Efficient paecilomyces cicadae fermentation process based on composite culture medium

Abstract

The invention relates to the technical field of paecilomyces cicadae, in particular to an efficient paecilomyces cicadae fermentation process based on a composite culture medium, which comprises the following steps: inoculating paecilomyces cicadae strain into the composite culture medium; then regulating and controlling the temperature and the ventilatory capacity according to the fermentation stage; meanwhile, adding a metabolism accelerant after fermenting for 72 hours; and after fermentation is completed, obtaining fermentation liquor, and separating to obtain paecilomyces cicadae. Nutrient substances are rapidly conveyed through the liquid phase to promote rapid proliferation of hyphae, meanwhile, the adsorption and expansion capacity of the hyphae is enhanced through the porous structure of the solid phase, and therefore the density of the hyphae and the synthesis efficiency of metabolic active substances are improved; a precise temperature control strategy is matched with physiological transformation from hypha growth to product accumulation, enzyme activity is guaranteed, and heat damage is reduced; and the metabolism accelerant is added to synergistically stimulate the high-efficiency expression of the target metabolite, so that the yield and quality of the target product are improved.

Description

Technical Field

[0001] The invention relates to the technical field of Paecilomyces cicadae, in particular to an efficient fermentation process of Paecilomyces cicadae based on a composite culture medium. Background Art

[0002] Paecilomyces cicadae is a microorganism with important industrial applications. With the development of biotechnology in recent years, its application value has received increasing attention. This fungus can produce a variety of valuable enzymes, such as cellulases, chitinases, and proteases, as well as bioactive substances such as cicadoidins and chitosan. These products show broad application prospects in many fields such as food processing, textile printing and dyeing, bioenergy, medicine and health, and agriculture and forestry protection.

[0003] In order to efficiently obtain Paecilomyces cicadae and its products, the fermentation process is a key link; however, the existing technology generally adopts traditional single-component liquid culture medium and relatively fixed fermentation parameters, which may not fully meet the complex nutritional needs of Paecilomyces cicadae at different growth stages, resulting in problems such as limited microbial growth, low metabolite yield, and long fermentation cycle. In view of this, we propose an efficient fermentation process for Paecilomyces cicadae based on a composite culture medium. Summary of the Invention

[0004] The purpose of the present invention is to provide an efficient fermentation process of Paecilomyces cicadae based on a composite culture medium, so as to solve the problem proposed in the above background technology that the conventional technology generally adopts a traditional single-component liquid culture medium and relatively fixed fermentation parameters, which may not be able to fully meet the complex nutritional requirements of Paecilomyces cicadae at different growth stages, resulting in limited microbial growth, low metabolite yield, and long fermentation cycle.

[0005] The present invention provides an efficient fermentation process of Paecilomyces cicadae based on a composite culture medium, comprising the following steps: S1.1. Inoculate 8-12% of the Paecilomyces cicadae strain into the composite culture medium with an initial stirring speed of 150 rpm, an aeration rate of 0.5 vvm, and a tank pressure of 0.01-0.03 MPa. The temperature and aeration rate are then adjusted according to the fermentation stage. A metabolic accelerator is added after 72 hours of fermentation. S1.2. After the fermentation is completed, a fermentation liquid is obtained and Paecilomyces cicadae is isolated.

[0006] Preferably, in said S1.1, the method for inoculating the Paecilomyces cicadae species is as follows: inoculating the activated Paecilomyces cicadae species into a sterilized seed liquid culture medium for cultivation, and then inoculating into a composite culture medium.

[0007] Preferably, the activated Paecilomyces cicadae strain is inoculated into a PSA slant culture medium and cultured at 22-25° C. for 5-7 days.

[0008] Preferably, the method of inoculating the activated Paecilomyces cicadae species into the sterilized seed liquid culture medium is as follows: scrape the fungal moss on the PSA slant culture medium, inoculate it into the seed liquid culture medium at a 5% inoculation rate, and culture it with shaking at 25°C and 150rpm for 48-72h until the diameter of the mycelial ball reaches 2-3mm.

