KR100881210B1 - Silk peptide manufacturing method - Google Patents

Abstract

A silk peptide manufacturing method is provided to remove fire hazard and water pollution and to obtain low-molecularized silk peptide by being solubilized by using calcium chloride solution without ethanol at a solubilization step. A silk peptide manufacturing method comprises steps of: preparing calcium chloride solution of 40 ~ 60% with calcium chloride for food addition and purified water; injecting cocoon into the calcium chloride dissolution and solubilizing it; injecting and diluting the purified water of 150±10% based on weight of the solution in the soluble solution; filtering, desalinating and sterilizing it; controlling pH using acetic acid for food addition or sodium acetate; dissolving and injecting enzyme protease-N with the purified water and hydrolyzing it for a certain time; processing inactive of enzyme, cooling, decompressing and concentrating it and manufacturing powder of silk peptide.

Description

Silk peptide manufacturing method

The present invention relates to a method for producing silk peptides, and more particularly, to prepare a silk peptide of a new process that can solve the problem of fire risk and water pollution that can occur when working with ethanol in a conventional solubilization step and reduce manufacturing costs. It is about a method.

Silk peptide refers to the decomposition of silk silk, which is a raw material of silk, into a binding state of low molecular weight amino acids and several amino acids. After 3 days (38 days) of healthy silkworms, the silkworms in the silk thread are used to make cocoons. The cocoon thread, or silk thread, is made up of 98% protein.

Research to produce high value-added proteins from protein sources has attracted much attention and achievements in recent years. Silk from silkworms (Bombyx mori) is a great natural protein that can be obtained from nature, and consists of fibroin, a fibrous protein, and sericin, a rubbery protein that attaches them. The silk protein consists of 18 amino acids, the main components of fibroin are glycine and alanine, and these two amino acids account for 65% of the total components of fibroin. Account for about 60%. In addition, since it is not synthesized in the human body, the essential amino acid to be consumed from the outside contains about 6% of fibroin and about 19% of sericin.

Free amino acids, oligopeptides and low molecular weight proteins obtained as hydrolysis products of proteins have inherent functions and are therefore likely to be used as new bioactive materials.

Methods of making silk peptides from silk fibroin are known. Generally, sericin is removed from silk protein to prepare silk fibroin, and the silk fibroin is dissolved in ethanol and salt solution to prepare a neutral salt lysate. However, the manufacturing method as described above has a problem of not only causing fire risk and water pollution by working with ethanol, but also having high manufacturing cost.

In addition, another general method for producing silk peptides is a method for hydrochloric acid hydrolysis. However, the production method as described above is difficult to produce functional peptides by working with hydrochloric acid, so that the amino acid is not a peptide in the hydrolysis step.

The problem to be achieved in the present invention is a new process that can obtain a low molecular weight silk peptide while solving the fire hazard and water pollution problems that may occur when working with ethanol in the conventional solubilization step and induces a reduction in manufacturing cost To provide a method for producing silk peptides.

Silk peptide production method according to the present invention to achieve the above technical problem,

(a) preparing a calcium chloride solution of 40 to 60% with calcium chloride and purified water for food addition;

(b) solubilizing the cocoon for the calcium chloride solution prepared in step (a);

(c) adding, diluting 150 ± 10% of the purified water with the dissolved solution to the solubilized solution by filtration, desalting and sterilizing;

(d) adjusting the pH using acetic acid or sodium acetate for food addition, dissolving the enzyme protease-N in purified water, and hydrolyzing for a predetermined time; And

(e) treating the hydrolyzate of step (d) with an enzyme inert process, cooling, concentrating under reduced pressure, and lyophilizing to prepare a silk peptide powder.

Here, the cocoon may be selected from the group consisting of articulated silkworm cocoons, sheep manureul, subjamsa, or a combination thereof.

In addition, the step (b) is preferably (b ') solubilizing by adding a cocoon of 80 ~ 100 kg to 1000 L of the calcium chloride solution prepared in the step (a).

In addition, in step (d), (d ') is added to dissolve 0.25% of enzyme protease-N relative to the substrate with purified water while maintaining pH 6.75 ± 0.25 using food or sodium acetate for food addition and 53 to 55 ° C. It is preferable that the step of hydrolysis at a certain time.

