Cell preservative, method for producing cell preservative, cell-cultured food, and method for preserving cell-cultured food

Abstract

To provide a cell preservative that can suppress deterioration of a cell-cultured food which contains cultured cells.SOLUTION: A cell preservative contains an extract of tea leaf residue. The tea leaf residue is preferably a residue remaining after tea leaves are subjected to hot-water extraction treatment. The protein content in the extract is preferably 5 wt.% or more. Furthermore, the catechin content in the extract is preferably less than 2 ppm.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell-cultured food, and more particularly to a cell preservative for inhibiting deterioration of a cell-cultured food. [Background technology]

[0002] In recent years, technological developments related to the production of cell-cultured foods such as cultured meat have progressed. Cell-cultured foods are not only expected to help resolve issues such as food shortages and food waste, but also to reduce livestock production and contribute to preventing global warming. On the other hand, the technology to preserve the quality of cell-cultured foods has not yet been fully developed, and the general understanding is that the quality of cell-cultured foods can be maintained by keeping them in a sterile environment. For example, it is believed that by packaging cell-cultured foods in a sterile environment to prevent quality deterioration caused by microorganisms, the quality of the cell-cultured foods can be maintained without additional sterilization treatment, the addition of food additives, or freezing. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2023-519478 [Patent Document 2] Special Publication No. 2022-513441 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-50356 [Patent Document 4] Japanese Patent Publication No. 2020-5517 Summary of the Invention [Problem to be solved by the invention]

[0004] However, cell-cultured foods containing muscle cells have the problem that the muscle cells break down proteins present in the cell-cultured foods in order to secure a source of nutrients, which can affect the maintenance of viable cells in the cell-cultured foods and result in a decrease in cell number. Furthermore, when cell-cultured foods are stored in a low-temperature environment, gelation inside the cells can cause the lipid bilayer on the cell surface to separate, destroying the surface, dissolving the cytoplasm and reducing the number of cells. In such cases, the cell-cultured food may end up with an unintended texture or taste.

[0005] Therefore, the present inventors conducted extensive research and succeeded in developing a cell preservative that can solve the above problems, thereby completing the present invention. Specifically, we selected materials that are rich in protein and branched-chain amino acids (valine, leucine, isoleucine), methionine, and lysine, which suppress the expression of protein degradation pathways.

[0006] We also selected materials containing proteins that can suppress the loss of viable cells in cell-cultured foods. For example, bovine serum albumin has the ability to bind to the cell surface, effectively covering the entire cell surface and suppressing swelling, and has been reported to be effective in preventing cytoplasmic elution when cells are stored in a low-temperature environment.

[0007] The inventors then discovered that the residue left after hot water extraction of tea leaves, many of which are disposed of as waste, is suitable as the above-mentioned material, and completed the cell preservative of the present invention. Tea leaves contain catechins such as epigallocatechin, which are cytotoxic, so in order to prevent the loss of viable cells in cell-cultured foods using tea leaves as a material, it was necessary to reduce the amount of catechins in the cell preservative of the present invention.

[0008] Here, Patent Document 1 describes that because the processes of packaging and sealing the cultured tissue are carried out under aseptic conditions, no additional sterilization procedures or preservative additives are required, making it possible to produce packaged cultured meat with a long shelf life. Furthermore, Patent Document 2 describes edible foods that use cultured cells, stating that the shelf life of the edible foods can be significantly extended because they are grown in a completely sterile environment, and that they are free of bacteria and do not require freezing due to their aseptic packaging. However, these patent documents did not describe or suggest a cell preservative made from tea leaves that can suppress the decrease in viable cells in cell-cultured foods.

[0009] Furthermore, Patent Document 3 describes an agent for inducing differentiation of stem cells into ectodermal cells using a black tea extract. Furthermore, Patent Document 4 describes a cell culture medium using black tea infusion extract. However, these patent documents do not describe or suggest any cell preservatives that can suppress the decrease of viable cells in cell-cultured foods.