[0009] Preferably, in S1.1, the composite culture medium is a mixture of a liquid phase and a solid phase in a mass ratio of 7:3; The liquid phase comprises 200-250 mL of potato boiled juice, 10-20 g of sucrose, 2-5 g of peptone, 1.5-3 g of potassium dihydrogen phosphate, 1-2.5 g of magnesium sulfate, and 200-300 mL of bamboo shoot residue cellulose enzymatic hydrolyzate per 1000 mL, and deionized water is added to make up the volume to 1000 mL; after mixing, the pH is adjusted to 5.8-6.2 with 0.5 mol / L sodium hydroxide, and then sterilized at 121°C for 20 minutes; Every 100g of the solid phase includes 3-8g of bagasse, 5-10g of rice husk powder, 0.2-0.6g of shell powder and 2-5g of astragalus residue, and perlite is added to make up to 100g; after mixing, ultrasonic treatment is performed at a frequency of 40kHz for 30min, and then sterilized at 121℃ for 20min.

[0010] Preferably, the preparation method of the bamboo shoot residue cellulose enzymatic hydrolyzate is as follows: The bamboo shoot heads remaining after processing are rinsed with running water and chopped into 3-5 mm particles; a 0.5 mol / L sodium hydroxide solution is added at a mass ratio of 1:10, and the mixture is treated in an 80°C water bath for 1 hour to remove lignin, filtered, and washed with clean water until neutral to obtain a pretreated bamboo shoot head residue; The pretreated bamboo shoot residue was placed in an autoclave at 121°C for 20 min, quickly cooled to room temperature, and centrifuged to dehydrate to a moisture content of 65% to obtain a dehydrated material; To every 100 g of dehydrated material, 1.5 g of cellulase, 0.5 g of xylanase, and 400 mL of 0.05 mol / L sodium citrate buffer were added; the mixture was shaken at 50°C and 150 rpm for 48 h, and 0.2 g of cellulase was added every 12 h during the shaking reaction to obtain an enzymatic hydrolyzate; The enzymatic hydrolysate was boiled for 10 minutes to terminate the reaction, and centrifuged at 5000 rpm for 15 minutes. The supernatant was filtered through a 0.45 μm filter membrane to obtain the enzymatic hydrolysate of bamboo shoot residue cellulose.

[0011] Preferably, in S1.1, temperature control is performed during the fermentation stage: Stage 1: Control the temperature at 25°C from 0 to 48 hours; Stage 2: Temperature drops to 22°C within 49-120 hours; Stage 3: The temperature was further lowered to 20°C for more than 120 h.

[0012] Preferably, in S1.1, ventilation volume is regulated during the fermentation stage: Stage 1: aeration volume of 0.5 vvm and stirring speed of 150 rpm; Stage 2: aeration volume of 1 vvm and stirring speed of 100 rpm; Stage 3: The aeration rate was 0.5 vvm and the stirring speed was reduced to 20 rpm.

[0013] Preferably, in said S1.1, the added amount of the metabolic promoter is 0.5-1.0% of the mixed volume of the Paecilomyces cicadae species and the composite culture medium; including 0.1mmol / L adenosine, 0.1mmol / L sodium iron ethylenediaminetetraacetic acid and 0.01-0.05% w / v cicada slough extract.

[0014] Preferably, in S1.2, the fermentation broth is first filtered through a 100-mesh sieve, then pre-filtered through a 10 μm microfiltration membrane, and then finely filtered through a 300 nm ceramic membrane.

[0015] Compared with the prior art, the present invention has the following beneficial effects: In the efficient fermentation process of Paecilomyces cicadae based on the composite culture medium of the present invention, nutrients are quickly transported through the liquid phase to promote the rapid proliferation of mycelium, and the porous structure of the solid phase is used to enhance the adsorption and expansion capabilities of the mycelium, thereby improving the mycelium density and the synthesis efficiency of metabolically active substances; the three-stage precise temperature control strategy matches the physiological transition from mycelial growth to product accumulation, ensuring enzyme activity and reducing thermal damage; metabolic promoters are added to synergistically stimulate the efficient expression of target metabolites, thereby improving the yield and quality of the target product. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] The deposit number of the Paecilomyces cicadae strain is CICC 41799.

