CN108250170B

CN108250170B - Gibberellic acid isomeric compound and application thereof

Info

Publication number: CN108250170B
Application number: CN201810084897.9A
Authority: CN
Inventors: 刘春凤; 孙伟康; 王旭; 李崎; 李永仙; 郑飞云; 王金晶; 钮成拓
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-01-29
Filing date: 2018-01-29
Publication date: 2020-10-09
Anticipated expiration: 2038-01-29
Also published as: CN108250170A; WO2019144503A1

Abstract

The invention discloses a gibberellic acid isomeric compound and application thereof, belonging to the field of beer quality control. The invention adopts the high performance liquid chromatography, preparative chromatography, mass spectrum and nuclear magnetic resonance spectrum technology simultaneously, thereby achieving the purpose of separating and identifying the gibberellic acid degradation products in the beer brewing process. The highest level of this compound is produced at the beginning of the brewer's malt manufacturing process, undergoes saccharification and wort boiling. The substance A is slightly soluble in neutral aqueous solution, is easily soluble in an acidic aqueous solution system, and can stably exist after high-temperature treatment. The invention provides a new direction for the safety evaluation of beer and other related foods, and can evaluate the use and residual condition of the gibberellic acid by detecting the gibberellic acid isomeric compound A in the beer brewing process.

Description

Gibberellic acid isomeric compound and application thereof

Technical Field

The invention relates to a gibberellic acid isomeric compound and application thereof, belonging to the field of beer quality control.

Background

Gibberellin is a highly efficient and widely used diterpenoid plant growth regulator. Molecular formula C₁₉H₂₂O₆The relative molecular mass was 346.37. In 1926, Japanese scientists isolated from rice for the first time, mainly existed in higher plants and microorganisms, wherein Gibberellic Acid (GA) has the strongest activity and is most widely used in agriculture and agricultural product processing industry₃)。

Beer is one of the longest-history alcoholic beverages. Barley is an essential raw material for beer brewing and plays an essential role in the brewing process, so the quality of barley plays a crucial role in the quality of finished beer. In china, the increase in beer production has led to an increased demand for raw barley. However, the profile affects the quality of the malt due to climate change which leads to inconsistent growth of the barley, poor quality stability, and uneven water sensitivity and germination rate of the barley. The gibberellic acid is added in the malting process, so that the barley which does not meet the requirements in quality originally can obtain malt products which can meet the requirements of the beer industry under the action of the exogenous gibberellic acid, and the utilization rate of the barley is improved.

Albeit GA₃The gibberellic acid is widely applied to agriculture including the malting industry, however, as a plant growth regulator, the gibberellic acid belongs to a toxic chemical pesticide, and according to the toxicity data of Sigma company, the gibberellic acid can cause tumors of respiratory system and liver, and belongs to a suspected carcinogenic reagent meeting the RTECS standard. GA3 has potential toxicity to mammalian organs, especially the breast, lung, liver and kidney. In addition, it was shown that GA3 can induce chromosome aberration in human and mouse, and is associated with oxidative stress and cell damage in soft organs of mammals, and can also induce respiratory deficiency and liver cancer. Considering GA₃And thus, when left over by improper use, it will pose a certain degree of harm to human health. The highest residual quantity of gibberellic acid in fruits and vegetables is 0.2mg/kg, and the highest residual quantity of gibberellic acid in grains is 0.02 mg/kg. But there are currently no legal regulations in china related to GA 3. Some scientists have studied influencing GA in aqueous solutions₃Degradation factors, pH, temperature, sterilization mode, ion radiation and the like. At present, GA is involved in beer brewing at home and abroad₃The residual study of (A) is rarely reported.

Disclosure of Invention

Previous studies by the inventors have shown that gibberellic acid is decomposed during beer brewing to produce a thermal decomposition product a, and thus the problem of gibberellic acid residues during beer brewing is transformed into a thermal decomposition product a residue problem. The separation, preparation and structure identification of the substance A lay a solid foundation for the subsequent research of the safety of beer products, provide methods and theoretical references for the evaluation of other potential risk substances in the beer brewing process, and have important theoretical significance and actual reference value for ensuring the beer quality, promoting the formulation of safety regulations of related food and agricultural products and maintaining the human health.

