A method for preparing paraherquamide A by biological fermentation culture

Abstract

The present invention discloses a method for preparing paraherquamide A by biological fermentation culture. The penicillium fungus producing paraherquamide A of the present invention is characterized in that it is named Penicillium sp., strain number KWF31, and deposit number CCTCC NO.M 20221625. The present invention screens and obtains a penicillium strain capable of producing paraherquamide A, and the present invention optimizes the culture conditions so that the yield of paraherquamide A during biological fermentation culture is improved, and the maximum yield is increased by about 6 times compared with the initial yield (42 mg / L). The separation and purification method is further optimized to obtain a relatively pure product.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing paraherquamide A through biological fermentation and cultivation. Background Art

[0002] It is a new type of oral short-acting insecticide for sheep developed by Zoetis, an animal health drug and vaccine company. The active ingredients of this drug are Derquantel (10 mg / mL) and Avermectin (1 mg / mL). It can kill worms that have developed resistance to insecticides such as levamisole, benzimidazole, macrolide and triclobendazole, such as Haemonchus contortus, Teledorsagia circumcincta, Trichostrongylus colubriformis, etc. The standard dosage is 1mL / 5kg (volume of drug solution / sheep weight). For an adult sheep weighing 50kg, 0.5g of Dequent is required for each sheep. According to statistics from the Food and Agriculture Organization of the United Nations, the number of sheep in the world exceeds 1 billion. Therefore, it is conservatively estimated that the potential demand for Dequent is hundreds of tons or even higher, and the market demand is huge.

[0003]

[0004] Dequent is a chemical derivative of fungal metabolite paraherquamide A (abbreviated as PHQ A, CAS No. 77392-58-6, chemical formula as shown in Formula I), which can be obtained by paraherquamide A through amide carbonyl reduction and deoxygenation (Mauragis Micheal A, Lipton Micheal F, Veley Micheal F. Process Development and Preparation of 2-Deoxyparaherquamide: Implementation of a Selective Reduction of Secondary Amides on a Kilogram Scale. Organic Process Research & Development, 2002.6, 192-196.), which is the current process route for industrial production of dequent. The yield of this chemical reaction reaches more than 75%, so the industrial raw material supply of dequent is mainly affected by the output of paraherquamide A. The chemical structure of paraherquamide natural products is complex. Although there is a method for chemically synthesizing paraherquamide A using industrial raw materials isoprene or isoprene epoxide, the chemical reaction steps are as many as 46 (Williams Robert M, Cao Jianhua, Tsujishima H, et al. Asymmetric, stereocontrolled total synthesis of paraherquamide A. Journal of American Chemistry Society, 2003, 125, 12172-12178), the yield is extremely low, and it is not suitable for industrial production. The production of paraherquamide A by microbial fermentation is currently the only way to obtain this compound on a large scale. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for preparing paraherquamide A through biological fermentation culture.

[0006] The invention firstly provides a paraherquamide A-producing penicillium, which is named as Penicillium sp., with strain number KWF31 and preservation number CCTCC NO.M 20221625.

[0007] The present invention further provides a method for preparing paraherquamide A by biological fermentation culture, comprising the following steps: inoculating the Penicillium KWF31 in a culture medium and performing fermentation culture, and after the fermentation culture is completed, separating and purifying to obtain the compound paraherquamide A,

[0008] Wherein, the carbon source in the culture medium is at least one of sucrose, glucose, maltose and soluble starch;

[0009] The nitrogen source is at least one of peptone, soybean cake powder, yeast powder and nitrate;

[0010] The inoculum size was such that the final spore concentration was 10 5 ~10 7 sp / mL;

[0011] The fermentation temperature is 20-30°C, and the initial pH during the fermentation is 6.0-9.0.

[0012] The carbon source in the culture medium includes soluble starch, and the mass volume ratio of the soluble starch is 0.5% to 4%; preferably, the mass volume ratio of the soluble starch is 2% to 2.5%. More preferably, the carbon source in the culture medium includes soluble starch and glucose, wherein the mass volume ratio of the glucose is 0.5% to 4%.

[0013] The nitrogen source includes yeast powder, and the mass volume ratio of the yeast powder is 0.3% to 2.4%; preferably, the mass volume ratio of the yeast powder is 0.9% to 1.8%. More preferably, the nitrogen source includes yeast powder and nitrate, wherein the mass volume ratio of the nitrate is 0.2% to 1.6%; preferably, the mass volume ratio of the nitrate is 0.6% to 1.2%; more preferably, the mass volume ratio of the nitrate is 0.6% to 0.8%.

[0014] Preferably, the culture medium further includes inorganic salts, the inorganic salts include at least one of MgSO4, MnCl2, ZnSO4 and CoCl2, the mass volume ratio concentration of MgSO4 is 0.0125% to 0.2000%; the mass volume ratio concentration of MnCl2 is 0.02% to 0.04%; the mass volume ratio concentration of ZnSO4 is 0.04% to 0.08%; the mass volume ratio concentration of COCl2 is 0.0025% to 0.0400%. More preferably, the mass volume ratio concentration of CoCl2 is 0.0050% to 0.0100%.