[0018] The preparation method of bamboo shoot residue cellulose enzymatic hydrolyzate is as follows: The bamboo shoot heads remaining after processing were rinsed with running water and chopped into 4 mm particles; a 0.5 mol / L sodium hydroxide solution was added at a mass ratio of 1:10, and the mixture was treated in an 80°C water bath for 1 hour to remove lignin, and the mixture was filtered and washed with clean water until neutral to obtain a pretreated bamboo shoot head residue; The pretreated bamboo shoot residue was placed in an autoclave at 121°C for 20 min, quickly cooled to room temperature, and centrifuged to dehydrate to a moisture content of 65% to obtain a dehydrated material; To every 100 g of dehydrated material, 1.5 g of cellulase, 0.5 g of xylanase, and 400 mL of 0.05 mol / L sodium citrate buffer were added; the mixture was shaken at 50°C and 150 rpm for 48 h, and 0.2 g of cellulase was added every 12 h during the shaking reaction to obtain an enzymatic hydrolyzate; The enzymatic hydrolysate was boiled for 10 minutes to terminate the reaction, and centrifuged at 5000 rpm for 15 minutes. The supernatant was filtered through a 0.45 μm filter membrane to obtain the enzymatic hydrolysate of bamboo shoot residue cellulose.

[0019] The bamboo shoots were purchased from moso bamboo shoots harvested in the spring from the high mountains of Lin'an, Zhejiang.

[0020] Cellulase CAS No. 9012-54-8 and xylanase CAS No. 9025-57-4 were both obtained from Shanghai Yuanye Biotechnology Co., Ltd.

[0021] Preparation method of cicada slough extract: cicada slough powder is added to 60% ethanol at a ratio of 1:10 (g / mL), heated to boiling and refluxed for 2 hours, filtered and concentrated to 1 / 10 of the original volume.

[0022] The main active ingredients of cicada slough extract include chitin, amino acids and proteins.

[0023] Example 1: An efficient fermentation process of Paecilomyces cicadae based on a composite culture medium, comprising the following steps: S1.1. Inoculate the activated Paecilomyces cicadae into the sterilized seed culture medium at a 5% inoculum size and culture at 25°C and 150 rpm for 60 h. After the mycelial pellets reach 3 mm, inoculate the culture medium at a 10% inoculum size. The composite culture medium is a mixture of liquid phase and solid phase in a mass ratio of 7:3. The specific composition is as follows: Liquid phase: 250 mL of potato juice, 20 g of sucrose, 5 g of peptone, 3 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate, and 300 mL of bamboo shoot residue cellulose hydrolyzate, add deionized water to 1000 mL; adjust the pH to 6.0 with 0.5 mol / L sodium hydroxide, and sterilize at 121°C for 20 min. Solid phase: Every 100 g includes 5 g of sugarcane bagasse, 8 g of rice husk powder, 0.5 g of shell powder and 4 g of astragalus residue, and perlite is added to make up to 100 g; after mixing, ultrasonic treatment is carried out at a frequency of 40 kHz for 30 minutes, and then sterilized at 121°C for 20 minutes.

[0024] The fermentation process conditions are as follows: Initially: temperature 25°C, ventilation volume 0.5 vvm, stirring speed 150 rpm, tank pressure 0.03 MPa; Stage 2 (49-120 h): temperature 22°C, aeration 1 vvm, stirring speed 100 rpm; Stage 3 (more than 120 h): temperature 20 °C, ventilation 0.5 vvm, stirring speed 20 rpm.

[0025] After 72 h of fermentation, 0.7% (the volume of the mixture of Paecilomyces cicadae and the composite culture medium) of metabolic promoters: 0.1 mmol / L adenosine, 0.1 mmol / L sodium ferric ethylenediaminetetraacetic acid, and 0.01% w / v cicada slough extract were added; S1.2. After the fermentation is completed, the fermentation broth is first filtered through a 100-mesh sieve, then pre-filtered through a 10 μm microfiltration membrane, and then finely filtered through a 300 nm ceramic membrane to obtain Paecilomyces cicadae.

