METHOD FOR LINKING BIOPOLYMERS

Description

[0001] The present invention relates to a method for crosslinking biopolymers with sea buckthorn extracts and to the use of sea buckthorn extracts as a non-toxic agent for crosslinking biopolymers such as proteins, peptides, or polysaccharides. Such biopolymers are used, among other things, in the food, feed, pharmaceutical, and medical industries, as well as in other industrial applications.

[0002] Cross-linked biopolymers, such as proteins or polysaccharides, are widespread in nature and find diverse applications in the production of food, animal feed, enzymes, packaging materials, and in biomedical and industrial applications. Furthermore, the cross-linking of biopolymers is used in the tanning of hides and skins, as well as in textile finishing.

[0003] Various methods are used to immobilize or crosslink biopolymers, employing, for example, aldehydes or dialdehydes such as formaldehyde or glutaraldehyde, acetic acid, or enzymes, or crosslinking via metal salt complexes as in the tanning of hides. Natural components for crosslinking biopolymers include iridoids and seicoiridoids from olive leaves (EP 1 489 135 A1).

[0004] EP 1 489 135 A1 describes in detail the disadvantages of the prior art. In particular, the toxicological properties of substances such as formaldehyde or glutaraldehyde limit their use in the food, feed, cosmetics, and medical sectors. The use of heavy metals in leather tanning has significant disadvantages from a human and environmental toxicology perspective and necessitates complex wastewater treatment and waste disposal processes.

[0005] There is a growing need for natural, especially plant-based, alternatives for protein cross-linking that can be produced in sufficient quantities and at affordable costs.

[0006] CN 1 696 1 83 A concerns the use of condensed polyphenols, such as catechins and / or tannins formed therefrom, as starch crosslinking agents for the production of crosslinked starch and the natural crosslinking agent obtained from plant polyphenol for the production of crosslinked starch, as well as the pure production of plant polyphenol as a crosslinking agent.

[0007] US 2013 / 108733 A1 describes a chewing gum product based on a cross-linked gelatin matrix and a filler consisting of erythritol, mannitol, or mixtures thereof. The cross-linked gelatin matrix gum base includes a cross-linking agent that is a polyphenol or a combination of polyphenols. Green tea extract, which contains more than 90% polyphenols, primarily catechins, is given as an example of a polyphenol-containing plant extract. KR 2013 0000782 A describes a protein-polysaccharide hybrid membrane in which a mixture of a lipophilic drug and a solid lipid is cross-linked with tannic acid, the membrane surrounding the drug carrier. Membrane cross-linking is achieved by natural polyphenols, such as tannic acid, using 0.3% by weight of isolated tannic acid, specifically gallotannins with a purity of over 95%.

[0008] Sea buckthorn grows on more than 2 million hectares worldwide. The uses of the berries are well documented. Harvesting the berries yields considerable quantities of wood, leaves, and bark. These raw materials are currently only used to a limited extent for commercial purposes, but they also have the potential to generate additional value for farmers. DE 694 20 522 T2 discloses a nucleating agent for ice cream made from an extract of sea buckthorn berries or leaves, as well as a process for producing the nucleating agent. The nucleating agent preferably consists of a mixture of amino acids and lipids. The process for producing the nucleating agent includes extracting the berries or leaves of sea buckthorn. DE 697 09 316 T2 describes a protein-rich, dietetic ice cream containing up to 0.1% by mass of oil or oil extract of Happophae rhamnoides L.. KR 2008 0025479 A also reveals the production of ice cream with a sea buckthorn extract.

[0009] CN 103 181 414 A describes a yogurt containing natural flavonoid compounds, in particular a sea buckthorn extract with a flavonoid content of approximately 50%, and its production, wherein the color and bitter taste of the flavonoids are reduced by encapsulation. The encapsulation of the flavonoid composition is achieved using cyclodextrin to prevent the addition of the flavonoids from negatively affecting the color, emulsion structure, and taste of the yogurt.

