JP5437691B2 - 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, but naturally it is desired to produce a denatured peptide with good yield. Further, in the conventional method for producing a modified peptide, an unpleasant specific odor considered to be based on a by-product is recognized from the obtained water-soluble modified peptide, and it is desired to eliminate this specific odor. Yes.

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

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for producing a water-soluble denatured peptide that is good in yield and can suppress a specific odor.

  As a result of intensive studies to promote the use of keratin contained in wool, etc., the present inventors mixed citric acid in the pH adjustment step of the modified peptide production process in which keratin is modified with thioglycolic acid. The present inventors have found that when an oxidant is mixed before mixing of citric acid, a modified peptide with a suppressed specific odor can be produced with good yield. In addition, in this production method, it was found that keratin can be modified with mercaptopropionic acid in the same manner as keratin modification with thioglycolic acid, and in this case, the modified peptide can be produced with good 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, an alkaline compound, water, and mercaptoalkylcarboxylic acid and / or a salt thereof;
An oxidizing agent mixing step of mixing an oxidizing agent with the keratin mixture;
A pH adjustment step of mixing citric acid into the keratin mixture after the addition of the oxidizing agent,
As the mercaptoalkylcarboxylic acid and / or a salt thereof, one or more selected from the group consisting of thioglycolic acid, thioglycolate, mercaptopropionic acid and mercaptopropionate are used.

  In the modified peptide production method, a pH adjustment step is provided after the oxidizing agent mixing step, and citric acid is mixed in the pH adjustment step, whereby a water-soluble modified peptide can be produced with high yield, and the resulting modification The specific odor of the peptide can be suppressed.

  In the pH adjustment step, it is preferable to adjust the pH of the keratin mixture to 5.0 or more and 9.0 or less. By adjusting the pH of the keratin mixture in the pH adjustment step to this range, keratin denaturation can be performed efficiently.

  In the reduction step, it is preferable to adjust the pH of the keratin mixture to 9.0 or more and 13.0 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.

  As the alkaline compound in the reduction step, sodium hydroxide and / or potassium hydroxide is preferable. By selecting these alkaline compounds, the yield of the modified peptide is further improved.

  The thioglycolate and mercaptopropionate are preferably selected from the group consisting of sodium salts and potassium salts. This selection also improves the yield of the modified peptide.

  The temperature of the keratin mixture in the reduction step is preferably 20 ° C. or higher and 60 ° C. or lower. 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 citric acid is mixed in the pH adjusting step. It can be produced with good yield. And the modified | denatured peptide manufactured in this way becomes the thing by which the specific odor was suppressed. 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 one Embodiment of this invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate.
As shown in FIG. 1, the modified peptide production method is a method for producing a modified peptide using keratin as a raw material, and includes a reduction step (STP1), an oxidizing agent mixing step (STP2), and a pH adjustment step (STP3). And an insoluble matter removing step (STP4) and a collecting step (STP5) as optional steps.

(Reduction process)
The reduction step (STP1) is a step of preparing a keratin mixed solution by mixing keratin, an alkaline compound, water, and a mercaptoalkylcarboxylic acid and / or a salt thereof. In this reduction step, the disulfide group (—S—S—) of keratin is reduced to a mercapto group (—SH HS—).

  As raw material keratin, wool (merino wool, Lincoln wool, etc.), human hair, animal hair, feathers, nails, etc. containing this as a constituent protein are used. Among them, wool and feathers are inexpensive and stable. It is preferably used in terms of availability. About raw materials, such as this wool, it is good to process beforehand combining sterilization, degreasing, washing, cutting, crushing, and drying suitably.

  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 producing the modified peptide with high yield. In addition, the said alkaline compound can be used 1 type, or 2 or more types.

  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, 9.0 is preferred and 10.0 is especially preferred. On the other hand, the upper limit of the pH of the keratin mixture in the reduction step is preferably 13.0, and particularly preferably 12.0. The yield of the modified peptide can be 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 13.0.