[0026] Example 2: An efficient fermentation process of Paecilomyces cicadae based on a composite culture medium, comprising the following steps: S1.1. Inoculate the activated Paecilomyces cicadae into the sterilized seed culture medium at a 5% inoculum size and culture at 25°C and 150 rpm for 60 h. After the mycelial pellets reach 3 mm, inoculate the culture medium at an 8% inoculum size. The composite culture medium is a mixture of liquid phase and solid phase in a mass ratio of 7:3. The specific composition is as follows: Liquid phase: 200 mL of potato juice, 10 g of sucrose, 2 g of peptone, 1.5 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate, and 200 mL of bamboo shoot residue cellulose hydrolyzate, add deionized water to 1000 mL; adjust the pH to 6.0 with 0.5 mol / L sodium hydroxide, and sterilize at 121°C for 20 min. Solid phase: Every 100 g includes 3 g of sugarcane bagasse, 5 g of rice husk powder, 0.2 g of shell powder and 2 g of astragalus residue, and perlite is added to make up to 100 g; after mixing, ultrasonic treatment is carried out at a frequency of 40 kHz for 30 minutes, and then sterilized at 121°C for 20 minutes.

[0027] The fermentation process conditions are as follows: Initially: temperature 25°C, ventilation volume 0.5 vvm, stirring speed 150 rpm, tank pressure 0.03 MPa; Stage 2 (49-120 h): temperature 22°C, aeration 1 vvm, stirring speed 100 rpm; Stage 3 (more than 120 h): temperature 20 °C, ventilation 0.5 vvm, stirring speed 20 rpm.

[0028] After 72 h of fermentation, 0.5% (the volume of the mixture of Paecilomyces cicadae and the composite culture medium) of metabolic promoters: 0.1 mmol / L adenosine, 0.1 mmol / L sodium ferric ethylenediaminetetraacetic acid, and 0.01% w / v cicada slough extract were added; S1.2. After the fermentation is completed, the fermentation broth is first filtered through a 100-mesh sieve, then pre-filtered through a 10 μm microfiltration membrane, and then finely filtered through a 300 nm ceramic membrane to obtain Paecilomyces cicadae.

[0029] Example 3: An efficient fermentation process of Paecilomyces cicadae based on a composite culture medium, comprising the following steps: S1.1. Inoculate the activated Paecilomyces cicadae into the sterilized seed culture medium at a 5% inoculum size and incubate at 25°C and 150 rpm for 60 h. After the mycelial pellets reach 3 mm, inoculate the culture medium at a 12% inoculum size. The composite culture medium is a mixture of liquid phase and solid phase in a mass ratio of 7:3. The specific composition is as follows: Liquid phase: 250 mL of potato juice, 20 g of sucrose, 5 g of peptone, 3 g of potassium dihydrogen phosphate, 2.5 g of magnesium sulfate, and 300 mL of bamboo shoot residue cellulose hydrolyzate, add deionized water to 1000 mL; adjust the pH to 6.0 with 0.5 mol / L sodium hydroxide, and sterilize at 121°C for 20 min. Solid phase: Every 100 g includes 8 g of sugarcane bagasse, 10 g of rice husk powder, 0.6 g of shell powder and 5 g of astragalus residue, and perlite is added to make up to 100 g; after mixing, ultrasonic treatment is carried out at a frequency of 40 kHz for 30 minutes, and then sterilized at 121°C for 20 minutes.

[0030] The fermentation process conditions are as follows: Initially: temperature 25°C, ventilation volume 0.5 vvm, stirring speed 150 rpm, tank pressure 0.03 MPa; Stage 2 (49-120 h): temperature 22°C, aeration 1 vvm, stirring speed 100 rpm; Stage 3 (more than 120 h): temperature 20 °C, ventilation 0.5 vvm, stirring speed 20 rpm.

