Placenta hydrolysate and method for producing the same

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

[Technical field]

[0001] The present invention relates to a placental hydrolysate and a method for producing the same. [Background technology]

[0002] The placenta is an organ that forms in the uterus of pregnant mammals and provides nutrients to the fetus through the umbilical cord, sustaining its life. The placenta is expelled from the uterus at birth, and this expulsion is called the afterbirth.

[0003] 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, insulin-like growth factors, epidermal growth factors (EGFS), and unique senescent cell activating factors (SCAFS), as well as growth factors and cytokines, and is known to be useful for recovering from fatigue and boosting the immune system.

[0004] The method used to process the placenta has been to hydrolyze the placenta using strong acid (usually 3N hydrochloric acid) or strong alkali, and obtain the product. However, this method has the problem that the various amino acids and other physiologically active substances contained in the placenta are destroyed during the long-term high-temperature treatment process. In addition, the conventional placenta processing method using enzymes has the problem that it takes a long time to extract the active ingredients, and the reality is that a method that can improve this problem needs to be developed.

[0005] 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. It is described here 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 no detailed manufacturing method is described.

[0006] Korean Patent Application No. 2007-0065779 describes a method for producing a composition for cosmetics or health functional food raw materials derived from placenta, which comprises the steps of adding acetone and a fixed amount of purified water to placenta, degreasing reaction 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 protease and 30-60 parts by weight of water to 40-60 parts by weight of 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 on steps other than the protein decomposition process.

[0007] The specific components of the placenta are not precisely known, but in prior Korean Patent Application No. 2003-0000173, the placenta from a woman who had a normal delivery was degreased with acetone and hydrolyzed with hydrochloric acid to contain more than 56 milligrams of water-soluble substances per 1.0 milliliter, and when quantified, was arginine 0.01% by weight, lysine 0.02% by weight, phenylalanine 0.09% by weight, tyrosine 0.04% by weight, leucine 0.11% by weight, isoleucine 0.01% by weight, methionine 0.02% by weight, valine 0.02% by weight, glycine 0.02% by weight, proline 0.02% by weight, and glycine 0.02% by weight. It is described as containing the amino acids 0.01% by weight, 0.02% by weight glutamic acid, 0.03% by weight serine, 0.02% by weight threonine, and 0.21% by weight aspartic acid, and as containing 0.172 to 0.816 w / v% total nitrogen.

[0008] 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 electrophoretic device to separate proteins according to their molecular weight, and is a method to confirm the presence of major proteins. SDS-PAGE is applied to the separation of proteins and peptides using the content of acrylamide components and glycine or tricine as raw materials. Tris-glycine gels are useful for separating proteins with molecular weights of 10 kDa to 300 kDa, and Tris-tricine gels are useful for separating peptides with molecular weights of 2 kDa to 20 kDa. The presence or absence of final proteins is confirmed by Coomassie staining for proteins / peptides separated by SDS-PAGE. Recently, the amount of protein separated by PAGE can be quantified using a gel imaging program, but this is difficult to apply as an accurate method for analyzing protein amount due to band separation and band saturation in the staining method. The presence or absence of peptides with a molecular weight of 2 kDa or less can be confirmed by LC-MS analysis.

[0009] 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 human placenta. To date, mass spectrometry (ESI-MS / MS; Electrospray Ionization) is usually used to determine the sequence of known peptides. The proteins or peptides present in the placental hydrolysate can be confirmed and analyzed by the above method. Finally, the presence or absence of proteins or peptides contained in the placental hydrolysate is confirmed by SDS-PAGE, and the presence and content of low molecular weight peptides is analyzed by LC-MS, so that all proteins and peptides contained in the placental hydrolysate can be confirmed.

[0010] It is believed that the placenta contains 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 for analyzing the mineral content in powdered milk, health functional foods, and pharmaceuticals. A typical method, referring to the general test method in the Korean Pharmacopoeia, is to prepare standard solutions by concentration, obtain a calibration curve with absorbance values ​​corresponding to each concentration, and read the absorbance value of the sample on the calibration curve graph to confirm the content of the mineral component in the sample.