[0010] The present invention has been made in consideration of the above circumstances, and aims to provide a cell preservative that can suppress deterioration of a cell-cultured food containing cultured cells, a method for producing a cell preservative, a cell-cultured food, and a method for preserving a cell-cultured food. [Means for solving the problem]

[0011] In order to achieve the above object, the cell preservative of the present invention is configured to contain an extract of used tea leaves. It is also preferable that the cell preservative of the present invention be configured such that the used tea leaves are the residue remaining after tea leaves have been subjected to a hot water extraction treatment.

[0012] Furthermore, the cell preservative of the present invention is preferably configured so that the protein content in the extract is 5% by weight or more. It is also preferable that the cell preservative of the present invention is configured so that the content of catechins in the extract is less than 2 ppm.

[0013] The cell preservative of the present invention is also preferably configured to be used as a cell deterioration inhibitor. It is also preferable that the cell preservative of the present invention is used as an additive for cell-cultured foods. Furthermore, the cell preservative of the present invention is preferably composed of various combinations of the above-mentioned cell preservatives.

[0014] The method for producing the cell preservative of the present invention is a method for extracting used tea leaves and incorporating the obtained extract. Furthermore, in the method for producing a cell preservative of the present invention, the extraction treatment is preferably an alkali extraction treatment, an acid extraction treatment, or a subcritical water extraction treatment. Furthermore, the method for producing the cell preservative of the present invention is also preferably a method in which used tea leaves are stored at room temperature or in a refrigerator for one week or more and then subjected to the extraction treatment.

[0015] Furthermore, the cell-cultured food of the present invention is composed of a cell preservative containing an extract of used tea leaves and cultured cells. Furthermore, the method for preserving a cell-cultured food of the present invention is a method for preserving cultured cells by adding a cell preservative containing an extract of used tea leaves to the cultured cells. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a cell preservative that can suppress deterioration of a cell-cultured food containing cultured cells, a method for producing a cell preservative, a cell-cultured food, and a method for preserving a cell-cultured food. DETAILED DESCRIPTION OF THE INVENTION

[0017] Hereinafter, embodiments of the cell preservative, the method for producing the cell preservative, the cell-cultured food, and the method for preserving the cell-cultured food of the present invention will be described in detail, although the present invention is not limited to the specific details of the following embodiments and examples.

[0018] The cell preservative of the present invention is characterized by containing an extract of used tea leaves (hereinafter, sometimes referred to as used tea leaves extract). In addition, the cell preservative of the present invention is preferably made from used tea leaves, which are residues remaining after tea leaves are subjected to hot water extraction treatment.

[0019] The residue left after hot water extraction of tea leaves is generally treated as waste, although some of it is used as fertilizer or animal feed. In such a situation, the cell preservative of this embodiment makes it possible to suppress deterioration of cell culture foods containing cultured cells by using the residue (tea leaves) remaining after hot water extraction of tea leaves.

[0020] The hot water extraction treatment can be selected as appropriate, but for example, treatment can be carried out in heated water (pure water) at 70°C to 90°C for 2 minutes to 180 minutes. Furthermore, it is preferable to use as used tea leaves the residue obtained by drying after tea leaves are extracted with hot water (dried used tea leaves).

[0021] Furthermore, it is preferable to use as used tea leaves the residue after hot water extraction of tea leaves that has been stored at room temperature or in a refrigerator for at least one week. Tea leaves contain catechins such as epigallocatechin, and catechins are known to be cytotoxic. On the other hand, catechins contained in tea leaves are gradually removed from the tea leaves by oxidation, so the amount of catechins contained in used tea leaves can be reduced by storing the residue after hot water extraction at room temperature or in a refrigerator for a long period of time.

[0022] From this perspective, it is more preferable to use used tea leaves that have been stored at room temperature or in a refrigerator for more than one week after hot water extraction, even more preferable to use used tea leaves that have been stored at room temperature or in a refrigerator for more than one month, even more preferable to use used tea leaves that have been stored at room temperature or in a refrigerator for more than two months, and particularly preferable to use used tea leaves that have been stored at room temperature or in a refrigerator for more than three months.

[0023] Furthermore, in the cell preservative of the present invention, the content of catechins in the extract of used tea leaves is preferably less than 2 ppm. As described above, catechins are cytotoxic, and it is desirable that the cell preservative of this embodiment, which suppresses deterioration of cell culture foods containing cultured cells, does not contain catechins or contains very little of them.

