JP5431025B2 - Method for producing modified peptide and modified peptide - Google Patents

Description

  The present invention relates to a method for producing a water-soluble denatured peptide and a denatured peptide obtained by the production method.

  Wool, feathers and the like are widely used in many fields such as clothing, bedding, and interior products. It is said that the manufacturing process of such clothing and the waste wool generated as used waste exceeds about 50,000 tons per year. Such waste wool has been disposed of by landfill or incineration, but there are concerns about environmental problems associated with this disposal.

  On the other hand, regardless of whether wool or the like is a target for disposal, research and development for dissolving the wool or the like to separate and extract a predetermined protein and use it for a film, fiber, or the like is being carried out.

As a result of such research and development, for example, in JP-A-7-126296, a disulfide bond (—S—S—) in a water-insoluble protein (keratin) such as wool is reduced to a mercapto group (—SH). converted, solubilized proteins (denaturation peptide) and a manufacturing method thereof obtained by converting all or part of the mercapto groups in carboxymethyl disulfide group (-SSCH ₂ COOH) are disclosed.

  Japanese Unexamined Patent Application Publication No. 2009-23924 discloses a method for producing soluble keratin (modified peptide) in which α-keratin that is insoluble in water is modified with sodium thioglycolate. Specifically, the method for producing soluble keratin includes a reduction step of bringing α-keratin into contact with sodium thioglycolate in the presence of water, and an oxidant mixing step of adding an oxidant to the treatment liquid after the reduction step. And a pH adjusting step for adjusting the pH of the treatment liquid to 5.0 to 8.0. As this pH adjustment process, the form performed before the oxidant mixing process and the form performed after the oxidant mixing process are disclosed, and the form performed before the oxidant mixing process can improve the yield of soluble keratin. Is shown in the experimental example.

  As described above, it is possible to produce a denatured peptide. However, in the conventional method for producing a denatured peptide, the obtained water-soluble denatured peptide is colored (yellowed) and unpleasant sulfur-like. Odor is observed. Since such yellowing and sulfur-like odor significantly impair the commercial value of the modified peptide, it is desired to provide a production technique for solving such inconvenience. Moreover, it cannot be said that it is practical in the manufacturing method of the modified peptide with a yield of several percent, and naturally the modified peptide which can implement | achieve a practical yield is desired.

JP-A-7-126296 JP 2009-23924 A

  The present invention has been made in view of the above circumstances, and aims to provide a method for producing a water-soluble modified peptide capable of reducing yellowing and sulfur-like odor and realizing a practical yield. is there.

  As a result of intensive studies to promote the use of keratin contained in wool and the like, the present inventors, in the process of producing a modified peptide for modifying keratin with thioglycolic acid or the like, have a pH of 11 to 9 in the presence of an oxidizing agent. It was found that when the pH was adjusted with an acid so that the time was longer than the time of pH 9-7, yellowing and sulfur-like odor could be reduced, and the modified peptide could be produced in a practical yield.

As a result, the invention relating to the method for producing a denatured peptide, which has been made to solve the above problems,
A reduction step of preparing a keratin mixture by mixing keratin, water and a reducing agent;
An oxidizing agent mixing step of mixing an oxidizing agent with the keratin mixture;
In the method for producing a modified peptide comprising: mixing an acid with the keratin mixed solution after addition of the oxidizing agent, and adjusting the pH of the keratin mixed solution to near neutrality.
The time of pH 11-9 in the said pH adjustment process is longer than the time of pH 9-7, It is characterized by the above-mentioned.
In addition, a pH adjustment process can be performed after starting or simultaneously with completion of an oxidizing agent mixing process.

  In the modified peptide production method, a pH adjustment step is provided after the oxidant mixing step, and in the pH adjustment step, the pH of the keratin mixture is adjusted so that the time of pH 11-9 is longer than the time of pH 9-7. By doing so, a water-soluble modified peptide can be produced in a practical yield, and the yellowing and sulfur-like odor of the resulting modified peptide are reduced.

  In particular, the time of pH 11 to 10 in the pH adjusting step may be longer than the time of pH 9 to 7. Thus, the effect of reducing yellowing and sulfur-like odor of the modified peptide is improved by making the time of pH 11 to 10 in the pH adjustment step longer than the time of pH 9 to 7.

  Before the oxidizing agent mixing step, it is preferable to further include a preliminary pH adjusting step of mixing an acid with the keratin mixed solution and adjusting the pH of the keratin mixed solution to 9 or more and 11 or less. Thus, by adjusting the pH of the keratin mixture to 9 or more and 11 or less before mixing the oxidizing agent, the yellowing and sulfur-like odor of the above-mentioned modified peptide can be more reliably reduced. The coloring of the denatured peptide that occurs when hydrogen water is used and the pH exceeds 11 is also effectively reduced.

