JPH03285687A - Recovery of vegetable metabolic substance - Google Patents

Abstract

PURPOSE:To enable the recovery of metabolic substances independent of the kind of the substance by subjecting the cell or tissue of a plant containing metabolic substance to electric field treatment in the state of an aqueous liquid and secreting the metabolic substances from the cell, etc., into the aqueous liquid. CONSTITUTION:Cell or tissue of a plant (e.g. root or a stalk) is cultured in a medium such as Murashige-Skoog medium to produce a metabolic substance such as rose oil in the cell or tissue and the obtained cultured product or aqueous liquid containing the cultured product is subjected to electric field treatment to force the secretion of the metabolic substance into the medium or the aqueous liquid (electrolytic treatment process). The treated culture product or the aqueous liquid is cultured to allow the cell or tissue to produce the metabolic product (culture process). The above electrolytic treatment process and the culture process are repeated and the metabolic substance is collected from the liquid containing the secreted metabolic product directly or by resin adsorption, etc. The metabolic substance can easily be recovered in high efficiency by this process.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention involves culturing plant cells or tissues to secrete metabolites produced and accumulated in the cells or tissues into an aqueous solution or a medium, and collect them. Regarding how to.

[Conventional technology]

Conventionally, it has been known that differentiated plant tissues such as dedifferentiated cells, roots, stems, and leaves of plants can be used as a means of producing useful metabolites produced by plants, but many of the metabolites are not contained within cells. In such cases, it is usually necessary to collect all the cells or tissues grown in the culture tank in order to collect the metabolites. Therefore, conventionally, metabolites could only be collected once in one culture. Furthermore, when metabolites accumulate within cells, there is a drawback that the maximum production amount of the metabolites is rate-limited by the amount of cell growth. Examples of secretion of metabolites into the culture medium (Saga et al., Abstracts of the 34th Annual Meeting of the Japanese Society of Herbal Pharmaceutical Sciences, p. 62 (1987); Kobayashi et al., Current Status and Future Prospects of Plant Tissue Culture Technology, pp. 102-103 (1987)
8)) is also known, but since most metabolites are accumulated within tissues, they cannot be collected directly from the culture medium.

A method is also being considered in which metabolites are secreted into the medium and repeatedly collected.

For example, a method of increasing ionic strength (Japanese Patent Laid-Open No. 60-1805
No. 94) is known, but the substance liberated into the medium is a proteinaceous enzyme. Other methods of adding membrane-permeable substances such as organic solvents and surfactants to the culture solution have been studied (Special Patent No. 1-243991), but most organic solvents and surfactants cannot be used. However, only a few less toxic solvents such as butyl alcohol, dimethyl pol J, amide, dimethyl sulfoxide, and liquid paraffin were used, and the recovered metabolites were efficiently collected from the basic category. Although it is collected,
Some components in the acidic and neutral categories are secreted, but some are absorbed into cells through processing, and the components that can be secreted and recovered are limited (Koizumi et al., Current Status of Plant Tissue Culture Technology). and Future Prospects, pp. 108-109 (1988)).

[Problem to be solved by the invention]

The object of the present invention is to provide a practical method for recovering metabolites produced by culturing plant cells or tissues by secreting and releasing them into a culture medium, regardless of the type of metabolites.

[Means to solve the problem]

According to the present invention, plant cells or tissues containing metabolites are treated with an electric field in an aqueous solution, the cells or tissues secrete the metabolites into the aqueous solution, and the metabolites are collected from the aqueous solution. Metabolites can be easily recovered.

The above method can also be applied to a culture obtained by culturing plant cells or tissues in a medium as an aqueous solution containing plant cells or tissues containing metabolites.

Furthermore, by performing the electric field treatment under conditions that do not cause loss of metabolic activity and respiratory activity of cells or tissues, a large amount of metabolites can be efficiently collected by repeating the culture and electric field treatment.

That is, plant cells or tissues are cultured in a medium to produce metabolic substances in the cells or tissues, and (a) cells or tissues are separated from the resulting culture or culture and an aqueous solution containing the same. is treated with an electric field under conditions that the cells or tissues do not completely lose their metabolic and respiratory activities to secrete metabolites into the medium or aqueous solution (electric field treatment step), and (b) the treated culture or Cells or tissues are separated from an aqueous solution or these, and a new aqueous solution containing the same is cultured with nutrients necessary to maintain metabolic and respiratory activity, and metabolic substances are added to the cells or tissues. (culturing process), repeating the electric field treatment process and culturing process, and collecting the metabolites from the secreted metabolite-containing liquid, thereby making it possible to efficiently recover a large amount of plant metabolites.

