JP2011234682A - Method for preparing complex containing peptide and heme iron derived from heme protein obtained from fish blood - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an alternative raw material for enzyme-treated heme iron (HIP) as a substitute for mammal blood.SOLUTION: There is provided a method for preparing, from fish blood, a complex containing a peptide and heme iron derived from a heme protein. The method includes the steps of producing an aqueous solution of the heme protein by denaturation treatment of fish blood containing heme protein, decomposing the denatured heme protein by protease treatment of the aqueous solution to obtain a protease treated liquid containing a complex containing the peptide and heme iron derived from the heme protein, and performing ultrafiltration of the treated liquid.

Description

  The present invention relates to a method for producing a complex containing a peptide derived from heme protein and heme iron from fish blood containing heme protein.

  Iron, an essential mineral for humans, is usually taken from food and absorbed into the body. Here, heme iron contained in animal foods and non-heme iron contained in many plant foods have different absorption mechanisms (Non-Patent Documents 1 and 2). Heme iron is absorbed from intestinal mucosal cells in the form of an iron-porphyrin complex, whereas non-heme iron is digested and decomposed to become free iron ions and is absorbed into the body for the first time. Heme iron is not absorbed by other foods, whereas non-heme iron is not easily absorbed by dietary fiber, calcium, phosphate, etc. in addition to tannins contained in tea or coffee. Absorption rate in the body decreases.

  In the case of hemoglobin, the absorption rate of heme iron is five times that of non-heme iron. However, in the case of only heme iron from which the protein portion (globin) of hemoglobin is removed, the absorption rate in the body is significantly reduced. To do. This is thought to be due to the polymerization (stacking) of heme iron molecules.

  A peptide that maintains the stability of hemoglobin derived from hemoglobin and heme iron obtained by removing unnecessary peptides from the group of peptides derived from hemoglobin contained in the degradation product after degrading hemoglobin with protease Enzyme-treated heme iron (HIP) is defined as a complex.

  After decomposing hemoglobin with a protease, the heme iron content of HIP can be increased by removing peptides that are not bound to heme iron from the degradation product. For this reason, when HIP is added to food, a small amount of addition is sufficient compared with the case where hemoglobin itself is added. Therefore, when HIP is added to food, there is an advantage that the taste and flavor of the food are hardly impaired.

  In addition, when preparing HIP, if ultrafiltration is used to separate unwanted peptides that are not bound to heme iron from HIP, the resulting HIP has high solubility in water and absorbability in the intestinal tract It is known to show the characteristic of being high (Patent Document 1 and Non-Patent Documents 3 and 4). The above feature is thought to be due to the improvement in water solubility of HIP as a whole by forming HIP so that hemoglobin-derived peptide protects poorly water-soluble heme iron.

  Lebrun et al. Perform separation by ultrafiltration of a degradation product obtained by degrading bovine-derived hemoglobin with pepsin, and perform detailed analysis of the obtained separated fraction (Non-patent Documents 4 and 5). In this study, it was clarified that a peptide having a molecular weight of about 3,000 obtained by the decomposition of hemoglobin forms a complex with heme iron, and that heme iron is water-soluble.

  Currently marketed heme iron is HIP produced from the blood of mammals such as pigs. However, there are people who cannot take mammal-derived heme iron for religious reasons. In addition, mammal-derived heme iron may not be able to be ingested due to pathogenic reasons such as the occurrence of infections through the blood of mammals.

Japanese Patent Publication No. 01-24137

Conrad et al., J. Lab. Clin. Med., 68, p. 659-668 (1966) Uzel et al., Semin. Hematol., 35, p. 27-34 (1998) Piot et al., J. Chem. Technol. Biotechnol., 42, p. 147-156 (1988) Lebrun et al., J. Membr. Sci., 146, p. 113-124 (1998) Lebrun et al., J. Agric. Food Chem., 46, p. 5017-5025 (1998)

  As described above, HIP produced using mammalian blood as a raw material may be incapable of being ingested for religious reasons or incapable of being ingested for pathogenic reasons. Therefore, there is a need for alternative HIP materials that replace mammalian blood.

  The amount of hemoglobin contained in the blood of teleosts (fish blood) is reported to be 8-15 g / 100 ml (Saitoh, Nippon Suisan Gakkaishi, 29, 1104-1107 (1963)). For this reason, if HIP can be produced using fish blood as a raw material, it can be used with peace of mind even for those who cannot take it for religious reasons, or who cannot take it for pathogenic reasons. Heme iron can be ingested. As a result, it becomes possible to use it as a raw material for dietary supplements, etc. as HIP having a new added value, which is not found in conventional products using blood derived from mammals as a raw material.