  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, whereby the reduction reaction is favorably performed.

  Mercaptoalkylcarboxylic acid and / or a salt thereof is a reducing agent that converts a disulfide group of keratin into a mercapto group. 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. Among these, sodium thioglycolate and potassium thioglycolate are preferable and sodium thioglycolate is more preferable from the viewpoint of improving the yield of the modified peptide. Examples of mercaptopropionate include sodium mercaptopropionate, potassium mercaptopropionate, lithium mercaptopropionate, and ammonium mercaptopropionate. Among these, from the viewpoint of improving the yield of the modified peptide, sodium mercaptopropionate and potassium mercaptopropionate are preferable, and sodium mercaptopropionate is more preferable.

  The amount of mercaptoalkylcarboxylic acid and its salt used is preferably 0.005 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. The amount used is preferably from 0.1 mol / L to 0.4 mol / L, particularly preferably from 0.15 mol / L to 0.2 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.

  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, potassium perborate, hydrogen peroxide, and the like. In addition, the said oxidizing agent is used 1 type, or 2 or more types.

  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 order to avoid local concentration of the oxidizing agent in the keratin mixed solution, an oxidizing agent solution of about 1 mol / L to 5 mol / L may be gradually mixed, for example, over 30 minutes to 6 hours.

  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 citric acid into the keratin mixture after the oxidant is added in the oxidant mixing step (STP2). Thus, by performing the pH adjustment step (STP3) after the oxidizing agent mixing step (STP2), the modified peptide can be obtained with good yield, and the specific odor of the resulting modified peptide can be suppressed. In addition, the pH of the keratin mixture decreases with the mixing of citric acid, and the mercapto group of keratin can be sufficiently converted to a carboxylatoalkyl disulfide group.

  The amount of citric acid mixed is not particularly limited, but may be blended so as to adjust the pH of the keratin mixed solution to the following range. The final pH of the keratin mixed solution is preferably 5.0 or more and 9.0 or less, and particularly preferably 6.0 or more and 8.0 or less. In this way, by adjusting the final pH of the keratin mixed solution to the above range, introduction of a modifying group into keratin and generation of a disulfide group due to mercapto groups of keratin are suppressed. In addition, since the mercapto group of keratin may become a disulfide group when pH falls locally in a keratin liquid mixture, it is preferable to mix a citric acid gradually with a keratin liquid mixture.

  In addition, in a pH adjustment process, you may use together an organic acid and an inorganic acid other than a citric acid. Examples of the organic acid include lactic acid and succinic acid. Examples of inorganic acids include hydrochloric acid and phosphoric acid. Even when an organic acid or an inorganic acid other than citric acid is used in combination, the final pH of the keratin mixed solution is preferably adjusted to the above range.

  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 citric acid is completed in the pH adjustment step, the keratin mixture may be left for, for example, 1 to 48 hours. 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.

  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. 5 g of this wool containing keratin, 15.4 g of 30% by weight sodium thioglycolate aqueous solution and 8.5 g of 6 mol / L sodium hydroxide aqueous solution are mixed, and water is further mixed to give a total amount of 150 ml of keratin mixed solution of pH 11.0. Prepared. This keratin mixture was stirred at 45 ° C. for 1 hour. Next, water was further mixed to make a total volume of 200 ml, left under conditions of 45 ° C. for 2 hours, and then naturally cooled until the liquid temperature reached room temperature.

(Oxidizing agent mixing process)
25 g of an aqueous solution containing 2.05 g of sodium bromate was mixed with the keratin mixed solution after the reduction step over about 60 minutes. During mixing, the keratin mixture was constantly stirred.

(PH adjustment step)
80 g of an aqueous solution containing 5.7 g of citric acid was mixed with the stirred keratin mixture after the oxidizing agent mixing step over about 85 minutes. The keratin mixed solution after mixing with citric acid was a denatured peptide solution, and its pH was 7.

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

[Example 2]
A solid modified peptide was obtained in the same manner as in Example 1 except that the temperature of the keratin mixture in the reduction step was 32 ° C.

