CA2362948A1 - Method for preparing an albumin isolate from a substance containing albumin - Google Patents
Method for preparing an albumin isolate from a substance containing albumin Download PDFInfo
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- CA2362948A1 CA2362948A1 CA002362948A CA2362948A CA2362948A1 CA 2362948 A1 CA2362948 A1 CA 2362948A1 CA 002362948 A CA002362948 A CA 002362948A CA 2362948 A CA2362948 A CA 2362948A CA 2362948 A1 CA2362948 A1 CA 2362948A1
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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
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
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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
- A23J1/125—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from cereals, wheat, bran, or molasses by treatment involving enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention relates to a method for preparing an albumin isolate from a substance containing albumin, according to which the substance is first grou nd to a flour and the flour suspended in an aqueous solution. The albumin is th en extracted from the flour and introduced into the solution by means of at lea st one countercurrent protease. The albumin is extracted from the solution by precipitation using a mineral acid and then neutralized. The aim of the invention is to improve said method in such a way that the albumin is extracted from the solution and introduced into the solution by way of a process comprising at least two steps and carried out using the at least one protease, at a pH greater than 8 and at a temperature of between 30 and 60 ~ C. In the first step the flour is treated at a lower concentration of the at least one protease, in relation to albumin weight, at a lower pH and at a higher temperature than in the second step. After the first step a first upp er flow of a fraction containing the flour is separated and the albumin separat ed from said fraction by precipitation, and after the second step a second uppe r flow of a fraction containing the flour is separated and fed back to the fir st step.
Description
Title METHOD FOR PREPARING AN ALBUMIN ISOLATE FROM A SUBSTANCE
CONTAINING ALBUMIN
Field of the Invention The invention relates to a method for preparing an albumin isolate from a substance containing albumin, the substance being ground to a flour, the flour being suspended in an aqueous solution, the albumin being extracted from the flour into the solution using at least one countercurrent protease, and the albumin being precipitated from the solution using mineral acid and being neutralized.
Background of the Invention Several methods are known to extract albumin from vegetable raw materials, an alkaline extraction of the albumin at a pH of between 7 and 9 and at a temperature of 40 to 50 °C being the basis of these methods. The extracted albumin is then purified by centrifugation and acidified with a mineral acid until the isoelectric point of the albumin is reached. The albumin precipitated at the isoelectric point is again concentrated and purified by means of centrifugation. In this way yields of 70 % as related to the albumin contained in the raw materials are possible. However, it is a serious drawback that only such raw materials may be used in which the albumin has a low level of denaturation. Especially in exploring the most important vegetable albumin isolate, i. e.
soya albumin isolate, the limitation to so called "white flakes" as a raw material is an economic drawback. White flakes are only obtained by means of a special drying method for the remainders of Soya oil production. Normally, the remainders are dried less carefully, and they are then present in a form of so called "toasted flakes" in which the albumin is strongly denatured.
From DE I 203 588 it is known to enhance the alkaline extraction process in exploring albumins from an albumin containing substance by means of a pre-treatment of an aqueous suspension of the substance with hydrogen peroxide within the alkaline range and with proteolytic enzymes. To this end, the pH of the suspension of the albumin containing substance is at first raised, then hydrogen peroxide is added and the temperature is raised to initiate peroxidation. Then, the pH is adjusted to 4 to 9, and enzymes are added to the suspension, the activity minimum of which is within this pH-range. Afterwards, the suspension is stirred for two hours.
Preferably, vegetable enzymes like bromelaine, ficine and papaine are used. After the enzymatic hydrolysis, the albumin is dissolved in that the pH is raised to 9 to 12. At the same time, high temperatures are applied from which it is known that they result in albumin damages particularly in combination with the high pH. The multiple change of the pH in this known method requires great efforts in large scale applications and results in a high consumption of alkali and acid followed by a strong formation of salt.
It is also known from DE 44 29 787 C2 to use proteolytic enzymes for enhancing the solubility of albumins. Here, the starting material is at first extracted with alcohol, only afterwards it is mechanically broken up and then further enzymatically broken up in the acid to neutral range.
The extraction of attendant material to the albumin is effected by means of a countercurrent process. A treatment with hydrogen peroxide can take place after the extraction of the albumin in this known method, too. Due to the use of alcohol for the extraction, the method requires high efforts which are disadvantageous. The explosion danger of the suspension and the inactivation of the used enzymes by the presence of alcohol have to be permanently considered. In the known method, the proteolyses is per definitionem limited to a pH-range of < 9,5, only the pH-range of < 7,5 being concretely described in DE 44 29 787 C2. On the other hand it is known to those skilled in the art that the solubility of albumin is particularly great, if the pH is over 9. All at all, the efficiency of the known method as compared to the cost to be spend for its application is only low so that the prepared albumin isolates are not competitive.
A method according the preamble of claim 1 is known from DE 43 39 743 C 1.
This method does without the use of alcohol. For dissolving the protein, however, a pH of over 11,5, particularly of about 12,5, is needed which is associated with a high consumption of alkali and of acid for a later neutralization. Prior to the proteolysis, the albumin containing substance is treated with a protease at a pH > 10.5. The pre-treatment requires a comparatively large amount of enzyme, which results in a drawback with regard to the production cost. The comparatively high salt content at the end of the albumin extraction of this known method, which in praxis requires an additional washing step, is a further drawback.
CONTAINING ALBUMIN
Field of the Invention The invention relates to a method for preparing an albumin isolate from a substance containing albumin, the substance being ground to a flour, the flour being suspended in an aqueous solution, the albumin being extracted from the flour into the solution using at least one countercurrent protease, and the albumin being precipitated from the solution using mineral acid and being neutralized.
