WO2018192529A1 - Method for preparing s-abscisic acid - Google Patents

Abstract

Disclosed in the present invention is a method for preparing S-abscisic acid, comprising the steps of: (1) performing seed culture and fermentation culture using a strain capable of generating the S-abscisic acid by fermentation to obtain a fermentation liquor containing the S-abscisic acid; (2) performing plate and frame filtration, film filtration, acid precipitation and extraction on the fermentation liquor containing the S-abscisic acid of step (1), so as to obtain an extraction liquid; and (3) performing concentration crystallization, recrystallization and drying for the extraction liquid obtained in the step (2), so as to obtain an S-abscisic acid finished product with a purity of 95% or more. By using the preparation method, the fermentation period can be shortened, the content of the S-abscisic acid generated by fermentation is increased, waste of the S-abscisic acid is avoided by means of the extraction and refining treatment, the purity of the S-abscisic acid is increased at the same time, the purity of the S-abscisic acid in the final product is 95% or more, and the product quality is improved. Moreover, the preparation process conditions are mild, the preparation process is environmentally friendly, and industrial production of the S-abscisic acid is facilitated by same.

Description

Preparation method of S-excitation

The present application claims priority to Chinese Patent Application No. 200910254199.4, entitled "A Preparation Method of S-Elicitin", filed on April 18, 2017, the entire contents of which is incorporated by reference. In this application.

Technical field

The invention relates to a method for preparing S-expressor.

Background technique

S-excitation (natural abscisic acid), together with auxin, ethylene, gibberellin and cytokinin, is a plant's five major natural growth regulators. It is a plant's "resistance-inducing factor" and can initiate plant resistance. The reverse gene induces the activation of the anti-reverse immune system in plants, and enhances the plant's resistance to cold, drought, pests, and salinity. By applying S-excitation, the use of chemical pesticides can be reduced, and the quality of agricultural products can be improved. Many aspects have important physiological activities and application value.

At present, the preparation methods of S-Elicitin mainly include direct extraction method, chemical synthesis method and biological fermentation method. The direct extraction method refers to direct extraction from plants, but due to the low content in the plant, the production method has a low yield and a small scale; the chemical synthesis method produces S-excitation, and the purity of the synthesis is low because The products obtained by chemical synthesis are non-natural abscisic acid and natural abscisic acid (S-excitation). Only natural abscisic acid (S-excitation) has the above physiological activity, and the chemical synthesis method has extremely high production cost, so currently Only developed countries such as Japan and the United States are used in large-scale agricultural production; the production of S-Elicitin produced by biological fermentation method is high, and there is no non-natural abscisic acid production. It is a comprehensive preparation method of S-Elicitor. However, there are still some technical problems in this method. For example, the existing fermentation process has a long cycle, and the content of S-Elicitin produced needs to be improved. The by-products produced by fermentation are more, which causes certain difficulties in extraction. And the extracted process parameters are not easy to control, resulting in a large amount of S-exposed melanin waste. In addition, because of the defects of the process and the control of the process parameters, the final S-exposed hormone product is not high in purity, only To 95%, the product quality can not be improved.

Summary of the invention

In view of this, the present application provides a preparation method of S-excitation, which can shorten the fermentation cycle, increase the content of S-expressor produced by fermentation, and avoid the waste of S-excitation by extracting and refining treatment, and simultaneously improve The purity of S-Elicitin makes the purity of S-Elicitin in the final product reach over 95%, which improves product quality. In addition, the preparation process is mild, and the preparation process is environmentally friendly, which is beneficial to the industrial production of S-excitation.

In order to solve the above technical problem, the technical solution provided by the present invention is a preparation method of S-excitation, and the preparation method comprises the following steps:

(1) using a strain capable of fermenting S-expressor, seed culture, and fermentation to obtain a fermentation liquid containing S-expressin;

(2) the fermentation broth containing the S-Ecalin in the step (1) is subjected to plate-frame filtration, membrane filtration, acid precipitation, and extraction to obtain an extract;

(3) The extract obtained in the step (2) is concentrated, crystallized, recrystallized, and dried to obtain a S-expressant product having a purity of 95% or more.