[Comparative Example 1]
A solid modified peptide was obtained in the same manner as in Example 1 except that the pH adjustment step was set before the oxidation step.

[Comparative Example 2]
Solid modified peptide in the same manner as in Example 1 except that the temperature of the keratin mixed solution in the reduction step was 32 ° C. and the citric acid-containing water in the pH adjustment step was changed to acetic acid-containing water (acetic acid 34 g, total amount 825 g). Got.

[Evaluation of characteristics]
The yield results of the modified peptides obtained in the examples and comparative examples are shown in Table 1 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

  As shown in Table 1, the yield of the modified peptide produced by the method of Comparative Example 1 in which citric acid was mixed and then the oxidizing agent was mixed was low (dispersion of wool by dispa in Comparative Example 1). If it is increased, the yield of Comparative Example 1 is expected to increase.) In contrast, the yield of the modified peptide produced by the method of Example 1 and Example 2 in which citric acid was mixed after the oxidant was mixed is the yield of the modified peptide produced by the method of Comparative Example 1. It can be confirmed that it was significantly higher than that. In addition, the modified peptide produced by the method of Comparative Example 2 showed a specific odor, but the modified peptides produced by the methods of Example 1, Example 2 and Comparative Example 1 showed no specific odor. It was.

[Example 3]
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. The yield of the modified peptide calculated according to the above formula was 59%.

(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 sodium thioglycolate aqueous solution and 42.5 g of 6 mol / L sodium hydroxide aqueous solution were mixed, and water was further mixed to prepare a total amount of 750 ml of keratin mixed solution of pH 11.0. Prepared. This keratin mixture was stirred at 45 ° C. for 1 hour. Next, water was further mixed to make a total volume of 1000 ml, left under conditions of 45 ° C. for 2 hours, and then naturally cooled until the liquid temperature reached room temperature.

(Oxidizing agent mixing process)
To the keratin mixed solution after the reduction step, 178 g of an 8.6% by mass hydrogen peroxide aqueous solution was mixed over about 60 minutes. During mixing, the keratin mixture was constantly stirred.

(PH adjustment step)
500 g of an aqueous solution containing 35.4 g of citric acid was mixed with the stirred keratin mixture after the oxidizing agent mixing step over about 85 minutes. The keratin mixed solution after mixing with citric acid was a denatured peptide solution, and its pH was 7.

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

Claims (7)

A reduction step of preparing a keratin mixture by mixing keratin, an alkaline compound, water, and mercaptoalkylcarboxylic acid and / or a salt thereof;
An oxidizing agent mixing step of mixing an oxidizing agent with the keratin mixture;
A pH adjustment step of mixing citric acid into the keratin mixture after the addition of the oxidizing agent,
A method for producing a modified peptide, wherein one or more selected from the group consisting of thioglycolic acid, thioglycolate, mercaptopropionic acid and mercaptopropionic acid salt is used as the mercaptoalkylcarboxylic acid and / or salt thereof.   The method for producing a modified peptide according to claim 1, wherein in the pH adjustment step, the pH of the keratin mixed solution is adjusted to 5.0 or more and 9.0 or less.   The method for producing a modified peptide according to claim 1 or 2, wherein in the reduction step, the pH of the keratin mixed solution is adjusted to 9.0 or more and 13.0 or less.   The method for producing a modified peptide according to any one of claims 1 to 3, wherein the alkaline compound in the reduction step is sodium hydroxide and / or potassium hydroxide.   The method for producing a modified peptide according to any one of claims 1 to 4, wherein the thioglycolate and mercaptopropionate in the reduction step are selected from the group consisting of a sodium salt and a potassium salt.   The method for producing a modified peptide according to any one of claims 1 to 5, wherein the temperature of the keratin mixed solution in the reduction step is 20 ° C or higher and 60 ° C or lower.   The modified | denatured peptide obtained by the manufacturing method of the modified | denatured peptide of any one of Claims 1-6.

Images