Method for producing fermented placenta extract

Abstract

To provide a novel production method for fermented placenta extract having predetermined efficacy.SOLUTION: A production method for fermented placenta extract includes: a fermentation step for fermenting placenta using the koji mold Aspergillus luchuensis; an extraction step for extracting a fermented placenta extract from the fermented product obtained through the fermentation step, in the presence of water at 30-50°C; and a separation step for separating the fermented placenta extract obtained in the extraction step from the fermented product.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a fermented placenta extract, etc. [Background technology]

[0002] Placenta is the placenta of mammals. Since placenta contains many kinds of proteins and nutritional components such as vitamins, placenta and placenta extracts obtained by extracting active ingredients from placenta have been attracting attention in the fields of cosmetics and health foods. Various methods for producing placenta extracts have been reported (for example, the method for producing placenta extracts by subcritical extraction described in Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2017-100998 A [Patent Document 2] International Publication No. 2014 / 181769 Summary of the Invention [Problem to be solved by the invention]

[0004] By the way, hyaluronic acid is known as a high molecular weight polysaccharide with high water retention. In recent years, the promotion of hyaluronic acid production has been attracting attention, for example, in the field of cosmetics, from the viewpoint of improving wrinkles and sagging skin. In addition to this manufacturing method, it is required to establish a manufacturing method for placenta extract that has a certain efficacy, for example, the ability to promote the production of hyaluronic acid.

[0005] An object of the present invention is to provide a new method for producing a fermented placenta extract having a predetermined efficacy. [Means for solving the problem]

[0006] (1) In order to achieve the above object, a method for producing a fermented placenta extract according to one embodiment comprises the steps of: A fermentation process in which the placenta is fermented using Aspergillus luchuensis. An extraction step of extracting a fermented placenta extract from the fermented product obtained by the fermentation step in the presence of water at 30 to 50°C; A separation step of separating the fermented placenta extract obtained in the extraction step from the fermentation treatment product; Includes. (2) In the method for producing a fermented placenta extract according to another embodiment, preferably, the molecular weight of the protein contained in the fermented placenta extract may be 240,000 or less. (3) In the method for producing a fermented placenta extract according to another embodiment, the extraction step may be preferably carried out at a pH in the range of 2.0 to 6.5. (4) In the method for producing a fermented placenta extract according to another embodiment, preferably, the placenta is derived from a pig. (5) In the method for producing a fermented placenta extract according to another embodiment, preferably, the placenta is a dried placenta. Effect of the Invention

[0007] According to the present invention, for example, a new method for producing a fermented placenta extract having a predetermined efficacy can be provided. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows a flow diagram of the method for producing a fermented placenta extract according to this embodiment. [Diagram 2] FIG. 2 shows the results of the HPLC analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Next, an embodiment of the present invention will be described with reference to the drawings. Note that the embodiment described below does not limit the invention according to the claims, and all of the elements and combinations thereof described in the embodiment are not necessarily essential to the solution of the present invention.

[0010] 1.Definition (Acorbiculatus) The koji fungus used in the present invention is Aspergillus luchuensis (hereinafter, sometimes simply referred to as "koji fungus"). As an example of Aspergillus luchuensis, Aspergillus luchuensis NBRC 4314 is also called black koji fungus, and is a filamentous fungus that has been traditionally used in brewing Okinawan awamori (The scientific name of black koji fungus has become Aspergillus luchuensis, Yamada Osamu, Vol. 110, No. 2, Jyokyo 2015, p. 64-67). Aspergillus luchuensis is known to produce decomposition enzymes such as amylase, cellulase, and protease, as well as a variety of metabolites. The strain of koji fungus used in this embodiment is not particularly limited as long as it is possible to carry out the present invention, and may include, for example, a mutant strain such as Aspergillus luchuensis mut.kawachii.

