CN118638651B - Fermentation process of medicinal silkworm Beauveria bassiana spore powder - Google Patents

Fermentation process of medicinal silkworm Beauveria bassiana spore powder Download PDF

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CN118638651B
CN118638651B CN202411121052.4A CN202411121052A CN118638651B CN 118638651 B CN118638651 B CN 118638651B CN 202411121052 A CN202411121052 A CN 202411121052A CN 118638651 B CN118638651 B CN 118638651B
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罗远鸿
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Sichuan Gongyao Technology Co ltd
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Abstract

本发明提供了一种药用僵蚕的球孢白僵菌孢子粉的发酵工艺，属于微生物培养技术领域。本发明药用僵蚕的球孢白僵菌孢子粉的发酵工艺，它包括液体发酵和固体发酵两个发酵环节，包括如下步骤：a、菌株活化；b、配制灭菌液体培养基；c、一级发酵液的制备；d、二级接种液的制备；e、固体培养基的制备；f、固体产孢的处理；g、干燥、过筛，获得孢子粉。本发明建立了液固双相发酵方法，并利用该方法生产出的白僵菌孢子粉内在质量无显著性差异。将孢子粉应用到僵蚕养殖生产中，所得到药材质量差异较小，并且该方法无需大型发酵罐设备，操作简便，更适用于僵蚕养殖企业大规模生产，降低生产成本。

The present invention provides a fermentation process of Beauveria bassiana spore powder of medicinal silkworm, belonging to the technical field of microbial culture. The fermentation process of Beauveria bassiana spore powder of medicinal silkworm of the present invention includes two fermentation links of liquid fermentation and solid fermentation, including the following steps: a. strain activation; b. preparation of sterilized liquid culture medium; c. preparation of primary fermentation liquid; d. preparation of secondary inoculation liquid; e. preparation of solid culture medium; f. treatment of solid spore production; g. drying and sieving to obtain spore powder. The present invention establishes a liquid-solid biphasic fermentation method, and the intrinsic quality of Beauveria bassiana spore powder produced by the method has no significant difference. The spore powder is applied to the production of silkworm breeding, and the quality difference of the obtained medicinal materials is small, and the method does not require large-scale fermentation tank equipment, is easy to operate, and is more suitable for large-scale production of silkworm breeding enterprises, reducing production costs.

Description

Fermentation process of beauveria bassiana spore powder of medicinal stiff silkworm
Technical Field
The invention relates to a fermentation process of beauveria bassiana spore powder of medicinal stiff silkworm, belonging to the technical field of microbial culture.
Background
The stiff silkworm is a dry body which is dead by infecting (or artificially inoculating) stiff silkworm Beauveria bassiana (bals.) Vuillant with larvae of Bombyx moriLinnaeus to Bombyx moriLinnaeus years old of silkworm of the silkworm moth family, and has the effects of calming endogenous wind, relieving spasm, dispelling wind, relieving pain, resolving phlegm, resolving masses and the like. As one of typical common animal medicines, the medicine has definite curative effect and wide application.
The beauveria bassiana is taken as one of the sources of the batryticated silkworm seeds, and is critical to the yield and quality of the batryticated silkworm. The beauveria bassiana production mode mainly comprises three modes of liquid fermentation, solid fermentation and liquid-solid double-phase fermentation, and researches show that the liquid-solid double-phase fermentation mode combines the dual advantages of liquid-phase fermentation and solid-phase fermentation, so that the fermentation period is obviously shortened, and the stress resistance and environmental stability of the obtained conidium are obviously superior to those of the single fermentation mode of the two, thus the beauveria bassiana production technology is the most main production technology of the beauveria bassiana. At present, the artificially produced stiff silkworm strain mainly comes from conidium of beauveria bassiana carried by silkworm bodies which are collected in silkworm cocoon production, but is easy to be polluted by a large amount of mixed bacteria, and the next production is inoculated with silkworm, so that the risk of bacterial silkworm diseases and insufficient rigidification exists, and the cultivation loss is caused.
Patent application number: 201910703859.1, name of invention: a method for culturing beauveria bassiana spore powder by fungus bags, discloses a method for culturing beauveria bassiana spore powder by using fungus bags. Firstly, in the liquid culture stage, preparing nutrient components such as a carbon source, a nitrogen source and the like, and allowing beauveria bassiana to grow until the spore concentration reaches 10 8～109 spores/mL, and transferring the beauveria bassiana to a solid culture medium. In the solid culture stage, rice is used as a culture medium, and after liquid bacteria are inoculated, an edible fungus cultivation bag, a fruiting ring and an aseptic breathable sealing film are combined by using rubber bands to form a cylindrical miniature fermentation tank. The fungus bag is used for culturing, isolating the environmental microbial pollution, well ventilating and utilizing the inner wall of the fungus bag to adsorb the water metabolized by the beauveria bassiana so as to promote the beauveria bassiana to produce spores rapidly. The method has the spore production period of 8-10 days, no need of large-scale fermentation tank equipment, simple operation, low cost of raw materials, equipment, manpower and time, and suitability for workshop production of agricultural cooperation. The method disclosed in the document has low spore content (100 hundred million/g) and large inoculation amount (2.0+/-0.5 g for each silkworm to 5 years).
Disclosure of Invention
In order to solve the technical problems, the invention provides a production process method of beauveria bassiana spore powder, and the beauveria bassiana spore powder is applied to the breeding production of stiff silkworms, so that a foundation is laid for the breeding production technology, and the quality of medicinal materials is ensured from the source.