[0011] Generally, ion exchange chromatography is used in the process of purifying amino acids, peptides, or compositions containing proteins. Proteins and peptides are composed of amino acids that are zwitterions, so the net charge of proteins and peptides changes depending on the pH of the surrounding environment, but ions and polar substances are separated by using the difference in pI (isoelectric point) that makes them neutral. This separation process is called ion exchange chromatography, and is classified into chromatography using anion exchangers and cation exchangers based on the surface charge of the proteins to be separated. In the case of anion exchange chromatography, proteins with high isoelectric points bind relatively weakly to the column and can be eluted even at low salt concentrations, so proteins with high isoelectric points start eluting from the column first. The ion exchanger used in ion exchange chromatography, i.e., the type and equilibrium conditions of the ion exchange resin, the loading conditions of the sample and the washing procedure, or the pH gradient range of the eluent, and the type and elution conditions of the buffer, determine the elution rate. The resulting product will have different content.

[0012] The inventors of the present invention established optimal conditions for ion exchange chromatography in the process of processing 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. Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a method for producing a placental hydrolysate that contains a large amount of amino acids and oligopeptides that can exhibit pharmacological activity. [Means for solving the problem]

[0014] 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.

[0015] 2. The method for producing a placenta hydrolysate according to item 1 above, wherein the defatting is carried out by adding an organic solvent selected from the group consisting of acetone, alcohols, and esters to the placenta disruption.

[0016] 3. A method for producing a placenta hydrolysate according to item 1 above, wherein the defatting is carried out by adding one of acetone, an alcohol, or an ester-containing organic solvent to the placenta disruption material and defatting for 2 to 8 hours, and drying the material at 60 to 100°C for 10 to 24 hours to remove the organic solvent.

[0017] 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.

[0018] 5. The method for producing a placenta hydrolysate according to item 1 above, wherein the proteolytic enzyme is papain, pepsin, bromelain, pronase, or alcalase.

[0019] 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%.

[0020] 7. The method for producing a placenta hydrolysate according to item 1, wherein during the enzymatic hydrolysis, 10 kg to 25 kg of the defatted powder of crushed placenta is treated with 0.5 kg to 3 kg of a proteolytic enzyme.

[0021] 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 hydrolyzate or by passing the hydrolyzate through an anion exchange resin column.

[0022] 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. Effect of the Invention

[0023] The present invention relates to a method for producing placenta powder from placenta, a method for producing a placenta hydrolysate from placenta powder, and a method for producing a placenta 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. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows the results of SDS-PAGE protein separation after TCA precipitation concentration of the placental hydrolysate of the present invention. [Diagram 2] FIG. 2 shows the results of SDS-PAGE protein separation after Speedvac concentration of a placental hydrolysate of the present invention. [Diagram 3] FIG. 3 shows the results of peptide separation by Tris-tricine gel electrophoresis after Speedvac concentration of the placental hydrolysate of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention will be described in detail below.

[0026] The present invention relates to a method for producing a placental hydrolysate.

[0027] The method of the present invention includes the steps of defatting and drying crushed animal placenta to obtain crushed animal placenta powder, treating the defatted powder of crushed animal placenta with an acidic solution of a proteolytic enzyme to enzymatically hydrolyze the product, and contacting the hydrolyzed product with an anion exchange resin to perform ion exchange.

[0028] The placenta may be a mammalian placenta, including a human placenta, and in particular a human placenta.

[0029] For the placenta, chorionic or umbilical tissue can be used.

[0030] The defatting can be carried out 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.

[0031] The solvent can be added in an amount of, for example, 2 to 20 v / w times or 4 to 12 v / w times the amount of the placenta disruption product, but is not limited thereto. When the amount of the solvent is within the above range, a sufficient degreasing effect is exhibited and subsequent solvent removal is easy.

[0032] The 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 cannot be obtained, and if the stirring time is too long, the total process time becomes long, which is inefficient.

[0033] The degreasing can be carried out by adding one of acetone, alcohol, or ester-containing organic solvents to the placenta disruption material 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.

[0034] The degreasing can be carried out multiple times from the viewpoint of completely removing 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 in multiple degreasing can be carried out for the time exemplified above, but is not limited thereto.

[0035] When degreasing is performed multiple times, the next degreasing time can be longer than the previous degreasing time, for example, the second degreasing time can be longer than the first degreasing time.

[0036] After degreasing, the precipitating solvent is removed, which can be done by centrifugation.

[0037] If degreasing is performed multiple times, a solvent removal step can be performed between each degreasing step.

[0038] The drying temperature may be, for example, 60° C. to 100° C. The temperature 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.

[0039] 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, or 24 hours.

[0040] Thereafter, the defatted powder of the crushed animal placenta is treated with an acid solution of a protease to carry out enzymatic degradation.

[0041] As the proteolytic enzyme, for example, papain, pepsin, bromelain, pronase or alcalase can be used.

[0042] 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.

[0043] The acid concentration of the acid solution may be 0.1 to 10%, and the content thereof can be used in an amount of 0.2 to 20 times (W / W) the dry content of the placenta powder.

