JPS6368094A - Production of glycoside of phenols - Google Patents

Abstract

PURPOSE:To efficiently and industrially enable production of glycoside of phenols, by carrying out a plant tissue culture in the coexistence of phenols by adjusting concentrations of cultured cell and phenols to specific concentrations. CONSTITUTION:When a plant tissue culture in the coexistence of phenols is carried out, concentrations of cultured cell and phenols in a liquid medium are adjusted to be respectively >=250g/l (based on fresh wt.) and 5-20mM/l to produce glycoside of phenols. In order to continuously preferably carry out above-mentioned production, the cultured above-mentioned production, the cultured cell is continuously propagated and incubate, beforehand in a cell growth zone under updated conditions of culture medium, so that a cultured cell concentration in a liquid medium become 100-500g/l (based on fresh weight). Then, the cultured cell is continuously subjected to tissue culture, adjusting the resultant cultured cell concentration and concentration of phenols to be, respectively, >=250g/l (based on fresh wt.) and 5-20mM/l.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for efficiently producing phenolic glycosides by tissue culturing cultured plant cells in the presence of phenols.

[Conventional technology and problems]

It is well known that phenolic glycosides can be produced by culturing plant tissue in the presence of phenols. For example, it is known that arbutin, which is useful in cosmetics, can be obtained using plant tissue culture. For example, phytochemistry
s-try) + Vol. 15, p. 1225 (1976) states that when culturing Datura cells, 10% of hydroquinone, resorcinol, or catechol is added to the culture medium.
- It has been shown that phenolic glycosides of arbutin or m- or 0-hydroxyphere β-glucoside can be obtained by culturing in the presence of a concentration of The concentration of is calculated to be less than 3.5mM, which is small, and the concentration of cultured cells in the medium is also 240g, expressed as fresh cell weight.
There is a problem that the production amount of phenol glycosides such as arbutin is low and inefficient, probably because it is calculated to be less than / It. One possible method for increasing the productivity of glycosides is to increase the concentration of phenols corresponding to the non-sugar portions of glycosides in the medium.

However, in conventional culture methods, if the concentration of phenols is too high, there is a risk that the cells will die or growth will be inhibited. 3.5
No attempt has been made to obtain phenolic glycosides by raising the concentration above mM.

Furthermore, a method of culturing at a high cell concentration has not generally been attempted.

[Purpose of the invention]

The present inventors have developed a method for producing phenolic glycosides using plant tissue culture, which enables industrial production of phenolic glycosides more efficiently than conventionally known methods. We investigated a method for efficiently producing the glycoside by significantly increasing the yield of the glycoside per unit volume of the tank.

[Summary of the invention]

As a result, they found that the above object could be achieved by employing the following method, and completed the present invention.

That is, according to the first method of the present invention, in a method for producing phenolic glycosides by performing tissue culture of plants in the coexistence of phenols, the cultured cell concentration in the liquid medium is set to 250 g/l or more (fresh Weight conversion), phenol frequency concentration 5-20mM/7! A method for producing a phenolic glycoside is provided, which is characterized by adjusting the phenolic glycoside so that the following is achieved.

Further, as a preferred embodiment of the first invention in the present invention, a continuous manufacturing method is provided as a second invention. That is, in a method for producing phenolic glycosides by culturing plant tissue in the presence of phenol, the culture medium is previously renewed in a cell growth culture zone. The cultured cells are continuously grown and cultured so that the cultured cell concentration in the liquid medium is 100 to 500g# (fresh weight conversion), and the cultured cells thus obtained are grown in the reaction culture zone to a cultured cell concentration of 250g/1 or more. The second invention provides a method for continuous production of phenolic glycosides, which comprises continuously culturing cultured cells while adjusting the phenolic concentration to 5 to 20 mM/l (based on fresh weight). provided as.

[Detailed description of the invention]

Day 1 ・According to method 1 for producing phenolic glycosides according to the Japanese invention, it is carried out by culturing cultured cells in the presence of phenols using plant tissue culture. is not particularly limited, for example, phytochemistry (P
Hytochemistry), Volume 15.

