KR20190130714A - Extraction method of silk peptide by enzymatic hydrolysis under ultra high pressure - Google Patents

Abstract

The present invention relates to a silk protein extraction method by enzymatic hydrolysis under ultra-high pressure, including: a step of preparing a silk protein solution solubilizing silk in calcium chloride, a mixed solvent of ethanol and water, an aqueous lithium bromide solution, or an aqueous calcium chloride solution; a silk decomposition step of adding protease to the silk protein solution; and an ultrahigh pressure treatment step of pressurizing the silk protein solution at a pressure of 100 to 300 MPa.

Description

Extraction of silk protein using ultrahigh-pressure enzyme hydrolysis. {EXTRACTION METHOD OF SILK PEPTIDE BY ENZYMATIC HYDROLYSIS UNDER ULTRA HIGH PRESSURE}

The present invention relates to a silk protein extraction method using ultra-high pressure hydrolysis, and more particularly, to a method for extracting low-molecular weight silk protein by using proteolytic enzymes under ultra-high pressure conditions.

Silk, a silk, is a protein complex composed of fibroin (75%) and sericin (25%). However, fibroin has been used as silk, a natural fiber as a protein that is insoluble and maintains constant strength due to its β-sheet structure such as cellulose due to its structural properties. Entering the 21st century, it has been put into practical use for various food and human body materials such as diabetes, hypertension, hangover removal, as well as functional daily necessities such as shampoos using silk decomposition products.

Applicants have been developing a method for producing silk protein using high pressure enzyme decomposition, not acid treatment, which is a method for decomposing existing silk protein. In the production of silk protein using high-pressure enzymatic digestion, the decomposition process of silk protein becomes an important factor. To this end, the pressure conditions and proteolytic characteristics should be considered.

Natural silk protein is composed of about 50% glycine, about 30% alanine and about 10% serine, and the quality of silk protein products is determined by the content of other amino acids that can be obtained through the decomposition process. There is a need for optimization of such protein degradation efficiency.

Conventionally, a method for preparing cheonma extract using high-pressure enzymatic digestion (Korea Patent Publication No. 10-1740519, 10-1643245) or a method for preparing ginseng extract (Korea Patent Publication No. 10-1328413) is disclosed. However, these methods are applied to high pressure enzymatic decomposition, but not optimized for the decomposition process of silk protein. Therefore, it is necessary to develop a silk protein extraction method for silk protein.

Republic of Korea Patent Registration No. 10-1740519 Republic of Korea Patent Publication No. 10-1643245 Republic of Korea Patent Publication No. 10-1328413

The present invention has been made in view of the prior art as described above, silk protein extraction method using enzymatic hydrolysis under ultra-high pressure conditions to improve the production yield by improving the production method or high pressure enzyme decomposition method of the existing acid-treated hydrolysis. To provide that purpose.

In addition, the present invention provides a method for extracting low-molecular weight silk protein by using an ultrahigh-pressure enzyme hydrolysis method, and aims to provide a method for preparing an edible silk protein.

Silk protein extraction method using the ultrahigh-pressure enzyme hydrolysis of the present invention for achieving the above object is a silk protein solution manufacturing step of solubilizing silk in a mixed solvent of calcium chloride, ethanol and water, aqueous lithium bromide, or calcium chloride aqueous solution, the silk Silk decomposition step of adding a protease to the protein solution, characterized in that it comprises an ultra-high pressure treatment step to press the silk protein solution at a pressure of 100 to 300MPa.

At this time, the ultra-high pressure treatment step is preferably treated for 5 to 24 hours at a temperature of 40 to 70 ℃.

In addition, the protease is any one of Alcalase, FoodPro Alkaline Protease, Alphalase, and Delvolase, it is preferable to add 0.1 to 5% by weight of the protease relative to the silk protein.

In addition, the step of desalting the silk protein solution after the ultra-high pressure treatment step using a micro filter or desalting machine may be further included, and the step of drying the silk protein solution after the ultra-high pressure treatment step through lyophilization or spray drying It may further include.

Silk protein extraction method using the ultrahigh-pressure enzyme hydrolysis according to the present invention can improve the production yield by improving the conventional acid-treated hydrolysis method or high-pressure enzyme decomposition method.

In addition, it is possible to extract a low-molecular weight silk protein by using an ultrahigh-pressure enzyme hydrolysis method, thereby producing an edible silk protein.

1 is a graph showing the change in yield with time of the ultra-high pressure treatment process in the silk protein extraction method of the present invention.
Figure 2 is a graph showing the change in yield according to the pressure of the ultrahigh pressure treatment process in the silk protein extraction method of the present invention.