  In Japan, a large amount of fish blood is discharged due to the aliveness of farmed fish. At present, such fish blood has no use and is disposed of as industrial waste in the farm. Therefore, if fish blood can be recovered and used as a raw material for HIP, not only can industrial waste be effectively utilized as an unused resource, but also compared to conventional products that use blood derived from mammals as a raw material, HIP can be produced at a lower cost. Therefore, an object of the present invention is to provide a method for producing a complex containing a peptide derived from heme protein and heme iron by using fish blood containing heme protein as a raw material.

  As a result of various studies on means for solving the above problems, the present inventor has denatured heme protein contained in fish blood, and then treated with protease to produce a complex containing a peptide derived from heme protein and heme iron It was found that a complex containing peptide and heme iron could be efficiently prepared from fish blood by forming a body and separating it by ultrafiltration, and completed the present invention.

That is, the gist of the present invention is as follows.
(1) A method for producing a complex comprising a peptide derived from heme protein and heme iron,
(I) a hemoprotein denaturation step of denaturing fish blood containing heme protein to denature the heme protein to obtain a denatured heme protein aqueous solution;
(Ii) a protease treatment step in which an aqueous solution of the denatured heme protein is treated with a protease to decompose the denatured heme protein to obtain a protease treatment solution containing a complex containing a peptide derived from heme protein and heme iron;
(Iii) Ultrafiltration step of ultrafiltration of the protease treatment solution to obtain a complex containing a peptide derived from heme protein and heme iron from the retained fraction;
Including said method.

(2) The method according to (1) above, wherein the ultrafiltration step is performed using an ultrafiltration membrane having a molecular weight cut off of 20,000 to 50,000.
(3) The method according to (1) or (2) above, wherein the denaturation treatment is carried out by mixing fish blood with alkali.

  According to the present invention, it is possible to produce a complex containing a peptide derived from heme protein and heme iron using fish blood obtained by slaughtering cultured fish as a raw material.

It is a schematic diagram which shows the method of this invention. It is a figure which shows the gel filtration chromatogram (detection wavelength 230nm) of the retentate fraction dilution liquid of yellowtail blood. It is a figure which shows the gel filtration chromatogram (detection wavelength 230nm) of the protease processing liquid obtained by the protease processing process. It is a figure which shows the gel filtration chromatogram (detection wavelength 230nm) of the retentate fraction of the ultrafiltration obtained by the ultrafiltration process. It is a figure which shows the gel filtration chromatogram (detection wavelength 230 nm) of the permeate | liquid fraction of the ultrafiltration obtained by the ultrafiltration process. It is a figure which shows the gel filtration chromatogram (detection wavelength 400nm) of the retentate fraction of the ultrafiltration obtained by the ultrafiltration process. It is a figure which shows the gel filtration chromatogram (detection wavelength 400nm) of the permeate | liquid fraction of the ultrafiltration obtained by the ultrafiltration process. Presence of iron in the retentate fraction, 1 permeate fraction, and 2 permeate fractions obtained by separating untreated yellowtail fish blood by ultrafiltration (based on the total iron weight of all fractions) It is a figure which shows (percentage). Presence of iron in the retentate fraction, 1 permeate fraction, and 2 permeate fractions obtained by separating the denatured hemoprotein aqueous solution by ultrafiltration (percentage of the total iron weight of all fractions) FIG. The abundance of iron in the retentate fraction, permeate 1 fraction, and permeate 2 fraction obtained by separating the protease treatment liquid by ultrafiltration (percentage of the total iron weight of all fractions) FIG. It is a figure which shows the flow rate of the permeation | transmission liquid at the time of isolate | separating a protease processing liquid using the ultrafiltration membrane of a different fraction molecular weight.

Hereinafter, preferred embodiments of the present invention will be described in detail.
In the present specification, “fish blood” means fish blood. In the present invention, fish blood derived from either cartilaginous fish or teleost fish can be used. Cultured fish with a large production volume such as yellowtails or Thailand is more preferable because a certain amount can always be secured.

By using fish blood derived from the above fishes, it is possible to stably supply raw materials.
In this specification, “heme protein” means a protein containing heme iron as a constituent component, and examples thereof include proteins such as hemoglobin and myoglobin.

  As used herein, “heme iron” means an iron complex of porphyrin, such as protoporphyrin IX iron complex. In the blood of animals, including fish blood, heme iron exists in the form of a complex with heme protein.

  The method for producing a complex comprising a heme protein-derived peptide and heme iron from fish blood containing heme protein provided by the present invention includes a heme protein denaturation step, a protease treatment step, and ultrafiltration. Process. Each step will be described below.

1. Heme protein denaturation step This step aims to obtain a denatured heme protein by denaturing heme protein contained in fish blood.

  In the present specification, the “denaturation treatment” means a treatment that destroys the higher-order structure of the protein. The “denatured hemoprotein” means a hemoprotein whose higher-order structure is destroyed by denaturation treatment. The denaturation treatment can be carried out by heat treating fish blood containing heme protein or mixing it with acid or alkali. When denaturation treatment is performed by mixing fish blood with acid or alkali, the acid or alkali may be mixed with fish blood in a form that does not contain a medium, or mixed in the form of a solution dissolved in a medium. May be. It is preferably mixed with fish blood in the form of an acid or alkaline aqueous solution. Degeneration of heme protein contained in fish blood can promote the degradation of heme protein in the protease treatment step described below.

