CN114057753A - Method for separating and purifying antifungal active substance HSAF in zymogen fermentation liquor - Google Patents

Method for separating and purifying antifungal active substance HSAF in zymogen fermentation liquor Download PDF

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CN114057753A
CN114057753A CN202010781788.XA CN202010781788A CN114057753A CN 114057753 A CN114057753 A CN 114057753A CN 202010781788 A CN202010781788 A CN 202010781788A CN 114057753 A CN114057753 A CN 114057753A
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汤宝
刘凤权
孙伟波
赵延存
徐会永
陈贤
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Jiangsu Academy of Agricultural Sciences
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Abstract

The invention discloses a method for separating and purifying antifungal active substances HSAF in zymogen fermentation liquor, which comprises the steps of firstly degrading a byproduct ATB in OH11 fermentation liquor by illumination, then carrying out static adsorption or dynamic adsorption on the HSAF in the fermentation liquor by using macroporous resin, and evaporating, concentrating and drying desorbed desorption liquid to obtain an HSAF crude extract. The method has the advantages of simple process, strong operability, low cost and safe and pollution-free production process, and provides reference for large-scale production.

Description

Method for separating and purifying antifungal active substance HSAF in zymogen fermentation liquor
Technical Field
The invention belongs to the technical field of separation and purification of microbial natural products, and particularly relates to a method for selectively separating and purifying an antifungal active substance HSAF from zymogen lysobacter zymogenes.
Background
A thermostable Antifungal Factor (HSAF) is a micromolecule Antifungal substance generated by lysobacter enzymogenes, and has the characteristics of high thermostability, wide antibacterial spectrum, low toxicity and the like. The mode of action of the antifungal agent on pathogenic fungi is completely different from that of the commercial fungicide reported at present, and the polar growth of fungal hyphae is influenced mainly by inhibiting the activity of ceramide synthetase of pathogenic fungi and changing the composition of sphingolipid compounds in the cell membranes of filamentous fungi. Therefore, HSAF has the potential to be developed as a novel bactericide.
The research on HSAF, a novel antifungal active substance, has been receiving global attention and has made some important progress. However, these methods mainly focus on the analysis of biosynthetic genes and pathways, the identification of regulatory factors, the investigation of antagonistic mechanisms, the improvement of fermentation yield, and the like, and little progress has been made in the research of the isolation and extraction of HSAF. At present, the traditional ethyl acetate extraction method is still adopted for separating and purifying the HSAF from the fermentation liquor, the steps are multiple, a large amount of time and labor are consumed, and a large amount of organic solvent (ethyl acetate) with high toxicity is used, so that the physical health hazard and the environmental pollution of operators are easily caused. And the ATB is also a metabolite generated by lysobacter enzymogenes, coexists with the HSAF in an OH11 fermentation liquid, has the content of 258.81mg/L, is a main byproduct in the fermentation production process of the HSAF, is extremely difficult to remove by a conventional method due to the similar structure and chemical properties with the HSAF, seriously influences the separation purity (only about 8 percent) of the final HSAF, and cannot meet the research requirements at all. This is also an important factor limiting its industrial production. Therefore, it is necessary to develop a simple, economical and efficient extraction method to separate and purify the HSAF from the fermentation broth, thereby accelerating the subsequent industrial work of HSAF.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for separating and purifying an antifungal active substance HSAF in zymogen fermentation liquor, which is a method for extracting HSAF and is safe, pollution-free, high in resource utilization rate, low in production cost and beneficial to environmental protection.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for separating and purifying antifungal active substance HSAF in zymogen producing lysobacter comprises the steps of firstly degrading a by-product ATB in OH11 zymogen through illumination, then carrying out static adsorption or dynamic adsorption on the HSAF in the zymogen by utilizing macroporous resin, and evaporating, concentrating and drying desorbed desorption liquid to obtain an HSAF crude extract.
