JPH05227983A - Production of peptide from rice bran protein - Google Patents

Abstract

PURPOSE:To produce a peptide free from the browning and the insolubilization troubles by using defatted rice bran as a starting material. CONSTITUTION:Defatted rice bran is treated with a dilute acid under heating to remove soluble carbohydrates and the insoluble component is suspended in water and hydrolyzed with a proteinase. After removing the insoluble residue, the soluble component is desalted and purified by electrodialysis to obtain the objective peptide induced from rice bran protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a rice bran protein-derived peptide. More specifically, defatted rice bran is used as a starting material, and the defatted rice bran is heat-treated with a dilute acid to remove soluble sugars, and then the insoluble portion is suspended in water and hydrolyzed by a proteolytic enzyme to remove the insoluble matter. The present invention relates to a method for producing a rice bran protein-inducing peptide capable of preventing a browning phenomenon and insolubilization by purifying a soluble portion by desalting by electrodialysis.

[0002]

BACKGROUND OF THE INVENTION Proteins are usually hydrolyzed by acids, alkalis, or enzymes. When plants are used as raw materials, hydrolysis with acids or alkalis leads to the presence of a large amount of fiber and storability in plants. Since the sugar becomes a soluble sugar and is mixed in the hydrolyzed liquid, there is a problem that a phenomenon of discoloring to brown, that is, a so-called browning phenomenon is caused.

Therefore, as a plant-derived protein raw material for cosmetic raw materials and nutrient enhancers, purified ones having a high protein content such as wheat gluten and defatted soybean protein have been used (see, for example, JP-A-62-62). 53969, JP-B-63-50998, JP-A-63-287462, etc.).

[0004] By the way, rice bran has been used as a bran bag for bathing the skin for a long time, and it is mainly intended to utilize oil and fat components and granular fiber in rice bran. , As its application, fermenting rice bran and using the fermented component as a bathing agent or a hair wash has also been studied, but all of them contain an oil and fat component and a fiber material. No attempt has been made to produce peptides from proteins.

[0005]

As mentioned above, as described above, the raw materials have been limited to obtain peptides from plant-derived proteins. In addition, it is also necessary to utilize an unused protein source or a discarded protein source from the viewpoint of advanced utilization of resources.

In view of such circumstances, the present invention focuses on rice bran as a new plant peptide source, and an object thereof is to provide a method for producing a peptide capable of preventing browning phenomenon and insolubilization from this rice bran. To do.

[0007]

Means for Solving the Problems As a result of various studies on a method for producing a peptide from rice bran, the present invention shows that after defatted rice bran is heat-treated with a dilute acid to remove soluble sugars,
A rice bran protein-derived peptide that can prevent browning phenomenon and insolubilization by suspending the insoluble portion in water and hydrolyzing it with a proteolytic enzyme, removing the insoluble matter, and then desalting the soluble portion by electrodialysis for purification. It is made possible to manufacture.

The composition of the present invention will be described below in terms of (1) raw rice bran,
(2) Removal of soluble sugars, (3) enzymatic hydrolysis, (4)
The insoluble matter removal and (5) desalination purification will be described in detail in this order.

(1) Raw Rice Bran The rice grain mainly contains two proteins, glutelin and prolamin, and the content of brown rice is about 7% by weight. There is no detailed report on the type and content of protein in rice bran, but the protein content in commercially available defatted rice bran is 18-25.
It is included in the weight percentage.

In the production of the rice bran protein-derived peptide, non-defatted rice bran can be used as a raw material.
In that case, a degreasing step must be included. in this way,
Even when non-defatted rice bran is used, a degreasing step is required, and once the non-defatted rice bran is used, the case where non-defatted rice bran is used as a raw material is also included in the scope of the present invention. It will be.

Since defatted rice bran is commercially available in large quantities and can be easily obtained, it is preferable to use defatted rice bran as a starting material from the viewpoint that the process can be simplified in the production of a protein derived from rice bran protein.