Background of the Invention Several methods are known to extract albumin from vegetable raw materials, an alkaline extraction of the albumin at a pH of between 7 and 9 and at a temperature of 40 to 50 °C being the basis of these methods. The extracted albumin is then purified by centrifugation and acidified with a mineral acid until the isoelectric point of the albumin is reached. The albumin precipitated at the isoelectric point is again concentrated and purified by means of centrifugation. In this way yields of 70 % as related to the albumin contained in the raw materials are possible. However, it is a serious drawback that only such raw materials may be used in which the albumin has a low level of denaturation. Especially in exploring the most important vegetable albumin isolate, i. e.
soya albumin isolate, the limitation to so called "white flakes" as a raw material is an economic drawback. White flakes are only obtained by means of a special drying method for the remainders of Soya oil production. Normally, the remainders are dried less carefully, and they are then present in a form of so called "toasted flakes" in which the albumin is strongly denatured.
From DE I 203 588 it is known to enhance the alkaline extraction process in exploring albumins from an albumin containing substance by means of a pre-treatment of an aqueous suspension of the substance with hydrogen peroxide within the alkaline range and with proteolytic enzymes. To this end, the pH of the suspension of the albumin containing substance is at first raised, then hydrogen peroxide is added and the temperature is raised to initiate peroxidation. Then, the pH is adjusted to 4 to 9, and enzymes are added to the suspension, the activity minimum of which is within this pH-range. Afterwards, the suspension is stirred for two hours.
Preferably, vegetable enzymes like bromelaine, ficine and papaine are used. After the enzymatic hydrolysis, the albumin is dissolved in that the pH is raised to 9 to 12. At the same time, high temperatures are applied from which it is known that they result in albumin damages particularly in combination with the high pH. The multiple change of the pH in this known method requires great efforts in large scale applications and results in a high consumption of alkali and acid followed by a strong formation of salt.
It is also known from DE 44 29 787 C2 to use proteolytic enzymes for enhancing the solubility of albumins. Here, the starting material is at first extracted with alcohol, only afterwards it is mechanically broken up and then further enzymatically broken up in the acid to neutral range.
The extraction of attendant material to the albumin is effected by means of a countercurrent process. A treatment with hydrogen peroxide can take place after the extraction of the albumin in this known method, too. Due to the use of alcohol for the extraction, the method requires high efforts which are disadvantageous. The explosion danger of the suspension and the inactivation of the used enzymes by the presence of alcohol have to be permanently considered. In the known method, the proteolyses is per definitionem limited to a pH-range of < 9,5, only the pH-range of < 7,5 being concretely described in DE 44 29 787 C2. On the other hand it is known to those skilled in the art that the solubility of albumin is particularly great, if the pH is over 9. All at all, the efficiency of the known method as compared to the cost to be spend for its application is only low so that the prepared albumin isolates are not competitive.
A method according the preamble of claim 1 is known from DE 43 39 743 C 1.
This method does without the use of alcohol. For dissolving the protein, however, a pH of over 11,5, particularly of about 12,5, is needed which is associated with a high consumption of alkali and of acid for a later neutralization. Prior to the proteolysis, the albumin containing substance is treated with a protease at a pH > 10.5. The pre-treatment requires a comparatively large amount of enzyme, which results in a drawback with regard to the production cost. The comparatively high salt content at the end of the albumin extraction of this known method, which in praxis requires an additional washing step, is a further drawback.
A method for extraction of albumin from an albumin containing substance which operates with high amounts of enzyme is also know from JP 2 076 597 A. An alkaline protease is added to an alkaline suspension of the albumin containing substance, the pH of which is between 10 and 13, and incubated at a temperature of 30 to 50 °C for 1 to 20 hours.
Afterwards, a neutralization and a filtration of the suspension take place. These indeed dramatic conditions of the albumin extraction result in extended albumin damages. As a result, it is only possible to obtain hydrolysates for cosmetic applications by the known method, it is unsuitable, however, for the preparation of albumin isolates as food.
The application of albumin cleaving enzymes prior to the alkaline extraction which enhances the extractability of the albumins in the following alkaline extraction may be regarded as the common step of the methods which have been described in detail above.
The present invention is based on the problem to disclose a method for preparing an albumin isolate from a substance containing albumin which results in a better albumin yield with at the same time limited use of the agents water, alkali, acid and enzyme, without reducing the quality of the prepared albumin isolate both with albumin containing substances with high and with low albumin solubility.
Summary of the Invention The invention discloses a method for preparing an albumin isolate from a substance containing albumin, the method comprising the steps of grinding the substance to a flour;
suspending the flour being suspended in an aqueous solution; extracting the albumin from the flour into the solution using at least one countercurrent protease; precipitating the albumin from the solution using mineral acid; and neutralizing the precipitated albumin; wherein the step of extracting the albumin from the flour into the solution comprises the further step of subjecting the flour suspended in the aqueous solution to an at least two stage treatment with the at least one protease, with a pH greater than 8 and with heat of temperatures between 30 and 60 °C; the flour being treated in the first stage with a lower concentration of the at least one protease in relation to the albumin weight, with a lower pH and with a higher temperature than in the second stage; at the end of the first stage, a first over flow being separated from a fraction containing the flour, from which over flow the albumin is precipitated; and at the end of the second stage, a second over flow being separated from a fraction containing the flour, which over flow is fed back to the fist stage.
In a preferred embodiment of the method, the albumin containing substance is ground to a flour with an average particle size of 30 to 100 Vim.
Further, it is preferred that the flour suspended in the aqueous solution is homogenized by energy input at the beginning of the second stage.
Further, it is preferred that the flour suspended in the aqueous solution is subjected to a three stage treatment with the at least one protease, with pH-values of greater than 8 and with heat of temperatures between 30 and 60 °C; the flour being treated in the third stage with a higher concentration of the at least one protease in relation to the albumin weight and with a lower temperature than in the second stage; and a third over flow being separated from a fraction containing the flour after the third stage, which over flow is fed back into the second stage.
Further, it is preferred that the protease is added to the solution in the third stage.
Further, it is preferred that the at least one protease is selected from the group comprising serine, cysteine, asparagine and metal proteases.
Further, it is preferred that the at least one protease is inactivated in the albumin by means of a heat treatment.
Further, it is preferred that the albumin is extracted from the flour into the solution using at least one alcohol and/or hydrogen peroxide.