The lysobacter enzymogenes fermentation broth according to the present invention can be obtained by conventional methods in the art, such as, but not limited to: inoculating the activated lysobacter enzymogenes OH11 strain into LB seed liquid, after overnight culture, transferring the strain into a fermentation medium according to the inoculum size of 2-5%, and placing the strain in a shaking table for fermentation culture to obtain HSAF fermentation liquid; the fermentation medium used may be one commonly used in the art, for example: 6.00-10.00 g/L of soybean meal, 7-8 g/L of glucose and CaCl20.65-0.80 g/L; the fermentation medium conditions are 25-30 ℃, 160-200 rpm and 55-60 h.
The lysobacter enzymogenes fermentation broth of the present invention is not particularly required, and in some embodiments, the pH of the fermentation broth is in the range of 6-8, and the concentration of HSAF is 200-500 mg/L.
In some embodiments, the method for degrading the byproduct ATB in the OH11 fermentation broth by using light is specifically as follows: the zymogen fermentation liquid is irradiated for 1 to 5 days under the illumination condition, and the illumination intensity is 1000-. The light according to the invention can be obtained by conventional methods of the prior art, such as direct solar illumination or by means of fluorescent lamps; the invention can degrade ATB in the fermentation liquor in advance by illumination, thereby achieving the purpose of improving HSAF purity.
In a specific embodiment of the invention, the NKA type macroporous resin with the aperture of 20-22 nm is selected as the macroporous resin.
In some embodiments, the macroporous resin in the process of the invention is pretreated before being used for adsorption, and the invention provides a specific pretreatment method: soaking the macroporous resin with absolute ethyl alcohol, removing floating resin fragments, repeatedly soaking for 3-5 times until no white turbidity exists, finally cleaning with deionized water until no ethyl alcohol smell exists, and drying for later use.
In some embodiments, the specific method for performing static adsorption of HSAF in a fermentation broth by using macroporous resin is as follows: adding macroporous resin and fermentation liquid into a container, sealing, adsorbing on a shaking table, and filtering to separate resin and filtrate.
In some embodiments of the invention, when performing static adsorption, the volume to mass ratio of fermentation broth to macroporous resin is 30-60 ml: 0.62g in one particular embodiment is 50 ml: 0.62 g.
In some embodiments of the invention, when static adsorption is performed, the adsorption conditions are 120-200rpm, 27-37 deg.C, 4-8h, and in one embodiment 180rpm, 37 deg.C, 6 h.
In some embodiments of the invention, when performing static adsorption, the adsorption formula is:
Figure BDA0002620521750000031
Figure BDA0002620521750000032
in the formula: q. q.se-adsorption capacity, mg/g; e-adsorption rate,%; c0-initial concentration, mg/L; ce-equilibrium concentration, mg/L; v is volume of fermentation liquor, mL; w-weight of dry resin, g.
When static adsorption is employed, in one embodiment of the invention, the invention also provides a desorption process of static adsorption: and (3) washing the resin collected by filtration with deionized water until no sample liquid remains on the surface, then sucking water by using filter paper, putting the resin into the container again, adding absolute ethyl alcohol, sealing and putting the resin into a shaking table for desorption.
In some embodiments of the invention, when performing static adsorption, the volume-to-mass ratio of the absolute ethanol added to the macroporous resin during desorption is 30-50 mL: 0.62g, preferably 50 mL: 0.62 g.
In some embodiments of the invention, when static adsorption is performed, desorption conditions are 120-200rpm, 27-37 ℃ and 0.5-3h, and in a specific embodiment 180rpm, 37 ℃ and 2 h.
In some embodiments of the invention, when performing static adsorption, the desorption formula:
Figure BDA0002620521750000033
Figure BDA0002620521750000034
in the formula: q. q.sd-desorption capacity, mg/g; d-desorption rate,%; cdThe concentration of HSAF in the stripping liquid is mg/L; vdDesorption solution volume, mL.
In some embodiments, the specific method for dynamic adsorption of HSAF in fermentation broth using macroporous resin is: and (3) loading the macroporous resin into a chromatographic column by a wet method, balancing with deionized water, uniformly loading a fermentation liquid sample, collecting effluent liquid every other column volume (BV), and stopping loading when the concentration of HSAF in the effluent liquid reaches 6-15% of the initial loading concentration.