Preferably, the strain capable of fermenting the S-expressor according to the step (1) comprises Botrytis cinerea.

Preferably, the fermentation culture in the step (1) is specifically: maintaining the pH of the fermentation medium at 4-7, and performing fermentation culture.

Preferably, the medium component of the fermentation culture in the step (1) comprises soy protein, lactose, glucose, maltose, sucrose, sucrose, maltodextrin, starch, zein, cottonseed cake powder, potassium dihydrogen phosphate, and heptahydrate. At least one of magnesium sulfate, manganese sulfate monohydrate, ferrous sulfate heptahydrate, zinc sulfate heptahydrate, ammonium sulfate, and salad oil.

Wherein, the fermentation culture temperature is 20-35 ° C, and the fermentation culture time is 12-25 d.

More preferably, the pH of the maintenance fermentation medium is 4-7, specifically: the liquid alkali is continuously replenished when the pH of the fermentation medium is lower than 4; and the fermentation medium liquid is continuously replenished when the pH is higher than 7.

Preferably, before the plate frame of step (2) is filtered, solid NaOH is added to the fermentation broth containing S-exposed hormone to adjust the pH to 4-7, and the plate frame is filtered, and the control pressure is 0.05-0.5. MPa.

Preferably, the membrane filtration according to the step (2) comprises a microfiltration, ultrafiltration and nanofiltration process.

More preferably, the microfiltration step in the step (2) is specifically: performing microfiltration under the conditions of a pressure of 0.05-0.6 MPa and a filtration temperature of not more than 60 °C.

More preferably, the ultrafiltration step in the step (2) is specifically: performing ultrafiltration under the conditions of a pressure of 0.05-1.0 MPa and a filtration temperature of not more than 60 °C.

More preferably, the nanofiltration process in the step (2) comprises a primary nanofiltration and a secondary nanofiltration, and the primary nanofiltration is specifically: a pressure of 0.1-2.0 MPa and a filtration temperature of not more than 60 ° C. The first-stage nanofiltration is carried out; the second-stage nanofiltration is specifically: performing two-stage nanofiltration under the conditions of a pressure of 0.5-10.0 MPa and a filtration temperature of not more than 60 °C.

Preferably, in the extraction in the step (2), the extracting agent used is at least one selected from the group consisting of petroleum ether, ethyl acetate, butyl acetate, n-pentanol, ethanol, acetone, methanol, and the pH of the extraction system is 1. -4.

Preferably, the temperature of the concentrated crystallization, recrystallization, and drying in the step (3) is 40-80 ° C.

The purity of the S-Ecalin is a purity in mass percent.

The technical solution of the present application provides a preparation method of S-expressor, in particular, a method for preparing S-expressant by using a fermentation method to produce S-expressant, and using the fermentation method to produce S-excitation, The yield is high, and the S-Epossin product obtained by extraction and purification from the fermentation liquid has a purity of 95% or more.

In the above technical solution of the present application, in the step (1), a fermentation broth containing S-excitation is obtained by seed culture and fermentation culture using a strain capable of fermenting S-expressor, wherein the fermentation can be produced by S- The strain of the inducer is selected from the group consisting of glucose, lactose, maltose, sucrose, sucrose, maltodextrin, starch, soy protein, zein, cottonseed cake powder as a carbon source and nitrogen. The source, using ammonium sulfate as the nitrogen source, increases the proportion of beneficial elements, promotes the fermentation of strains, and promotes the rapid formation of S-excitation; the natural oil salad oil is used as a carbon source, and the medium liquid is thickened at the same time. Defoaming effect, and prevent contamination during fermentation and culture; adding trace element manganese to promote rapid propagation of strains, adding in the form of manganese sulfate monohydrate; adding magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, seven Zinc sulphate water increases the magnesium, iron and zinc elements in the fermentation process to promote the formation of S-excitation. In addition, the supplemental liquid base is continuously replenished when the pH value is lower than 4 in the fermentation medium; when the pH is higher than 7, the fermentation medium feed medium is continuously replenished, and the pH of the fermentation medium is maintained at 4-7. The pH of the fermentation medium is finely adjusted by adding a liquid alkali and a fermentation medium solution, which does not change the composition of the fermentation medium, nor interferes with the fermentation of the strain. Through the medium setting suitable for the production of S-excitation strains, and the combination of culture conditions, the fermentation cycle can be significantly shortened (from 29 days to 18 days), while increasing the content of S-Ecalin in the fermentation broth (from 3000ppm increased to 8000ppm).

