WO2002060273A1 - Method for preparing a protein preparation having a reduced content of phenolic compounds - Google Patents

Method for preparing a protein preparation having a reduced content of phenolic compounds Download PDF

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Publication number
WO2002060273A1
WO2002060273A1 PCT/NL2002/000067 NL0200067W WO02060273A1 WO 2002060273 A1 WO2002060273 A1 WO 2002060273A1 NL 0200067 W NL0200067 W NL 0200067W WO 02060273 A1 WO02060273 A1 WO 02060273A1
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Prior art keywords
protein
phenolic compounds
preparation
water
organic solvent
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PCT/NL2002/000067
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French (fr)
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WO2002060273A8 (en
Inventor
Gerrit Anne Van Koningsveld
Harm Gruppen
Gerrit Wijngaards
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Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
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Publication of WO2002060273A1 publication Critical patent/WO2002060273A1/en
Publication of WO2002060273A8 publication Critical patent/WO2002060273A8/en

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    • 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
    • A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
    • 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
    • A23J1/142—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 by extracting with organic solvents

Definitions

  • This invention relates to a method for preparing from a protein source containing phenolic compounds a preparation of water-soluble proteins having a reduced content of phenolic compounds.
  • Preparation of water-soluble proteins is meant a protein preparation, the proteins of which possess a solubility in aqueous medium of at least 5 g/1.
  • Aqueous medium is defined herein as water or an aqueous solution.
  • preparation is meant a protein-containing composition, which preferably contains more than 95%, more preferably more than 99% protein; the protein content in the preparation, however, is at least 80 w/w %.
  • Phenolic compounds are defined herein as any chemical compound in which at least a phenol group is present.
  • polyphenols are thereby meant, such as, for instance, chlorogenic acid, caffeic acid and quinic acid.
  • protein preparations from vegetable base materials are substantially prepared by aqueous extraction of the protein in large volumes, followed by precipitation of the protein from the resulting extract. It has been found that in particular during the extraction and precipitation binding between protein and phenolic compounds occurs, which has the result that the proteins in the resulting protein preparation are poorly water-soluble and the functionality, color and nutritional value are adversely affected.
  • the above-mentioned problem is solved according to the invention by providing a method for preparing from a protein source containing phenohc compounds a preparation of water-soluble proteins having a reduced content of phenolic compounds, comprising the steps of a) ⁇ ?recipitating the protein by bringing the protein source into a mixture of an aqueous medium and at least a water-miscible organic solvent, such that substantially no denaturation of the protein occurs, while the pH of the mixture deviates at most 1 pH value from the isoelectric point of the protein to be isolated, b) purifying the precipitate obtained in a).
  • a reduced content of phenohc compounds is defined herein as a decrease of the content of phenolic compounds in the resulting preparation with respect to the content thereof in the protein source. This decrease in the resulting protein preparation is preferably at least a factor of 1.5-2, preferably a factor of 2.5-3 with respect to the content in a protein preparation obtained under comparable conditions when the same protein source is obtained without organic solvent, that is to say from only an aqueous medium. It has been found that during the precipitation according to the invention the content of phenolic compounds can be reduced by a factor of 3 or more with respect to a similar precipitation without organic solvent.
  • substantially no denaturation of the protein takes place, by which is meant that after the precipitation at least 80% of the protein to be obtained in the preparation is in undenatured condition.
  • the precipitation is carried out near the isoelectric point of the proteins, optimum precipitation of the relevant proteins is obtained, while, surprisingly, the binding between protein and the phenolic compounds is highly reduced during such a precipitation step.
  • the pH of the mixture is as close to the isoelectric point of the proteins to be isolated as possible. The pH deviates therefrom as less as possible, however, at most 1 pH value, preferably at most 0.5 pH value.
  • the purification of the protein precipitate can take place in many ways known in the art and therefore requires no further explanation.
  • the protein is purified under non-denaturing conditions to obtain a protein preparation in which the protein is substantially in undenatured form.
  • the protein is obtained in concentrated or solid form, for instance in the form of a powder, which is advantageous from a point of view of stability, shelf life and transport.
  • the isolated protein can also be stored in a suitable storage buffer. Those skilled in the art will be able to simply select the composition of such a storage buffer.
  • the temperature during the isolation of the proteins according to the invention is preferably 20°C or less, even more preferably 15°C or less.
  • the method according to the invention can also be carried out in cooled conditions, such as at 0°C, 4°C or 15°C. Those skilled in-the art, however, will wish to keep the temperature as close to the ambient temperature as possible, because cooling involves additional expenses.
  • the volume ratio of aqueous medium to organic solvent is preferably at least 1.
  • the content of aqueous medium in the mixture is preferably equal to or greater than that of the organic solvent. It is self-evident that the maximum amount of solvent, in addition to the requirement that in the relevant mixture substantially no denaturation of the protein occurs, is also determined by the maximum amount thereof still miscible with the volume of water; a phase separation in the mixture should thus preferably be prevented.