  When performing denaturation treatment by heat-treating fish blood, leave the container containing fish blood in a predetermined temperature environment or shake the container in a predetermined temperature environment to The contained heme protein can be heat denatured. Here, the temperature at which the above mixture is heat-treated is preferably in the range of 80 to 130 ° C, and more preferably in the range of 90 to 100 ° C. The time for heat-treating the above mixture is preferably in the range of 0.1 to 24 hours, and more preferably in the range of 0.1 to 4 hours. By performing the heat treatment under the above conditions, the hemoprotein can be denatured in a short time to obtain a denatured hemoprotein aqueous solution.

When the fish blood is denatured by acid or alkali treatment, after mixing the fish blood with acid or alkali, the mixture obtained in a predetermined temperature environment is allowed to stand, or the mixture is subjected to a predetermined treatment. By shaking in a temperature environment, the heme protein contained in fish blood can be denatured with acid or alkali. Here, the temperature at which the above mixture is treated with an acid or an alkali is preferably in the range of 0 to 80 ° C. In the case of acid treatment, fish blood and acid may be mixed so that the pH of the mixture after treatment is in the range of 0-2. Examples of the acid used include acids such as hydrochloric acid, sulfuric acid and nitric acid. Hydrochloric acid is preferred. When used in the form of an aqueous acid solution, the concentration of the aqueous acid solution is preferably at least 1 × 10 ⁻³ mol dm ⁻³ . In the case of alkali treatment, fish blood and alkali may be mixed so that the pH of the mixture after treatment is in the range of 12-14. Examples of the alkali used include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide. When used in the form of an aqueous alkaline solution, the concentration of the aqueous alkaline solution is preferably at least 1 × 10 ⁻³ mol dm ⁻³ .

  The mixture obtained by the acid or alkali treatment in the above procedure can be used as it is in the next step as a denatured hemoprotein aqueous solution. Alternatively, after mixing fish blood with acid or alkali according to the above procedure, the resulting mixture is mixed with alkali or acid to neutralize the mixture, whereby a denatured heme protein aqueous solution exhibiting a neutral pH is obtained. May be obtained. The alkali or acid used for neutralization may be mixed with the mixture after the acid or alkali treatment in a form not containing the medium, or may be mixed in the form of a solution dissolved in the medium. It is preferable to mix with the denatured hemoprotein aqueous solution in the form of an alkali or acid aqueous solution. As the alkali or acid used for neutralization, the above alkali or acid can be used. By performing an acid or alkali denaturation treatment under the above conditions, the hemoprotein can be completely denatured to obtain a denatured hemoprotein aqueous solution.

  In the present specification, “denatured hemoprotein aqueous solution” means an aqueous solution containing a modified hemoprotein.

  By carrying out this step under the above conditions, a denatured heme protein aqueous solution containing a denatured fish blood-derived heme protein can be obtained.

2. Protease treatment step In this step, the aqueous solution of the denatured heme protein obtained in the above step is treated with a protease to hydrolyze the denatured heme protein to obtain a complex containing a peptide derived from heme protein and heme iron. For the purpose.

  In the present specification, “hemoprotein hydrolysis” means that at least one peptide bond contained in the polypeptide chain of the heme protein is hydrolyzed by a protease, resulting in two shorter chains derived from the heme protein. It means the treatment for forming the above peptides.

  In the present specification, the “complex containing a peptide derived from a heme protein and heme iron” is a complex containing at least one peptide having a shorter chain length and heme iron obtained by hydrolyzing the heme protein. Means the body.

  In this step, various endoproteases commonly used in the art can be used. An alkaline endoprotease having an optimum pH in the alkaline region is preferred. Examples of suitable proteases include, but are not limited to, Alcalase (registered trademark) 2.4L FG (Novozymes Japan) and Alloase XA-10 (Yakult Pharmaceutical Co., Ltd.). The protease may be used in a form (solution or dispersion) substantially dissolved or dispersed in a medium, or in a form (immobilized enzyme) bound to a carrier that is substantially insoluble in water. May be used. It is preferably used in the form of an aqueous solution. The amount of protease used is preferably in the range of 0.0001-20 U / mg protein, preferably in the range of 0.01-10 U / mg protein, based on the total protein weight contained in the denatured heme protein aqueous solution to be treated. It is more preferable that By using the above protease, the denatured heme protein can be efficiently hydrolyzed.