As shown on page 1225 (1976), the method of the present invention can be applied to plants that have been known to produce phenolic glycosides as metabolites in plant cells in the presence of phenols. Applicable.

In the present invention, among such plants, specifically, American datura (Datura 1nnox+a)
, plants of the Solanaceae family such as tobacco (Nicotiana tabacum), and periwinkle (Vinca maj).
or)), Vinca m1n
Most plant calli, including plants belonging to the Apocynaceae family such as Apocynaceae (or), and other plants with thread count, have such a glycosylation ability, but among these, American Datura is particularly preferred.

In the present invention, in order to obtain phenolic glycosides, cultured cells of the plant are tissue cultured in the presence of phenols using a liquid medium. Tissue culture in this case will be described in detail below.

As the liquid medium, a conventionally known medium used for plant tissue culture can be used.

Specifically, the medium is a medium containing inorganic components, carbon sources, and plant hormones as essential components, to which vitamins are added, and amino acids are further added as necessary. Inorganic components of the medium include inorganic salts containing elements such as nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, ammonium nitrate,
Ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, Examples include compounds such as molybdenum oxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

Examples of carbon sources for the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

The plant hormone in the medium includes indole acetic acid (IA
A), naphthaleneacetic acid (NAA), P-chlorophenoxyisobutyric acid and 2,4-dichlorophenoxyacetic acid (2
, 4-D), and cytokinins such as kinetin, zeatin, and benzyladenine.

The vitamins in the medium include biotin, thiamine (vitamin BI), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate,
Examples include ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin B2).

Examples of amino acids in the medium include glycine, alanine, glutamic acid, cysteine, tyrosine, and lysine.

The liquid medium used in the present invention usually contains about 0.1 μM to about 100 mM of the inorganic component and about 1 μM of the carbon source.
g/l to about 100 g/l, usually 0.01 μM to 1000 μM, preferably 1
Preferably, the vitamins are contained in the range of μM to 100 μM, the vitamins are contained in the range of about 0.1 mg/It to about 150 mg/It, and the amino acids are used in the range of 0 to about 1000 mg/It.

Specifically, media containing these components include conventionally known media used for plant tissue culture;
For example, Murashige Skoog ('62) CMurashi
ge & Skoog) medium, Rinse My 1? −・
Skoog (RM-1965) CLinsmajer
& Skoog) medium, White ('63)
ite), Gamborg's B-5 medium, Mitsui's M-9 medium, etc., the carbon source and plant hormones described above are added, and if necessary, the vitamins and amino acids described above are added. Examples of the culture medium prepared include, but in the present invention, a medium prepared using a Linsmeyer-Skoog or Murashige-Skoog medium is particularly preferred. Regarding the composition of the above-mentioned conventionally known culture medium, for example, "New Plant Tissue Culture J P3B6-P391," written by Tenshi, Nakataka, and Furuya, Asakura Shoten, 19
It was written in 1979.

In the present invention, the cultured cells of the plant are cultured in the presence of phenols using the liquid medium, and the culture in this case is carried out under the following conditions. That is, in the present invention, the concentration of cultured cells in the liquid medium is 250 g#! expressed in fresh weight of cells. As mentioned above, the concentration of phenols in the liquid medium must be adjusted to 5 to 20mM/It, and for this reason, phenols are appropriately supplied to the culture tank continuously or discontinuously to culture the tissue. will be held. Here, the phenols are taken up and consumed by the cultured cells as described below, and react biochemically with sugars possessed by the cells to produce phenolic glycosides.

The cultured cells used in the present invention include leaves, stems, roots,
It can be obtained by culturing tissues such as flower buds using the above-mentioned medium according to a conventional method. Furthermore, cultured cells obtained by subjecting the thus obtained cultured cells to tissue culture in the same manner can also be used.

In the present invention, the concentration of the cultured cells during culture is expressed as the fresh weight of the cells, and the culture is carried out under conditions of 250 g/It or more. It's weight.

That is, the entire amount of cultured cells in the culture tank is placed in a Nutsche covered with a filter cloth, filtered for 5 minutes using a water pump, etc.
This is the weight of the tissue culture after removing the culture medium other than the cultured cells. The fresh weight of cultured cells determined in this way varies depending on the plant, but usually has a moisture content of 80 to 95.
It is wt%.

