Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
  • A23J1/007—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials from leafy vegetables, e.g. alfalfa, clover, grass
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/12—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/185—Vegetable proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention relates to a method for increasing the stress protein content in a liquid, to a stress protein product and to the use thereof.
• Stress proteins are formed by microorganisms, plants and animals when, as a result of a change in the environment such as exposure to heat, radiation or chemicals, so-called stress- susceptible genes are expressed. According to current insights, such proteins can contribute to a protection against detrimental effects resulting from such environmental changes. For that reason, the use of stress proteins is in the center of interest of, inter alia, medicine, molecular biology, the cosmetic industry and the industry producing plant protection products.
• stress proteins have a number of important natural therapeutic functions in plants and animals, people included. These proteins are involved as regulator in protein synthesis and protein folding. In addition, an important function lies in the regulation of gene function during growth, but also during cell death.
• the stress proteins are capable of stimulating both human and animal immune systems, and clinical research has shown that the chaperone proteins contribute positively in the fight against cancer.
• HSP 70 stress proteins
• BSE low-density protein
• HSP 70 can also be produced from microorganisms and cancer cells, but these production methods too are expensive and only give a low yield.
• Stress proteins of vegetable origin could offer an alternative. As the production of stress proteins depends on the condition the cell is in, a change in the circumstances outside the cell can tremendously increase the synthesis of the stress proteins in the plant cell.
• a disadvantage of the use of plants as a source of stress proteins is the large amount of other proteins (bulk proteins) and other bulk compounds present.
• the purification of stress proteins is difficult and also difficult to scale up, because the standard purification techniques such as ultrafiltration and column chromatography are hindered by the high concentrations of other proteins.
• the stress proteins / bulk proteins ratio is so low that a thorough separation of bulk proteins from the stress protein fraction is required to obtain a product with a suitable amount of stress protein without obtaining an excess of bulk proteins.
• the amount of product to be administered to obtain a desired amount of stress protein must be so high that an excess of bulk proteins and other bulk compounds such as polyphenols would be introduced into the food product or pharmaceutical preparation.
• bulk proteins can have a negative effect on. the activity of the stress protein, for instance because the stress protein binds to a bulk protein.
• bulk proteins (such as enzymes) can reduce the stability of a stress protein product. It is an object of the present invention to provide an economically attractive method for increasing the content of stress protein (as percentage of the total solid matter content, preferably as percentage of the total protein content) in a liquid containing protein.
• the present invention relates to a method for increasing the stress protein content (as percentage of the total solid matter content, preferably as percentage of the total protein content) in a liquid that contains at least one stress protein and at least one bulk compound, wherein
• the precipitate formed is separated from the supernatant containing the stress protein.
• a method according to the invention is particularly suitable for removing inconvenient bulk compounds and in particular inconvenient bulk proteins in a simple and industrially attractive manner.
• a method is particularly suitable for the purification of a stress protein product, optionally in combination with one or more other purification steps. It has also been found that it is possible by means of a method according to the invention to remove bulk compounds while maintaining a high nativity and/or activity of one or more stress proteins.
• a method proves to be very suitable for separating chlorophyll (in the form of chlorophyll protein complex and/ or as free chlorophyll) and/or rubisco as precipitate from a liquid that contains vegetable proteins. By removing these two components, a next concentration or isolation of the stress protein proves to be highly facilitated.
• stress protein can be efficiently isolated from the supernatant or concentrated with conventional techniques such as, for instance, (preparative) column chromatography, which technique was unsuitable because of binding to the column material (for instance binding of chlorophyll), damage to and/or clogging of the column by chlorophyll particles and overloading because of the high concentration of protein (for instance a high concentration of rubisco).
• a stress protein is a protein that under the influence of an exposure to a harmful environmental factor, not internally determined by the organism, ("stress") is expressed by a gene inducible by "stress".
