of the respective value for "EE (total)" of 42.5 (corresponding to 100%). "E ₅₁₃ cumulative" represents the total amount of anthocyanins bound during the experiment. Table 3: sample E _Frak EE (total) in the fraction E ₅₁₃ (%) of the starting solution Tied total amount of anthocyanins at the time of measurement AL 0.85 - 100 0 V / ml in the fraction E ₅₁₃ cumulative 1 50 0.19 9.5 22.4 33 33 2 100 0.37 18.5 43.5 24 57 3 150 0.42 21 49.4 21.5 78.5 4 200 0.44 22 51.8 20.5 99 5 250 0.45 22.5 52.9 20 119 6 300 0.47 23.5 55.3 19 138 7 350 0.48 24 56.5 18.5 156.5 8th 400 0.48 24 56.5 18.5 175 9 450 0.50 25 58.8 17.5 192.5 V / ml denotes the cumulative sample volume at the end of the experiment with the number given in Table 3.
EE: extinction units

45.3% (= (192.5 EE / 425 EE) × 100%) of the amount of anthocyanin (425 EU) used in the experiment, determined by way of the extinction units (EE), are bound.

The data show that the binding of the anthocyanins to the membrane adsorber M1 progressively slows down in the course of the experiment and that the adsorbed amount tends to a limit value.

The adsorber was washed with buffer until the absorbance value of the liquid in the effluent had dropped to virtually zero. The bound anthocyanins were eluted from the adsorbent with 200 ml of eluant as indicated above by cycling and subsequent drainage.

A mass balance was created. Since the specific extinction coefficient (s) of the anthocyanins / anthocyanins present was not known, the total textinctions were calculated, ie with E _tot (starting solution) = 0.85 = 500 ml = 425 and with E ₂ = E _tot (respective fraction) / E _tot (starting solution) × 100%.

Table 4 shows the mass balance of Example 4 with linear overflow: Table 4: fraction ml E ₅₁₃ E _dead E ₂ (%) starting solution 500 0.85 425 100 pass 475 0.4 190 45 Bound - - 235 55 To wash 300 0.23 69 16 Rest after washing - - 166 39 eluate 200 0.61 122 29 The amount of anthocyanide eluted by the adsorber is thus 122/166 = 73%.

Example 5: Adsorption of anthocyanins on a strongly acidic membrane adsorber by controlled cyclic overflow of the membrane adsorber M1

50 ml of the crude extract was added to 450 ml of buffer and mixed by means of a magnetic stirrer, which was in operation during the entire experiment (speed 500 revolutions / min). The original Erlenmeyer flask had a nominal volume of 500 ml, the magnetic stir bar was 3 cm long.

This slightly cloudy suspension was passed by means of a peristaltic pump at a flow rate of 50 ml / min from bottom to top through the vertical adsorber, while the exiting liquid at the outlet was returned to the template and mixed with their contents continuously. At the times indicated in Table 5A, samples were taken from the original and the extinction of the suspension in a spectrophotometer at 513 nm against the buffer was determined as a blank value. Tables 5A and 5B show the values obtained, wherein E ₁ and the bound amount of anthocyanins are analogously defined as for Table 3: TABLE 5A t (min) E ₅₁₃ E ₁ in% of the starting solution Bound amount of anthocyanins in the effluent at the time of measurement AL (0) 0.82 100 0 5 0.66 80 20 10 0.54 66 34 16 0.45 55 45 23 0.39 48 52 29 0.35 43 57 36 0.32 39 61 41 0.31 38 62 47 0.30 37 63 53 0.29 35 65

The adsorbent was drained by draining the depleted crude extract, washed with buffer and then eluted with the eluent. Table 5B: Template content at the end of the Volume / ml E ₅₁₃ - Try it 500 0.29 - washing 250 0.09 - the elution 210 0.9

A mass balance was prepared analogously to Example 4 (Table 6). Table 6: Mass Balance of Example 5: fraction V / ml E ₅₁₃ E _dead E ₂ (%) 1) starting solution 500 0.82 410 100 2) pool 500 0.29 145 35 3) washing 250 0.09 22.5 5.5 4) Bound - - 265 - 5) Remainder after washing - - 242.5 100 6) eluate 210 0.9 189 78 E ₂ = E _tot (respective fraction) / E _tot (starting solution) × 100% for entries 1) to 4) or E ₂ = E _tot (eluate) / E _tot (remainder after washing) × 100%
The amount eluted by the adsorber is thus 189 / 242.5 = 78%

Example 6: Serial Connection of the Membrane Adsorber Modules M1, M2 and M3

Further experiments were carried out in a similar manner as in Examples 4 and 5, using different amounts of extract and a different number of modules connected in series, so that the liquid emerging from the first module M1 was passed through the second downstream module M2 and the liquid leaving the second module was passed through the third downstream module M3.