The invention provides a fermentation process of beauveria bassiana spore powder of medicinal stiff silkworm, which comprises two fermentation links of liquid fermentation and solid fermentation, and comprises the following steps:
a. Activating the strain; inoculating purified slant strain to SDY solid culture medium by streaking method, standing in constant temperature incubator under semi-illumination to culture, and collecting when the culture medium grows light yellow white conidium powder;
b. preparing a sterilized liquid culture medium:
the liquid medium is SDY medium with agar removed: 40g of glucose, 10g of yeast extract powder, 10g of peptone, 1000mL of distilled water and 0.05% tween-80, weighing the raw materials according to a formula, mixing, dissolving, packaging and sterilizing;
c. preparation of primary fermentation liquor:
Under the aseptic condition, preparing spore powder in the step a into spore suspension by using 0.05% Tween-80 aseptic aqueous solution, sucking the spore suspension to ensure that the initial inoculation concentration is 2.4X10 6 spores/mL, and culturing for 72 hours on a shaking table with the temperature of 26.2 ℃ and the shaking table rotating speed of 174r/min to obtain beauveria bassiana primary fermentation liquor;
d. Preparation of a secondary inoculation liquid: transferring 5% of the primary fermentation liquid in the step c under the aseptic condition, and culturing for 60 hours on a shaking table with the rotation speed of 174r/min at the temperature of 26.2 ℃ to obtain a beauveria bassiana secondary inoculation liquid;
e. Preparation of solid medium: loading rice into a stainless steel tray, adding 0.4% glucose and potassium nitrate, adding water with a material-water ratio of 2:1W:V, sterilizing with high-pressure steam at 121deg.C for 15min, taking out, and cooling to room temperature;
f. Solid spore production treatment: under the aseptic condition, adding 25% of the secondary inoculation liquid in the step d into a solid culture medium, uniformly mixing the bacterial liquid and rice, sealing with a preservative film, and culturing for 10d under the condition that the temperature is 26 ℃ and the humidity RH=85%; the inoculation amount is 25% v/w based on the total rice; culturing for 10d, sealing the front 3d for dark culture, and opening the rear 7d for illumination culture;
g. drying and sieving: and (5) drying and sieving after the culture is finished, and separating rice matrixes to obtain spore powder.
Wherein the temperature of the constant temperature incubator in the step a is 26 ℃; the semi-illumination conditions were: l: d=12:12; incubation time 14d.
Wherein, the size of the stainless steel shallow tray in the step e is as follows: 24 cm. Times.18 cm. Times.3 cm.
Wherein, the drying conditions in the step g are as follows: drying at 35 ℃ for 2d; sieving with 100 mesh sieve.
The invention also provides the beauveria bassiana spore powder prepared by fermentation of the beauveria bassiana spore powder fermentation process of the medicinal stiff silkworm, wherein the spore content is 9.36 multiplied by 10 10 spores/g-9.56 multiplied by 10 10 spores/g, the viable count is 8.81 multiplied by 10 10 spores/g-8.95 multiplied by 10 10 spores/g, and no mixed bacteria are detected.
The invention also provides an application of the beauveria bassiana spore powder prepared by fermentation of the beauveria bassiana spore powder of medicinal stiff silkworm in preparation of stiff silkworm.
The beneficial effects of the invention are as follows:
1. The invention determines the fermentation liquor and fermentation time of the liquid fermentation link, and determines four key technical parameters of fermentation temperature, rotation speed of a shaking table, liquor loading quantity and initial inoculation concentration, so that strains fully grow in the liquid culture stage under optimal conditions, and good inoculation liquor is provided for the solid fermentation link.
2. The invention adopts a tray as a fermentation container and combines closed fermentation (mycelium growing period) and open fermentation (spore producing period), which can effectively solve the problem of heat accumulation in the fermentation process and ensure the growth and spore production of strains.
3. The beauveria bassiana spore powder produced by the invention has stable quality, the spore content reaches 9X 10 10 spores/g after quality detection, the number of viable spores is above 8X 10 10 spores/g, and no mixed bacteria are detected.
4. The spore powder obtained by the invention only needs about 0.5g of silkworm inoculated with one piece of paper, the obtained stiff silkworm has a bar shape which is thick and full, the dead rate of the silkworm is over 95 percent, the yield is over 15 kg/paper, the first grade product is over 50 percent, the sum of the first and second grade medicinal materials is over 90 percent, the ratio of 4 silk gland rings is over 70 percent, the moisture, ash content and extract meet the specification of Chinese pharmacopoeia of 2020 edition, and the beauveria bassiana content is over 0.030 percent.
The invention aims at examining the beauveria bassiana liquid-solid double-phase fermentation production process, defines the key technical parameters of two continuous links of liquid fermentation and solid fermentation, examines the transfer quantity and time of the two-stage expansion culture of the liquid fermentation, provides good fermentation liquid for the solid fermentation link, has strong operability, ensures stable quality of the produced spore powder, and can obtain good economic benefit when being applied to stiff silkworm cultivation.
Drawings
FIG. 1 shows a graph of blastospore concentration under different culture conditions;
FIG. 2 shows a graph of blastospore concentration at various fermentation temperatures;
FIG. 3 shows a graph of blastospore concentration at different shaking table rotational speeds;
FIG. 4 shows a graph of blastospore concentration at various loading levels;
FIG. 5 shows a graph of blastospore concentration at various seed concentrations;
FIG. 6 is a graph of blastospore concentration for different transfer amounts of secondary liquid fermentation;
FIG. 7 is a pilot plant flow chart of the beauveria bassiana production process;
FIG. 8 is a diagram of the appearance of the spore powder;
FIG. 9 shows a test chart of spore powder contamination.
Detailed Description
EXAMPLE 1 fermentation Process of beauveria bassiana spore powder of medicinal Bombyx Batryticatus
According to the invention, the blastospore concentration and the mycelium dry weight of liquid fermentation are used as indexes, different culture solutions and culture time are inspected by utilizing a single factor test, key technical parameters such as fermentation temperature, liquid loading amount, shaking table rotation speed, inoculation concentration and the like in the liquid fermentation process of the beauveria bassiana are screened, and optimal technological parameters of the liquid fermentation of the beauveria bassiana are preferably obtained; and meanwhile, taking the spore production number as an index, screening out the basic composition of the solid fermentation matrix and the proportion of the supplementary carbon and nitrogen sources by utilizing a single factor test, and screening key technical parameters such as shallow tray sample loading quantity, material-water ratio, inoculation quantity and the like in the solid fermentation process of the beauveria bassiana to obtain optimal technological parameters of solid fermentation of the beauveria bassiana, and combining the two parameters to obtain the liquid-solid biphasic fermentation production process of the beauveria bassiana.