The invention carries out preparation purification and structure identification on the gibberellic acid degradation products, and adopts the technologies of high performance liquid chromatography, preparative chromatography, mass spectrum and nuclear magnetic resonance spectrum, thereby achieving the purpose of separating and identifying the gibberellic acid degradation products in the beer brewing process.

The present invention provides a novel gibberellic acid isomeric compound A which is produced in the highest amount after the beginning of a beer wort production process, saccharification and wort boiling in a beer brewing process. The gibberellic acid isomerate A has strong stability, and can still stably exist in finished beer samples after being treated at the high temperature of 115 ℃. The relative molecular mass 346 of substance A, the molecular formula is shown in formula I:

the invention also provides a preparation method of the compound shown in the formula I, which comprises the following steps:

(1) preparation of beer malt and wheat juice

Micro malting: weighing barley, and preparing malt by adopting a process of soaking four times, soaking eight times, soaking four times, soaking eight times and soaking four times;

preparing wort: pulverizing fructus Hordei Germinatus, adding water for saccharification, filtering, washing the residue with hot water, mixing the filtrate with the mash, and boiling the wort;

(2) preparation of gibberellic acid isomerate A

a. Taking boiled raw wort, evaporating and concentrating to one tenth of the volume of the raw wort, then adding acetonitrile of four tenths of the volume of the raw wort, carrying out homogeneous extraction for a period of time, carrying out suction filtration, washing filter residues with acetonitrile, combining two filtrates after suction filtration, adding 25g/100mL NaCl of the raw wort, shaking for a period of time, standing in a separating funnel until obvious layering occurs, discarding the water phase, concentrating the obtained extract at 40 ℃ to dryness, dissolving with ultrapure water, and adjusting the pH to 2.5 with formic acid to obtain a pretreated sample for later use;

b. before the sample loading analysis of the preparative chromatography, the pretreated sample is taken for LC-MS detection analysis, and the retention time of the substance A is determined according to an LC-MS liquid chromatography ion-flow diagram.

c. Preparative chromatography experiments: and (3) taking the pretreated sample in the step a to prepare a substance A, collecting eluent of each section, performing LC-MS detection according to the analysis conditions in the step b respectively, further determining the elution peak of the substance A, removing acetonitrile by rotary evaporation at room temperature, and then freeze-drying overnight to obtain purified solid powder.

In one embodiment of the present invention, step (1)

Micro malting: weighing 1000g of barley, adopting a process of soaking four, soaking eight, soaking four and soaking four, wherein the temperature of soaking wheat and water cut is 18 ℃, and performing in a constant temperature and humidity incubator. The germination temperature is controlled at 16 ℃, the humidity is kept at 95%, and the germination is stopped when the leaf bud length is 3/4 of the wheat grain length; then baking and drying in an electric heating constant temperature blast drying oven by adopting a gradient heating method: firstly, blowing air at 35 ℃ for 24 hours to remove moisture so that the moisture content is below 20 percent, then drying at 55 ℃ for 5 hours so that the moisture content is reduced to 8 to 12 percent, finally raising the temperature to 85 ℃, and baking for 3 hours so that the moisture content is controlled to 2 to 5 percent; the dried malt is rubbed to remove the root buds.

Preparing wort: crushing the weighed malt on a crusher, feeding at 48 ℃, keeping the temperature of the malt and the feed water at a ratio of 1:6 at 48 ℃ for 30min, heating to 63 ℃ for 60min, heating to 72 ℃ for 20min, keeping the temperature of the malt and the feed water at 78 ℃ for 10min after the heat preservation is finished, filtering malt mash after the saccharification stage is finished, and washing the lees with hot water after the filtration is finished; mixing the filtrate and mash, and boiling the wort.

In one embodiment of the invention, 100mL of boiled wort is taken in step a, concentrated to 10mL by a rotary evaporator at 40 ℃, then 40mL of acetonitrile is added to make the proportion of acetonitrile to be 80%, and the mixture is extracted for 30min in a homogeneous way. And (3) performing suction filtration, washing filter residues by using 10mL of acetonitrile (80%), combining two filtrates after suction filtration, adding 25g of NaCl, shaking for 15min, standing in a separating funnel for 10min until obvious layering is achieved, and discarding the water phase. The extract was concentrated to dryness at 40 ℃ and then 1.0mL of ultrapure water was added and dissolved with formic acid (pH 2.5) to obtain a pretreated sample.