• stress is exposure to temperature change (exposure to a warm or cold environment), to radiation, for instance UV-radiation, to a changed atmosphere such as hypoxia or anoxia, to an osmolarity change or a combination thereof. It is also possible to induce stress protein production by exposure to an agent inducing stress protein, for instance a transition metal ion (for instance silver or copper) or an organic xenobiotic compound (for instance paraquat).
• an agent inducing stress protein for instance a transition metal ion (for instance silver or copper) or an organic xenobiotic compound (for instance paraquat).
• stress proteins particularly suitable to be produced through the invention are the so-called "heat-shock” proteins (proteins induced by a heat treatment), such as HSP 20-30, HSP 40, HSP 60 (Chaperonin) HSP 70, HSP 90, HSP 130 and ubiquitin, which are described in S. Lewis et al., Ecotoxicology 8, 351-368 (1999).
• stress proteins that can be produced according to the invention are cytochrome p450 proteins, metallothioneins and heme oxygenases.
• bulk compounds are compounds that are contained rri a large amount in the liquid from which the desired stress protein can be produced, such as proteins and other peptidic compounds and other contaminants tnaj; are not stress proteins.
• Typical examples of bulk compo nds are chlorophyll and proteins such as complexes of chlorophyll with "chlorophyll binding proteins", rubisco, enzymes (for instance proteases and polyphenol oxidases) and other proteins present in the liquid to such a degree that they impede efficient production of the stress protein.
• Inconvenient proteins, such as rubisco, "chlorophyll binding protein” and chlorophyll protein complexes are referred to herein as bulk proteins.
• the bulk proteins rubisco, "chlorophyll binding protein", chlorophyll protein complexes and optionally also other inconvenient bulk compounds are removed from the liquid by a pH reduction and separation of the formed precipitate.
• the stress protein can be recovered from the resultant supernatant optionally by means of a next purification step, wherein a residue of the mentioned bulk proteins and/or other components such as polyphenols, water and/or organic solvents are removed from the stress protein fraction.
• Very good results are achieved by increasing the stress protein content in a hquid, the stress protein coming from an organism in which one or more stress proteins have been induced by a heat treatment between 20 and 90°C, preferably between 30 and 60°C, and more preferably between 35 and 45°C.
• the duration of the treatment can be chosen within a wide range, preferably between 1 minute and 24 hours, more preferably 30 min. - 12 hours, and still more preferably between 1 — 4 hours.
• the invention is used for a hquid with stress proteins coming from a plant, such as for instance lucerne (alfalfa), a cereal (for instance barley), soy, a grass (for instance oat), beet, potato, clover or a water plant (for instance an alga).
• a plant such as for instance lucerne (alfalfa), a cereal (for instance barley), soy, a grass (for instance oat), beet, potato, clover or a water plant (for instance an alga).
• This technique can also be used for stress protein (such as HSPs) from yeasts, fungi and bacteria. Particularly good results are obtained with stress proteins from lucerne, clover, oat and barley.
• the leaf of the plant is used as source for one or more stress proteins.
• stress proteins particularly suitable are beet tops, lucerne leaves, barley leaves, oat leaves and potato tops.
• leaf material which is normally a waste product, in a useful manner.
• the plant can be grown in a conventional manner. Very good results have also been obtained with a plant grown in the dark in light- deficient conditions, or, conversely, in conditions rich in light. The choice of the light conditions can influence the formation of specific bulk proteins.
• any liquid with vegetable proteins can be used as source from which a stress protein according to the invention can be produced.
• Particularly suitable is for instance the sap of the plant obtained by pressing vegetable material and an extract of a vegetable material.
• Suitable preparation methods for such a hquid are known, for instance from
• Extraction can for instance take place with water, a buffer (for instance Tris, borate, phosphate, optionally with additives such as polyvinyl pyrrolidone or EDTA) a saline solution (for instance NaCl, KC1) or another solvent in which stress proteins dissolve.
