KR101706754B1 - residual product of fish protein having reduced antigencity and improved antioxidant activity - Google Patents

Abstract

The present invention relates to a glycosylated protein degradation product having reduced antigenicity and excellent antioxidation activity and a method for producing the same, which comprises hydrolyzing a fish by-product or a fish protein from which the intestines have been removed into protease; And a step of reacting the hydrolyzed hydrolyzate with a saccharide to perform a Maillaird reaction, thereby having an excellent antioxidative activity and an allergy reducing effect.

Description

[0001] The present invention relates to a protein degradation product of a saccharified fish having reduced antigenicity and an antioxidative activity and a method for producing the protein product having reduced antigencity and improved antioxidant activity,

The present invention relates to a fish protein degradation product having an excellent antioxidative activity and an allergy reducing effect, and a process for producing the same.

Of the fish in Korea, the flounder aquaculture industry accounts for half of the total production of the sea fish culture industry and accounts for the largest production volume in the world (50,000 tons per year, about 500 billion won per year) because it has the world's best production technology .

Recently, the perception of depletion of biological resources has increased, and food processing by-products, which are considered as environmental pollution, have been recognized in many fields.

The total supply of aquatic products in Korea is about 32 million tons (Statistics Korea: 2012), of which about 85% is used as processing material and 70% of the total is by-products such as viscera, hair, skin, bones, It is used as feed or discarded. Although a large amount of fish processing by-products occur each year, there are insufficient researches to utilize them efficiently, resulting in waste of useful resources and environmental pollution.

In an effort to utilize the fish processing by-products, efforts are being made to recover these protein resources as bioactive proteins and peptides through enzymatic degradation. In addition, decomposition of fish processing by-products by enzymatic biological techniques is known to be advantageous in terms of stability of the production process, reduction of by-products, selective acquisition of functional materials, and human safety.

In addition, the fish processing by-products have many kinds of tastes including proteins, peptides, amino acids and nucleic acids, and thus they can be good raw materials for food materials and bases. However, due to the characteristic odor and taste of fish, If this resource is converted into high value added material, it will contribute not only to increase profit of company but also to accumulate technology of material development, and it will be recognized as national competitiveness enhancement and environment friendly process.

On the other hand, high-pressure treatment technology is mainly used in the food industry for the purpose of sterilizing harmful microorganisms in foods. However, recently, the advantages of non-heating technology that can provide water-soluble and extracting effect while keeping the color, flavor and nutrients of the useful ingredient from the animal and plant raw materials centering on domestic and Japan have been highlighted. Recently, due to the development of high-pressure processing equipment of commercial scale, the utilization rate has been gradually increasing mainly in the manufacture of small-sized food material and beauty material.

In addition, Maillal reaction (MR) is a series of reactions initiated by the condensation of the amino groups of amino acids, peptides, and proteins with the carbonyl group of the reducing sugar in a nonenzymatic browning reaction. This reaction produces a number of reactive substances called so-called MRPs, which play an important role in the quality and taste of stored or processed food.

Korean Patent No. 0729255 Korea Patent Publication No. 2011-0011387

An object of the present invention is to provide a hydrolyzate of a saccharified fish protein having an excellent antioxidative activity and an allergy reducing effect by hydrolyzing a fish by-product from which the intestines have been removed with an enzyme and saccharifying it by a Maial reaction.

It is another object of the present invention to provide a method for producing the protein hydrolyzate of the saccharified fish.

In order to accomplish the above object, the present invention provides a method for producing a proteinaceous hydrolyzate of saccharified fish having reduced antigens and antioxidative activity, which comprises hydrolyzing a fish by-product or a fish protein with a protease by protease; And a step of reacting the hydrolyzed hydrolyzate with a saccharide to perform a Maillaird reaction.

And further desalting the hydrolyzate between the step of hydrolyzing and the step of performing myial reaction. Further, the step of separating the desalted hydrolyzate may further include separating the desalted hydrolyzate so as to have a desired molecular weight between the desalting step and the Myial reaction step.

The proteolytic enzyme may be selected from the group consisting of Protamex, Flavourzyme, Neutrase, Alcalase,? -Chymotrypsin, Trypsin and Papain. And may be at least one selected from the group consisting of

The weight ratio of the fish by-product or fish protein to the proteolytic enzyme may be 40-100: 1.