The cells or tissues used in the present invention can be any type of plant, but they are usually cells or tissues grown by culture (undifferentiated cell masses, roots with differentiated structures, stems, leaves, etc.). flower vases or their mixed tissues) are used.

These cells or tissues are sterilized using ethanol, sodium hypochlorite, etc. by a method known per se, cut into sections of appropriate size (usually 1 to 20 mm), and cultured and propagated for use.

The invention is applicable to all plants. For example, roses,
Sokei, lavender, iris barida, saffron, perilla, pokeweed, belladonna, Indian jaboku,
It can be applied to plants such as periwinkle, yam, foxglove, senna, badge yam, podophyllum, purple, Chinese, ginseng, hemlock, japonica, stevia, and candela.

Furthermore, all metabolites metabolized by plants can be targeted. For example, 1. Rose oil, jasmine oil, lavender oil, iris butter, crocetin, anthocyanin, betacyanin, scopolamine, hyoscyamine, reserpine, vincristine, vincamine, diosgenin, digitoxin, digoxin, sennoside A, dopa, bodophyllotgycin, shikonin, Examples include Sannin, Ginsside, Pyl/)rin, Haedokirin, Stevioside, Rebaudioside, and Glycyrrhizin.

As the medium for culturing and propagating plant cells or tissues, any burrowing medium can be used as long as the cells or tissues can be cultured and propagated. That is, as a medium, 10 to
Any natural or synthetic medium can be used as long as it contains 100 g/A of sugar, 0.1 to 10 μ/L of plant hormones, nitrogen sources, inorganic substances, vitamins, etc. in an appropriate amount.

As the sugar, sucrose, glucose, lactose, flu-1-ose, etc. are used.

As plant hormones, α-naphthalene acetic acid, 2.4
- Auxins such as dichlorophenoxyacetic acid, indoleacetic acid, and indolebutyric acid, cytokinins such as kinetin, N-(2-chloro-4-pyridyl)-N-phenylurea mibenzyladenine, and zeatin, gibberellin A3, gibberellin A4, gibberellin Gibberellins such as A7, absisins, ethyl/one, etc. are used.

As the nitrogen source, potassium nitrate, sodium nitrate, ammonium nitrate, calcium nitrate, ammonium sulfate, amino acids such as glycine, glutamic acid, lysine, and aspartic acid, yeast extract, meat extract, peptone, and the like are used.

Inorganic substances include potassium chloride, calcium chloride, manganese chloride, nickel chloride, cobalt chloride, aluminum chloride, iron chloride, magnesium sulfate, monolithium sulfate,
Nickel sulfate, iron sulfate, manganese sulfate, titanium sulfate, zinc sulfate, copper sulfate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, potassium iodide, boric acid, thorium molybdate, and the like are used.

In addition, vitamin B1, inositol, pyridoxine hydrochloride, nicotinic acid, thiamine hydrochloride, biotin, etc. may be added to the medium as necessary.

Specific media include Murashige-Skoog's medium, Linsmeyer-Skoog's medium, White's medium, and Kutsubu's medium.

Cultivation is carried out at a temperature of 10 to 35°C, an illuminance of O to 20,000 lux, and a pH of 3.5 to 8.5, usually 10 to 100.
The culture is completed within a few days, but the culture may be continued further.