• a buffer for instance Tris, borate, phosphate, optionally with additives such as polyvinyl pyrrolidone or EDTA
• a saline solution for instance NaCl, KC1
• another solvent in which stress proteins dissolve for instance
• the liquid with vegetable proteins preferably has a pH in the range of pH 6 - 9, more preferably pH 6.5 - 8.
• the pH is preferably reduced to a target value ("set point").
• set point a target value
• the pH to which the reduction takes place and the speed with which the pH is preferably reduced is dependent, inter aha, on the bulk compounds present, the type of stress protein that is isolated and can be routinely determined by the skilled person.
• the pH is reduced step by step or gradually with an average speed of approximately 0.1 - 2 pH unit per hour, more preferably with an average speed of approximately 0.2 - 1 pH unit per hour, most preferably approximately 0.3 - 0.5 pH unit per hour, for instance step by step each half hour with a step-by-step reduction of 0.2 pH unit.
• the precipitate that forms can optionally be separated from the supernatant during the pH reduction or at the end.
• the hquid is stirred during addition of the acid, and the stirring is temporarily discontinued between two reductions.
• HSP 20 - 30, HSP 40, HSP 60 (Chaperonin), HSP 70, HSP 90, HSP 130 Cytoehrome p450, metallothionein, heme oxygenase and/or ubiquitin from a liquid further containing chlorophyll and/or rubisco proteins is, for instance, a method in which chlorophyll and/or rubisco is precipitated from the liquid by step-by-step or gradual reduction of the pH )f the hquid with the proteins by adding an acid of a pH of approximately 6.5 or higher to a pH of approximately 4.8 - 5.2, the precipitate formed is separated from the supernatant containing the stress protein, and optionally the stress protein in the supernatant is isolated or concentrated.
• Particularly good results are obtained with such a method in which HSP70 was produced from lucerne, clover, oat or barley.
• any acid can be used for the pH reduction through a method according to the invention.
• Very suitable are (relatively) strong inorganic or organic acids such as hydrochloric acid, nitric acid, phosphoric acid, boric acid, sulfuric acid, citric acid, acetic acid, lactic acid and the like.
• hydrochloric acid and acetic acid are particularly preferred because of economic reasons.
• lactic acid and citric acid are also preferred in human and animal uses.
• the pH reduction In addition to the pH reduction, other parameters, such as the salt concentration, the concentration of organic solvents, the protein concentration (by dilution or concentration) and the temperature, can be changed for separating bulk compounds in a desired manner.
• a method according to the invention can be carried out within a broad temperature range, which temperature, if desired, can be kept constant or can be changed.
• the precipitation point pH at which precipitation takes place
• the temperature lies between approximately 1 and 50°C, more preferably between 5 and 30°C, and still more preferably between 5 and 20°C.
• the precipitate can be separated from the supernatant in any suitable manner, for instance by centrifugation, filtration or decantation.
• the stress protein in the supernatant can be isolated or concentrated in any suitable manner.
• Very suitable techniques are, inter alia, gel filtration, ultrafiltration, ultracentrifugation, cross-flow filtration, precipitation (complete or partial) drying, membrane filtration, ion exchange, chromatography (for instance gel filtration, gel permeation or ion exchange chromatography) and electrophoresis.
• the stress protein is concentrated or isolated with chromatography or a filtration technique.
• chromatography for instance with ATP agarose as column material.
• affinity chromatography other contaminants such as polyphenols can be removed very rapidly and effectively after removal of rubisco and chlorophyll protein complex without much column contamination, so that in an economically attractive manner an end product with a high degree of purity can be obtained.
• the stress protein is additionally purified from polyphenolic compounds and/or other contaminants, particularly when the stress protein comes from leaf material. As a result, a higher activity of the stress protein is preserved.
• Suitable methods for removing polyphenols are known.
• a very suitable method for removing polyphenohc compound from a protein product is described in Dutch patent application NL 1017241. This application describes a method in which protein is precipitated by mixing a solution of the protein in an aqueous medium with a water-miscible organic solvent at a pH about the isoelectric point ( ⁇ 1 pH unit), and the protein is then purified. It has now been found that such a method is also suitable for removing polyphenols from a stress protein product while preserving the stress protein activity.