  When the protease is used in a form dissolved or dispersed in a medium, after mixing the aqueous solution of the denatured heme protein and the above protease, the resulting mixture is allowed to stand or 200 rpm or less, preferably 10 to 200 rpm Protease treatment can be performed by shaking at a rate of When the protease is used in the form of an immobilized enzyme, it may be treated in the same manner as described above, or may be treated with the protease by passing a denatured heme protein aqueous solution through a column preliminarily packed with the immobilized enzyme. Good. The temperature when the protease treatment is performed by the above method is preferably in the range of 10 to 50 ° C, and more preferably in the range of 15 to 40 ° C. The time for protease treatment by the above method is preferably in the range of 0.5 to 24 hours, and more preferably in the range of 0.5 to 12 hours. By treating with protease under the above conditions, the denatured heme protein can be hydrolyzed in a short time.

  When a protease is used in a form dissolved or dispersed in a medium, it is derived from the heme protein by hydrolyzing the denatured heme protein by the above procedure and then heat-treating the mixture containing the protease to inactivate the protease. A protease treatment solution containing a complex containing a peptide and heme iron can be obtained. The heat treatment temperature is preferably in the range of 70 to 130 ° C, and more preferably in the range of 70 to 100 ° C. The heat treatment time is preferably in the range of several seconds to 12 hours, and more preferably in the range of several seconds to 1 hour. By performing heat treatment under the above conditions, it is possible to inactivate the protease and prevent undesirable hydrolysis.

  In the present specification, the “protease treatment solution” is derived from a heme protein obtained by this step and containing a heme protein-derived peptide and heme iron, as well as a heme protein that does not form the complex. An aqueous solution containing at least one peptide is meant.

  The present inventor has found that when hemoprotein is hydrolyzed by the above procedure, a specific peptide derived from heme protein and heme iron form a complex having an estimated molecular weight in the range of 2,000 to 10,000. Since the molecular weight of hemoglobin is about 64,500, the molecular weight of myoglobin is about 16,000, and the molecular weight of heme iron (protoporphyrin IX iron complex) is 616.5, this complex is not a complex of heme protein itself and heme iron It is considered to be a complex comprising a peptide having a shorter chain length derived from heme protein and heme iron.

  In addition, said estimated molecular weight can be determined by performing a gel filtration chromatography (GFC) analysis on the same conditions about an analytical sample and a sample with a known molecular weight.

  By carrying out this step under the above conditions, it is possible to obtain a protease treatment solution containing a complex containing a specific peptide having a shorter chain length derived from heme protein and heme iron.

3. Ultrafiltration step This step involves ultrafiltration of the protease-treated solution obtained in the above step to separate a retentate fraction and a permeate fraction, and the peptide and heme derived from the heme protein from the retentate fraction. The object is to obtain a complex containing iron.

  In the present specification, the “retained fraction” means a fraction retained on the ultrafiltration membrane after performing ultrafiltration. The “permeate fraction” means a fraction that permeates the ultrafiltration membrane and is collected in the receiver.

  When the present inventor ultrafilters the protease-treated solution obtained in the above step and separates it into a retentate fraction and a permeate fraction, it originates from a heme protein having an estimated molecular weight in the range of 2,000 to 10,000. It has been found that a complex containing a peptide and heme iron can be recovered in the retentate fraction. In particular, when using an ultrafiltration membrane with a molecular weight cut off of 20,000 to 50,000, the iron weight in the range of 85 to 95% with respect to the total iron weight of the retentate fraction and permeate fraction obtained by ultrafiltration. Can be recovered in the retentate fraction. In addition, when using an ultrafiltration membrane with a molecular weight cut off of 20,000 to 200,000, compared to the case of using an ultrafiltration membrane with a molecular weight cut off of 10,000, the complex is separated into the retentate fraction at 2 to 3 times the separation rate. It can be recovered.

  Therefore, in this step, it is preferable to use an ultrafiltration membrane having a fractional molecular weight of 20,000 to 200,000, and it is more preferable to use an ultrafiltration membrane having a fractional molecular weight of 20,000 to 50,000. The material of the ultrafiltration membrane is preferably aromatic polyamide, polysulfone or cellulose acetate. By using the above ultrafiltration membrane, a high-purity composite can be produced in a short time.

  The above iron weight is determined by treating the lyophilized residue of the retentate fraction and permeate fraction obtained by ultrafiltration with aqua regia and then using an ICP emission spectrophotometer to measure the iron content in the aqua regia treatment liquid. It can be determined by quantifying the concentration.

  The protease treatment solution obtained in the protease treatment step is poured into the ultrafiltration device equipped with the ultrafiltration membrane described above, and stirred while being pressurized, or centrifuged, so that the retentate fraction and the permeate It can be separated into fractions. When performing ultrafiltration by stirring while applying pressure, the applied pressure is preferably in the range of 0.1 to 1 MPa, and more preferably in the range of 0.1 to 0.3 MPa. When ultrafiltration is performed by centrifugation, the centrifugal acceleration is preferably in the range of 1,000 to 100,000 × g, and more preferably in the range of 1,000 to 10,000 × g. The ambient temperature when performing ultrafiltration is preferably in the range of 0 to 50 ° C, more preferably in the range of 10 to 30 ° C. By performing ultrafiltration under the above conditions, a complex containing a peptide derived from heme protein and heme iron can be separated from a low molecular weight component contained in the protease treatment solution.