For culturing, first cut tissues such as leaves, stems, and roots of plants into small pieces (5 x 5 to 50 x 50 mm), sterilize the surface with sodium hypochlorite, ethyl alcohol, etc., and then rinse with sterile water. Wash well. After placing the surface-sterilized small pieces on a sterilized solid medium at a ratio of 1 small piece per 2 to 10 ml of medium, and culturing them for 20 to 50 days at 10 to 35°C, clusters of differentiated tissues such as stems, leaves, roots, etc. can get. The thus obtained differentiated tissue mass is transplanted into a flask or culture tank containing a sterilized medium and cultured. For culture in a medium, for example, when using a 300 mR Erlenmeyer flask, transplant 1 to 5 of the above differentiated tissue masses per 100 females into a liquid medium containing approximately 30 to 200 females, and
Perform shaking culture at 10-35°C and 60-250 revolutions per minute. In addition, when using a 31-volume culture tank, 1 to 2
1 to 5 of the above-mentioned differentiated tissue masses per 100 m2 of the medium are placed in a liquid medium of 1, and cultured at 10 to 35°C while aerating sterile air at a rate of 0.5 to 3 ρ per minute. When the differentiated tissue grows to 2 to 20 times the transplanted amount, remove it from the culture medium and
Divide into 0 pieces and add 100mR of liquid medium in the same manner as above.
The operation of transplanting 1 to 5 divided tissues into a liquid medium and culturing them under the same conditions is repeated.

By repeating the culturing in this way, differentiated tissue that has grown 2 to 20 times the amount transplanted is obtained as a material for secreting and producing metabolic substances. In addition, there are known methods for obtaining plant differentiated tissue materials that produce metabolites, such as JP-A-54-40138, JP-A-055-1.5734, JP-A-5541.8319,
The method described in JP-A No. 61-56022 and the like can be used as is, and differentiated sterile plant tissue can be cultured in large quantities. That is, the differentiated tissues of plants classified as ferns, gymnosperms, and angiosperms, such as roots, stems, leaves, or a mixture thereof, are cultured by a known method using the above-mentioned plant tissue culture medium. The differentiated plant tissues can be propagated by this method, and metabolites can be produced and used as materials for recovery. In order to improve the production of metabolites and the recovery efficiency by electric field treatment, it is preferable to carry out the treatment after the differentiated tissue has grown sufficiently in the culture container. For example, once 70% to 100% of the carbon source (usually sucrose) and inorganic salts added to the culture medium have been consumed and the differentiated plant tissue has grown to 10% to 50% of the total volume in the culture vessel, processing can begin. Good.

By applying an electric field in an aqueous solution to the thus obtained plant cells or tissues containing the target metabolite, the cells or tissues are perforated and the metabolites are easily secreted.

A culture of plant cells or tissues may be treated with an electric field, but in some cases, cells or tissues may be separated from the culture, placed in an appropriate aqueous solution, and treated with an electric field to easily secrete metabolites. I can do it.

Distilled water, deionized water, general water,
Examples include various buffer solutions, various inorganic salt solutions, various sugar solutions, and mixed or diluted solutions thereof.

For example, electric field treatment is carried out by providing electrodes in an aqueous solution with a structure that allows cells or tissues to pass between the electrodes, and applying any one of direct current, rectification, alternating current, or capacitor discharge to the electrodes. The voltage and treatment time are selected so that the current can perforate the cells. The treatment is performed by single or multiple pulses or continuous energization.

The number of times the pulse is processed is not particularly important, but the total processing time of the pulse is important regardless of the number of times the pulse is processed.

When the processing time is 100 milliseconds or less, it is preferable to avoid electric field processing using AC 12 current and rectification because it is unstable, and to perform processing using DC pulses or attenuated pulses using covert discharge. Approximate electric field treatment strength and treatment time per cell or tissue: 3000V/cm or higher, 10 nanoseconds to 500 microseconds at 20000V/cm, microseconds to 5 milliseconds at 1500V/cm, 75
50 microseconds to 50 milliseconds at 0V/cm, 350V
/cm: 500 microseconds to 500 milliseconds, 200V
5 milliseconds to 5 seconds at /cm, 50 at 100V/cm
0 milliseconds to 50 seconds, 5 seconds to 500 seconds at 50V/cm,
For IV/crn1 or higher of 25 V/cm or less, the treatment time is 50 seconds to 5,000 seconds, but the actual treatment conditions must be determined by actually measuring secretion and respiratory activity through experiments to determine the optimum conditions.

In order to set conditions that promote the secretion and release of metabolites from cells and tissues by electric field treatment without losing respiratory activity, electric fields are treated at various intensities during culture.
After ~5 days or more have elapsed, the respiratory activity of cells and tissues can be measured using an oxygen electrode, and treatment conditions with respiratory activity can be determined.

