JP2025072544A - Placental hydrolysate and method for producing same - Google Patents

Abstract

To provide a method of producing a placenta hydrolysate.SOLUTION: A method of producing a placenta hydrolysate containing large amounts of amino acids and oligopeptides that are capable of exhibiting pharmacological activity comprises the steps of: degreasing and drying an animal placenta lysate to obtain animal placenta lysate powder; treating the degreased animal placenta lysate powder with an acidic protease solution to cause enzymatic degradation; and bringing the degraded product into contact with an anion exchange resin to cause ion exchange.SELECTED DRAWING: Figure 1

Description

The present invention relates to a placental hydrolysate and a method for producing the same.

The placenta is an organ that forms in the uterus of pregnant mammals, and it supports life by supplying nutrients to the fetus through the umbilical cord. The placenta is expelled from the uterus at birth, and this expulsion of the placenta is called the afterbirth.

The placenta contains essential amino acids, melatonin, nucleic acid components such as RNA and DNA, the antioxidant enzyme SOD (super oxide dismutase), hyaluronic acid, antioxidants, immune cofactors (cytokines), placental peptides, growth factors such as insulin-like growth factors (EGFS), epidermal growth factors (EPFs), and unique senescent cell activating factors (SCAFSs), as well as cytokines, and is known to be useful for recovering from fatigue and boosting the immune system.

The method used to process the placenta has been to hydrolyze the placenta using a strong acid (usually 3N hydrochloric acid) or strong alkali, and obtain the product. However, this method has the problem that the long-term high-temperature treatment process destroys the various amino acids and other physiologically active substances contained in the placenta. In addition, conventional placenta processing methods that use enzymes have the problem that it takes a long time to extract the active ingredients, and there is a need to develop a method that can improve this problem.

In Korean Patent Application No. 2000-0043588, a method for treating and preventing liver cancer was developed.
The product uses a hydrolyzate of Shikawasha (placenta), which is made by extracting various bioactive components such as cytokines, amino acids, nucleic acid bases, and carbohydrates from human placenta. The product describes that Shikawasha is treated with acetone to defatted, and then treated with pepsin and hydrochloric acid for sufficient hydrolysis to prevent the production of an incomplete hydrolyzate, but does not describe the detailed manufacturing method.

Korean Patent Application No. 2007-0065779 describes a method for producing a composition for cosmetics or health functional food ingredients derived from placenta, which comprises the steps of adding acetone and a fixed amount of purified water to the placenta, degreasing it at 40°C for 3-5 hours, and drying the product of the degreasing reaction at 80°C for 8 hours to obtain defatted placenta powder, i) hydrolyzing the placenta powder by adding 0.5-8 parts by weight of a protease and 30-60 parts by weight of water to 40-60 parts by weight of the placenta powder, ii) concentrating the hydrolysis product under reduced pressure, and iii) freeze-drying the vacuum concentrate. However, in the examples of the prior invention, porcine placenta, not human placenta, is used, and it is described that the placenta extract obtained by the protease method has a higher amino acid content than the placenta extract obtained by the acid addition hydrolysis method, but no comparison or analysis is made of steps other than the protein decomposition process.