[0015] More preferably, the inorganic salt includes MgSO4, MnCl2, ZnSO4 and CoCl2, wherein the mass volume ratio concentration of MgSO4 is 0.1%, the mass volume ratio concentration of MnCl2 is 0.004%, the mass volume ratio concentration of ZnSO4 is 0.004%, and the mass volume ratio concentration of CoCl2 is 0.0005%.

[0016] The inoculum size for fermentation culture was 10 5 ~10 6 sp / mL; the fermentation culture temperature is 20-27°C; the initial pH during fermentation culture is 7.0-7.5.

[0017] During separation and purification, a macroporous adsorption resin is used for adsorption, and then ethanol with a volume ratio concentration of 20% is used to wash away impurities, and then ethanol with a volume ratio concentration of 80% is used for desorption; the model of the macroporous adsorption resin is SP825L.

[0018] Each component is calculated by mass volume ratio. The optimal medium formula after optimization is SGY (%): soluble starch 3.1%, glucose 1.28%, yeast powder 1.44%, sodium nitrate (NaNO3) 0.8%, MgSO4 0.1%, MnCl2 0.004%, ZnSO4 0.004%, CoCl2 0.0005%. The culture condition is the final inoculation concentration of 10 5 ~10 6 sp / mL, fermentation temperature 25℃, initial pH 7.0.

[0019] The present invention screened and obtained a Penicillium strain capable of producing paraherquamide A, and the present invention optimized the culture conditions so that the yield of paraherquamide A during biological fermentation culture was increased, and the maximum yield was increased by about 6 times compared with the initial yield (42 mg / L). The separation and purification method was further optimized to obtain a relatively pure product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony picture of strain KWF31.

[0021] Figure 2 This is a graph showing the effect of different carbon sources on the fermentation level.

[0022] Figure 3 This is a graph showing the effect of soluble starch content on fermentation level.

[0023] Figure 4 This is a graph showing the effect of glucose content on fermentation level.

[0024] Figure 5 This is a graph showing the effects of different nitrogen sources on fermentation levels.

[0025] Figure 6 This is a graph showing the effect of yeast powder content on fermentation level.

[0026] Figure 7 This is the result graph of the effect of NaNO3 content on fermentation level.

[0027] Figure 8 This is a graph showing the effects of different inorganic salts on the fermentation level.

[0028] Fig. 9 This is the result graph showing the effect of MgSO4 content on fermentation level.

[0029] Fig.10 This is a graph showing the effect of different trace element contents on the fermentation level, where A is the Mn element, B is the Zn element, and C is the Co element.

[0030] Fig.11 This is a graph showing the effect of inoculation spore concentration on fermentation level.

[0031] Fig.12 This is a graph showing the effect of temperature on fermentation level.

[0032] Fig.13 This is the result graph of the effect of initial pH on the fermentation level.

[0033] Fig.14 This is the result graph showing the effect of different ethanol elution ratios on MAR desorption PHQA.

[0034] Fig.15 The liquid phase analysis diagram after elution with different ethanol concentrations, wherein A: bacterial solution; B: 20% ethanol concentration; C: 80% ethanol concentration. The peak indicated by the arrow is the target product paraherquamide A. DETAILED DESCRIPTION

[0035] The strain used in the fermentation of this application is Penicillium sp. KWF31, which was isolated from a marine sediment sample and has been deposited in the China Center for Type Culture Collection (CCTCC) on October 21, 2022, with the deposit number CCTCCNO.M 20221625.

[0036] In the present application, the components added to the culture medium, including carbon sources, nitrogen sources, and inorganic salts, are all measured by mass-to-volume ratio.

[0037] Example 1

[0038] Collection, isolation and identification of strain KWF31

[0039] Sediment samples were collected from the South China Sea (16°43′43.64″N, 110°28′23.84″E, depth 1352m). The sediments were diluted with sterile seawater and spread on 2216E solid culture medium and cultured at 25°C. Single colonies were picked according to the shape and color of the plaques, and a filamentous fungus was purified by plate streak separation, named KWF31, and then moved to a slant and stored at 4°C for future use.

[0040] On the culture medium, the colony of strain KWF31 grew rapidly, with a loose, flat texture, velvety radial shape, and neat edges; the conidia were spherical, dark green, and purple-pink on the outermost edge, such as Figure 1 shown.

[0041] The strain DNA was extracted, and the ITS fragment was amplified from the extracted DNA gene, and the primers used were ITS1: TCCGTAGGTGAACCTGCGG, and ITS4: TCCTCCGCTTATTGATATGC. The amplified ITS sequence was 546 bp, and the sequence was shown in SEQ ID No.1.

[0042] The ITS sequence was compared with the NCBI database data, and the similarity between KWF31 and multiple strains of Penicillium sp. in the database was over 98%. It was identified as Penicillium sp. and named Penicillium sp., strain number KWF31. It was deposited in the China Center for Type Culture Collection (CCTCC) in Wuhan, China, with a deposit date of October 21, 2022, and a deposit number of CCTCC NO.M 20221625.