In addition, the step (e) is (e ') to the hydrolyzate of the step (d) after the enzyme inert treatment at 90 ± 3 ℃ for at least 30 minutes to cool, concentrated under reduced pressure and lyophilized to prepare a silk peptide powder It is preferable.

According to the method for producing silk peptide of the present invention, the weight ratio of cocoon: calcium chloride: purified water is 9:50:50 to solve the problem of fire risk and water pollution which may occur when working with ethanol in the conventional solubilization step. This can lead to a reduction in manufacturing costs.

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

Figure 1 shows the main process of the silk peptide production method according to an embodiment of the present invention as a flow chart. Referring to Figure 1, in the method for producing silk peptides according to the present invention, the purchase and selection of raw materials is preceded (not shown). Purchasing and screening raw materials conforms to the prescribed standard, and the raw materials with good quality and freshness removed from foreign substances are used in the self-quality test. Each raw material is accurately weighed using an electronic balance (2 kg to 150 kg) at a predetermined ratio. However, purified water is measured in a separate weighing tank.

Next, according to a preferred embodiment of the present invention, first, a 50% calcium chloride (CaCl ₂ ) solution is prepared (S100). Table 1 shows the process inspection items before the input of raw materials. Referring to Table 1, calcium chloride and pH and conductivity are evaluated.

Inspection items Calcium chloride pH conductivity importance unit w / w% - S / m kg / ℓ standard 37.37 ± 0.5 7.9 ± 1 11.3 ± 1 1.393 ± 0.03 Red Judgment Reference Reference Reference

Next, raw material input (1st process) is performed. At the time of input of raw materials, the operator shall check the contents of the instructions for using the raw materials in the manufacturing instructions and records, as well as the name and weight of the raw materials. In particular, according to the present invention, a calcium chloride (CaCl ₂ ) solution is prepared by first adding purified water to a glass lined hydrolysis tank and stirring the solution while slowly adding calcium chloride while operating a stirrer. Check the process result of calcium chloride solution and, if appropriate, put cocoon dispensed into the preparation tank.

Next, a solubilization process takes place. In the solubilization process, solubilization is carried out by adding 90 kg of cocoon, preferably carved silkworm cocoon, sheep manure water, sub-naxa, or a combination thereof (hereinafter referred to as cocoon) per 1000 liters of 50% calcium chloride solution (S102). Here, the cocoon is corrected by the water test result value of the test report and applied as a reference quantity for input raw materials (calcium chloride, purified water). Calcium chloride shall be weighed by adjusting the lower limit of the content to 74%, correcting the amount calculated as the input ratio to the net weight of Cocoon by using the result of the content test of calcium chloride to be used. Purified water is preferably measured by subtracting the water content of the cocoon from the amount calculated as the input ratio to the cocoon net weight. In the solubilization process, the tank is warmed and maintained at 115 ± 5 ° C. and solubilized for 6 hours to bring the cocoon protein molecular weight to about 15,000 Da. Then, to confirm the dissolved state and to facilitate the operation, distilled purified water is added most preferably 150 ± 3%, preferably 150 ± 10% by weight of the dissolved solution. If the ratio of purified water is too low, the viscosity increases, so the filtration efficiency is lowered. If the ratio of the purified water is too high, the working time of each process may be increased due to the increase of the liquid amount. The process inspection items and criteria in this process are shown in Table 2.

Inspection items Average molecular weight pH Brix conductivity importance unit Dalton -  %  S / m kg / ℓ standard Stock solution 15,000 ± 1,000 4.2 ± 0.3 63 ± 1 11.5 ± 1 1.40 ± 0.02 diluent - 5.0 ± 0.2 31 ± 1 17.2 ± 1 1.17 ± 0.02 Red Judgment Reference Reference Reference Reference

Next, in the solubilized solution, purified water is diluted with 140 to 160% of the dissolved solution by weight, and then filtered, desalted and sterilized (S104). In the filtration process, the dissolved solution is first removed from particulate insoluble matter and foreign matter in a 20 mesh bag filter. Next, diatomaceous earth is added to about 0.5% of the solution as a filter aid and filtered using a precoated candle filter (70 μm) within a pressure of 2 kgf / cm 2, or a filter press (Filter press: 1 μm) or Primary filtration is performed using a Nutsche filter (200 mesh). Then, the mixture is passed through M / F (Micro Filter, 10 μm) to secondary filtration and transferred to a desalination tank. The process inspection items and criteria in this process are shown in Table 3.