[0010] CN 104 256 375 A discloses an instant noodle dish containing sea buckthorn and a method for its production.

[0011] CN 104 365 803 A discloses the use of sea buckthorn berries for the production of an extract, in particular a polysaccharide-concentrated sea buckthorn berry extract, which is used for baking cakes.

[0012] KR 2013 0031590 A discloses functional foods, pharmaceutical compositions and cosmetic compositions for preventing skin aging and reducing wrinkles containing sea buckthorn fruit extracts, collagen, blueberry extract, hyaluronic acid and honey.

[0013] RU 2 028 796 C1 describes the addition of sea buckthorn extracts to cosmetic products for skin softening, cleansing and anti-inflammatory effects.

[0014] CN 101 327 232 A discloses the production and purification of a sea buckthorn flavonoid extract. The extract is described for medicinal applications and healthy nutrition, including for treating coughs, raising blood pressure, relieving congestion, preventing clots, and lowering blood pressure.

[0015] Tulsawani et al. disclose the treatment of cells, particularly hepatocytes or liver epithelial cells, with an aqueous sea buckthorn extract to prevent hypoxia-induced cell death. The sea buckthorn extract contains a total phenol content of approximately 120 mg / g extract, which corresponds to about 12% (Tulsawani R, Gupta R, Misra K (2013) Efficacy of aqueous extract of Hippophae rhamnoides and its bio-active flavonoids against hypoxia-induced cell death. Indian J Pharmacol. 45(3): 258-263). Tulsawani et al. describe the cause of cell death as the formation of reactive oxygen species (ROS), which can lead to the release of the enzymes LDH, ALT, and AST. Preventing cell death by blocking ROS formation with the aqueous sea buckthorn leaf extract prevents the release of these enzymes.

[0016] CN 102 058 631 A describes the production of a sea buckthorn extract for use in the treatment of cardiovascular diseases, specifying an optimal flavonoid content of 10 to 200 mg per unit (tablet, capsule, etc.). The formulation further describes the mixing of a powdered sea buckthorn extract with 50 g of oil, 2 g of gelatin, and 8 g of glycerin.

[0017] The object of the invention is to provide a method for crosslinking biopolymers using plant extracts. A further object of the invention is to provide natural sea buckthorn extracts for crosslinking biopolymers.

[0018] The problem is solved by a process for crosslinking biopolymers, in which the biopolymers are brought into contact with sea buckthorn extracts to crosslink the aforementioned biopolymers, wherein the sea buckthorn extracts are obtained from sea buckthorn plant residues or unused plant parts, wood, leaves, bark and / or shoots resulting from sea buckthorn fruit processing and contain polyphenols with a content of 10-95 wt% based on the dry mass in the extract, wherein the polyphenols originate from the group of tannins, flavonoids and catechins, wherein the crosslinking of the biopolymers takes place in an aqueous environment, wherein the biopolymers are selected from proteins, peptides and polysaccharides, wherein the crosslinking of the biopolymers is achieved by adding 0.1 wt% to 100 wt% of the sea buckthorn extract based on the biopolymer.

[0019] Sea buckthorn extracts are derived from the aerial parts of the sea buckthorn plant or from residues of sea buckthorn fruit processing. Residues from sea buckthorn fruit processing include leftover sea buckthorn plant parts or unused plant components generated during fruit processing. Sea buckthorn berries are reserved for fruit processing. Small amounts of berries present during the extraction of the aerial parts of the sea buckthorn plant do not negatively affect the cross-linking properties of the extract.

[0020] According to the invention, residual components of sea buckthorn plants or unused plant components arising during the processing of sea buckthorn fruit are wood, leaves, bark and / or shoots.

[0021] In one embodiment, the sea buckthorn extracts are selected from a sea buckthorn variety chosen from Ascola, Hergo, Leikora, Pollmix 1 and / or Pollmix 4, preferably from the sea buckthorn variety Hergo.