[0043] Example 2

[0044] Single factor optimization experiment of fermentation medium: carbon source optimization.

[0045] The carbon source in the culture medium is not only the main energy source for microbial growth, but also the main element in the composition of microbial cells and metabolites. In this experiment, six carbon sources commonly used in microbial culture: sucrose, glucose, soluble starch, maltose, lactose, and mannitol were used to replace the carbon source in the basal culture medium (Czapek medium: sucrose 30.0g, sodium nitrate 3.0g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate (MgSO4·7H2O) 0.5g, potassium chloride 0.5g, ferrous sulfate 0.01g, fixed to 1L, pH natural or 7.0-7.2). The carbon source concentration was set to 3.0% (w / v, the same below), and the other components of the basal culture medium remained unchanged. The effects of different carbon sources on the fermentation production of Paraherquamide A by KWF31 were investigated, and the control carbon source was sucrose in the basal culture medium formula. In this experiment, the fermentation titer of Paraherquamide A in the control group was taken as 100%, and the fermentation titer of other experimental groups was calculated in a standardized manner. The fermentation results are shown in the figure. Figure 2 shown.

[0046] Depend on Figure 2 It can be seen that not all carbon sources can produce Paraherquamide A. When the carbon source is lactose, it is basically not produced. When soluble starch is used as the carbon source, the yield of Paraherquamide A is the highest. Therefore, soluble starch is selected as the best carbon source. On this basis, the formula content of soluble starch is further investigated, and 0.5% soluble starch content is added as a control. The fermentation results are shown in Figure 2. Figure 3 shown.

[0047] Depend on Figure 3 It can be seen that as the starch content increases, the titer also increases. When the soluble starch content is 2.5%, the titer is the highest, and when the content continues to increase, the titer gradually decreases. It may be because too high a content of soluble starch inhibits the production of secondary metabolites by the bacteria, which is not conducive to the accumulation of Paraherquamide A. Therefore, the soluble starch content is selected to be 2.5%.

[0048] Microorganisms need different carbon sources during the fermentation process, fast carbon sources and slow carbon sources. Fast carbon sources are mostly small molecule carbon sources, which are easily absorbed and utilized, and are conducive to the rapid growth of microorganisms in the early stage, but the composition is simple and not conducive to the synthesis of metabolites. Although slow carbon sources are used more slowly, they can provide more energy under the same amount, which is conducive to the synthesis of secondary metabolites. Soluble starch is a large molecular carbon source. As a slow carbon source in KWF31 fermentation, it is conducive to the synthesis of Paraherquamide A and consists of Figure 2It can be seen that the effect of glucose on fermentation is second only to soluble starch, so it is necessary to examine the effects of soluble starch and glucose on fermentation. The control group was 2.5% soluble starch and 0% glucose. The experimental results are as follows Figure 4 shown.

[0049] Figure 4 The results showed that adding fast carbon source glucose on the basis of adding 2.5% soluble starch as the main carbon source was beneficial to the synthesis of Paraherquamide A. The titer at 1.0% glucose content was 50% higher than that without adding glucose. However, with the increase of glucose, the synthesis of Paraherquamide A did not increase significantly. Taking into account the economic and environmental aspects, the final combination of composite carbon source addition was determined to be 2.5% soluble starch and 1.0% glucose.

[0050] Example 3

[0051] Single factor optimization experiment of fermentation medium: nitrogen source optimization.

[0052] Nitrogen source is mainly used by microorganisms to synthesize their own proteins and genetic material, and is also one of the sources of nitrogen-containing metabolites. Common nitrogen sources can be divided into organic nitrogen sources and inorganic nitrogen sources. The organic nitrogen sources explored in this experiment are peptone, soybean cake powder, urea, and yeast powder; the inorganic nitrogen sources are NaNO3, (NH4)2SO4, and NH4C1. On the basis of not adding nitrogen source to the basal culture medium, nitrogen sources with the same mass concentration of 0.3% (w / v, the same below) were added to investigate the effects of different nitrogen sources on KWF31 fermentation, where the control is NaNO3 in the basal fermentation formula. The fermentation results are as follows: Figure 5 shown.

[0053] Depend on Figure 5 It can be seen that when only urea, (NH4)2SO4, and NH4C1 were added, almost no paraherquamide A was produced, indicating that the strain has a certain selectivity for the nitrogen source of Paraherquamide A. Not all nitrogen sources can produce this compound. When yeast powder was added as a nitrogen source, the yield of Paraherquamide A was the highest. Therefore, yeast powder was selected as the best nitrogen source. On this basis, the formula content of yeast powder was further investigated, and 0.3% yeast powder was added as a control. The fermentation results are shown in Figure 2. Figure 6 shown.