In this case, a module M1 or three modules M1, M2 and M3 were operated as follows: "Linear" means that the specified volume was passed only once through an adsorber or the three adsorbers connected in series, "circular" means that the Liquid was circulated through the adsorber. "Anz Mod" means the number of modules used (M1, M2 and M3 or only M1)

A summary of the experiments carried out is shown in Table 7. Table 7: Results of Experiments Nos. 1 to 5 from Example 6 No. kind Num. Mod V / ml E _dead (513) bound * elution ** 1 Linear 1 500 166 73 2 circularly 1 500 242 78 3 Linear 3 1500 495 65 4 Linear 3 1000 331 94 5 circularly 3 1000 400 76 * indicated in extinction units EE
** Extinction value of the eluate indicated in EE

With the two methods of linear and circular extraction of the extract through the adsorber anthocyanins are extracted and can be eluted. Regardless of the mode of operation, increasing amounts of anthocyanins were bound with increasing supply of anthocyanin-containing crude extract. The largest capacity was achieved by loading 1.5 l of diluted crude extract in linear mode (test No. 3).

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

• DE 102009024410 A1 [0006]
• WO 2008/136741 A1 [0007]
• WO 2008/097154 A1 [0008]
• US 5141611 [0010]
• EP 0806474 A1 [0011, 0011]
• DE 10236664 A1 [0028, 0076]
• EP 0527992 B1 [0031]

Cited non-patent literature

• Z. Borneman et al. in Journal of Membrane Science 134 (1997), 191-197 [0012]
• K. Shetty, G. Paliyath, AL Pometto, RE Levin, "Functional Foods and Biotechnology", CRC Press LLC 2005, Taylor & Francis Group, pp. 152-159 [0022]
• H.-D. Belitz and W. Grosch, "textbook of food chemistry", 4th edition, H.-Springer-Verlag Berlin, Heidelberg, New York, 1992, pp. 738-754, ISBN 3-540-55449-1 [0023]
• L. Jurd, Food Sci. 29, 1964, pp. 16-19 [0056]

Claims (12)

A method for isolating positively charged phenolic secondary plant ingredients from plant material comprising the steps of: (a) providing an extract of plant material containing positively charged phenolic phytochemicals, (b) contacting the extract with a microporous membrane having cation exchange groups for interaction with the positively charged phenolic phytochemicals, thereby adsorbing the positively charged phenolic phytochemicals to the membrane, and (c) eluting the positively charged phenolic phytochemicals from the membrane to yield a solution containing the positively charged phenolic phytochemicals. The method of claim 1, wherein the plant material is selected from the group consisting of fruits, especially berries, vegetables, legumes, tubers, onions, beets, tea, cocoa, coffee, wood, flowers, seeds, leaves and cones of coniferous trees , The method of claim 1 or 2, wherein the positively charged phenolic phytochemicals are selected from the group consisting of flavonoids, especially flavones, flavonols, flavanones, flavanonols, anthocyanins, proanthocyanins, isoflavonoids and biflavonoids. The method of claim 3 wherein the positively charged phenolic phytochemicals are selected from the group consisting of flavonols selected from kaempferol, quercetin, myricetin, and their arabinosides, galactosides, glucosides, glycosides, rhamnosides and xylosides; Flavanones selected from isosakuranetin, naringenin, hesperitin, eriodictyol and their glycosides, rutinosyl derivatives and neohesperidosyl derivatives; Anthocyanins selected from the glycosides of pelargonidin, cyanidin, päonidin, delphinidin, petunidin and malvidin; Proanthocyanins selected from the glycosides of Procyanidine and Prodelphinidine; Isoflavones selected from genistein, daidzein, glycetein, and their glycosides; are selected. A method according to any one of claims 1 to 4, wherein the cation-exchanging groups are selected from the group of weak or strong cation-ion exchange ligands. The method of claim 5, wherein the weak cation exchange ligand is selected from the group of methacrylate, carboxymethyl or orthophosphate groups. The method of claim 5, wherein the strong cation exchange ligand is selected from the group consisting of sulfoalkyl, sulfonate, and sulfate groups and from the group of heparin and its derivatives. A process according to any one of claims 1 to 7, wherein the eluent used for elution in step (c) is water or selected from the group of aqueous solutions of alkali metal or alkaline earth metal salts of organic or inorganic acids. A process according to any one of claims 1 to 8, wherein the cation-exchanging groups are sulphonate groups and the microporous membrane consists of cellulose hydrate and uses as eluent in step c) a solution of sodium chloride and potassium phosphate in a mixture of water, ethanol and 1- or iso-butanol becomes. Method according to one of claims 1 to 9, after step (a) and before step (b) further comprising the step: (a2) homogenizing the plant extract such that the particles contained in the plant extract have a size of not more than 0.5 mm. Method according to one of claims 1 to 10, after step (b) and before step (c) further comprising the step: (b2) the removal of remaining on the membrane plant residues from the plant extract by washing with an aqueous medium. A food additive obtainable by the process according to any one of claims 1 to 11.

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