[0024] Furthermore, in the cell preservative of this embodiment, the extract of used tea leaves is preferably obtained by alkaline extraction treatment or acid extraction treatment, and is also preferably obtained by subcritical water extraction treatment. That is, the extract of used tea leaves is preferably a protein that is water-insoluble and soluble in acid or alkali, and is also preferably a protein that is soluble in subcritical water. Furthermore, it is also preferable that the extract of used tea leaves is obtained by finely crushing used tea leaves and then extracting the same.

[0025] In the cell preservative of this embodiment, examples of the alkali used in the alkaline extraction treatment include sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, ammonium carbonate, lithium chloride, tripotassium phosphate, dipotassium hydrogen phosphate, trisodium phosphate, disodium hydrogen phosphate, sodium gluconate, potassium gluconate, and trisodium citrate. In the cell preservative of this embodiment, examples of the acid used in the acid extraction treatment include hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, and formic acid. Furthermore, in the case where subcritical water extraction treatment is used in the cell preservative of this embodiment, subcritical water is used for extraction.

[0026] Furthermore, in the cell preservative of the present invention, the protein content in the extract of used tea leaves is preferably 5% by weight or more. The cell preservative of the present invention is desirably rich in protein because it solves the problem that in cell culture foods containing muscle cells, muscle cells may decompose proteins present in the cell culture food in order to secure a source of nutrients, which may affect the maintenance of viable cells in the cell culture food and result in a decrease in cell number. From this perspective, it is more preferable that the protein content in the tea leaves extract of the cell preservative of the present invention is 5% by weight or more, even more preferably 10% by weight or more, even more preferably 15% by weight or more, and particularly preferably 20% by weight or more.

[0027] Here, the proteins in the extract of used tea leaves include albumin, globulin, glutelin, and prolamin, as shown in Table 1 below. Furthermore, proteins in the extract of used tea leaves that are water-insoluble and soluble in acid or alkali include globulin, glutelin, and prolamin, with glutelin and / or prolamin being preferred.

[0028] [Table 1]

[0029] The cell preservative of the present embodiment is preferably used as a cell deterioration inhibitor. Furthermore, the cell preservative of this embodiment is also preferably used as an additive for cell culture foods. The cell preservative of this embodiment can suitably suppress deterioration of a cell-cultured food product containing cultured cells.

[0030] The method for producing a cell preservative of this embodiment is characterized by carrying out an extraction treatment of used tea leaves and containing the obtained extract. In the method for producing the cell preservative of this embodiment, the extract of used tea leaves can be obtained specifically, for example, as follows.

[0031] The residue obtained after the hot water extraction of tea leaves is mixed with an aqueous solution of sodium hydroxide to dilute the mixture, and the mixture is stirred for a predetermined period of time using a stirrer. The resulting composition is then centrifuged and the supernatant is collected to obtain an extract of used tea leaves comprising the supernatant.

[0032] In addition, in the method for producing the cell preservative of this embodiment, the extraction treatment of used tea leaves is preferably an alkaline extraction treatment, an acid extraction treatment, or a subcritical water extraction treatment. In the method for producing the cell preservative of this embodiment, examples of the alkali used in the alkaline extraction treatment include sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, ammonium carbonate, lithium chloride, tripotassium phosphate, dipotassium hydrogen phosphate, trisodium phosphate, disodium hydrogen phosphate, sodium gluconate, potassium gluconate, and trisodium citrate.

[0033] In the method for producing the cell preservative of this embodiment, examples of the acid used in the acid extraction treatment include hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, and formic acid. Furthermore, in the method for producing a cell preservative of this embodiment, when subcritical water extraction treatment is used, subcritical water is used for extraction.

[0034] In addition, in the method for producing the cell preservative of this embodiment, it is preferable to store used tea leaves at room temperature or in a refrigerator for one week or more before carrying out the extraction treatment. According to the method for producing a cell preservative of this embodiment, it is possible to produce a cell preservative that is rich in tea leaf-derived proteins such as glutelin and prolamin. Furthermore, according to the method for producing a cell preservative of this embodiment, it is possible to produce a cell preservative that does not contain catechins or that contains almost no catechins.