The temperature for the hydrolysis may be 40 to 80 占 폚, and the pH for hydrolysis may be 6.0 to 7.5.

The saccharide may be at least one pentane selected from the group consisting of ribose, xylose and arabinose, or at least one 6-valent sugar selected from the group consisting of glucose, galactose, mannose and fructose.

The hydrolyzate and the saccharide may be mixed in a weight ratio of 1: 1 to 5.

The Maillard reaction temperature may be from 100 to 150 ° C, the Maillard reaction pressure may be from 0.2 to 3 kg / cm 2, the Maillal reaction pH may be from 7 to 9, and the Maillet reaction time may be from 20 to 60 minutes.

The fraction may be an ultrafiltration membrane, and the molecular weight of the fractionated hydrolyzate may be 5 KDa or less.

In order to accomplish the above-mentioned other objects, the saccharified fish protein degradation product of the present invention may be a Maillaird-reacted hydrolyzed fish protein degradation product.

The saccharified fish protein degradation product may be used at a concentration of 0.5 to 20 mg / ml.

The protein hydrolyzate of the saccharified fish protein is 10 to 13%, the degree of browning is 550 to 650, the HMF (hydroxymethylfural) content is 30 to 120 ug _HMF / mg _{saccharified fish protein degradation product} , and the molecular weight is 80 Da to 100 Da and 7 kDa To 47 kDa can be distributed in a weight ratio of 2: 8.

The fish protein degradation product of the present invention has excellent antioxidative activity and allergy reducing effect by producing fish by hydrolysis of fish at high pressure and saccharification by Meal reaction.

In addition, it uses all of the by-products that are discarded and does not contain chemical additives, so it is an eco-friendly food additive material.

1 is a graph showing the molecular weight of a fish protein degraded product prepared according to an embodiment of the present invention by high performance liquid chromatography equipped with a vaporization light scattering detector.

The present invention relates to a fish protein degradation product having an excellent antioxidative activity and an allergy reducing effect, and a process for producing the same.

Hereinafter, the present invention will be described in detail.

The method for producing a fish protein degradation product of the present invention comprises the steps of hydrolyzing a fish by-product or a fish protein from which the intestines have been removed into a protease; And a step of reacting the hydrolyzed hydrolyzate with a saccharide to perform Maillaird reaction. Further, the method may further include desorbing the hydrolyzate sequentially between the hydrolysis step and the Myial reaction step, and further dividing the desalted hydrolyzate.

First, the fish by-product or fish protein from which the intestines have been removed and the protease are subjected to a pressure of 80 to 300 MPa, preferably 100 to 150 MPa; A temperature of 40 to 80 캜, preferably 50 to 60 캜; Hydrolysis is carried out for 1 to 6 hours under a pH of 6.0 to 7.5, preferably 6.0 to 7.0.

At this time, the weight ratio of the fish by-product (or fish protein) and the enzyme is 40 to 100: 1, preferably 45 to 60: 1. When the weight ratio of the fish by-product (or fish protein) and the enzyme is less than the lower limit based on the enzyme, the fraction yield of the desired molecular weight may be significantly lowered at the following fractionation, and the hydrolysis efficiency may be lowered above the upper limit.

If the pressure is lower than the lower limit, the hydrolysis efficiency may be lowered. If the pressure exceeds the upper limit, the yield of the desired molecular weight fraction may be significantly lowered.

In addition, when the temperature is lower than the lower limit, hydrolysis efficiency is lowered, and the yield of fraction of desired molecular weight at the subsequent fractionation can be significantly lowered. When the temperature is above the upper limit, the yield of fraction of desired molecular weight is significantly lowered .

When the pH is outside the above range, the hydrolysis efficiency is lowered and the yield of the desired molecular weight fraction may be significantly lowered at the subsequent fractionation.

If any of the fish by-products and enzymes, pressure, temperature, pH, and time are out of the above range during the hydrolysis, the hydrolysis efficiency and the yield of the desired molecular weight fraction may be significantly lowered.

The fishes are not particularly limited as long as they have a crude protein content of 18% by weight or more, and preferably one species selected from the group consisting of flounder (flounder), brassiere, and abalone.