  • the organic solvent is preferably selected from the group consisting of dioxane, methanol, isopropanol and ethanol or from a mixture of two or more thereof.
  • ethanol is preferred to select organic solvent, because the other solvents are not accepted for this purpose under the prevailing legislation.
  • the mixture of the aqueous medium and the water- miscible organic solvent preferably contains between 10-30 vol.% ethanol, preferably between 15-25 vol.%. At such an ethanol content a good precipitation and redissolvability of the protein is obtained, and the binding with phenolic compounds during the precipitation is largely removed.
  • the mixture contains between 1-10 vol.% dioxane, preferably between 3-7 vol.% dioxane.
  • Dioxane is the most apolar water-miscible organic solvent known at present and has proved to be very effective for the prevention of the binding between phenolic compounds and protein (Sastri and Rao, J. Agric. Food Chem. 1990, 38, 2103-2110).
  • the optimum dioxane concentration in the mixture is lower than that of ethanol, because it has been found that higher dioxane concentrations have a negative effect on the redissolvability of the protein in water. Most preferably, the dioxane content in the mixture is therefore 5 vol.% or lower.
  • protein sources containing phenolic compounds are conceivable, for which vegetable material is particularly considered; the protein source most preferably originates from potatoes, maize, beet leaves, sunflower seeds; other materials, such as clover, leaves, pulses, nuts, beet chips, brewer's draff and vegetables are also suitable.
  • Such base materials contain valuable proteins suitable for a large number of uses in, for instance, food technology, as in the preparation of meat products and of vegetarian products.
  • the method according to the invention has particularly proved very useful with base materials having a relatively low protein content of 5 wt.% or less, and with base materials having a relatively high ratio of phenolic compounds with respect to protein, such as vegetable leaf material and fruits.
  • potatoes and beet leaves contain about 2 g phenolic compounds per kg protein; sunflower seeds about 60 g/kg protein.
  • the protein source particularly when of vegetable origin is subjected to an extraction step, previous to the precipitation step (step a).
  • a considerable enrichment as regards the protein content can be obtained.
  • Suitable extraction methods are known to those skilled in the art; it is important that the extraction method should be selected such that the intended protein to be isolated is extracted from the base material substantially in undenatured form. An extraction in an aqueous medium is therefore preferred.
  • other and additional known per se methods for enriching the protein content of the protein source such as, for instance, diafiltration, may be used according to the invention.
  • the protein source previous to or during the precipitation step (step a), is contacted with a material absorbing phenolic compounds.
  • a material absorbing phenolic compounds Such a material will hereinafter also be referred to as "absorbing material”. Due to this, the amount of phenolic compounds present is reduced, so that less organic solvent needs to be included in the mixture. Even at very high contents of phenolic compounds in the protein source the method according to the invention can thus be carried out effectively, without requiring the use of such a high concentration of organic solvent to counteract the binding between the phenolic compound and the protein, at which the intended protein to be isolated might denature.
  • Materials absorbing phenolic compounds are known in the art.
  • the absorbing material is selected from the group consisting of polyvin l polypyrrolidone, aluminum oxide and polyst rols, preferably of the Amberlite XAD-series (Rohm & Haas, USA), anion exchangers, dextrans and cyclodextrans.
  • the protein source previous to or during the precipitation, is contacted with a material decreasing the binding between the protein and the phenolic compounds present in the protein source. This may take place, for instance, by weakening or preventing the binding.
  • a material decreasing the binding between the protein and the phenolic compounds present in the protein source This may take place, for instance, by weakening or preventing the binding.
  • an additional binding-reducing material can thus be included in the mixture, which results in an effect comparable to the effect obtained by the above-described use of a material absorbing phenolic compounds. It is of course also possible to use a combination of an absorbing material and a binding-reducing material.
  • the binding-reducing material is preferably an inorganic salt, preferably selected from the group consisting of NaCl, metal salts, such as AICI3, borate and germanate salts, chaotropric salts, such as, for instance, chlorate and thiocyanate salts.
  • concentration of such salts is usually in the range of 5-50 mM. For NaCl this range is preferably between 0.1-1 M. What remains important is that the conditions in the mixture are substantially not denaturing to the intended protein to be isolated.
  • the purification step b) comprises washing the resulting precipitate in aqueous medium, which medium is substantially free from organic solvent under conditions in which the precipitated protein substantially does not redissolve.
  • substantially free from organic solvent is meant a maximum content of 0.5 v/v %, preferably at most 0.1 v/v %. Most preferably, the medium is completely free from organic solvent. By this washing step any possible undesirable rest of organic solvent is removed from the precipitate.
  • the pH and/or the temperature can be adjusted accordingly, and the conditions are preferably selected such that substantially no denaturation of the protein occurs.
  • substantially does not dissolve is meant that at most 0.5 g protein per 1 aqueous medium dissolves. If desired, this step may be repeated a number of times.
  • the purification step b) comprises dissolving the precipitate in an aqueous medium and separating the dissolved protein from the aqueous medium.