  As the reducing agent, one or more selected from the group consisting of thioglycolic acid, thioglycolate, mercaptopropionic acid and mercaptopropionic acid may be used. By using these reducing agents, the yield of the modified peptide is further improved.

  As the oxidizing agent, one or more selected from the group consisting of sodium bromate, potassium bromate, sodium perborate and hydrogen peroxide may be used. Any of these oxidizing agents can be used to improve the yield of the modified peptide and reduce yellowing and sulfur-like odor.

  As the acid in the pH adjustment step and / or the preliminary pH adjustment step, citric acid or acetic acid is preferable. In particular, by using citric acid, it is possible to effectively reduce the generation of a specific odor like thioglycolic acid.

  In the reduction step, it is preferable to adjust the pH of the keratin mixture to 9 or more and 13 or less. By adjusting the pH of the keratin mixed solution in the reduction step to this range, the disulfide group can be reduced while inhibiting the cleavage of the keratin main chain, and a high molecular weight modified peptide can be produced with high yield.

  In the reduction step, an alkaline compound may be mixed into the keratin mixture. Thus, the yield of a modified | denatured peptide improves more by mixing an alkaline compound with a keratin liquid mixture.

  As temperature of the keratin liquid mixture in the said reduction | restoration process, 20 to 60 degreeC is preferable. Thus, by setting the temperature of the keratin mixed solution in the reduction step within this range, the reduction of the keratin disulfide group can proceed smoothly, and the keratin main chain can be prevented from being broken.

  The modified peptide according to the present invention is obtained by the method for producing the modified peptide.

  As described above, according to the method for producing a modified peptide according to the present invention, a pH adjusting step is provided after the oxidizing agent mixing step, and in the pH adjusting step, the time of pH 11 to 9 is longer than the time of pH 9 to 7. By adjusting the pH of the keratin mixture so as to be long, a water-soluble modified peptide can be produced in a practical yield while reducing yellowing and sulfur-like odor. As a result, according to the method for producing a modified peptide, the productivity and low cost are improved, and the utility and versatility of the modified peptide obtained in addition are improved.

The flowchart which shows the manufacturing method of the modified | denatured peptide which concerns on 1st embodiment of this invention. The flowchart which shows the manufacturing method of the modified | denatured peptide which concerns on 2nd embodiment of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate.

<First embodiment>
As shown in FIG. 1, the method for producing a modified peptide according to the first embodiment of the present invention is to produce a modified peptide using keratin as a raw material. The reduction step (STP1) and the oxidizing agent mixing step (STP2) And a pH adjustment step (STP3), and an optional insoluble matter removal step (STP4) and a recovery step (STP5).

(Reduction process)
The reduction step (STP1) is a step of adjusting the keratin mixture by mixing keratin, water and a reducing agent. In this reduction step, the disulfide group (—S—S—) of keratin is reduced to a mercapto group (—SH HS—).

  Examples of the raw material keratin include wool (merino wool, Lincoln wool, etc.), human hair, animal hair, feathers, nails and the like containing this as a constituent protein. Among these, wool or feather is preferably used because it is inexpensive and can be stably obtained. About raw materials, such as this wool, it is good to process beforehand combining sterilization, degreasing, washing, cutting, crushing, and drying suitably.

  The amount of water is not particularly limited. For example, the amount of water is preferably 20 parts by volume or more and 200 parts by volume or less with respect to 1 part by mass of raw materials such as wool.

  The reducing agent has an action of converting a disulfide group of keratin into a mercapto group. Examples of the reducing agent include mercaptoalkylcarboxylic acid and / or a salt thereof, thiolactic acid and / or a salt thereof, dithiothreitol, 2-mercaptoethanol, glutathione, and thiourea. In the modified peptide production method of the present invention, one or more reducing agents can be used.

  Among the reducing agents, mercaptoalkyl carboxylic acids and / or salts thereof are preferable, and serve as denaturing agents for converting mercapto groups generated in the reduction step into carboxylatoalkyl disulfide groups. As this mercaptoalkylcarboxylic acid and / or salt thereof, one or more selected from the group consisting of thioglycolic acid, thioglycolate, mercaptopropionic acid, and mercaptopropionate are used. Examples of the thioglycolate include sodium thioglycolate, potassium thioglycolate, lithium thioglycolate, and ammonium thioglycolate. Of these, sodium thioglycolate and potassium thioglycolate are preferable, and sodium thioglycolate is more preferable from the viewpoint of efficiently modifying keratin. Examples of mercaptopropionate include sodium mercaptopropionate, potassium mercaptopropionate, lithium mercaptopropionate, and ammonium mercaptopropionate. Among these, sodium mercaptopropionate and potassium mercaptopropionate are preferable, and sodium mercaptopropionate is more preferable from the viewpoint of efficiently modifying keratin.