[0035] The cell-cultured food of this embodiment is characterized by comprising a cell preservative containing an extract of used tea leaves and cultured cells. The cultured cells in the cell-cultured food product of this embodiment are not particularly limited, but may be, for example, induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). Such a cell-cultured food product can suitably inhibit the deterioration of cultured cells in the cell-cultured food product.

[0036] The method for preserving a cell-cultured food product of this embodiment is characterized by adding a cell preservative containing an extract of used tea leaves to the cultured cells and storing them. According to such a method for preserving a cell-cultured food, it is possible to store the cell-cultured food while suitably suppressing deterioration of the cultured cells in the cell-cultured food. [Example]

[0037] [Test 1] A test was conducted as follows to confirm the amount of a specific amino acid component contained in the residue (used tea leaves) after hot water extraction of the tea leaves used as a material for the cell preservative of this embodiment.

[0038] <Quantitative analysis of valine, leucine, isoleucine, and lysine> Sencha (green tea, unfermented tea) was used as the tea leaves, and the residue after hot water extraction was dried to obtain dried tea leaves. Next, 0.5 g of the obtained used tea leaves and 20 mL of 20% hydrochloric acid containing 0.04% 2-mercaptoethanol were sealed in a container and stirred, and then the container was degassed and sealed.

[0039] Next, the sealed container containing the tea leaves and hydrochloric acid was heated at 110°C for 24 hours to hydrolyze the proteins in the tea leaves. The mixed solution in the container was allowed to cool, and then purified water was added to bring the total volume to 100 mL. Next, 25 mL of the supernatant of the mixed solution in the container was taken and concentrated to dryness under reduced pressure, and then dissolved in 5 mL of sodium citrate buffer solution (pH 2.2) to obtain a measurement solution.

[0040] Using this measurement solution, the amount of amino acids in the used tea leaves was measured using an automatic amino acid analyzer (LA8080, Hitachi High-Tech Science Corporation). The content of valine, leucine, isoleucine, and lysine per 100 g of used tea leaves was then calculated using calculation software installed in the automatic amino acid analyzer.

[0041] <Quantitative analysis of methionine> As in the above, sencha tea was used as the tea leaves, and the residue after hot water extraction was dried to obtain dried tea leaves. Next, 0.5 g of the resulting used tea leaves and 10 mL of performic acid solution were sealed in a container, stirred, and then stored in a refrigerator for 16 hours.

[0042] Next, the solution in the container was concentrated to dryness under reduced pressure, and 50 mL of 20% hydrochloric acid was added and stirred, and then heated at 130°C for 20 hours to hydrolyze the protein in the used tea leaves. The mixed solution in the container was allowed to cool, and then purified water was added to bring the total volume to 100 mL. Next, 25 mL of the supernatant of the mixed solution in the container was taken and concentrated to dryness under reduced pressure, and then dissolved in 5 mL of sodium citrate buffer solution (pH 2.2) to obtain a measurement solution.

[0043] Using this measurement solution, the amount of amino acids in the used tea leaves was measured using an automatic amino acid analyzer (LA8080, Hitachi High-Tech Science Corporation). The methionine content per 100g of used tea leaves was then calculated using calculation software installed in the automatic amino acid analyzer.

[0044] The results of Test 1 are shown in Table 2. For comparison with these results, data for food number 04026 (soybean) and food number 01015 (wheat) from the Ministry of Education, Culture, Sports, Science and Technology's Food Composition Database are also shown in Table 2.

[0045] [Table 2]

[0046] As shown in Table 2, the amount of amino acids per 100 g of used tea leaves was 2620 mg of valine, 2140 mg of leucine, 1390 mg of isoleucine, 1750 mg of lysine, and 670 mg of methionine. On the other hand, the amino acid content per 100g of edible portion of soybeans was 1700mg of valine, 2700mg of leucine, 1600mg of isoleucine, 2300mg of lysine, and 490mg of methionine, while the amino acid content per 100g of edible portion of wheat was 380mg of valine, 610mg of leucine, 320mg of isoleucine, 190mg of lysine, and 150mg of methionine.

