Method for producing proteolytic enzyme derived from cudrania tricuspidata fruit, hydrolytic enzyme prepared thereby, hydrolyzate prepared using the same, and food composition containing the same

Abstract

The present invention relates to a method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, the hydrolyzing enzyme produced thereby, a hydrolysate produced using the same, and a food composition containing the same. More specifically, the present invention relates to a method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, comprising the steps of: freeze-drying the Cudrania tricuspidata fruit to produce Cudrania tricuspidata fruit powder; dissolving the Cudrania tricuspidata fruit powder in a sodium phosphate buffer to produce a lysate; centrifuging the lysate to produce a first supernatant; diluting the first supernatant in ethanol to produce a diluted product; and centrifuging the diluted product and removing the second supernatant to produce a precipitate, which is then redissolved in a sodium phosphate buffer to produce a Cudrania tricuspidata fruit-derived fraction. The present invention can improve the protein degradation activity of a hydrolyzing enzyme by optimizing the ethanol dilution ratio used in the production of a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit.

Description

Technology Field

[0001] The present invention relates to a method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, the hydrolyzing enzyme produced thereby, a hydrolysate produced using the same, and a food composition containing the same. More specifically, the present invention relates to a method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, comprising the steps of: freeze-drying the Cudrania tricuspidata fruit to produce Cudrania tricuspidata fruit powder; dissolving the Cudrania tricuspidata fruit powder in a sodium phosphate buffer to produce a lysate; centrifuging the lysate to produce a first supernatant; diluting the first supernatant in ethanol to produce a diluted product; and centrifuging the diluted product and removing the second supernatant to produce a precipitate, which is then redissolved in a sodium phosphate buffer to produce a Cudrania tricuspidata fruit-derived fraction. Background Technology

[0002] Proteolytic enzymes are primarily used in the food industry for the production of meat tenderizers, dairy products, and bioactive peptides derived from food proteins. In particular, research on plant-derived proteolytic enzymes is actively underway because their production process is simple and they offer excellent industrial applicability regarding conditions such as temperature, pH, and substrate affinity. Currently, most commercially utilized plant-derived proteolytic enzymes are enzymes such as bromelain (derived from pineapple), papain (derived from papaya), ficin (derived from fig), and actinidin (derived from kiwi). Since the degradation capabilities of each enzyme vary, the search and identification of novel plant-derived proteolytic enzymes is a major research area for the development of functional food materials.

[0003] Cudrania tricuspidata has been reported to possess various physiological effects, including antioxidant activity, and there are instances where it has been utilized as an ingredient in processed foods. Conventional methods for producing Cudrania tricuspidata fruit extracts have primarily utilized hot water extraction or reflux extraction; however, these methods have the disadvantage of requiring complex and time-consuming processes for each step, as they involve subdividing the types of solvents used to extract beneficial components.

[0004] Ethanol is used as a solvent for extracting Cudrania tricuspidata, which is highly effective for extracting physiologically active substances such as antioxidants. However, there are problems in that extraction efficiency is limited due to the long extraction time required, and it is difficult to expect an efficient extraction method because extraction is performed in a batch without separation based on ethanol ratios. Prior art literature

[0005] Republic of Korea Registered Patent Publication No. 10-1407355 The problem to be solved

[0006] The objective of the present invention is to provide a method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit with enhanced protein degradation activity by optimizing the ethanol dilution ratio used in the production of the protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit.

[0007] Another objective of the present invention is to provide a hydrolyzing enzyme prepared according to the above-described manufacturing method and a hydrolysate prepared using the same.

[0008] Another objective of the present invention is to provide a food composition comprising the above hydrolysate as an active ingredient.

[0009] The technical problems that the invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems can be clearly understood by those skilled in the art from the description of the invention. means of solving the problem

[0010] The present invention provides a method for producing a protein hydrolyzing enzyme derived from Cudrania fruit, characterized by comprising the steps of: freeze-drying the Cudrania fruit to produce Cudrania fruit powder; dissolving the Cudrania fruit powder in a sodium phosphate buffer to produce a lysate; centrifuging the lysate to produce a first supernatant; diluting the first supernatant in ethanol to produce a diluted product; and centrifuging the diluted product and removing the second supernatant to obtain a precipitate, which is then redissolved in a sodium phosphate buffer to produce a Cudrania fruit-derived fraction.