The macroporous resin is filled into a chromatographic column by a wet method, and the deionized water can be used for balancing.
In some embodiments of the invention, when the dynamic adsorption is performed, the flow rate of the fermentation broth when the macroporous resin adsorbs the HSAF in the fermentation broth is 1-2 BV/h; in a specific embodiment, the flow rate is 2 BV/h.
In some embodiments of the invention, when dynamic adsorption is performed, the loading is 15-28BV, preferably 20-22 BV.
In some embodiments of the invention, the macroporous resin has an adsorption capacity of 20mg/g to 25mg/g for adsorption of the HSAF in the fermentation broth when the dynamic adsorption is performed.
When dynamic adsorption is employed, in one embodiment of the invention, the invention also provides a desorption process of dynamic adsorption: washing the resin which is adsorbed and saturated with deionized water until no sample liquid remains, and desorbing by adopting a two-stage elution procedure; the first stage is as follows: eluting with low concentration ethanol water solution to remove polar or medium polar compounds adsorbed on the resin; and a second stage: desorbing HSAF with high concentration ethanol water solution, and collecting eluate according to column volume.
In some embodiments, the concentration of the low-concentration ethanol aqueous solution is 20-40%, the elution volume is 2-5BV, and the elution flow rate is 1-4 BV/h; in a specific example, the concentration of the aqueous ethanol solution was 40%, the elution volume was 4BV, and the elution flow rate was 2 BV/h.
In some embodiments, the concentration of the high-concentration ethanol aqueous solution is 80-100%, the elution volume is 8-15BV, and the elution flow rate is 1-4 BV/h; in a specific example, the concentration of the aqueous ethanol solution was 80%, the elution volume was 10BV, and the elution flow rate was 2 BV/h. The inventors have found that under such elution concentrations and conditions, the concentration of HSAF in the eluate is at its highest.
In some embodiments of the invention, when dynamic adsorption is performed, desorption adopts a two-stage elution procedure, after washing with 1-3 BV of deionized water, washing with 20-40% by volume of an ethanol aqueous solution with an elution volume of 2-5BV and an elution flow rate of 1-4BV/h, and then eluting with 80-100% by volume of an ethanol aqueous solution with an elution volume of 8-15BV and an elution flow rate of 1-4 BV/h; in a specific example, the column was washed with 40% ethanol in water at an elution volume of 4BV and an elution flow rate of 2BV/h, and then eluted with 80% ethanol in water at an elution volume of 10BV and an elution flow rate of 2 BV/h.
The desorption solution after desorption is evaporated, concentrated and dried to obtain the HSAF crude extract, and the HSAF crude extract can be obtained by adopting the conventional method in the field of evaporation, concentration and freeze drying, for example, by rotary evaporation and concentration. In some embodiments of the invention, the desorption solution evaporation temperature is from 30 to 60 ℃.
The invention also provides a method for calculating the adsorption capacity and the adsorption rate after static adsorption, which comprises the following steps: after filtering and separating the resin and the filtrate, performing ethyl acetate extraction and HPLC detection on the HSAF in the filtrate, and calculating the adsorption capacity and the adsorption rate according to a formula; in one example, the HPLC detection conditions: InterSustain swift C185 μm, 250X 4.6 mm. Mobile phase: solution a (0.025% TFA in water) and solution B (0.025% TFA in acetonitrile), flow rates: 1 mL/min; the sample size is 20 μ L, ultravioletAbsorption value: 318 nm; sample introduction procedure: 0-10min, and mixing the solution B from 5% to 25%; 25min, increasing to 80% B; 26min, increasing to 100%; returning to 5% in 30min, wherein the total proportion of the solution A and the solution B is 100%; the peak area was recorded by establishing a linear equation between the concentration of HSAF and the peak area of 4E-05X +12.46, R2The amount of HSAF in ethyl acetate was calculated as 0.9996.