Examples of the protease include Protamex, Flavourzyme, Neutrase, Alcalase,? -Chymotrypsin, trypsin and papain. Papain), preferably protamex, flavozymes, or a mixed enzyme thereof, more preferably, protamex.

Next, the hydrolyzed hydrolyzate is desalted to enhance bioavailability.

The method of desalting the hydrolyzate is not particularly limited, but desalting is preferably carried out by a forced dialysis method using an ion exchange membrane and an electric force using an electrodialyzer.

Specifically, the desalting method is a method in which a cation exchange membrane and an anion exchange membrane are combined, the hydrolyzate is put between the two exchange membranes, and then a DC voltage is applied to electrically remove ions from the hydrolyzate.

Next, in order to improve the bioavailability and subsequent efficiency of the Maillaird reaction, the desalted hydrolyzate is fractionated to obtain a hydrolyzate having a desired molecular weight, for example, a molecular weight of 5 KDa or less, preferably 0.1 to 3 KDa Respectively.

When the molecular weight of the fractionated hydrolyzate is more than 5 KDa, the yield (yield) is low and the bioavailability such as antioxidation and allergy reduction is low and the Maillard reaction may not be performed smoothly.

The step of fractionating the hydrolyzate is carried out by fractionation using an ultrafiltration system equipped with an ultrafiltration membrane, followed by concentration and lyophilization.

Next, the hydrolyzate (or the fractionated hydrolyzate) and the saccharide are mixed and saccharified by a Maial reaction.

The hydrolyzate and the saccharide are mixed at a weight ratio of 1: 1 to 5, preferably 1: 2 to 4, and water is added. The mixture is then heated at 100 to 150 ° C, preferably 110 to 130 ° C, Cm 2, preferably 0.6 to 1.5 kg / cm 2 for 20 to 60 minutes, preferably 30 to 45 minutes, to produce a glycosylated hydrolyzate such as glycated fish protein degradation product. The pH at this time is 7 to 9, preferably 8.2.

When the weight ratio of the hydrolyzate and the saccharide is lower than the lower limit of the hydrolyzate, the antioxidant activity and the antiallergic effect are low. If the weight ratio of the hydrolyzate and the saccharide is higher than the upper limit, a large amount of side reactions may occur at the time of the myelin reaction.

When the temperature and the pressure are lower than the lower limit, the antioxidant ability and the anti-allergic effect are low. If the temperature and the pressure are higher than the upper limit, toxic substances may be generated.

When the pH is out of the above range, antioxidant activity and anti-allergic effect may not occur.

Examples of the saccharides include pentose and hexose, and specifically include pentose, which is at least one selected from the group consisting of ribose, xylose and arabinose; Glucose, glucose, galactose, mannose, and fructose, and is preferably ribose, which is pentane. In the case of using the ribose as the saccharide, the myelin reaction occurs well and the browning and antioxidant ability are better than the case of using the saccharide per 6 carbon atoms.

The saccharified fish protein solution prepared by the above method is used at a concentration of 0.5 to 20 mg / ml, preferably 1 to 5 mg / ml, more preferably 1 mg / ml.

When the amount of the saccharified fish protein degradation product is less than the lower limit, the expected effect can not be obtained. If the upper limit is exceeded, toxicity may occur.

In addition, the fish protein degradation product of the present invention has a hydrolyzed degree of 10 to 13%, a browning degree of 550 to 650, a HMF (hydroxymethylfural) content of 30 to 120 ug _HMF / mg _{saccharified fish protein degradation product} and a molecular weight of 80 Da to 100 Da And 7 kDa to 47 kDa in a weight ratio of 2: 8 (Fig. 1). The molecular weight of the saccharified protein hydrolyzate of FIG. 1 was analyzed by gel filtration chromatography using high performance liquid chromatography (1200 series, Agilent, USA) equipped with an evaporating light scattering detector. The analytical conditions were as follows: After injecting 50 μl of sample, 0.02% sodium azide was eluted at a flow rate of 0.5 ml / min on a Agilent PL aquagel-OH 30 column (8 μm, 300 × 7.5 mm) / PEO (Agilent, USA) were compared with standard materials to confirm the molecular weight distribution.