Table 1 shows an example of analysis of the composition ratio of amino acids by complete hydrolysis of rice bran. Usually, in the case of amino acid analysis of proteins, the sample is completely hydrolyzed using 6N hydrochloric acid before the analysis, so that the amide bond of asparagine and glutamine is hydrolyzed into aspartic acid and glutamic acid, respectively. Therefore, in Table 1, asparagine and glutamine are added to aspartic acid and glutamic acid, respectively. In addition, tryptophan cannot be quantified because it is decomposed and disappears by hydrolysis, and therefore is not shown in Table 1.

[0013]

[Table 1]

(2) Removal of Soluble Carbohydrate Degreased rice bran is suspended in a 3 to 7% (wt%, hereinafter, wt% unless otherwise noted for concentration) hydrochloric acid aqueous solution, and the suspension is placed at 30 to 75 ° C. for 1 hour. Continue stirring for ~ 4 hours to extract soluble carbohydrates. After extraction, the insoluble portion is separated by filtration, and the insoluble portion is subjected to the operation of (3) and below.

The concentration of hydrochloric acid used here can achieve the purpose to some extent outside the above range, but when the concentration of hydrochloric acid is lower than 3%, the soluble sugar cannot be sufficiently removed and the soluble sugar is not removed. Quality may remain in the peptide, causing browning.

Further, when the concentration of hydrochloric acid becomes higher than 7%,
Although the effect of removing soluble carbohydrates is improved, the protein is also decomposed and extracted at the same time, resulting in a poor yield of the obtained peptide.

In addition to hydrochloric acid, sulfuric acid may be used, but in the case of sulfuric acid, the concentration is preferably 1.5 to 4%.

When defatted rice bran contains phytin, phytin is also removed in this soluble sugar removal step. Therefore, the turbidity due to phytin is eliminated.

(3) Enzymatic hydrolysis After the soluble sugar is removed from the defatted rice bran by heat treatment with a dilute acid, the insoluble portion is suspended in water and hydrolyzed by a proteolytic enzyme.

Examples of the proteolytic enzyme include acidic proteolytic enzymes such as pepsin, proctase A and proctase B, papain, bromelain, thermolysin,
Neutral or alkaline proteolytic enzymes such as trypsin, pronase and chymotrypsin can be used.

Further, fungal neutral or alkaline proteolytic enzymes such as subtilisin and staphylococcus protease can also be used.

The pH during hydrolysis is preferably adjusted within the range of pH 1 to 4 in the case of acidic proteolytic enzyme, and within the range of pH 4 to 10 in the case of neutral or alkaline proteolytic enzyme.

Generally, the pH is preferably adjusted appropriately with a buffer solution such as acetic acid / sodium acetate buffer solution, phosphate buffer solution, or by addition of acid, alkali or the like. The reaction temperature during hydrolysis is preferably 30 to 60 ° C., and the reaction time is generally 3 to 24 hours.

Whichever enzyme is used, the molecular weight of the obtained peptide can be controlled by changing the amount of the enzyme used, the reaction temperature, the reaction time, etc., so that the molecular weight of the target rice bran protein-derived peptide can be controlled. In addition, it is preferable to determine the optimum conditions by examining the molecular weight distribution of the obtained peptide for each condition of the amount of enzyme used, reaction temperature, and reaction time by gel filtration method or the like.

After enzymatic hydrolysis, at 70-80 ° C for 30 minutes-
Continue stirring for 1 hour to deactivate the proteolytic enzyme.

By this enzymatic hydrolysis, a wide range of peptides having an average molecular weight in the range of 200 to 4000 can be obtained. Particularly, those having an average molecular weight of 200 to 2000 are easily dissolved in water, are easy to handle, and can be applied to the skin and skin. Since it has good adsorption to hair, it can be used as a raw material for cosmetics.
Alternatively, it can be preferably used as a compounding agent for cosmetics.

(4) Removal of insoluble matter After deactivating the proteolytic enzyme, the hydrolyzed solution is filtered to separate it from the decomposition residue. The rice bran protein-inducing peptide can be obtained by purifying the obtained solution by the method described in (5) below, but during long-term storage, temporarily dissolved polymer substances associate and become insoluble. Therefore, it is necessary to remove the polymer substance that causes this by performing a thermal decomposition treatment with an alkali or an acid.