[0031] After 72 h of fermentation, 1.0% (the volume of the mixture of Paecilomyces cicadae and the composite culture medium) of metabolic promoters: 0.1 mmol / L adenosine, 0.1 mmol / L sodium ferric ethylenediaminetetraacetic acid, and 0.05% w / v cicada slough extract were added; S1.2. After the fermentation is completed, the fermentation broth is first filtered through a 100-mesh sieve, then pre-filtered through a 10 μm microfiltration membrane, and then finely filtered through a 300 nm ceramic membrane to obtain Paecilomyces cicadae.

[0032] Spore yield determination: Take 1 mL of fermentation broth sample, mix thoroughly and then dilute it 10-fold with sterile saline (usually dilute to 10 -4 -10 -6 ), inoculate 0.1 mL of the diluted solution onto PDA or potato dextrose agar plates and incubate at 25-28°C for 3-5 days. Select plates with 30-300 colony counts and calculate the spore yield based on the dilution factor using the formula: spore yield = colony count × dilution factor × 10.

[0033] Determination of active product concentration: Centrifuge the fermentation broth and collect the supernatant. If it is a hydrophobic metabolite of the toxin, add an equal volume of acetonitrile / methanol for extraction. Filter (0.22 μm filter membrane) to remove impurities. Analyze by high-performance liquid chromatography (HPLC) and prepare a standard curve. Use pure toxin or active metabolite standard solution to prepare samples of different concentrations and draw a standard curve for quantitative analysis.

[0034] Conversion rate determination: The formula for calculating the product conversion efficiency based on the total dry weight of the raw materials is: conversion rate = (total mass of product / dry mass of substrate) × 100%, where total mass of product = concentration of active product × volume, dry mass of substrate = total dry weight of the input organic raw materials (such as bamboo shoot hydrolysate, sugarcane bagasse, astragalus residue, etc.).

[0035] Among the above-mentioned embodiments 1-3, embodiment 1 is selected as the optimal embodiment for comparative analysis with the comparative example.

[0036] Comparative Example 1: Compared with Example 1, this comparative example compares the effects of different liquid-solid mass ratios of the composite culture medium on the fermentation process of Paecilomyces cicadae; specifically, the liquid-solid mass ratio of Comparative Example 1-A is 10:0; the liquid-solid mass ratio of Comparative Example 1-B is 5:5; and the liquid-solid mass ratio of Comparative Example 1-C is 3:7. The measurement results are shown in Table 1.

[0037] Table 1 Data of Example 1 and Comparative Example 1 The results in Table 1 show that when the liquid-solid composite culture medium was 7:3, the spore yield and active product concentration both reached the optimal values, while the spore yield and product concentration of the pure liquid culture medium were significantly lower than those of the composite culture medium; specifically, the spore yield of the liquid-solid composite culture medium was 84% ​​higher than that of the pure liquid culture medium, while the product concentration was 59% higher.

[0038] This difference indicates that the liquid-solid composite culture medium can more effectively promote the growth and metabolism of Paecilomyces cicadae, probably because the presence of the solid phase provides additional surface area and nutrient sources, promoting the growth of mycelium and spore formation, while the liquid phase ensures better gas exchange and dissolved oxygen supply; this liquid-solid combination can better simulate the nutrient transfer in the natural ecological environment and promote the efficient fermentation of Paecilomyces cicadae.

[0039] Through this set of comparative data, it can be concluded that the liquid-solid composite culture medium provides a more superior growth environment during the efficient fermentation process of Paecilomyces cicadae, which can significantly increase the spore yield and the concentration of active products, thereby improving the overall fermentation efficiency.

[0040] Comparative Example 2: Compared with Example 1, this comparative example evaluates the effect on the fermentation process of Paecilomyces cicadae by removing different raw material components in the composite culture medium; specifically, Comparative Example 2-A removes the bamboo shoot hydrolyzate in the composite culture medium; Comparative Example 2-B removes the astragalus residue in the composite culture medium; Comparative Example 2-C uses a traditional culture medium, and the measurement results are shown in Table 2.