• the invention further relates to a stress protein product obtainable through a method according to the invention.
• a stress protein product can for instance be a solution, a dispersion, an emulsion or a dry product.
• a product according to the invention can be obtained without genetic modification and without the possible health risks that may be attached to stress protein such as HSPs from bovine brain.
• a product according to the invention is characterized by a high nativity of the stress protein and/or a high activity of the stress protein.
• a product according to the invention is very suitable in numerous uses. Therefore, the invention also relates to a food product for human or animal consumption, a pharmaceutical or cosmetic preparation or a plant protection product comprising a stress protein, which stress protein is obtainable according to a method of the present invention.
• a human food product according to the invention a low content of chlorophyll is highly desirable.
• rubisco is an interesting protein for human food, a high dose of stress protein will result in an excess of rubisco.
• a human food product according to the invention proves to have a very high stress protein content with regard to the rubisco and chlorophyll content.
• a pharmaceutical, cosmetic and plant protection product_according to the invention it has been found that a very low degree of complexing of stress protein (such as HSPs) with rubisco occurs. As a result, the activity of the stress protein remains high. This can prevent a part of the effect of the HSP from being undone as a result of the undesired complexing of HSP to rubisco, which would cause a reduced dose of the free HSP. Additionally, the other components (rubisco and chlorophyll) can have a negative effect.
• the invention also relates to its use in animal food or human food, in a pharmaceutical preparation such as an immune system stimulating agent or a preparation for the treatment of cancer, or cosmetic preparation or in a plant protection product.
• Example 1 HSP 70 from lucerne
• the heat-treated lucerne was frozen with liquid nitrogen and then reduced in a mortar.
• a 20 ml buffer 25 g 1 polyvinyl pyrrolidone; 50 mM Tris pH 8, prepared 24 hours in advance and kept at 4°C.
• 2mM EDTA and 5 mM DTT dithiothreitol.
• the extract was centrifuged for 15 min at 10.000 g). The supernatant contains both the HSP 70 and the chlorophyll and rubisco.
• pH precipitation The liquid fraction was brought to pH 6.5, and at a temperature of
• the leaf materials was frozen with liquid nitrogen and then reduced in a mortar. After this was added a two times larger buffer (with the composition as indicated in Example 1), and then the extract was centrifuged.
• the liquid fraction contained HSP 70 as well as some other proteins.
• the liquid fraction could optionally be treated further as described in Example 1.
• Leaf material of lucerne was pressed with a sap press, and the sap samples obtained were exposed to a temperature in the range of from 0 to 60°C for 30 minutes. Denaturated proteins were removed by means of centrifugation. The supernatant with soluble proteins was separated on an SDS/page gel and then blotted. With antibodies it was determined whether HSP 70 was still present. The results are shown in Fig. 1. The HSP band is indicated with the arrow. From the results it is clear that HSP is not heat stable. Already at 30 minutes exposure to 40°C, no detectable amount of HSP 70 is present anymore.
• Example 4 HSP from clover or oat.
• Clover leaves or oat leaves were heated directly (within half an hour) after mowing at 40°C for 2 hours and then pressed with a sap press.
• sap obtained were added 3.9 g/1 of sodium metabisulfite, and then the pH precipitation was carried out as described in Example 1.
• the pellet contained chlorophyll and the rubisco and the supernatant HSP 70 protein. (Determined by means of western blotting and gel electrophoresis.)

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• 2001
  • 2001-06-05 NL NL1018211A patent/NL1018211C2/nl not_active IP Right Cessation
• 2002
  • 2002-06-04 WO PCT/NL2002/000365 patent/WO2002098910A1/en not_active Application Discontinuation
  • 2002-06-04 EP EP02741520A patent/EP1392727A1/en not_active Withdrawn

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