  The amount of the mercaptoalkylcarboxylic acid and its salt used is preferably 0.0050 mol or more and 0.02 mol or less, particularly preferably 0.0075 mol or more and 0.01 mol or less, based on 1 g of raw materials such as wool. Further, the amount used is preferably from 0.10 mol / L to 0.40 mol / L, particularly preferably from 0.15 mol / L to 0.20 mol / L, based on the volume of the keratin mixture. By making the usage-amount of this mercaptoalkylcarboxylic acid into the said range, the reduction reaction of keratin can be performed favorably.

  In addition, it is good to mix an alkaline compound with the keratin liquid mixture in a reduction | restoration process. An alkaline compound is a compound that can be made alkaline by adding it to water. Examples of the alkaline compound include lithium hydroxide, sodium hydroxide, potassium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate, ammonia and the like, and other basic compounds such as monoethanolamine, diethanolamine, arginine, and lysine. Amino acids, sodium bicarbonate, ammonium bicarbonate and the like are also included. Among these, sodium hydroxide and potassium hydroxide are preferable and sodium hydroxide is particularly preferable from the viewpoint of efficiently modifying keratin. In the reduction step, one or more of the above alkaline compounds can be used.

  The mixing amount of the alkaline compound is not particularly limited, but it may be blended so that the pH of the keratin mixture in the reduction step is adjusted to the following range. As a minimum of pH of a keratin liquid mixture in a reduction process, 9 are preferred and 10 is especially preferred. On the other hand, the upper limit of the pH of the keratin mixture in the reduction step is preferably 13, preferably 12 and particularly preferably 11. The yield of the modified peptide is improved by adjusting the pH of the keratin mixed solution to be equal to or higher than the above lower limit. On the other hand, the upper limit of the mixing amount can be set according to the molecular weight of the resulting modified peptide. Specifically, by adjusting the pH of the keratin mixed solution to be not more than the above upper limit, cleavage of the keratin main chain can be suppressed and a high molecular weight modified peptide can be produced. When the purpose is to promote the cleavage of the keratin main chain and to produce a low molecular weight modified peptide, it is preferable to adjust the pH of the keratin mixed solution to exceed 13.

  As a minimum of the temperature of a keratin liquid mixture in a reduction process, 20 ° C is preferred, 30 ° C is more preferred, and 40 ° C is still especially preferred. On the other hand, the upper limit of the temperature of the keratin mixture is preferably 60 ° C. If the temperature of the keratin mixture is lower than the lower limit, the reduction time for converting the disulfide group into a mercapto group becomes long, and there is a possibility that sufficient reduction cannot be performed. On the other hand, when the temperature of the keratin mixture exceeds the upper limit, the keratin main chain may be cleaved. The set time for maintaining the temperature of the keratin mixture is set longer as the temperature of the keratin mixture is lower, and is set shorter as the temperature is higher. The time is, for example, 20 minutes or more and 120 minutes or less.

(Oxidizing agent mixing process)
The oxidant mixing step (STP2) is a step of mixing an oxidant with the keratin mixture after the reduction step (STP1). The mixing of the oxidizing agent is performed to promote an oxidation reaction for modifying the mercapto group of keratin.

  Examples of the oxidizing agent include sodium bromate, potassium bromate, sodium perborate, hydrogen peroxide, and the like. In the oxidizing agent mixing step, one or two or more oxidizing agents selected from the group consisting of sodium bromate, potassium bromate, sodium perborate and hydrogen peroxide are preferably used to improve the yield of the modified peptide. Yellowing and reduction of sulfur-like odor can be achieved.

  The amount of the oxidizing agent used is not particularly limited, but is preferably 0.001 mol or more and 0.02 mol or less based on 1 g of raw materials such as wool, and 0.02 mol / L or more and 1 mol based on the volume of the keratin mixture. / L or less is preferable. If the amount of the oxidizing agent used exceeds the above range, cystine monooxide, cystine dioxide and cysteic acid may be generated, and the yield of the modified peptide may be reduced. On the other hand, when the amount of the oxidizing agent used is smaller than the above range, the keratin may be insufficiently modified. In mixing the oxidizing agent, in order to avoid local concentration of the oxidizing agent in the keratin mixture, an oxidizing agent solution of about 1 mol / L or more and 5 mol / L or less is gradually added over, for example, 30 minutes to 6 hours. Mix well.