  • This isolation step may comprise, for instance, one or more precipitation, evaporation, chromatography steps.
  • the precipitate When the precipitate is, for instance, dissolved in an aqueous medium under suitable conditions, it may be precipitated again and, if desired, be redissolved in an aqueous medium. If desired, this procedure may be repeated a number of times to obtain the desired purity.
  • Those skilled in the art will be able without any inconvenience to find and use the right separation conditions, in which connection it is again important that in such precipitation steps preferably substantially no denaturation of the protein occurs.
  • the invention further relates to a water-soluble potato protein preparation, in which the weight ratio of phenolic compounds to potato protein is at most 1.0 g, preferably at most 0.5 g, even more preferably at most 0.1 g phenolic compounds per kg potato protein.
  • a water-soluble potato protein isolate can be obtained in which the weight ratio of phenolic compounds to potato protein of 0.35-0 ⁇ g phenolic compounds per kg potato protein can be obtained.
  • the content of phenolic compounds can thus be reduced by a factor of 3 or more in comparison with a potato protein preparation obtained in a comparable manner without use of an organic solvent (this contains about 1.4 g phenolic compounds per kg potato protein).
  • the invention also relates to a water-soluble beet protein preparation in which the weight ratio of phenolic compounds to beet protein is at most 1.0 g, preferably at most 0.5 g, even more preferably at most 0.1 g phenolic compounds per kg beet protein.
  • a beet protein preparation having a weight ratio of 0.35-0.5 g phenolic compounds per kg beet protein can also be obtained, which in this case, too, means a decrease of the content of phenolic compounds by a factor of 3 or more in comparison with a beet protein preparation prepared in a comparable manner without use of an organic solvent.
  • beet leaves are preferably used as protein source. A decrease by a factor of 10 is possible according to the invention, see Example 3.
  • the invention relates to a water-soluble sunflower seed protein preparation in which the weight ratio of phenolic compounds to sunflower seed protein is at most 6.0 g, preferably at most 2.5 g, most preferably at most 0.8 g phenolic compounds per kg sunflower seed protein.
  • a sunflower seed protein preparation can be obtained having a weight ratio of 1.5-2.5 g phenolic compounds per kg protein, which in this case, too, means a decrease of the content of phenolic compounds by a factor of 3 or more with respect to a comparable preparation method without use of an organic solvent.
  • the invention also relates to any preparation of water- soluble proteins originating from a polyphenol-containing protein source, which preparation has such a low polyphenol content that a factor of 3 or more is lower than the polyphenol content of a protein preparation from the same protein source, when the latter preparation has been obtained by precipitation without use of an organic solvent.
  • the invention further relates to the use of a preparation of water- soluble proteins, obtainable according to the method of the invention, preferably of vegetable origin, in foods, coatings, films and glues.
  • the invention relates to a food and a food ingredient containing a preparation of water-soluble proteins, preferably of vegetable origin, obtainable according to the invention, or an above-described vegetable protein preparation.
  • the steps in the method according to the invention are preferably selected such that the intended protein to be isolated is thus substantially not denatured, so that the isolated protein is obtained in undenatured from.
  • the protein source and the protein base material should therefore also contain the protein to be isolated preferably substantially in undenatured form.
  • Fig. 2 shows that for protein from sugar beet leaves a volume percentage of 15-20% (v/v) 2-propanol during precipitation leads to an optimum protein yield.
  • the protein sediment was dissolved in a third volume of 6 M urea having therein 1% SDS and 0.03%
  • Fig. 3 shows that for potato protein a volume percentage of 15-30 % ethanol, 15-20 % (v/v) 2-propanol and 10 % dioxane during precipitation leads to a protein yield optimal for that solvent.
  • the protein sediment was dissolved in a third volume of 6 M urea having therein 1% SDS and 0.03% . NaHS ⁇ 3 to dissolve the phenolic compounds bound to the protein. The solution was then liberated from proteins by filtration over a membrane having an MWCO of 1 kDa. In the filtrate the total content of phenolic compounds was then determined by the Folin-Ciocalteu method, in which chlorogenic acid served as reference.
  • the content of protein-bound phenolic compounds is plotted in Fig. 4 as a percentage of the content when no ethanol was added during precipitation.
  • Fig. 4 shows that the residual content of phenolic compounds is minimal at concentrations of ethanol and 2-propanol of 15% (v/v) and higher and dioxane concentrations of 10% (v/v) and higher.
  • organic solvents such as ethanol, 2-propanol or dioxane

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Abstract

Described is a method for preparing from a protein source containing phenolic compounds a water-soluble protein preparation having a reduced content of phenolic compounds, comprising the steps of a) precipitating the protein by bringing the protein source into a mixture of an aqueous medium and at least a water-miscible organic solvent, such that substantially no denaturation of the protein occurs, while the pH of which mixture deviates at most 1 pH value from the isoelectirc point of the protein, b) purifying the precipitate obtained in a).