[0022] Sea buckthorn extracts contain polyphenols with a content of 10–95% by weight (based on the dry mass of the extract), with the polyphenols belonging to the groups of tannins, flavonoids, and catechins. The polyphenol content can be determined using the modified Folin-Ciocalteu method (Singleton and Rossi (1965) Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vitic. 16: 144–158).

[0023] In a preferred embodiment, the sea buckthorn extracts contain polyphenols with a content of 25 wt.% to 50 wt.% (based on the dry mass of the sea buckthorn extract).

[0024] Advantageously, the polyphenols, especially the hydrolyzable and condensed tannins, lead to the cross-linking of the biopolymers.

[0025] In one embodiment, the sea buckthorn extracts consist exclusively of natural ingredients, whereby natural ingredients are understood to be naturally occurring, plant-based substances without modification.

[0026] The sea buckthorn extracts for crosslinking biopolymers are obtained by extracting sea buckthorn plant material in dried or fresh form with water, an organic solvent or a mixture of water and organic solvents as the extraction agent and / or solvent.

[0027] In one embodiment, the process for obtaining sea buckthorn extracts for crosslinking biopolymers is carried out by extracting sea buckthorn plant material in dried or fresh form with water, an organic solvent, or a mixture of water and organic solvents as the extraction agent and / or solvent. In another embodiment, the process for obtaining sea buckthorn extracts for crosslinking biopolymers is carried out by extracting sea buckthorn plant material in frozen form with water, an organic solvent, or a mixture of water and organic solvents as the extraction agent and / or solvent.

[0028] The extracts are obtained from the above-ground parts of the sea buckthorn plant, such as leaves, wood, bark, or shoots, or from residues of sea buckthorn fruit processing. The material can be processed fresh, but dried is preferable. Residues from sea buckthorn fruit processing include leftover sea buckthorn plant components or unused plant parts generated during fruit processing. Sea buckthorn berries are reserved for fruit processing. Small amounts of berries during the extraction of the above-ground parts of the sea buckthorn plant do not negatively affect the cross-linking properties of the extract.

[0029] Water, polar organic solvents, or mixtures of polar organic solvents with water can be used as extraction agents. According to one embodiment of the present invention, polar organic solvents are alcohols or ketones. Alcohols with 1 to 4 carbon atoms or ketones with 1 to 4 carbon atoms are particularly preferred. Examples of organic solvents are methanol, ethanol, 1-propanol, isopropanol, 1-butanol, 2-butanol, ethylene glycol, acetone, butanone, or any mixtures of these solvents.

[0030] Preferably, the extraction agent or solvent is a mixture of an aqueous solution and at least one polar organic solvent. The polar organic solvent typically comprises 10 to 95 vol.% in the mixture. A proportion of 10 to 90 vol.% is preferred, more preferably 20 to 80 vol.%, and most preferably 30 to 70 vol.%. Preferred polar organic solvents in the mixture correspond to those listed above. A water / alcohol or a water / ketone mixture with a solvent content of 20 to 80 vol.% is particularly preferred.

[0031] The extraction solvent may contain additives for pH adjustment or to reduce surface tension. Extraction takes place at temperatures between 20 °C and 95 °C and at pH values ​​from 2 to 11.

[0032] According to one embodiment of the invention, additives, in particular acids or alkalis and / or anionic, non-ionic or amphoteric surfactants, can be added to the extraction agent, in particular the water.

[0033] Extraction can be carried out continuously, discontinuously, in a single stage or in multiple stages under normal or positive pressure using generally known methods such as maceration, percolation or digestion.

[0034] The processing of the crude extracts for polyphenol enrichment can be carried out using membrane processes or adsorption. Furthermore, the processing of the crude extracts for polyphenol enrichment can be carried out using liquid-liquid extraction.

[0035] In one embodiment, the polyphenols are enriched using an adsorbent resin.

[0036] After the process is carried out, the extracts are subjected to enzymatic or acid-catalytic treatment.