In one embodiment of the present invention, in step b, before performing preparative chromatography sample loading analysis, 10 μ L of the pretreated sample is taken for LC-MS detection analysis, and the analyzer: agilent 1200HPLC System/6110 MS; a chromatographic column: waters sunfire, 50X 40mm, 3.5 μm; column temperature: 50 ℃; mobile phase A: water + 0.01% TFA, mobile phase B: acetonitrile + 0.01% TFA; flow rate: 2.0 mL/min; detection wavelength: 214 nm; gradient elution: 0.0min (95% A + 5% B) -1.2min (5% A + 95% B) -2.5min (95% A + 5% B). And determining the retention time of the substance A according to the LC-MS liquid chromatography ion flow diagram.

In one embodiment of the invention, step c. preparative chromatography experiment: and (3) taking 800 mu L of the pretreated sample in the step a to prepare a substance A, wherein the apparatus is as follows: gilson 281; a chromatographic column: waters sunfire, 150X 30mm, 5 μm; mobile phase A: water + 0.01% TFA, mobile phase B: acetonitrile; flow rate: 30 mL/min; detection wavelength: 214 nm; and (3) 0.0min (90% A + 10% B) to 11.0min (60% A + 40% B) to 15.0min (5% A + 95% B) to 15.2min (95% A + 5% B) to 18.0min (95% A + 5% B), collecting eluates of all sections, respectively carrying out LC-MS detection according to the analysis conditions in the step B, further determining the elution peak of the substance A, carrying out rotary evaporation at room temperature to remove acetonitrile, and then carrying out freeze drying overnight to obtain purified solid powder.

The present inventors have found that a novel gibberellic acid isomeric compound A is present during beer brewing, and that the highest level of gibberellic acid isomeric compound A is produced during the initial production of beer malt, saccharification and wort boiling, at which time the presence of the exogenous additive gibberellic acid can no longer be followed. Therefore, if the substance A is detected in the subsequent corresponding process of beer brewing or in the finished beer, the plant growth promoter gibberellic acid is indirectly reflected in the beer brewing material. The invention provides a new direction for the safety evaluation of beer and other related foods, and can evaluate the use and residual condition of the gibberellic acid by detecting the gibberellic acid isomeric compound A in the beer brewing process.

Drawings

FIG. 1 LC-MS Bar Chart

Structural formula of substance in figure 2A

Detailed Description

Extraction and High Performance Liquid Chromatography (HPLC) analysis of gibberellic acid and substance A:

a. pretreatment of malt samples: weighing 10g of malt, crushing, filling into a 100mL conical flask with a plug, adding 10mL of water, shaking uniformly, adding 40mL of acetonitrile to make the concentration of the acetonitrile be 80%, and carrying out ultrasonic extraction for 30 min. Filtering to obtain filtrate, adding 5g of NaCl, shaking for 15min, transferring the mixed solution into a separating funnel, standing for 10min to obviously separate layers, discarding the water phase, taking the upper organic phase into a round-bottomed flask, concentrating to dryness at 40 ℃ on a rotary evaporator, adding 1mL of ultrapure water (the pH value is adjusted to be 2.5 by formic acid) to fully dissolve, and filtering through a 0.45-micron filter membrane in a sample injection bottle to be tested.

b. Pretreatment of a wort sample: 10mL of wort was weighed into a 100mL conical flask with a stopper, 40mL of acetonitrile was added to make the acetonitrile concentration 80%, and ultrasonic extraction was performed for 30 min. The subsequent steps are the same as the pretreatment operation of the malt.

HPLC chromatographic analysis conditions: a chromatographic column: Zorbax-Eclipse XDB-C18 Analytical 4.6X 250mm5-Micron (Agilent Corp., USA); mobile phase: 40% methanol (formic acid to pH 4.0); flow rate: 0.4 mL/min; and (3) an elution mode: isocratic elution; column temperature: 40 ℃; sample introduction amount: 10 μ L.

Mass spectrometric (LC/MS) detection of gibberellic acid isomerate a:

the instrument comprises the following steps: agilent 1200HPLC System/6110 MS; a chromatographic column: waters sunfire, 50X 40mm, 3.5 μm; column temperature: 50 ℃; mobile phase A: water (0.01% TFA), B: acetonitrile (0.01% TFA); flow rate: 2.0 mL/min; detection wavelength: 214 nm; gradient elution: 0.0min (95% A + 5% B) -1.2min (5% A + 95% B) -2.5min (95% A + 5% B).