[0044] 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 is not performed sufficiently, which causes a problem of the hydrolysis time being extended and the process time being unnecessarily extended. On the other hand, if the content of the protease is too high, the molecular weight of the placenta component is reduced by hydrolysis, which may cause a problem of the desired effect not being fully exerted.

[0045] 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.

[0046] Thereafter, the decomposition product is brought into contact with an anion exchange resin to carry out ion exchange.

[0047] The treatment with an anion exchange resin can be carried out by adding an anion exchange resin to the hydrolyzed liquid or by passing the hydrolyzed liquid through an anion exchange column.

[0048] This can then be followed by filtration, pH adjustment and heat sterilization.

[0049] This is a process in which the temperature of the hydrolysate solution is raised to inactivate the enzyme, and the residue is removed by filtration. The temperature at this time may be 70°C to 100°C.

[0050] 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, for example, to a range of 5.5 to 7.5. This is because it is preferable for the composition of the present invention to have the above pH range in order to use it as an injection or for oral administration. The concentration and liquid volume 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. The preservative can be a conventional one within the range acceptable for the composition of the present invention (e.g., pharmaceutical composition or food composition).

[0051] The sterilization process can be preferably carried out at 100 to 140° C. for 20 to 120 minutes.

[0052] 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 therefore can be used as a raw material for functional health foods.

[0053] The health functional food produced from the hydrolysate can help improve liver function.

[0054] 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 Health Functional Foods Act, Korea Act No. 7428). And "function" or "functionality" refers to obtaining beneficial effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. In other words, it means that it can be used for health purposes in healthy or unhealthy people.

[0055] The dry content of the 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 is not fully expressed, and the dosage is increased, which is inconvenient to administer. If the content is too high, problems arise in solubility and product formulation.

[0056] For ease of administration, the placental hydrolysate-containing health functional food is preferably used as a functional food having a formulation such as a tablet, sugar-coated tablet, capsule, or drink.

[0057] Working Example 1. Manufacturing of placenta powder 200 kg of frozen human placenta was washed, thawed and crushed to obtain placenta homogenate.

[0058] The obtained placenta disruption was added with a degreasing solvent and stirred to defat the mixture. The degreasing solvent may be acetone, alcohol or ester.

[0059] The delipidation solvent was used in a volume (v / w) that was 4 to 12 times the placenta disruptant content and stirred for 2 to 8 hours.

[0060] 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 2 to 24 hours. The solvent was then removed and the mixture was dried to obtain a placenta disruption powder.

[0061] 2. Production of placental hydrolysate The placenta disruption powder was subjected to acid hydrolysis by adding an acid solution, in which a protease was also used.

[0062] As acids hydrochloric acid, nitric acid or acetic acid could be used, and as proteolytic enzymes papain, pepsin, bromelain, pronase or alcalase were used.

[0063] In this step, 0.5 kg to 3 kg of proteolytic enzyme was used for 10 to 25 kg of placenta powder.

[0064] Acids were used in the concentration range of 0.1–10%, and their contents were 0.2–20 times (W / W) the dry content of placenta powder.

[0065] Hydrolysis was continued until no further protein reaction occurred via the Biuret reaction. After hydrolysis, an ion exchange process was performed.

[0066] Ion exchange was carried out using anion exchange chromatography or by adding an ion exchange resin to the resulting hydrolysate.

[0067] Specific examples using anion exchange chromatography are as follows:

[0068] (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 onto a Source Q (XK50 / 25, 500 ml) column equilibrated with 20 mM Tris (pH 7.5) buffer at a flow rate of 12 ml / min for binding, and then the placental hydrolysate was eluted using a linear concentration gradient of 18 column volumes (sodium chloride concentration 0 M → 1 M).

[0069] (2) Anion exchange example 2 The fraction obtained in the previous step was adjusted to pH 5.8 with 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 solution 2 (20 mM sodium acetate, pH 5.8), and the placental hydrolysate was eluted by sequentially increasing the concentration of sodium chloride in 10 mM increments.

[0070] (3) Anion Exchange Example 3 Generally, the matrix of the resin used to purify the mixture is a mixture of polystyrene or polyacrylate with DVB (Divinylbenzene) and, in the case of a cation exchange resin, a sulfonate (-SO) is added to the functional group. 3- ), and in the case of anion exchange resin, the exchange group is -N3(CH3)3Cl - (trimethylammonium), -N + (CH3)2C2H4OHCl - (dimethylethanolammonium), -(CH2) n N(CH3)2(Tertiary Amine), or -CH2NH(CH2CH2NH) n A resin called H(CH2)2NH2 (Secondary Amine) is used.