In the step (2), the fermentation liquid containing the S-excitation is subjected to plate-plate filtration, membrane filtration, acid precipitation, and extraction to obtain an extract. Before filtering the plate frame, the solid pH is added to adjust the pH value to 4-7 to facilitate the filtration; in addition, the filtrate filtered by the plate frame is further filtered by membrane filtration, and the membrane filtration includes microfiltration arranged in sequence. , ultrafiltration and nanofiltration, in the microfiltration process, suspended particles, strains and other large particle impurities in the filtrate after the plate frame filtration; in the ultrafiltration process, the macromolecule in the filtrate after microfiltration Protein, glucose, etc. are filtered out; in the nanofiltration process, the ultrafiltration filtrate is further filtered, and some small molecules are filtered out to obtain a relatively pure S-excitation solution. Controlling the pressure during microfiltration, ultrafiltration, and nanofiltration can increase filtration efficiency while avoiding waste of filtrate. The membrane-filtered solution is further separated by extraction, and at least one of petroleum ether, ethyl acetate, butyl acetate, n-pentanol, ethanol, acetone, and methanol is used as an extractant to control the pH of the extraction system. The acidic environment of 1-4 realizes the separation of S-excitation with oils and esters. The content of S-excitation in the solution is increased by the above filtration and extraction steps.

In the step (3), the extract is concentrated, crystallized, recrystallized, and dried to obtain a finished product of S-excitation having a weight percentage of 95% or more. In the process of concentrated crystallization and recrystallization, the crystal of S-excitation is obtained, which is separated from the soluble inorganic salt in the extract, the purity thereof is improved, and the temperature of controlling concentrated crystallization, recrystallization and drying is 40-80. Between °C, the speed of concentrated crystallization, recrystallization and drying can be accelerated, and the structure of S-excitation can be prevented from being damaged and its physiological activity is affected.

Compared with the prior art, the preparation method of the S-Ecalonin described in the technical solution of the present application can shorten the fermentation cycle, increase the content of S-Ecalin produced by fermentation, and further improve S by extracting and refining treatment. - The purity of the inducer is such that the purity of the S-expressin in the final product is over 95%. In addition, the preparation process is mild, and the preparation process is environmentally friendly, which is beneficial to the industrial production of S-excitation.

detailed description

In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

The preparation method of the S-Ecalonin described in the present application is specifically a preparation method using a fermentation method to produce a purity of 95% or more, comprising the following steps:

(1) using a strain capable of fermenting S-expressor, seed culture, and fermentation to obtain a fermentation broth containing S-expressin; wherein the strain capable of fermenting S-expressant includes Botrytis cinerea Mold, fermentation culture medium components include: soy protein, lactose, glucose, maltose, sucrose, sucrose, maltodextrin, starch, zein, cottonseed cake powder, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, sulfuric acid monohydrate Manganese, ferrous sulfate heptahydrate, zinc sulfate heptahydrate, ammonium sulfate, salad oil; the temperature of the fermentation culture is 20-35 ° C, and the fermentation culture time is 12-25 d. In the fermentation culture, when the pH value in the fermentation medium is lower than 4, the liquid alkali is continuously replenished; when the pH value is higher than 7, the fermentation medium liquid is continuously replenished, and the pH of the fermentation medium is maintained at 4-7, wherein The liquid nitrogen source can be ammonia water.