[0011] In the present invention, the sodium phosphate buffer is characterized by being 0.05 to 0.2 M.

[0012] In the present invention, the sodium phosphate buffer is characterized by further comprising 4 to 6 mM ethylene-diamine-tetraacetic acid (EDTA).

[0013] In the present invention, the sodium phosphate solution is characterized by having a pH of 6 to 7.

[0014] In the present invention, the step of preparing a first supernatant by centrifuging the dissolved product is characterized by centrifuging the dissolved product at 3500 to 3900 xg for 50 to 70 minutes at 4°C.

[0015] In the present invention, the step of preparing a diluted product by diluting the first supernatant with ethanol is characterized by diluting the first supernatant with ethanol in a weight ratio of 1:3 to 5.

[0016] In the present invention, the ethanol is characterized as being 90 to 100% ethanol.

[0017] In addition, the present invention provides a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit prepared by the method for preparing the protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit.

[0018] In the present invention, the protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit is characterized by hydrolyzing casein, isolated soy protein, or whey protein.

[0019] In the present invention, the protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit is characterized by having a casein protein degradation activity of 3.5 to 4.0 (Units / mL protease), and the protein degradation activity is calculated by the following formula 1.

[0020] [Equation 1]

[0021] Protein degradation activity (Units / mL protease) = (μmol tyrosine equivalents × VT) / VE × T × VA

[0022] (In the above Equation 1,

[0023] VT = Total amount of solution used in the experiment (mL),

[0024] VE = Amount of added protease (mL),

[0025] T = Reaction time after addition of proteolytic enzyme (minutes),

[0026] VA = refers to the amount of supernatant (mL) used for absorbance measurement.

[0027] In addition, the present invention provides a method for producing a food protein hydrolysate, characterized by comprising the steps of: mixing a food protein with a protein hydrolysate derived from Cudrania tricuspidata fruit, prepared by the method for producing a protein hydrolysate derived from Cudrania tricuspidata fruit; hydrolyzing the mixture to produce a hydrolysate; heating the hydrolysate to inactivate the enzyme; and filtering the hydrolysate in which the enzyme has been inactivated.

[0028] In the present invention, the food protein is characterized as being one or more proteins selected from the group consisting of isolated soy protein and whey protein.

[0029] In the present invention, the step of hydrolyzing the mixture to produce a hydrolysate is characterized by hydrolyzing the mixture by reacting it at 34 to 40°C for 2 to 4 hours.

[0030] In the present invention, the step of heating the hydrolysate to inactivate the enzyme is characterized by heating the hydrolysate at 85 to 95°C for 10 to 30 minutes to inactivate the enzyme.

[0031] In addition, the present invention provides a food composition comprising a food protein hydrolysate prepared by the above method for preparing a food protein hydrolysate as an active ingredient.

[0032] In the present invention, the food composition is characterized by being prepared in the form of a powder, granule, pill, tablet, capsule, candy, beverage, or dairy product. Effects of the invention

[0033] The present invention can provide a method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit with enhanced protein degradation activity by optimizing the ethanol dilution ratio used in the production of the protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit.

[0034] In addition, the present invention may provide a hydrolyzing enzyme prepared according to the above-described manufacturing method and a hydrolysate prepared using the same.

[0035] In addition, the present invention can provide a food composition comprising the above hydrolysate as an active ingredient.

[0036] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description in the claims. Brief explanation of the drawing

[0037] Figure 1 is a schematic diagram showing a method for preparing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit. Figure 2 is a figure showing the results of analyzing the protein size of protein hydrolyzing enzymes derived from Cudrania tricuspidata fruit at different ethanol dilution ratios. Figure 3 is a graph showing the degree of hydrolysis of protein hydrolyzing enzymes derived from Cudrania tricuspidata fruit at different ethanol dilution ratios. Figure 4 is a figure showing the results of analyzing the protein size of a hydrolysate using a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit. Specific details for implementing the invention

[0038] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0039] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0040] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

[0042] The present invention will be described in detail below.