[0071] After that, filtration, pH adjustment, and heat sterilization were performed.

[0072] The process involves increasing the temperature of the hydrolysate solution to inactivate the enzymes and filtering out any residues. The temperature at this time was 70° C. to 100° C. Thereafter, the hydrolysate solution was concentrated and filtered.

[0073] 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 the above pH range in order to use it as an injection or for oral administration. The concentration and volume of the composition obtained by the above step can be adjusted by adding purified water. This process is carried out by heat treatment, and it is preferable to add a preservative such as sodium benzoate in a high-temperature, high-pressure sterilizer and heat sterilize the composition. The preservative can be a conventional one within the range acceptable for the pharmaceutical or food composition of the present invention.

[0074] In the sterilization process, heat sterilization is preferably performed at 100 to 140° C. for 20 to 120 minutes.

[0075] Experimental Example 1. Confirmation of the presence of proteins and peptides in placental hydrolysates Proteins and peptides in placental hydrolysate samples 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, but these two concentration methods were used to confirm trace amounts of proteins or peptides present in the placental hydrolysate samples of the present invention. The concentrated placental hydrolysate samples were separated into proteins and peptides using Tris-glycine gel for protein separation and Tris-tricine for peptide separation, respectively.

[0076] 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 numbers for distinguishing samples from different production lots.

[0077] A band pattern different from the usual protein migration pattern was observed. To confirm the presence or absence of protein, the band was excised, subjected to in-gel digestion, and then subjected to MALDI-TOF / TOF analysis.

[0078] 1.2 Speedvac concentration FIG. 2 shows the results of SDS-PAGE analysis of placental hydrolysate samples of the present invention concentrated using a Speedvac system.

[0079] When 10 μL of the placental hydrolysate sample was loaded, a molecular weight of 7 kDa was confirmed, while the 50 μL loaded sample and the 100 μL loaded sample had molecular weights of approximately 15 kDa and 20 kDa, respectively (see FIG. 2).

[0080] Usually, this can be confirmed by SDS-PAGE as a band of a specific molecular weight, and the more the amount of sample loaded, the thicker the band becomes at the same molecular weight. The concentrated sample c 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.

[0081] 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 with a Speedvac system are shown in FIG.

[0082] 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).

[0083] 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 were excised and subjected to in-gel digestion and MALDI-TOF / TOF analysis.

[0084] 2. Analysis of peptide sequences present in placental hydrolysates 2.1 Peptide sequence analysis using LC-MS / MS In the above 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 could not be confirmed by the above method. Therefore, in this Example, LC-MS analysis was performed. 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.

[0085] As a result of analyzing more than 100 peptides, all of them were found to have a molecular weight of 1.5 kDa or less. Peptides with a molecular weight of 5 amino acids or less (5mer) to 2 mers or more are summarized in Table 1.

[0086] [Table 1]

[0087] 2.2 Comparison of peptide content using the 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.

[0088] The original solution of placental hydrolysate and the control sample were compared based on the same peptide content. The EIC analysis was performed three times on the same sample to confirm the reproducibility of the EIC analysis method. The average total area of ​​each peptide and the standard deviation confirmed by the three repeated tests were within ±3%.

[0089] When the peptide contents of the two samples were compared, 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 a content of 1% or more, peptides contained at a content of 0.5% to less than 1%, and peptides contained at a content of less than 0.5% of the total peptides in the placental hydrolysate of the present invention.

[0090] Experimental Example 3. Analysis of mineral types and content The results of analyzing the minerals in the placental hydrolysate are shown in Table 2. The analysis of minerals was performed according to the general test methods in the Korean Pharmacopoeia, and the plasma concentrations in healthy adults are shown as reference values.

[0091] [Table 2]

Claims

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 placental hydrolysate according to claim 1, wherein the defatting is carried out by adding an organic solvent selected from the group consisting of acetone, alcohols and esters to the placenta disruption.

3. 2. The method of claim 1, wherein the defatting comprises the steps of adding any one of acetone, alcohol, and ester-containing organic solvents to the placenta disruptant and defatting for 2 to 8 hours, and drying at 60 to 100° C. for 10 to 24 hours to remove the organic solvent.

4. 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.

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

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

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

8. 2. 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 hydrolyzate or by passing the hydrolyzate through an anion exchange resin column.

9. 2. The method of claim 1, further comprising the step of adding a base to the ion exchanged hydrolysate to adjust the pH.

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