The method comprises the following steps:
(1) Strain activation:
taking out purified slant strain in a refrigerator at-4deg.C, inoculating to SDY solid culture medium by streaking method, standing in a constant temperature incubator at 26deg.C under half-illumination L:D=12:12, culturing for 14D, and collecting when the culture medium grows light yellow white conidium powder;
(2) Preparing a sterilized liquid culture medium:
The liquid medium is SDY medium with agar removed: 40g of glucose, 10g of yeast extract powder, 10g of peptone, 1000mL of distilled water and 0.05% tween-80, weighing the raw materials according to a formula, mixing and dissolving, split charging, and carrying out primary fermentation: 250mL triangular flask liquid 72mL, secondary fermentation flask: 288mL of 1000mL triangular flask liquid and sterilizing;
(3) Preparation of primary fermentation liquor:
Under the aseptic condition, preparing spore powder in the step a into spore suspension by using 0.05% Tween-80 aseptic aqueous solution, sucking a proper amount of spore suspension to ensure that the initial inoculation concentration of a primary fermentation bottle is 2.4X10 6 spores/mL, and culturing the spore powder on a shaking table with the temperature of 26.2 ℃ and the shaking table rotating speed of 174r/min for 72 hours to obtain beauveria bassiana primary fermentation liquor;
(4) Preparation of a secondary inoculation liquid:
Under aseptic conditions, transferring 5% (14.4 mL) of the primary fermentation liquid in the step c into a secondary fermentation bottle, and culturing for 60 hours on a shaking table with the temperature of 26.2 ℃ and the shaking table rotating speed of 174r/min to obtain a beauveria bassiana secondary inoculation liquid;
(5) Preparation of solid medium:
Loading rice into stainless steel shallow tray (24 cm×18cm×3cm), loading rice sample 170g, adding 0.4% glucose and potassium nitrate, adding water to make the ratio of water to material be 2:1 (W: V), sterilizing with high pressure steam (121deg.C) for 15min, taking out, and cooling to room temperature;
(6) Solid spore production treatment:
Under the aseptic condition, adding the secondary inoculation liquid in the step d according to 25% of the weight of rice in a solid culture medium, uniformly mixing the bacterial liquid and the rice, sealing the mixture with a preservative film, and culturing the mixture for 10d under the condition of 26 ℃ and RH=85%, wherein the culture is performed in a sealed dark state for the first 3 days and in an open light state for the last 7 days. Continuously drying at 35deg.C for 2d after culturing, sieving with 100 mesh sieve, and separating rice matrix to obtain spore powder.
(7) Spore powder quality detection
The quality detection is carried out on the spore powder obtained by production, and the drying decrement, the spore content, the viable spore number and the mixed bacterial rate are respectively detected. The spore powder has a drying decrement of below 7%, a spore content of 9.36×10 10 spores/g-9.56×10 10 spores/g, a viable count of 8.81×10 10 spores/g-8.95×10 10 spores/g, and no foreign bacteria are detected.
EXAMPLE 2 application of spore powder to Bombyx Batryticatus culture
The spore powder obtained by production is used for uniformly diluting the beauveria bassiana spore powder with sterile water of 0.05% Tween-80 to ensure that the concentration is 1 multiplied by 10 8 spores/mL, the silkworm grows to the early stage of five years old, is inoculated after being fed for 1 time, is uniformly sprayed for three times, maintains the temperature and humidity environment of 90% RH and 26 ℃ for 2h, is subsequently cultivated normally to dead under the environment of 23-28 ℃ and 70-80% RH, and is used for counting the dead rate and yield, and then the quality of the stiff silkworm medicinal material is measured. The spore powder obtained by the invention only needs about 0.5g of silkworm inoculated with one piece of paper, the obtained stiff silkworm has a bar shape which is thick and full, the dead rate of the silkworm is over 95 percent, the yield is over 15 kg/paper, the first grade product is over 50 percent, the sum of the first and second grade medicinal materials is over 90 percent, the ratio of 4 silk gland rings is over 70 percent, the moisture, ash content and extract meet the specification of Chinese pharmacopoeia of 2020 edition, and the beauveria bassiana content is over 0.030 percent.
Table 1 comparison of the present invention with patent application number 201910703859.1
EXAMPLE 3 fermentation Process parameter screening of beauveria bassiana spore powder of the medicinal stiff silkworm of the present invention
1. Liquid fermentation of beauveria bassiana
1. Materials and instruments
1.1 Test strains and Medium
The tested strain is beauveria bassiana strain with stronger pathogenicity to silkworm, and the inclined plane is stored at 4 ℃.
Test medium:
SDAY solid medium (peptone 10g, yeast extract 10g, glucose 40g, distilled water 1000mL, agar 20 g); PDA culture (200 g of potato, 20g of glucose, 1000mL of distilled water, 0.05% Tween-80), PPDA culture (200 g of potato, 5g of peptone, 20g of glucose, 1000mL of distilled water, 0.05% Tween-80), PDAY culture (200 g of potato, 5g of yeast extract, 20g of glucose, 1000mL of distilled water, 0.05% Tween-80), MEA culture (3 g of soybean peptone, 30g of malt extract, 1000mL of distilled water, 0.05% Tween-80), SDY culture (10 g of peptone, 10g of yeast extract, 40g of glucose, 1000mL of distilled water, 0.05% Tween-80), SMY culture (10 g of peptone, 10g of yeast extract, 40g of maltose, 1000mL of distilled water, 0.05% Tween-80).