[0054] Depend on Figure 6It can be seen that when the nitrogen source content is low, the increase in nitrogen source content can provide sufficient nutrients for the bacteria to produce metabolites, so the fermentation titer increases with the increase in nitrogen source content, but when the nitrogen source content increases to a certain extent, the increase in nitrogen source content will reduce the fermentation titer. The experiment found that too high a yeast powder content will seriously inhibit the growth of the bacteria, which is not conducive to the accumulation of Paraherquamide A, so the yeast powder content is selected to be 1.2%.

[0055] Organic nitrogen source yeast powder is a delayed nitrogen source that is beneficial to the synthesis of Paraherquamide A. In the early stage of microbial growth, fast-acting nitrogen sources are usually beneficial to the growth of bacteria. Therefore, this experiment considers adding fast-acting nitrogen sources and long-acting nitrogen sources together. Figure 5 It can be seen that the effect of NaNO3 on fermentation is second only to yeast powder. The effect of yeast powder and NaNO3 on fermentation was investigated. The control group was 1.2% yeast powder and 0% NaNO3. The experimental results are as follows Figure 7 shown.

[0056] from Figure 7 It can be seen that the fermentation titer of the strain is significantly improved by adding NaNO3 on the basis of yeast powder as the main nitrogen source, and the titer reaches the highest when the NaNO3 content is 0.8%. Therefore, 1.2% yeast powder content and 0.8% NaNO3 content are selected as the best composite carbon source combination. The existing medium SGY-1.0 after optimizing carbon and nitrogen sources: 2.5% soluble starch, 1.0% glucose, 1.2% yeast powder, and 0.8% sodium nitrate (NaNO3).

[0057] Example 4

[0058] Single factor optimization experiment of fermentation medium: inorganic salt optimization.

[0059] Inorganic salts have a certain influence on the growth of microorganisms and the production of metabolites during the fermentation process. They participate in the composition of cell structural substances, constitute the active groups of enzymes, activate enzyme activity, and regulate cell osmotic pressure. Inorganic salts are divided into two categories: trace elements and macroelements. + , P 5+ , Ca 2+ Mg 2+ etc. are major elements; Co 2+ , Fe 2+ , Mn 2+ , Cu 2+ 、Zn 2+ , B 3 + 、Mo 6+In this experiment, different types of inorganic salts were added, with the concentration of macroelements at 0.05% (w / v) and trace elements at 0.001% to the previously optimized medium SGY-1.0. The control group was set without inorganic salts to investigate the effect of inorganic salt types on fermentation results. Figure 8 shown.

[0060] Depend on Figure 8 It is known that the macronutrient Mg 2+ The most significant promoting effect may be related to the fact that magnesium is a coenzyme or activator of some enzymes. MnCl2, ZnSO4 and CoCl2 also have a positive effect on the fermentation synthesis of Paraherquamide A. The other inorganic salts have an inhibitory effect on the synthesis of Paraherquamide A. Therefore, based on the medium SGY-1.0, the optimal addition amount of the macroelement MgSO4 and the trace elements MnCl2, ZnSO4 and CoCl2 was considered respectively, and the control group was set without adding inorganic salts. The experimental results are as follows Fig. 9 , Fig.10 shown.

[0061] The experimental results showed that adding Mg to the culture medium 2+ 、Co 2+ , Mn 2+ and Zn2 + It can significantly improve the fermentation titer of Paraherquamide A. It has been verified experimentally that Mg 2+ Addition amount 0.1%, Mn 2+ 、Zn 2+ The addition amount is 0.004%, Co 2+ The fermentation titer of Paraherquamide A increased the most when the addition amount was 0.0005%. The medium SGY-2.0 was obtained by single factor optimization, and its formula was 2.5% soluble starch, 1.0% glucose, 1.2% yeast powder, 0.8% sodium nitrate (NaNO3), 0.1% MgSO4, 0.004% MnCl2, 0.004% ZnSO4, and 0.0005% CoCl2.

[0062] Example 5

[0063] Response surface optimization experiment of fermentation medium.

[0064] The optimal fermentation medium screened out by single-factor experiments is only a collection of the optimal conditions of each single factor. It is impossible to clarify the interaction between the factors and obtain the optimal ratio of the fermentation medium components. Therefore, it is necessary to further optimize the fermentation medium through response surface experiments.

[0065] 1. Plackeet-Burman experiment

[0066] In this experiment, eight factors (soluble starch, glucose, yeast powder, NaNO3, MgSO4, MnCl2, ZnSO4, CoCl2) in the culture medium were selected and numbered X1 to X8. Two levels were selected for each influencing factor, where "1" represents a high level and "-1" represents a low level. The software Design expert 8.0.2 was used to design the experiment, and the first-order optimization was performed through the eight-factor two-level Plackett-Burman experimental design (N=12). Taking the fermentation titer of Paraherquamide A as the investigation target, the main factors affecting the fermentation production of Paraherquamide A by strain KWF31 were screened. The process and results of the experiment are shown in Tables 1, 2, 3 and 4.