[0047] Thus, the residue (used tea leaves) left after hot water extraction of tea leaves, which is used as a material for the cell preservative of this embodiment, is found to be rich in branched-chain amino acids (valine, leucine, isoleucine), methionine, and lysine, which inhibit the expression of protein degradation pathways, even though much of it is considered waste. Furthermore, it was found that the content of these amino acids is comparable to that of soybeans and far exceeds that of wheat.

[0048] [Test 2] A test was conducted as follows to confirm the amount of a specific amino acid component contained in the extract of used tea leaves used in the cell preservative of this embodiment.

[0049] <Extraction of proteins from used tea leaves> The residue (used tea leaves) remaining after hot water extraction of tea leaves was subjected to alkaline extraction to obtain a used tea leaves extract containing the protein. Specifically, sencha tea was used as the tea leaves, and the residue after hot water extraction was stored in a refrigerator for two months and dried to obtain dried tea leaves.

[0050] Next, 5 g of the used tea leaves were mixed with 70 mL of a 0.05 M aqueous solution of NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.), and the mixture was stirred at 60° C. for 3 hours using a hot stirrer (AS ONE Corporation). Furthermore, the obtained composition was centrifuged (6000×g, 60 minutes) and the supernatant was collected to obtain a used tea leaves extract containing proteins derived from used tea leaves.

[0051] <Quantitative analysis of valine, leucine, isoleucine, and lysine> 0.5 mL of the obtained used tea leaves extract and 20 mL of 20% hydrochloric acid containing 0.04% 2-mercaptoethanol were sealed in a container and stirred, after which the container was degassed and sealed.

[0052] Next, the sealed container containing the tea leaves and hydrochloric acid was heated at 110°C for 24 hours to hydrolyze the proteins in the tea leaves. The mixed solution in the container was allowed to cool, and then purified water was added to bring the total volume to 100 mL. Next, 25 mL of the supernatant of the mixed solution in the container was taken and concentrated to dryness under reduced pressure, and then dissolved in 5 mL of sodium citrate buffer solution (pH 2.2) to obtain a measurement solution.

[0053] Using this measurement solution, the amount of amino acids in the used tea leaves extract was measured using an automatic amino acid analyzer (LA8080, Hitachi High-Tech Science Corporation). The contents of valine, leucine, isoleucine, and lysine per 100 g of used tea leaves extract were then calculated using calculation software installed in the automatic amino acid analyzer.

[0054] The results of Test 2 are shown in Table 3. [Table 3]

[0055] As shown in Table 3, the amount of amino acids per 100 g of used tea leaves extract was 480 mg valine, 720 mg leucine, 375 mg isoleucine, 555 mg lysine, and 180 mg methionine.

[0056] As such, it was found that the tea leaves extract used in the cell preservative of this embodiment also contains relatively high amounts of branched-chain amino acids (valine, leucine, isoleucine), methionine, and lysine, which suppress the expression of protein degradation pathways.

[0057] [Test 3] Tests to measure the protein concentration and catechin concentration contained in the used tea leaves extract used in the cell preservative of this embodiment were carried out as follows.

[0058] <Extraction of proteins from used tea leaves> As in Test 2, the residue (used tea leaves) after the hot water extraction treatment of the tea leaves was subjected to alkaline extraction treatment to obtain a used tea leaves extract containing the obtained protein. Specifically, sencha tea was used as the tea leaves, and the residue after hot water extraction was stored in a refrigerator for two months and dried to obtain dried tea leaves.

[0059] Next, 5 g of the used tea leaves was mixed and diluted with 70 mL of a 0.05 M aqueous solution of NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.), and the mixture was stirred at 60°C for 3 hours using a hot stirrer (AS ONE Corporation). The resulting composition was centrifuged (6000×g, 60 minutes) to recover the supernatant, yielding a used tea leaves extract containing proteins derived from used tea leaves. The protein concentration in this supernatant was then measured as follows.