Further, it is preferred that, before the step of extracting the albumin into the solution, the flour is subjected to a pre-treatment using at least one substance selected from the group comprising lyes, acids and enzymes.
Further, it is preferred that at least one of the over flows is separated from the fraction containing the flour in that the flour suspended in the solution is centrifugated on a decanter under vacuum.
In the new method, the dissolution of the albumins is effected out with the aid of an alkaline countercurrent extraction by means of a temperature gradient, different pH-values and a simultaneous application of albumin cleaving enzymes during the extraction cascade. Within an individual case, this extraction can be supported by the application of hydrogen peroxide and/or alcohols.
At first, the starting material, if it does not already have such a small particle size, is milled into a flour with a particle size of 50 to 100 Vim, and mixed with water at a ratio of 1:5 to 1:8, the suspension being raised to a temperature of 30 to 60 °C, particularly of about 50 °C. The pH of the suspension is adjusted to 8 to 10, particularly to about 9, by means of alkaline or alkaline earth hydroxides. To enhance the albumin solubility in this stage of extraction, a protease may be added already here. Preferably, however, only the cleared over flow or the filtrate of the following stage of extraction is added. The enzyme/albumin-ratio obtained should be between 1 to 2000 and 1 to 6000. After an incubation time of 10 to 60 min., particularly of about 20 min, a first separation takes place.
In general, all common preparation techniques are useable. The use of a Sedikanter obtained from Flottweg GmbH, Germany providing a force field of 6000 x g for the separation is preferred.
During the separation, a formation of foam may be essentially avoided by means of a vacuum surrounding. A suitable vacuum is in the range of an absolute pressure of 300 to 500 mbar. In this way, processing of albumin suspensions without an antifoam agent is successful. If it is not possible to carry out the separation under vacuum it has to be cared for a sufficient degassing of the suspension prior to the separation, if no antifoam agents shall be used.
After the separation has been completed, the non-dissolved solid matter is dispersed in water, then homogenized and then again subjected to an alkaline extraction. To this end, the temperature is adjusted to between 30 to 45 °C, particularly to about 40 °C, and the pH is adjusted to between 10,5 and 11,5, particularly to about 11,2. Generally, homogenization may take place at each extraction stage. Particularly effective, however, is the homogenization prior to the stage of extraction described here, which may thus be regarded as sufficient for carrying out the new method. It is important to add the cleared over flow or the filtrate of the following stage of extraction to the stage of extraction described here so that an enzyme/albumin-ratio of between 1:
500 and 1:2000, particularly of about 1:1250, is present. After an extraction time of 10 to 60 min., a second separation takes place.
Afterwards, the solid matter is suspended with water for a third time. The pH
self adjusts to between 10,2 and 10,5 depending of the water quality. The temperature is adjusted to between 25 and 40 °C, particularly to about 35 °C. To this suspension a protease is originally added. The enzyme/albumin-ratio here is between 1:100 to and 1:500, particularly about 1:400. After adding the protease which also may be a protease mixture, an enzymatic hydrolysis takes place for 3 to 30 min. The hydrolysis times depends of the used amount of enzyme, the respective enzyme activity and the kind of protease. Suitable proteases may by selected from the groups of the serine proteases, the cysteine proteases, the asparagine proteases and/or the metal proteases.
Some albumin containing substances as raw material for the new method show sensorics which make the resulting albumin isolate inedible, if no further measures are taken.
Thus, it has been proved advantageous to add hydrogen peroxide to the second alkaline stage of extraction. The amount of hydrogen peroxide as related to a 35 % hydrogen peroxide solution may be between 5 and 50 ml per kg albumin, particularly 20 ml/kg albumin. The concentration has to be adjusted in such a way that damages to the used proteases do not occur.
If the albumin containing starting material contains organic substances which may not be removed by means of an aqueous pre-treatment, it is advantageous to carry out the alkaline extraction in the presence of 5 to 20 % alcohol, particularly of 10 % alcohol.
Herein, the solubility of the organic substances is raised so that they remain in the aqueous solution when the albumins are precipitated at the isoelectric point. It is also sufficient to add the alcohol within the second stage of extraction, if it is cared for that the desired alcohol concentration after feeding back the clear over flow of the second separation into the first stage of extraction is not below 5 %.
In the new method, the clear over flow of the first stage of extraction is, in a way which is known as such, adjusted to the product depending isoelectric point of the albumin which is generally in the pH-range of 4,2 to 4,6 by means of mineral acids or organic acids. By means of this, the albumin is precipitated, and a separation can be carried out with known separation techniques, especially with decanters. The quark-like albumin isolate obtained in this way can be washed and neutralized before drying, to adjust a desired pH for the end product.
Surprisingly, the step by step alkaline extraction using proteases with feeding back the extraction agents results in an albumin quality which is clearly above of the products of known methods.
This is the case both with regard to the yield and the sensorics of the method products. At the same time, the process course in the new method can be tuned in such a way, that a water consumption of about 81 fresh water per kg raw material used is sufficient for all steps of the procedure. Only 25 % of this have to be warm water. The arising sewage water is only about 5 1 per kg raw material used.
Because of the comparatively high solid matter contents of the processed suspensions, despite the higher pH no greater amounts of acid and/or alkali are used in the new method as compared to classic extraction methods. Instead, a reduction of the amounts of acid and alkali and thus of the resulting salt of up to 20 % may be obtained as compared to commercial methods presently used to prepare albumin isolates.
The albumin isolates obtained by the method according to the invention have a typical high albumin content of 90 to 94 % as related to the dry matter content. Depending of the kind of drying applied, the remaining water may be between 4 and 8 %. The yield as related to the albumin content of the starting material is typically between 80 and 87 %.
With a tuned process course, the taste of the method products is neutral. A special taste resulting from the starting material can be removed without problems. The protein damages are low. This is documented, for example, by a very low lysinoalanine content of only about 50 to 150 ppm as related to the albumin in the obtained albumin isolate.
Short description of the Drawing Fig. l shows a flow diagram of an embodiment of the method for preparing an albumin isolate from a substance containing albumin according to the invention.