[0041] Table 2 Data of Example 1 and Comparative Example 2 The results in Table 2 show that removing any one of the raw materials will lead to a significant decrease in spore yield and active product concentration. Specifically, under the complete raw material combination (bamboo shoot enzymatic hydrolyzate, astragalus residue) used in Example 1, the spore yield was 4.8×10 7 CFU / mL, and the active product concentration was 215 mg / L. When the bamboo shoot hydrolyzate, astragalus residue and traditional culture medium were removed, the spore yield and active product concentration were significantly lower than those in Example 1.

[0042] These results indicate that the enzymatic hydrolysate of bamboo shoots and astragalus residue played an important synergistic role in the fermentation process; the enzymatic hydrolysate of bamboo shoots may have provided additional carbon source and enzyme activity, promoting the growth of mycelium and the synthesis of metabolites; and the plant compounds in astragalus residue may have helped enhance the metabolic activity of Paecilomyces cicadae and further optimized the growth environment.

[0043] Therefore, it was proved that the agricultural and sideline products such as bamboo shoot hydrolysate and astragalus residue had an important synergistic effect in the culture medium, which could significantly improve the fermentation performance of Paecilomyces cicadae, thereby increasing the spore yield and active product concentration.

[0044] Comparative Example 3: Compared with Example 1, this comparative example evaluates the influence of metabolic promoters used alone and in combination on the fermentation process of Paecilomyces cicadae; specifically, Comparative Example 3-A only adds cicada slough extract as a metabolic promoter; Comparative Example 3-B only uses adenosine and sodium iron ethylenediaminetetraacetic acid (NaFeEDTA); and Comparative Example 3-C does not add any metabolic promoter. The measurement results are shown in Table 3.

[0045] Table 3 Data of Example 1 and Comparative Example 3 The results in Table 3 show that the synergistic effect of the metabolic promoter significantly increased the spore yield and active product concentration. Specifically, in Example 1, a combination of adenosine, sodium ferric EDTA, and cicada slough extract was used, and the spore yield reached 4.8×10 7 CFU / mL, the active product concentration was 215 mg / L, and the conversion rate was 89%, showing the best effect; in comparison, when only cicada slough extract or only adenosine and sodium ferric EDTA were used, the spore yield, active product concentration and conversion rate were significantly lower than those in Example 1; and when no metabolic promoter was added, these indicators were significantly reduced.

[0046] Adenosine, as a nucleoside compound, is known to promote the metabolic activity of fungi and increase their metabolic rate. However, its effect is limited when used alone, probably because it only plays a certain metabolic induction role and lacks the necessary minerals and growth factors. 2 ⁺) has a certain promoting effect on the metabolic activity of microorganisms, especially playing an auxiliary role in enzyme reactions, which can significantly improve product synthesis; however, when used alone, due to the lack of support from other factors, its effect cannot be compared with the synergistic effect when used in combination; cicada shell extract is rich in bioactive ingredients and trace elements, which helps to enhance the resistance and activity of microorganisms, but lacks adenosine and Fe 2 ⁺ support, failed to fully exert its metabolism-promoting effect.

[0047] By combining these three metabolic promoters, the growth environment of Paecilomyces cicadae has been comprehensively optimized; adenosine provides nucleotide precursors, promoting cell division and metabolism; sodium ferric EDTA provides essential metal ions, enhancing enzyme activity; and cicada slough extract provides natural phytochemicals and trace elements, further activating the cell's metabolic pathways; this synergistic effect significantly improves the spore yield, active product concentration, and conversion rate of Paecilomyces cicadae.

[0048] Comparative Example 4: Compared with Example 1, this comparative example compares the effects of different fermentation control parameters (temperature and ventilation control) on the fermentation process of Paecilomyces cicadae; specifically, Comparative Example 4-A is kept at a constant temperature of 25°C; Comparative Example 4-B is kept at a constant ventilation of 0.5 vvm. The measurement results are shown in Table 4.