[0060] <Measurement of protein concentration in used tea leaves extract> The protein concentration in the used tea leaves extract was measured by the BCA method. Specifically, a protein concentration measurement kit (TaKaRa BCA Protein Assay Kit, Takara Bio Inc.) was used. Following the manufacturer's instructions, equal volumes of the prepared BCA reaction solution and the target substance, appropriately diluted with Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque Inc.), were mixed in a 96-well plate (351172, Corning Incorporated) and incubated at 37°C for 2 hours. The absorbance was then measured using a microplate reader (Corona Electric Co., Ltd.) at a dominant wavelength of 562 nm and a secondary wavelength of 447 nm to determine the protein concentration. Bovine serum albumin (BSA) included in the kit was used for the calibration curve. As a result, the protein concentration in the used tea leaves extract was found to be 20.4 mg / mL.

[0061] <Measurement of catechin concentration> The catechin concentration in the used tea leaves extract was measured by liquid chromatography-mass spectrometry. Specifically, the used tea leaves extract was filtered through a 0.2 μm PTFE membrane filter. Next, a commercially available green tea catechin mixture (032-18231, Fujifilm Wako Pure Chemical Corporation) was mixed with 10 times the amount of ascorbic acid and dissolved in ultrapure water to obtain an arbitrary catechin concentration. This was used as a calibration curve, and the catechin concentration in the used tea leaves extract was calculated using an LC / MS measurement system (ACQUITY UPLC / SynaptG2, Waters Corporation).

[0062] The column temperature was set to 40°C, and the measurement was performed using an electron spray ionization method. The mobile phase (liquid A: 0.1% formic acid aqueous solution, liquid B: acetonitrile) and a UV detector (PDA detector, scan mode 210-400 nm) were used. As a result, the catechin concentration in the used tea leaves extract was found to be below 2 ppm (below the detection limit).

[0063] As described above, it has been found that the used tea leaves extract used in the cell preservative of this embodiment is rich in protein, even though it is obtained from the residue left after hot water extraction of tea leaves, many of which are considered waste. Furthermore, it was found that the used tea leaves extract used in the cell preservative of this embodiment has a catechin concentration of 2 ppm or less (below the detection limit) and contains almost no catechins that are cytotoxic.

[0064] [Test 4] A test was conducted as follows to confirm the effect of the cell preservative of this embodiment in inhibiting deterioration of a cell-cultured food product containing cultured cells.

[0065] <Extraction of proteins from used tea leaves> As in Test 2, the residue (used tea leaves) after the hot water extraction treatment of the tea leaves was subjected to alkaline extraction treatment to obtain a used tea leaves extract containing the obtained protein. Specifically, sencha tea was used as the tea leaves, and the residue after hot water extraction was stored in a refrigerator for two months and dried to obtain dried tea leaves.

[0066] Next, 5 g of the used tea leaves was mixed and diluted with 70 mL of a 0.05 M aqueous solution of NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.), and the mixture was stirred at 60°C for 3 hours using a hot stirrer (AS ONE Corporation). Furthermore, the obtained composition was centrifuged (6000×g, 60 minutes) to collect the supernatant, and the supernatant was used tea leaves extract containing proteins derived from used tea leaves.

[0067] <Storage of cell-cultured foods> In this test, cultured cells and a culture medium were enclosed in a packaging container, and the composition containing the cultured cells and the culture medium was used as a cell culture food. Then, various concentrations of used tea leaves extract, which is the cell preservative of this embodiment, were added to this cell-cultured food, and the viable cell rate in the cell-cultured food after storage for a predetermined period of time was measured.

[0068] For comparison, the viable cell rate of a cell-cultured food after storage for a predetermined period was measured without adding the used tea leaves extract, which is the cell preservative of this embodiment, to the cell-cultured food. Furthermore, without adding the used tea leaves extract, which is the cell preservative of this embodiment, to the cell culture food, bovine serum albumin or a protease inhibitor, which is said to be effective in preventing cytoplasmic elution when cells are stored in a low-temperature environment, was added, and the viability of the cell culture food after storage for a specified period of time was measured.