Hereinafter, the present invention will be described in more detail. The terms or words used in this specification and claims are not to be construed as being limited to their ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to best describe their invention. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that the present invention.

Silk protein extracting method using the ultrahigh-pressure enzyme hydrolysis according to the present invention is a silk protein solution preparation step of solubilizing silk in a mixed solvent of calcium chloride, ethanol and water, aqueous lithium bromide, or calcium chloride solution, proteolytic enzymes in the silk protein solution Adding silk decomposition step, characterized in that it comprises an ultra-high pressure treatment step to press the silk protein solution to a pressure of 100 to 300MPa.

The degradation of silk protein can generally be degraded by enzymatic digestion using proteolytic enzymes, but the effective ingredient can be extracted more effectively by ultra-high pressure treatment. In particular, the extraction of low molecular weight silk protein through the decomposition of silk protein is a very important technology in the use of food, medicine, etc. of the silk protein.

Therefore, in the present invention, in addition to the conventional enzymatic method of adding proteolytic enzymes to the silk protein solution for the decomposition of the silk protein, the yield for these low molecular weight silk proteins is greatly increased through the treatment under ultra-high pressure conditions.

Generally, proteolytic enzymes are classified into methods of decomposing proteins with endo type protease and exo type aminopeptidase. Depending on the function of amino acids in the active site of the enzyme, it may be classified into Serine protease, thiol (cysteine) protease, and Aspartyl protease. Bacterial protease and Fungal protease are classified into acidic, neutral, and alkaline protease according to the reaction conditions of pH.

In the present invention, a protease was selected to increase the degradation efficiency of the silk protein and used in combination thereof, and tested for a protease suitable for the treatment of ultra-high pressure conditions. In particular, the use of a system approved as a food additive as a protease has the advantage that the silk protein produced by the enzyme treatment can be safely used in food or health functional food.

In the first process, a silk protein solution was prepared, and the optimum solvent for solubilizing silk was searched for. That is, by solubilizing in a mixed solvent of calcium chloride, ethanol and water, an aqueous lithium bromide solution, or an aqueous solution of calcium chloride, it was shown that a high yield of low molecular weight silk protein can be obtained through the respective process conditions. That is, silk (silk silk) is treated with sodium carbonate to remove sericin, and the obtained silk protein is solubilized using the solvent as described above, and then the excess protein is removed through a dialysis process to obtain a silk protein solution.

In addition, in the second step, the silk protein solution is decomposed using a protease approved for food, and the low molecular weight soluble silk protein is obtained through this decomposition process.

In addition, the yield of the low molecular weight silk protein is greatly improved through the ultrahigh pressure treatment in the third process.

When explaining the decomposition method of the silk protein of the present invention in detail through Examples.

First step: refining process to remove sericin

The silk (silk yarn) was immersed sufficiently in 0.02M sodium carbonate solution and boiled at 100 ° C. for 20 minutes, and this process was repeated three times. Thereafter, it is dried to obtain silk protein from which sericin is removed.

Second Process: Dissolution and Dialysis of Silk Proteins

100 parts by weight of a calcium chloride solution prepared by mixing calcium chloride, ethanol, and water in a molar ratio of 1: 2: 8 or a 10M lithium bromide solution or a 10M calcium chloride solution was added to the refined 20 parts by weight of the silk protein. A high viscosity silk protein solution of 20% (w / v) is prepared by dissolving it at 80 to 90 ° C. for 1 to 3 hours.

Excess salt is removed while the solution is replaced by distilled water for 3 days or more using a dialysis membrane (Sigma) having a limit molecular weight of 14,000. In this process, the viscosity of the silk protein solution is lowered.

Upon completion of dialysis, the silk protein solution is centrifuged to remove solids and filtered using a 0.45 μm cellulose acetate membrane (Advantec). At this time, the concentration of silk fibroin is reduced to about 4-5% (w / v) level after dialysis-centrifugation-filtration from 20% (w / v) before dialysis.

Third Step: Preparation and Filtration of Protease

Protease applied to the present invention is used to purchase a variety of protease sold for use as a food additive. Proteolytic enzymes are prepared using a phosphate buffer or glycine buffer using an inorganic salt for foods corresponding to the appropriate pH, and then filtered using a 0.45 μm cellulose acetate membrane (Advantec) and then refrigerated. The protease used in the present invention can be applied to the proteolytic enzymes shown in Table 1 as approved for food.