Description

Title: Method for preparing a protein preparation having a reduced content of phenolic compounds.
This invention relates to a method for preparing from a protein source containing phenolic compounds a preparation of water-soluble proteins having a reduced content of phenolic compounds.
When preparing protein preparations from protein sources containing phenolic compounds, the problem occurs that the phenolic compounds bind to the proteins during purification. Owing to this, the solubility of the proteins m water is highly reduced, which has the result that such preparations have inferior functional properties and are therefore less interesting from a commercial point of view. Moreover, phenolic compounds have a negative effect on the color of the protein preparation to be obtained and, when food proteins are concerned, on the nutritional value thereof. The above-mentioned problem especially occurs when preparing protein preparations from vegetable material, because vegetable material usually has a high content of phenolic compounds (up to about 50 mg/g protein). By "preparation of water-soluble proteins" is meant a protein preparation, the proteins of which possess a solubility in aqueous medium of at least 5 g/1. "Aqueous medium" is defined herein as water or an aqueous solution. By "preparation" is meant a protein-containing composition, which preferably contains more than 95%, more preferably more than 99% protein; the protein content in the preparation, however, is at least 80 w/w %.
Phenolic compounds are defined herein as any chemical compound in which at least a phenol group is present. In particular, but not exclusively, polyphenols are thereby meant, such as, for instance, chlorogenic acid, caffeic acid and quinic acid. According to the state of the art protein preparations from vegetable base materials are substantially prepared by aqueous extraction of the protein in large volumes, followed by precipitation of the protein from the resulting extract. It has been found that in particular during the extraction and precipitation binding between protein and phenolic compounds occurs, which has the result that the proteins in the resulting protein preparation are poorly water-soluble and the functionality, color and nutritional value are adversely affected. It is also known in the art that an increased binding between protein and phenolic compounds occurs in an aqueous solution that is near the isoelectric point of the intended protein to be isolated. For this reason it has been decided in the art not to use an aqueous medium, in particular at a pH near the isoelectric point of the proteins to be obtained. Therefore, a solution has been sought to prevent and/or counteract binding between protein and phenolic compounds when preparing protein preparations.
Thus, for instance, it is known to isolate sunflower seed protein after sunflower seed meal has been extracted with organic solvents to remove phenolic compounds. The concentration of the solvent, however, is so high (40 vol.% ethanol, methanol or acetone) that in this extraction the protein is denatured (Sripad and Rao, J. Agric. Food Chem. 1987, 35, 962-967). Denaturation of protein usually has a strong negative effect on the solubility thereof.
Extraction of phenolic compounds from sunflower seed meal with acidic 1-butanol (92 vol.% butanol and 8 vol.% HC1) has also been described (Sodini and Canella, J. Agric. Food Chem. 1977, 25, 822-825). Under these conditions, too, the protein is denatured.
Rahma and Rao (J. Food Science, 1981, 46, 1521-1522) describe extraction of phenolic compounds from sunflower seed meal with 40% ethanol and acidic 1-butanol. Here, too, the protein is denatured. Vaughn et al (Cereal Chemistry 1976, 53(1), 118-125) describe sunflower meal extraction of phenolic compounds with 70% ethanol, in which protein also denatures.
It has hitherto not been known in the art to satisfactorily provide a preparation of water-soluble proteins from a protein source containing phenolic compounds, in which the protein is substantially free from phenohc compounds and is obtained in undenatured condition.
The above-mentioned problem is solved according to the invention by providing a method for preparing from a protein source containing phenohc compounds a preparation of water-soluble proteins having a reduced content of phenolic compounds, comprising the steps of a)τ?recipitating the protein by bringing the protein source into a mixture of an aqueous medium and at least a water-miscible organic solvent, such that substantially no denaturation of the protein occurs, while the pH of the mixture deviates at most 1 pH value from the isoelectric point of the protein to be isolated, b) purifying the precipitate obtained in a).
By combining the right pH and the presence of organic solvent in an aqueous medium it has been found that phenolic compounds substantially do not bind to proteins and a possible physical interaction already present is counteracted and that the protein is effectively precipitated. "A reduced content of phenohc compounds" is defined herein as a decrease of the content of phenolic compounds in the resulting preparation with respect to the content thereof in the protein source. This decrease in the resulting protein preparation is preferably at least a factor of 1.5-2, preferably a factor of 2.5-3 with respect to the content in a protein preparation obtained under comparable conditions when the same protein source is obtained without organic solvent, that is to say from only an aqueous medium. It has been found that during the precipitation according to the invention the content of phenolic compounds can be reduced by a factor of 3 or more with respect to a similar precipitation without organic solvent.