[0044] Method for manufacturing protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit

[0045] The present invention provides a method for producing a protein hydrolyzing enzyme derived from Cudrania fruit, characterized by comprising the steps of: freeze-drying the Cudrania fruit to produce Cudrania fruit powder; dissolving the Cudrania fruit powder in a sodium phosphate buffer to produce a lysate; centrifuging the lysate to produce a first supernatant; diluting the first supernatant in ethanol to produce a diluted product; and centrifuging the diluted product and removing the second supernatant to obtain a precipitate, which is then redissolved in a sodium phosphate buffer to produce a Cudrania fruit-derived fraction.

[0046] In the step of preparing a solution by dissolving the above Cudrania tricuspidata fruit powder in a sodium phosphate buffer, the sodium phosphate buffer may be 0.05 to 0.2 M, preferably 0.1 M. Additionally, the sodium phosphate buffer may further include 4 to 6 mM ethylene-diamine-tetraacetic acid (EDTA), preferably 5 mM EDTA. Furthermore, the sodium phosphate solution may have a pH of 6 to 7, preferably 6.6.

[0047] The step of preparing a first supernatant by centrifuging the above-mentioned solution may be characterized by centrifuging the above-mentioned solution at 3500 to 3900 xg at 4°C for 50 to 70 minutes, and preferably, may be characterized by centrifuging the above-mentioned solution at 3700 xg at 4°C for 60 minutes.

[0048] The step of preparing a diluted product by diluting the first supernatant in ethanol may be characterized by diluting the first supernatant in ethanol at a weight ratio of 1:3 to 5, and preferably at a weight ratio of 1:4. If the first supernatant is diluted in an amount of ethanol less than 1:3 or in an amount of ethanol greater than 1:5, the protein degradation activity of the hydrolytic enzyme may be lowered.

[0049] In the step of preparing a diluted product by diluting the first supernatant with ethanol, the ethanol may be 90 to 100% ethanol, and preferably 99.9% ethanol.

[0050] In the step of preparing a fraction derived from Cudrania tricuspidata fruit by centrifuging the above diluted product and removing the second supernatant to obtain a precipitate, and redissolving the precipitate in a sodium phosphate buffer, the sodium phosphate buffer may be 0.05 to 0.2 M, preferably 0.1 M. Additionally, the sodium phosphate buffer may further contain 4 to 6 mM ethylene-diamine-tetraacetic acid (EDTA), preferably 5 mM EDTA. Additionally, the sodium phosphate solution may have a pH of 6 to 7, preferably 6.6.

[0052] Protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit

[0053] The present invention may provide a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit prepared according to the method for preparing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit described above. The protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit may be characterized by hydrolyzing proteins, and preferably may be characterized by hydrolyzing casein, isolated soy protein, or whey protein.

[0054] The above-mentioned protease derived from Cudrania tricuspidata fruit may be characterized by having a casein protease activity of 3.5 to 4.0 (Units / mL protease), and the protease activity can be calculated by the following Formula 1.

[0056] [Equation 1]

[0057] Protein degradation activity (Units / mL protease) = (μmol tyrosine equivalents × VT) / VE × T × VA

[0058] (In the above Equation 1,

[0059] VT = Total amount of solution used in the experiment (mL),

[0060] VE = Amount of added protease (mL),

[0061] T = Reaction time after addition of proteolytic enzyme (minutes),

[0062] VA = refers to the amount of supernatant (mL) used for absorbance measurement.

[0064] Method for preparing a hydrolysate using a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit

[0065] The present invention may provide a method for producing a protein hydrolysate, characterized by comprising the steps of: mixing a protein hydrolysate derived from Cudrania tricuspidata fruit, prepared according to the method for producing a protein hydrolysate derived from Cudrania tricuspidata fruit described above, with a protein to produce a mixture; hydrolyzing the mixture to produce a hydrolysate; heating the hydrolysate to inactivate the enzyme; and filtering the hydrolysate in which the enzyme has been inactivated.