[0067] Table 1 Plackett-Burman experimental design factors and levels

[0068]

[0069]

[0070] Table 2 Plackett-Burman experimental design and results

[0071] serial number <![CDATA[X1]]> <![CDATA[X2]]> <![CDATA[X3]]> <![CDATA[X4]]> <![CDATA[X5]]> <![CDATA[X6]]> <![CDATA[X7]]> <![CDATA[X8]]> Fermentation titer (μg / mL) 1 1 1 -1 1 1 1 -1 -1 220.36±3.65 2 1 -1 1 1 -1 1 1 1 234.26±0.51 3 1 -1 1 1 1 -1 -1 -1 248.00±6.72 4 1 1 -1 -1 -1 1 -1 1 218.06±8.85 5 -1 -1 1 -1 1 1 -1 1 207.86±2.07 6 -1 1 -1 1 1 -1 1 1 215.33±4.61 7 -1 -1 -1 1 -1 1 1 -1 192.46±6.57 8 1 -1 -1 -1 1 -1 1 1 221.71±13.34 9 -1 1 1 -1 1 1 1 -1 230.13±11.33 10 -1 -1 -1 -1 -1 -1 -1 -1 192.95±2.97 11 1 1 1 -1 -1 -1 1 -1 245.97±15.04 12 -1 1 1 1 -1 -1 -1 1 234.05±2.49

[0072] Table 3 Statistical analysis of regression equation errors

[0073] Statistical items Numeric Standard Deviation (Std.Dev) 5.43 Mean 221.77 CV% 2.45 Prediction Error Sum of Squares (PRESS) 1417.84 <![CDATA[R 2 (R-Squared)]]> 0.9753 <![CDATA[Adjusted R 2 (Adj R-Squared)]]> 0.9095 <![CDATA[Predicted R 2 (Predicted R-Squared)]]> 0.6051 Signal-to-noise ratio (Adeq Precision) 12.349

[0074] The P value (0.0243) of the regression model obtained in Table 4 is less than 0.05, indicating that the model is significant, that is, the model has a good fit in the entire regression area studied; R 2 is the multiple correlation coefficient. The larger its value is, the better the experimental correlation is. 2 =0.9753>0.9, indicating that the experimental correlation is relatively good; the correction determination coefficient R 2 adj =0.9095, indicating that this regression model can explain 90.95% of the changes in the response values; the reliability and accuracy of the experiment are related to the coefficient of variation (CV), the smaller the latter value, the higher the former, and the CV value is 2.45%, indicating that the reliability and accuracy of the Plackeet-Burman experiment are very high; precision (AdeqPrecision) refers to the ratio of effective signal to noise, and a value greater than 4 is considered reasonable. The precision of this experiment reached 12.349.

[0075] Table 4 Significance analysis of Plackett-Burman experiment

[0076]

[0077]

[0078] Note: If P <= 0.05, it means that the influence of the factors in the model is significant.

[0079] As can be seen from Table 2, the fermentation titer of group 3 was the highest, reaching 248.00 μg / mL; the fermentation titer of group 10 was the lowest, only 192.95 μg / mL. The experimental data were analyzed by regression analysis using Design expert software, and the multivariate linear regression equation was obtained:

[0080] Y=221.77+9.63X1+5.55X2+11.62X3+2.31X4+2.14X5-4.57X6+1.55X7+0.12X8 (1)

[0081] From the experimental significance analysis results in Table 4, it can be seen that the factors that have the greatest impact on the fermentation titer are yeast powder X3 (P = 0.0051), soluble starch X1 (P = 0.0087), and glucose X2 (P = 0.0384), while the P values ​​of the other five factors are all greater than 0.05, and have no significant effect on the fermentation titer. It can be seen that yeast powder, soluble starch and glucose are the key factors affecting the fermentation titer. If the regression coefficient in the regression equation is a positive value, it indicates a positive effect, and if it is a negative value, it indicates a negative effect. Therefore, among the factors affecting the fermentation titer in this experiment, MnCl2 (X6) has a negative effect, that is, the fermentation titer decreases with the increase of X6; except for X6, all other factors have positive effects, that is, as each factor increases, the fermentation titer also increases. Therefore, in the subsequent design experiments, the value of the positive effect factor should be increased, and the value of the negative effect factor should be reduced.

[0082] 2. Steepest Climbing Experiment

[0083] Through the Plackeet-Burman experimental design, the significant factors affecting the production of Paraherquamide A by marine fungus KWF31 were screened as yeast powder, soluble starch and glucose. After obtaining the contribution of each significant factor, in order to ensure the accuracy and reliability of the response surface experiment, it is necessary to approach the center point through the steepest climbing test. Taking the three significant factors as the investigation objects, the climbing step length was designed according to the effect coefficient of each factor in the multivariate linear equation (1) fitted by Design expert. The values ​​of other influencing factors adopted the optimal conditions of single factor experiment, among which NaNO3 was 8.0g / L, MgSO4 was 1.0g / L, CoCl2 was 0.005g / L, MnCl2 and ZnSO4 were both 0.04g / L. The experimental design and results are shown in Table 5.