By now including such an amount of organic solvent in the aqueous medium, and by selecting the right conditions, such as temperature and salt concentration, substantially no denaturation of the protein takes place, by which is meant that after the precipitation at least 80% of the protein to be obtained in the preparation is in undenatured condition. When the precipitation is carried out near the isoelectric point of the proteins, optimum precipitation of the relevant proteins is obtained, while, surprisingly, the binding between protein and the phenolic compounds is highly reduced during such a precipitation step. During the protein precipitation the phenolic compounds largely remain in the liquid phase, while the protein is substantially precipitated. Preferably, the pH of the mixture is as close to the isoelectric point of the proteins to be isolated as possible. The pH deviates therefrom as less as possible, however, at most 1 pH value, preferably at most 0.5 pH value.
The skilled worker will be able without any inconvenience to determine the maximum allowable amount of organic solvent in the mixture and the right precipitation conditions, such as temperature, at which the protein remains in undenatured condition.
The purification of the protein precipitate can take place in many ways known in the art and therefore requires no further explanation. Preferably, the protein is purified under non-denaturing conditions to obtain a protein preparation in which the protein is substantially in undenatured form. By carrying out a purification step the protein is obtained in concentrated or solid form, for instance in the form of a powder, which is advantageous from a point of view of stability, shelf life and transport. The isolated protein can also be stored in a suitable storage buffer. Those skilled in the art will be able to simply select the composition of such a storage buffer. The temperature during the isolation of the proteins according to the invention is preferably 20°C or less, even more preferably 15°C or less. Above 20°C there is a risk that the protein will denature in aqueous medium in the presence of organic solvent. Since at higher temperature the tendency towards denaturation of protein is greater, at high temperatures the maximum allowable content of organic solvent will be lower in comparison with the situation at a lower temperature. At higher temperature the organic solvent content may thus be too low to obtain an effective decrease of the binding between phenolic compounds and the intended protein to be isolated. The method according to the invention can also be carried out in cooled conditions, such as at 0°C, 4°C or 15°C. Those skilled in-the art, however, will wish to keep the temperature as close to the ambient temperature as possible, because cooling involves additional expenses. To this end, the volume ratio of aqueous medium to organic solvent is preferably at least 1. This means that the content of aqueous medium in the mixture is preferably equal to or greater than that of the organic solvent. It is self-evident that the maximum amount of solvent, in addition to the requirement that in the relevant mixture substantially no denaturation of the protein occurs, is also determined by the maximum amount thereof still miscible with the volume of water; a phase separation in the mixture should thus preferably be prevented.
The organic solvent is preferably selected from the group consisting of dioxane, methanol, isopropanol and ethanol or from a mixture of two or more thereof. To isolate proteins used in foods, it is preferred to select ethanol as organic solvent, because the other solvents are not accepted for this purpose under the prevailing legislation.
The mixture of the aqueous medium and the water- miscible organic solvent preferably contains between 10-30 vol.% ethanol, preferably between 15-25 vol.%. At such an ethanol content a good precipitation and redissolvability of the protein is obtained, and the binding with phenolic compounds during the precipitation is largely removed.
In other embodiments of the invention, in particular suitable for non-food uses of the protein preparation to be obtained, the mixture contains between 1-10 vol.% dioxane, preferably between 3-7 vol.% dioxane. Dioxane is the most apolar water-miscible organic solvent known at present and has proved to be very effective for the prevention of the binding between phenolic compounds and protein (Sastri and Rao, J. Agric. Food Chem. 1990, 38, 2103-2110). The optimum dioxane concentration in the mixture, however, is lower than that of ethanol, because it has been found that higher dioxane concentrations have a negative effect on the redissolvability of the protein in water. Most preferably, the dioxane content in the mixture is therefore 5 vol.% or lower.
Many protein sources containing phenolic compounds are conceivable, for which vegetable material is particularly considered; the protein source most preferably originates from potatoes, maize, beet leaves, sunflower seeds; other materials, such as clover, leaves, pulses, nuts, beet chips, brewer's draff and vegetables are also suitable. Such base materials contain valuable proteins suitable for a large number of uses in, for instance, food technology, as in the preparation of meat products and of vegetarian products.
The method according to the invention has particularly proved very useful with base materials having a relatively low protein content of 5 wt.% or less, and with base materials having a relatively high ratio of phenolic compounds with respect to protein, such as vegetable leaf material and fruits. Thus, potatoes and beet leaves contain about 2 g phenolic compounds per kg protein; sunflower seeds about 60 g/kg protein.
Preferably, the protein source, particularly when of vegetable origin is subjected to an extraction step, previous to the precipitation step (step a). Thus, a considerable enrichment as regards the protein content can be obtained. Suitable extraction methods are known to those skilled in the art; it is important that the extraction method should be selected such that the intended protein to be isolated is extracted from the base material substantially in undenatured form. An extraction in an aqueous medium is therefore preferred. Also, other and additional known per se methods for enriching the protein content of the protein source, such as, for instance, diafiltration, may be used according to the invention.