The specific components of the placenta are not precisely known, but in prior Korean Patent Application No. 2003-0000173, the placenta of a woman who had a normal delivery was degreased with acetone and hydrolyzed with hydrochloric acid to contain 56 mg or more of water-soluble substances per 1.0 milliliter, and when quantified, the results were 0.01% by weight of arginine, 0.02% by weight of lysine, 0.09% by weight of phenylalanine, 0.04% by weight of tyrosine, 0.11% by weight of leucine, 0.01% by weight of isoleucine, 0.02% by weight of methionine, 0.02% by weight of valine, 0.02% by weight of glycine, 0.02% by weight of proline, 0.02% by weight of glycine, 0.02 ...glycine, 0.02% by weight of glycine, 0.02% by weight of glycine, 0.02% by weight of glycine, 0.02% by weight of glycine, 0.02% by weight of glycine, 0.02% by weight of glycine, 0.02% by weight of glycine, 0.02% by weight of glyc
It is described that the extract contains the following amino acids by weight: 0.01% by weight of glutamic acid, 0.02% by weight of serine, 0.03% by weight of threonine, and 0.02% by weight of aspartic acid, and that the extract contains 0.172 to 0.816 w/v% of total nitrogen.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is generally used to analyze the presence or absence of proteins and peptides. This is a qualitative analysis method that uses an electrophoresis device to separate proteins according to their molecular weight, and is a method to confirm the presence of major proteins. SDS-PAGE is used to separate proteins and peptides using the content of acrylamide components and glycine or tricine as raw materials. Tris-glycine gel is useful for separating proteins with a molecular weight of 10 kDa to 300 kDa, and Tris-tricine gel is useful for separating peptides with a molecular weight of 2 kDa to 20 kDa. The presence or absence of the final protein is confirmed for the proteins/peptides separated by SDS-PAGE using Coomassie staining. Recently, the amount of protein separated by PAGE has been quantified using a gel imaging program, but due to band separation and band saturation in the staining method, it is difficult to apply this method for accurate analysis of the amount of protein. The presence or absence of peptides with a molecular weight of 2 kDa or less can be confirmed using LC-MS analysis.

During the production of placental hydrolysate, an acid hydrolysis process is carried out, which means that a large number of peptides or amino acids may be present in the human placenta. To date, a mass spectrometer (ESI-MS/MS; Electrospray Ionization) is usually used to determine the sequence of known peptides. Proteins or peptides present in the placental hydrolysate can be confirmed and analyzed using the above method. Finally, the presence or absence of proteins or peptides contained in the placental hydrolysate is confirmed using SDS-PAGE, and the presence and content of low molecular weight peptides is analyzed using LC-MS, allowing all proteins and peptides contained in the placental hydrolysate to be identified.

The placenta is believed to contain minerals that are known to affect various physiological functions of the human body. Typical minerals include iron powder, magnesium, calcium, zinc, and selenium. There are various methods known for analyzing the mineral content in powdered milk, functional health foods, and pharmaceuticals. A typical method, referring to the general test method in the Korean Pharmacopoeia, involves preparing standard solutions by concentration, obtaining a calibration curve with absorbance values corresponding to each concentration, and reading the absorbance value of the sample on the calibration curve graph to confirm the content of the mineral component in the sample.

Generally, ion exchange chromatography is used in the process of purifying a composition containing amino acids, peptides, or proteins. Proteins and peptides are composed of amino acids that are zwitterions, so the net charge of the protein and peptide changes depending on the pH of the surrounding environment, but ions and polar substances are separated using the difference in pI (isoelectric point) that makes them neutral. This separation process is called ion exchange chromatography, and is divided into chromatography using anion exchangers and cation exchangers based on the surface charge of the protein to be separated. In the case of anion exchange chromatography, proteins with high isoelectric points are relatively weakly bound to the column and can be eluted even at low salt concentrations, so proteins with high isoelectric points start to elute from the column. The content of the product obtained varies depending on the type and equilibrium conditions of the ion exchanger of ion exchange chromatography, i.e., the ion exchange resin, the loading conditions and washing procedure of the sample, or the pH gradient range of the eluent, and the type and elution conditions of the buffer.

The inventors of the present invention established optimal conditions for ion exchange chromatography in the process of processing the placenta to hydrolyze the placenta or extracting a pharmaceutical composition having effective effects from the placenta, and confirmed the content of proteins, peptides, or minerals in the placental hydrolysate or placental extract obtained thereby, thereby completing the present invention.

The present invention aims to provide a method for producing a placenta hydrolysate that contains a large amount of amino acids and oligopeptides that can exhibit pharmacological activity.