That is, an alkaline agent was added to the above-mentioned hydrolyzed liquid so as to be 8 to 25% with respect to defatted rice bran,
Thermal decomposition at ~ 70 ° C for 1-2 hours. As the above-mentioned alkaline agent, sodium hydroxide, potassium hydroxide, calcium hydroxide, etc. are used, but the molecular weight of the peptide obtained by enzymatic hydrolysis can be changed by changing the concentration of this alkaline agent solution or the thermal decomposition time. It can be further reduced. For example, if the average molecular weight of the peptide is 200-
This thermal decomposition may be used to adjust to 2000.

The concentration of the alkaline agent in the defatted rice bran in the above-mentioned thermal decomposition by heat is specified. When the amount of the alkaline agent is less than 8% with respect to the defatted rice bran, the polymer substance cannot be sufficiently removed. As described above, there is a risk that the polymer substance may become insoluble during storage and precipitate.
When it is more than 5%, the peptide is further decomposed into amino acids, and the yield of the peptide is deteriorated.

Further, the thermal decomposition with alkali decomposes the oligosaccharides into monosaccharides when the oligosaccharides remain, so that the saccharides can be completely removed in the subsequent purification step by electrodialysis. There is also an effect.

After alkaline thermal decomposition, the mixture is filtered, the pH of the filtrate is adjusted to 3 to 4 with an acid such as hydrochloric acid or sulfuric acid to generate a precipitate, and the precipitate is removed by filtration.

The insoluble matter can be removed by thermal decomposition with an acid as well as the above-mentioned alkaline thermal decomposition. In the case of this acid thermal decomposition, the hydrolyzed solution obtained in (3) is made into a 5 to 15% solution with hydrochloric acid, and the hydrolysis is carried out at 60 to 70 ° C.
The method of thermal decomposition for 2 hours is adopted.

After the above-mentioned acid thermal decomposition, it is filtered and the filtrate is adjusted to pH 9-10 with an alkaline agent such as sodium hydroxide or potassium hydroxide to form a precipitate on the alkaline side, and the precipitate is filtered. Remove.

(5) Desalting and Purification Since the peptide solution obtained by the above method contains an inorganic salt or an organic salt due to pH adjustment during hydrolysis or removal of insolubles. In order to mix the obtained peptide solution with cosmetics and foods to obtain a stable composition, it is necessary to remove these salts.

As a desalting method, an ion exchange resin method,
Although an electrodialysis method or the like can be adopted, it is preferable to employ an electrodialysis method that can easily desalt in a short time and has a small peptide loss. As this electrodialysis method, the method described in Japanese Patent Application Laid-Open No. 61-183295 previously filed by the present applicant can be applied.

That is, the peptide solution after removing the insoluble matter in the above (4) is neutralized with an acid or an alkali, and then an anion exchange membrane and a cation exchange membrane are alternately separated from each other, and a DC power supply is applied to the ion exchange membrane to load the ions. The electrodialysis is carried out by accelerating the migration of the peptide and causing the ions of the inorganic salt or the organic salt mixed in the peptide to diffuse out of the membrane.

The electrodialysis is terminated after confirming that the electric conductivity is 3 ms / cm or less, but the electric conductivity is 3 or less.
"Ms / cm or less" means that the salt concentration in the liquid is already sufficiently low.

The rice bran protein-derived peptide liquid thus obtained is subjected to treatments such as decolorization, deodorization, pH adjustment, concentration adjustment, sterilization filtration, heat sterilization, etc., after being treated in a liquid state, or It is made into a dry powder by spray drying or the like and used as a raw material or a compounding agent for cosmetics and foods.

[0039]

EXAMPLES Next, the present invention will be described more specifically by way of examples.

Example 1 250 g of defatted rice bran in 1.5 liter of 3.5% hydrochloric acid aqueous solution
Was added and suspended, and stirring was continued for 3 hours on a water bath at 65 ° C., soluble sugar was extracted with hydrochloric acid, and then filtered with a Tetron (registered trademark) cloth.