[0037] Improved activation of the extracts occurs through acid-catalytic or enzymatic cleavage of the bound monosaccharides.

[0038] In one embodiment of the process, the bound monosaccharides are cleaved by acid-catalytic or enzymatic treatment of the extracts, yielding aglycones. Aglycones are defined as organic compounds that represent the non-monosaccharide component of a glycoside. For example, isorhamnetin can be formed from isorhamnetin-3-O-glycoside and / or quercetin from quercetin-3-O-rutinoside.

[0039] Following the execution of the process, enzymatic treatment is carried out using glucosidases. Glucosidases are a group of enzymes that catalyze the hydrolysis of glycosidically bound glucose.

[0040] In a further embodiment of the process, the extracts are subjected to fermentation with microorganisms. In one embodiment, the fermentation is carried out with microorganisms that produce glucosidases.

[0041] The extracts can be used in solution or as a powder. Drying of the extracts can be carried out using known drying methods.

[0042] Biopolymers, as defined in the invention, are polymers synthesized within the cells of living organisms. According to the invention, biopolymers are selected from proteins, peptides, and polysaccharides; preferably, the biopolymers are proteins.

[0043] In one embodiment, proteins may be selected from milk proteins, preferably casein or β-lactoglobulin; and / or cereal proteins, preferably gliadins, glutenin, albumins or globulins; collagen or gelatin.

[0044] Advantageously, with the inventive method, no modification of the biopolymers is necessary before crosslinking by bringing the biopolymers into contact with the sea buckthorn extracts.

[0045] According to the invention, the crosslinking of the biopolymers takes place in an aqueous environment. An environment is understood to be the chemical surroundings in which the biopolymer is located and the crosslinking of the biopolymer occurs. In one embodiment of the process, the aqueous environment comprises water or an aqueous buffer solution.

[0046] In another embodiment of the process, the cross-linking of the biopolymers is achieved by adding the sea buckthorn extract to the aqueous environment.

[0047] According to the invention, the crosslinking of the biopolymers is carried out by adding 0.1 wt.% to 100 wt.% of the sea buckthorn extract based on the biopolymer, preferably 0.5 wt.% to 50 wt.% of the sea buckthorn extract based on the biopolymer.

[0048] In another embodiment of the process, the crosslinking of the biopolymers takes place over a period of 1 h to 72 h.

[0049] The invention also relates to the use of the sea buckthorn extracts according to the invention, alone or in mixtures, for crosslinking biopolymers in the fields of food, animal feed, cosmetics and pharmaceuticals, as well as medical products or for industrial application, for tanning hides and skins, as well as for textile finishing, wherein the sea buckthorn extracts were obtained from sea buckthorn plant residues or unused plant parts, wood, leaves, bark and / or shoots resulting from the processing of sea buckthorn fruit and contain polyphenols with a content of 10-95 wt% based on the dry mass in the extract, wherein the polyphenols originate from the group consisting of tannins, flavonoids and catechins, and wherein the biopolymers are selected from proteins, peptides and polysaccharides.

[0050] According to the invention, extracts from sea buckthorn are used for crosslinking biopolymers in the fields of food, feed, cosmetics and pharmaceuticals, as well as medical products or in industrial applications, for tanning hides and skins, and for textile finishing.

[0051] According to the invention, extracts from above-ground components of the sea buckthorn plant or from residues of sea buckthorn fruit processing are used as sea buckthorn extracts.

[0052] In one embodiment, extracts from sea buckthorn are used to crosslink biopolymers by extracting sea buckthorn plant material in dried or fresh form with water, an organic solvent or a mixture of water and organic solvents as the extraction agent and / or solvent.

[0053] In one embodiment, extracts from sea buckthorn are used for crosslinking biopolymers, wherein the mixture of water and organic solvent used as extraction agent contains 10 to 95 vol% organic solvent.

[0054] In one embodiment, extracts from sea buckthorn are used for crosslinking biopolymers, wherein additives, in particular acids or alkalis and / or anionic, non-ionic or amphoteric surfactants, are added to the extraction agent.