In a preferred embodiment of the present invention, the target product can be harvested in large quantities by alternately repeating culture and electric field treatment. To carry out this method, a culture of plant cells or tissues capable of producing a metabolite is isolated, or cells or tissues are separated from the culture, and an aqueous solution containing the same is added to the cell or tissue so that the cells or tissues exhibit metabolic activity and respiration. The metabolites are secreted by electric field treatment under conditions that do not completely lose the activity, and the treated culture or aqueous solution or cells or tissues are separated from these,
If necessary, nutrients necessary to maintain metabolic activity and respiratory activity are added to the new aqueous solution containing this, cultured to produce and accumulate metabolic substances in cells or tissues, and then subjected to electric field treatment under the above conditions, Thereafter, the cell or tissue culture and electric field treatment may be repeated until metabolic activity is lost.

In the above method, if a liquid plant culture medium is newly used as the aqueous liquid, culture can be performed as is after electric field treatment.

Since no significant increase in cells or tissues is observed in culture after electric field treatment, the culture period may be determined by experimentally determining changes in the production of metabolites, and then electric field treatment may be performed.

Once the metabolites are secreted into the aqueous fluid upon treatment with the electric field, they are collected. As a collection method, for example, a method of directly recovering from an aqueous liquid, a method of recovering metabolites by adsorption with a resin that adsorbs metabolites, etc. can be used.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
These examples do not limit the scope of the invention in any way.

Example 1 The stem of Helladonna was cut into lengths of about 5 cm and sterilized with 70% ethyl alcohol for 2 minutes, then with an aqueous sodium hypochlorite solution (available chlorine amount 0.5%) for 100 minutes, and then
Cut into ~10 mm sections. The sections were mixed with N(2-chloro-4-pyridyl)-N-phenylurea in the Murashige-Skoog medium shown in Table 1 in medium II2. Agar was added at a concentration of 1 g per 1 kg and 8 g per 12 medium (hereinafter referred to as
It is called medium a. ) was transplanted into a test tube with a diameter of 24 mm and a length of 125 mm, containing 100 ml of microorganisms, and cultured at 22° C. under continuous illumination of 12,500 lux for 30 days.

After 30 days of culture, the grown tissue was aseptically removed, and only the roots that had developed from the tissue were aseptically collected using tweezers and a scalpel. These roots were transplanted into a conical beaker containing 100 mR of fresh medium having the composition shown in Table 2, and cultured at 22°C for 30 days to obtain a mass of grown roots.

This root mass was aseptically divided using tweezers and a scalpel, transplanted again into a conical beaker containing 100 mR of fresh medium having the composition shown in Table 2, and subcultured in the same manner as described above. only grew. The roots that had grown in this manner were removed aseptically, and only the roots that had developed from the tissues were aseptically collected using tweezers and a scalpel. These roots contain 6 sucrose in the composition shown in Table 1 above.
0.0g, and further added 0.3g of α-naphthaleneacetic acid.
Liquid medium (hereinafter referred to as medium) changed to 10 mg
0mf! 300mj containing! Transplant an average of 2 g per 100 mm of medium into a wide-capacity flask, and culture with rotational shaking at 120 revolutions per minute at 22°C5G for 35 days to obtain medium 1.
00mj! The average weight per plant was about 8g. Insert sterilized electrodes (electrode size: 30 x 12 mm, electrode spacing: 2 cm) into the flask in which the roots grown in this way were cultured, sandwich the roots between the electrodes, and then apply a maximum voltage of 3000.
V, a decaying wave pulse with a time constant of 5 microseconds was processed 20 times. After treatment, the electrode was removed from the flask and placed at 22° again.
After culturing with shaking at 120 revolutions per minute for 3 hours, only the medium was collected aseptically in a clean bench, hyoscyamine and scoboretin were quantified, and sucrose in the composition in Table 1 was changed to 10 g. Then, 100 mR of liquid medium (hereinafter referred to as medium C) in which all salt concentrations were diluted 1710 times was added, and the rotary shaking culture was continued again at 22°C and 120 revolutions per minute. Thereafter, the same electric field treatment and cultivation were repeated four times at 5-day intervals, and the culture medium was collected for each time to quantify hyoscyamine and scopoletin, and to measure the respiratory activity of the roots after the cultivation was completed.