(2) The S-exducin-containing fermentation broth according to the step (1) is subjected to plate-plate filtration, membrane filtration, acid precipitation, and extraction to obtain an extract, wherein the S-inducing reaction is carried out before the plate frame is filtered. The solid fermentation NaOH is added to the fermentation broth to adjust the pH to 4-7, and the plate frame is filtered, and the control pressure is 0.05-0.5 MPa; the membrane filtration includes microfiltration, ultrafiltration and nanofiltration processes arranged in sequence, in the microfiltration In the process, the pressure is 0.05-0.6 MPa, the filtration temperature is not more than 60 ° C; in the ultrafiltration process, the pressure is 0.05-1.0 MPa, the filtration temperature is not more than 60 ° C; the nanofiltration process includes primary nanofiltration and secondary nanofiltration. In the first-stage nanofiltration, the pressure is 0.1-2.0 MPa, the filtration temperature is not more than 60 ° C; in the second-stage nanofiltration, the pressure is 0.5-10.0 MPa, and the filtration temperature is not more than 60 ° C. In the extraction process, the extractant is at least one selected from the group consisting of petroleum ether, ethyl acetate, butyl acetate, n-pentanol, ethanol, acetone, methanol, and the extraction system has a pH of 1-4.

(3) The extract obtained in the step (2) is subjected to concentrated crystallization, recrystallization, and drying at a temperature of 40 to 80 ° C to obtain the S-excitation finished product.

In order to verify the technical effects of the technical solutions of the present application, based on the requirements of the above specific embodiments, test verification is performed using specific parameters, and the following specific embodiments are obtained.

Example 1

Effect of Fermentation Medium on Fermentation Cycle and S-Ecalin Production

The preparation method is:

(1) Botrytis Cinerea-Cercospoxa Rosicola FD338 (abbreviated as B.C. FD338) is subjected to seed culture and fermentation culture to obtain a fermentation broth containing S-expressin.

The medium composition and fermentation cycle of the fermentation culture are shown in Table 1. The fermentation temperature is 20 to 35 °C.

The ammonia water was continuously replenished when the pH value was lower than 4 in the fermentation medium; when the pH was higher than 7, the fermentation medium liquid was continuously replenished, and the pH of the fermentation medium was maintained at 4.5.

(2) The S-exducin-containing fermentation broth obtained in the step (1) is subjected to plate-plate filtration, membrane filtration, acid precipitation, and extraction to obtain an extract.

Before filtering the plate frame, solid NaOH was added to the fermentation broth containing S-Ecalin to adjust the pH to 5.5 and then filtered.

Then, the above-mentioned pH-adjusted fermentation broth was filtered through a plate frame, and the filtration pressure was controlled to 0.5 MPa, and the filtration efficiency was 5 L/min to obtain a filtrate.

The filtrate is subjected to membrane filtration, wherein the membrane filtration comprises microfiltration, ultrafiltration and nanofiltration arranged in sequence;

During the microfiltration process, the suspended particles, strains and other large particle impurities in the filtrate after the plate frame filtration are filtered out; the pressure of the microfiltration is 0.6 MPa, the filtration efficiency is 4 L/min, and the pore diameter of the microfiltration membrane is 50 nm. ;

In the ultrafiltration process, the macromolecular protein, glucose, etc. in the filtrate after microfiltration are filtered; the ultrafiltration pressure is 0.7 MPa, the filtration efficiency is 3 L/min, and the ultrafiltration membrane pore diameter is 20000 D;

In the nanofiltration process, the ultrafiltration filtrate is further filtered, and some small molecules are filtered out to obtain a relatively pure S-excitation solution; wherein the nanofiltration is divided into two stages, respectively, a primary nanofiltration and a secondary Nanofiltration; the pressure of the first nanofiltration is 2 MPa, the filtration efficiency is 3.2 L/min, and the pore size of the primary nanofiltration and the secondary nanofiltration is 100D;

The filtrate after membrane filtration is further separated by extraction, the extractant is selected from ethyl acetate, and the pH of the extraction system is controlled to 3.0, thereby realizing the separation of S-excitation with oils and esters, and obtaining S-inducing An extract of antibiotics.