1.2 Instruments
SW-CJ-2F double vertical air supply purification workbench (Shanghai Langxuan laboratory equipment Co., ltd.); an electric thermostatic water bath (Test instruments Co., tianjin); YJY-2102C constant-temperature shaking incubator (Shanghai medical instruments Co., ltd.); PRX-1000A intelligent artificial climate box (Ningbopride instruments limited); LS-75LD vertical pressure steam sterilizer (Jiangyin coast river medical equipment Co., ltd.).
2. Test method
2.1 Strain activation
Inoculating purified slant strain to SDY culture medium by streaking method, culturing in a constant temperature incubator at 26deg.C under half-illumination L:D=12:12 for 14D, and collecting when the culture medium grows light yellow white conidium powder.
2.2 Test liquid Medium
Preparing culture solution according to the test strain 1.1 and culture medium, sterilizing with high pressure steam (121deg.C) for 20min, transferring conidium powder into 100mL sterilized culture solution after the strain is fully produced, and preparing into seed solution with concentration of 10 6 spores/mL with conical bottle liquid volume of 100 mL/250 mL.
3. Liquid fermentation single factor test screening
3.1 Culture Medium and culture time screening
Inoculating the prepared spore suspension into different kinds of fermentation liquid, placing the fermentation liquid in a shaking table with the liquid loading amount of 100/250mL and the rotating speed of 160r/min at 26 ℃ for 5d, measuring the spore concentration and the mycelium dry weight every 24h, repeating each treatment for 3 times, and screening out proper culture liquid and culture time.
3.2 Fermentation temperature screening
Inoculating the prepared spore suspension into the screened optimal fermentation liquid, setting the temperature of a shaking table at 20 ℃, 23 ℃, 26 ℃, 29 ℃, 32 ℃ and the rest conditions unchanged, culturing for 3 days, measuring the spore concentration of the spore and the dry weight of the mycelium every 12 hours, repeating each treatment for 3 times, and screening out the proper fermentation temperature.
3.3 Liquid Loading amount screening
Inoculating the prepared spore suspension into the screened optimal fermentation liquor, setting the liquid loading amount to be 50/250mL, 75/250mL, 100/250mL, 125/250mL and 150/250mL, culturing for 3 days under the same conditions, measuring the spore concentration and the dry weight of mycelium every 12 hours, repeating each treatment for 3 times, and screening out the proper liquid loading amount.
3.4 Shaking table rotational speed Screen
Inoculating the prepared spore suspension into the screened optimal fermentation liquor, setting the rotation speed of a shaking table to be 120r/min, 140r/min, 160r/min, 180r/min and 200r/min, culturing for 3 days under the same conditions, measuring the spore concentration and the dry weight of mycelium every 12 hours, repeating each treatment for 3 times, and screening out the proper rotation speed.
3.5 Seed concentration screening
Inoculating the prepared spore suspension into the screened optimal fermentation broth, setting the inoculation concentration to be 10 5、5*105、106、5*106、107 spores/mL, culturing for 3 days under the same conditions, measuring the spore concentration and the dry weight of mycelium every 12 hours, repeating each treatment for 3 times, and screening out the proper initial PH.
3.6 Determination of the biological Mass
Determination of blastospore concentration: 1mL of the cultured bacterial liquid is diluted by water with proper proportion, the diluted liquid is placed in a blood cell counting plate, and the bacterial liquid is subjected to microscopic examination and counting under a 40-time mirror, so that the spore concentration of the bacterial liquid is calculated.
Determination of mycelium dry weight: the biomass is measured by adopting a dry matter weighing method, different repeated liquid fermentation broths are taken every day, 2mL of each bottle is taken in a centrifuge tube every time, the centrifuge tube is centrifuged for 5min at 12000r/min, the supernatant is discarded, the sediment is poured into a dry filter paper and weighed to be W1, the sediment is dried at 80 ℃ until the constant weight is weighed again to be W2, and the dry weight of the mycelium is calculated according to the following formula:
Hypha dry weight= (W2-W1)/2×1000 g/L
3.7 Optimization of liquid phase culture conditions Using response surface method
According to the experimental design principle of Box-Behnken center combination, a result is obtained by combining single factors, 4 factors including fermentation temperature (A), liquid loading amount (B), shaking table rotating speed (C) and inoculation concentration (D) are selected, a 4-factor 3 horizontal response surface analysis test is designed, a center point is repeated 5 times, a blastospore concentration (X1) and mycelium dry weight (X2) of 72h are used as indexes, the comprehensive score of the two indexes (the maximum value of mass fractions of the indexes is used as a reference to carry out standardized treatment on data, and a comprehensive score formula is obtained, wherein the comprehensive score (Y) = (X1/X1 max) ×0.3+ (X2/X2 max) ×0.7) is used as a response value, and each group of test is repeated 3 times. And analyzing the obtained quadratic regression fit equation by using Design-Expert to obtain the optimal liquid culture condition, and carrying out verification test on the fitted optimal condition according to the single-factor test screening result. The test factors and horizontal designs are shown in Table 2.
TABLE 2 response surface test factors and levels for liquid fermentation
3.8 Data statistics and analysis
All data were analyzed using Excel 2019, and using SPSS 22.0 software and Design experert 13.0 software for saliency analysis and response surface analysis, respectively.
4. Results and analysis
4.1 Liquid fermentation Medium Screen
4.1.1 Blastospore concentration under different culture conditions
The measurement results of the spore concentration under different culture solution conditions are shown in figure 1, and the spore concentration of beauveria bassiana under different culture solution conditions is remarkably different. The spore concentration increases most rapidly from day 1 to day 2, and gradually increases after day 3 and begins to decrease after day 4. In the SDY culture solution, the spore concentration is highest in each time period, the spore concentration reaches (44.63+/-3.36) multiplied by 10 7 spores/mL in the third day, which is extremely obviously higher than that of the rest treatment, the SMY culture solution is used next, the spore concentration reaches (19.98+/-0.15) multiplied by 10 7 spores/mL in the third day, the spore concentration in the PDA culture medium is lowest, and the analysis reason is that the nutrition is relatively lack, so that the beauveria bassiana spore development and spore production capacity are limited.