[0066] The above protein may be a food protein, and preferably, it may be one or more proteins selected from the group consisting of casein, isolated soy protein, and whey protein.

[0067] The step of preparing a hydrolysate by hydrolyzing the above mixture may be characterized by reacting the mixture at 34 to 40°C for 2 to 4 hours to hydrolyze it, and preferably by reacting it at 37°C for 3 hours to hydrolyze it.

[0068] The step of heating the hydrolysate to inactivate the enzyme may be characterized by heating the hydrolysate at 85 to 95°C for 10 to 30 minutes to inactivate the enzyme, and preferably by heating at 90°C for 20 minutes to inactivate the enzyme. If the step of inactivating the enzyme is not performed, the action of the protein hydrolyzing enzyme does not stop, and the protein is excessively hydrolyzed, which may make it difficult to utilize in subsequent processes such as food protein processing.

[0070] Food composition containing a hydrolysate produced using a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit

[0071] The present invention may provide a food composition comprising, as an active ingredient, a hydrolysate produced using the protein hydrolyzing enzyme derived from the Cudrania tricuspidata fruit described above. The food composition may be characterized by being prepared in the form of a powder, granule, pill, tablet, capsule, candy, beverage, or dairy product.

[0073] The embodiments of the present invention are described in detail below, but it is obvious that the present invention is not limited by the following embodiments.

[0075] Example 1. Preparation of a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit

[0076] Cudrania tricuspidata fruits were harvested from a farm in Sancheong, Gyeongsangnam-do. After an initial wash and visual inspection for foreign matter, the fruits were washed 1-2 times. The washed Cudrania tricuspidata fruits were frozen and then freeze-dried for 60 hours to obtain Cudrania tricuspidata fruit powder. 10 g of the Cudrania tricuspidata fruit powder was dissolved in 190 mL of 0.1 M sodium phosphate buffer (pH 6.6) containing 5 mM EDTA to prepare a lysate. The lysate was refrigerated at 4°C for at least 12 hours to induce the elution of water-soluble components, after which the lysate was centrifuged for 60 minutes in a centrifuge set at 3,700 × g and 4°C to separate the insoluble residue. The first supernatant obtained from the centrifugation was diluted with 99.9% ethanol at a weight ratio of 1:4 to induce protein precipitation and prepare a diluted product. The above diluted mixture was centrifuged for 60 minutes in a centrifuge set at 3,700 × g and 4°C to remove the second supernatant. The precipitate remaining after removing the second supernatant was redissolved in 0.1 M sodium phosphate buffer (pH 6.6) containing 5 mM EDTA to prepare a fraction derived from Cudrania tricuspidata fruit. The fraction derived from Cudrania tricuspidata fruit was used as a Cudrania tricuspidata fruit-derived protease and was frozen and stored at -20°C. The preparation process of the Cudrania tricuspidata fruit-derived protease of Example 1 is schematically illustrated in Fig. 1.

[0078] Comparative Example 1. Preparation of a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit prepared with an ethanol dilution ratio of 1:1

[0079] A protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit was prepared in the same manner as in Example 1, except that the first supernatant was prepared in the same manner as in Example 1 and the first supernatant was diluted in 99.9% ethanol at a weight ratio of 1:1.

[0081] Comparative Example 2. Preparation of a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit prepared with an ethanol dilution ratio of 1:2

[0082] A protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit was prepared in the same manner as in Example 1, except that the first supernatant was prepared in the same manner as in Example 1 and the first supernatant was diluted in 99.9% ethanol at a weight ratio of 1:2.

[0084] Comparative Example 3. Preparation of a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit prepared with an ethanol dilution ratio of 1:6

[0085] A protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit was prepared in the same manner as in Example 1, except that the first supernatant was prepared in the same manner as in Example 1 and the first supernatant was diluted in 99.9% ethanol at a weight ratio of 1:6.