[0084] Table 5 Steepest climbing test design and results

[0085]

[0086]

[0087] According to the results in Table 5, the fermentation titer of Paraherquamide A reached the highest value when the initial value increased by 5 steps, and the fermentation titer reached 257.58 mg / mL, and the yield was further increased by 12.3%, which was 6.13 times the yield of the original Czapek medium. This is consistent with the expectations of the previous PB experimental results. As the step length increased, the fermentation titer decreased instead, which may be because the culture medium was too thick and not conducive to the growth of strain KWF31. Therefore, 30 g / L soluble starch, 13.5 g / L glucose, and 14.5 g / L yeast powder were used as the center points of the response surface experiment.

[0088] 3. Box-Behnken Design Experiment

[0089] According to the results of the steepest climbing experiment, 30g / L soluble starch, 13.5g / L glucose, and 14.5g / L yeast powder were used as the zero level of the response surface experiment design. The three factors were used in the Box-Behnken design experiment method. By constructing a multiple regression equation model, the relationship between the response value and the variable was fitted, and the interaction between the yield and the variable was intuitively displayed in a 3D graphic manner to optimize the fermentation medium components of the wild strain KWF31, so that the fermentation unit of Paraherquamide A was higher. This experiment was designed with the help of the statistical analysis software Design expert 8.0.2. In the experiment, three factors were taken at 3 levels, respectively represented by "-1, 0, 1". The true value and coded value of each factor are shown in Table 6. The fermentation efficiency values ​​of 17 groups of samples after fermentation are shown in Table 7.

[0090] Table 6 Experimental factor level coding and level setting

[0091]

[0092] Table 7 Box-Behnken design experiment results

[0093]

[0094]

[0095] Table 8 ANOVA analysis of Box-Behnken design experimental results

[0096]

[0097] It can be seen from Table 8 that the P values ​​of soluble starch in the first square term are all less than 0.01, indicating that soluble starch has a very significant effect on the fermentation titer of Paraherquamide A; the determination coefficient R 2 =0.8329, which means that 83.29% of the experimental results can be explained by this model, so the model has a high degree of fit. According to the design-expert 8.02 analysis software, the following regression equation is obtained:

[0098] Y=215.49+11.5X1+1.06X2+0.85X3-4.23X1X12-3.53X1X3+0.94X2X3+7.79X1 2 +3.48X2 2 -14.94X3 2 (2)

[0099] The best optimization conditions and prediction results were obtained using design-expert8.02 software. The optimal conditions are when X1=31.00, X2=12.80, and X3=14.44, and the Y value of the optimal point is 241.642. In order to facilitate the experimental operation, X3 is taken as 14.4. Therefore, the predicted optimal culture medium is: soluble starch 3.1%, glucose 1.28%, yeast powder 1.44%, sodium nitrate (NaNO3) 0.8%, MgSO4 0.1%, MnCl2 0.004%, ZnSO4 0.004%, CoCl2 0.0005%.

[0100] 4. Optimization of Fermentation Conditions

[0101] (1) Optimization of inoculum size

[0102] This experiment investigated the effect of different inoculation amounts on fermentation. The fermentation results were as follows: Fig.11 shown.

[0103] Depend on Fig.11 It can be seen that the final inoculation concentration is 10 5 ~10 6 sp / mL had little difference, but the yield decreased with the increase of concentration. This may be because the inoculation amount was large, more nutrients were needed for growth, and the sugar carbon source was consumed prematurely, which reduced the synthesis of Paraherquamide A. Therefore, the final concentration of inoculated spores was controlled at 10 5 ~10 6 sp / mL is enough.

[0104] (2) Temperature optimization

[0105] Each microorganism has its optimum and tolerable range. The optimum temperature of microorganisms in their growth and metabolic activities is also different, mainly because various enzymes in microorganisms have their optimum temperatures. This experiment examines the effect of different temperatures on fermentation, and the results are shown in Fig.12 .

[0106] Depend on Fig.12 It can be seen that as the temperature increases, the fermentation titer first increases and then decreases, and the fermentation titer is the highest at 25°C. Although the fermentation titer at 23°C is not much different from that at 25°C, considering the economic and environmental protection perspectives, 25°C was finally selected as the fermentation culture temperature.

[0107] (3) Initial pH optimization

[0108] This experiment investigated the effect of initial fermentation pH on the synthesis of Paraherquamide A. The experimental results are as follows: Fig.13 .

[0109] Depend on Fig.13 It can be seen that the initial pH of the fermentation broth has a significant effect on the fermentation results. As the pH increases, the fermentation titer of Paraherquamide A shows a trend of first rising and then falling, reaching the highest point at the initial pH of 7.0. It is speculated that it may be more conducive to the growth of this strain in a neutral environment.

[0110] In summary, through single factor experiments and response surface optimization experiments, the optimized medium formula was determined to be SGY (%): soluble starch 3.1%, glucose 1.28%, yeast powder 1.44%, sodium nitrate (NaNO3) 0.8%, MgSO4 0.1%, MnCl2 0.004%, ZnSO4 0.004%, CoCl2 0.0005%. The culture conditions were inoculated with a final concentration of 10 5 ~10 6 sp / mL, fermentation temperature 25℃, initial pH 7.0, under this condition, the yield exceeded 240μg / mL, which was about 6 times higher than the initial yield (42mg / L).