  In addition, the temperature of the keratin mixed solution in the oxidizing agent mixing step is not particularly limited, but can be set to be equal to or lower than the temperature in the reducing step, for example.

(PH adjustment step)
The pH adjustment step (STP3) is a step of mixing an acid with the keratin mixture after the addition of the oxidant in the oxidant mixing step (STP2), and lowering the pH of the keratin mixture to near neutrality. This pH adjustment step (STP3) can be performed after the start or simultaneously with the completion of the mixing of the oxidant into the keratin mixture in the oxidant mixing step (STP2). By reducing the pH of the keratin mixture by mixing the acid in this step, the mercapto group of keratin in the mixture can be sufficiently converted to a carboxylatoalkyl disulfide group.

  In the pH adjustment step (STP3), the pH of the keratin mixture is adjusted so that the time of pH 11-9 is longer than the time of pH 9-7. By such pH adjustment, the modified peptide can be obtained in a practical yield, and the yellowing and sulfur-like odor of the resulting modified peptide can be reduced. In the pH adjusting step (STP3), the time of pH 11 to 9 is preferably 5 minutes or longer and more preferably 10 minutes or longer compared to the time of pH 9 to 7. By making the time of pH 11-9 longer than the time of pH 9-7, the effect of reducing the yellowing and sulfur-like odor of the above-mentioned modified peptide can be reliably exhibited.

  In particular, the time of pH 11 to 10 in the pH adjustment step (STP3) may be longer than the time of pH 9 to 7. Thus, the effect of reducing the yellowing and sulfur-like odor of the above-mentioned modified peptide is further improved by making the time of pH 11 to 10 in the pH adjustment step (STP3) longer than the time of pH 9 to 7.

  The time of pH 11 to 9 in the pH adjustment step (STP3) is not particularly limited as long as it is longer than the time of pH 9 to 7, but preferably 20 minutes or more and 120 minutes or less, more preferably 40 minutes or more and 90 minutes or less. It can be. Further, the time of pH 9 to 7 in the pH adjusting step (STP3) is not particularly limited as long as it is shorter than the time of pH 11 to 9, but preferably 1 minute to 90 minutes, more preferably 2 minutes to 60. It can be less than a minute. By controlling the time of pH 11 to 9 and / or the time of pH 9 to 7 in such a preferable range, while suppressing disulfation between mercapto groups due to local pH decrease in the keratin mixture, It is possible to obtain a modified peptide with a good yield and a good reduction in yellowing and sulfur-like odor.

  As the acid to be mixed in the pH adjustment step, one or more selected from organic acids and inorganic acids may be used. Examples of the organic acid include citric acid, lactic acid, succinic acid, and acetic acid, and examples of the inorganic acid include hydrochloric acid and phosphoric acid. Of these, acetic acid or citric acid is preferred. If acetic acid is used, a specific odor like thioglycolic acid may be generated from the modified peptide, but if citric acid is used, the specific odor is reduced.

  The mixing amount of the acid is not particularly limited, and the final pH is in the vicinity of neutrality (specifically, 5 or more, preferably 9 or less, preferably such that the pH 11 to 9 is longer than the pH 9 to 7). Is preferably blended so as to decrease to 6 or more and 8 or less. Thus, by adjusting the final pH of the keratin mixed solution to near neutrality, introduction of a modifying group into keratin is promoted, and generation of disulfide groups by keratin mercapto groups is suppressed.

  As temperature of the keratin liquid mixture in a pH adjustment process, 10 to 60 degreeC is preferable and 20 to 40 degreeC is especially preferable. In this way, by controlling the temperature of the keratin mixed solution within the above range, production of cystine monooxide and the like can be suppressed.

  In addition, after mixing of an acid is complete | finished by a pH adjustment process, it is good to leave a keratin liquid mixture for 1 to 48 hours, for example. Thus, by leaving the keratin mixture for a predetermined time after pH adjustment, the modifying group can be sufficiently introduced into keratin.

A modified peptide solution is obtained through the reduction step (STP1), the oxidizing agent mixing step (STP2), and the pH adjustment step (STP3). When one or two or more kinds selected from thioglycolic acid and salts thereof are used as a reducing agent in the reduction step, the resulting modified peptide has a keratin mercapto group as an ionic group (-S-SCH ₂ COO) of carboxymethyl disulfide. ^-) in which it has been converted to. The reaction formula for the conversion is as follows.