Inspection items pH Brix conductivity Viscosity importance unit -  %  S / m  cp kg / ℓ standard 5.0 ± 0.2 31 ± 1 17.2 ± 1 6.5 ± 1 1.17 ± 0.02 Red Reference Reference Reference Reference Reference

Desalting process, the solution of calcium chloride (CaCl ₂₎ 0.02% desaltor until less (dry powder basis salinity 0.5%) (the ion exchange membrane method electric dialyzer: Ion Exchange Membrane Method Electrodialyzer, ASAHI KASEI Co., LTD four SV-1 / 2 products). The process inspection items and criteria in this process are shown in Table 4.

Inspection items Salinity pH Brix conductivity importance unit % - %  S / m kg / ℓ standard 0.02 or less 6.1 ± 0.1  6.0 ± 0.5  Less than 0.08 1.013 ± 0.03 Red Judgment Reference Reference Reference Reference

To measure the salinity of CaCl ₂ , potentiometric titration can be used, and 0.05 M AgNO ₃ is used as a titration solution. The equation used to calculate salinity is shown in Equation 1.

In the sterilization process, the desalting solution is maintained at 90 ± 3 ° C. for 30 minutes to sterilize, and the sterilization solution is cooled to 53 to 55 ° C.

Next, using a food additive or sodium acetate for pH 6.75 ± 0.25 environment while maintaining the environment of pH 6.75 ± 0.25 dissolved 0.25% enzyme protease-N (protease-N, AMANO Enzyme Inc.) with purified water and 53 ~ 55 An enzymatic hydrolysis process is performed to hydrolyze at a predetermined time (S106).

Example 1 (Enzyme Protease-N)

According to the first embodiment of the present invention, the enzymatic digestion is performed in an environment of pH 6.75 ± 0.25. Preferably out of this range is dissolved or diluted with acetic acid or sodium acetate (C ₂ H ₃ NaO ₂ ) 5 to 10 times in purified water to adjust the amount added to the enzyme hydrolysis tank. In addition, according to the present invention, the enzyme protease-N (protease-N, AMANO Enzyme Inc.) is dissolved in 0.25% of the substrate (cocoon). For example, 1,125 grams of enzyme protease-N is charged in one batch to 450 kilograms of cocoon. Then, the stirrer is operated to add an enzyme solution, followed by hydrolysis at 53 to 55 ° C. for 3 hours.

Comparative Example 1 (Enzyme Protease-M)

In order to examine the difference from the manufacturing process of the present invention, the enzyme is used at a pH of 4.5 and 50 ° C., which is an optimal condition for the enzyme Protease-M. Digested. The remaining conditions are the same as in Example 1.

Comparative Example 2 (Enzyme Protease-P)

In addition, in order to examine the difference from the manufacturing process of the present invention, the enzyme is used at pH 8.0 and the temperature of 45 ° C., which is the optimum condition for the enzyme Protease-P, and 1.26% of the substrate is added in consideration of the enzyme titer. Hydrolysis. The remaining conditions are the same as in Example 1.

Comparative Example 3 (Thermoase-PC)

In addition, in order to examine the difference from the manufacturing process of the present invention, the enzyme usage at pH 7.4, which is an optimum condition for the thermoase-PC (F10) enzyme, and the temperature of 65 ° C. is 0.53% of the substrate in consideration of the enzyme titer. It was added and hydrolyzed. The remaining conditions are the same as in Example 1.

Enzyme Type Protease-N Protease-M Protease-P Thermoase-PC 10F Desalting Solution 50 ml (9.5 Brix) 50 ml (9.5 Brix) 50 ml (9.5 Brix) 50 ml (9.5 Brix) pH 7.0 4.5 8.0 7.4 Temperature (℃) 55 50 45 65 Enzyme Consumption (mg) 12 324 60 25 % Of substrate 0.25 6.82 1.26 0.53

Comparative Example 4

The solution was prepared with a ratio of calcium chloride and purified water at 30%. Other conditions are controlled by dissolving / diluting to the same pH environment as above and adding an appropriate amount to the enzyme hydrolysis tank, and dissolving the enzyme Protease-N in purified water by 0.25% relative to the substrate (cocoon). The remaining conditions are the same as in Example 1.