In the present invention, the concentration of cultured cells in the liquid medium is set to 250
When culturing is carried out at a concentration of less than 42 g/g, the amount of phenols to be added is determined depending on the objective and the yield per effective unit volume of the culture tank of phenolic glycosides, which are metabolic products of the cultured cells. In the present invention, the cell concentration is increased to 250 g/l or higher and the culture is carried out.

Although the present invention does not particularly specify the upper limit of the concentration of cultured cells, it is usually 500 g/j! This will be done below. This is because if the concentration of cultured cells is increased to more than 500 g/l, the slurry concentration will increase and the stirring and mixing of the culture solution will become insufficient, making it difficult to efficiently supply oxygen. This is because necrosis of the cultured cells may occur during the process, making normal culture difficult. In addition, vigorous stirring to ensure sufficient oxygen supply may damage the cultured cells, so it is usually carried out at 500 g/l or less.

According to the method of the present invention in which the culture cells are cultured at a concentration of 250 g/# or more, there is an advantage that the reaction time for producing phenolic glycosides can be shortened.

In other words, when looking at the relationship between the rate at which raw material phenols are converted to glycosides (conversion rate) and culture time, for example, the first
As shown in the figure, when culturing at a high cell concentration, it is possible to shorten the substantially effective culture time until the conversion rate reaches a plateau. When producing glycosides, the number of batch cycles can be increased compared to the low concentration cell culture method even within the same amount of time, and therefore phenolic glycosides can be produced efficiently.

In the present invention, phenols are continuously or discontinuously supplied to the culture tank so that the concentration in the liquid medium is 5 to 20 mM/II, preferably 8 to 18 mM/1.

Phenol concentration is 5mM/ji! If the concentration is less than 250 g/l, the yield of phenolic glycosides per unit volume of light in the culture tank will not improve even if the cell concentration is increased to 250 g/l or more, If the concentration exceeds the above range, the cultured cells will suffer growth problems and die. Therefore, in the present invention, the concentration of phenol [?W] is maintained within the above range for culturing.

In the present invention, the method for administering phenols to the culture tank is to charge the phenols into the culture tank at the beginning of the culture so that the concentration falls within the above range, and then continuously as the phenols are consumed as the culture progresses. A method may also be used in which an appropriate amount of phenol is added periodically or discontinuously to maintain the concentration within the above range. Alternatively, immediately after the start of culture, the phenol concentration is lowered to 5mM/2 or less to allow the cultured cells to acclimate to the phenol coexistence environment for an appropriate period of time, and then the phenol concentration is gradually increased to within the concentration range of the present invention. It is also possible to use a method in which phenols are appropriately added continuously or discontinuously depending on the amount consumed. In the present invention, phenols can be dissolved in a liquid medium to a concentration of 5 to 20 mM or more, and this solution can be added to the culture tank as appropriate depending on the consumption of phenols during liquid culture. Furthermore, in the culture tank of the present invention, it is also possible to use a method of culturing while renewing a liquid medium containing phenols in the above-mentioned concentration range, as in the case of the first cell growth culture tank described later.

The cultivation time in the culture tank is usually several hours to several days, and the temperature during cultivation is usually 20 to 30°C.

To explain a little about the phenolic glycoside of the present invention,
The glycosides are produced when phenols added to the culture medium are taken up into the cultured cells, and the phenols react biochemically with sugars in the cultured cells catalyzed by enzymes contained in the cultured cells, and the sugars are converted into phenols. It is a glycosylated metabolite. Since the types of saccharides contained in the cultured cells may differ depending on the type of cultured cells used, it is possible to obtain different phenolic glycosides even if the same phenol is used as a substrate.

Specifically, the phenols used in the method of the present invention include m-phenols such as phenol, salicylic acid, and cresol, and dihydric phenols such as hydroquinone, resorcinol, and tecol. Among them, hydroquinone is particularly preferred. In addition, specific examples of the sugar moiety of the phenolic glycoside according to the present invention include sugars contained in cultured cells such as β-D-glucose, α-D-glucose, mannose, galactose, and fructose. Among these, β-D-glucose is preferred. Specifically, the phenolic glycosides according to the present invention include arbutin (p-hydroxyphenyl-β-glucoside), salicin (salicyl alcohol β-loglucoside), isosalicin (0
-hydroxybenzyl β-D-glucoside), hydroxyphenyl β-glucoside, and carboxyphenyl β-glucoside. Among these, arbutin is preferred.