In addition, the fish protein degradation product of the present invention has 190-300% histamine release (%), 90-130% beta-histamine release (%), DPPH radical scavenging activity (0.4-1.2 mg / and a FRAP (Ferric reducing ability of plasma) activity of 2.00 to 3.00 mM FeSO ₄ eq./mg extract.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

Example 1.

100 parts by weight of flounder flour milled with a wet crusher and 2 parts by weight of protamex were mixed and then hydrolyzed at a pressure of 100 MPa and a temperature of 57 DEG C and pH 6.8 for 6 hours and then concentrated and lyophilized 100 parts by weight of the lyophilized hydrolyzate and 100 parts by weight of ribose were mixed and water was added thereto. The mixture was subjected to a Maillard reaction at 121 ° C, 1.0 kg / cm 2 pressure and pH 8.2 for 38 minutes to prepare a saccharified fish protein degradation product Respectively.

Example 2.

100 parts by weight of flounder flour milled with a wet grinder and 2 parts by weight of protamex were mixed and then hydrolyzed at a pressure of 100 MPa, a temperature of 57 DEG C and a pH of 6.8 for 6 hours, -acilyzer S3, Creation Techno, Korea) to obtain a desalted hydrolyzate. The desalted hydrolyzate was fractionated using an ultrafiltration system (QuixStandtm benchtop system, GE healthcare, USA) to obtain a fractionated hydrolyzate having a molecular weight of 3 KDa or less, which was concentrated and lyophilized. 100 parts by weight of the fractionated hydrolyzate and 100 parts by weight of ribose were mixed, and water was added thereto. The mixture was subjected to a Maillard reaction at 121 ° C, 1.0 kg / cm 2 pressure and pH 8.2 for 38 minutes to prepare saccharified fish protein degradation product.

Example 3.

The hydrolyzate of the saccharified fish protein was prepared using the fractionated hydrolyzate having a molecular weight of 3.1 to 5.0 KDa as the fractionated hydrolyzate.

Example  4.

The same procedure as in Example 2 was carried out except that the saccharified fish protein degradation product was prepared using glucose instead of the ribose.

Example 5.

The same procedure as in Example 2 was conducted except that 5 parts by weight of protamex was used for 100 parts by weight of flounder flour to prepare saccharified fish protein degradation products.

Example 6.

The hydrolyzate of the saccharified fish protein was prepared using the fractionated hydrolyzate having a molecular weight of 10 KDa or more as the fractionated hydrolyzate.

Example 7.

The protein hydrolysates of saccharified fish were prepared using 1000 parts by weight of ribose per 100 parts by weight of the hydrolyzate obtained in Example 2. The pH at this time is 4.2 to 4.9.

Example  8.

The protein hydrolyzate of Saccharomyces cerevisiae was prepared in the same manner as in Example 2 except that the temperature at the time of the Maial reaction was 80 ° C.

Example  9.

The same procedure as in Example 2 was carried out except that the saccharified fish protein degradation product was prepared at a pressure of 6 kg / cm 2 during the Maial reaction.

Comparative Example  One.

The same procedure as in Example 2 was carried out except that the saccharified fish protein degradation product was prepared without carrying out the Myal reaction.

Test Example  One. Allen  Active measurement

In order to measure the allergen activity of Examples and Comparative Examples, histamine and β-hexosaminidase release experiments were conducted. The histamine and? -Hisosaminidase were secreted to the outside of the cell as the allergic reaction progressed, and the cells were measured using RBL-2H3 cells.

1-1. Histamine release: To measure histamine release, RBL-2H3 was dispensed into a 96-well plate at 5 × 10 ⁴ cells / well and cultured for 24 hours. After the medium was removed, the cells were washed twice with PBS, and the sample was diluted with the release solution (116.9 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.6 mM Glucose, 25 mM HEPES, 2 mM CaCl2, 1 mg / mL BSA), treated with the cell line, and cultured for 2 hours. Add 100 μL of the cell culture supernatant, add 60 μL of 0.5N NaOH and 2.5 mg / mL o-phthalaldehyde in a ratio of 2: 1, incubate at 37 ° C for 30 minutes, add 10 μL of 3 N HCl, Terminated. Fluorescence was measured at excitation 355 nm / emission 460 nm and the degree of release of histamine was calculated using the following equation (1).