product name Reaction condition Enzyme classification pH Temperature (° C) type source Active site pH Alcalase ^ⓡ AF2.4 L 7.0-9.0 30-65 Endo Bacteria B. licheniformis Serin Protease Alkaline Alphalase ^ⓡ NP 6.0-7.5 50-60 Endo Bacteria B. amyloliquefaciens Neutral Delvolase ™ 7.0-10.5 45-75 Endo Bacteria B. licheniformis Serin Protease Alkaline FoodPro ^ⓡ Alakline Protease 7.0-9.0 45-70 Endo Bacteria B. licheniformis Serin Protease Alkaline Maxazyme ^ⓡ NNP DS 6.5 ~ 7.5 40-50 Endo Bacteria B. subtilis Neutral Neutrase ™ 0.8 L 7.0 40-50 Endo Bacteria B. amyloliquefaciens Metallo (Zn) Enzyme Neutral Protamex ^ⓡ 7.0-8.0 50 Endo Bacteria Bacillus protease Complex Serin Protease Alkaline Bromelain BR1200 6.0-8.0 45-50 Endo Plant Pineapple
(Ananas comosus) Thiol (Cys) Protease Neutral Collupulin ™ MG 5.0 ~ 7.5 50-70 Endo Plant Carica papaya Thiol (Cys) Protease Acidic Flavorzyme ^ⓡ 500 MG 5.5 ~ 7.5 50-55 Exo / Endo Fungi A. oryzae Neutral Promod ™ 192P 4.0-6.0 40-55 Exo Fungi A. oryzae Acidic Promod ™ 279MDP 4.0-6.0 50-60 Exo Fungi Aspergillus sp. Acidic Promod ™ 278MDP 6.0-8.5 50-70 Endo Plant + Bacteria Carica papaya
+ B. subtilis Neutral Pancreatin 7.0-8.0 40-50 Endo Animal Porcine pancreas Serin Protease Alkaline BC Pepsin 2.0-5.5 40-55 Endo Animal Porcine gastric mucosa Aspartyl Protease Acidic

Fourth Step: Selection and Application of Protease

The protein solution of silk protein was diluted to 2% by weight (20 g / L), and the proteolytic enzyme of Table 1 was subjected to the enzyme digestion under the same reaction conditions as those of Table 2.

Protease Silk Fibroin (%) Enzyme throughput
Enzyme: Protein Buffer
(0.25 M) Reaction condition pH Temperature (° C) Alcalase ^ⓡ AF2.4 L 2 7.3% Phosphate Buffer 8.0 50 Alphalase ^ⓡ NP 2 5.5% Phosphate Buffer 7.0 50 Delvolase ™ 2 4.7% Phosphate Buffer 7.0 50 FoodPro ^ⓡ Alakline Protease 2 4.7% Phosphate Buffer 8.0 50 Maxazyme ^ⓡ NNP DS 2 5.2% Phosphate Buffer 7.0 50 Neutrase ™ 0.8 L 2 4.1% Phosphate Buffer 7.0 50 Protamex ^ⓡ 2 4.3% Phosphate Buffer 7.6 50 Bromelain BR1200 2 4.7% Phosphate Buffer 7.0 50 Collupulin ™ MG 2 2.7% Phosphate Buffer 6.0 50 Flavorzyme ^ⓡ 500 MG 2 1.5% Phosphate Buffer 7.0 50 Promod ™ 192P 2 4.7% Phosphate Buffer 6.0 50 Promod ™ 279MDP 2 1.9% Phosphate Buffer 6.0 50 Promod ™ 278MDP 2 4.4% Phosphate Buffer 7.0 50 Pancreatin 2 5.5% Phosphate Buffer 7.6 50 BC Pepsin 2 3.0% Glycine Buffer 2.2 50

In order to quantitatively confirm the degradation of proteins by enzymatic reaction, the Lowry method was used. In the Lowry method, 1 ml of a solution containing 2% Na ₂ CO ₃ (0.1M NaOH), 1% CuSO _4, and 2% sodium sodium stannate tetrahydrate in a weight ratio of 98: 1: 1 was added to 0.2 ml of the enzyme reaction solution. After standing at room temperature for 15 minutes, 0.1 mL of a solution of Folin-Ciocaiteu Reagent (Sigma) and distilled water mixed at a weight ratio of 1: 1 was added thereto, mixed at room temperature for 30 minutes, and the absorbance was measured at a wavelength of 595 nm. Protein standard (Sigma) is used as a standard for measuring protein concentration.

Peptides and amino acids, the products of degradation by enzymatic reaction, were used in the ninhydrin method which reacts quantitatively with amino groups. That is, 0.1 ml of Ninhydrin Reagent (Sigma) is mixed with 0.2 ml of the enzyme reaction solution, heated for 10 minutes, cooled to room temperature, 0.5 ml of 95% ethanol is added thereto, and the absorbance is measured at a wavelength of 570 nm. . A standard for quantifying amino acids was used by dissolving glycine (Samchun), a major amino acid of silk fibroin, in 0.05% acetic acid.