The invention also provides a purity detection method of the HSAF extract, which comprises the steps of weighing a certain amount of the extract and preparing the extract into a methanol solution (C)m) And the actual content (C) was determined by HPLCh) The purity (%) of HSAF in the extract is equal to Ch/Cm×100％。
The invention also provides a method for removing ATB in HSAF, which is characterized in that ATB in HSAF is degraded by illumination.
In one embodiment of the present invention, the degradation of ATB in HSAF by light irradiation is carried out by exposing HSAF liquid containing ATB to light irradiation for 1-5 days with light intensity of 1000-.
The ATB-containing HSAF liquid of the invention can be any HSAF liquid containing ATB impurities, such as fermentation liquid of lysobacter enzymogenes strain OH 11.
The invention has the beneficial effects that:
(1) the method avoids the defect that the traditional process adopts ethyl acetate and other organic solvents which pollute the environment, selects conventional water and ethanol as the eluent, has safe and pollution-free production process, high resource utilization rate and low production cost, is beneficial to environmental protection, and is a green and environment-friendly method for extracting HSAF;
(2) the technology has the advantages of simple and convenient operation process, stable physical and chemical properties by using NKA type resin, simple and convenient regeneration, repeated recycling, long service cycle and the like, and is more suitable for large-scale industrial extraction of HSAF;
(3) the invention can improve the purity of HSAF by degrading ATB in the fermentation liquor in advance by light, and the content of HSAF obtained after separation and extraction is about 31 percent, which is 3.58 times of that of the prior separation method.
Drawings
FIG. 1 comparison of HPLC chromatograms of fermentation broths after illumination;
FIG. 2 shows adsorption/desorption of HSAF from fermentation broth by different resins;
FIG. 3 static adsorption/desorption kinetics of NKA resin on HSAF;
FIG. 4 is a graph of leakage at different sample flow rates;
FIG. 5 elution profiles at different elution concentrations;
FIG. 6 comparison of HSAF appearance, HPLC chromatograms and purity obtained by different extraction methods; (A) pure HSAF products; (B) NKA adsorption-80% ethanol elution; (C) NKA adsorption-two stage elution; (D) extracting with ethyl acetate; (E) the fermentation liquor which is not irradiated by light is subjected to NKA adsorption-two-stage elution.
Detailed Description
The present invention is described below with reference to specific examples, wherein the reagents used in the examples are those conventionally purchased and the methods used are those conventionally used in the art unless otherwise specified.
The fermentation broth used in the following examples is specifically: inoculating the activated lysobacter enzymogenes OH11 strain into LB seed liquid, culturing overnight, transferring into a fermentation culture medium according to the inoculum size of 2.5%, and fermenting and culturing in a shaking table to obtain HSAF fermentation liquid, wherein the pH range is 6-8, and the concentration of HSAF is about 300 mg/L; fermentation medium used: 8g/L of soybean meal, 7.89g/L of glucose and CaCl20.72 g/L; the fermentation medium conditions were 28 ℃, 180rpm, 58 h.
Example 1: photodegradation for removing ATB in fermentation liquor
800mL of the fermentation broth (HSAF concentration 300mg/L) was placed in a 1L serum bottle and placed in a light incubator with a light intensity of 15000LX for 2 days. Through extraction detection, it can be seen from fig. 1 that after light treatment, ATB in the fermentation broth is gradually degraded and completely disappears after 2 days. The content of HSAF in the fermentation liquor is kept unchanged all the time. Therefore, the ATB in the fermentation liquor can be removed by light degradation, and the method is simple and convenient.