1. Defatting and drying the animal placenta fragments to obtain animal placenta fragment powder;
a step of treating the defatted powder of the crushed animal placenta with a protease acid solution to enzymatically decompose the protease;
and contacting the hydrolyzate with an anion exchange resin to exchange the hydrolyzate.

2. The method for producing a placenta hydrolysate according to item 1, wherein the defatting is carried out by adding an organic solvent selected from the group consisting of acetone, alcohol, and esters to the placenta disruption.

3. The method for producing a placenta hydrolysate according to item 1, wherein the defatting step includes the steps of adding one of acetone, an organic solvent containing an alcohol or an ester to the placenta disruptant and defatting for 2 to 8 hours, and drying the placenta disruptant at 60 to 100°C for 10 to 24 hours to remove the organic solvent.

4. The method for producing a placenta hydrolysate according to item 3, wherein the defatting and removal of the organic solvent are carried out multiple times, and the second defatting is carried out for a longer period of time than the first defatting.

5. The method for producing a placenta hydrolysate according to item 1, wherein the protease is papain, pepsin, bromelain, pronase, or alcalase.

6. The method for producing a placenta hydrolysate according to item 1, wherein the acid concentration of the acid solution is 0.1 to 10%.

7. The method for producing a placenta hydrolysate according to item 1, wherein 10 kg to 25 kg of defatted powder of crushed placenta is treated with 0.5 kg to 3 kg of proteolytic enzyme during the enzymatic hydrolysis.

8. The method for producing a placenta hydrolysate according to item 1, wherein the contact with the anion exchange resin is carried out by adding an anion exchange resin to the hydrolysate or by passing the hydrolysate through an anion exchange resin column.

9. The method for producing a placental hydrolysate according to item 1, further comprising the step of adding a base to the ion-exchanged hydrolysate to adjust the pH.

The present invention provides a method for producing placental powder from placenta, and a method for producing a placental hydrolysate from placenta powder. The placental hydrolysate obtained by the production method of the present invention contains 2mer to 5mer peptides and a large amount of minerals with high physiological activity, so that a pharmaceutical composition or a functional health food composition containing the placental hydrolysate of the present invention can alleviate symptoms and diseases related to fatigue, and is particularly effective for liver diseases. In addition, a cosmetic composition containing the placental hydrolysate of the present invention can be useful for preventing skin aging and improving wrinkles.

FIG. 1 shows the results of SDS-PAGE protein separation after TCA precipitation concentration of the placental hydrolysate of the present invention. FIG. 2 shows the results of SDS-PAGE protein separation following Speedvac concentration of a placental hydrolysate of the present invention. FIG. 3 shows the results of peptide separation by Tris-tricine gel electrophoresis after Speedvac concentration of the placental hydrolysate of the present invention.

The present invention is described in detail below.

The present invention relates to a method for producing placental hydrolysate.

The method of the present invention includes the steps of defatting and drying crushed animal placenta to obtain crushed animal placenta powder, treating the crushed animal placenta defatted powder with a proteolytic enzyme acid solution to enzymatically decompose the powder, and contacting the decomposed product with an anion exchange resin to perform ion exchange.

The placenta may be a mammalian placenta, including a human placenta, and may specifically be a human placenta.

For the placenta, chorionic or umbilical tissue can be used.

Defatting can be performed by adding a solvent selected from the group consisting of acetone, alcohol, and ester to the placenta disruption. Specifically, acetone or alcohol can be used. As the alcohol, ethanol can be used.

For example, the solvent can be added in an amount of 2 to 20 v/w times or 4 to 12 v/w times the amount of the placenta disruption, but is not limited to these amounts. When the amount of the solvent is within the above range, a sufficient degreasing effect is obtained and subsequent solvent removal is easy.

Degreasing can be carried out under stirring. The stirring time is preferably 2 to 8 hours. If the stirring time is too short, a sufficient amount of precipitate will not be obtained, and if the stirring time is too long, the total process time will be long and inefficient.