Description of the preferred Embodiment of the Invention The flow diagram of the method according to the invention which is shown in the accompanying Fig. 1 indicates the preferred ranges of pH and temperature, and starts with a mash which is prepared by mixing the albumin containing starting material having a small particle size with the clear over flow of a separation step. In the right hand part of Fig. 1, the three alkaline/enzymatic stages of extraction of the preferred embodiment of the invention are indicated. In the left hand part, the processing of the over flow 1 of the separation l, i. e. the separation after the first stage of extraction, is shown. At the beginning, this over flow contains the albumin in a dissolved form.
Non dissolved substances within the over flow are removed by clearing.
Afterwards, a precipitation of the albumins at the isoelectric point takes place. The precipitate of this precipitation is removed by separation and again suspended in water. By means of a following heat deactivation the enzymes from the extraction of the albumins are inactivated. In the following separation, the over flow is obtained with which the mash is prepared. This over flow does not contain relevant enzyme activity. The downstream neutralization and drying of the solid matter results into the desired albumin isolate.
Examples In the following, the invention is further explained and described by means of examples.
1. Soya Albumin Isolate Toasted soya meal is at first processed into a albumin concentrate. The albumin concentrate having a particle size of about 50 ~m is suspended in 50 °C warm water to a solid matter concentration of the resulting suspension of 12,5 %. This corresponds to the mash in Fig. 1.
Then, this suspension is added with the fed back flow of the downstream second stage of extraction. Afterwards, the pH is adjusted to 9 with 25 % sodium hydroxide.
The enzyme/albumin-ratio is 1:5500. The suspension is stirred for 15 min at 45 °C. Then, the suspension is separated over a Sedikanter at 6000 x g, a vacuum of 300 mbar absolute pressure being present in the Sedikanter. The over flow of the Sedikanter contains the already dissolved albumin, the under flow contains the solid matter with the still bonded albumin.
The under flow is again suspended in water so that a solid matter content of 8 % is adjusted. The water may be cold. The suspension obtained in this way is homogenized by means of a pressure drop of 80 bar and added with the cleared over flow of the third stage of extraction. The self adjusted temperature is 38 °C, the enzyme/albumin ratio is 1:1250.
Then, the pH is adjusted to 11,2 again with 25 % sodium hydroxide, and it is stirred for 15 min.
Afterwards, a second separation via a Sedikanter under vacuum takes place. The over flow of this Sedikanter is fed back into the first stage of extraction. The solid matter output, i. e. the under flow is dispersed with cold water and adjusted to a solid matter content of 6 %. The self adjusting temperature should be below 40 °C. Now, proteases having a total activity of 6 Anson Units/kg albumin are added to this suspension, which corresponds to an enzyme/albumin-ratio of 1:400. This suspension reacts for 5 min., and is then centrifugated in a Sedikanter in the presence of vacuum in a third separation. The over flow is fed back to the second stage of extraction. The solid matter output or under flow may be neutralized and used as a fibre containing swelling agent, for example. Within the method described here, however, it is not further used.
The over flow of the first stage of extraction is adjusted to pH 4,3 by means of 15 % hydrogen chloride and then centrifugated in a Sedikanter at 300 mbar. The temperature of the input to the Sedikanter is 45 °C. The solid matter output has a dry matter content of 28 %, i. e. the under flow of the Sedikanter contains the albumin, the over flow contains the attendant material and soluble albumin, which is directed to the sewage water. The solid matter is then mixed with water of best quality to 15 % and for a short time heated up to 110 °C. By means of this, the solid matter is washed, and the enzymes contained in it are inactivated. After cooling down to 50 °C a separation of the solid matter from the added water takes place in a standard decanter at about 4000 x g. The over flow of this separation is used for preparing the mash for the first stage of extraction. The under flow is again diluted with water to 15 % dry matter content, neutralized with sodium hydroxide and then spray dried.
2. Potato Albumin Isolate Albumin containing byproducts occurring in the production of starch are at first milled down to a particle size of 50~m and then suspended in 50 °C warm water in such a way that a suspension having a solid matter content of 15 % is produced. A protease having an activity of 5 Anson Units/kg protein is added to this suspension. After 5 min. of stirring, the pH
is adjusted to 8,5, and then it is stirred for further 10 min. Afterwards, the pH of the suspension is adjusted to 4,2 by means of hydrogen chloride, and the suspension is centrifugated on a decanter at 4000 x g. The over flow is removed; the solid matter is again suspended in 50 °C warm water and then process according to the steps of the new method shown in Fig. 1. To this end, the over flow from the second stage of extraction is added to the solid matter suspended in water.
The enzyme/albumin-ratio is 1:6500, then. The solid matter content is 10 %. The pH
is adjusted to 9, and afterwards it is stirred for 15 min. Here, the temperature should be 45 °C. Afterwards a centrifugation in a Sedikanter at 6000 x g takes place. Here, the over flow contains the dissolved protein. The under flow, i. e. the solid matter output having a solid matter content of 26 %, is suspended in cold water and added with the over flow of the following third stage of extraction.
The temperature self adjusts to 38 °C. The pH is raised to 11,2 by a 25 % sodium hydroxide solution. The enzyme/albumin-ratio is 1:1600. This suspension is stirred for l5min, 20 pl of 25 hydrogen peroxide per kg of solid matter being added after 10 min. After the stirring time of 15 min, the suspension is homogenised by via a pressure drop of 80 bars, and then separated in a second Sedikanter under vacuum of 300 mbar. The over flow is added to the first stage of extraction; the under flow containing the solid matter is again suspended in cold water. The temperature of the dispersed solid matter self adjusts to 32 °C. The pH
reaches 10,5. Now, proteases having an activity of 4 Anson Units/kg proteins are added, resulting in an enzyme/albumin-ratio of 1:500. After 5 min., this dispersion is centrifugated in a Sedikanter for a third time, again under vacuum of 300 mbar absolute pressure. The over flow of this separation is fed back to the second stage of extraction. The solid matter output, i. e. the under flow, is disposed.
The over flow of the first stage of extraction is adjusted to pH 4,3 by means of hydrogen chloride.