[0087] Experimental Example 1. Analysis of Protein Size of Protein Hydrolyzing Enzyme Derived from Cudrania tricuspidata Fruit

[0088] Protein size was analyzed by electrophoresis of the proteolytic enzymes derived from Cudrania tricuspidata fruit prepared in Example 1 and Comparative Examples 1 to 3. First, all of the proteolytic enzymes derived from Cudrania tricuspidata fruit prepared in Example 1 and Comparative Examples 1 to 3 were diluted to a protein concentration of 2.5 mg / mL. Next, the diluted enzymes were diluted in a 4:1 ratio with 5× sample buffer (60 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 14.1 mM β-mercaptoethanol, 0.1% bromophenol blue), heat-denatured in a water bath preheated to 100°C for 5 minutes, and electrophoresis was performed.

[0089] The electrophoresis method is described in detail. 20 μL of the sample diluted in the above 5x sample buffer was injected into a 12% polyacrylamide gel. The 12% gel was prepared using a 5% stacking gel and a 12% separating gel. For the electrophoresis, the stacking gel was passed at a voltage of 80 V for 10 minutes, followed by the separating gel at 120 V.

[0090] The Coomassie blue staining method is described in detail. After the electrophoresis was completed, the gel was stained by immersing it in a staining solution (0.25% (w / v) Coomassie blue R-250, 50% (v / v) methanol, 40% (v / v) distilled water, 10% (v / v) acetic acid), and then destained by immersing it in a destaining solution (50% (v / v) methanol, 40% (v / v) distilled water, 10% (v / v) acetic acid). Finally, a standard substance (Precision plus protein standards, 1610373, BIO-Rad, Hercules, CA, USA) was used to confirm the molecular weight of the Cudrania tricuspidata fruit-derived proteolytic enzymes prepared in Example 1 and Comparative Examples 1 to 3. The results of Experimental Example 1 are shown in Figure 2.

[0091] As a result of Experimental Example 1, a band at approximately 65 kDa could be observed in all the enzymes prepared in Example 1 and Comparative Examples 1 to 3 (Fig. 2).

[0093] Experimental Example 2. Analysis of Casein Degradation Activity of Protein Hydrolyzing Enzymes Derived from Cudrania tricuspidata Fruit

[0094] The casein degradation activity of the Cudrania tricuspidata fruit-derived proteolytic enzymes prepared in Example 1 and Comparative Examples 1 to 3 was compared, and a commercial enzyme, alkalase, was used as a control group. First, all Cudrania tricuspidata fruit-derived proteolytic enzymes prepared in Example 1 and Comparative Examples 1 to 3 were diluted to a protein concentration of 2.5 mg / mL. Next, a 0.65% casein solution was prepared by equilibrating it by incubating at 37°C for 10 minutes. 1.75 mL of the casein solution was mixed with 100 μL of the enzymes from Example 1 and Comparative Examples 1 to 3 and incubated at 37°C for 10 minutes. Subsequently, 500 μL of 110 mM Trichloroacetic acid (TCA) was added, and the supernatant was obtained after centrifugation at 21,000×g for 20 minutes. To 200 μL of the above supernatant, 500 μL of 500 mM sodium carbonate and 100 μL of 0.5 M Folin & Ciocalteu's phenol reagent were added and incubated at 37°C for 30 minutes. After centrifugation at 21,000×g for 20 minutes, the absorbance of the supernatant was measured at 660 nm and converted into protein degradation activity in Units / mL, and the protein degradation activity was calculated using Equation 1 below. The results of Experimental Example 2 are listed in Table 1, and the relative casein degradation activity against commercial enzyme alkalase is shown as a graph in Figure 3.

[0096] [Equation 1]

[0097] Protein degradation activity (Units / mL protease) = (μmol tyrosine equivalents × VT) / VE × T × VA

[0098] (In the above Equation 1,

[0099] VT = Total amount of solution used in the experiment (mL),

[0100] VE = Amount of added protease (mL),

[0101] T = Reaction time after addition of proteolytic enzyme (minutes),

[0102] VA = refers to the amount of supernatant (mL) used for absorbance measurement.