[0049] Table 4 Data of Example 1 and Comparative Example 4 In Comparative Example 4, Example 1 adopted multi-stage temperature control and stage-by-stage ventilation rate regulation, while the control group used fermentation conditions of a constant temperature of 25° C. and a constant ventilation of 0.5 vvm.

[0050] The results in Table 4 show that the spore yield of Example 1 is 4.8×10 7 CFU / mL, the active product concentration was 215 mg / L, and the fermentation period was 156 hours. The spore yield of the control group using constant temperature conditions was 3.5×10 7 CFU / mL, the active product concentration was 160 mg / L, and the fermentation period was 180 h.

[0051] The specific analysis results are as follows: Example 1 can simulate the temperature changes in the natural fermentation process through staged temperature control, and promote the metabolic regulation of P. cicadae at different growth stages; the high temperature in the early stage (25°C) is conducive to rapid growth and mycelial expansion; the medium temperature stage (22°C) promotes the sporulation process, while the low temperature stage (20°C) helps promote the accumulation and secretion of metabolites; compared with the constant temperature of 25°C, multi-stage temperature control significantly increases the spore yield and the concentration of metabolites.

[0052] In Example 1, the initial high ventilation volume (1 vvm) helps provide more oxygen to support rapid cell growth; while the later reduction in ventilation volume (0.5 vvm) and the lowering of the stirring speed help promote the accumulation of metabolites while reducing the side effects that may be caused by excessive oxygen. This combination of ventilation volume regulation and temperature changes provides an ideal environment for the fermentation of Paecilomyces cicadae.

[0053] The control group used constant temperature and constant ventilation. Although a stable growth environment was ensured, the secondary metabolic pathway of Paecilomyces cicadae was not fully stimulated. The results showed that the spore yield and product concentration were significantly lower than those in Example 1, and the fermentation cycle was also longer, indicating that constant conditions could not optimize the metabolic activity of the bacteria like multi-stage regulation.

[0054] By comparing the data, it can be concluded that multi-stage temperature control and ventilation volume regulation have a significant synergistic effect in the fermentation process of P. cicadae, which can effectively promote the growth of microorganisms, the synthesis of metabolites and their ultimate yield increase; compared with constant fermentation conditions, this dynamic control strategy can greatly improve the fermentation efficiency and enhance the yield and quality of the target product.

[0055] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. An efficient fermentation process of Paecilomyces cicadae based on a composite culture medium, characterized in that: The following steps are involved: S1.

1. Inoculate 8-12% of the Paecilomyces cicadae strain into the composite culture medium with an initial stirring speed of 150 rpm, an aeration rate of 0.5 vvm, and a tank pressure of 0.01-0.03 MPa. The temperature and aeration rate are then adjusted according to the fermentation stage. A metabolic accelerator is added after 72 hours of fermentation. S1.

2. After the fermentation is completed, a fermentation liquid is obtained and Paecilomyces cicadae is isolated.

2. The efficient fermentation process of Paecilomyces cicadae based on composite culture medium according to claim 1, characterized in that: In the above S1.1, the method for inoculating the Paecilomyces cicadae species is as follows: the activated Paecilomyces cicadae species is inoculated into a sterilized seed liquid culture medium for cultivation, and then inoculated into a composite culture medium.

3. The efficient fermentation process of Paecilomyces cicadae based on composite culture medium according to claim 2, characterized in that: The activated Paecilomyces cicadae strain is inoculated into a PSA slant culture medium and cultured at 22-25° C. for 5-7 days.

4. The efficient fermentation process of Paecilomyces cicadae based on composite culture medium according to claim 2, characterized in that: The method for inoculating the activated Paecilomyces cicadae species into the sterilized seed liquid culture medium is as follows: scrape the fungal moss on the PSA slant culture medium, inoculate it into the seed liquid culture medium at a 5% inoculation rate, and culture it with shaking at 25°C and 150 rpm for 48-72 hours until the diameter of the mycelial ball reaches 2-3 mm.

