KR20120135451A - Additive composition of culture medium for protein producing cells, which contains black garlic extract as an active ingredient - Google Patents

Abstract

PURPOSE: A medium additive composition containing black garlic extract is provided to enhance cell ability of producing proteins and to produce bio pharmaceutical products. CONSTITUTION: A medium additive composition for culturing protein producing cells contains black garlic extract as an active ingredient. The cells are hydridoma cells for producing proteins. The black garlic extract is prepared using water or low carbon number alcohol. The black garlic extract is a fraction which is prepared by extracting black garlic with ethanol and performing ultrafiltration. A cell culture method for enhancing protein production comprises a step of adding the composition to a medium for culturing the cells. [Reference numerals] (AA) Black garlic+solvent; (BB) Producing garlic concentrate liquid by mixing with mixer(20%, w/v); (CC) Centrifuging(3,000rpm, 30 minutes); (DD) Removing precipitate; (EE) Aseptic black garlic concentrate(BAG); (FF) Germ filtration; (GG) Ultrafiltration(10kDa); (HH,LL,QQ,RR) Freeze-drying; (II) Black garlic extract fraction liquid; (JJ) Filtered liquid(under 10kDa); (KK) Supernatant(10kDa or more); (MM) Nanofiltration(3kDa); (NN) Black garlic extract fraction liquid(>10kDa); (OO) Filtered liquid(under 30kDa); (PP) Supernatant(under 3kDa); (SS) Black garlic extract fraction liquid(3kDa); (TT) Black garlic extract fraction liquid(3-10kDa)

Description

ADDITIVE COMPOSITION OF CULTURE MEDIUM FOR PROTEIN PRODUCING CELLS, WHICH CONTAINS BLACK GARLIC EXTRACT AS AN ACTIVE INGREDIENT}

The present invention provides a composition for adding a medium for culturing a protein-producing cell containing black garlic extract as an active ingredient, and a method for preparing the composition. The present invention also relates to a cell culture method for enhancing protein production, comprising the step of adding the composition to the medium.

In the past, the disease treatment agent used a material obtained from a natural product separation or chemical synthesis method, but is currently gaining through the development of protein therapeutics using genetic engineering technology. Since most protein pharmaceutical ingredients exist in the natural world, it is difficult to develop a large amount of time and expenses. However, the development of genetic engineering and the development of monoclonal antibody production techniques have opened the way for mass production of these therapeutic proteins.

Currently, about 60-70% of the recombinant drugs on the market are approved by the FDA and produced using animal cell culture methods. In particular, most of the high value-added glycoproteins are produced by cell culture methods. R & D is a fast-growing industrial technology sector that is attracted by industry.

Production of proteins using cell lines adds precise conformation and sugar chains, but has slow growth rates, difficult control of the culture environment, and high prices of incubators and culture media. Therefore, researches on how to overcome these shortcomings and maximize productivity and efficacy.

Most black garlic is aged raw garlic at a temperature of 40-90 ℃ for tens of days.In the process of aging, allicin, a garlic's unique fragrance, is reduced, reducing garlic's unique taste and aroma, increasing sweetness, Imparts and increases the activity of antioxidants and polyphenols, flavonoids. Until recently, studies on the efficacy of black garlic and its potential as a composition for food and beverage and cosmetics have been conducted. However, studies on the effect of black garlic in cell line culture or the increase in productivity of recombinant protein produced by animal cell lines have been insufficient.

An object of the present invention is to provide a composition for adding a medium for culturing a protein-producing cell, and a method for producing the same, which is characterized by enhancing the protein production capacity of the cell.

In order to achieve the above object, the present invention provides a composition for adding a medium containing the black garlic extract as an active ingredient and a method for producing the composition.

The present invention also provides a cell culture method for enhancing protein production of a cell, comprising the step of adding the composition to the medium.

The composition for the addition of a culture medium for culturing proteins for protein production using the black garlic extract of the present invention shows excellent efficacy in enhancing the protein production capacity of the cells. It can be usefully used as a composition for adding a medium for the production of biopharmaceuticals as described above.

Figure 1 is an example of a low molecular weight bioactive material fractionation process obtained by extracting aged dry black garlic using ethanol and water and stepwise nanofiltration and lyophilization.
FIG. 2 shows DNA after 3 days and 5 days when black garlic extract extracted with water (A, B) and ethanol (C, D) was added to the culture medium at a concentration of 0.25 g / L, 0.5 g / L and 1.0 g / L. It is a graph evaluating proliferative power.