[0104]

[0105] (In Table 1 above, all values ​​are mean ± standard error, and different initials within the same row are considered significant (p<0.05).)

[0107] As a result of Experimental Example 2, the casein degradation activities of the hydrolases prepared in Comparative Example 1, Comparative Example 2, Example 1, and Comparative Example 3 were 2.25, 3.28, 3.17, and 2.13 Units / mL protease, respectively. In addition, the casein degradation activity of the alkalase was 26.82 Units / mL protease, and the relative casein degradation activities of the hydrolases prepared in Comparative Example 1, Comparative Example 2, Example 1, and Comparative Example 3 to the alkalase were 8.38, 12.23, 13.82, and 7.92%, respectively. That is, the best casein degradation activity was observed at the 1:4 ethanol precipitation ratio of Example 1.

[0109] Example 2. Preparation of isolated soy protein hydrolysate using protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit

[0110] Isolated soy protein hydrolysate was prepared using the Cudrania tricuspidata fruit-derived proteolytic enzyme prepared in Example 1 above. First, isolated soy protein was used as the substrate for the hydrolysate. The isolated soy protein and the Cudrania tricuspidata fruit-derived proteolytic enzyme of Example 1 were mixed. The mixture was hydrolyzed by reacting in a 37°C constant temperature water bath for 3 hours, and heated at 90°C for 20 minutes to inactivate the enzyme. After cooling the enzyme-inactivated hydrolysate to room temperature, the isolated soy protein hydrolysate was obtained by filtering through a test sieve (885705, CHUNG GYE GONG SA, Seoul, Korea) with a mesh size of 500 μm / 35 and a line thickness of 351 μm.

[0112] Example 3. Preparation of whey protein hydrolysate using proteolytic enzyme derived from Cudrania tricuspidata fruit

[0113] A whey protein hydrolysate was prepared using the Cudrania tricuspidata fruit-derived proteolytic enzyme prepared in Example 1 above. The substrate for the whey protein hydrolysate was whey protein, and all other processes were carried out in the same manner as in Example 2 above.

[0115] Experimental Example 3. Analysis of the degree of hydrolysis of hydrolysate using protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit

[0116] The degree of hydrolysis of the hydrolysates prepared in Examples 2 and 3 above was measured, respectively. The degree of hydrolysis was determined by applying the bicinchoninic acid (BCA) protein quantification method. The hydrolysates were measured and calculated using the BCA protein quantification method. 30 μL of the hydrolysate sample and 200 μL of the prepared BCA solution were mixed and incubated at 37°C for 30 minutes, after which the absorbance was measured at 560 nm and calculated. Protein concentration was calculated by substituting into a standard curve of bovine serum albumin (BSA) diluted to different concentrations, and the degree of hydrolysis was calculated using Equation 2 below.

[0118] [Equation 2]

[0119] Degree of hydrolysis (%) = Protein concentration after hydrolysis (mg / mL) / Protein concentration before hydrolysis (mg / mL) × 100

[0121] As a result of Experimental Example 3, the degree of hydrolysis of the isolated soy protein hydrolysate prepared in Example 2 was 54.42%, and the degree of hydrolysis of the whey protein hydrolysate prepared in Example 3 was 40.83%.

[0123] Experimental Example 4. Analysis of protein size of hydrolysate using proteolytic enzyme derived from Cudrania tricuspidata fruit

[0124] The hydrolysates prepared in Examples 2 and 3 above were subjected to electrophoresis to analyze the protein size. The electrophoresis was performed in the same manner as in Experimental Example 1, and the results of Experimental Example 4 are shown in Figure 4. For reference, the major protein bands of isolated soy protein were identified as β-conglycinine (50-75 kDa) and glycinin (10-37 kDa), and the major protein bands of whey protein were identified as β-lactoglobulin (15-20 kDa), casein (30-43 kDa), lactoperoxidase (70-100 kDa), lactoferrin (70-100 kDa), and bovine serum albumin (70-100 kDa).