The principal ion assignment observed for ESI-MS was M/z 369.1([ M + Na)]⁺)，m/z＝715.2([2M+Na]⁺)，m/z＝329.2([M-H₂O+H]⁺)，m/z＝283.1([M-H₂O-HCO₂H+H]⁺) The substance A has a relative molecular mass of 346, and is an isomer of gibberellic acid.

Nuclear magnetic resonance structural analysis of gibberellic acid isomer a:

the instrument model is as follows: bruker, AVANCE III 500MHz UltraShield-PlusTM fully digital NMR spectrometer; solvent: CDCl₃(ii) a And (3) testing items:¹H-NMR,¹³C-NMR,¹H-¹h COSY, HSQC, HMBC, NOESY. The structural formula of the finally obtained substance A is shown in the attached figure 1 of the specification.

Degradation of gibberellic acid during preparation of wheat, saccharification and boiling of wheat juice

(1) Micro malting:

weighing 1000g of barley (added with wheat, FAQ and SCO), adopting a process of soaking four times, soaking eight times, soaking four times, soaking eight times and soaking four times (adding 3g of gibberellic acid into the last wheat soaking water), taking the malt without adding the gibberellic acid as a blank control, and performing wheat soaking and water cutting in a constant temperature and humidity incubator at the temperature of 18 ℃. The germination temperature is controlled at 16 deg.C, humidity is maintained at 95%, and germination is stopped when the leaf bud length is 3/4 of wheat grain length. Then baking and drying in an electric heating constant temperature blast drying oven by adopting a gradient heating method: firstly, blowing air at 35 ℃ to remove moisture for 24 hours to ensure that the moisture content is below 20 percent, then drying at 55 ℃ for 5 hours to ensure that the moisture content is reduced to 8 to 12 percent, finally increasing the temperature to 85 ℃, and baking for 3 hours to ensure that the moisture content is controlled to 2 to 5 percent. The dried malt is rubbed to remove the root buds.

Weighing 10g of malt samples which are roasted at 35 ℃ for 24h, roasted at 55 ℃ for 5h and roasted at 85 ℃ for 3h after germination is finished, respectively, crushing, filling into a 100mL conical flask with a plug, adding 10mL of water, shaking uniformly, adding 40mL of acetonitrile to make the concentration of the acetonitrile be 80%, and carrying out ultrasonic extraction for 30 min. Filtering to obtain filtrate, adding 5g NaCl, shaking for 15min, transferring the mixed solution into a separating funnel, standing for 10min, obviously layering, discarding the water phase, taking the upper organic phase into a round-bottomed flask, concentrating to dryness at 40 ℃ on a rotary evaporator, adding 1mL of ultrapure water (pH adjusted to 2.5 by formic acid) for full dissolution, filtering with a 0.45 μm filter membrane in a sample injection bottle, and observing the degradation of gibberellic acid and the generation of A substances in different stages of wheat production by high performance liquid chromatography, wherein the results are shown in Table 1.

TABLE 1 degradation of gibberellic acid and formation of substance A in different stages of malting

Note: wheat, FAQ and SCO are 3 kinds of different barley materials.

As can be seen from Table 1, neither gibberellic acid nor substance A was detected in the blank sample; the formation of substance A was not observed in the sample after the completion of germination; the content of gibberellic acid in the three kinds of malt dried at 35 ℃ for 24h is not much different from that after germination is finished, and the residual content of the gibberellic acid in the wheat, FAQ and SCO is respectively reduced by 2.2%, 1.4% and 1.2%, and the generation of substance A is accompanied; after the malt was dried at 55 ℃ for 5 hours, the gibberellic acid content of the three malt was further reduced, and the residual gibberellic acid content in the wheat, FAQ and SCO was reduced by 9.8%, 13.8% and 9.9%, respectively, while the amount of substance A produced in the three samples reached 18.2, 9.8 and 5.5 times that in the 24-hour drying at 35 ℃. After drying at 85 ℃ for 3h, the residual amount of gibberellic acid is obviously reduced, the residual amounts of gibberellic acid in wheat, FAQ and SCO are respectively reduced by 31.7%, 34.0% and 40.5%, and the generation amounts of corresponding A substances in the three samples are respectively 34.3, 20.8 and 13.5 times as high as that in drying at 35 ℃ for 24 h.