After removing the soluble sugars from the defatted rice bran as described above, the insoluble portion, that is, the residue after the filtration is added to 1.5 liters of ion-exchanged water and suspended, and the mixture is stirred for 20 minutes. % Sodium hydroxide aqueous solution to adjust the pH to 8.
5, and add to this suspension biopulase bulk powder [630,000 units,
Bioprase was hydrolyzed by adding 250 mg of proteolytic enzyme subtilisin (trade name, manufactured by Nagase & Co., Ltd.) and continuing stirring for 3 hours in a water bath at 45 ° C. After the hydrolysis, the temperature of the water bath was adjusted to 85 ° C., and stirring was continued for 30 minutes to inactivate the bioprase.

The hydrolysis liquid was 2 filter paper [filter paper No. manufactured by Toyo Roshi Kaisha, Ltd. 2], the residue was washed with 500 ml of ion-exchanged water at 40 ° C., the filtrate and the washing solution were combined, 25 g of sodium hydroxide was added, and the mixture was stirred for 2 hours on a water bath at 65 ° C. for thermal decomposition. did.

After cooling, the hydrogen peroxide solution with a concentration of 35% was
ml was added and left for 12 hours for decolorization. The insoluble matter that was not decomposed was No. 2 Filter out with filter paper to remove 6
The pH was adjusted to 4 with N hydrochloric acid, and the insoluble matter was precipitated. This insoluble matter is also No. After removal by filtration using 2 filter paper, the filtrate was neutralized to pH 7 with a 20% sodium hydroxide solution, and then electrodialyzed by the electrodialysis equipment shown below.

Model: DO-Cb type [manufactured by Teijin Engineering Ltd.] Membrane name: Selemion CMV and Seledion AM
V [all are trade names of electrodialysis membranes manufactured by Asahi Glass Co., Ltd.] Membrane size: 18 × 12 cm Number of incorporated membranes: 10 vs. voltage: 30V Positive / catholyte solution: 5% sodium sulfate aqueous solution 2 liters

The initial concentration of the circulating fluid between the membranes is 3
% Of saline solution, the electric conductivity was measured 2 hours after the start of electrodialysis, and when it was confirmed to be 3 ms / cm or less, electrodialysis was terminated and the filtrate was recovered.

The filtrate was concentrated under reduced pressure to give a solution having a concentration of 25%, and the pH was adjusted to 6.5 with a 20% aqueous sodium hydroxide solution. 5C filter paper [No. 5 manufactured by Toyo Roshi Kaisha, Ltd. 5C
Filter paper], sterilization filtration is performed using a membrane filter having a membrane pore of 0.45 μm, and the mixture is filled in a glass sterilized container, and a 25% aqueous solution of rice bran protein-derived peptide is added to 155
g was obtained.

When this peptide solution was analyzed by a total carbon and total nitrogen analyzer, the nitrogen content was 2.562%. The defatted rice bran used as a raw material had a nitrogen content of 4.268%, and thus the yield determined from the total nitrogen content was 37.22%.

When the peptide solution was subjected to gel filtration analysis under the following conditions, the average molecular weight of the obtained rice bran protein-derived peptide was 521.

Gel filtration analysis conditions Column: Diameter × length = 7.5 mm × 300 mm Stationary phase: TSK gel G3000pw (manufactured by Tosoh Corporation) Mobile phase: 45% containing 0.1% trifluoroacetic acid
Acetonitrile solution Flow rate: 0.3 ml / min Detector: UV detector Detection wavelength: 210 nm

Standard sample for calibration curve Aprotinin: Molecular weight 6500 α-MSH (melanophore stimulating hormone): Molecular weight 1655 Pradykinin: Molecular weight 1060 Glutathione: Molecular weight 307

Example 2 250 g of defatted rice bran was added to 1.5 liters of a 3% aqueous hydrochloric acid solution to suspend it, and the mixture was continuously stirred in a hot water bath at 65 ° C. for 3 hours to extract soluble sugars with hydrochloric acid. The mixture was filtered through a Tetoron (registered trademark) cloth, the residue was washed with 1 liter of ion-exchanged water at 50 ° C., and filtered through a Tetoron (registered trademark) cloth again.