[0111] Example 6

[0112] Optimization and screening of separation and purification processes.

[0113] (1) Macroporous resin pretreatment method:

[0114] a. Take an appropriate amount of macroporous resin and place it in a container, soak it in anhydrous ethanol for 24 hours, then filter it and wash it with water until there is no alcohol smell;

[0115] b. Soak in 5% HCI solution for 4-6 hours, then filter and wash with water until neutral;

[0116] c. Soak in 5% NaOH solution for 4-6 hours, then filter and wash with water until neutral.

[0117] d. If the macroporous resin is not used for a long time, it needs to be stored in 95% ethanol to prevent bacterial growth and contamination.

[0118] (2) Determination of water content of macroporous resin

[0119] Weigh 2.00g (wet weight) of six pre-treated macroporous resins and place them in a constant weight sample bottle, dry them in a drying oven at 70°C for 36 hours, take them out and place them in a dryer to cool for 0.5 hours, and weigh them; dry them again for 30 minutes under the same temperature conditions, take them out and place them in a dryer to cool for 0.5 hours, and weigh them. Repeat the previous weighing process until the difference in mass before and after is less than 0.003g. Calculate its moisture content based on the mass difference before and after drying. Take three parallel samples of each resin when measuring the moisture content to ensure the accuracy of the weighing. The moisture content results are shown in Table 9.

[0120] Table 9 Macroporous adsorption resin moisture content %

[0121] MAR Model Moisture content (%) D101 32.16±0.02 HP20 38.83±0.08 XAD4 41.48±0.03 XAD16 31.88±0.06 SP825L 40.92±0.06 SP850 47.64±0.05

[0122] (3) Screening of macroporous resin models

[0123] a. Prepare a crude solution of Paraherquamide A with an initial concentration of about 0.22 mg / mL C0.

[0124] b. Weigh 0.5000g (dry weight) of the six pretreated resins respectively and place them in 500mL conical flasks. Add 250mL (V1) of 0.22mg / mL PHQ A crude solution to each conical flask. Place them in a constant temperature oscillator and adsorb for 24h at room temperature (25℃) and 180r / min to saturate the resin adsorption. (Three replicates)

[0125] c. After the adsorption is completed, take 500 μL of the upper clear liquid in the conical flask, add 500 μL of liquid methanol and mix thoroughly, then pass through a 0.45 μm organic filter membrane and detect its peak area by HPLC to obtain the mass concentration C1 of the PHQ A solution after adsorption.

[0126] d. The adsorbed resin was then transferred to another clean 500 mL conical flask, 250 mL of 80% ethanol aqueous solution (V2) was added, and the flask was placed in a constant temperature oscillator for desorption at room temperature (25°C) and 180 r / min for 24 h.

[0127] e. After desorption is completed, take 500 μL of the upper clear liquid in the conical flask, add 500 μL of liquid methanol and mix thoroughly, then pass through a 0.45 μm organic filter membrane and detect its peak area by HPLC to obtain the mass concentration C2 of the PHQ A solution after desorption.

[0128] f. Calculate the specific adsorption capacity (mg / g), adsorption rate (%) and desorption rate (%) of the six resins for PHQA. Calculate according to formula (3) to formula (5).

[0129]

[0130] Where: C0 is the concentration of PHQA before adsorption, mg / mL;

[0131] C1 is the concentration of PHQ A after adsorption, mg / mL;

[0132] C2 is the concentration of PHQ A after desorption, mg / mL;

[0133] V1 is the volume of the adsorption liquid, mL;

[0134] V2 is the volume of the desorption liquid, mL;

[0135] m is the mass of wet resin, g.

[0136] Table 10 Adsorption performance of different resins for Paraherquamide A

[0137] MAR Model <![CDATA[Specific adsorption capacity / (mg·g -1 )]]> Adsorption rate / % Desorption rate / % D101 40.62±0.18 37.46±0.16 97.33±0.26 HP20 52.92±0.47 48.8±0.43 95.47±0.4 XAD4 55.05±0.38 50.76±0.35 94.69±0.42 XAD16N 54.63±0.52 50.37±0.48 95.36±0.82 SP825L 70.62±0.47 65.12±0.43 94.66±0.19 SP850 78.81±0.28 72.67±0.26 92.65±0.17

[0138] As shown in Table 10, after comprehensive consideration of the adsorption capacity and resolution, it was finally decided to use the SP825L model to optimize the adsorption purification process of the compound PHQ A.

[0139] (4) Ethanol elution concentration

[0140] a. Prepare 9L bacterial solution, take 500μL of the supernatant, add 500μL of liquid methanol, mix thoroughly, pass through a 0.45μm organic filter membrane, and use HPLC to detect the PHQA peak area.

[0141] b. Weigh 30 portions (1.0000 g (wet weight)) of pretreated SP825L respectively and place them in 500 mL conical flasks. Add 300 mL of bacterial solution to each conical flask and place it in a constant temperature oscillator for adsorption at room temperature (25°C) and 180 r / min for 3 hours.