[0055] In one embodiment, extracts from sea buckthorn are used for crosslinking biopolymers, wherein the extracts are subjected to enzymatic or acid-catalytic treatment.

[0056] The invention will be explained in more detail using the following exemplary embodiments. Determination of polyphenols:

[0057] The polyphenols are determined using a modified method according to Folin-Ciocalteu (Singleton and Rossi (1965) Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vitic. 16: 144-158). Determination of flavonoids:

[0058] The flavonoids are measured by HPLC on an RP 18 column. The concentrations are calculated using external standards (quercetin-3-O-rutinoside, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-glucoside, quercetin, and isorhamnetin).

[0059] The determination of the protein, sugar, fat and ash content was carried out in accordance with the methods of the German Food, Consumer Goods and Feed Code (LFBG, 2013). Example 1:

[0060] Dried and ground sea buckthorn leaves are exhaustively extracted with water at a temperature of 40 °C. The extract is filtered, concentrated, and dried. The polyphenol content of the resulting extract is 20% by weight (based on the dry mass of the extract).

[0061] In an alternative embodiment of Example 1, sea buckthorn leaves of the Hergo variety are used, and the ratio of the mass of the dried and ground sea buckthorn leaves to the mass of the extraction solvent is 1:10. The polyphenol content of the obtained extract is 15 wt% (based on the dry mass in the extract), and the extract yield is 17%.

[0062] Extract yield refers to the extracted dry mass relative to the mass of the dried and ground sea buckthorn leaves. Example 2

[0063] Dried and ground sea buckthorn leaves are exhaustively extracted with water at a temperature of 40 °C. After filtration of the extract, the pH is adjusted to 7, followed by the addition of β-glucosidase (from almonds). Incubation lasts for 3 hours at 25 °C. The degradation of the glycosidically bound polyphenols is monitored by HPLC, specifically by measuring the conversion of isorhamnetin-3-O-glucoside to isorhamnetin and quercetin-3-O-rutinoside to quercetin. More than 80% of the glycosides were degraded. The extract is then filtered, concentrated, and dried. Example 3

[0064] Dried and ground sea buckthorn leaves of the Hergo variety are exhaustively extracted with an ethanol / water mixture, where the mass fraction of ethanol is 60%, at a temperature of 40°C. The ratio of the mass of the dried and ground sea buckthorn leaves to the mass of the extraction solvent is 1:10. The extract is filtered, concentrated, and dried. The polyphenol content of the resulting extract is 20% by weight (based on the dry mass of the extract). The extract yield is 20%. Table 1. Polyphenol, flavonoid, protein, total sugar and ash content of sea buckthorn extracts according to examples 1 and 3: values ​​based on dry mass of the extract. Polyphenols Flavonoids protein Total sugar ash Extract according to alternative formulation Example 1 15% by weight 2.9 wt.% 7 wt.% 21% by weight 12 wt.% Extract according to example 3 20 wt.% 3.8 wt.% 5 wt.% 16% by weight 9 wt.% Example 4

[0065] Fat-free natural yogurt is mixed with 15 g / L of the dry extract according to Example 1. After an incubation period of 24 h at 11 °C, the viscosity was tested using a Bostwick consistometer. The result of the experiment showed that the addition of the sea buckthorn extract according to the invention leads to an increase in viscosity. Example 5

[0066] To demonstrate the crosslinking properties of the sea buckthorn extract according to Example 1, a casein solution is used. A solution of 10 g / l casein in sodium phosphate buffer (pH 7) is mixed with 10 g / l sea buckthorn extract according to Example 1. Incubation takes place at room temperature for 45 min. The crosslinking of the casein solution is confirmed by the resulting turbidity. Example 6