(2) Saccharification process

Crushing the weighed malt on a crusher, feeding at 48 ℃, keeping the ratio of malt to feed water at 1:6 at 48 ℃ for 30min, heating to 63 ℃ for 60min, heating to 72 ℃ for 20min, keeping the temperature to 78 ℃ for 10min after the heat preservation is finished and iodine test does not develop color, filtering the malt mash after the saccharification stage is finished, washing the mash with hot water after the filtration is finished, combining the filtrate and the mash to obtain a saccharified wort sample, and observing the degradation of gibberellic acid and the generation of substance A in different stages of the wheat production by adopting a high performance liquid chromatography, wherein the results are shown in table 2.

TABLE 2 degradation of gibberellic acid and formation of substance A at different saccharification stages

Note: wheat, FAQ and SCO are 3 kinds of different barley materials.

As can be seen from Table 2, the degradation rates of gibberellic acid in the four stages of the saccharification process are all faster, and after the heat preservation at 63 ℃ for 60min, the content of gibberellic acid in the mash added with wheat, FAQ and SCO is respectively reduced by 47.0%, 36.1% and 39.4%; after the temperature is kept at 72 ℃ for 20min, the content of gibberellic acid in the mash added with wheat, FAQ and SCO is respectively reduced by 70.9 percent, 70.0 percent and 80.2 percent; after the temperature is kept at 78 ℃ for 10min, the content of gibberellic acid in the mash added with wheat, FAQ and SCO is respectively reduced by 85.5 percent, 81.9 percent and 91.3 percent. In contrast, after the temperature is kept at 48 ℃ for 30min, the area of the gibberellic acid peak is higher than that of the substance A peak, which means that the content of the gibberellic acid is higher than that of the substance A; the production of A substance gradually increases with the progress of saccharification process, and after 10min of saccharification and heat preservation at 78 ℃, the A substance content of three barley malt juices is higher than the residual gibberellic acid.

(3) Boiling process

The boiling of wort can achieve the purpose of evaporating water and concentrating wort, and simultaneously inactivate all enzymes and kill all microorganisms harmful to beer fermentation. The experiment selects six stages of the boiling process of three barley malts to measure the residual gibberellic acid, namely boiling the barley malts for 10min, 20min, 30min, 40min, 50min and 60 min. The degradation of gibberellic acid and the formation of A-species at different stages of boiling were examined by HPLC, and the results are shown in Table 3.

TABLE 3 degradation of gibberellic acid and formation of substance A at different boiling stages

Note: wheat, FAQ and SCO are 3 kinds of different barley materials.

As can be seen from table 3, the content of gibberellic acid decreased with the increase of time during boiling, and the amount of substance a produced gradually increased. After boiling for 20min, the concentrations of gibberellic acid in the wheat, Australian wheat FAQ and SCO wheat juices are respectively reduced by 33.6%, 28.0% and 39.1%; after boiling for 30min, the gibberellic acid concentration in the wheat, FAQ and SCO added wheat juice is respectively reduced by 67.0%, 51.3% and 51.6%; when the wort is boiled for more than 40min, the residual content of gibberellic acid in three wort is less than 0.01mg/(ml 12 ° P), and when the boiling time reaches 60min, the residual content of gibberellic acid is reduced to 0, and the production of substance A reaches the maximum.

In summary, the following steps: a new type of gibberellic acid isomeric compound A appears in the beer brewing process, and the compound A is generated in the highest amount after the beer malt manufacturing process is started, and is saccharified and wort is boiled, and at the moment, the existence of the exogenous additive gibberellic acid can not be tracked and detected any more. Therefore, if the substance A can be detected in the subsequent corresponding process of beer brewing or in the finished beer, the plant growth promoter gibberellic acid is indirectly reflected in the production process of the beer brewing raw material.