The present invention includes a black garlic extract as an active ingredient and provides a composition for adding a medium for adding to a culture medium of cells for protein production.

The present invention provides a cell culture method for enhancing protein production of a cell, comprising adding a composition for adding a medium containing a black garlic extract as an active ingredient to a culture medium of cells for protein production.

In another aspect, the present invention provides a method for producing a composition for adding a medium for adding to the culture medium of cells for protein production, comprising the step of extracting black garlic.

Hereinafter, the present invention will be described in detail.

The black garlic of the present invention is prepared by using general garlic as black garlic.

The garlic may be used as it is raw garlic, germinated garlic, dried garlic, chilled and frozen garlic, etc., the garlic stored after the harvest can be used regardless of the storage period. The garlic envelope of the present invention includes both a whole garlic shell and a side garlic shell, and the endothelial means a thin translucent membrane that wraps garlic inside the garlic coat. In order to facilitate the removal of the outer skin and the inner skin of the garlic, garlic can be peeled and dried, or the growth and eradication of various microorganisms and molds that can be contaminated primarily by water can be expected to be sterilized. By blocking the chemical and enzymatic reaction of various enzymes of garlic, there is an effect that can prevent further changes in the components.

The black garlic of the present invention may be prepared through general methods well known in the art. For example, the black garlic of the present invention is a pre-treatment step of classifying the garlic by state, and cleans the appearance;

Putting garlic in a plastic pack in 10 kg units and then sealing the garlic, and storing the garlic in a tray;

Putting the rice into a black garlic aging device, and steaming at a high temperature and high humidity condition by applying steam and heat of 100% RH for 1 to 3 hours while maintaining a temperature of 80 to 100 ° C. in the aging device;

Main ripening step of ripening the garlic by applying heat for 198 hours while maintaining the temperature of 72 ~ 77 ℃ in the steamed garlic in the aging device;

After ripening the garlic after the main ripening step, by applying steam and heat for 35 hours while maintaining the temperature 60 ~ 69 ℃ in the aging device;

Drying the garlic for 51 hours while maintaining the temperature of 50 to 58 ° C. in the aging device after the garlic has been aged; And

The garlic after the drying step is cooled to a low temperature condition for 168 hours while maintaining a temperature of 0 ~ 5 ℃ in the aging device; the garlic after low temperature aging; can be prepared by the black garlic production method comprising a. However, the black garlic of the present invention is not limited by the above manufacturing method, and those skilled in the art can select the appropriate method to manufacture the black garlic of the present invention.

Unlike raw milky white garlic, garlic matured sufficiently by the manufacturing method has a black color, which is a component contained by aging. There is no discomfort peculiar to garlic, in which the smell of garlic vibrates in the body after eating because the volatile sulphide that causes odor is reduced through aging. Black garlic is less hydrated and moist than raw garlic and can be eaten without objection. SOD content (antioxidant activity) of black garlic is 8.7 times higher than ordinary garlic and 79 times higher DPPH content (antioxidant power). When lysine (fat-soluble) is fermented, it is converted into S-arylcysteine (water-soluble), which increases the absorption rate in the body.

S-arylcysteine (SAC) is a physiologically active substance in garlic, and when fresh garlic goes through fermentation process, it creates a new ingredient called antioxidant that does not exist in raw garlic. S-arylcysteine (SAC) has antioxidant activity and prevents liver disorders. It has various pharmacological effects such as cancer prevention, cancer cell proliferation inhibition.

The black garlic extract may be a fraction obtained by ultrafiltration of black garlic after ethanol extraction. Preferably, the black garlic extract may be a fraction of less than 3,000 Daltons obtained by ultrafiltration after ethanol extraction of black garlic.

In addition, the composition for adding a medium of the present invention is characterized by enhancing the protein production capacity of the cell. The cell is preferably a hybridoma cell for protein production.

The composition for adding a medium of the present invention

1) crushing the semi-dried aged black garlic with a solvent to liquefy;

2) centrifuging the black garlic liquefied in step 1) to obtain a supernatant; And

3) filtering the supernatant obtained in step 2) to remove the residue; It can be prepared through.