2. Shellfish ・i゛According to the present invention, phenolic glycosides can be produced by the method described above using plant tissue culture.

In this case, the culture may be carried out batchwise or continuously.

Industrially, it is preferable to carry out the culture in a continuous manner, and the invention related to the second method of the present invention described above is provided. The method will be described in detail below.

In the method for continuous production of phenolic glycosides according to the present invention, the cultured cells in the cell proliferation culture zone are cultured using, for example, a cell proliferation culture tank (first tank), and the culture in the reaction culture zone is carried out using, for example, a reaction culture tank. This can be carried out using a tank (second tank).

This method will be explained below. First, a cell growth culture tank (
1st tank) will be explained.

In a cell growth culture tank, a liquid medium with a nutrient substrate concentration adjusted to a predetermined concentration is continuously or discontinuously supplied to the culture tank, while a culture solution is continuously or discontinuously supplied from the other side of the culture tank. While renewing the culture solution in the culture tank, the cultured cells are grown by culturing so that the concentration of cultured cells in the culture tank is in the range of 100 to 500 g/6 expressed as fresh weight of cells. Cultivated.

The culturing method using the cell growth culture tank according to the present invention can be carried out by the same method as the high-density culturing method for plants proposed in Japanese Patent Application No. 16838/1983 by the present applicant. In the present invention, the concentration of cultured cells in the first tank is within the above range, but the cultured cells obtained in the first tank are used as raw materials in the reaction culture tank (second tank).
The cultured cells were supplied to the second tank under the same conditions as those related to the first method of the invention described above, that is, the concentration of cultured cells in the second tank was set to 2.
50 g/1) Since phenolic glycosides are produced by culturing cultured cells under the above conditions, the first
The concentration of cultured cells in the tank should be 2 within the above range.
It is preferable to set it as 50 g/1 or more. When cultured at a concentration of 100 to 250 g/l in the first tank, the cultured cells in the second tank can be removed by removing an appropriate amount of the culture solution when transferring the cultured cells in the first tank to the second tank. The concentration can be <250 g/I1 or more as described above. The culture solution removed at this time can be recycled to the first tank.

In the present invention, the concentration of cultured cells in the first tank is as described above.
Culture is carried out at a concentration in the range of 100 to 500 g/12. In the method of culturing at a low concentration, the yield of cultured cells per unit volume of the culture tank is low. Therefore, when attempting to produce phenolic glycosides by transferring the cultured cells obtained in the first cell growth culture tank to the second reaction culture tank, the amount of cultured cells used as raw materials is insufficient, and In order to ensure the weight, it is necessary to increase the number or volume of the first culture tank, which deteriorates the efficiency of culture. For this reason, it is not preferable for the cultured cell concentration in the first tank to be less than 100 g/l in terms of industrial production of phenolic glycosides. In addition, in the first tank, the concentration of cultured cells was
0g/7! If the concentration exceeds 1, it is not preferable for the same reasons as described above regarding the concentration of cultured cells during production of phenolic glycosides in the first method of the invention, such as necrosis of the cells. From the above points, in the cell growth culture tank according to the present invention, the concentration of cultured cells is 100 to 500 g/f.
It is carried out within the range of

In the first cell growth culture tank according to the present invention, a liquid medium in which the concentration of cultured cells is within the above range and the nutrient substrate concentration of the medium is adjusted to a predetermined concentration is continuously supplied to the cell growth culture tank. Alternatively, tissue culture is performed while supplying the culture solution discontinuously, while continuously or discontinuously extracting the culture solution from the other side of the cell growth culture tank, and renewing the culture solution in the culture tank. Renewal of the culture solution may be continuous or discontinuous.

Here, the nutrient substrate is the same medium component of the liquid medium used in the first method of the invention described above, and includes the above-mentioned inorganic components, carbon sources, plant hormones, vitamins, and amino acids.