[0069] Specifically, mouse-derived muscle cells (C2C12, KAC Corporation) were used as culture cells, and DMEM high glucose (043-30085, Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS (S-FBS-NL-025, Cosmo Bio Co., Ltd.) was used as culture medium. The packaging container used was a bag made of linear low-density polyethylene (LLDPE) with the inside surface corona-treated.

[0070] The cell suspension containing the cultured cells and the culture medium were then poured into a packaging container, which was then sealed and stored at 37°C for one day, allowing the cultured cells to adhere to the inner surface of the packaging container. At this time, the amount of cultured cells seeded into the packaging container was 400,000 cells per bag. The amount of culture medium poured into the packaging container was 4 mL. Seven such packaging containers were prepared.

[0071] Next, the culture medium in one of the containers was drained and replaced with 1.4 mL of Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque, Inc.) and 0.1 mL of RM-50 (RM-Gelatin, Jellice Co., Ltd.), and 0.5 mL of used tea leaves extract, a cell preservative, was added to the container (Example 1). The concentration of used tea leaves extract in the solution in the container in Example 1 was 25%.

[0072] In addition, the culture medium in one packaging container was drained and replaced with 1.65 mL of Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque, Inc.) and 0.1 mL of RM-50 (RM-Gelatin, Jellice Co., Ltd.), and 0.25 mL of used tea leaves extract, a cell preservative, was added to the packaging container (Example 2). The concentration of used tea leaves extract in the solution in the packaging container in Example 2 was 12.5%.

[0073] In addition, the culture medium in one packaging container was drained and replaced with 1.775 mL of Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque, Inc.) and 0.1 mL of RM-50 (RM-Gelatin, Jellice Co., Ltd.), and 0.125 mL of used tea leaves extract, a cell preservative, was added to the packaging container (Example 3). The concentration of used tea leaves extract in the solution in the packaging container in Example 3 was 6.25%.

[0074] Furthermore, the culture medium in one of the containers was drained and replaced with 1.85 mL of Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque, Inc.) and 0.1 mL of RM-50 (RM-Gelatin, Jellice Co., Ltd.), and 0.05 mL of used tea leaves extract, a cell preservative, was added to the container (Example 4). The concentration of used tea leaves extract in the solution in the container in Example 4 was 2.5%.

[0075] In addition, the culture medium in one packaging container was drained and replaced with 1.9 mL of Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque Inc.) and 0.1 mL of RM-50 (RM-Gelatin, Jellice Co., Ltd.), and a packaging container without tea leaves extract was prepared (Comparative Example 1).

[0076] In addition, the culture medium in one packaging container was drained and replaced with 0.9 mL of Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque Inc.) and 0.1 mL of RM-50 (RM-Gelatin, Jellice Co., Ltd.); no tea leaves extract was added to the packaging container; instead, 1 mL of a solution containing bovine serum albumin (017-15146, Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 20 mg / mL was added as an additive (Comparative Example 2).

[0077] Furthermore, the culture medium in one packaging container was drained and replaced with 1.88 mL of Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque Inc.) and 0.1 mL of RM-50 (RM-Gelatin, Jellice Co., Ltd.); no tea leaves extract was added to the packaging container; instead, 0.02 mL of a protease inhibitor (P2714-1BTL, Sigma-Aldrich Japan LLC) was added as an additive (Comparative Example 3). These packaging containers were then stored at 5°C for 5 days.

[0078] <Measurement of viable cell count> The number of viable cells in each packaging container was measured twice: once when the cultured cells were seeded into the packaging container (day 0) and once after the cultured cells had been stored in the packaging container for five days (day 5).

[0079] The viable cell count at the time of seeding the cultured cells into the packaging container (day 0) was measured as follows. The cell suspension containing the cultured cells and the culture solution were poured into a packaging container and stirred, and then 0.1 mL of the cell-containing solution was taken from the packaging container and poured into a 1.5 mL Eppendorf tube. Then, several tens of μL of cell-containing liquid was collected from the Eppendorf tube using a cell counting cartridge (941-0012, Chemometec), and this cartridge was set in a cell counting device (NC-200, Chemometec), and the number of cells was measured to calculate the number of viable cells in each packaging container.