  In order to improve the purity of the complex containing peptide and heme iron, water is added to the retentate fraction collected by the above procedure, and ultrafiltration is performed by the same procedure, and the retentate fraction and permeate The operation of separating into fractions may be repeated a plurality of times. Thereby, it becomes possible to further improve the purity of the complex containing the peptide and heme iron collected in the retentate fraction.

In addition, a process similar to the ultrafiltration process described above may be performed between the heme protein denaturing process and the protease treatment process as the first ultrafiltration process. Therefore, the method of the present invention
(I) a hemoprotein denaturation step of denaturing fish blood containing heme protein to denature the heme protein to obtain a denatured heme protein aqueous solution;
(Ii) a first ultrafiltration step in which a denatured heme protein aqueous solution is ultrafiltered to obtain an ultrafiltered denatured heme protein aqueous solution from the retentate fraction;
(Iii) Protease treatment step of obtaining a protease treatment solution containing a complex containing a peptide derived from heme protein and heme iron by decomposing the denatured heme protein by subjecting the ultrafiltered denatured heme protein aqueous solution to protease treatment ;
(Iv) a second ultrafiltration step in which a protease-treated solution is ultrafiltered to obtain a complex containing a peptide derived from heme protein and heme iron from the retained fraction;
It can also be implemented as a method including

  A complex produced by the method of the present invention and containing a peptide derived from a heme protein and heme iron has a low molecular weight compared to the heme protein itself, and is water-soluble, unlike fat-soluble heme iron. Therefore, by using the above complex produced by the method of the present invention as a food additive, for example, it is possible to produce a highly functional dietary supplement for preventing anemia.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.
[Example 1-1]
The weight of the enzyme-treated heme iron recovered by ultrafiltration and the distribution of iron ions The yellowtail blood provided by the fisheries company was used as a raw material. In accordance with the flow of FIG. 1, alkaline denaturation of yellowtail blood, hydrolysis of hemoglobin by protease, concentration and recovery by ultrafiltration were performed in the following steps. Then, the abundance ratio of dry weight and iron ion was analyzed for the obtained retentate fraction and permeate fraction.

(i) Protein denaturation step Fish blood was filtered through quantitative filter paper to remove solids. 50 cm ³ of this fish blood was mixed with 50 cm ³ of 0.2 mol dm ^-3 NaOH. The mixture was shaken at 120 rpm for 1 hour in a 30 ° C. constant temperature bath to denature the protein component. A small amount of 4 mol dm ^-3 HCl was added to the denatured fish blood for neutralization to obtain a denatured heme protein aqueous solution.

(ii) Protease treatment step The denatured heme protein aqueous solution obtained in the above step was weighed by 20 cm ³ into a 30 cm ³ sample tube. To each sample tube, add 0.20 cm ³ of a stock solution of protease (Alcalase (registered trademark) 2.4L FG (Novozymes Japan)) or a 10-1000 times (volume ratio) dilution thereof to prepare protease-treated samples. Was shaken at 120 rpm in a constant temperature bath at 30 ° C. for 24 hours. Here, 0.20 cm ³ of the stock solution contains 0.5 U of protease. Further, 20 mg ³ of the denatured heme protein aqueous solution contains 18 mg protein. In addition, the sample which added the same amount of water instead of protease as a blank was prepared, and the same process was performed also about this. After 24 hours, each sample was heat-treated in a 90 ° C. oil bath for 1 hour to inactivate the enzyme to obtain a protease treatment solution.

(iii) Ultrafiltration step A half amount (10 cm ³ each) of the total volume was taken from each sample and blank sample of the protease-treated solution obtained in the above step, and ultrafiltration was performed. The ultrafiltration device used was a stirred ultra holder (manufactured by ADVANTEC) with a cell capacity of 10 cm ³ , and the ultrafiltration membrane was an ultra filter (manufactured by ADVANTEC) with a molecular weight cut off of 20,000 (material: aromatic polyamide). is there. Ultrafiltration was carried out under a temperature of 25 ° C. under a pressure of 0.3 MPa, and the ultrafiltration was terminated when the permeate reached 5 cm ³ . The permeate fraction and the retentate fraction were collected and lyophilized. Then, the dry weight of the freeze-dried residue of both fractions was weighed, and the weight ratio of the freeze-dried residue was calculated. Further, after treating the residue of both fractions with aqua regia for 12 hours, the iron concentration in the aqua regia treatment solution was quantified using an ICP emission spectrophotometer (Shimadzu ICPS-7000). The results are shown in Table 1.