The degreasing process can include the steps of adding one of the organic solvents, including acetone, alcohol, or ester, to the placenta fragments and degreasing for 2 to 8 hours, and drying at 60°C to 100°C for 10 to 24 hours to remove the organic solvent.

Degreasing can be carried out multiple times in order to completely remove residual fat. For example, it can be carried out two or more times, three or more times, or four or more times. The upper limit may be, for example, six, five, four, etc. Each degreasing step during multiple degreasing can be carried out for the time exemplified above, but is not limited to these.

When degreasing is performed multiple times, the next degreasing time can be made longer than the previous one. For example, the second degreasing time can be made longer than the first degreasing time.

After degreasing, the precipitating solvent is removed. This can be done by centrifugation.

If degreasing is performed multiple times, a solvent removal step can be performed between each degreasing step.

The drying temperature may be, for example, 60°C to 100°C. Temperatures within the above range may be 60°C to 100°C, 70°C to 100°C, 80°C to 100°C, 90°C to 100°C, etc. When the drying temperature is within the above range, sufficient drying is possible and damage to the placental tissue can be prevented.

The drying time may be, for example, 10 hours or more, 12 hours or more, 14 hours or more, or 16 hours or more. The upper limit may be, for example, 48 hours, 36 hours, 24 hours, etc.

Then, the defatted powder of crushed animal placenta is treated with an acid solution of proteolytic enzymes to carry out enzymatic degradation.

Examples of proteolytic enzymes that can be used include papain, pepsin, bromelain, pronase, and alcalase.

The protease acid solution is an acid solution containing a protease, and the acid may be, for example, hydrochloric acid, nitric acid, sulfuric acid, acetic acid, etc., and specifically, hydrochloric acid.

The acid concentration of the acid solution may be 0.1-10%, and the acid content may be 0.2-20 times (w/w) the dry content of the placenta powder.

In this step, 0.5 kg to 3 kg of protease can be used for 10 to 25 kg of placenta powder. The content of the protease is preferably 0.01 to 5 times (w/w) the dry content of the placenta powder. If the content of the protease is too low, hydrolysis will not be sufficient, which will lengthen the hydrolysis time and unnecessarily lengthen the process time. On the other hand, if the content of the protease is too high, the molecular weight of the placenta components will decrease due to hydrolysis, which may result in the desired effect not being fully achieved.

The enzymatic degradation can be carried out until no further degradation reactions occur, for example until no further protein reactions occur via the Biuret reaction.

The decomposition product is then brought into contact with an anion exchange resin to carry out ion exchange.

Anion exchange resin treatment can be performed by adding anion exchange resin to the hydrolyzate or by passing the hydrolyzate through an anion exchange column.

After that, filtration, pH adjustment, and heat sterilization can be performed.

This is the process of increasing the temperature of the hydrolysate solution to inactivate the enzymes and then filtering out the residue. The temperature at this time may be between 70°C and 100°C.

The next step is to increase the pH by adding a base such as sodium hydroxide as a pH adjuster. The pH can be adjusted to, for example, a range of 5.5 to 7.5. This is because it is preferable for the composition of the present invention to have a pH in this range in order to use it as an injection or for oral administration. The concentration and volume of the liquid can be adjusted by adding purified water to the composition obtained by the above step. This process is carried out by heat treatment, and it is preferable to add a preservative such as sodium benzoate and heat sterilize it in a high-temperature, high-pressure sterilizer. Conventional preservatives can be used within the range acceptable for the composition of the present invention (e.g., pharmaceutical composition or food composition).

The sterilization process is preferably carried out at 100-140°C for 20-120 minutes.

The placental hydrolysate obtained by the method of the present invention contains a large amount of amino acids and oligopeptides that can exhibit pharmacological activity, and can therefore be used as a raw material for functional health foods.

Health functional foods made from the hydrolysate can help improve liver function.