In addition, the culture solution in which the nutrient substrate concentration has been adjusted to a predetermined concentration refers to the culture solution containing inorganic components of about 0.1 μM to about 100 μM, as described above.
M, carbon source about 1 to about 100 g/l plant hormone about 0
．． The liquid medium is prepared in a concentration range of 0.01 to 1000 μM, preferably 1 to 100 μM, vitamins of about 0.1 to about 150 mg/12, and amino acids of O to about 1000 mg/l. In the present invention, it is usually preferable to use a fresh liquid medium with the concentration of medium components adjusted to the above range to be supplied to the cell growth culture tank. It is also possible to adjust the nutrient substrate concentration of the medium and then recycle it. In the case of cyclic use, it is preferable to appropriately remove waste products generated by cell metabolism in the culture solution.

In the present invention, when carrying out tissue culture while renewing the culture medium, the amount of culture medium in the culture tank is assumed to be ■ [l], and the amount of liquid medium supplied to the culture tank is FCl/day).
The relationship between the medium renewal rate defined as /V (day-') and the specific growth rate μ (dar') of cultured cells is μ≦F/V
Preferably, the tissue culture of the plant is carried out in a range of <20μ. Here, the specific growth rate μ (dar') is an amount defined by the following method. When the amount of cultured cells in the culture tank is maintained as Xo and the proliferated cultured cells are discharged outside the tank, and the amount of cultured cells discharged outside the tank after time (days) is Yt, the specific growth rate μ is defined by the following equation.

The physical meaning of Yt and μ has the dimension of (time-'), and the larger the μΦ value, the faster the cultured cells proliferate.

In the present invention, the specific growth rate μΦ value varies depending on the type of plant to be tissue cultured, but is usually 0.02 to 0.4 [day-
1], preferably in the range of 0.05 to 0.2 (day-'). In the present invention, the concentration of the nutrient substrate in the medium is adjusted to a predetermined concentration. The supply amount FCβ/day when the medium is continuously or discontinuously supplied to the cell growth culture tank is μ≦F/V<
As mentioned above, it is preferable to decide so as to satisfy the equation of 20μ. In the present invention, the medium renewal rate F/
The value of V is usually in the range of 0.02 to 8 (day-').

In the present invention, when the culture solution is extracted continuously or discontinuously from the other side of the culture tank, the concentration of cultured cells in the cell growth tank during culturing is 100 to 500 (g/7
The culture medium is extracted and renewed so that it is within the range.

Furthermore, in the present invention, not only the culture solution but also the cultured cells are extracted from the first cell growth tank and supplied to the second reaction culture tank. When extracting the culture solution, special application
It is also possible to use a method as shown in No. 1-16838, in which only the culture solution is drained into the culture tank using a discharge pipe equipped with a filter. Moreover, an appropriate amount of the culture solution can be transferred to the second tank together with the cultured cells.

In the present invention, when the medium renewal rate (F/V) is 20μ≦F/V, which is extremely larger than the specific growth rate μ, adverse effects such as blackening and necrosis of the tissue culture are likely to occur. Therefore, in the present invention, the culture medium renewal rate (F/'V
It is particularly preferable to perform tissue culture by controlling µ≦F/V<20μ.

In the present invention, the amount of culture solution that is continuously or discontinuously extracted from the other side of the cell growth culture tank is usually the culture volume F (12/day) supplied to the above-mentioned culture tank.
), but it is not necessarily limited to this amount; for example, when culturing under the above-mentioned conditions of F/V
(j!/day) may be increased as appropriate.

In the present invention, the cultured cells obtained from the cell growth culture tank by the above method have a concentration of cultured cells in the reaction culture tank (second tank) of 250 g/1 expressed as the fresh weight of the cells.
Transfer an appropriate amount continuously or discontinuously to the second tank so that the concentration of phenols in the second tank is 5 to 20.
By culturing the transferred cultured cells in the presence of phenols in the range of mM/1), glycosides in which sugars contained in the cultured cells are glycosidated to the phenols can be obtained. The culture method in the second tank in this case is carried out by the same method as explained above in the first invention method.