[0125] As a result of Experimental Example 4, it was confirmed that the proteins of the isolated soy protein and whey protein hydrolysates prepared in Examples 2 and 3 were reduced to a low molecular weight of 37 kDa or less (Fig. 4). That is, the protein hydrolyzing enzyme derived from the Cudrania fruit of Example 1 is considered to be a natural plant-based protein hydrolyzing enzyme capable of effectively hydrolyzing isolated soy protein and whey protein, which are widely used in the bio and food industries.

[0127] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A step of preparing Cudrania fruit powder by freeze-drying Cudrania fruit; a step of preparing a solution by dissolving the Cudrania fruit powder in a 0.05 to 0.2 M sodium phosphate buffer; a step of preparing a first supernatant by centrifuging the solution at 3500 to 3900 xg at 4℃ for 50 to 70 minutes; a step of preparing a diluted solution by diluting the first supernatant in ethanol at a weight ratio of 1:3 to 5; A method for preparing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, characterized by comprising the step of centrifuging the above diluted product and removing the second supernatant to obtain a precipitate, and redissolving the precipitate in a second sodium phosphate buffer to prepare a fraction derived from Cudrania tricuspidata fruit, wherein the first sodium phosphate buffer and the second sodium phosphate buffer further comprise 4 to 6 mM ethylene-diamine-tetraacetic acid (EDTA). Claim 2 delete Claim 3 delete Claim 4 A method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, characterized in that, in claim 1, the first sodium phosphate buffer and the second sodium phosphate buffer have a pH of 6 to 7. Claim 5 delete Claim 6 delete Claim 7 A method for producing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, characterized in that, in claim 1, the ethanol is 90 to 100% ethanol. Claim 8 Protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, prepared by the method according to Paragraph 1, Paragraph 4, or Paragraph 7. Claim 9 In claim 8, the above-mentioned protein hydrolyzing enzyme derived from Cudrania fruit is characterized by hydrolyzing casein, isolated soy protein, or whey protein. Claim 10 In claim 8, the protease derived from Cudrania tricuspidata fruit is characterized by having a casein protease activity of 3.5 to 4.0 (Units / mL protease), and the protease derived from Cudrania tricuspidata fruit is characterized by the protease being calculated by the following Formula 1. [Formula 1] Protease activity (Units / mL protease) = (μmol tyrosine equivalents × VT) / VE × T × VA (in Formula 1 above, VT = amount of total solution used in the experiment (mL), VE = amount of added protease (mL), T = reaction time after addition of protease (min), VA = amount of supernatant used for absorbance measurement (mL).) Claim 11 A method for producing a protein hydrolysate, characterized by comprising: a step of preparing a mixture by mixing a protein hydrolyzing enzyme derived from Cudrania tricuspidata fruit, prepared by the method according to claim 1, with a protein; a step of preparing a hydrolysate by hydrolyzing the mixture; a step of inactivating the enzyme by heating the hydrolysate; and a step of filtering the hydrolysate in which the enzyme has been inactivated. Claim 12 A method for producing a protein hydrolysate according to claim 11, characterized in that the protein is one or more proteins selected from the group consisting of isolated soy protein and whey protein. Claim 13 A method for producing a protein hydrolysate according to claim 11, wherein the step of producing a hydrolysate by hydrolyzing the mixture is characterized by reacting the mixture at 34 to 40°C for 2 to 4 hours. Claim 14 A method for preparing a protein hydrolysate according to claim 11, wherein the step of heating the hydrolysate to inactivate the enzyme is characterized by heating the hydrolysate at 85 to 95°C for 10 to 30 minutes to inactivate the enzyme. Claim 15 A food composition comprising a protein hydrolysate prepared by the method according to Clause 11 as an active ingredient. Claim 16 In claim 15, the food composition is characterized by being manufactured in the form of a powder, granule, pill, tablet, capsule, candy, beverage, or dairy product.

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