At this time, the supernatant from which the residue was removed may be sterilized by filtration, the sterile filtered supernatant may be lyophilized at −80 ° C., and the obtained supernatant may be liquefied in PBS.

In addition, the method for producing a composition for adding a medium of the present invention

1) crushing the semi-dried aged black garlic with a solvent to liquefy;

2) centrifuging the black garlic liquefied in step 1) to obtain a supernatant;

3) filtering the supernatant obtained in step 2) to remove the residue;

4) ultrafiltration of the black garlic concentrate obtained by removing the residue in step 3);

5) sterilizing the fraction obtained in step 4);

6) freeze-drying the supernatant sterile filtered in step 5) at -80 ℃; And

7) liquefying the supernatant obtained in step 6) in PBS; It may include (Figure 1).

The solvent is water or lower alcohol having 1 to 4 carbon atoms. The lower alcohol is preferably ethanol. In addition, the weight of the solvent may be 1 to 20 times, preferably 4 to 10 times the weight of the sample weight.

The obtained supernatant of step 2) is filtered through a 1-5 μm filtration membrane, preferably 3-5 μm filtration membrane to remove the residue.

In step 4), the black garlic concentrate obtained by removing the residue in step 3) is ultra-filtration and fractionated to less than 10,000 Daltons ii) less than 10,000 Daltons to more than 3000 Daltons and less than 3000 Daltons.

The fraction obtained in step 5) is sterilized by filtration using a 0.22 μm filtration membrane.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

<Experimental Materials and Equipment>

1) hybridoma cells

The hybridoma (anti-My-10) cell line used in this experiment was purchased from Korea Cell Line Bank, and DMEM medium (JBI, Korea) was added to CDM4MAb medium (Hyclone, Logan, UT, USA). The ratio was 7: 3 and used as a basal medium. 5% CO ₂ , 37 ℃, was incubated at 80 rpm in an orbital shaker (Orbital shaker) in a carbon dioxide incubator maintained at 95% humidity was used for the experiment. The mouse IgG1 ELISA kit used for the experiment was purchased from BETHYL, USA, and the remaining reagents were purchased from Sigma chemical Co., St. Louis, MO, USA.

2) Chinese Hamster Ovary (CHO) Cells

The Chinese hamster ovary (CHO DG44 / dhfr-/-) cell line used in this experiment was purchased from ATCC and CDM4CHO medium (Hyclone, Logan, UT, USA) was used as the basal medium. 5% CO ₂ , 37 ℃, was incubated at a speed of 110 rpm in an orbital shaker (Orbital shaker) in a carbon dioxide incubator maintained at 95% humidity was used for the experiment.

3) Experiment equipment

CO ₂ for cell experiments Incubator, Clean bench, Inverted microscope (olympus, Japan), Centrifuge (Hanil Centrifuge), Hemocytometer, Water bath, Pump Ultrafiltration membrane (Centricon ^® Plus Filter units, nominal molecular weight limits of 10,000, and 3,000 Da, Millipore Co., USA), lyophilizer (DRC-1000, EYELA), sterile filtration membrane 0.2 μm Durapore filter, Millipore, USA).

[ Example  One] Black garlic  Extract extracted with water

200 g / L semi-dried aged black garlic It was added to water (1kg) to (20%) and pulverized three times for 10 minutes in the stirrer to liquidate. The high concentration of black garlic liquid liquefied with water was centrifuged three times for 30 minutes at 3,000 rpm to obtain an extract. The extract of the black garlic concentrate was filtered through a pretreatment filtration membrane having a size of 3-5 μm to obtain a supernatant from which the residue was removed.

The obtained supernatant was filtered using a 0.22 μm filtration membrane, lyophilized (DRC-1000, EYELA) at -80 ° C. for 48 hours, and in PBS at a concentration of 200 g / L (20% w / v). Liquid fractionated (Example 1).

[ Example  2 to 4] Black garlic  Fractionated water extract Fraction  Produce

Extracted with water prepared in Example 1 described above, the supernatant from which the residue was removed was fractionated into a supernatant of 10,000 Daltons or more and a filtrate of less than 10,000 Daltons using an ultrafiltration membrane of 10k Dalton size. It was. The fractionated supernatant of 10,000 Daltons or more was filtered using a 0.22 ㎛ filter membrane, lyophilized (DRC-1000, EYELA) for 48 hours at -80 ℃, 200 g / L (20% w / v) of The solution was fractionated by liquefaction in PBS (Example 2).