A "health functional food" is one that meets the criteria of "foods manufactured or processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients with functional properties that are beneficial to the human body" (Article 3, Paragraph 1 of the Act on Health Functional Foods (Korea Act No. 7428)). And "function" or "functionality" refers to obtaining a beneficial effect for health purposes, such as regulating nutrients or physiological effects on the structure and functions of the human body. In other words, it means that it can be used for health purposes in healthy or unhealthy people.

The dry content of placental hydrolysate in the health functional food is preferably 0.1 to 50% by weight of the total content. If the content is too low, the efficacy of the placental hydrolysate described above will not be fully expressed, and the dosage will be too high, making administration inconvenient. If the content is too high, problems will arise in solubility and product formulation.

For ease of administration, it is preferable to use functional health foods containing placental hydrolysates as functional foods in the form of tablets, sugar-coated tablets, capsules, drinks, etc.

Examples 1. Production of Placenta Powder 200 kg of frozen human placenta was washed, thawed and crushed to obtain placenta crushed material.

A degreasing solvent was added to the resulting placenta fragments and the mixture was stirred to degrease the fragments. Acetone, alcohol, or esters can be used as the degreasing solvent.

The degreasing solvent was used at a volume (v/w) of 4 to 12 times the placenta fragment content and stirred for 2 to 8 hours.

The degreasing was carried out in two separate steps. The repeated degreasing step (second degreasing step) is a process in which acetone, alcohol or ester is added again to the precipitate obtained in the previous degreasing step (first degreasing step) and stirring is continued to obtain a precipitate. Here, the amount of acetone is the same as that used in the previous degreasing step, but the stirring time is set to 2 to 24 hours.
The solvent was then removed and the mixture was dried to obtain a placenta disruption powder.

2. Preparation of placenta hydrolysate The placenta disruption powder was added with an acid solution to carry out acid hydrolysis in combination with a protease.

The acid used could be hydrochloric acid, nitric acid or acetic acid, and the proteolytic enzyme used was papain, pepsin, bromelain, pronase or alcalase.

In this step, 0.5 kg to 3 kg of proteolytic enzyme was used for 10 to 25 kg of placenta powder.

The acid was used in a concentration range of 0.1-10%, and the content was 0.2-20 times (w/w) the dry content of the placenta powder.

Hydrolysis was continued until no further protein reactions occurred via the Biuret reaction. After hydrolysis, an ion exchange process was performed.

Ion exchange was performed using anion exchange chromatography or by adding an ion exchange resin to the resulting hydrolysate.

The following are three specific examples of using anion exchange chromatography, each of which can be used selectively.

(1) Anion Exchange Example 1
The placental hydrolysate treated with 20 mM Tris (pH 7.5) buffer diafiltration (ILDF, In-line diafiltration module) and ethanol was diluted 10-fold and loaded at a flow rate of 12 ml/min onto a Source Q (XK50/25, 500 ml) column equilibrated with 20 mM Tris (pH 7.5) buffer, and bound to the column. The placental hydrolysate was then eluted using a linear concentration gradient of 18 column volumes (sodium chloride concentration 0 M→1 M) by the linear concentration gradient method.

(2) Anion Exchange Example 2
The fraction obtained in the previous step was adjusted to pH 5.8 using NaOH and then subjected to DEAE-Sepharose fast flow column chromatography as follows: the fraction was adsorbed onto a DEAE-Sepharose column pre-equilibrated with Buffer 2 (20 mM sodium acetate, pH 5.8), and the placental hydrolysate was eluted by sequentially increasing the concentration of sodium chloride by 10 mM increments.

(3) Anion Exchange Example 3
Generally, the matrix of the resin used to purify the mixture is a matrix of polystyrene or polyacrylate mixed with DVB (Divinylbenzene), and in the case of a cation exchange resin, the functional group is sulfonate (-SO ^3- ), and in the case of an anion exchange resin, the functional group is -N ₃ (CH ₃ ) ₃ Cl ^- (trimethylammonium), -N ⁺ (CH ₃ ) ₂ C ₂ H ₄ OHCl ^- (dimethylethanolammonium), -(CH ₂ ) _n N (CH ₃ ) ₂ (Tertiary Amine), or -CH ₂ NH (CH ₂ CH ₂ NH) _n H (CH ₂ ) ₂ NH ₂ (Secondary Amine) resin is used.