Example 1 preparation and structural resolution of gibberellic acid degradation products

(1) Preparation of gibberellic acid isomerate A

a. Weighing 1000g of barley, and preparing the malt by adopting a process of soaking four times, soaking eight times, soaking four times, soaking eight times and soaking four times; pulverizing fructus Hordei Germinatus, adding water for saccharification, filtering, washing the residue with hot water, mixing the filtrate with the mash, and boiling the wort; collecting boiled wort 100mL, concentrating to 10mL at 40 deg.C with rotary evaporator, adding acetonitrile 40mL to make acetonitrile ratio 80%, homogenizing and extracting for 30 min. And (3) carrying out suction filtration, washing filter residues by using 10mL of acetonitrile (80%), combining two filtrates after suction filtration, adding 25g of NaCl, shaking for 15min, standing in a separating funnel for 10min until obvious layering is achieved, and discarding the water phase. The extract was concentrated to dryness at 40 ℃ and dissolved in 1.0mL of ultrapure formic acid (pH 2.5) for further use.

b. Before the preparative chromatography sample loading analysis, 10 μ L of the pretreated sample is subjected to LC-MS detection and analysis by Agilent 1200HPLC System/6110MS, chromatographic column of Waters sunfire, 50 × 40mm, 3.5 μm, column temperature of 50 deg.C, mobile phase A of water (0.01% TFA), mobile phase B of acetonitrile (0.01% TFA), flow rate of 2.0mL/min, detection wavelength of 214nm, gradient elution of 0.0min (95% A + 5% B)1.2min (5% A + 95% B) -2.5min (95% A + 5% B). And determining the retention time of the substance A according to the LC-MS liquid chromatography ion flow diagram. The results are shown in figure 1 of the specification. As can be seen from fig. 1, the principal ion assignment observed for ESI-MS is 369.1([ M + Na) ([ M + Na ])]⁺)，m/z＝715.2([2M+Na]⁺)，m/z＝329.2([M-H₂O+H]⁺)，m/z＝283.1([M-H₂O-HCO₂H+H]⁺) The substance A has a relative molecular mass of 346, and is an isomer of gibberellic acid.

c. Preparative chromatography experiments: and (3) taking 800 mu L of the pretreated sample in the step a to prepare a substance A, wherein the apparatus is as follows: gilson 281; a chromatographic column: waters sunfire, 150X 30mm, 5 μm; mobile phase A: water (0.01% TFA), mobile phase B: acetonitrile; flow rate: 30 mL/min; detection wavelength: 214 nm; gradient elution: 0.0min (90% A + 10% B) to 11.0min (60% A + 40% B) to 15.0min (5% A + 95% B) to 15.2min (95% A + 5% B) to 18.0min (95% A + 5% B). Collecting the eluates of each section, performing LC-MS detection according to the analysis conditions in b, determining the elution peak of the substance A, removing acetonitrile by rotary evaporation at room temperature, and freeze-drying overnight to obtain 20mg of purified solid powder with purity of 100%.

(2) Nuclear magnetic resonance structural analysis of gibberellic acid isomer a

10mg of the solid powder of substance A prepared above was taken and 0.5mL of deuterated reagent CDCl was added₃After dissolving in the nuclear magnetic tube, carry out nuclear magnetic resonance analysis, instrument model: bruker, AVANCE III 500MHz UltraShield-PlusTM fully digital NMR spectrometer; solvent: (ii) a And (3) testing items:¹H-NMR，¹³C-NMR，¹H-¹h COSY, HSQC, HMBC, NOESY, DEPT 135. The results of the NMR measurements are shown in Table 4, and the structural formula of the substance A is shown in figure 1 of the specification.

EXAMPLE 2 physicochemical parameters of Compound A

a, solubility: the substance A is slightly soluble in neutral aqueous solution, and can be dissolved by adjusting pH of the aqueous solution to 2.5 with formic acid.

b, stability: after the wort prepared according to the above example 1 and the beer brewed with the corresponding wort were sterilized at high temperature (115 ℃, 15min) and left at room temperature for 180 days (beer shelf life), respectively, the product A was found to be still stable.

Therefore, although the low-toxicity chemical pesticide gibberellic acid cannot be detected in the finished beer, the existence of the substance A of the degradation product reduces the food safety index of the beer to some extent, and provides a new direction for safety evaluation of relevant foods such as beer.