4.1.2 Mycelium dry weight under different culture conditions
The measurement results of mycelium dry weight under different culture solution conditions are shown in Table 3, and the results show that the types of the culture solution have obvious influence on mycelium dry weight, the mycelium dry weight of beauveria bassiana J-2 after three days of culture in SDY and SMY culture solutions is highest and is extremely higher than that of other treatment groups (F=170.550 and P is less than 0.001), the change in the fourth day and the fifth day is small, the color is deepened in the later period of fermentation, the viscosity is reduced, and the cells are easy to have layering autolysis phenomenon, so that three days of culture are preferable.
TABLE 3 hyphal dry weight under different culture conditions
Note that: the same column of different lowercase letters indicates that P < 0.05 differs significantly, as follows.
The two indexes of spore concentration and mycelium dry weight are combined, and the culture solution is preferably SDY culture solution, namely glucose is used as a carbon source, peptone and yeast powder are used as nitrogen sources, and the spore concentration and mycelium dry weight reach the maximum value after liquid fermentation for three days under the condition.
4.2 Fermentation temperature screening
4.2.1 Blastospore concentration at different fermentation temperatures
The results of the determination of the spore concentration at different fermentation temperatures are shown in FIG. 2, and the spore concentration is significantly different at different temperatures. The strain has the greatest blastospore concentration at 26 ℃ and 23 ℃ and reaches (44.63+/-3.36) multiplied by 10 7、（44.25±2.97）×107 spores/mL respectively, which is extremely higher than other treatment groups (F=146.39 and P < 0.0001), and in addition, the strain is inhibited from growing under high temperature and low temperature conditions, and the high temperature conditions are more serious than the low temperature conditions.
4.2.2 Dry weight of mycelium at different fermentation temperatures
The measurement results of the dry weight of the mycelium under different temperature conditions are shown in Table 4, and the dry weight of the mycelium of the strain shows a tendency of increasing and then decreasing with the increase of temperature in a certain temperature range; at the temperature of 23-29 ℃, the hyphae of the strain develop well, the dry weight of the hyphae reaches the maximum value for 72 hours, wherein the dry weight of the hyphae is extremely higher than that of other treatment groups (F=25.732 and P is less than 0.001) at the temperature of 26 ℃, the dry weight of the hyphae is the lowest at the temperature of 32 ℃, the growth is seriously delayed at 12-48 hours, and the dry weight of the hyphae is basically unchanged.
TABLE 4 hyphal dry weight at different fermentation temperatures
In view of the above, the fermentation temperature was selected at 23℃and 26℃at 29℃as the level of investigation for the subsequent experiments.
4.3 Rotation speed screening of shaker
4.3.1 Blastospore concentration at different shaking table rotational speeds
As shown in FIG. 3, the growth rate of the blastospore concentration of the strain at different rotation speeds of the shaking table is the same as that of the strain at different rotation speeds of the shaking table, the growth rate is the fastest at 36-48 h, and gradually slows down to 72h, the blastospore concentration is greater than 4×10 8 spores/mL, and no significant difference exists between treatment groups (F=1.114, P=0.402).
4.3.2 Dry weight of mycelium at different shaking table rotational speeds
The dry weight measurement results of the mycelia at different rotation speeds of the shaker are shown in Table 5. In a certain rotating speed range, the mycelium dry weight of the strain tends to increase and decrease with the increase of the rotating speed; and it has a significant difference at different shaking table rotational speeds, ferment until the dry weight of mycelium reaches a maximum value for 72h, at which time the dry weight of mycelium at 160r/min is significantly higher than 140r/min and 180r/min (f=127.326, p < 0.001).
TABLE 5 hyphae dry weight at different shaker rotational speeds
In the above situation, the rotation speed of the shaking table is 140r/min, 160r/min and 180r/min, which are selected as the investigation level of the subsequent experiment.
4.4 Liquid Loading Screen
4.4.1 Blastospore concentration under different liquid Loading conditions
The results of the blastospore concentration measurement under different liquid loading amounts are shown in FIG. 4, and the blastospore concentrations of the strains have significant differences under different liquid loading amounts. By 72h, the blastospore concentration at 75/250mL was significantly higher than that of the other treatment groups (f=206.837, p < 0.0001), reaching (69.90 ±1.56) ×10 7 spores/mL.
4.4.2 Dry weight of mycelium under different liquid Loading conditions
The dry weight measurement results of the mycelia at various liquid loadings are shown in Table 6. In a certain liquid loading range, the mycelium dry weight of the strain is in a trend of increasing and then decreasing along with the increase of the liquid loading; and it has a significant difference at different shaker speeds, fermenting until the dry weight of the mycelium reaches a maximum value for 72h, at which time the dry weight of the mycelium at 50/250mL, 75/250mL is significantly higher than that of the other treatment groups (f=275.858, p < 0.001).
TABLE 6 hyphae dry weight under different fluid loading conditions
In summary, the liquid loading amounts of 50/250mL, 75/250mL and 100/250mL were selected as the investigation levels of the subsequent experiments.
4.5 Seed concentration screening
4.5.1 Blastospore concentration under different seed concentration conditions
The results of the blastospore concentration measurements at the different inoculation concentrations are shown in FIG. 5. The inoculation concentration is too large, the consumption of the strain on nutrient substances of the culture solution is relatively fast, and the spore production is incomplete; the inoculation concentration is lower, so that the growth rate of the spore concentration is lower and the time is longer; the spore concentration of the strain was significantly different at different inoculation concentrations. By 72h, the initial inoculation concentration was 5×10 5、106、5×106 spores/mL of blastospores concentration without significant difference, but very significantly higher than 10 5、107 spores/mL (f=6.431, p=0.008).