In a special embodiment of the method according to the invention the protein source, previous to or during the precipitation step (step a), is contacted with a material absorbing phenolic compounds. Such a material will hereinafter also be referred to as "absorbing material". Due to this, the amount of phenolic compounds present is reduced, so that less organic solvent needs to be included in the mixture. Even at very high contents of phenolic compounds in the protein source the method according to the invention can thus be carried out effectively, without requiring the use of such a high concentration of organic solvent to counteract the binding between the phenolic compound and the protein, at which the intended protein to be isolated might denature. Materials absorbing phenolic compounds are known in the art. Preferably, the absorbing material is selected from the group consisting of polyvin l polypyrrolidone, aluminum oxide and polyst rols, preferably of the Amberlite XAD-series (Rohm & Haas, USA), anion exchangers, dextrans and cyclodextrans.
In another preferred embodiment of the method according to the invention the protein source, previous to or during the precipitation, is contacted with a material decreasing the binding between the protein and the phenolic compounds present in the protein source. This may take place, for instance, by weakening or preventing the binding. In addition to the organic solvent, or a combination of different organic solvents, an additional binding-reducing material can thus be included in the mixture, which results in an effect comparable to the effect obtained by the above-described use of a material absorbing phenolic compounds. It is of course also possible to use a combination of an absorbing material and a binding-reducing material. The binding-reducing material is preferably an inorganic salt, preferably selected from the group consisting of NaCl, metal salts, such as AICI3, borate and germanate salts, chaotropric salts, such as, for instance, chlorate and thiocyanate salts. The concentration of such salts is usually in the range of 5-50 mM. For NaCl this range is preferably between 0.1-1 M. What remains important is that the conditions in the mixture are substantially not denaturing to the intended protein to be isolated. Preferably, the purification step b) comprises washing the resulting precipitate in aqueous medium, which medium is substantially free from organic solvent under conditions in which the precipitated protein substantially does not redissolve. By "substantially free from organic solvent" is meant a maximum content of 0.5 v/v %, preferably at most 0.1 v/v %. Most preferably, the medium is completely free from organic solvent. By this washing step any possible undesirable rest of organic solvent is removed from the precipitate. Those skilled in the art will be able to simply determine conditions in which the protein substantially does not dissolve; thus, the pH and/or the temperature can be adjusted accordingly, and the conditions are preferably selected such that substantially no denaturation of the protein occurs. By "substantially does not dissolve" is meant that at most 0.5 g protein per 1 aqueous medium dissolves. If desired, this step may be repeated a number of times.
In a next embodiment of the invention the purification step b) comprises dissolving the precipitate in an aqueous medium and separating the dissolved protein from the aqueous medium. This isolation step may comprise, for instance, one or more precipitation, evaporation, chromatography steps. When the precipitate is, for instance, dissolved in an aqueous medium under suitable conditions, it may be precipitated again and, if desired, be redissolved in an aqueous medium. If desired, this procedure may be repeated a number of times to obtain the desired purity. Those skilled in the art will be able without any inconvenience to find and use the right separation conditions, in which connection it is again important that in such precipitation steps preferably substantially no denaturation of the protein occurs.
The invention further relates to a water-soluble potato protein preparation, in which the weight ratio of phenolic compounds to potato protein is at most 1.0 g, preferably at most 0.5 g, even more preferably at most 0.1 g phenolic compounds per kg potato protein. With the method according to the invention a water-soluble potato protein isolate can be obtained in which the weight ratio of phenolic compounds to potato protein of 0.35-0 δ g phenolic compounds per kg potato protein can be obtained.
As already indicated above, the content of phenolic compounds can thus be reduced by a factor of 3 or more in comparison with a potato protein preparation obtained in a comparable manner without use of an organic solvent (this contains about 1.4 g phenolic compounds per kg potato protein).
Thus, the invention also relates to a water-soluble beet protein preparation in which the weight ratio of phenolic compounds to beet protein is at most 1.0 g, preferably at most 0.5 g, even more preferably at most 0.1 g phenolic compounds per kg beet protein. With the method according to the invention a beet protein preparation having a weight ratio of 0.35-0.5 g phenolic compounds per kg beet protein can also be obtained, which in this case, too, means a decrease of the content of phenolic compounds by a factor of 3 or more in comparison with a beet protein preparation prepared in a comparable manner without use of an organic solvent. To this end, beet leaves are preferably used as protein source. A decrease by a factor of 10 is possible according to the invention, see Example 3.
Moreover, the invention relates to a water-soluble sunflower seed protein preparation in which the weight ratio of phenolic compounds to sunflower seed protein is at most 6.0 g, preferably at most 2.5 g, most preferably at most 0.8 g phenolic compounds per kg sunflower seed protein. According to the method of the invention a sunflower seed protein preparation can be obtained having a weight ratio of 1.5-2.5 g phenolic compounds per kg protein, which in this case, too, means a decrease of the content of phenolic compounds by a factor of 3 or more with respect to a comparable preparation method without use of an organic solvent.
Because with the method according to the invention a decrease of the content of phenolic compounds by a factor of 3 or more can be obtained in comparison with the conventional preparation method from the state of the art, in which in the absence of an organic solvent the protein is precipitated and then purified, the invention also relates to any preparation of water- soluble proteins originating from a polyphenol-containing protein source, which preparation has such a low polyphenol content that a factor of 3 or more is lower than the polyphenol content of a protein preparation from the same protein source, when the latter preparation has been obtained by precipitation without use of an organic solvent.