In the case of a blank sample in which the same amount of water is added instead of protease, 63% is recovered as the lyophilized residue of the retentate fraction by weight ratio to the total weight of the lyophilized residue of the permeate fraction and retentate fraction. It was. In contrast, the higher the protease concentration, the lower the weight of the lyophilized residue in the retentate fraction, while the weight of the lyophilized residue in the permeate fraction increases. It was suggested that the degradation of the protein was more advanced. However, no iron ions were detected in the permeate fraction even in the case of a sample with a high protease concentration, which suggested that protein degradation had progressed.
From the above results, it was shown that the desired heme iron can be concentrated by the above process.

[Example 1-2]
In the method of Example 1-1, the denatured heme protein aqueous solution obtained in the protein denaturation step was ultrafiltered using an ultrafiltration membrane having a fractional molecular weight of 20,000 in the same procedure as in the ultrafiltration step. The obtained retentate fraction was collected and diluted to the same volume as that before the ultrafiltration to obtain a denatured protein retentate fraction dilution. The obtained retentate fraction diluted solution was subjected to protease treatment in the same procedure as in the protease treatment step of Example 1-1. The protease used was a stock solution. Then, the 1st ultrafiltration was performed in the procedure similar to the ultrafiltration process of Example 1-1. The process of washing the retentate fraction by adding water to the first retentate fraction obtained and performing ultrafiltration again was repeated twice. The weight percentage of the lyophilized residue in each fraction relative to the total weight of the lyophilized residue in the third retentate fraction and permeate fraction was 17.6 wt% in the retentate fraction and 82.4 wt% in the permeate fraction. %Met.

  From the above results, by adding an ultrafiltration step of the denatured heme protein aqueous solution between the protein denaturation step and the protease treatment step, and / or by repeating the treatment of washing the retentate fraction in the ultrafiltration step, It was shown that the purity of the resulting enzyme-treated heme iron can be further improved.

[Example 2]
Molecular weight analysis of enzyme-treated heme iron After removing low molecular weight components contained in fish blood in advance, fish blood was first ultrafiltered in order to measure the molecular weight of the protein degradation product by protease treatment. The fish blood filtered through the quantitative filter paper was subjected to ultrafiltration using an ultrafiltration membrane (ultrafilter, material: aromatic polyamide) having a molecular weight cut off of 20,000. The process of washing the retentate fraction by adding water to the obtained retentate fraction and performing ultrafiltration again was repeated.

The retentate fraction obtained by the above treatment was collected and diluted to the same volume as the volume before ultrafiltration to obtain a retentate fraction dilution of fish blood. Using this fish blood retentate fraction dilution, a protein denaturation step and a protease treatment step were performed in the same procedure as in Example 1-1. The protease used was a stock solution. 10 cm ³ was collected from the protease treatment solution obtained. Using this, an ultrafiltration process using an ultrafiltration membrane having a fractional molecular weight of 20,000 was carried out in the same procedure as in Example 1-1 to obtain a retentate fraction and a permeate fraction.

  Using the fish blood retentate fraction dilution solution, protease-treated solution, and ultrafiltration retentate fraction and permeate fraction obtained in the above steps as analysis samples, each analysis sample was subjected to gel filtration chromatography ( Analysis was performed using HPLC (pump: Shimadzu LC-10ADvp, UV-visible light detector: Shimadzu SPD-10ADvp) equipped with a GFC column (Shimadzu Shim-pack Diol-300). The ultraviolet (UV) detection wavelength was set to 230 nm or 400 nm. Analysis was performed under the same conditions using a sample with a known molecular weight, and a calibration curve was prepared based on the elution time of the peak of each sample detected on a gel filtration chromatogram with a detection wavelength of 230 nm. Using this calibration curve, the estimated molecular weight of the component corresponding to each peak was calculated from the elution time of each peak detected on the gel filtration chromatogram (detection wavelength 230 nm) of each analysis sample. The gel filtration chromatogram (detection wavelength: 230 nm) of the fish blood retentate fraction dilution is shown in FIG. 2, and the elution time and area percentage of the peak detected on the gel filtration chromatogram and the components corresponding to the peak are shown. The estimated molecular weight is shown in Table 2, respectively.

  As shown in FIG. 2 and Table 2, the maximum area peak was detected at an elution time of 17.18 minutes. Since the estimated molecular weight of the component corresponding to this peak was calculated to be about 60,000, this component is presumed to be hemoglobin. As other components, a component considered to be a protein having an estimated molecular weight of about 17,500 (elution time 18.4 minutes) and a component considered to be a low molecular weight compound having an estimated molecular weight of about 1,000 or less (elution time 21.1 minutes) were confirmed.

  The gel filtration chromatogram (detection wavelength: 230 nm) of the protease treatment liquid obtained by the protease treatment step is shown in FIG. 3, and the elution time and area percentage of the peak detected on the gel filtration chromatogram and the components corresponding to the peak Table 3 shows the estimated molecular weight of each.