[0080] Furthermore, the number of viable cells after the cultured cells had been stored in the packaging container for 5 days (day 5) was measured as follows. The Dulbecco's phosphate-buffered saline was removed from the packaging and washed once with Dulbecco's phosphate-buffered saline (14249-95, Nacalai Tesque, Inc.). Next, 0.4 mL of Actase (12679-54, Nacalai Tesque, Inc.) was added to the entire bag, after which all the liquid was removed and the bag was stored in a CO2 incubator (MC018AIC, Panasonic Corporation) at 37°C for 4 minutes.

[0081] Next, the cells were detached with 1 mL of DMEM high glucose (043-30085, Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% FBS (S-FBS-NL-025, Cosmo Bio Co., Ltd.), and the detached cells were collected in a 15 mL tube (430791, Corning Incorporated). Furthermore, 0.1 mL was collected from the cell-containing solution collected in the 15 mL tube and poured into a 1.5 mL Eppendorf tube. Then, several tens of μL of cell-containing liquid was collected from the Eppendorf tube using a cell counting cartridge (941-0012, Chemometec), and this cartridge was set in a cell counting device (NC-200, Chemometec), and the number of cells was measured to calculate the number of viable cells in each packaging container.

[0082] The viable cell count was measured three times and the average was calculated. The viable cell rate (%) was calculated by calculating the ratio of the number of viable cells on day 5 to the number of viable cells on day 0 (viable cell number on day 5 / viable cell number on day 0 × 100). The results are shown in Table 4.

[0083] [Table 4]

[0084] As shown in Table 4, the viable cell rate of the extract of used tea leaves stored for 5 days was 9.4% in Example 1, in which 25% extract of used tea leaves was added, 12.2% in Example 2, in which 12.5% ​​extract of used tea leaves was added, 7.6% in Example 3, in which 6.25% extract of used tea leaves was added, and 6.8% in Example 4, in which 2.5% extract of used tea leaves was added. On the other hand, the concentration was 5.3% in Comparative Example 1, in which no additive was added, 1.6% in Comparative Example 2, in which bovine serum albumin was added, and 6.8% in Comparative Example 3, in which a protease inhibitor was added.

[0085] As described above, when the extract of used tea leaves was added, the viability of cells increased by approximately 28% to 130% compared to when the extract of used tea leaves was not added. This indicates that the addition of the extract of used tea leaves has the effect of inhibiting the deterioration of cell-cultured foods containing cultured cells. It was also revealed that the addition of used tea leaves extract was more effective in inhibiting the deterioration of cell-cultured foods than the addition of protease inhibitors.

[0086] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the present invention. For example, it is possible to use tea leaves that have not been subjected to hot water extraction treatment. [Industrial Applicability]

[0087] The present invention can be suitably used in cases such as producing a cell preservative that can suppress the deterioration of cell-cultured foods by effectively utilizing the residue after hot water extraction of tea leaves, much of which is considered waste.

Claims

1. A cell preservative characterized by containing an extract of tea leaves.

2. 2. The cell preservative according to claim 1, wherein the used tea leaves are the residue remaining after tea leaves have been extracted with hot water.

3. 3. The cell preservative according to claim 1, wherein the extract contains 5% by weight or more of protein.

4. 3. The cell preservative according to claim 1, wherein the content of catechins in the extract is less than 2 ppm.

5. 3. The cell preservative according to claim 1, which is used as an agent for inhibiting cell deterioration.

6. 3. The cell preservative according to claim 1, which is used as an additive for cell culture foods.

7. A method for producing a cell preservative, comprising extracting used tea leaves and incorporating the resulting extract.

8. 8. The method for producing a cell preservative according to claim 7, wherein the extraction treatment is an alkaline extraction treatment or an acid extraction treatment.

9. 9. The method for producing a cell preservative according to claim 7 or 8, characterized in that the extraction treatment is carried out after storing used tea leaves at room temperature or in a refrigerator for at least one week.

10. A cell culture food comprising cultured cells and a cell preservative containing an extract of used tea leaves.

11. A method for preserving cell-cultured foods, characterized by adding a cell preservative containing an extract of used tea leaves to cultured cells and storing them.

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