The conditions in the comparative example 4 generate | occur | produce the silkworm cocoon which does not melt | dissolve, and the problem that the manufacturing yield falls, it turned out that it was unpreferable.

Comparative Example 5

The solution was prepared with a ratio of calcium chloride and purified water at 70%. Other conditions are controlled by dissolving / diluting to the same pH environment as above and adding an appropriate amount to the enzyme hydrolysis tank, and dissolving the enzyme Protease-N in purified water by 0.25% relative to the substrate (cocoon). The remaining conditions are the same as in Example 1.

However, in this condition, the excessive use of calcium chloride increased the viscosity and increased the filtration time, such as not only a problem in the filtration process but also a problem in that the cost of wastewater treatment was found to be undesirable.

Comparative Example 6

This time, the hydrolysis was performed in an environment outside of 6.75 ± 0.25 in order to grasp the change according to the pH condition during the hydrolysis. Other conditions are controlled by dissolving / diluting to the same environment as above and adding an appropriate amount to the enzymatic hydrolysis tank, and dissolving the enzyme Protease-N in purified water by 0.25% relative to the substrate (cocoon). The remaining conditions are the same as in Example 1.

Under the above conditions, the enzyme degradation rate was lowered, which caused the problem of lowering the yield.

The process inspection items and criteria in this process are shown in Table 6.

Inspection items Molecular Weight pH brix conductivity importance unit Dalton - % mS / m kg / ℓ After enzymatic digestion 1,000-1,500 6.75 ± 0.25 6.5 ± 0.5  110 ± 10 1.013 ± 0.03 Red Judgment Reference Reference Reference Reference

Next, the hydrolyzate of step S106 is heated to 90 ± 3 ° C. and maintained for at least 30 minutes to inactivate the enzyme (S108). After sterilization by elevated temperature, it is cooled to 50 ° C before and after, and the sterilization liquid is transferred to the concentration tank. Concentrated enzyme hydrolyzate was concentrated to 715 ± 10 mmHg under reduced pressure at 35 ± 5 ℃, and Brix of the concentrate was measured to finish concentration at 20 ± 5% (32% of the stock solution). Cool. When the concentration is completed, the intermediate package is released or freeze-dried (S110) and can be packaged and released after the description is omitted.

The peptide molecular weight was measured for the hydrolyzate obtained by the above process in order to control the quality of the silk peptide prepared by the above process. The molecular weight is analyzed by gel chromatography (Gel permeation chromatography) method and the average molecular weight is preferably in the range of 1000 ~ 1500 Da.

Peptide molecular weight measurement of the hydrolyzate was analyzed using MALDI / TOF / MS (voagertm DE-STR, PerSeptive Biosystems Inc., USA), and protease-N (( Protease-N) was used to instantaneously irradiate and crystallize the peptide and matrix mixed crystals, and then the ions were passed through a mass spectrometer to obtain molecular weight data.

Table 7 shows the distribution of the molecular weight thus obtained. Referring to Table 7, the molecular weight of the composition ranged from 1000 to 1500 Da was 67.2%, which was the largest, 11.6% for 1501-2000 Da, 6.8% for 2001-2500 Da, and 9.2% for 2501 to 3000 Da. Showed. More than 3000 Da showed a content of 5.2%.

Molecular Weight Range (Da) 1000-1500 1501-2000 2001 to 2500 2501 to 3000 3000- Composition ratio 67.2% 11.6% 6.8% 9.2% 5.2%