  As shown in FIG. 3, the peak (elution time 17.18 minutes) estimated to be hemoglobin detected on the gel filtration chromatogram of the fish blood retentate fraction dilution solution has completely disappeared, and most of the peaks It eluted later. From this result, it was confirmed that hemoglobin in the fish blood retentate fraction dilution was decomposed by protease treatment. In addition, as shown in Table 3, the main peaks detected on the gel filtration chromatogram of the protease treatment solution are peaks at elution times of 19.26 minutes, 19.71 minutes, and 20.34 minutes, and components corresponding to these peaks. The estimated molecular weight was calculated to be about 7,200, 4,500 and 2,400, respectively. From the above results, it was confirmed that a decomposition product close to the desired molecular weight was obtained.

  The gel filtration chromatograms (detection wavelength 230 nm) of the ultrafiltration retentate fraction and permeate fraction obtained by the ultrafiltration step are shown in FIGS. 4A and 4B, and the retentate fraction and permeate fraction gels. The elution times and area percentages of the peaks detected on the filtration chromatogram and the estimated molecular weights of the components corresponding to the peaks are shown in Tables 4A and B, respectively.

  As shown in FIG. 4A, peaks corresponding to components of estimated molecular weights of about 7,200, 4,500, and 2,400 detected in the protease treatment solution were detected on the gel filtration chromatogram of the ultrafiltration retentate fraction. From this result, it was confirmed that the above components did not permeate the ultrafiltration membrane and remained in the retentate fraction.

  As shown in Tables 3 and 4A, the area percentage of the peak corresponding to the above components detected on the gel filtration chromatogram of the ultrafiltration retentate fraction increased as compared with the analysis result of the protease treatment solution. However, the area percentage of the peak corresponding to the component with an estimated molecular weight of 1,000 or less decreased. From this result, it was confirmed that the component which has a desired molecular weight was concentrated by the ultrafiltration process.

  On the other hand, as shown in FIG. 4B, the gel filtration chromatogram of the permeate fraction of ultrafiltration corresponds to the estimated molecular weight components of about 7,200, 4,500, and 2,400, which were the main components of the retentate fraction. A peak was detected. The reason why the above components were detected in both the ultrafiltration retentate fraction and the permeate fraction was because, in this example, an ultrafiltration membrane with a molecular weight cut off of 20,000 was used. It is presumed that most of the low molecular weight peptides produced by degradation by protease treatment permeated through the ultrafiltration membrane and were collected in the permeate fraction. For this reason, in the permeate fraction, the proportion of low molecular weight components increased compared to the protease treatment solution (Table 4B).

  Gel filtration chromatograms (detection wavelength: 400 nm) of the above retentate fraction and permeate fraction are shown in FIGS. 5A and 5B, respectively. When GFC analysis is performed at a detection wavelength of 230 nm in the ultraviolet region, various components are detected. However, when GFC analysis is performed at a detection wavelength of 400 nm, only a colored component having characteristic absorption in the visible light region is detected. For this reason, in the GFC analysis of the above retentate fraction and permeate fraction, when detection is performed at a wavelength of 400 nm, components containing enzyme-treated heme iron are mainly detected.

  As shown in FIG. 5A, on the gel filtration chromatogram of the ultrafiltration retentate fraction detected at a wavelength of 400 nm, peaks of colored components were detected at retention times of 10.4 minutes and 18.8 minutes. From this result, it is estimated that the component considered to have an estimated molecular weight of around 10,000 corresponding to the peak of 18.8 minutes elution is enzyme-treated heme iron, which is a degradation product of hemoglobin.

  On the other hand, as shown in FIG. 5B, almost no colored component was detected on the gel filtration chromatogram of the permeate fraction of the ultrafiltration detected at a wavelength of 400 nm. From this result, it was shown that most of the enzyme-treated heme iron is present in the retained fraction of ultrafiltration.

[Example 3]
Heme Iron Permeation Behavior of Heme Iron Before and After Enzymatic Degradation Alkaline denaturation of yellowtail blood and hemoglobin hydrolysis by protease were sequentially performed in the same steps as in Example 1-1. Using untreated fish blood, a denatured heme protein aqueous solution obtained in the protein denaturation step, and a protease treatment solution obtained in the protease treatment step, a molecular weight cut-off of 10,000, 50,000 or 200,000 (material: polysulfone) or fraction An ultrafiltration step using an ultra filter (manufactured by ADVANTEC) having a molecular weight of 20,000 (material: aromatic polyamide) was performed.