4.5.2 Hyphae dry weight at different inoculum concentrations
The results of mycelium dry weight measurements at different inoculation concentrations are shown in Table 7. In a certain inoculation concentration range, the dry weight of hypha of the strain is in a trend of increasing and then decreasing with the increase of inoculation concentration; fermentation was carried out until the dry weight of mycelia reached a maximum value of 72h, at which time the dry weight of mycelia at an inoculation concentration of 10 6 spores/mL was significantly higher than in the other treatment groups (f=356.868, p < 0.001).
TABLE 7 hyphal dry weight at different inoculum concentrations
In summary, the inoculation concentrations were selected as 5X 10 5 spores/mL, 10 6 spores/mL, and 5X 10 6 spores/mL for the study level of the subsequent experiments.
5. Liquid fermentation Box-Behnken response surface test design
According to the single factor test result, the comprehensive score of 2 indexes of the blastospore concentration and the mycelium dry weight of 72 hours is used as a dependent variable, a four-factor three-level response surface analysis experiment is designed, the design of the test scheme is shown in table 8, multiple regression fitting is carried out on the fermentation temperature (A), the shaking table rotating speed (B), the liquid loading amount (C) and the inoculation concentration (D), the complex correlation coefficient R 2 = 0.9642 of the binomial fitting is obtained according to the test result of the liquid fermentation Box-Behnken in table 9, the model P is less than 0.0001, the significance level is reached, the fitting degree is good, the analysis can be carried out by using the equation instead of a real test point, and the growth change of the liquid fermentation of beauveria bassiana can be better reflected in the investigation range. Model regression equation mismatching test p= 0.0624 > 0.05, no significant difference, shows that the equation fits well with the actual situation, the residual error of the model may be random error, and the model can be used for analyzing and predicting the optimal liquid fermentation process. The multiple regression fit equation obtained from the anova is: comprehensive score Y＝0.9189-0.0762A+0.0594B-0.1372C+0.0798D-0.0502AB+0.003AC-0.0378AD-0.0207BC+0.0220BD+0.0166CD-0.0766A2-0.0575B2+0.0066C2-0.2367D2.
TABLE 8 liquid fermentation Box-Behnken experimental design Table
TABLE 9 liquid fermentation Box-Behnken experimental results
TABLE 10 liquid fermentation response surface analysis of variance
According to the square difference results in table 10, the primary and secondary orders of the process parameters affecting the comprehensive score value are C > D > A > B, namely, the liquid loading amount > the inoculation concentration > the fermentation temperature > the rotation speed of the shaking table, and the significance analysis finds that the four factors affect the comprehensive score to a very significant level (< 0.01), the interaction item AC, BC, BD, CD affects the comprehensive score value not significantly, and the AB and AD affect the comprehensive score value to a significant level (< 0.05), thus indicating that the factors affect the growth of beauveria bassiana more complex.
6. Determination of optimal liquid fermentation process
Solving a fitting equation by adopting Design Expert 13.0 software to obtain the optimal technological parameters when the comprehensive score value of the beauveria bassiana liquid fermentation is predicted to be maximum, wherein the optimal technological parameters are as follows: the fermentation temperature is 26.2 ℃, the rotation speed of a shaking table is 174r/min, the liquid loading amount is 72mL (250 mL triangular flask), the inoculation concentration is 2.4X10 6 spore/mL, and the predicted comprehensive liquid fermentation score value is 0.91.
7. Verification of optimal liquid fermentation process
And carrying out verification experiments according to the optimal liquid fermentation conditions obtained by the design of the response surface curve, and repeating the experiments three times, wherein the experimental results are shown in Table 11. The average value of the comprehensive scores is 0.89 (RSD is 0.79%), which is closer to the predicted comprehensive score value of 0.91, and no obvious difference exists. Therefore, the model has accurate predictability and reliability, and can also prove the feasibility of optimizing the beauveria bassiana liquid fermentation process by using the response surface. The comprehensive score is slightly different from the regression equation predicted value under the optimal condition. The analysis reasons may be that the temperature, oxygen content, etc. of the culture medium during fermentation are changed with the growth of hyphae and the increase of spore production, thereby influencing the experimental result and generating a difference from the predicted value.
TABLE 11 results of liquid fermentation validation experiments
Numbering device Blastospore concentration (10 8 spores/mL) Hypha dry weight (g/L) Comprehensive scoring
S1 7.68 14.08 0.88
S2 7.58 14.20 0.89
S3 8.30 13.78 0.90
Average value of 7.72 14.02 0.89
RSD（%） 4.97 1.54 0.79
2. Solid fermentation of beauveria bassiana
1. Materials and instruments
1.1 Test strains
The tested strain is beauveria bassiana strain with stronger pathogenicity to silkworm, and the inclined plane is stored at 4 ℃.
1.2 Instruments and reagents
1.2 Instrument for liquid fermentation of beauveria bassiana.
2. Test method
2.1 Strain activation
The same method for activating 2.1 strain of beauveria bassiana liquid fermentation.
2.2 Preparation of liquid fermentation broth
Culture medium: SDY broth (peptone 10g, yeast extract 10g, glucose 40g, distilled water 1000mL,0.05% Tween-80).
Adding a small amount of activated spore powder into sterile water with 0.05% Tween-80, shaking to obtain spore suspension, inoculating into a 250mL triangular flask containing 72mL SDY culture solution to make initial inoculation concentration 2.40X10 6 spores/mL, and culturing at constant temperature of 26.2deg.C and rotation speed of 174r/min for 72 hr to obtain fermentation broth for solid inoculation culture.
3. Solid fermentation substrate screening
3.1 Basal fermentation substrate screening
Two agricultural and sideline products, namely rice and corn nibs, are selected as a solid fermentation base matrix, and different proportions are set, and the specific proportions are shown in table 12. Respectively weighing 100g of the 5 matrixes, placing the matrixes into a stainless steel shallow tray (16.5 cm is 11.5cm is 4.3cm, about 700 mL), adding water according to the proportion of 3:1 of water, uniformly mixing, sterilizing at a high temperature of 121 ℃ for 20min, cooling, inoculating 20% of fermentation liquor, uniformly mixing, enabling the matrixes to be tiled, culturing for 10d under the condition of the same thickness, at the temperature of 26 ℃ and the humidity RH=85%, sealing the front 3d, culturing in dark, and culturing in 7d open light. And continuously drying for 2d at 35 ℃ after the culture is finished, collecting spore powder, and measuring the spore production number.