5. The efficient fermentation process of Paecilomyces cicadae based on composite culture medium according to claim 1, characterized in that: In S1.1, the composite culture medium is a mixture of a liquid phase and a solid phase in a mass ratio of 7:3; The liquid phase comprises 200-250 mL of potato boiled juice, 10-20 g of sucrose, 2-5 g of peptone, 1.5-3 g of potassium dihydrogen phosphate, 1-2.5 g of magnesium sulfate, and 200-300 mL of bamboo shoot residue cellulose enzymatic hydrolyzate per 1000 mL, and deionized water is added to make up the volume to 1000 mL; after mixing, the pH is adjusted to 5.8-6.2 with 0.5 mol / L sodium hydroxide, and then sterilized at 121°C for 20 minutes; Every 100 g of the solid phase includes 3-8 g of bagasse, 5-10 g of rice husk powder, 0.2-0.6 g of shell powder and 2-5 g of astragalus residue, and perlite is added to make up to 100 g; after mixing, ultrasonic treatment is performed at a frequency of 40 kHz for 30 minutes, and then sterilized at 121° C. for 20 minutes.

6. The efficient fermentation process of Paecilomyces cicadae based on composite culture medium according to claim 5, characterized in that: The preparation method of the bamboo shoot residue cellulose enzymatic hydrolyzate is as follows: The bamboo shoot heads remaining after processing are rinsed with running water and chopped into 3-5 mm particles; a 0.5 mol / L sodium hydroxide solution is added at a mass ratio of 1:10, and the mixture is treated in an 80°C water bath for 1 hour to remove lignin, filtered, and washed with clean water until neutral to obtain a pretreated bamboo shoot head residue; The pretreated bamboo shoot residue was placed in an autoclave at 121°C for 20 min, quickly cooled to room temperature, and centrifuged to dehydrate to a moisture content of 65% to obtain a dehydrated material; To every 100 g of dehydrated material, 1.5 g of cellulase, 0.5 g of xylanase, and 400 mL of 0.05 mol / L sodium citrate buffer were added; the mixture was shaken at 50°C and 150 rpm for 48 h, and 0.2 g of cellulase was added every 12 h during the shaking reaction to obtain an enzymatic hydrolyzate; The enzymatic hydrolysate was boiled for 10 minutes to terminate the reaction, and centrifuged at 5000 rpm for 15 minutes. The supernatant was filtered through a 0.45 μm filter membrane to obtain the enzymatic hydrolysate of bamboo shoot residue cellulose.

7. The efficient fermentation process of Paecilomyces cicadae based on composite culture medium according to claim 1, characterized in that: In S1.1, temperature control is performed during the fermentation stage: Stage 1: Control the temperature at 25°C from 0 to 48 hours; Stage 2: Temperature drops to 22°C within 49-120 hours; Stage 3: The temperature was further lowered to 20°C for more than 120 h.

8. The efficient fermentation process of Paecilomyces cicadae based on composite culture medium according to claim 1, characterized in that: In S1.1, the ventilation volume is regulated during the fermentation stage: Stage 1: aeration volume of 0.5 vvm and stirring speed of 150 rpm; Stage 2: aeration volume of 1 vvm and stirring speed of 100 rpm; Stage 3: The aeration rate was 0.5 vvm and the stirring speed was reduced to 20 rpm.

9. The efficient fermentation process of Paecilomyces cicadae based on a composite culture medium according to claim 1, characterized in that: In said S1.1, the added amount of the metabolic promoter is 0.5-1.0% of the mixed volume of the Paecilomyces cicadae species and the composite culture medium; it includes 0.1mmol / L adenosine, 0.1mmol / L sodium ferric ethylenediaminetetraacetic acid and 0.01-0.05% w / v cicada slough extract.

10. The efficient fermentation process of Paecilomyces cicadae based on a composite culture medium according to claim 1, characterized in that: In S1.2, the fermentation broth is first filtered through a 100-mesh screen, then pre-filtered through a 10 μm microfiltration membrane, and then finely filtered through a 300 nm ceramic membrane.

Images