In general, when a high molecular weight protein is decomposed into a low molecular weight protein (or peptide) by proteolytic enzymes, the amount of protein by the Lowry method is reduced, and at the same time, an amino group is exposed from the decomposed peptide bonds, resulting in an enzymatic reaction. The amount of amino acids will increase. When a protein is degraded by an endotype protease, the amount of protein decreases while the amount of amino acids increases.

On the other hand, in the case of the exotype (aminopeptidase) protease sequester the amino acid of the terminal constituting the protein in the protein body, the amount of protein decreases while the amount of amino acids increases while enzymatic reaction.

Experimental results showed that the application of Alcalase, FoodPro Alkaline Protease, Alphalase, and Delvolase as protease was superior to the degradability of silk protein. In particular, when the proteolytic enzyme is added 0.1 to 5% by weight compared to the silk protein, good results were obtained, and when it was out of the above range, the protein degradation was insufficient.

Fifth Process: Desalination Process

After the enzymatic reaction, the silk protein solution was desalted through a demineralizer to remove the used calcium chloride.

Sixth Process: Ultra High Pressure Treatment Process

The desalted silk protein was subjected to ultra high pressure treatment under a pressure condition of 100 to 300 MPa. At this time, the ultrahigh pressure treatment conditions are preferably 5 to 24 hours at a temperature of 40 to 70 ℃. The temperature is a temperature attained when the ultra-high pressure reactor is pressurized without heating, and thus the destruction of the components is suppressed by the separate heating treatment of the silk protein solution. In addition, if the ultra-high pressure treatment time is too short outside the above range, silk protein degradation efficiency similar to that of the sample without ultra-high pressure treatment was obtained. It is preferable to process in the range.

In order to confirm the effect of the ultra-high pressure treatment, the change of the yield of silk protein when the pressure was changed to 100 MPa and the pressurization time was as shown in FIG. 2, and the yield of the silk protein when the pressure was changed to 20 hours Is shown in FIG. 2. In Figures 1 and 2 the extraction amount shows an increased content based on serine in the silk protein obtained by conventional acid treatment. The serine content when the acid treatment through the experiment was 8.7 mol% of the total amino acids, the serine content of the total amino acids when the degradation by applying the protease according to the present invention was 10.31 mol%.

As a result, in the ultrahigh pressure condition, the yield according to the pressure or pressurization time showed a tendency to increase gradually with the pressurization time or pressure. In particular, when comparing the ultra-high pressure condition and the hydrothermal conditions over 100MPa in Figure 3, the yield difference of more than 15% in the ultra-high pressure condition was confirmed that the increase in the yield of low-molecular weight silk peptides according to the ultra-high pressure treatment. In addition, once the pressure reached 100MPa or more, the increase in yield was not significant, and it was found that there was no sudden change in yield under ultrahigh pressure conditions of 100 to 300MPa.

The present invention has been described with reference to preferred embodiments as described above, but is not limited to the above embodiments and various modifications by those skilled in the art to which the present invention pertains without departing from the spirit of the present invention. Changes are possible. Such modifications and variations are intended to fall within the scope of the invention and the appended claims.

Claims (5)

Preparing a silk protein solution by solubilizing silk in a mixed solvent of calcium chloride, ethanol and water, an aqueous lithium bromide solution, or an aqueous calcium chloride solution;
Silk decomposition step of adding a protease to the silk protein solution;
Silk protein extraction method using ultra-high pressure hydrolysis, characterized in that it comprises an ultra-high pressure treatment step to pressurize the silk protein solution at a pressure of 100 to 300MPa.
The method according to claim 1,
The ultra-high pressure treatment step is a silk protein extraction method using ultra-high pressure enzyme hydrolysis, characterized in that the ultra-high pressure treatment for 5 to 24 hours at a temperature of 40 to 70 ℃.
The method according to claim 1,
The protease is any one of Alcalase, FoodPro Alkaline Protease, Alphalase, and Delvolase, silk protein extraction method using ultrahigh-pressure enzyme hydrolysis, characterized in that the addition of the protease 0.1 ~ 5% by weight compared to the silk protein.
The method according to claim 1,
Silk protein extraction method using ultra-high pressure hydrolysis of the enzyme, characterized in that further comprising the step of desalting the silk protein solution after the ultra-high pressure treatment step using a micro filter or demineralizer.
The method according to claim 1,
Silk protein extraction method using ultra-high pressure enzyme hydrolysis characterized in that it further comprises the step of drying the silk protein solution after the ultra-high pressure treatment step by lyophilization or spray drying.

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