[0142] c. After the adsorption is completed, take 500 μL of the upper clear liquid in the conical flask, add 500 μL of liquid methanol and mix thoroughly, then pass through a 0.45 μm organic filter membrane and use HPLC to detect its peak area.

[0143] d. The adsorbed resin was then transferred to another clean 500 mL conical flask, and 100 mL (v / v) 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% ethanol aqueous solutions (3 parallel portions) were added respectively. The resin was placed in a constant temperature oscillator and desorbed at room temperature (25°C) and 180 r / min for 24 h.

[0144] e. After desorption, take 1 mL of the supernatant in the conical flask, filter it through a 0.45 μm organic filter membrane, and then perform HPLC to detect its peak area.

[0145] Table 11 Effect of different ethanol elution ratios on MAR desorption of PHQ A

[0146] Ethanol elution ratio PHQ A desorption rate % 10％ 1.92±0.06 20％ 2.96±0.09 30％ 7.74±0.60 40％ 22.20±0.70 50％ 50.55±1.79 60％ 74.94±1.26 70％ 87.38±1.33 80％ 94.25±0.69 90％ 96.06±0.51 100％ 98.63±0.72

[0147] From Table 11 and Fig.14 As shown, 10%, 20%, and 30% ethanol concentrations were selected as washing concentrations, and 80%, 90%, and 100% ethanol concentrations were selected as desorption concentrations. The experiments were combined to obtain Table 12, which determined that impurities were washed away with 20% ethanol concentration, and then desorbed with 80% ethanol solution.

[0148] Table 12 Effect of different ethanol elution ratios on MAR desorption PHQA

[0149] Ethanol elution ratio Desorption rate % 10％-80％ 92.75723797 20％-80％ 93.64206613 30％-80％ 91.94575714 10％-90％ 91.85124157 20％-90％ 92.79420593 30％-90％ 89.14451054 10％-100％ 90.86569796 20％-100％ 91.98642095 30％-100％ 91.39227642

[0150] like Fig.15 As shown, it was determined that the impurities were washed away with 20% ethanol concentration and then PHQ A was desorbed with 80% ethanol solution.

Claims

1. A paraherquamide A-producing Penicillium, characterized in that: Named as Penicillium sp., strain number KWF31, preservation number CCTCC NO.M 20221625.

2. A method for preparing paraherquamide A by biological fermentation culture, characterized in that: The method comprises the following steps: inoculating the Penicillium according to claim 1 into a culture medium and performing fermentation culture, and after the fermentation culture is completed, separating and purifying to obtain the compound paraherquamide A, Wherein, the carbon source in the culture medium is at least one of sucrose, glucose, maltose and soluble starch; The nitrogen source is at least one of peptone, soybean cake powder, yeast powder and nitrate; The inoculum size was such that the final spore concentration was 10 5 ~10 7 sp / mL; The fermentation temperature is 20-30°C, and the initial pH during fermentation is 6.0-9.0; The culture medium also includes inorganic salts, which are MgSO4, MnCl2, ZnSO4 and CoCl2, among which the mass volume ratio concentration of MgSO4 is 0.1%, the mass volume ratio concentration of MnCl2 is 0.004%, the mass volume ratio concentration of ZnSO4 is 0.004%, and the mass volume ratio concentration of CoCl2 is 0.0005%.

3. The method according to claim 2, characterized in that The carbon source in the culture medium includes soluble starch, and the mass volume ratio concentration of the soluble starch is 0.5% to 4%.

4. The method according to claim 3, characterized in that The mass volume ratio concentration of soluble starch is 2% to 2.5%.

5. The method according to claim 3, characterized in that: The carbon source in the culture medium includes soluble starch and glucose, wherein the mass volume ratio concentration of glucose is 0.5% to 4%.

6. The method according to claim 2, characterized in that The nitrogen source includes yeast powder, and the mass volume ratio concentration of the yeast powder is 0.3% to 2.4%.

7. The method according to claim 6, characterized in that The mass volume ratio concentration of yeast powder is 0.9% to 1.8%.

8. The method according to claim 6, characterized in that The nitrogen source includes yeast powder and nitrate, wherein the mass volume ratio of the nitrate is 0.2% to 1.6%.

9. The method according to claim 8, characterized in that The mass volume ratio of nitrate is 0.6% to 1.2%.

10. The method according to claim 9, characterized in that The mass volume ratio of nitrate is 0.6% to 0.8%.

11. The method according to claim 2, characterized in that The inoculum size for fermentation culture was 10 5 ~10 6 sp / mL; the fermentation culture temperature is 20-27°C; the initial pH during fermentation culture is 7.0-7.

5.

12. The method according to claim 2, characterized in that: During separation and purification, a macroporous adsorption resin is used for adsorption, and then ethanol with a volume ratio concentration of 20% is used to wash away impurities, and then ethanol with a volume ratio concentration of 80% is used for desorption; the model of the macroporous adsorption resin is SP825L.

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