The invention further relates to the use of a preparation of water- soluble proteins, obtainable according to the method of the invention, preferably of vegetable origin, in foods, coatings, films and glues.
Moreover, the invention relates to a food and a food ingredient containing a preparation of water-soluble proteins, preferably of vegetable origin, obtainable according to the invention, or an above-described vegetable protein preparation.
The steps in the method according to the invention are preferably selected such that the intended protein to be isolated is thus substantially not denatured, so that the isolated protein is obtained in undenatured from. The protein source and the protein base material should therefore also contain the protein to be isolated preferably substantially in undenatured form.
The invention will hereinafter be explained in more detail with reference to some examples. EXAMPLES EXAMPLE 1
To an aqueous extract of sunflower meal as protein source were added at 4°C different amounts of ethanol. Subsequently, the pH of the mixture was brought to 5 by means of 0.5 M sulfuric acid. After stirring at 4°C for one hour the flocculated protein was removed by centrifugation (30 min.,
5000 x g, 4°C). After removal of the supernatant the sediment was dissolved in a similar volume of 50 M sodium phosphate buffer, pH 7, to which 100 mM NaCl were added. After centrifugation the protein concentration in the resulting supernatant was determined. From this was calculated the protein yield, which indicates how much redissolvable protein is obtained as a percentage of the amount of protein originally present in the extract. Fig. 1 shows that for sunflower protein a volume percentage of 5-15% ethanol during precipitation leads to an optimum protein yield. In a second embodiment of this experiment the protein sediment was dissolved in a third volume of 6 M urea having therein 1% SDS and 0.03%
NaHSO3 to dissolve the phenohc compounds bound to the protein. The solution was then liberated from proteins by filtration over a membrane having an MWCO (molecular weight separation limit) of 1 kDa. In the filtrate the total content of phenolic compounds was then determined by the Folin-Ciocalteu method, in which chlorogenic acid served as reference. The content of protein-bound phenolic compounds is plotted in Fig. 1 as a percentage of the content when no ethanol was added during precipitation. Fig. 1 shows that the residual content of phenolic compounds is minimal at ethanol concentrations of 15% (v/v) and higher. This example shows that acid precipitation of protein from aqueous sunflower extract in 15% ethanol results in a protein yield of 75%. The content of phenolic compounds in the protein preparation thus obtained is 3 times lower than in the protein preparation obtained without addition of ethanol, which results in a higher protein solubility. EXAMPLE 2
In this experiment different amounts of 2-propanol were added at 4°C to an aqueous extract of sugar beet leaves as protein source. Subsequently, the pH of the mixture was brought to 5 by means of 0.5 M sulfuric acid. After stirring at 4°C for one hour the flocculated protein was removed by centrifugation (30 min., 5000 x g, 4°C). After removal of the supernatant the sediment was dissolved in a similar volume of 50 mM sodium phosphate buffer, pH 7, to which 100 mM NaCl were added. After centrifugation the protein concentration in the resulting supernatant was determined. From this was calculated the protein yield, which indicates how much redissolvable protein is obtained as a percentage of the amount of protein originally present in the extract. Fig. 2 shows that for protein from sugar beet leaves a volume percentage of 15-20% (v/v) 2-propanol during precipitation leads to an optimum protein yield. In a second embodiment of this experiment the protein sediment was dissolved in a third volume of 6 M urea having therein 1% SDS and 0.03%
NaHSOβ to dissolve the phenolic compounds bound to the protein. The solution was then liberated from proteins by filtration over a membrane having an MWCO of 1 kDa. In the filtrate the total content of phenolic compounds was then determined by the Folin-Ciocalteu method, in which chlorogenic acid served as reference. The content of protein-bound phenolic compounds is plotted in Fig. 2 as a percentage of the content when no ethanol was added during precipitation. Fig. 2 shows that the residual content of phenolic compounds is minimal at concentrations of 2-propanol of 20% (v/v) and higher. This example shows that acid precipitation of protein from an aqueous extract of sugar beet leaves in the presence of 20% 2-propanol results in a protein yield of 45%. The content of phenolic compounds in this protein preparation is 10 times lower than that of the protein preparation in the absence of 2-propanol, which results in a higher protein solubility. EXAMPLE 3
In this experiment different amounts of ethanol, 2-propanol and dioxane were added at 4°C to potato extract. Subsequently, the pH of the mixture was brought to 5 by means of 0.5 M sulfuric acid. After stirring at 4°C for one hour the flocculated protein was removed by centrifugation (30 min., 5000 x g, 4°C). After removal of the supernatant the sediment was dissolved in a similar volume of 50 mM sodium phosphate buffer, pH 7, to which 100 mM^TaCl were added. After centrifugation the protein concentration in the resulting supernatant was determined. From this was calculated the protein yield, which indicates how much redissolvable protein is obtained as a percentage of the amount of protein originally present in the extract.