[0011] (Placenta) The placenta used in the present invention is a placenta of a mammal. The mammal is not particularly limited, but may be, for example, a pig, a cow, a horse, a human, a sheep, etc., but from the viewpoint of traceability, animals that are listed as livestock (for example, a pig, a cow, a horse, a sheep) are preferred.

[0012] (Fermented product) In this specification, the "fermentation product" may include placenta after the fermentation step described below, Aspergillus oryzae fungus bodies, placenta, metabolic products by Aspergillus oryzae, etc. The form of the fermentation product is not particularly limited, but is mainly solid.

[0013] (Fermented placenta extract) In this specification, the "fermented placenta extract" is a mixture containing placenta-derived proteins, peptides, amino acids, various vitamins, metabolic products by Aspergillus oryzae, etc. The fermented placenta extract can be made into a powder by removing water by spray drying, freeze drying, etc.

[0014] The fermented placenta extract produced by the production method according to the present embodiment described below (production method for a fermented placenta extract) has, for example, a hyaluronic acid production promoting activity.

[0015] 2. Manufacturing method of fermented placenta extract Next, the method for producing the fermented placenta extract according to this embodiment will be described with reference to Fig. 1. Fig. 1 shows a flow diagram of the method for producing the fermented placenta extract according to this embodiment.

[0016] (1) Pretreatment process (S50) The pretreatment step (S50) is a step for treating the placenta in order to smoothly proceed with fermentation in the fermentation step described later. The pretreatment step is not an essential step in the method for producing a fermented placenta extract according to this embodiment, but is a step that is preferably performed from the viewpoint of performing the fermentation step more effectively. As an example, the pretreatment step may include a water absorption process in which water is added to the placenta to allow it to absorb water, a steaming process in which the absorbed placenta is steamed, and a cooling process of the steamed placenta. The water absorption process, the steaming process, and the cooling process will be described below.

[0017] The placenta used in this step is preferably a dried product obtained by a freeze-drying method or the like, from the viewpoint of making the processing easier for workers.

[0018] The water added to the placenta in the water absorption treatment may contain yeast extract etc. from the viewpoint of promoting the growth of the Aspergillus oryzae to be inoculated in the fermentation process, although this is not essential. The standing time for water absorption is preferably 1 to 24 hours, more preferably 2 to 18 hours.

[0019] The water content may be within a range suitable for inoculating the Aspergillus oryzae, and is preferably 25 to 50% by mass, more preferably 30 to 45% by mass, and more preferably 35 to 40% by mass, based on the total mass of the placenta, water, and other additives.

[0020] The steaming treatment is a treatment for facilitating the fermentation of the placenta in the fermentation step. Various known methods can be used for the steaming method, and for example, steaming under normal pressure is preferred. The steam discharge pressure in the steamer is preferably 0.01 to 0.09 MPa, more preferably 0.02 to 0.06 MPa, and even more preferably 0.02 to 0.05 MPa. The temperature of the steam during steaming is preferably 90 to 100°C, more preferably 95 to 100°C, and even more preferably 98 to 100°C.

[0021] The cooling treatment (also called the cooling treatment) is a treatment for cooling the placenta after the steaming process. The placenta after the steaming process is cooled to preferably 25 to 45°C, more preferably 28 to 40°C, and even more preferably 30 to 35°C by this cooling treatment.

[0022] (2) Fermentation process (S100) The fermentation step (S100) is a step of fermenting the placenta using the Aspergillus luchuensis fungus to obtain a fermented product. The fermentation step includes an inoculation treatment of inoculating the placenta with the Aspergillus fungus, and a fermentation treatment of fermenting the inoculated placenta.

[0023] The amount of the inoculated Aspergillus oryzae is preferably 0.10 to 0.30% by mass, more preferably 0.15 to 0.25% by mass, and even more preferably 0.15 to 0.20% by mass, based on the total mass of the placenta and the like present in the fermentation process.

[0024] The temperature of the water in the fermentation treatment is preferably 25 to 45°C, more preferably 28 to 42°C, and even more preferably 30 to 40°C.