Afterwards, it is centrifuged in a Sedikanter at an under pressure of 300 mbar absolute. The over flow of the Sedikanter is disposed. The quark-like under flow is adjusted to a solid matter content of 15 % with demineralised water. This dispersion is heated up to 110 °C for a short time, then cooled down to 50 °C and centrifuged in a decanter at 4000 x g. The cleared flow or over flow obtained here is used for preparing the mash from the flour of the starting materials for the first stage of extraction. The solid matter output, i. e. the under flow, is neutralized and dried.
Instead of hydrogen peroxide also ethanol can be used in the preparation of potato albumin isolate for extracting undesired organic attendant materials.
Afterwards, a neutralization and a filtration of the suspension take place. These indeed dramatic conditions of the albumin extraction result in extended albumin damages. As a result, it is only possible to obtain hydrolysates for cosmetic applications by the known method, it is unsuitable, however, for the preparation of albumin isolates as food.
The application of albumin cleaving enzymes prior to the alkaline extraction which enhances the extractability of the albumins in the following alkaline extraction may be regarded as the common step of the methods which have been described in detail above.
The present invention is based on the problem to disclose a method for preparing an albumin isolate from a substance containing albumin which results in a better albumin yield with at the same time limited use of the agents water, alkali, acid and enzyme, without reducing the quality of the prepared albumin isolate both with albumin containing substances with high and with low albumin solubility.
Summary of the Invention The invention discloses a method for preparing an albumin isolate from a substance containing albumin, the method comprising the steps of grinding the substance to a flour;
suspending the flour being suspended in an aqueous solution; extracting the albumin from the flour into the solution using at least one countercurrent protease; precipitating the albumin from the solution using mineral acid; and neutralizing the precipitated albumin; wherein the step of extracting the albumin from the flour into the solution comprises the further step of subjecting the flour suspended in the aqueous solution to an at least two stage treatment with the at least one protease, with a pH greater than 8 and with heat of temperatures between 30 and 60 °C; the flour being treated in the first stage with a lower concentration of the at least one protease in relation to the albumin weight, with a lower pH and with a higher temperature than in the second stage; at the end of the first stage, a first over flow being separated from a fraction containing the flour, from which over flow the albumin is precipitated; and at the end of the second stage, a second over flow being separated from a fraction containing the flour, which over flow is fed back to the fist stage.
In a preferred embodiment of the method, the albumin containing substance is ground to a flour with an average particle size of 30 to 100 Vim.
Further, it is preferred that the flour suspended in the aqueous solution is homogenized by energy input at the beginning of the second stage.
Further, it is preferred that the flour suspended in the aqueous solution is subjected to a three stage treatment with the at least one protease, with pH-values of greater than 8 and with heat of temperatures between 30 and 60 °C; the flour being treated in the third stage with a higher concentration of the at least one protease in relation to the albumin weight and with a lower temperature than in the second stage; and a third over flow being separated from a fraction containing the flour after the third stage, which over flow is fed back into the second stage.
Further, it is preferred that the protease is added to the solution in the third stage.
Further, it is preferred that the at least one protease is selected from the group comprising serine, cysteine, asparagine and metal proteases.
Further, it is preferred that the at least one protease is inactivated in the albumin by means of a heat treatment.
Further, it is preferred that the albumin is extracted from the flour into the solution using at least one alcohol and/or hydrogen peroxide.
Further, it is preferred that, before the step of extracting the albumin into the solution, the flour is subjected to a pre-treatment using at least one substance selected from the group comprising lyes, acids and enzymes.
Further, it is preferred that at least one of the over flows is separated from the fraction containing the flour in that the flour suspended in the solution is centrifugated on a decanter under vacuum.
In the new method, the dissolution of the albumins is effected out with the aid of an alkaline countercurrent extraction by means of a temperature gradient, different pH-values and a simultaneous application of albumin cleaving enzymes during the extraction cascade. Within an individual case, this extraction can be supported by the application of hydrogen peroxide and/or alcohols.
At first, the starting material, if it does not already have such a small particle size, is milled into a flour with a particle size of 50 to 100 Vim, and mixed with water at a ratio of 1:5 to 1:8, the suspension being raised to a temperature of 30 to 60 °C, particularly of about 50 °C. The pH of the suspension is adjusted to 8 to 10, particularly to about 9, by means of alkaline or alkaline earth hydroxides. To enhance the albumin solubility in this stage of extraction, a protease may be added already here. Preferably, however, only the cleared over flow or the filtrate of the following stage of extraction is added. The enzyme/albumin-ratio obtained should be between 1 to 2000 and 1 to 6000. After an incubation time of 10 to 60 min., particularly of about 20 min, a first separation takes place.
In general, all common preparation techniques are useable. The use of a Sedikanter obtained from Flottweg GmbH, Germany providing a force field of 6000 x g for the separation is preferred.
During the separation, a formation of foam may be essentially avoided by means of a vacuum surrounding. A suitable vacuum is in the range of an absolute pressure of 300 to 500 mbar. In this way, processing of albumin suspensions without an antifoam agent is successful. If it is not possible to carry out the separation under vacuum it has to be cared for a sufficient degassing of the suspension prior to the separation, if no antifoam agents shall be used.
After the separation has been completed, the non-dissolved solid matter is dispersed in water, then homogenized and then again subjected to an alkaline extraction. To this end, the temperature is adjusted to between 30 to 45 °C, particularly to about 40 °C, and the pH is adjusted to between 10,5 and 11,5, particularly to about 11,2. Generally, homogenization may take place at each extraction stage. Particularly effective, however, is the homogenization prior to the stage of extraction described here, which may thus be regarded as sufficient for carrying out the new method. It is important to add the cleared over flow or the filtrate of the following stage of extraction to the stage of extraction described here so that an enzyme/albumin-ratio of between 1:
500 and 1:2000, particularly of about 1:1250, is present. After an extraction time of 10 to 60 min., a second separation takes place.