Phenol glycosides can be separated from the culture mixtures cultured by the first and second methods of the present invention, for example, by the following method.

After drying the callus separated from the culture solution by filtration using a blow dryer, it is soaked in methanol and subjected to ultrasonic treatment to extract the phenolic glycoside arbutin.The extract is concentrated to dryness to remove solids. Dissolve arbutin in hot water or hot ethanol to make a saturated solution, filter out insoluble matter when heated,
Arbutin crystals can be obtained by lowering the temperature of the filtrate.

〔Effect of the invention〕

According to the method for producing phenolic glycosides using plant tissue culture according to the method of the present invention, the yield of phenolic glycosides per unit volume of light in a culture tank can be increased compared to conventional methods. Moreover, since the culture time can be shortened, the glycoside can be efficiently produced. Furthermore, according to the method of the present invention, continuous production of the phenolic glycoside is possible.

〔Example〕

The method of the present invention will be specifically illustrated below with reference to Examples.

Example 1 Cultured cells of American Datura were grown at a cell concentration of 300 g/L using a Linsmeyer-Skoog liquid medium with a hydroquinone concentration of 10 mM and an aerated stirring type culture tank.
When cultured in the dark at 25°C for 12 hours, arbutin increased by 2 hours per unit volume of the culture medium.
593 g/n was produced. At this time, the conversion rate of hydroquinone to arbutin was 95%, and the yield of arbutin per dry cell mass was 216 mg/g.

Example 2 During culture, when the concentration of initially charged hydroquinone decreased to 2mM, the concentration was further increased to 10mM/7! Table 1 shows the results obtained in the same manner as in Example 1, except that a corresponding amount of vitroquinone was added and the culture was continued for a total of 12 hours. The maximum concentration of hydroquinone in the medium during the culture period at this time was 12 mM.

Also, the conversion rate of hydroquinone (HO) to arbutin at this time (what percentage of the total amount of HQ initially supplied to the culture tank was converted to arbutin at any given time after the start of culture) Fig. 1 fa) shows the change over time of .

Example 3 In Example 2, the concentration of hydroquinone in the culture solution was 4mM.
When the concentration of hydroquinone decreased to 8.0 μl, hydroquinone equivalent to 10 mM/l was added and culture was continued.
Table 1 shows the results of culturing for a total of 12 hours in the same manner as in Example 2, except that when the concentration was reduced to mM, hydroquinone was further added in an amount equivalent to 10 mM/l.

Examples 4 to 6 Cultures were carried out in the same manner as in Example 3 except that hydroquinone was changed to catechol, resorcinol, or salicylic acid, and 0-hydroxyphenyl β-〇-glucoside, m-
Hydroxyphenyl β-D-glucoside or salicin was obtained in the yield shown in Table 1.

Comparative Examples 1 to 2, 5 Table 1 shows the results of culturing in the same manner as in Example 1, except that the cell concentration was 200 g/4 and the hydroquinone concentration of the medium was 5+++M, 10mM, and 30mM, respectively.

In addition, in Comparative Example 2, the conversion rate of hydroquinone to arbutin is shown in FIG. 1 (bl) similarly to Example 2.

Comparative Examples 3 and 4 Example 2 except that the cell concentration was 200 g/A
Alternatively, the same amount of hydroquinone was added at the same time interval as the time required for the hydroquinone concentration to decrease to the same predetermined concentration in Example 3, and culture was performed for 12 hours in the same manner as in Example 3. Table 1 shows the results. Shown below. In the case of Comparative Example 4, HQ was 20 mM or more immediately after adding hydroquinone three times, which is thought to have caused cell damage and reduced the yield of arbutin.

Comparative Example 6 Table 1 shows the results of culturing in the same manner as in Comparative Example 1, except that the cell concentration was 300 g/J and the hydroquinone concentration was 3 mM.

Comparative Example 7 Table 1 shows the results of culturing in the same manner as Comparative Example 5 except that the cell concentration was 300 g/1.

2. Removal of dishes from the ceiling The effects of the present invention shown in the above-mentioned comparative examples and examples will be further clarified by the following explanation.