Further, when one or more selected from mercaptopropionic acid and salts thereof are used as a reducing agent in the reduction step, the resulting modified peptide has a keratin mercapto group as an ionic group (-S-SCH) of carboxyethyl disulfide. ₂ CH ₂ COO ⁻ ). The reaction formula for the conversion is as follows.

(Insoluble matter removal process)
A water-soluble modified peptide is produced through a pH adjustment step, but the obtained modified peptide solution contains insoluble matter. Therefore, in the insoluble matter removing step (STP4), insoluble matters are removed from the modified peptide solution obtained by the end of the pH adjusting step (STP3). In the insoluble matter removing step (STP4), known solid-liquid separation means such as centrifugation, squeezing separation, sedimentation separation, and flotation separation can be employed, and desalting by ion exchange, electrodialysis, etc., if necessary. Good to do.

(Recovery process)
The next recovery step (STP5) is a step of recovering the solid denatured peptide from the denatured peptide solution. As a method for recovering the solid modified peptide in this recovery step (STP5), (1) recovery by freeze-drying the modified peptide solution, (2) recovery by spray drying the modified peptide solution, (3) hydrochloric acid, etc. And the like, and the like. For example, recovery of a modified peptide precipitate generated by adding the acid of the above to a modified peptide solution and lowering the pH of the solution from about 2.5 to about 4.0. In addition, about the collect | recovered solid modified | denatured peptide, it is good to perform washing | cleaning with water or acidic aqueous solution, drying, etc. as needed.

  The modified peptides thus obtained are water-soluble and can be used as cosmetic raw materials, fiber surface treatment agents, protein film raw materials and the like.

  The modified peptide contained in the insoluble matter removed in the insoluble matter removing step (STP5) also has reduced yellowing and sulfur-like odor. The modified peptide has a higher sulfur content than the modified peptide obtained in the recovery step. And if the modified | denatured peptide contained in the said insoluble matter is hydrolyzed, the water-soluble modified | denatured peptide with many sulfur content and reduced yellowing and sulfur-like odor can be obtained. Examples of the hydrolysis method include (a) hydrolysis with an enzyme, (b) hydrolysis with an acid, and (c) hydrolysis with an alkali. In the method (c), since β-elimination reaction of the carboxylate alkyl disulfide group of the peptide may proceed, (a) hydrolysis with an enzyme or (b) hydrolysis with an acid is particularly preferable.

  Examples of the enzyme in the hydrolysis by the enzyme (a) include acidic proteolytic enzymes such as pepsin, protease A, and protease B; neutral proteolytic enzymes such as papain, promelain, thermolysin, pronase, trypsin, chymotrypsin, etc. Is mentioned. As a commercially available proteolytic enzyme, “Proteerizer A” manufactured by Daiwa Chemical Industry Co., Ltd. is preferably used for hydrolysis of wool keratin. Examples of the acid in the hydrolysis with the acid (b) include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid and hydrobromic acid; organic acids such as formic acid and oxalic acid. Examples of alkali in the hydrolysis with (c) alkali include sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide, sodium carbonate, potassium carbonate, lithium carbonate, sodium silicate, sodium borate and the like. Is mentioned.

  Thus, since the modified peptide obtained by hydrolyzing impurities in the insoluble matter removing step is water-soluble and has a high sulfur content, a wide range of cosmetic raw materials, fiber surface treatment agents, protein film raw materials, etc. Can be used in the field.

<Second Embodiment>
As shown in FIG. 2, the method for producing a modified peptide according to the second embodiment of the present invention is to produce a modified peptide using keratin as a raw material, and includes a reduction step (STP1) and a preliminary pH adjustment step (STP6). ), An oxidizing agent mixing step (STP2), and a pH adjustment step (STP3), and an optional insoluble matter removing step (STP4) and a recovery step (STP5).

  The reduction step (STP1), the oxidizing agent mixing step (STP2), the pH adjustment step (STP3), the insoluble matter removal step (STP4) and the recovery step (STP5) in the modified peptide production method according to the second embodiment include: Since it is the same as that of the manufacturing method of the modified | denatured peptide which concerns on said 1st embodiment, detailed description regarding these processes is abbreviate | omitted here. In addition, the modified peptide contained in the insoluble matter removed in the insoluble matter removing step in the present embodiment also has a high sulfur content and is yellowed by hydrolysis, as in the first embodiment. A water-soluble modified peptide with reduced sulfur-like odor can be obtained.

(Preliminary pH adjustment step)
In the preliminary pH adjustment step (STP6), an acid is mixed with the keratin mixture after the reduction step (STP1) and before the oxidant mixing step (STP2), and the pH is 9 to 11, preferably 10 to 11. It is the process of adjusting to the following. As an example of the acid in this preliminary pH adjustment process, the acid similar to the acid in the pH adjustment process of 1st embodiment can be mentioned.