Example 2: comparison of the types of macroporous adsorbent resins
0.62g (dry weight) of pretreated macroporous resins (strong polar resin S-8 and nonpolar resin NKA) with different properties are respectively weighed and placed in a 250mL triangular flask with a plug, 50mL (the concentration is 300mg/L) of HSAF fermentation liquor with ATB in the fermentation liquor removed by photodegradation is respectively added at room temperature, and the mixture is placed in a shaking table, and the temperature is 37 ℃, the rotation speed is 180rpm and the shaking is carried out for 6 hours, so that the full adsorption is achieved. Separating resin and filtrate by using 3 layers of gauze, and extracting and detecting HSAF from the filtrate. Desorption experiments were performed on the adsorbed resins: washing with deionized water for 3 times, drying with filter paper, transferring resin again, placing in a triangular flask, adding 50mL of anhydrous ethanol, placing in a shaking table, desorbing at 37 deg.C and 180rpm for 2 h. The eluate was filtered through a 0.22 μm filter and the concentration of HSAF was determined by HPLC. And finally, calculating the resin adsorption/desorption capacity, the adsorption rate and the desorption rate according to a formula.
As can be seen from FIG. 2, the strongly polar resin S-8 and the nonpolar resin NKA both have a relatively high adsorption capacity for HSAF in the fermentation broth, while for the desorption process, the desorption capacity (1.91mg/g) and the desorption rate (12.81%) of S-8 are very low, and HSAF adsorbed on S-8 is difficult to elute. The NKA type macroporous resin used by the invention has higher adsorption capacity and desorption capacity to HSAF in fermentation liquor, and the adsorption capacity and the desorption capacity are respectively 19.59mg/g and 15.86mg/g, and the adsorption rate and the desorption rate are 80.99 percent and 80.92 percent.
Example 3: NKA static adsorption and desorption kinetics study
Weighing 0.62g of dry resin NKA in a 250mL triangular flask, adding 50mL of HSAF fermentation liquor from which ATB in the fermentation liquor is removed by photodegradation, placing the HSAF fermentation liquor in a shaking table at 37 ℃, oscillating at 180rpm, sampling once every 20min, performing HSAF extraction detection, and drawing an HSAF static adsorption kinetic curve; and simultaneously, washing the adsorbed NKA resin with deionized water for 3 times, and after the water is absorbed by filter paper, adding 50mL of absolute ethyl alcohol, placing the mixture in a shaking table, oscillating the mixture at 37 ℃ and 180rpm, sampling every 10min, carrying out HPLC (high performance liquid chromatography) detection, and drawing an NKA static desorption kinetic curve.
As can be seen from FIG. 3, HSAF adsorbs more rapidly on NKA resin for the first 20min, and then enters a slow process until 240min reaches adsorption equilibrium. The desorption process is relatively quick, and the HSAF desorption reaches the balance in 60 min.
Example 4: comparison of different sample loading flow rates on HSAF in NKA type resin dynamic adsorption fermentation liquor
A glass column (10 mm. times.300 mm) was packed with 1.55g of NKA dry resin and the column volume was 7.2 mL. And (3) enabling the fermentation liquor subjected to photodegradation to remove ATB in the fermentation liquor to pass through a chromatographic column at different flow rates (0.5 BV/h, 1.0 BV/h, 2.0 BV/h, collecting effluent according to the column volume (BV), and performing HSAF extraction detection.
As can be seen from FIG. 4, when the concentration of HSAF in the effluent reached 10% of the initial concentration, i.e. the leakage point, the loading was stopped, and thus it was determined that the loading volumes of the fermentation broths were 28, 22, 20 and 15BV, respectively, at which time the adsorption capacities of NKA to HSAF were 33.37, 25.53, 24.14 and 17.52mg/g, respectively. The results show that: when the sample feeding flow rate is too high, the adsorption capacity is reduced; when the flow rate is too slow, a lot of time is consumed. The flow rate adopted by the invention is 1BV/h-2BV/h, the reduction of the adsorption capacity is not large, particularly when the sample loading flow rate is 2BV/h, the sample loading volume is 20BV, namely 144mL, and the adsorption capacity is 24.14 mg/g.