Table 12 solid fermentation substrate formulations
3.2 Nutrient Screen for supplementing carbon and nitrogen to Medium
To further increase the spore production level of the screened solid medium, 0.1%, 0.4%, 0.7% glucose and sucrose were added as supplemental carbon sources to the screened basal medium rice, respectively, 0.1%, 0.4%, 0.7% peptone and KNO3 were added as supplemental nitrogen sources, respectively, and the rice was repeated three times with respect to the blank, and solid culture was performed according to the "3.1 basal fermentation substrate screening" method, and a single factor test was performed to screen the optimal supplemental carbon and nitrogen sources.
3.3 Single factor screening of solid fermentation conditions
3.3.1 Shallow tray sample Loading Screen
The optimal solid substrate is used as a solid culture medium, sample loading amounts of 50g, 75g, 100g, 125g and 150g are respectively set, the bacterial strain is cultivated in a spore production mode according to the treatment mode of screening the basic fermentation substrate of 3.1, and the spore production number is measured. Each treatment was repeated 3 times.
3.3.2 Feed to Water ratio screening
Taking the optimal solid substrate as a solid culture medium, adding a certain amount of water into the substrate to ensure that the ratio of the water to the water is 1:1, 2:1, 3:1, 4:1 and 5:1 respectively, carrying out spore production culture on the strain according to the treatment mode of screening the basic fermentation substrate of 3.1, and measuring the spore production number. Each treatment was repeated 3 times.
3.3.3 Inoculum size selection
The optimal solid substrate is used as a solid culture medium, the inoculum sizes of the fermentation broths are respectively set to be 15%, 20%, 25%, 30% and 35%, and the bacterial strains are cultivated for spore production according to the treatment mode of screening the basic fermentation substrate of 3.1, so as to determine the spore production number. Each treatment was repeated 3 times.
3.3.4 Determination of spore production number
Weighing 0.5g of solid fermentation substrate after culturing and drying, adding into a centrifuge tube filled with 25ml of 0.05% Tween-80 solution, shaking and mixing uniformly by using a vortex oscillator to prepare spore suspension, performing microscopic examination by using a blood cell counting plate, calculating the concentration of the spore suspension, and converting into the number of spores (per gram) generated per gram of substrate.
4. Optimization of solid phase culture conditions using corresponding surface method
According to the experimental design principle of Box-Behnken center combination, a result is obtained by combining single factors, 3 factors including sample loading quantity (A), material-water ratio (B) and inoculation quantity (C) are selected, a 3-factor 3 horizontal response surface analysis test is designed, a center point is repeated 5 times, the spore yield after 10 days is taken as a response value, and each group of test is repeated 3 times. And analyzing the obtained quadratic regression fit equation by using Design-Expert 13.0 to obtain the optimal solid-phase culture condition, and carrying out verification test on the fitted optimal condition according to the screening result of the single-factor test. The test factors and horizontal designs are shown in Table 13.
TABLE 13 response surface test factors and levels for solid fermentation
5. Data statistics and analysis
All data were analyzed by processing with Excel 2019, origin 2021, and by performing saliency analysis and response surface analysis with SPSS 22.0 software and Design Expert 13.0 software, respectively.
6. Results and analysis
6.1 Solid fermentation Medium Screen
6.1.1 Solid fermentation basal Medium Screen
The rice and corn kernels were selected for solid fermentation in different proportions, and the experimental results are shown in Table 14. There was a significant difference in spore production numbers for the different solid fermentation substrates (f=14.71, p < 0.001). When the rice is used as the solid fermentation substrate, the spore yield reaches 2.15X10 9/g, which is extremely higher than the other treatment groups, so the rice is the best for the solid fermentation substrate.
TABLE 14 influence of solid culture Medium on the spore production of the strains±s，n=3）
6.1.2 Screening of carbon and Nitrogen Source supplemented solid fermentation Medium
Sucrose and glucose are respectively added into the solid fermentation substrate rice as supplementary carbon sources, peptone and KNO3 are added as supplementary nitrogen sources, and the results of spore production numbers under different treatments are shown in tables 15 and 16.
The addition of different carbon sources has a significant influence on the spore production number (F=8.626, P < 0.001), the spore production number of the strain is increased and then reduced along with the increase of the concentration of the supplementary carbon sources, when 0.4% of glucose is added, the spore production number reaches 2.98X10 9/g, no significant difference exists between the spore production number and 0.4% of sucrose, but the spore production number is significantly higher than that of other treatment groups, and the price of sucrose is higher than that of glucose in terms of market price, so that the carbon sources are preferably glucose.
The addition of different nitrogen sources has the same significant influence on the spore production number (F=26.236, P is less than 0.001), the spore production number of the strain is increased and then reduced along with the increase of the concentration of the supplementary nitrogen sources, and when 0.4% KNO 3 is added, the spore production number is 2.99X10 9/g and is obviously higher than that of a blank group and other treatment groups, so that the nitrogen source is preferably KNO 3.