Meanwhile, the filtrate of less than 10,000 Daltons was again filtered through an ultrafiltration membrane of 3,000 Daltons (3k Dalton) size and fractionated into a supernatant of less than 3000 Daltons and a filtrate of less than 3000 Daltons. The fractionated supernatant of less than 10,000 Daltons or more than 3,000 Daltons was filtered through a 0.22 ㎛ filter membrane, and lyophilized (DRC-1000, EYELA) for 48 hours at -80 ℃, 200 g / L (20% fractionated by liquefying in PBS at a concentration of w / v) (Example 3).

In addition, the fractionated filtrate of less than 3,000 Daltons was filtered through a 0.22 ㎛ filter membrane, and lyophilized (DRC-1000, EYELA) for 48 hours at -80 ℃, 200 g / L (20% w / fractionated by liquefying in PBS at a concentration of v) (Example 4).

[ Example  5] Black garlic  Extract extracted with ethanol

Semi-dried ripened black garlic was added to ethanol (1 kg) to 200 g / L (20%) and pulverized three times for 10 minutes in a stirrer to liquefy. The high concentration of black garlic liquid liquefied with ethanol was centrifuged three times for 30 minutes at 3,000 rpm to obtain an extract. The extract of the black garlic concentrate was filtered through a pretreatment filtration membrane having a size of 3-5 μm to obtain a supernatant from which the residue was removed.

The obtained supernatant was filtered using a 0.22 μm filtration membrane, lyophilized (DRC-1000, EYELA) at -80 ° C. for 48 hours, and in PBS at a concentration of 200 g / L (20% w / v). Fractionated by liquefaction (Example 5).

[ Example  6 to 8] Black garlic  Fractionated ethanol extract Fraction  Produce

The supernatant extracted with ethanol prepared in Example 5 described above, and the residue was removed, was used as a supernatant of 10,000 Daltons or more and a filtrate of less than 10,000 Daltons using an ultrafiltration membrane of 10k Dalton size. Fractionated.

The fractionated supernatant of 10,000 Daltons or more was filtered using a 0.22 ㎛ filter membrane, lyophilized (DRC-1000, EYELA) for 48 hours at -80 ℃, 200 g / L (20% w / v) of The solution was fractionated by liquefaction in PBS (Example 6).

Meanwhile, the filtrate of less than 10,000 Daltons was again filtered through an ultrafiltration membrane of 3,000 Daltons (3k Dalton) size and fractionated into a supernatant of less than 10,000 Daltons and more than 3000 Daltons and a filtrate of less than 3000 Daltons. The fractionated supernatant of less than 10,000 Daltons or more than 3000 Daltons was filtered through a 0.22 ㎛ filter membrane, and lyophilized (DRC-1000, EYELA) for 48 hours at -80 ℃, 200 g / L (20% fractionated by liquefying in PBS at a concentration of w / v) (Example 7).

In addition, the fractionated filtrate of less than 3,000 Daltons was filtered through a 0.22 ㎛ filter membrane, and lyophilized (DRC-1000, EYELA) for 48 hours at -80 ℃, 200 g / L (20% w / fractionated by liquefying in PBS at a concentration of v) (Example 8).

[ Experimental Example 1 ] Hybridoma ( anti - My -10) survival Cell number  Measure

Survival cell numbers of hybridoma (anti-My-10) cells producing mouse IgG1 antibody were measured when the concentration of black garlic extract was added to the culture medium of the hybridoma cells.

Carbon dioxide incubator with 5% CO ₂ , 37 ° C and 95% humidity for culturing hybridoma (anti-My-10) cells, Erlenmeyer flask and suspension shaker for suspension culture ) Was prepared. The medium in which the cells were cultured was used by mixing at a ratio of 7: 3 with CDM4MAb and DMEM medium, and Examples 1 to 8 were added at 0.25 g / L, 0.5 g / L, and 1.0 g / L, respectively. The hybridoma cells were inoculated at a concentration of 2.5 × 10 ⁵ cells / mL and cultured for 7 days at a suspension rate of 80 rpm in a suspension culture.