     Composition of free amino acid (percentage) by enzyme Unit:% Analysis Content  Free amino acids       Enzyme components protease -N (Example 1) protease -M (Comparative Example 1) protease -P (Comparative Example 2) Thermoase PC 10F (Comparative Example 3) Average GLY 1.004 14.968 10.259 5.101 7.833 SER 2.321 9.072 6.900 8.992 6.821 TYR 7.718 12.003 10.492 15.440 11.413 ALA 14.400 18.520 12.409 3.959 12.322 ARG 16.764 15.244 23.450 2.027 14.371 THR 21.524 12.876 9.507 9.114 13.255 PRO 26.468 1.038 4.902 31.492 15.975 ASP 0.237 1.761 0.508 2.163 1.167 GLU 0.443 2.138 0.487 1.292 1.090 HIS 0.131 1.613 1.655 1.265 1.166 CYS 3.994 2.099 11.272 8.761 6.531 VAL 0.658 2.688 3.117 1.809 2.068 MET 0.140 0.109 0.457 1.184 0.472 LYS 3.001 2.050 1.442 5.142 2.909 ILE 0.842 1.194 0.909 1.224 1.042 LEU 0.227 1.247 0.866 0.694 0.758 PHE 0.130 1.378 1.368 0.340 0.804 TRP 0.000 0.000 0.000 0.000 SUM 100.000 100.000 100.000 100.000 100.000

Table 8 shows the distribution of free amino acid composition by enzyme. Referring to Table 8, GLY, SER, TYR, ALA, ARG, THR, PRO as components of free amino acids by enzymes of protease-N, protease-M, protease-P, and thermoase PC 10F. Data evaluating composition distributions for ASP, GLU, HIS, CYS, VAL, MET, LYS, ILE, LEU, PHE, and TRP are shown in% and are advantageous when digested with protease-N enzyme. Among the amino acid components, the composition ratio of PRO, THR, ARG, and ALA was as high as 79%, indicating the substrate specificity of the protease-N enzyme.

 Enzyme Type Highest in composition cost Composition ratio (%) Sum Hydrolysis rate  Protease-N (Example 1)  PRO> THR> ARG> ALA  26.5> 21.5> 16.8> 14.4  79.2  88.9%  Protease-M (Comparative Example 1)  ALA> ARG> GLY> THR  18.5> 15.2> 15.0> 12.9  61.6  62.4%  Protease-P (Comparative Example 2)  ARG> ALA> CYS> TYR  23.5> 12.4> 11.3> 10.5  57.7  88.3%  Thermoase-PC 10F (Comparative Example 3)  PRO> TYR> THR> SER  31.5> 15.4> 9.1> 9.0  65.0  66.7%

In addition, Table 9 shows the results obtained by arranging the data in the order of high free amino acid composition ratio by enzyme. Referring to Table 9, the composition ratio was in order of PRO> THR> ARG> ALA in order of 26.5> 21.5> 16.8> 14.4.

Silk peptide production method according to the present invention as described above can be used to reduce the production cost because the protease-N enzyme in the calcium chloride solution without working with ethanol in the solubilization step can increase the production yield high hydrolysis rate. In addition, the risk of fire is small.

1 is a flow chart showing the main process of the silk peptide production method according to an embodiment of the present invention.

Claims (5)

(a) preparing a calcium chloride solution of 40 to 60% with calcium chloride and purified water for food addition; (b) solubilizing the cocoon for the calcium chloride solution prepared in step (a); (c) adding, diluting 150 ± 10% of the purified water with the dissolved solution to the solubilized solution by filtration, desalting and sterilizing; (d) adjusting the pH using acetic acid or sodium acetate for food addition, dissolving the enzyme protease-N in purified water, and hydrolyzing for a predetermined time; And (e) treating the hydrolyzate of step (d) with an enzyme inert process, cooling, concentrating under reduced pressure, and lyophilizing to prepare a silk peptide powder. According to claim 1, wherein the cocoon, Silk peptide manufacturing method, characterized in that selected from the group consisting of silkworm cocoon, sheep manureul, sub-salam, or a combination thereof. According to claim 1, wherein step (b), (b ') step of solubilizing the cocoon of 80 ~ 100 kg with respect to 1000 L of the calcium chloride solution prepared in step (a); solubilizing silk peptide production method. The method of claim 1, wherein step (d) (d ') dissolving 0.25% of enzyme protease-N in purified water with purified water while maintaining a pH of 6.75 ± 0.25 using food additives or sodium acetate for food addition, and hydrolyzing at 53 to 55 ° C for a predetermined time; Silk peptide production method, characterized in that. The method of claim 1, wherein step (e) (e ') preparing a silk peptide powder by cooling and concentrating under reduced pressure after lyophilization after enzymatic treatment at 90 ± 3 ° C. for 30 minutes or more with respect to the hydrolyzate of step (d); Way.

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