TABLE 4 NMR results of gibberellic acid isomerate A

Note: "/" indicates no detection.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing a gibberellic acid isomeric compound A, wherein the molecular structural formula of the gibberellic acid isomeric compound A is shown as a formula I:

the method for preparing the gibberellic acid isomeric compound A comprises the following steps:

(1) preparation of beer malt and wheat juice

(2) preparation of gibberellic acid isomerate A

a. Taking boiled wort, evaporating and concentrating to one tenth of the volume of the original wort, then adding acetonitrile of four tenths of the volume of the original wort, wherein the proportion of the acetonitrile is 80%, homogenizing and extracting for a period of time, then carrying out suction filtration, washing filter residues by using the acetonitrile with the concentration of 80%, merging two filtrates after suction filtration, adding 25g/100mL NaCl of the original wort, shaking for a period of time, standing in a separating funnel until obvious layering occurs, discarding a water phase, concentrating the obtained extracting solution at 40 ℃ until the extracting solution is dry, dissolving by using ultrapure water, adjusting the pH to 2.5 by using formic acid, and obtaining a pretreated sample for later use;

b. before carrying out sample loading analysis of preparative chromatography, the pretreated sample is taken for LC-MS detection analysis, and an analysis instrument: agilent 1200HPLC System/6110 MS; a chromatographic column: waters sunfire, 50X 40mm, 3.5 μm; column temperature: 50 ℃; mobile phase A: water + 0.01% TFA, mobile phase B: acetonitrile + 0.01% TFA; flow rate: 2.0 mL/min; detection wavelength: 214 nm; gradient elution: 0.0min, 95% A + 5% B-1.2min, 5% A + 95% B-2.5min, 95% A + 5% B; determining the retention time of the gibberellic acid isomerate A according to an LC-MS liquid chromatography ion flow diagram;

c. preparative chromatography experiments: taking the pretreated sample in the step a to prepare the gibberellic acid isomerate A, wherein the instrument is as follows: gilson 281; a chromatographic column: waters sunfire, 150X 30mm, 5 μm; mobile phase A: water + 0.01% TFA, mobile phase B: acetonitrile; flow rate: 30 mL/min; detection wavelength: 214 nm; gradient elution: 0.0min, 90% A + 10% B-11.0 min, 60% A + 40% B-15.0 min, 5% A + 95% B-15.2 min, 95% A + 5% B-18.0 min, 95% A + 5% B, collecting each section of eluent, performing LC-MS detection according to the analysis conditions in B respectively, further determining the elution peak of the gibberellic acid isomeride A, removing acetonitrile by rotary evaporation at room temperature, and then freeze-drying overnight to obtain purified solid powder, namely the gibberellic acid isomeride A.

2. The method of claim 1, wherein step (1):

micro malting: weighing 1000g of barley, adopting a process of 'four-soaking, eight-soaking, four-soaking, eight-soaking and four-soaking', wherein the temperature of wheat soaking and water cut is 18 ℃, the wheat soaking and water cut are carried out in a constant-temperature constant-humidity incubator, the germination temperature is controlled at 16 ℃, the humidity is kept at 95%, and the germination is stopped when the length of leaf buds is 3/4 of the length of wheat grains; then baking and drying in an electric heating constant temperature blast drying oven by adopting a gradient heating method: firstly, blowing air at 35 ℃ for 24 hours to remove moisture so that the moisture content is below 20 percent, then drying at 55 ℃ for 5 hours so that the moisture content is reduced to 8 to 12 percent, finally raising the temperature to 85 ℃, and baking for 3 hours so that the moisture content is controlled to 2 to 5 percent; the dried malt is rubbed to make root buds fall off;

preparing wort: crushing the weighed malt on a crusher, feeding at 48 ℃, keeping the material-liquid ratio of the malt to water at 1:6 for 30min at 48 ℃, then heating to 63 ℃ and keeping the temperature for 60min, then heating to 72 ℃ and keeping the temperature for 20min, after the heat preservation is finished, keeping the temperature for 10min when the iodine test does not develop color, namely heating to 78 ℃, and after the saccharification stage is finished, filtering the malt mash, and washing the lees with hot water; mixing the filtrate and mash, and boiling the wort.

3. The method of claim 1, wherein step a: taking 100mL of boiled wort, concentrating to 10mL by using a rotary evaporation instrument at 40 ℃, then adding 40mL of acetonitrile to enable the proportion of the acetonitrile to be 80%, homogenizing and extracting for 30min, carrying out suction filtration, washing filter residues by using 10mL of acetonitrile with the concentration of 80%, merging two filtrates after suction filtration, adding 25g of NaCl, oscillating for 15min, standing for 10min in a separating funnel until obvious layering is achieved, and removing a water phase; concentrating the above extractive solution at 40 deg.C to dry, adding 1.0mL ultrapure water, adjusting pH to 2.5 with formic acid, and dissolving to obtain pretreated sample.