The viable cell number of hybridoma (anti-My-10) was measured by hemocytometer and trypan blue dye exclusion method by taking 0.5 mL of the culture solution daily.

As a control, a mixed medium (HyQCDM4 MAb: DMEM = 7: 3) without black garlic extract was used.

As a result, Examples 2 to 4 increased the growth of the cells, and especially when the composition of Example 4 was added, it can be seen that the total number of viable cells than the control group.

Specifically, Examples 1 to 4 show a higher total number of viable cells of hybridoma cells than Examples 5 to 8, and as a whole, compared to using a composition for adding medium by extract of black garlic extracted with ethanol , It can be seen that using the composition for the addition of the medium by the extract extracted with water brings a better effect on the cell growth (Table 1).

[ Experimental Example 2 ] CHO Cell survival Cell number  Measure

The viable cell number of CHO cells was measured when black garlic extract was added to the culture medium of Chinese hamster ovary (CHO) cells by concentration.

For culturing CHO cells, a carbon dioxide incubator maintained at 5% carbon dioxide, 37 ° C. and 95% humidity and an Erlenmeyer flask were prepared. The medium to which the cells were cultured was CDM4CHO medium, and Example 1 To 8 were added at 0.25 g / L, 0.5 g / L and 1.0 g / L, respectively. CHO cells were inoculated at a concentration of 3.0 × 10 ⁵ cells / mL and incubated for 7 days at a suspension speed of 110 rpm in a suspension incubator.

Cell number [survival cell number, cell viability (%)] was measured using trypan blue dye exclusion method (trypan blue dye exclusion method).

As a control, a basic medium (CDM4CHO) without black garlic extract was used.

That As in [Experimental Example 1], All of Examples 1 to 8 increased the growth of CHO cells, it can be seen that using the composition for the addition of the medium by the extract extracted with water also CHO cells have a better effect on cell growth (Table 1).

Concentration of composition
(g / L) Cell growth rate ( Example / Relevant control group * 100,%) Hybridoma CHO Example 1 0.25 94.87 92.45 0.5 103.53 104.01 1.0 90.03 89.33 Example 2 0.25 96.24 95.14 0.5 103.84 96.60 1.0 104.63 97.89 Example 3 0.25 108.22 103.12 0.5 109.59 104.44 1.0 108.22 121.02 Example 4 0.25 112.40 119.07 0.5 115.43 123.83 1.0 107.42 121.90 Example  5 0.25 88.03 85.43 0.5 89.74 87.76 1.0 91.45 89.05 Example  6 0.25 94.41 92.14 0.5 88.71 88.62 1.0 83.81 85.33 Example  7 0.25 103.99 98.51 0.5 94.30 97.64 1.0 80.62 83.96 Example  8 0.25 106.96 103.32 0.5 101.95 106.67 1.0 92.82 99.14

Cell growth rate by concentration

[ Experimental Example 3 ] Hybridoma ( anti - My -10) of the cell DNA  Proliferation Capacity Measurement

Effects of Examples 1 to 8 on Various Levels (0.25, 0.5, 1.0 g / L) in Culture Media of Hybridoma (anti-My-10) Cells on Cell DNA Proliferation Ability at 3 and 5 Days The effect was investigated.

The suspended culture solution was centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded and only cells were collected. After adding the PBS solution again, the solution was sufficiently washed and centrifuged at 1,000 rpm and 4 ° C. for 5 minutes. The supernatant was discarded, and 0.2 mL of cold PBS was added to the remaining cells to release pellets of cells. 3 mL of cold 70% ethanol prepared was added slowly to fix the cells and stored at 4 °. After the fixed cells were centrifuged at 1,000 rpm and 4 ° C, the supernatant was removed and washed with 5 mL of cold PBS. The washed cells were centrifuged at 1,000 rpm and 4 ° C to remove supernatant, and the sunk cells were removed by adding 1 mL of PI staining solution, and then transferred to FACS tube, followed by DNA flow cytometry (Becton Dickinson, San). Jose, CA, USA).

As a control, a mixed medium (HyQCDM4 MAb: DMEM = 7: 3) without black garlic extract was used.

As a result, the DNA proliferation ability of the hybridoma cells on the 3rd day of culture showed increased proliferative capacity compared to the control groups of Examples 1 to 8, but the difference was not significantly different for each fraction. Also on day 5 of culture, all of Examples 1 to 8 showed increased proliferative capacity compared to the control group. Therefore, it can be seen that the black garlic solution increases the DNA proliferation ability of the hybridoma cells at the 3rd and 5th days of culture (FIG. 2).