Fig. 3 shows that for potato protein a volume percentage of 15-30 % ethanol, 15-20 % (v/v) 2-propanol and 10 % dioxane during precipitation leads to a protein yield optimal for that solvent. In a second embodiment of this experiment the protein sediment was dissolved in a third volume of 6 M urea having therein 1% SDS and 0.03% . NaHSθ3 to dissolve the phenolic compounds bound to the protein. The solution was then liberated from proteins by filtration over a membrane having an MWCO of 1 kDa. In the filtrate the total content of phenolic compounds was then determined by the Folin-Ciocalteu method, in which chlorogenic acid served as reference. The content of protein-bound phenolic compounds is plotted in Fig. 4 as a percentage of the content when no ethanol was added during precipitation. Fig. 4 shows that the residual content of phenolic compounds is minimal at concentrations of ethanol and 2-propanol of 15% (v/v) and higher and dioxane concentrations of 10% (v/v) and higher. This example shows that the presence of organic solvents, such as ethanol, 2-propanol or dioxane, during the acid precipitation of protein from an aqueous potato extract substantially reduces the content of phenolic compounds of the resulting protein preparation, which strongly improves the solubility of the protein.

Claims

1. A method for preparing from a protein source containing phenolic compounds a preparation of water-soluble proteins having a reduced content of phenolic compounds, comprising the steps of a) precipitating the protein by bringing the protein source into a mixture of an aqueous medium and at least a water-miscible organic solvent, such that substantially no denaturation of the protein occurs, while the pH of the mixture deviates at most 1 pH value from the isoelectric point of the protein, b)^purifying the precipitate obtained in a).
2. A method according to claim 1, characterized in that the temperature in step a) is 20°C or less, preferably 15°C or less.
3. A method according to claim 1 or 2, characterized in that the volume ratio of aqueous medium to organic solvent is at least 1.
4. A method according to any one of the preceding claims, characterized in that the organic solvent is selected from dioxane, methanol, isopropanol, ethanol or a mixture of two or more thereof.
5. A method according to any one of the preceding claims, characterized in that the mixture contains between 10-30 vol.% ethanol, preferably between 15-25 vol.%.
6. A method according to any one of the preceding claims, characterized in that the mixture contains between 1-10 vol.% dioxane, preferably between 3-7 vol.%.
7. A method according to any one of the preceding claims, characterized in that the protein source comprises vegetable material, preferably originating from potatoes, maize, beet leaves and sunflower seeds.
8. A method according to any one of the preceding claims, characterized in that previous to step a) the protein source is subjected to an extraction step.
9. A method according to any one of the preceding claims, characterized in that previous to or during step a) the protein source is contacted with a material absorbing phenolic compounds.
10. A method according to claim 9, characterized in that the material absorbing phenolic compounds is selected from the group consisting of polyvinyl polypyrrolidone, aluminum oxide, anion exchangers, dextrans, cyclodextrans, polystyrols.
11. A method according to any one of the preceding claims, characterized in that that previous to or during step a) the protein source is contacted with a material reducing the binding between the protein and the phenolic compounds present in the protein source.
12. A method according to claim 11, characterized in that the binding- reducing material is an inorganic salt, preferably selected from the group consisting of NaCl, metal salts, borate salts, germanate salts and chaotropric salts, or a combination of two or more thereof.
13. A method according to any one of the preceding claims, characterized in that step b) comprises washing the precipitate obtained in step a) in an aqueous medium, which is substantially free from organic solvent under conditions in which the precipitated protein substantially does not dissolve.
14. A method according to any one of the preceding claims, characterized in that step b) comprises dissolving the resulting precipitate in an aqueous medium and separating the protein from the aqueous medium.
15. A water-soluble potato protein preparation, in which the weight ratio of phenolic compounds to potato protein is at most 1.0 g, preferably at most 0.5 g, even more preferably at most 0.1 g phenolic compounds per kg potato protein.
16. A water-soluble beet protein preparation, in which the weight ratio of phenolic compounds to beet protein is at most 1.0 g, preferably at most 0.5 g, even more preferably at most 0.1 g phenolic compounds per kg beet protein.
17. A water-soluble sunflower seed protein preparation, in which the weight ratio of phenolic compounds to sunflower seed protein is at most 6.0 g, preferably at most 2.5 g, most preferably at most 0.8 g phenolic compounds per kg sunflower seed protein.
18. Use of a vegetable water-soluble protein preparation, obtainable according to any one of claims 1-14 or according to any one of claims 15-17, in foods, coatings, films and glues.
19. A4ϋood or food ingredient, comprising a water-soluble protein preparation, obtainable according to any one of the preceding claims 1-14 or according to any one of claims 15-17.
PCT/NL2002/000067 2001-01-30 2002-01-29 Method for preparing a protein preparation having a reduced content of phenolic compounds WO2002060273A1 (en)

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