Afterwards, the solid matter is suspended with water for a third time. The pH
self adjusts to between 10,2 and 10,5 depending of the water quality. The temperature is adjusted to between 25 and 40 °C, particularly to about 35 °C. To this suspension a protease is originally added. The enzyme/albumin-ratio here is between 1:100 to and 1:500, particularly about 1:400. After adding the protease which also may be a protease mixture, an enzymatic hydrolysis takes place for 3 to 30 min. The hydrolysis times depends of the used amount of enzyme, the respective enzyme activity and the kind of protease. Suitable proteases may by selected from the groups of the serine proteases, the cysteine proteases, the asparagine proteases and/or the metal proteases.
Some albumin containing substances as raw material for the new method show sensorics which make the resulting albumin isolate inedible, if no further measures are taken.
Thus, it has been proved advantageous to add hydrogen peroxide to the second alkaline stage of extraction. The amount of hydrogen peroxide as related to a 35 % hydrogen peroxide solution may be between 5 and 50 ml per kg albumin, particularly 20 ml/kg albumin. The concentration has to be adjusted in such a way that damages to the used proteases do not occur.
If the albumin containing starting material contains organic substances which may not be removed by means of an aqueous pre-treatment, it is advantageous to carry out the alkaline extraction in the presence of 5 to 20 % alcohol, particularly of 10 % alcohol.
Herein, the solubility of the organic substances is raised so that they remain in the aqueous solution when the albumins are precipitated at the isoelectric point. It is also sufficient to add the alcohol within the second stage of extraction, if it is cared for that the desired alcohol concentration after feeding back the clear over flow of the second separation into the first stage of extraction is not below 5 %.
In the new method, the clear over flow of the first stage of extraction is, in a way which is known as such, adjusted to the product depending isoelectric point of the albumin which is generally in the pH-range of 4,2 to 4,6 by means of mineral acids or organic acids. By means of this, the albumin is precipitated, and a separation can be carried out with known separation techniques, especially with decanters. The quark-like albumin isolate obtained in this way can be washed and neutralized before drying, to adjust a desired pH for the end product.
Surprisingly, the step by step alkaline extraction using proteases with feeding back the extraction agents results in an albumin quality which is clearly above of the products of known methods.
This is the case both with regard to the yield and the sensorics of the method products. At the same time, the process course in the new method can be tuned in such a way, that a water consumption of about 81 fresh water per kg raw material used is sufficient for all steps of the procedure. Only 25 % of this have to be warm water. The arising sewage water is only about 5 1 per kg raw material used.
Because of the comparatively high solid matter contents of the processed suspensions, despite the higher pH no greater amounts of acid and/or alkali are used in the new method as compared to classic extraction methods. Instead, a reduction of the amounts of acid and alkali and thus of the resulting salt of up to 20 % may be obtained as compared to commercial methods presently used to prepare albumin isolates.
The albumin isolates obtained by the method according to the invention have a typical high albumin content of 90 to 94 % as related to the dry matter content. Depending of the kind of drying applied, the remaining water may be between 4 and 8 %. The yield as related to the albumin content of the starting material is typically between 80 and 87 %.
With a tuned process course, the taste of the method products is neutral. A special taste resulting from the starting material can be removed without problems. The protein damages are low. This is documented, for example, by a very low lysinoalanine content of only about 50 to 150 ppm as related to the albumin in the obtained albumin isolate.
Short description of the Drawing Fig. l shows a flow diagram of an embodiment of the method for preparing an albumin isolate from a substance containing albumin according to the invention.
Description of the preferred Embodiment of the Invention The flow diagram of the method according to the invention which is shown in the accompanying Fig. 1 indicates the preferred ranges of pH and temperature, and starts with a mash which is prepared by mixing the albumin containing starting material having a small particle size with the clear over flow of a separation step. In the right hand part of Fig. 1, the three alkaline/enzymatic stages of extraction of the preferred embodiment of the invention are indicated. In the left hand part, the processing of the over flow 1 of the separation l, i. e. the separation after the first stage of extraction, is shown. At the beginning, this over flow contains the albumin in a dissolved form.
Non dissolved substances within the over flow are removed by clearing.
Afterwards, a precipitation of the albumins at the isoelectric point takes place. The precipitate of this precipitation is removed by separation and again suspended in water. By means of a following heat deactivation the enzymes from the extraction of the albumins are inactivated. In the following separation, the over flow is obtained with which the mash is prepared. This over flow does not contain relevant enzyme activity. The downstream neutralization and drying of the solid matter results into the desired albumin isolate.
Examples In the following, the invention is further explained and described by means of examples.
1. Soya Albumin Isolate Toasted soya meal is at first processed into a albumin concentrate. The albumin concentrate having a particle size of about 50 ~m is suspended in 50 °C warm water to a solid matter concentration of the resulting suspension of 12,5 %. This corresponds to the mash in Fig. 1.
Then, this suspension is added with the fed back flow of the downstream second stage of extraction. Afterwards, the pH is adjusted to 9 with 25 % sodium hydroxide.
The enzyme/albumin-ratio is 1:5500. The suspension is stirred for 15 min at 45 °C. Then, the suspension is separated over a Sedikanter at 6000 x g, a vacuum of 300 mbar absolute pressure being present in the Sedikanter. The over flow of the Sedikanter contains the already dissolved albumin, the under flow contains the solid matter with the still bonded albumin.
The under flow is again suspended in water so that a solid matter content of 8 % is adjusted. The water may be cold. The suspension obtained in this way is homogenized by means of a pressure drop of 80 bar and added with the cleared over flow of the third stage of extraction. The self adjusted temperature is 38 °C, the enzyme/albumin ratio is 1:1250.
Then, the pH is adjusted to 11,2 again with 25 % sodium hydroxide, and it is stirred for 15 min.
Afterwards, a second separation via a Sedikanter under vacuum takes place. The over flow of this Sedikanter is fed back into the first stage of extraction. The solid matter output, i. e. the under flow is dispersed with cold water and adjusted to a solid matter content of 6 %. The self adjusting temperature should be below 40 °C. Now, proteases having a total activity of 6 Anson Units/kg albumin are added to this suspension, which corresponds to an enzyme/albumin-ratio of 1:400. This suspension reacts for 5 min., and is then centrifugated in a Sedikanter in the presence of vacuum in a third separation. The over flow is fed back to the second stage of extraction. The solid matter output or under flow may be neutralized and used as a fibre containing swelling agent, for example. Within the method described here, however, it is not further used.