  The time for adjusting the pH to 9 or more and 11 or less by mixing the acid with the keratin mixed solution is preferably 5 minutes or more and 60 minutes or less, more preferably 10 minutes or more and 30 minutes or less. The amount of the acid mixed in the preliminary pH adjustment step is not particularly limited, but it is preferable that the acid is mixed so that the pH of the keratin mixture reaches 9 to 11 over a period of 5 minutes to 60 minutes. Further, the temperature of the keratin mixed solution in the preliminary pH adjustment step is preferably 10 ° C. or higher and 60 ° C. or lower, and particularly preferably 20 ° C. or higher and 40 ° C. or lower. In this way, by controlling the temperature of the keratin mixed solution within the above range, production of cystine monooxide and the like can be suppressed.

  The modified peptide production method according to the second embodiment includes a pH adjustment step after the oxidizing agent mixing step, as in the modified peptide production method according to the first embodiment. By adjusting the pH of the keratin mixture so that the time of -9 is longer than the time of pH 9-7, a water-soluble modified peptide is produced in a practical yield while reducing yellowing and sulfur-like odor. can do.

  The modified peptide production method includes a preliminary pH adjustment step (STP6) for adjusting the pH of the keratin mixture to 9 to 11 (preferably 10 to 11) before the oxidizing agent mixing step (STP2). When the pH adjustment step (STP3) is performed after or simultaneously with the mixing of the oxidizing agent in the oxidizing agent mixing step (STP2), and the pH of the keratin mixed solution in the oxidizing agent mixing step (STP2) is maintained at 9 or more and 11 or less Good. In this way, the pH of the keratin mixture before mixing of the oxidant is adjusted to 9 or more and 11 or less in the preliminary pH adjustment step (STP6), and the pH of the keratin mixture in the subsequent oxidant mixing step (STP3). By keeping any of the oxidants at 9 or more and 11 or less, the side reaction in the reaction system is suppressed in any of the oxidizing agents, and the resulting modified peptide causes yellowing and sulfur-like odor. In particular, coloring of the modified peptide that occurs when hydrogen peroxide is used as the oxidizing agent and the pH exceeds 11 is also effectively reduced.

  EXAMPLES Hereinafter, although this invention is explained in full detail based on an Example, this invention is not interpreted limitedly based on description of this Example.

[Example 1]
A solid denatured peptide was obtained according to the following reduction step, oxidizing agent mixing step, pH adjustment step, impurity removal step, and recovery step.

(Reduction process)
Merino wool, washed with neutral detergent and dried, was cut to about 5 mm. 25 g of this wool containing keratin, 76.8 g of 30% by weight aqueous sodium thioglycolate solution and 42.5 g of 6 mol / L aqueous sodium hydroxide solution were mixed, and water was further mixed to prepare a total amount of 750 ml, pH 11 keratin mixed solution. . This keratin mixture was stirred at 45 ° C. for 1 hour. Next, water was further mixed to make a total volume of 1000 ml, stirred at 45 ° C. for 2 hours, and then naturally cooled until the liquid temperature reached room temperature.

(Oxidizing agent mixing process)
125 g of an aqueous solution containing 10.25 g of sodium bromate was mixed with the keratin mixture after the reduction step over about 60 minutes. During mixing, the keratin mixture was constantly stirred.

(PH adjustment step)
About 300 ml of citric acid aqueous solution (500 g of aqueous solution containing 35.4 g of citric acid) is gradually mixed over about 50 minutes to the stirred keratin mixture after the oxidizing agent mixing step, and the pH of the keratin mixture gradually increases. Adjustments were made to be 11-9. Next, about 150 ml of the same citric acid aqueous solution was gradually mixed over about 30 minutes, and the pH of the keratin mixture was gradually adjusted to 9-8. Subsequently, about 50 ml of the same citric acid aqueous solution was gradually mixed over about 5 minutes, and the pH of the keratin mixture was adjusted to gradually become 8-7. In this way, a denatured peptide solution was obtained by mixing citric acid with an aqueous keratin solution.

(Insoluble matter removal process and recovery process)
The insoluble matter in the solution was removed by filtering the denatured peptide solution. Thereafter, 160 g of an aqueous solution containing 97.2 g of 36 mass% hydrochloric acid aqueous solution was added to the filtrate to adjust the pH of the modified peptide solution to 3.8, thereby causing the modified peptide to precipitate. This precipitate was recovered and washed with water to obtain a solid modified peptide.