1-2. Release of β-hysosomal DNA: To measure β-histamine release, RBL-2H3 was dispensed into a 96-well plate at 5 × 10 ⁴ cells / well and cultured for 24 hours. After the medium was removed, the cells were washed twice with PBS. The sample was diluted in a release solution (116.9 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.6 mM Glucose, 25 mM HEPES, 2 mM CaCl2, 1 mg / mL BSA) at a concentration of 1 mg / , And cultured for 2 hours. The cell culture supernatant (30 μL) was reacted with 70 μL of a substrate solution (5 mM 4-Nitrophenyl-N-acetyl-β-D-glucosaminide in 0.05 M citrate buffer, pH 4.5) at 37 ° C. for 1 hour. After incubation, 100 μL of 0.1 M sodium carbohydrate buffer (pH 10) was added to terminate the reaction and absorbance was measured at 405 nm using a multiplate reader. Then, the degree of? -Hisosamidine emission was calculated using the following formula (2).

division Histamine release (%) Hissosaminidis release (%) Example 1 256.4 99.7 Example 2 242.0 93.5 Example 3 280.7 109.8 Example 4 198.5 128.6 Example 5 410.5 160.8 Example 6 541.6 205.8 Example 7 397.3 158.7 Example 8 430.7 192.4 Example 9 450.9 198.8 Comparative Example 1 914.7 411.7

As shown in Table 1, it was confirmed that the amounts of histamine and hysosaminidase released from the fish protein degradation products prepared according to Examples 1 to 9 of the present invention were 1.5 to 3 times lower than those of Comparative Example 1. In particular, Examples 1 to 4 were found to have lower levels of histamine and hysosaminidase release than Examples 5 to 9.

Since the high release of histamine and hysosaminidase can induce an allergic reaction, it is confirmed that Examples 1 to 9 are superior to all Comparative Examples in allergy suppressing effect.

Test Example  2. Antioxidant activity measurement

2-1. DPPH radical scavenging activity: To 45 μL of 0.15 mM DPPH solution (in 94.5% ethanol) was added 10 μL of each GFPH diluted with distilled water to 45 μL of EtOH, vortexed for 10 seconds, After the reaction, the absorbance was measured at 515 nm. The measured values were calculated by substituting into the following equation (3). IC ₅₀ values were calculated by dilution factor (DF) when the DPPH radical scavenging rate of each sample was 50%, and the scavenging activity of each sample was compared.

2-2. Ferric reducing ability of plasma (FRAP) activity: 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) dissolved in 40 mM HCl and 20 mM FeCl ₃ 6H ₂ O 10: 1: 1 (v / v / v), and the mixture was heated at 37 ° C for 10 minutes and used as a FRAP reagent. 10 μL of a 5 mg / mL sample was mixed with 90 μL of FRAP reagent, reacted at 37 ° C. for 5 minutes, and the absorbance was measured at 593 nm. The calculated absorbance was calculated from a standard curve obtained using FeSO ₄ · 7H ₂ O as a standard FeSO ₄ eq. / Mg extract.

division DPPH scavenging activity (IC50, mg /) FRAP (mM FeSO ₄ eq./mg extract) Example 1 0.52 2.76 Example 2 0.48 2.98 Example 3 0.69 2.21 Example 4 1.12 2.01 Example 5 2.09 1.45 Example 6 2.11 1.19 Example 7 1.59 1.37 Example 8 1.48 1.39 Example 9 1.47 1.38 Comparative Example 1 9.02 0.87

As shown in Table 2 above, it was confirmed that the fish protein degradation products prepared according to Examples 1 to 9 of the present invention had better DPPH scavenging activity and FRAP activity than Comparative Example 1. In particular, Examples 1 to 4 were found to be more excellent in DPPH scavenging activity and FRAP activity than Examples 5 to 9.

Since the excellent DPPH scavenging ability and FRAP activity are excellent in antioxidant activity, it was confirmed that Examples 1 to 9 exhibit superior antioxidative activities as compared with Comparative Examples.