(1) Regarding the relationship between Comparative Example 2 and Example 1; the total amount of phenols supplied to the culture tank on both sides and the culture time were the same, but the cell concentration was changed from 200 g/l to 300 g/l.
g/7! In Example 1, where the conversion rate of phenols to arbutin was increased from 91% to 95%, the yield of arbutin per unit volume of the culture tank was 2477 m
g/l to 2593 mg/l. In addition, the arbutin yield per unit cell is from 310 mg/n to 216 m
g/It, but the low acid value means that the cultured cells have a surplus of arbutin production capacity.

Q (2) Regarding the relationship between Comparative Example 3 and Example 1; (1)
Although the total amount of phenol supplied and the culture time are the same as in the case of , the conversion rate increases from 60% to 80% when the cell concentration is increased, so the arbutin yield is 32%.
60 mg/I) to 4385 mg/p.

(3) Regarding the relationship between Comparative Example 4 and Example 3; Comparative Example 4
In this comparative example, the cell concentration was 200 g/i! Because of the low concentration of phenol in the medium, the concentration of phenol in the medium increases to a maximum of 22mM, which may cause cell damage, resulting in a phenol conversion rate of 27%.
It declined to . On the other hand, in Example 3 where the cell concentration was as high as 300 g/l, the consumption rate of phenol could be increased even if phenol was sequentially added three times as in Comparative Example 4, so the concentration of phenol in the medium was 18 mM or less. The phenol conversion rate was as high as 61%, and therefore the arbutin yield was 2191 mg/(5004 mg compared to 1 in Comparative Example 4).
/ j! I was able to more than double that amount.

Example 7 (1) Coptis japonic
The cultured cells in a) were cultured in Linsmeyer and Skoog's medium containing 2.4-D as a hormone at a concentration of 10 −6 M using the cell growth culture tank (first tank) shown in FIG. 1 .

The culture device is an ION aerated agitation type culture tank as shown in Figure 1, with a pore of about 0.4 mm at the culture solution outlet and a filtration area of about 10 mm.
A cIIY nylon mesh was attached and culture was carried out in such a manner that only the culture solution containing no cells could be discharged to the outside of the culture tank. The culture was continued at 25° C. for 2 weeks, during which time the cultured cells were accumulated in the culture tank without being discharged.

Also, the daily medium renewal rate F/V of the culture solution at this time
The value of w: concentration of the culture solution discharged from the culture tank is 0.5
0.3-1.0 depending on the growth of cultured cells so that
(day-'). During the two-week culture, Linsmeyer and Skoog's medium was supplied in an amount five times the amount of the culture solution in the cell growth tank. In 2 weeks, the cells grew approximately 6.6 times and reached a cell concentration of 395 g/Il.

(2) Next, using an aerated stirring type culture tank with a volume of 1.51, the hydroquinone concentration was lomM (1,1
1) Add 400 g of the cultured cells obtained in the cell growth culture tank (first tank) to the reaction culture tank containing the Linsmeyer-Stargu liquid medium (g/l). The concentration of cultured cells in the medium was 400 g/7 expressed in fresh weight. Bind, dark place, 12 at 25℃
The culture was continued for several hours with air aeration. After the culture was completed, the culture solution and cultured cells were separated by filtration, and the cultured cells were dried, immersed in methanol, and arbutin in the cells was extracted by ultrasonication. When a methanol solution of arbutin was analyzed using liquid chromatography, it was found that
It was found that 99% of the hydroquinone charged in the culture tank was converted to arbutin. In this case, the total weight of the dried cultured cells obtained by the treatment after completion of culture is 1
At 6.6 g, the arbutin content of this product was 16% by weight, and the arbutin yield was 2.7 g/A. Furthermore, the yield of arbutin per unit volume of culture solution and culture time is 5-4 g/j! -day, and the total yield of arbutin was 5.4 g.

Example 8 (1) By the same method as shown in Example 1, cultured cells of American datura were grown in the first cell growth culture tank (culture solution volume: 701), and the culture in the culture tank was When the cell concentration in the liquid reaches approximately 400 g/l, the newly proliferated and increased cultured cells are taken out of the culture tank, and the cell concentration in the first tank at this time is 350 to 4.
Cell growth continued to be in the range of 50 g/12. According to this growth method, cultured cells were produced from the first tank at a rate of 14 to 70 g/It-day expressed as fresh weight.