[Comparative Example 1]
In the pH adjustment step, about 300 ml of the same citric acid aqueous solution as in Example 1 is gradually mixed over about 5 minutes with the stirred keratin mixture after the oxidizing agent mixing step, and the pH of the keratin mixture gradually becomes 11 Next, about 150 ml of the same citric acid aqueous solution is gradually mixed over about 30 minutes, and the pH of the keratin mixture is gradually adjusted to 9 to 8, followed by the same citric acid. A solid denatured peptide was obtained in the same manner as in Example 1 except that about 50 ml of the acid aqueous solution was gradually mixed over about 50 minutes and the pH of the keratin mixture was adjusted to gradually become 8 to 7.

[Comparative Example 2]
The pH adjustment step is replaced with a preliminary pH adjustment step. In this preliminary pH adjustment step, about 300 ml of the same citric acid aqueous solution as in Example 1 is gradually added to the stirred keratin mixture after the reduction step over about 5 minutes. To adjust the pH of the keratin mixture gradually from 11 to 9, then about 150 ml of the same citric acid aqueous solution is gradually mixed over about 30 minutes, and the pH of the keratin mixture gradually increases from 9 Example 1 except that about 50 ml of the same citric acid aqueous solution was gradually mixed over about 50 minutes and adjusted so that the pH of the keratin mixture gradually became 8 to 7. In the same manner as above, a solid modified peptide was obtained.

[Example 2]
A solid modified peptide was obtained in the same manner as in Example 1 except that the following preliminary pH adjustment step, oxidizing agent mixing step, and pH adjustment step were followed.

(Preliminary pH adjustment step)
A citric acid aqueous solution (same concentration as the citric acid aqueous solution of Example 1) was mixed with the keratin mixed solution after the reduction step, and the pH of the keratin mixed solution was adjusted to gradually become 11 to 10.

(Oxidizing agent mixing process)
To the keratin mixture after the preliminary pH adjustment step, 178 g of an aqueous solution containing 15.26 g of 35% by mass hydrogen peroxide was mixed with stirring over about 30 minutes.

(PH adjustment step)
After starting the mixing of the oxidizing agent in the oxidizing agent mixing step, the keratin mixed solution was constantly stirred, and the citric acid aqueous solution was mixed so that the pH was maintained at 10 or more and 11 or less. In addition, about 50 ml of the citric acid aqueous solution was gradually mixed over about 5 minutes to the stirred keratin mixture after the oxidizing agent mixing step, and the pH of the keratin mixture was adjusted to gradually become 10 to 7. .

[Comparative Example 3]
The oxidizing agent in the oxidizing agent mixing step is changed to 178 g of an aqueous solution containing 15.26 g of 35% by mass hydrogen peroxide, and further, in the pH adjusting step, the keratin mixed solution that is stirred after the oxidizing agent mixing step is implemented. About 300 ml of the same citric acid aqueous solution as in Example 1 is gradually mixed over about 5 minutes, the pH of the keratin mixture is gradually adjusted to 11 to 9, and then about 150 ml of the same citric acid aqueous solution is added over about 30 minutes. Mix gradually and adjust so that the pH of the keratin mixture gradually becomes 9 to 8, and then gradually mix about 50 ml of the same aqueous citric acid solution over about 50 minutes, and gradually adjust the pH of the keratin mixture from 8 to 8. A solid modified peptide was obtained in the same manner as in Example 1 except that the amount was adjusted to 7.

[Example 3]
An acetic acid aqueous solution (825 g aqueous solution containing 34 g of acetic acid) is used as the acid in the preliminary pH adjusting step, the oxidizing agent mixing step, and the pH adjusting step, and 10.25 g of sodium bromate is added as the oxidizing agent in the oxidizing agent mixing step. A solid modified peptide was obtained in the same manner as in Example 2 except that 125 g of the aqueous solution was used.

[Comparative Example 4]
In the reduction step, a 30% by mass thioglycolic acid aqueous solution is used as the reducing agent, the temperature condition during reduction is 30 ° C., and in the pH adjustment step, acetic acid is added to the stirred keratin mixture after the oxidant mixing step. An aqueous solution (825 g of an aqueous solution containing 34 g of acetic acid) is gradually mixed over about 5 minutes to adjust the pH of the keratin mixture gradually from 11 to 9, and then the aqueous acetic acid solution is gradually added over about 30 minutes. To adjust the pH of the keratin mixture gradually from 9 to 8, followed by gradually mixing the aqueous acetic acid solution over about 50 minutes, so that the pH of the keratin mixture gradually becomes 8 to 7. A solid modified peptide was obtained in the same manner as in Example 1 except that the amount was adjusted to 1.