Table 3 shows the quantitative results of hyoscyamine and scoboletin obtained by the above five treatments in total. The quantitative results of hyoscyamine and scoboretin were expressed as mg//2 of the content per 1°C of the medium. In addition, the results of measuring the respiratory activity of the roots after 5 treatments showed that the amount of oxygen consumed was
It was expressed in mg as oxygen consumption per g per hour.

That is, in the electric field-treated plot, 3.7 to 8.3 mg/(l) of hyoscyamine and 0.9 to 3.2 mg/l of scoboletin were recovered on any culture medium collection day in terms of 1ffi of the medium. On the other hand, in the control plot that was not treated with the electric field, the secretion of both hyoscyamine and scoboletin was minimal or no secretion was observed.The respiration rate was measured by sampling the roots on the 54th day, and the results showed that the amount of respiration was measured by sampling the roots on the 54th day. The respiratory activity was 0.33 mg Oz/g FW/h in the control group, which was a 31% decrease compared to 0.48 mg O□/gFW/h in the control group, but the respiratory activity was strong.

Table 1 Murashige-Skoog medium composition Ammonium nitrate 1, 650 mg7 8 Potassium nitrate Calcium chloride Dihydrate Magnesium sulfate Heptahydrate Potassium phosphate Naz EDTA Dihydrate Ferrous sulfate Heptahydrate Boric acid Sulfuric acid Manganese / tetrahydrate Zinc sulfate / heptahydrate Potassium iodide Sodium molybdate / Cuprous sulfate Cobalt chloride Vitamin B1 Inositol Hydrochloride Pyridoxine Nicotinic acid Glycine Sucrose 1.900mg 40mg 70mg 70mg 37.3mg 27.8mg 6.2mg 22 .3mg 8.6mg 0.83mg Dihydrate 0.25mg 0.025mg 0.025mg 0.40mg 00mg 0.50mg 0.50mg 2.00mg 30.0g (Dissolve the above components in deionized water and bring to 1°C. ,0,1
Adjust the pH to 6.2 with a specified aqueous sodium hydroxide solution, dispense into culture containers, and sterilize at 121°C for 20 minutes. ) Table 2 Skoog modified medium composition 25mg 50mg 22On+g 85mg 5mg 18.65mg 13.9mg 3, 1mg ], 1.1.5mg 4.3mg 0.415mg 0.125mg 0.0125mg Murashige Ammonium nitrate Potassium nitrate Calcium chloride diwater Magnesium sulfate salt/Potassium phosphate heptahydrate Naz4DTA・Ferrous sulfate dihydrate・Boric acid heptahydrate Manganese sulfate tetrahydrate・Potassium iodide heptahydrate Sodium molybdate dihydrate Salt Cuprous sulfate 9 0 Cobalt chloride Vitamin B1 Inositol Hydrochloride Pyridoxine Nicotinic acid Glycine Sucrose Naphthalene Acetic acid 0.0125 mg o, 2 mg 50.0 mg 0.25 mg 0.25 mg 1.00 mg 30.0 g 0.1 mg Dissolve in deionized water and bring to a concentration of 0.1
Adjust the pH to 6.2 with a specified aqueous sodium hydroxide solution, dispense into culture containers, and sterilize at 121°C for 20 minutes. ) (Margins below this page) Table 3 Recovery of hyoscyamine and scoboletin from belladonna root culture 35 days 8.3 mg/ 1 0.9 mg/ 1 0.
2mg/ 4239 5.4 3.2
0.145 6.3 2.4
0.250 3.7 2.7
0.154 4.6 1.6
0.1 Example 2 Cut belladonna stems into lengths of about 5 cm, sterilize them in 70% ethyl alcohol for 2 minutes, then in a sodium hypochlorite aqueous solution (available chlorine amount 0.5%) for 100 minutes, and then sterilize them for 5 to 5 cm.
Cut into 10 mm sections. The section was transplanted into a test tube with a diameter of 24 mm and a length of 125 mm containing 10 ml of medium A, and cultured for 30 days 1 2 at 22° C. under continuous illumination of 12,500 lux.

After 30 days of culture, the grown tissue was aseptically removed, and only the roots that had developed from the tissue were aseptically collected using a vinset and a scalpel. These roots were transplanted into a conical beaker containing 100 ml of fresh medium having the composition shown in Table 2, and cultured at 22°C for 30 days to obtain a mass of grown roots.