(2) While growing the cultured cells in the first tank, the cultured cells produced in the first tank are supplied to the second reaction culture tank (culture solution amount: 101) and cultured to continuously produce arbutin. produced. That is, the concentration of hydroquinone in the second tank is 10
A Linsmeyer-Sfueg liquid medium 81 containing 10-6M of 2.4-D as a hormone was added, and 3200 g of the cultured cells obtained in the first tank was added thereto to bring the cell concentration to 400 g/Il. Culture was carried out at 25° C. in a dark place with aeration of air. During cultivation, hydroquinone is taken up by cells and consumed, and the concentration of hydroquinone in the culture medium decreases. During the cultivation period, fresh Linsmeyer-Skoog liquid medium with a hydroquinone concentration of 10 mM was added to the reaction culture tank at approximately 0. .674! /hr, while the same amount of culture solution was taken out from the other side of the culture tank [medium renewal rate (F/V) was about 2] and cultured for 1 hour. After the culture was completed, the cells were treated and analyzed in the same manner as in Example 1, and a total yield of 57.6 g of arbutin was obtained (dry cell amount: 128 g, arbutin content: 45%). When this amount is converted into the amount of culture solution occupied in the culture tank and per culture time, it is 7.2g/j! -day.

According to the method of this embodiment, a plurality of reaction culture tanks of the second tank are installed, and culture is carried out by appropriately shifting the time for charging the cultured cells obtained from the first tank to the second tank. And arbutin can be produced semi-continuously using a plurality of reaction culture vessels.

Comparative Example 8 When the cell concentration of the nuclide was about 90 g/l in the first cell growth culture tank (70#) shown on the Example day, the cultured cells produced in this culture tank were used to grow the second tank. An attempt was made to culture cells in a reaction culture tank (10β) at a cell concentration of 250 g/1 or higher, but in this case, the conditions could not be met because the amount of cultured cells produced in the first tank was small. 1st
It was recognized that it was necessary to further increase the capacity of the culture tank by 2 to 10 times, or to increase the number of culture tanks to two or more, making it impossible to economically and efficiently produce phenolic glycosides.

Comparative Example 9 In Comparative Example 8, when culturing was carried out at a cell concentration of approximately 700 g/l in the first tank, the cells inhibited growth and became necrotic, making it impossible to supply the cells to the second tank, resulting in phenolic glycosides. The body could not be manufactured.

[Brief explanation of drawings]

FIG. 1 shows the culture method according to the present invention ((al; Example 1)
and a culture method with a low cell concentration of 200g/4 ((
Fig. 2 is a diagram showing the change over time in l(Q conversion rate) when arbutin is produced from hydroquinone (IIQ) using Comparative Example 2. It is a diagram showing a method for continuous production of glycosides. 1. Culture medium inlet pipe 2, culture medium inlet 3, opening 4 equipped with a filter that discharges only the culture solution, culture solution discharge pipe 5, oxygen-containing gas Ventilation pipe 6, stirring blade 7, culture mixture (tissue culture + culture solution) discharge pipe 8, pressurized fluid inlet

Claims (2)

[Claims] (1) In a method for producing phenolic glycosides by culturing plant tissue in the coexistence of phenols, the concentration of cultured cells in a liquid medium is 250 g/l or more (fresh weight equivalent), Adjust the concentration to 5-20mM/
1. A method for producing a phenolic glycoside, the method comprising adjusting the phenolic glycoside so that (2) In a method for producing phenolic glycosides by performing tissue culture of plants in the coexistence of phenols, the concentration of cultured cells in a liquid medium in advance in a tissue culture zone under conditions of renewal of the culture medium. 100-500g/l
The cultured cells are continuously grown and cultured so that the concentration of the cultured cells is 250 g/l or more (fresh weight equivalent) in the reaction culture zone.
. A method for continuous production of phenolic glycosides, which comprises continuously culturing cultured cells while adjusting the phenol concentration to 5 to 20 mM/l.