The mixture was then filtered, pH adjusted, and heat sterilized.

The temperature of the hydrolysate solution was raised to inactivate the enzyme, and the residue was removed by filtration. The temperature was 70° C. to 100° C. The hydrolysate solution was then concentrated and filtered.

The next step is to add a pH adjuster such as sodium hydroxide to adjust the pH to 5.5 to 7.5. This is because it is preferable for the composition of the present invention to have a pH in the above range in order to use it as an injection or for oral administration. The concentration and volume of the liquid can be adjusted by adding purified water to the composition obtained in the above step. This process is carried out by heat treatment, and it is preferable to add a preservative such as sodium benzoate and heat sterilize in a high-temperature, high-pressure sterilizer. Conventional preservatives can be used within the range acceptable for the pharmaceutical or food composition of the present invention.

The sterilization process was preferably carried out at 100 to 140°C for 20 to 120 minutes.

Experimental Example 1. Confirmation of the presence of proteins and peptides in placental hydrolysate Proteins and peptides in a placental hydrolysate sample were concentrated by TCA (Trichloroacetic acid) precipitation and Speedvac. Coomassie staining, which is equivalent to the staining method of SDS-PAGE, is known to have a detection intensity of about 20 ng, and these two concentration methods were used to confirm the presence of trace amounts of proteins or peptides in the placental hydrolysate sample of the present invention. The concentrated placental hydrolysate sample was separated into proteins and peptides using Tris-glycine gel for protein separation and Tris-tricine for peptide separation, respectively.

1.1 TCA Precipitation Sigma T9159-100G was used as the TCA solution. When 10 μL of the placental hydrolysate sample concentrated by TCA precipitation was loaded, no specific protein bands were detected as in lanes (2), (5), and (8). In the samples loaded with 50 μL and 100 μL, vertical streaking bands with molecular weights of 5 kDa to 35 kDa were observed (see FIG. 1). In the figure, sample number 30620 corresponds to the placental hydrolysate of the present invention, and 677870 and K018 correspond to the control group. These numbers are used to distinguish samples from different production lots.

A band pattern was observed that was different from the usual protein migration pattern. To confirm the presence or absence of protein, the band was excised and subjected to in-gel digestion and MALDI-TOF/TOF analysis.

1.2 Speedvac Concentration FIG. 2 shows the results of SDS-PAGE analysis of placental hydrolysate samples of the present invention concentrated in a Speedvac system.

When 10 μL of the placental hydrolysate sample was loaded, a molecular weight of 7 kDa was confirmed. When 50 μL of the sample was loaded, a molecular weight of approximately 15 kDa was confirmed, and when 100 μL of the sample was loaded, a molecular weight of approximately 20 kDa was confirmed (see Figure 2).

Usually, it can be confirmed by SDS-PAGE from a band of a specific molecular weight, and the thickness of the band increases at the same molecular weight as the amount of the loaded sample increases.
The c-enriched sample showed a pattern in which the molecular weight increased as the sample loading amount increased, which is different from normal SDS-PAGE. The bands observed here were excised and subjected to in-gel digestion and MALDI-TOF/TOF analysis to confirm the presence or absence of protein.

1.3 Identification of low molecular weight proteins and peptides using Tris-tricine The results of peptide gel (Tris-tricine) analysis of placental hydrolysate samples of the present invention concentrated using the Speedvac system are shown in FIG.

In lane (2), where 10 μL of the placental hydrolysate sample was loaded, molecular weights of 0 to 7 kDa were confirmed. In lane (3), where 50 μL was loaded, molecular weights of 0 to 15 kDa were confirmed, and in lane (4), where 100 μL was loaded, molecular weights of approximately 0 to 25 kDa were confirmed. The morphology is similar to that of the SDS-PAGE in Figure 2, but it can be confirmed that the low molecular weights are more widely spread (see Figure 3).