(3) The S-exducin-containing extract obtained above is subjected to concentration crystallization, recrystallization, and drying to obtain a S-expressant finished product having a weight percentage of 95% or more.

Wherein, the temperature of concentrated crystallization is 40 ° C, and the time of concentrated crystallization is 45 min;

The temperature of recrystallization is 40 ° C, and the time of concentrated crystallization is 50 min;

The drying temperature was 40 ° C and the drying time was 4.5 hours.

The single factor experiment method was used to prepare the S-Ecalin by using the different fermentation medium and the above preparation method under the same conditions. The results are shown in Table 1.

Table 1 Effect of fermentation medium on fermentation cycle and S-Eryin production

Example 2

Effect of pH Adjustment Mode of Fermentation Medium on Fermentation Cycle and S-Ecalin Production

The preparation of S-expressin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and all other conditions were identical, only The pH adjustment method was changed (corresponding to Test 2 to Test 4 in Table 2), and the pH adjustment methods of different fermentation media were used. The results are shown in Table 2.

Table 2 Effect of pH adjustment method of fermentation medium on fermentation cycle and S-Ecalin production

Example 3

Effect of pH of Fermentation Medium on Fermentation Cycle and S-Ecalin Production

The preparation of S-expressin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and all other conditions were identical, only The pH of the fermentation medium was changed, and the pH values of the different fermentation media were used. The results are shown in Table 3.

Table 3 Effect of pH of Fermentation Medium on Fermentation Cycle and S-Ecalin Production

Example 4

Effect of pH value of fermentation broth on filtration of plate and frame

The preparation of S-expressin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and all other conditions were consistent. Before the plate frame was filtered, the pH of the fermentation broth was changed, and the pH values of the different fermentation broths were used. The results are shown in Table 4.

Table 4 Effect of pH of fermentation broth on filtration of plate and frame

Remarks: The weight of the fermentation broth in this experiment is 50L and the content is 8000ppm.

Example 5

Effect of board frame filtering parameters on the filtering effect of the frame

The preparation of S-expressin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and was changed under the same conditions. The board filter parameters are filtered by using different board and frame filter parameters. The results are shown in Table 5.

Table 5 Effect of board frame filtering parameters on the filtering effect of the frame

Remarks: The weight of the fermentation broth in this experiment is 50L and the content is 8000ppm.

Example 6

Effect of membrane filtration parameters on membrane filtration

The preparation of S-expressin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and was changed under the same conditions. Membrane filtration parameters were filtered using different membrane filtration parameters. The results are shown in Table 6.

Table 6 Effect of membrane filtration parameters on membrane filtration

Remarks: The weight of the plate and frame filtrate, microfiltration supernatant, ultrafiltration supernatant and primary nanofiltration residue used in this experiment are 100L, 90L, 80L and 70L respectively.

Example 7

Effect of extractant components on extraction

The preparation of S-expressin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and was changed under the same conditions. For the type of extractant, the filtrate was extracted with different extractants. The results are shown in Table 7.

Table 7 Effect of extractant components on extraction

Remarks: The concentrate content used in this experiment is 20000ppm.

Example 8

Effect of pH value of extraction system on extraction

The preparation of S-expressin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and was changed under the same conditions. The pH of the extraction system was extracted with different pH values of the extraction system. The results are shown in Table 8.

Table 8 Effect of pH value of extraction system on extraction

Remarks: The concentrate content used in this experiment is 20000ppm.