This root mass was aseptically divided using a bottle set and a scalpel, and again 100 mj of fresh medium having the composition shown in Table 2 was used! The roots were transferred to a conical beaker containing the following, and subculture was repeated in the same manner as described above to propagate only the roots. The roots that had grown in this manner were removed aseptically, and only the roots that had developed from the tissues were aseptically collected using a vinset and a scalpel.

These roots were grown in medium containing b8j2 at 10°! Culture tank for culturing plant organs (Japanese Patent Application No. 1-243985)
Electrode for electric field treatment (electrode size 42 x 17 mm)
, 1 pair of conical beakers per culture tank were installed with electrode spacing of 20 mm).
For 5 days, the loosening device was operated at 30 rotations per minute at 5-speed intervals to cultivate the roots, and the roots were uniformly dispersed throughout the culture tank without forming clumps. (equivalent to approximately 700 g) (this result was calculated from the amount of change in electrical conductivity of the medium).

For the 355th time of culture, a decaying pulse electric field of 3000 V (1500 V/cm) between the electrodes and a time constant of 8 microseconds was applied to the culture tank thus cultured at 1 second intervals for 1 hour and 30 minutes. The culture medium was sampled every hour until 5 hours after the start of the electric field treatment, and the amount of scoboletin secreted into the culture medium from the root cells was measured over time.

Five hours after the start of electric field treatment, the entire volume of the medium was collected, hyoscyamine and scopoletin were quantitatively determined, and the medium was heated at 8°C.
was added and the culture was continued for 7 days again under the same conditions, a second electric field treatment was performed under the same conditions as the first time, the total amount of the medium was recovered, and hyoscyamine and scoboretin in the medium were quantified.

As a result, changes in the secretion amount of scoboretin up to 5 hours after the start of electric field treatment were as shown in Table 4, and were 0.57 mg per medium 1 at 1 hour after the start of treatment, and 0.57 mg per medium at 2 hours after the start of treatment.
．． The amount was 84 mg, and thereafter it increased slowly and reached 1.07 mg at the 5th hour. The amount of scopoletin in the medium collected the second time was 3.
It was 02 mg. In addition, the amount of hyoscyamine in the culture medium collected 5 hours after the start of electric field treatment was 4.5 hours per 11 hours of culture medium.
3 mg, and the amount of hyoscyamine in the second medium collected was medium II! , 9.3 mg per serving.

As for the control group that is not treated with an electric field, many experimental results have shown that neither hyoscyamine nor scopoletin is substantially secreted outside the cells.

(Leaving space below next summer) Table 4: Changes over time in permeation of scopoletin into the medium from belladonna root culture in jar fermenters 0.02 0.57 0.84 0.95 1.01 1.07 [ [Effects of the Invention] According to the present invention, the cells or tissues of plants containing metabolites are treated with an electric field in an aqueous solution to secrete and release the metabolites produced and accumulated in the cells or tissues into the aqueous solution. can be collected efficiently and easily.

5 6

Claims (1)

[Claims] 1. Electric field treatment of plant cells or tissues containing metabolites in an aqueous solution, secretion of metabolites from the cells or tissues into the aqueous solution, and collection of the metabolites from the aqueous solution. A method for recovering plant metabolites characterized by: 2. The method for recovering plant metabolites according to claim 1, wherein the aqueous liquid containing plant cells or tissues containing the metabolites is a culture obtained by culturing plant cells or tissues in a medium. 3. Cultivating plant cells or tissues in a medium to produce metabolic substances within the cells or tissues; (a) the resulting culture or an aqueous solution containing the cultured cells or tissues separated; treated with an electric field under conditions that cells or tissues do not completely lose their metabolic and respiratory activities to secrete metabolites into the medium or aqueous solution (electric field treatment step) (b) Culture or aqueous solution after treatment Alternatively, cells or tissues can be separated from these and cultured in a new aqueous solution containing them, with nutrients necessary to maintain metabolic and respiratory activity added if necessary, to produce metabolic substances in the cells or tissues. 1. A method for recovering plant metabolites, which comprises collecting metabolites from a solution containing secreted metabolites by repeating an electric field treatment step and a culturing step.