The peptide gel in this experiment also showed a different migration pattern from that of normal proteins. To confirm the presence or absence of protein, the bands observed here were excised and subjected to in-gel digestion and MALDI-TOF/TOF analysis.

2. Analysis of peptide sequences present in placental hydrolysate 2.1 Analysis of peptide sequences using LC-MS/MS In Experimental Example 1, only the presence or absence of proteins and peptides in the placental hydrolysate was confirmed. However, peptides with a molecular weight of 2 kDa or less cannot be confirmed by the above method. Therefore, LC-MS analysis was performed in this example. The presence of more than 100 peptide sequences was confirmed by LC-MS analysis, and sequence information of more than 100 peptides was confirmed by MS/MS analysis.

After analyzing over 100 peptides, all were found to have molecular weights of 1.5 kDa or less. Peptides with sizes ranging from 5 amino acids (5mer) or less to 2mers or more are summarized in Table 1.

2.2 Comparison of Peptide Content Using EIC Method The peptides analyzed in Experimental Example 2.1 and the peptides obtained from the control sample were further subjected to EIC (Extracted Ion Chromatography) analysis.

The original placental hydrolysate solution and the control sample were compared based on the same peptide content. The EIC analysis was first performed three times on the same sample to confirm the reproducibility of the EIC analysis method. The average total area content of each peptide and the standard deviation confirmed in the three repeated tests were confirmed to be within ±3%.

When comparing the peptide content of the two samples, it was confirmed that the peptides contained in the placental hydrolysate of the present invention were higher than those in the control group when comparing peptides contained at 1% or more, peptides contained at 0.5% to less than 1%, and peptides contained at less than 0.5% of the total peptides in the placental hydrolysate of the present invention.

Experimental Example 3. Analysis of types and contents of minerals The results of analysis of minerals in the placenta hydrolysate are shown in Table 2. Mineral analysis was performed according to the general test method in the Korean Pharmacopoeia, and plasma concentrations in healthy adults are shown as reference values.

Claims (9)

defatting and drying the animal placenta fragments to obtain animal placenta fragment powder;
a step of treating the defatted powder of the crushed animal placenta with a protease acid solution to enzymatically decompose the protease;
and contacting the hydrolyzate with an anion exchange resin to exchange the hydrolyzate. The method for producing a placenta hydrolysate according to claim 1, wherein the defatting is carried out by adding an organic solvent selected from the group consisting of acetone, alcohol, and ester to the placenta disruption. The method for producing a placenta hydrolysate according to claim 1, wherein the degreasing step includes the steps of adding one of acetone, an organic solvent including an alcohol or an ester to the placenta disruptant and degreasing for 2 to 8 hours, and drying the placenta disruptant at 60 to 100°C for 10 to 24 hours to remove the organic solvent. The method for producing a placental hydrolysate according to claim 3, wherein the defatting and removal of the organic solvent are carried out multiple times, and the second defatting is carried out for a longer period of time than the first defatting. The method for producing a placental hydrolysate according to claim 1, wherein the protease is papain, pepsin, bromelain, pronase, or alcalase. The method for producing a placental hydrolysate according to claim 1, wherein the acid concentration of the acid solution is 0.1 to 10%. The method for producing a placenta hydrolysate according to claim 1, wherein 10 kg to 25 kg of the defatted powder of the crushed placenta is treated with 0.5 kg to 3 kg of proteolytic enzyme during the enzymatic hydrolysis. The method for producing a placental hydrolysate according to claim 1, wherein the contact with the anion exchange resin is carried out by adding an anion exchange resin to the hydrolysate or by passing the hydrolysate through an anion exchange resin column. The method for producing a placental hydrolysate according to claim 1, further comprising the step of adding a base to the ion-exchanged hydrolysate to adjust the pH.