[0025] The humidity in the space where the fermentation treatment is carried out is preferably 50 to 100%, more preferably 55 to 100%, and even more preferably 60 to 100%.

[0026] The carbon dioxide concentration in the space where the fermentation treatment is carried out is preferably 15% or less. There are no particular restrictions on the fermentation time, but it is preferably 24 to 72 hours.

[0027] Although not essential, the fermented product obtained in the fermentation step may be subjected to a drying treatment, for example, to reduce the moisture content and improve the storage stability until the extraction step is performed. The drying treatment is performed, for example, by blowing hot air onto the fermented product to evaporate the moisture contained in the fermented product (hot air drying).

[0028] In the present invention, the drying process may not be performed, for example, by performing the extraction process immediately after the fermentation process.

[0029] (3) Extraction process (S200) The extraction process (S200) is a process of extracting a fermented placenta extract (e.g., a fermented placenta extract liquid) from the fermentation product that has undergone the above-mentioned fermentation process while causing an enzymatic reaction by endogenous enzymes (such as proteases) in the fermentation product in the presence of water at 30 to 50°C. The amount of water used in this extraction step is not particularly limited as long as it is an amount that can extract the fermented placenta extract. The amount of water is preferably 1 to 50 times, more preferably 2 to 20 times, and even more preferably 5 to 15 times the mass of the fermented product.

[0030] The water used in the extraction step may or may not include the water used in the fermentation step. The amount of water used in the extraction step is preferably greater than the amount of water used in the fermentation step.

[0031] The temperature of water in the extraction step is an optimum temperature from the viewpoint of carrying out an enzymatic reaction by endogenous enzymes (proteases, etc.) in the fermentation product, and is preferably 30 to 50° C., more preferably 35 to 45° C. During extraction, it is preferable to stir while maintaining the temperature at preferably 30 to 50° C. (more preferably 35 to 45° C.).

[0032] The extraction time is not particularly limited as long as the fermented placenta extract can be extracted. For example, the extraction time is preferably 2.0 to 72 hours, more preferably 2.5 to 48 hours, more preferably 3 to 36 hours, and even more preferably 3.5 to 24 hours.

[0033] In the extraction step, the pH of the mixture of the fermentation product and water may be adjusted before extraction. The pH value during extraction is preferably 2.0 to 7.0, more preferably 2.5 to 6.5, and even more preferably 3.0 to 6.0. When the pH value is equal to or lower than the upper limit, alkaline denaturation of enzymes (proteases, etc.) is unlikely to occur, and the risk of the fermentation being hindered is reduced. On the other hand, when the pH value is lower than the lower limit, the acidic condition may be too strong and the reaction by the enzyme may not occur.

[0034] Between the extraction step and the separation step described below, sterilization using a method such as heating may be carried out, although this is not essential.

[0035] (4) Separation process (S300) The separation step (S300) is a step of separating the fermented placenta extract obtained through the above extraction step from the fermentation product. More specifically, in this step, for example, when the fermented placenta extract is a liquid (fermented placenta extract liquid), the aqueous solution of the fermented placenta extract is separated from the fermentation product. Through this step, for example, the placenta residue and most or all of the Aspergillus oryzae fungus bodies are removed.

[0036] The separation method used in the separation step is not particularly limited as long as it can separate the fermented placenta extract from the fermentation treatment product. Examples of the separation method include filtration, centrifugation, membrane treatment, etc. The separation step can be carried out by using one or more of the above separation methods in combination.

[0037] For the filtration, for example, filter separation using filter paper, a metal filter such as stainless steel, etc., can be adopted. In addition, in the filtration using filter paper, for example, a filter aid may be precoated on the filter paper. Examples of the filter aid include diatomaceous earth, cellulose, and combinations thereof. The amount of the aid used can be appropriately selected. In addition, filtration methods such as pressure filtration and suction filtration can also be adopted.

[0038] (others) When the fermented placenta extract is to be made into a powder, the fermented placenta extract obtained in the separation step may be optionally subjected to spray drying, freeze drying, or the like after the separation step.