Example 9

Effect of concentrated crystallization, recrystallization and drying temperature on the purity of S-Ecalin

The preparation of S-Elicitin was carried out according to the preparation method provided in Example 1 by a single factor experimental method. The fermentation medium was selected from the medium of Test C in Table 1 of Example 1, and the other conditions were identical. Different concentrated crystals, recrystallization, drying temperature, the results are shown in Table 9.

Table 9 Effect of concentrated crystallization, recrystallization, and drying temperature on the purity of S-Ecalin

The above is only a preferred embodiment of the present invention, and it should be noted that the above-described preferred embodiments are not to be construed as limiting the scope of the invention, and the scope of the invention should be determined by the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and scope of the invention.

Claims (11)

1. A method for preparing an S-expressor, characterized in that the preparation method comprises the following steps:

   (1) using a strain capable of fermenting S-expressor, seed culture, and fermentation to obtain a fermentation liquid containing S-expressin;

   (2) the fermentation broth containing the S-Ecalin in the step (1) is subjected to plate-frame filtration, membrane filtration, acid precipitation, and extraction to obtain an extract;

   (3) The extract obtained in the step (2) is concentrated, crystallized, recrystallized, and dried to obtain a S-expressant product having a purity of 95% or more.
2. The method for preparing an S-Ecalorin according to claim 1, wherein the fermentation culture in the step (1) is specifically: maintaining the pH of the fermentation medium at 4-7 for fermentation culture.
3. The method for preparing an S-Ecalorin according to claim 2, wherein the pH of the fermentation medium is maintained at 4-7, specifically: when the pH of the fermentation medium is lower than 4, continuous supplementation Liquid base; the pH of the fermentation medium is continuously replenished when the pH is higher than 7.
4. The method for preparing an S-Ecalonin according to claim 1, wherein before the plate frame filtration in the step (2), solid NaOH is added to the fermentation liquid containing the S-exposed hormone. The pH value is 4-7, and the plate frame filtration has a control pressure of 0.05-0.5 MPa.
5. The method for preparing an S-Ecalonin according to claim 1, wherein the membrane filtration in the step (2) comprises a microfiltration, an ultrafiltration and a nanofiltration step.
6. The method for preparing an S-Ecalonin according to claim 5, wherein the microfiltration step in the step (2) is specifically: a condition in which the pressure is 0.05-0.6 MPa and the filtration temperature is not more than 60 ° C. Perform microfiltration underneath.
7. The method for preparing an S-Ecalin according to claim 5, wherein the ultrafiltration step in the step (2) is specifically: a condition in which the pressure is 0.05-1.0 MPa and the filtration temperature is not more than 60 °C. Perform ultrafiltration.
8. The method for preparing an S-Ecalonin according to claim 5, wherein the nanofiltration step of the step (2) comprises a primary nanofiltration and a secondary nanofiltration, wherein the primary nanofiltration is : performing a first-stage nanofiltration under the condition of a pressure of 0.1-2.0 MPa and a filtration temperature of not more than 60 ° C; the second-stage nanofiltration is specifically: under the condition of a pressure of 0.5-10.0 MPa and a filtration temperature of not more than 60 ° C Perform a secondary nanofiltration.
9. The method for preparing an S-Ecalonin according to claim 1, wherein in the extracting step (2), the extracting agent used is at least one selected from the group consisting of petroleum ether, ethyl acetate and acetic acid. Ester, n-pentanol, ethanol, acetone, methanol, the pH of the extraction system is 1-4.
10. The method for preparing an S-Ecalorin according to claim 1, wherein the temperature of the concentrated crystallization, recrystallization and drying in the step (3) is 40-80 °C.
11. The method for preparing an S-Ecalorin according to claim 1, wherein the fermentation medium component of the step (1) comprises soy protein, lactose, glucose, maltose, sucrose, sucrose, malt At least one of dextrin, starch, zein, cottonseed cake powder, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, ferrous sulfate heptahydrate, zinc sulfate heptahydrate, ammonium sulfate, and salad oil.