[0067] To demonstrate the crosslinking properties of the sea buckthorn extract according to Example 3, a 2.5 wt% aqueous gelatin solution is prepared. The dried sea buckthorn extract according to Example 3 is dissolved in an aqueous solution at a concentration of 2.5 wt% and filtered through a 0.45 µm filter until clear. Both clear solutions are mixed in a 1:1 ratio at room temperature. The crosslinking of the gelatin upon addition of the sea buckthorn extract solution is visualized by the immediate turbidity of the solution. Example 7

[0068] When ensiling grass-based animal feed, sea buckthorn extract is added to the silage according to Example 1. From an addition of 5% by weight of sea buckthorn extract, based on the mass of the silage, the proteins in the silage can be protected from hydrolysis, thus improving its nutritional value. The addition of the sea buckthorn extract reduces the degree of protein hydrolysis in the animal feed by 18%. The degree of protein hydrolysis is determined using the OPA method (Frister H, Meisel H, Schlimme E (1990) Applications of the modified OPA method in protein analysis. Ernährungs-Umschau 37:442-445). Example 8

[0069] The sea buckthorn extract according to Example 2 is used for tanning and retanning sheepskins. The sea buckthorn extract is used at a concentration of 25% by weight, based on the mass of the sheepskins. It has been shown that the tanning of sheepskins is possible through the cross-linking of protein structures. The shrinkage temperatures achieved ranged from 68°C to 74°C.

Claims

1. Method for cross-linking biopolymers, characterised in that biopolymers are brought into contact with extracts from sea buckthorn in order to cross-link the biopolymers, wherein the sea buckthorn extracts are obtained from sea buckthorn plant residues accumulated during sea buckthorn fruit processing or unused plant components, wood, leaves, bark and / or shoots, and contain polyphenols with a content of 10-95% by weight based on the dry matter in the extract, wherein the polyphenols originate from the group of tannins, flavonoids and catechins, wherein the cross-linking of the biopolymers takes place in an aqueous environment, wherein the biopolymers are selected from proteins, peptides and polysaccharides, wherein the cross-linking of the biopolymers is effected by adding 0.1% by weight to 100% by weight of the sea buckthorn extract based on the biopolymer.

2. Method according to claim 1, characterised in that the sea buckthorn extracts for cross-linking biopolymers are obtained by extracting sea buckthorn plant material in dried or fresh form with water, an organic solvent or a mixture of water and organic solvents as extractant and / or solvent.

3. Method according to claim 2, characterised in that the mixture of water and organic solvent used as extractant contains 10 to 95% by volume of organic solvent.

4. Method according to claim 2 or 3, characterised in that additives, in particular acids or alkalis and / or anionic, non-ionic or amphoteric surfactants, are added to the extractant.

5. Method according to one of claims 2 to 4, characterised in that the extracts are subjected to enzymatic or acid-catalysed treatment.

6. Use of extracts from sea buckthorn for cross-linking biopolymers in the field of food, animal feed, cosmetics and pharmaceuticals, as well as medical products or in industrial applications, for tanning hides and furs, as well as for textile finishing, wherein the sea buckthorn extracts are obtained from sea buckthorn plant residues accumulated during sea buckthorn fruit processing or unused plant components, wood, leaves, bark and / or shoots, and contain polyphenols with a content of 10-95% by weight based on the dry matter in the extract, wherein the polyphenols originate from the group of tannins, flavonoids and catechins, wherein the biopolymers are selected from proteins, peptides and polysaccharides.

7. Use according to claim 6, characterised in that the sea buckthorn extracts for cross-linking biopolymers are obtained by extracting sea buckthorn plant material in dried or fresh form with water, an organic solvent or a mixture of water and organic solvent as extractant and / or solvent.

8. Use according to claim 7, characterised in that the mixture of water and organic solvent used as the extractant contains 10 to 95% by volume of organic solvent.

9. Use according to claim 7 or 8, characterised in that additives, in particular acids or alkalis and / or anionic, non-ionic or amphoteric surfactants, are added to the extractant.

10. Use according to any one of claims 7 to 9, characterised in that the extracts are subjected to enzymatic or acid-catalysed treatment.