[Example 4]
A solid modified peptide was obtained in the same manner as in Example 2 except that an acetic acid aqueous solution (825 g aqueous solution containing 34 g of acetic acid) was used as the acid in the preliminary pH adjusting step, oxidizing agent mixing step and pH adjusting step. It was.

[Evaluation of characteristics]
(Coloring and odor of liquid phase part after pH adjustment step)
The coloration and odor of the liquid phase part of the modified peptide solutions in the examples and comparative examples were observed. It is judged that the smaller the degree of yellowing, the better the coloring, and the better the odor, the smaller the unpleasant odor such as sulfur-like odor. The observation results of coloring and odor regarding each Example and Comparative Example are shown in Tables 1-1 and 1-2 below.

(Molecular weight distribution of denatured peptide)
The molecular weight distribution of the modified peptide in the modified peptide aqueous solution (liquid phase after the impurity removal step) was confirmed. This molecular weight distribution was confirmed by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method using “Protein Molecular Weight Marker (Low)” manufactured by Takara Bio Inc. as a reference substance. In the confirmation of the molecular weight distribution, the clearer the molecular weight distribution band, the more likely the desired modified peptide is obtained under the optimum conditions, and it is judged that the molecular weight distribution is better. The observation results of the molecular weight distribution regarding each Example and Comparative Example are shown in Tables 1-1 and 1-2 below.

(Yield of modified peptide)
The yield results of the modified peptides obtained in the Examples and Comparative Examples are shown in Tables 1-1 and 1-2 below. In addition, the yield in Table 1 is a value obtained by the following calculation formula.

Yield = 100 × (W ₀ −W _res ) / W ₀
yield : Yield of modified peptide (%)
W ₀ : Dry mass of wool used in the reduction step W _res : Dry mass of insoluble matter removed in the insoluble matter removal step

  When Table 1-1 and 1-2 are confirmed, in Comparative Examples 1 to 4 in which the time of pH 11 to 9 in the pH adjustment step is shorter than the time of pH 9 to 7, the liquid phase part is colored yellow and sulfur odor ( In Comparative Example 4, it was found that there was a thioglycolic acid odor. In Comparative Example 2 in which the pH was adjusted only before mixing with the oxidizing agent, almost no modified peptide was obtained. On the other hand, in Examples 1 to 4 in which the time of pH 11 to 9 in the pH adjustment step is longer than the time of pH 9 to 7, coloring and sulfur odor are hardly observed in the liquid phase part, and the yield is good. A denatured peptide was obtained.

Claims (10)

A reduction step of preparing a keratin mixture by mixing keratin, water and a reducing agent;
An oxidizing agent mixing step of mixing an oxidizing agent with the keratin mixture;
In the method for producing a modified peptide comprising: mixing an acid with the keratin mixed solution after addition of the oxidizing agent, and adjusting the pH of the keratin mixed solution to near neutrality.
The method for producing a denatured peptide, wherein the time of pH 11 to 9 in the pH adjusting step is longer than the time of pH 9 to 7.   The method for producing a modified peptide according to claim 1, wherein the time of pH 11 to 10 in the pH adjustment step is longer than the time of pH 9 to 7.   The modification | denaturation of Claim 1 or Claim 2 further equipped with the preliminary pH adjustment process of mixing an acid with a keratin liquid mixture before the said oxidizing agent mixing process, and adjusting pH of this keratin liquid mixture to 9-11. A method for producing a peptide.   The said reducing agent is 1 type, 2 types or more selected from the group which consists of thioglycolic acid, thioglycolic acid salt, mercaptopropionic acid, and mercaptopropionic acid salt, Claim 2, Claim 2 or Claim 3 is used. A method for producing a denatured peptide.   5. The method according to claim 1, wherein one or more selected from the group consisting of sodium bromate, potassium bromate, sodium perborate, and hydrogen peroxide is used as the oxidizing agent. A method for producing a denatured peptide.   The method for producing a modified peptide according to any one of claims 1 to 5, wherein citric acid or acetic acid is used as the acid in the pH adjustment step and / or the preliminary pH adjustment step.   The method for producing a modified peptide according to any one of claims 1 to 6, wherein the pH of the keratin mixed solution is adjusted to 9 or more and 13 or less in the reduction step.   The method for producing a modified peptide according to any one of claims 1 to 7, wherein an alkaline compound is mixed in the keratin mixed solution in the reduction step.   The method for producing a modified peptide according to any one of claims 1 to 8, wherein the temperature of the keratin mixture in the reduction step is 20 ° C or higher and 60 ° C or lower.   A modified peptide obtained by the method for producing a modified peptide according to any one of claims 1 to 9.

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