Ultrafiltration was started using each 10 cm ³ sample, and when the permeate reached 9 cm ³ , the permeate was collected and used as one permeate fraction. Water was added to the obtained retentate fraction, and the second ultrafiltration was started. When the permeate reached 9 cm ³ , the permeate was collected to obtain a permeate 2 fraction. The retentate fraction, the permeate 1 fraction, and the permeate 2 fraction obtained by the second ultrafiltration were each collected and lyophilized. The freeze-dried residue of each obtained fraction was treated with aqua regia, and then the iron concentration in the aqua regia treatment solution was quantified using an ICP emission spectroscopic analyzer (Shimadzu ICPS-7000). Based on the above quantitative value, the abundance of iron in each fraction (percentage relative to the total iron weight of all fractions) was calculated. FIG. 6A shows the results when using untreated fish blood, FIG. 6B shows the results when using a denatured heme protein aqueous solution, and FIG. 6C shows the results when using a protease-treated solution.

  As shown in FIG. 6A, in the case of untreated fish blood, even if an ultrafiltration membrane having a fractional molecular weight of 200,000 was used, 90% or more was permeabilized and recovered in the retentate fraction. This is presumably because heme iron contained in untreated fish blood was hemoglobin before alkali denaturation, and the permeability was low for ultrafiltration membranes of any fractional molecular weight.

  As shown in FIG. 6B, in the case of the denatured heme protein aqueous solution, a clear correlation was not confirmed between the fractionated molecular weight of the ultrafiltration membrane and the permeation blocking rate. When using an ultrafiltration membrane with a molecular weight cut off of 200,000, 84% was permeabilized and recovered in the retentate fraction.

  On the other hand, as shown in FIG. 6C, in the case of the protease treatment solution, most of the enzyme-treated heme iron having an estimated molecular weight of about 2,400 to 7,300 confirmed in Example 2 is not permeated by the ultrafiltration and the retentate. It was shown to be collected in fractions. Enzyme-treated heme iron recovered in the retentate fraction is a percentage of the iron weight relative to the total iron weight of all fractions, 98% for the ultrafiltration membrane with a molecular weight cut off of 10,000, and ultrafiltration with a molecular weight cut off of 20,000. 95% of the membrane, 88% of the ultrafiltration membrane with a molecular weight cut off of 50,000, and 70% of the ultrafiltration membrane with a molecular weight cut off of 200,000 were recovered in the retentate fraction.

[Example 4]
Permeation rate of enzyme-treated heme iron through ultrafiltration membrane A protease-treated solution was prepared in the same manner as in Example 1-1. Using this protease treatment solution, an ultrafiltration step using an ultrafiltration membrane having a fractional molecular weight of 10,000, 20,000, 50,000, or 200,000 as in Example 3 was performed. At this time, the time until the permeate was obtained every 1 cm ³ was measured, and the flow rate of the permeate when using ultrafiltration membranes with different fractional molecular weights was compared. The results are shown in FIG.

As shown in FIG. 7, when using ultrafiltration membranes with a molecular weight cut off of 20,000, 50,000, and 200,000, the permeation rate was not so different. On the other hand, when an ultrafiltration membrane with a molecular weight cut off of 10,000 was used, it was shown that the permeation rate was relatively low compared to the above case. The average permeation rate until the first 3 cm ³ liquid permeates is 0.57 × 10 ⁻³ cm ³ s ⁻¹ and 1.03 × 10 ⁻ for ultrafiltration membranes with a molecular weight cut off of 10,000, 20,000, 50,000 or 200,000, respectively. The permeation rate was ³ cm ³ s ⁻¹ , 1.12 × 10 ⁻³ cm ³ s ⁻¹ or 1.27 × 10 ⁻³ cm ³ s ⁻¹ . In particular, ultrafiltration membranes with a molecular weight cut off of 20,000 are semipermeable membranes with a relatively dense network structure, but they can be concentrated at nearly twice the rate of ultrafiltration membranes with a molecular weight cut off of 10,000. It was done.

  According to the method of the present invention, a complex containing a peptide derived from heme protein and heme iron can be produced from fish blood containing heme protein. Thereby, it becomes possible to use the fish blood disposed as industrial waste as a raw material of a highly functional dietary supplement for preventing anemia, for example.

Claims (3)

A method for producing a complex comprising a peptide derived from a heme protein and heme iron,
(I) a hemoprotein denaturation step of denaturing fish blood containing heme protein to denature the heme protein to obtain a denatured heme protein aqueous solution;
(Ii) a protease treatment step in which an aqueous solution of the denatured heme protein is treated with a protease to decompose the denatured heme protein to obtain a protease treatment solution containing a complex containing a peptide derived from heme protein and heme iron;
(Iii) Ultrafiltration step of ultrafiltration of the protease treatment solution to obtain a complex containing a peptide derived from heme protein and heme iron from the retained fraction;
Including said method.   The method of claim 1, wherein the ultrafiltration step is performed using an ultrafiltration membrane having a molecular weight cut off of 20,000 to 50,000.   The method according to claim 1 or 2, wherein the denaturing treatment is carried out by mixing fish blood with alkali.

Images