The over flow of the first stage of extraction is adjusted to pH 4,3 by means of 15 % hydrogen chloride and then centrifugated in a Sedikanter at 300 mbar. The temperature of the input to the Sedikanter is 45 °C. The solid matter output has a dry matter content of 28 %, i. e. the under flow of the Sedikanter contains the albumin, the over flow contains the attendant material and soluble albumin, which is directed to the sewage water. The solid matter is then mixed with water of best quality to 15 % and for a short time heated up to 110 °C. By means of this, the solid matter is washed, and the enzymes contained in it are inactivated. After cooling down to 50 °C a separation of the solid matter from the added water takes place in a standard decanter at about 4000 x g. The over flow of this separation is used for preparing the mash for the first stage of extraction. The under flow is again diluted with water to 15 % dry matter content, neutralized with sodium hydroxide and then spray dried.
2. Potato Albumin Isolate Albumin containing byproducts occurring in the production of starch are at first milled down to a particle size of 50~m and then suspended in 50 °C warm water in such a way that a suspension having a solid matter content of 15 % is produced. A protease having an activity of 5 Anson Units/kg protein is added to this suspension. After 5 min. of stirring, the pH
is adjusted to 8,5, and then it is stirred for further 10 min. Afterwards, the pH of the suspension is adjusted to 4,2 by means of hydrogen chloride, and the suspension is centrifugated on a decanter at 4000 x g. The over flow is removed; the solid matter is again suspended in 50 °C warm water and then process according to the steps of the new method shown in Fig. 1. To this end, the over flow from the second stage of extraction is added to the solid matter suspended in water.
The enzyme/albumin-ratio is 1:6500, then. The solid matter content is 10 %. The pH
is adjusted to 9, and afterwards it is stirred for 15 min. Here, the temperature should be 45 °C. Afterwards a centrifugation in a Sedikanter at 6000 x g takes place. Here, the over flow contains the dissolved protein. The under flow, i. e. the solid matter output having a solid matter content of 26 %, is suspended in cold water and added with the over flow of the following third stage of extraction.
The temperature self adjusts to 38 °C. The pH is raised to 11,2 by a 25 % sodium hydroxide solution. The enzyme/albumin-ratio is 1:1600. This suspension is stirred for l5min, 20 pl of 25 hydrogen peroxide per kg of solid matter being added after 10 min. After the stirring time of 15 min, the suspension is homogenised by via a pressure drop of 80 bars, and then separated in a second Sedikanter under vacuum of 300 mbar. The over flow is added to the first stage of extraction; the under flow containing the solid matter is again suspended in cold water. The temperature of the dispersed solid matter self adjusts to 32 °C. The pH
reaches 10,5. Now, proteases having an activity of 4 Anson Units/kg proteins are added, resulting in an enzyme/albumin-ratio of 1:500. After 5 min., this dispersion is centrifugated in a Sedikanter for a third time, again under vacuum of 300 mbar absolute pressure. The over flow of this separation is fed back to the second stage of extraction. The solid matter output, i. e. the under flow, is disposed.
The over flow of the first stage of extraction is adjusted to pH 4,3 by means of hydrogen chloride.
Afterwards, it is centrifuged in a Sedikanter at an under pressure of 300 mbar absolute. The over flow of the Sedikanter is disposed. The quark-like under flow is adjusted to a solid matter content of 15 % with demineralised water. This dispersion is heated up to 110 °C for a short time, then cooled down to 50 °C and centrifuged in a decanter at 4000 x g. The cleared flow or over flow obtained here is used for preparing the mash from the flour of the starting materials for the first stage of extraction. The solid matter output, i. e. the under flow, is neutralized and dried.
Instead of hydrogen peroxide also ethanol can be used in the preparation of potato albumin isolate for extracting undesired organic attendant materials.
Claims (10)
1. A method for preparing an albumin isolate from a substance containing albumin, the method comprising the steps of:
grinding the substance to a flour;
suspending the flour being suspended in an aqueous solution;
extracting the albumin from the flour into the solution using at least one countercurrent protease;
precipitating the albumin from the solution using mineral acid; and neutralizing the precipitated albumin;
wherein the step of extracting the albumin from the flour into the solution comprises the further step of:
subjecting the flour suspended in the aqueous solution to an at least two stage treatment with the at least one protease, with a pH greater than 8 and with heat of temperatures between 30 and 60 °C;
the flour being treated in the first stage - with a lower concentration of the at least one protease in relation to the albumin weight, - with a lower pH and - with a higher temperature than in the second stage;
at the end of the first stage, a first over flow being separated from a fraction containing the flour, from which over flow the albumin is precipitated; and at the end of the second stage, a second over flow being separated from a fraction containing the flour, which over flow is fed back to the fist stage.
grinding the substance to a flour;
suspending the flour being suspended in an aqueous solution;
extracting the albumin from the flour into the solution using at least one countercurrent protease;
precipitating the albumin from the solution using mineral acid; and neutralizing the precipitated albumin;
wherein the step of extracting the albumin from the flour into the solution comprises the further step of:
subjecting the flour suspended in the aqueous solution to an at least two stage treatment with the at least one protease, with a pH greater than 8 and with heat of temperatures between 30 and 60 °C;
the flour being treated in the first stage - with a lower concentration of the at least one protease in relation to the albumin weight, - with a lower pH and - with a higher temperature than in the second stage;
at the end of the first stage, a first over flow being separated from a fraction containing the flour, from which over flow the albumin is precipitated; and at the end of the second stage, a second over flow being separated from a fraction containing the flour, which over flow is fed back to the fist stage.
2. The method of claim 1, wherein the albumin containing substance is ground to a flour with an average particle size of 30 to 100 µm.
3. The method of claim 1, wherein the flour suspended in the aqueous solution is homogenized by energy input at the beginning of the second stage.