[0039] The molecular weight of the protein contained in the fermented placenta extract after the separation step is, for example, 450,000 or less, more preferably 240,000. EXAMPLES

[0040] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. In the following examples, "%" means "% by mass".

[0041] (Production Example) Production of Fermented Placenta Extract The following Table 1 shows the raw materials used in the pretreatment and fermentation processes and their respective content ratios. In the actual manufacturing process, 2986.8 g of placenta, 1826.3 g of water, 48.7 g of yeast extract RK (Ajinomoto Co., Inc.), and 8.2 g of authentic shochu bacteria rice powder (Aspergillus luchuensis seed koji) (Higuchi Matsunosuke Shoten Co., Ltd.) were used.

[0042] [Table 1]

[0043] (1) Pretreatment process A yeast extract solution was prepared by dissolving yeast extract in water. The yeast extract solution was added to the dried placenta. The placenta after the addition was left to stand for 2 to 16 hours. Thereafter, the placenta was steamed for 60 minutes under conditions of a steam discharge pressure of 0.02 to 0.04 MPa while maintaining a temperature of 98°C or higher using a steamer. The steamed placenta was allowed to cool (cool) until it reached 25 to 40°C.

[0044] (2) Fermentation process The placenta was then inoculated with Aspergillus luchuensis koji mold. After the inoculation, the placenta was cultured for 36 to 48 hours under conditions of a temperature of 32 to 38°C, a humidity of 60 to 100%, and a carbon dioxide concentration of 10% or less, to ferment the placenta. After the fermentation, the fermented product was dried with hot air until the moisture content was 10% or less.

[0045] (3) Extraction process 100 mL of water was added to 10 g of the fermented product to obtain a mixture of the fermented product and water. The pH value of the mixture was about 5.8 in an unadjusted state. The mixture was stirred at 35 to 45°C for 2.0 to 4.5 hours. Then, heat sterilization was performed at 90°C for 30 minutes.

[0046] In the extraction step, when pH adjustment was performed, a 90% aqueous lactic acid solution or a 25% aqueous citric acid solution was added to the mixture before heating and stirring to adjust the pH to about 3.0.

[0047] (4) Separation process The mixture after heat sterilization was filtered through filter paper using diatomaceous earth as an auxiliary agent. The filtrate was collected. The collected filtrate was treated with a 0.45 μm membrane filter (manufactured by ADVANTEC) to obtain a fermented placenta extract.

[0048] The fermentation placenta extract or fermentation placenta extract product described in Table 2 below was used in each of the following test examples.

[0049]

Table 2

[0050] (Test Example 1) HPLC Analysis of Fermentation Placenta Extract The fermentation placenta extract obtained in the above Production Example was diluted with purified water to make a 1 mL solution (measurement sample). Then, under the following HPLC conditions, the molecular weight distribution of proteins and the like contained in the measurement sample was evaluated.

[0051] <HPLC Conditions> Column: Superdex 200 Increase 10 / 300 GL (manufactured by GE Healthcare) Column temperature: 40 °C Sample injection volume: 10 μL Mobile phase: Tris-HCl buffer (pH 7.0) Flow rate: 0.5 mL / min Detector: UV detector (SPD-20A manufactured by Shimadzu Corporation) Detection wavelength: 214 nm

[0052] (Results) Figure 2 shows the results of the HPLC analysis. The arrow in Figure 2 indicates a peak with a molecular weight of approximately 240,000. The retention time of the compound at this peak was around 22 - 24 minutes. As shown in Figure 2, the peaks obtained by HPLC analysis were broad. From this result, it was suggested that the fermentation placenta extract contains proteins and the like with various molecular weights.