Example 5: comparison of ethanol aqueous solutions with different concentrations on HSAF in NKA-type resin dynamic desorption fermentation liquor
After completion of the adsorption in example 4, the resin was washed with 2BV of deionized water followed by elution with different concentrations of aqueous methanol (0%, 20%, 40%, 60%, 80%, 100%) for a total volume of 10 BV. The eluate was collected according to column volume and tested for concentration of HSAF and the elution curve was plotted as shown in FIG. 5. When the concentration of the ethanol aqueous solution is less than or equal to 40 percent, the HSAF on the NKA resin can hardly be eluted; the concentration range of the invention can well elute HSAF, particularly when the concentration is 80%, the HASF concentration in the eluent reaches the highest, and the desorption rate reaches the best.
Example 6: influence of two-stage elution program on dynamic desorption of HSAF in fermentation broth by NKA-type resin
After completion of the adsorption in example 4, the resin was washed with 2BV of deionized water, followed by washing with 4BV of 40% aqueous ethanol at an elution flow rate of 2BV/h to remove polar compounds adsorbed on the NKA resin, and finally eluting HSAF adsorbed on the NKA resin with 10BV of 80% aqueous ethanol at an elution flow rate of 2BV/h, and collecting the HSAFCollecting the eluent. And (3) rotationally evaporating the collected eluent at 40 ℃, removing an ethanol eluent, and placing the eluent in a freeze dryer to remove water to obtain an HSAF extract and carrying out purity detection: weighing 1mg of HSAF extract, preparing into 1000mg/L methanol solution, and detecting actual content (C) by HPLCh) The purity (%) of HSAF in the extract is equal to ChPer 1000X 100%. And compared to the 80% ethanol concentration elution of example 5.
As can be seen from fig. 5: using the two-stage elution procedure (40% -80%), the elution profile of HSAF was nearly identical to that of 80% ethanol, but the crude HSAF extract was slightly lighter in color and had a purity of 31.07% for HSAF, which was 54.12% higher than that of 80% ethanol (20.16) (fig. 6B, C).
Comparative example 1: extracting with ethyl acetate
Sucking 3-15 mL fermentation liquid into a centrifuge tube, dropwise adding concentrated HCl until the pH is 3.0, and adding 0.45g CaCl into the mixed solution2And 3mL of ethyl acetate, placing the mixture on a vortex mixing oscillator at 2000rpm for reaction for 1min, and fully contacting the organic solvent with the fermentation liquor; centrifuging at 8000rpm for 5min to separate the fermentation liquid and organic solvent into two phases, collecting supernatant as HSAF organic solvent layer, sucking 1mL, blow drying, adding 500 μ L methanol to dissolve, and detecting the content by HPLC.
Results of comparative example versus examples 5 and 6 the comparison is shown in figure 6: the appearance of the HSAF extracts obtained by the NKA resin adsorption of the embodiment 5 and the embodiment 6 is light yellow, and the ethyl acetate extract is dark brown, which shows that the ethyl acetate extraction can extract more pigment together with the HSAF; ② the purity of HSAF extracted by NKA adsorption method in the embodiment 6 can reach 31.07%, which is 3.58 times of that of ethyl acetate method (8.67%), which shows that the final HSAF content can be obviously improved by NKA resin adsorption.
Comparative example 2:
the same procedure as in example 4 and example 6 is followed, except that the fermentation broth is subjected to the dynamic adsorption only to remove ATB from the fermentation broth without photodegradation, but directly used for the dynamic adsorption. As a result, as shown in FIGS. 6(C) and (E), the HSAF product obtained without light treatment was deep yellow in appearance and only 18.38% pure HSAF, which was much lower than that obtained with light treatment.

Claims (10)

1. A method for separating and purifying antifungal active substances HSAF in zymogen fermentation liquor is characterized in that firstly, a by-product ATB in OH11 fermentation liquor is degraded through illumination, then, macroporous resin is utilized to carry out static adsorption or dynamic adsorption on the HSAF in the fermentation liquor, and desorbed desorption liquid is evaporated, concentrated and dried to obtain an HSAF crude extract.
2. The method according to claim 1, wherein the visible light degradation of the byproduct ATB in the OH11 fermentation broth is specifically: the zymogen fermentation liquid is irradiated for 1 to 5 days under the illumination condition, and the illumination intensity is 1000-.