[ Experimental Example 4 ] Mouse IgG1  Antibody Production Measurement

Hybridoma (anti-My-10) cells produce mouse IgG1 antibodies and can measure the amount of IgG1 antibodies produced by the mouse using a mouse IgG1 ELISA kit. The experiment was carried out as follows to measure the antibody production in the culture medium to which the black garlic concentrate and extract were added.

0.1 mL of the coating buffer was dispensed on the 96 well plate to coat goat anti-mouse IgG1 for 1 hour, and then the coating buffer was washed. 0.2 mL of blocking solution was added to the wells after washing, followed by reaction for 30 minutes, followed by washing. After centrifugation of the culture broth obtained at the same time every day for 7 days in the culture medium, the supernatant from which cells were removed was appropriately diluted, and 0.1 mL of each well of a 96 well plate was mixed with a standard solution. (washing). The reaction was performed by adding 0.1 mL of HRP enzyme-bound antibody and reacting for 1 hour, followed by washing and adding 0.1 mL of TMB solution (3,3 ', 5,5'Tetramethyl-Benzidine). When the color turned blue, 0.1 mL of 2M H ₂ SO ₄ was added to stop the reaction and the absorbance was measured at 450 nm.

As a control, a mixed medium (HyQCDM4 MAb: DMEM = 7: 3) without black garlic extract was used.

As a result, in the case of Examples 3 to 8, the amount of mouse IgG1 antibody produced for 7 days showed increased antibody production compared to the control group in all the groups added with black garlic solution. In particular, it could be seen that the low molecular weight bioactive material contained in the fraction of 3 kDa or less increased the antibody production amount, and in particular, the antibody production amount of Example 8 was large.

Specifically, in Examples 5 to 8, the cell production was higher than that of Examples 1 to 4. Therefore, it can be seen that the medium addition composition, which is extracted using ethanol, is more significant in cell production than the medium addition composition, which is characterized by extracting black garlic with water (Table 2).

Concentration of composition
(g / L) Hybridoma
Antibody Production Example / Relevant control group * 100,%) Example 1 0.25 96.32 0.5 34.43 1.0 102.71 Example 2 0.25 96.75 0.5 93.49 1.0 103.74 Example 3 0.25 94.74 0.5 98.51 1.0 114.64 Example 4 0.25 104.50 0.5 93.89 1.0 101.17 Example  5 0.25 111.22 0.5 103.40 1.0 123.21 Example  6 0.25 122.01 0.5 142.89 1.0 125.44 Example  7 0.25 107.78 0.5 130.47 1.0 118.79 Example  8 0.25 124.83 0.5 147.51 1.0 126.48

Cell Antibody Production of Hybridoma Cells Using Black Garlic Extract

Claims (10)

Comprising a black garlic extract as an active ingredient for adding to the medium for culturing cells for protein production, the composition for medium addition.
The method of claim 1,
The composition is a composition for adding a medium, characterized in that to enhance the protein production capacity of the cell.
The method of claim 1,
The cell is a medium-added composition, characterized in that the hybridoma cells for protein production.
The method of claim 1,
The black garlic extract is a composition for adding a medium, characterized in that the black garlic is extracted using water or lower alcohol having 1 to 4 carbon atoms.
The method of claim 1,
The black garlic extract is a composition for adding a medium, characterized in that to extract the black garlic using ethanol.
The method of claim 1,
The black garlic extract is a medium-added composition, characterized in that the fraction of ultrafiltration after ethanol extract of black garlic.
The method of claim 1,
The black garlic extract is a medium-added composition, characterized in that the fraction of less than 3,000 Daltons obtained by filtration of black garlic after ethanol extraction.
A cell culture method for enhancing protein production of a cell comprising the step of adding the composition of any one of claims 1 to 7 to the culture medium of the cell for protein production.
A method for preparing a composition for adding a medium for adding a black garlic to a culture medium of protein-producing cells, comprising extracting water or a lower alcohol having 1 to 4 carbon atoms.
The method of claim 9,
Method for producing a composition for adding a medium, characterized in that the black garlic is extracted with ethanol.

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