Test Example  3. Hydrolysis, browning and HMF  Measure

3-1. Glycine (0.3125 mM ~ 2.5 mM) was used as a standard. Dilute 100-fold diluted samples and standard in a volume of 20 μL into 96 wells, and add 50 μL of sodium bicarbonate buffer (pH 8.5) 50 uL TNBS was added. After incubation for 60 min in a shaking incubator at 50 ° C, the protein was stabilized by adding 10% SDS and the reaction was terminated with 25 μL of HCl. The reaction mixture was then measured by Spectrometer (OD, 340 nm) The degree of hydrolysis was determined by analyzing the molar number of amino acids using the TNBS (Trinitrobenzenesulfonic Acid) assay after considering the protein content of the flounder byproduct and the average molecular weight through amino acid analysis.

3-2. Browning degree: The prepared saccharified fish protein degradation product solutions were diluted (1 to 500 times) with distilled water so as to be within the range of 0.2 to 0.8 which can be measured by absorbance, and then subjected to spectrophotometer (DU650 spectrophotometer, Beckman, USA) The degree of browning was measured by measuring the absorbance at 420 nm, which is the measurement range of the brown pigment.

3-3. HMF content: The glycated product (HMF) of the protein hydrolysates of the saccharified fish prepared in Examples and Comparative Examples was measured using HPLC analysis method.

division Hydrolysis (%) Browning HMF (ug _HMF / mg _{saccharified fish protein degradation product)} Example 1 11.6 589.67 97.5 Example 2 11.7 612.73 110.1 Example 3 11.6 534.41 78.4 Example 4 10.3 522.84 67.5 Example 5 9.0 384.51 8.4 Example 6 11.5 399.72 26.4 Example 7 11.4 259.46 10.2 Example 8 11.3 402.46 13.8 Example 9 11.5 399.75 19.5 Comparative Example 1 11.4 112.10 0.1

As shown in Table 3 above, it was confirmed that the saccharified fish protein degradation products prepared according to Examples 1 to 9 of the present invention were superior in both the hydrolysis degree, the browning degree and the HMF content, as compared with Comparative Example 1. In particular, it was confirmed that Examples 1 to 4 are superior to Examples 5 to 9 in the degree of hydrolysis, browning and HMF content.

In the above Examples 1 to 4, cell viability was measured using HepG2 cell line, RBL-2H3 cell line, and NRK-52E cell line. As a result, no cytotoxicity was observed, and at 1 mg / .

Claims (22)

Mixing and hydrolyzing a fish by-product or a fish protein from which the intestines of the fish having a crude protein content of 18% or more are removed and a protease in a weight ratio of 40 to 100: 1;
Desalting the hydrolyzate;
Fractionating the desalted hydrolyzate to obtain a fractionated hydrolyzate having a molecular weight of 0.1 to 3 kDa; And
Mixing the fractionated hydrolyzate with a saccharide at a weight ratio of 1: 1 to 5 and then subjecting the mixture to a Maillaird reaction. The method for producing the protein hydrolyzate of a saccharified fish having excellent antioxidative activity with reduced antigenicity . delete delete The method according to claim 1, wherein the protease is selected from the group consisting of Protamex, Flavourzyme, Neutrase, Alcalase,? -Chymotrypsin, Trypsin, And Papain. The method for producing the protein hydrolyzate of a saccharified fish having excellent antioxidative activity, wherein the antigenicity is reduced. delete The method according to claim 1, wherein the hydrolysis temperature is 40 to 80 ° C. The method according to claim 1, wherein the pH during the hydrolysis is 6.0 to 7.5, wherein the antigenicity is reduced and the antioxidant activity is excellent. The method according to claim 1, wherein the saccharide is pentane or hexane. 2. The method according to claim 1, wherein the saccharide is pentane or hexane. 9. The method of claim 8, wherein the pentose is at least one selected from the group consisting of ribose, xylose and arabinose, and the hexose is at least one selected from the group consisting of glucose, galactose, mannose, and fructose. Wherein the antigenicity is reduced and the antioxidant activity is excellent. delete The method according to claim 1, wherein the Maillard reaction temperature is 100 to 150 ° C. [2] The method according to claim 1, wherein the Maillard reaction pressure is 0.2 to 3 kg / cm < 2 >. The method according to claim 1, wherein the pH at the time of the myelin reaction is 7 to 9, wherein the antigenicity is reduced and the antioxidant activity is excellent. The method according to claim 1, wherein the Meal reaction time is 20 to 60 minutes. The method according to claim 1, wherein the fraction is an ultrafiltration membrane. delete delete delete delete delete delete delete

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