[0053] (Test Example 2) Human Fibroblast Proliferation Test 200 μL of Dulbecco's modified Eagle medium (hereinafter referred to as DMEM) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 5% fetal bovine serum (hereinafter referred to as FBS) (manufactured by Thermo Trace) was added to each well of a 96-well plate. Normal human dermal fibroblasts (KF-4009, Kurabo) were added at 4 × 10 3 The cells were seeded in each well so that the number of cells was 100 / well. After seeding, the cells were cultured for 24 hours in an environment of 37°C and 5% CO2. After the 24-hour culture, the DMEM containing 5% FBS was replaced with DMEM containing 0.25% FBS in each well except for the well for the positive control group. After the replacement, the fermented placenta extract was added to the medium in each well so that the composition was as shown in Table 3 below. After the addition, the cells were cultured for 3 days in an environment of 37°C and 5% CO2. After the 3-day culture, the number of normal human dermal fibroblasts was measured using Cell Counting Kit-8 (CK04, DOJINDO). The measurement was performed according to the instruction manual attached to the Cell Counting Kit-8.

[0054] [Table 3]

[0055] (result) Table 3 shows the results of the human fibroblast proliferation test. The measurement results in Table 3 are relative values ​​calculated by calculating the average value of the measurements in each group (n=8) and setting the value of the negative control group to 1.00. * in Table 3 indicates a significant difference of p<0.001 compared to the negative control group in a Student's t-test of n=8. The results show that the addition of fermented placenta extract exerted a proliferation effect on human fibroblasts, similar to the positive control group.

[0056] (Test Example 3) Hyaluronic acid production promoting activity test 1000 μL of DMEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 5% FBS (manufactured by Thermo Trace) was added to each well of a 24-well plate. Normal human dermal fibroblasts (KF-4009, Kurabo) were added at 2 × 10 4 The cells were seeded in each well so that the number of cells was 1 / well. After seeding, the cells were cultured at 37°C and 5% CO2 until confluent. Then, the DMEM containing 5% FBS in each well was replaced with DMEM containing 0.25% FBS. The fermented placenta extract was added to the medium in each well so that the composition was as shown in Table 4 below. After addition, the cells were cultured for 3 days at 37°C and 5% CO2. 500 μL of cell culture supernatant was taken from the cells after culture. The supernatant was diluted 10 times with purified water. The amount of hyaluronic acid produced (ng / mL) contained in the diluted supernatant was calculated using Hyaluronan Quantification Kit (HA-96KIT, Cosmo Bio). The calculation was performed according to the instruction manual attached to the Hyaluronan Quantification Kit.

[0057] The calculated value of the amount of hyaluronic acid produced S was then substituted into the following formula 1 to calculate the amount of hyaluronic acid produced per cell X. X=(S / R)×10...(Formula 1) (X: amount of hyaluronic acid produced per cell, S: amount of hyaluronic acid produced in the diluted supernatant, R: percentage of cell number.)

[0058] [Table 4]

[0059] (result) Table 4 shows the results of the hyaluronic acid production promotion activity test. The measurement results in Table 4 show the average amount of hyaluronic acid produced (X) per cell in each group (n=4). * in Table 4 indicates a significant difference of p<0.01 compared to the control group in a Student's t-test of n=4. The results show that the addition of fermented placenta extract promotes hyaluronic acid production.

Claims

1. a fermentation step of fermenting the placenta using the koji fungus Aspergillus luchuensis; an extraction step of extracting a fermented placenta extract from the fermented product obtained by the fermentation step in the presence of water at 30 to 50°C; A separation step of separating the fermented placenta extract obtained in the extraction step from the fermentation treatment product; A method for producing a fermented placenta extract comprising the steps of:

2. 2. The method for producing a fermented placenta extract according to claim 1, wherein the molecular weight of the protein contained in the fermented placenta extract is 240,000 or less.

3. The method for producing a fermented placenta extract according to claim 1, wherein the extraction step is carried out at a pH in the range of 2.0 to 6.

5.

4. The method for producing a fermented placenta extract according to claim 1, wherein the placenta is derived from a pig.

5. The method for producing a fermented placenta extract according to claim 1, wherein the placenta is a dried placenta.

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