The residue after filtration was added to 1.5 liters of ion-exchanged water to suspend it, and while stirring, 20% aqueous sodium hydroxide solution was added to adjust the pH to 8.5, and the bulk biopulase powder [690,000 units, biopulase 200 mg of a proteolytic enzyme subtilisin manufactured by Nagase & Co., Ltd.] was added, and the mixture was stirred for 3 hours in a water bath at 45 ° C. for hydrolysis.

After hydrolysis, the water bath was heated to 85 ° C.
The biopulase was inactivated by continuing stirring for a minute.

The hydrolyzed solution was changed to No. The mixture was filtered through 2 filter papers, the residue was washed with 500 ml of ion-exchanged water at 40 ° C., and the filtrate and the washing liquid were combined to make 2 liters.

35% hydrochloric acid was added to this hydrolyzed solution so that the solution concentration became 10%, and the mixture was stirred for 1 hour in a hot water bath at 65 ° C. for thermal decomposition.

The decomposition liquid after the above thermal decomposition was No. The mixture was filtered through 2 filter paper, sodium hydroxide was added to the filtrate so that the pH became 10, 10 ml of hydrogen peroxide solution having a concentration of 35% was further added, and the mixture was left for 12 hours.

The insoluble matter generated by the addition of the above sodium hydroxide was added to No. 2 Remove by filtering with filter paper,
The filtrate was brought to pH 7 with hydrochloric acid.

The resulting neutralized solution was electrodialyzed under the same conditions as in Example 1, and after completion of electrodialysis, 5 g of activated carbon was added to deodorize. After removing the activated carbon by filtration, the filtrate was concentrated to give a solution having a concentration of 25%.

Methyl p-hydroxybenzoate and propyl p-hydroxybenzoate were added as preservatives to this aqueous solution so as to be 0.3% and 0.03%, respectively, and 0.4
Sterilization filtration was performed using a 5 μm membrane filter, and the mixture was filled in a glass sterilized container and the rice bran protein-derived peptide 2
184 g of a 5% aqueous solution was obtained.

The obtained rice bran protein-derived peptide had a nitrogen analysis value of 2.609%, and the yield determined from the total nitrogen content was 4
It was 4.99%.

When gel filtration analysis was performed under the same conditions as in Example 1, the average molecular weight of the obtained rice bran protein-derived peptide was 556.

A 25% aqueous solution of the rice bran protein-derived peptides obtained in Examples 1 and 2 described above changed in color when stored in a thermostatic chamber at 40 ° C. for 12 weeks and insoluble when stored in a refrigerator at 5 ° C. for 12 weeks. The presence or absence of the occurrence of a thing was investigated. The results are shown in Table 2.
Shown in. The color change was measured by the Gardner method, and the presence or absence of insoluble matter was visually observed.

[0063]

[Table 2]

As shown in Table 2, the rice bran protein-derived peptides obtained in Examples 1 and 2 showed almost no change in color upon storage, no generation of insoluble matter was observed, and browning phenomenon and insolubilization upon storage were observed. Did not occur.

[0065]

As described above, according to the present invention,
Peptides can now be produced from rice bran, which has never been used as a peptide source.

Since the rice bran protein-derived peptide obtained by the present invention does not cause browning phenomenon or insolubilization, it can be preferably used as a raw material or a compounding agent for cosmetics, foods and the like.

Claims (2)

[Claims] 1. The defatted rice bran is heat-treated with a dilute acid to remove soluble sugars, the insoluble portion is suspended in water and hydrolyzed by a proteolytic enzyme to remove the insoluble matter, and the soluble portion is then electrolyzed. A method for producing a rice bran protein-derived peptide, which comprises desalting by dialysis and purification. 2. The average molecular weight of the rice bran protein-derived peptide is 2
The method for producing a rice bran protein-inducing peptide according to claim 1, which is from 0 to 2000.