3. The method according to claim 1, wherein the macroporous resin is NKA-type macroporous resin with a pore size of 20-22 nm.
4. The method as claimed in claim 1, wherein the static adsorption of HSAF in the fermentation broth by macroporous resin is carried out by the following steps: adding macroporous resin and fermentation liquor into a container, sealing, placing on a shaking table for adsorption, and filtering to separate resin and filtrate; preferably, when the static adsorption is carried out, the volume-mass ratio of the fermentation liquor to the macroporous resin is 30-60 ml: 0.62 g; more preferably, the volume-to-mass ratio of the fermentation liquid to the macroporous resin is 50 ml: 0.62 g; preferably, when the static adsorption is carried out, the adsorption conditions are 120-200rpm, 27-37 ℃ and 4-8h, more preferably 180rpm, 37 ℃ and 6 h.
5. The method according to claim 1, characterized in that, when static adsorption is employed, the desorption process of static adsorption: washing the filtered and collected resin with deionized water until no sample liquid remains on the surface, then sucking water with filter paper, putting the resin into a container again, adding absolute ethyl alcohol, sealing and putting the resin into a shaking table for desorption; preferably, the volume mass ratio of the absolute ethyl alcohol to the macroporous resin added during desorption is 30-50 mL: 0.62g, more preferably 50 mL: 0.62 g; preferably, the desorption conditions are 120-200rpm, 27-37 ℃ and 0.5-3h, more preferably 180rpm, 37 ℃ and 2 h.
6. The method as claimed in claim 1, wherein the specific method for dynamic adsorption of HSAF in the fermentation broth by using macroporous resin is as follows: loading the macroporous resin into a chromatographic column by a wet method, balancing with deionized water, uniformly loading a fermentation liquid sample, collecting effluent liquid every other column volume (BV), and stopping loading when the concentration of HSAF in the effluent liquid reaches 6-15% of the initial loading concentration; preferably, the flow rate of the fermentation liquor is 1-2BV/h when the HSAF in the fermentation liquor is adsorbed by the macroporous resin; more preferably the flow rate is 2 BV/h; preferably, the loading is from 15 to 28BV, more preferably from 20 to 22 BV; preferably, the adsorption capacity of the macroporous resin for adsorbing the HSAF in the fermentation liquor is 20 mg/g-25 mg/g.
7. The method according to claim 1, characterized in that the desorption process of dynamic adsorption: washing the resin which is adsorbed and saturated with deionized water until no sample liquid remains, and desorbing by adopting a two-stage elution procedure; the first stage is as follows: eluting with low concentration ethanol water solution to remove polar or medium polar compounds adsorbed on the resin; and a second stage: desorbing HSAF with high concentration ethanol water solution, and collecting eluate according to column volume.
8. The method according to claim 7, wherein the concentration of the low-concentration ethanol aqueous solution is 20-40%, the elution volume is 2-5BV, and the elution flow rate is 1-4 BV/h; preferably, the concentration of the ethanol water solution is 40 percent, the elution volume is 4BV, and the elution flow rate is 2 BV/h; preferably, the concentration of the high-concentration ethanol aqueous solution is 80-100%, the elution volume is 8-15BV, and the elution flow rate is 1-4 BV/h; more preferably, the concentration of the ethanol aqueous solution is 80%, the elution volume is 10BV, and the elution flow rate is 2 BV/h.
9. The method as claimed in claim 1, wherein the pH of the fermentation broth is in the range of 6-8, and the concentration of HSAF is 200-500 mg/L; preferably, the desorbed desorption solution is subjected to rotary evaporation concentration and freeze drying to obtain an HSAF crude extract; more preferably, the desorption solution has an evaporation temperature of 30 to 60 ℃.
10. A method for removing ATB in HSAF is characterized in that ATB in HSAF is degraded by illumination; preferably, the HSAF liquid containing ATB is irradiated for 1-5 days under the illumination condition with the illumination intensity of 1000-.
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