4. The method of claim 1, wherein the flour suspended in the aqueous solution is subjected to a three stage treatment with the at least one protease, with pH-values of greater than 8 and with heat of temperatures between 30 and 60 °C; the flour being treated in the third stage with a higher concentration of the at least one protease in relation to the albumin weight and with a lower temperature than in the second stage; and a third over flow being separated from a fraction containing the flour after the third stage, which over flow is fed back into the second stage.
5. The method of claim 4, wherein the protease is added to the solution in the third stage.
6. The method of claim 1, wherein the at least one protease is selected from the group comprising serine, cysteine, asparagine and metal proteases.
7. The method of claim 1, wherein the at least one protease is inactivated in the albumin by means of a heat treatment.
8. The method of claim 1, wherein the albumin is extracted from the flour into the solution using at least one alcohol and/or hydrogen peroxide.
9. The method of claim 1, wherein, before the step of extracting the albumin into the solution, the flour is subjected to a pre-treatment using at least one substance selected from the group comprising lyes, acids and enzymes.
10. The method of claim 1 wherein at least one of the over flows is separated from the fraction containing the flour in that the flour suspended in the solution is centrifugated on a decanter under vacuum.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19907725A DE19907725A1 (en) | 1999-02-23 | 1999-02-23 | Production of protein isolates in good yield and purity using an extraction process which involves the use of a gradient of protease concentration, pH and temperature |
| DE19907725.8 | 1999-02-23 | ||
| PCT/EP2000/001485 WO2000049887A1 (en) | 1999-02-23 | 2000-02-23 | Method for preparing an albumin isolate from a substance containing albumin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2362948A1 true CA2362948A1 (en) | 2000-08-31 |
Family
ID=7898534
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002362948A Abandoned CA2362948A1 (en) | 1999-02-23 | 2000-02-23 | Method for preparing an albumin isolate from a substance containing albumin |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1154702B1 (en) |
| AT (1) | ATE221323T1 (en) |
| AU (1) | AU2913900A (en) |
| CA (1) | CA2362948A1 (en) |
| DE (2) | DE19907725A1 (en) |
| PL (1) | PL350586A1 (en) |
| RU (1) | RU2233097C2 (en) |
| WO (1) | WO2000049887A1 (en) |
Families Citing this family (8)
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|---|---|---|---|---|
| EP1264545A1 (en) * | 2001-06-08 | 2002-12-11 | Coöperatieve Verkoop- en Productievereniging van Aardappelmeel en Derivaten 'AVEBE' B.A. | A method for improving a protein product |
| DE10224257B4 (en) * | 2002-05-31 | 2008-11-27 | Neumüller, Waldemar, Dr. | Process for obtaining a protein isolate and a fiber fraction from a fibrous and protein-containing starting substance |
| FR2960129B1 (en) * | 2010-05-20 | 2014-10-10 | Roquette Freres | PROCESS FOR THE PREPARATION OF ALKALINE HYDROLYSATES OF PLANT PROTEINS |
| ES2915253T3 (en) | 2015-02-16 | 2022-06-21 | Cooeperatie Koninklijke Avebe U A | Method for preparing a food grade coagulated potato protein concentrate |
| RU2612151C1 (en) | 2016-05-05 | 2017-03-02 | Сергей Борисович Тришин | Method of producing soy protein isolate |
| RU2633501C1 (en) | 2016-07-12 | 2017-10-12 | Сергей Борисович Тришин | Plant for obtaining soy protein isolate |
| RU2709384C1 (en) * | 2019-04-30 | 2019-12-17 | Дмитрий Викторович Морозов | Method of producing soya isolated protein |
| EA202192702A1 (en) * | 2019-05-24 | 2022-03-05 | Кооперати Конинклейке Авебе У.А. | PROTEIN ISOLATION FROM PEELED TUBS |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH444879A (en) * | 1965-09-17 | 1967-10-15 | Nestle Sa | Process for preparing a protein hydrolyzate and hydrolyzate obtained by this process |
| US4443540A (en) * | 1980-05-09 | 1984-04-17 | University Of Illinois Foundation | Protein hydrolysis |
| US4559307A (en) * | 1983-10-17 | 1985-12-17 | Phillips Petroleum Company | Treatment of yeast cells with proteolytic enzymes |
| DE4339743C1 (en) * | 1993-11-22 | 1995-08-31 | Waldemar Dr Neumueller | Process for the preparation of proteins from a protein-containing substance |
| DE4429787C2 (en) * | 1994-08-23 | 1996-08-14 | Braunschweigische Masch Bau | Process for producing a food-grade protein |
-
1999
- 1999-02-23 DE DE19907725A patent/DE19907725A1/en not_active Ceased
-
2000
- 2000-02-23 CA CA002362948A patent/CA2362948A1/en not_active Abandoned
- 2000-02-23 DE DE50000337T patent/DE50000337D1/en not_active Expired - Lifetime
- 2000-02-23 EP EP00907609A patent/EP1154702B1/en not_active Expired - Lifetime
- 2000-02-23 AU AU29139/00A patent/AU2913900A/en not_active Abandoned
- 2000-02-23 PL PL00350586A patent/PL350586A1/en unknown
- 2000-02-23 AT AT00907609T patent/ATE221323T1/en not_active IP Right Cessation
- 2000-02-23 RU RU2001125936/13A patent/RU2233097C2/en not_active IP Right Cessation
- 2000-02-23 WO PCT/EP2000/001485 patent/WO2000049887A1/en active IP Right Grant
Also Published As
| Publication number | Publication date |
|---|---|
| AU2913900A (en) | 2000-09-14 |
| PL350586A1 (en) | 2003-01-13 |
| DE50000337D1 (en) | 2002-09-05 |
| RU2233097C2 (en) | 2004-07-27 |
| ATE221323T1 (en) | 2002-08-15 |
| EP1154702B1 (en) | 2002-07-31 |
| DE19907725A1 (en) | 2000-08-24 |
| WO2000049887A1 (en) | 2000-08-31 |
| EP1154702A1 (en) | 2001-11-21 |
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