TABLE 15 influence of different carbon source types and addition amounts on spore production of strains±s，n=3）
TABLE 16 influence of different nitrogen source types and addition amounts on spore production of bacterial strains±s，n=3）
Numbering device Treatment of Spore yield (10 9/g)
CK Without adding 2.15±0.03d
D1 0.1% Peptone 2.27±0.15d
D2 0.4% Peptone 2.79±0.08b
D3 0.7% Peptone 2.20±0.18d
K1 0.1%KNO3 2.58±0.11c
K2 0.4%KNO3 2.99±0.29a
K3 0.7%KNO3 2.71±0.15bc
6.2 Single factor screening of solid fermentation conditions
6.2.1 Sample Loading Screen
The effect of tray rice loading on solid fermentation spore production is shown in Table 17. The spore yield is obviously different under different sample loading treatments (F=134.529, P is less than 0.001), and the spore yield is increased and then decreased with the increase of the sample loading. The rice sample loading amount is mainly that the growth of the strain is influenced by influencing the oxygen demand of the strain in the fermentation process, the beauveria bassiana is aerobic bacteria, and certain oxygen is needed in the solid fermentation spore production process. In the growth process of microorganisms, bacterial films are formed on the solid surface to enable matrixes to be agglomerated, and with the growth of the microorganisms, excessive sample loading amount can cause poor ventilation performance of the matrixes, poor culture effect and influence on the growth and spore production; too little sample loading strain distributes water in the growth process, which is unfavorable for the water retention of the matrix. When the loading amount is 75g, namely the spore yield of the treatment group ZYL-2 reaches 3.76X10 9 pieces/g, the treatment group is obviously higher than the other treatment groups. Thus, the sample loading amounts were selected to be 50g, 75g, 100g as the examination level for the subsequent experiments.
TABLE 17 influence of different sample loadings on the spore production of the strains±s，n=3）
Group of Specific treatment of Spore yield (10 9/g)
ZYL-1 50G of rice 2.73±0.09b
ZYL-2 75G rice 3.76±0.27a
ZYL-3 100G rice 2.90±0.20b
ZYL-4 125G rice 1.87±1.07c
ZYL-5 150G rice 1.56±0.22d
6.2.2 Feed to Water ratio screening
The effect of the amount of water added to the solid fermentation substrate on the solid spore production of beauveria bassiana strain is shown in Table 18. The spore yield is obviously different (F=85.059, P < 0.001) under different feed water ratios, and the spore yield is increased and then decreased with the increase of the water addition amount. When the feed water ratio is 3:1, namely the spore yield of the treatment group LSB-3 reaches 2.90 multiplied by 10 9/g, which is obviously higher than that of the other treatment groups, when the feed water ratio is 1:1, the matrix can be bonded into blocks due to more water adding amount, and the matrix needs to be stirred uniformly with great effort, and meanwhile, in the drying process, the drying speed is slower and the efficiency is low due to higher feed water ratio. Thus, the feed water ratios were chosen as 2:1, 3:1, 4:1 as the levels of investigation for the subsequent experiments.
TABLE 18 influence of different feed water ratios on the spore production of the strains±s，n=3）
Group of Specific treatment of Spore yield (10 9/g)
LSB-1 The ratio of the feed to the water is 1:1 1.82±0.23c
LSB--2 The ratio of the feed to the water is 2:1 2.22±0.04b
LSB-3 The ratio of the material to the water is 3:1 2.90±0.20a
LSB-4 Feed water ratio 4:1 1.61±0.02d
LSB-5 The ratio of the feed to the water is 5:1 1.41±0.17e
6.2.3 Inoculum size selection
The effect of inoculum size on spore yield of solid fermentation is shown in Table 19. There was a significant difference in spore yield (f=21.246, p < 0.001) with different inoculum size treatments, with increasing inoculum size and decreasing spore yield. Too high or too low inoculation amount is not beneficial to the spore production of the strain, too high inoculation liquid is easy to cause overgrowth of hypha, too high humidity of the culture bag and occurrence of water drops in the bag in the later period are visible, and the spore production is not beneficial; the inoculation amount is too low, and the rice matrix is not fully utilized. When the inoculum size was 25%, i.e. the sporulation number of the treatment group JZL-3 reached 3.08X10 9/g, there was no significant difference from the inoculum size of JZL-2, i.e. 20%, but significantly higher than the other treatment groups. Thus, 20%, 25%, 30% of the inoculum size was chosen as the level of investigation for the subsequent experiments.
TABLE 19 influence of different inoculum sizes on the spore production of the strains±s，n=3）
Group of Specific treatment of Spore yield (10 9/g)
JZL-1 Inoculum size 15% 2.19±0.29b
JZL--2 Inoculum size 20% 2.90±0.20a
JZL-3 Inoculum size 25% 3.08±0.05a
JZL-4 Inoculum size 30% 2.36±0.31b
JZL-5 Inoculum size 35% 2.16±0.19b
7. Solid fermentation Box-Behnken response surface test design
According to the single-factor test result, a three-factor and three-level response surface analysis experiment is designed by taking the spore production number of solid fermentation as a dependent variable, and the design of the experimental scheme is shown in table 20.
Multiple regression fitting is carried out on the sample loading quantity (A), the feed water ratio (B) and the inoculation quantity (C), the complex correlation coefficients R 2 = 0.9667 of binomial fitting are obtained from the tables 20 and 21, the model P is less than 0.001, the fitting degree is good, the true test points can be replaced for analysis by using the equation, and the growth and spore production process of the beauveria bassiana solid fermentation can be reflected well in the investigation range. Model regression equation mismatch test p=0.9997 > 0.05, no significant difference, shows that the equation fits well with the actual situation, the residual error of the model may be random error generation, and the model can be used for analyzing and predicting so as to obtain the optimal solid fermentation process. The multiple regression fit equation obtained from the anova is: comprehensive score Y＝2.68+0.1579A-0.4966B+0.0472C+0.0572AB-0.1399AC+0.1537BC-0.3764A2-0.2684B2-0.3116C2.
TABLE 20 design of solid fermentation Box-Behnken experiment table
TABLE 21 solid fermentation Box-Behnken experimental results
TABLE 22 analysis of variance of response surface of solid fermentation
According to the analysis of variance results in table 22, the primary and secondary orders of the process parameters affecting the comprehensive score value are B > A > C, namely the feed-water ratio > sample loading amount > inoculation amount, the influence of two factors of the primary term B (feed-water ratio) and the primary term A (sample loading amount) on the spore yield in the regression model reaches a significant level (< 0.05), the secondary terms are significant, the influence of the interaction terms AC, AC and BC on the comprehensive score value is not significant, and the fact that different factor parameters and the solid fermentation spore yield are not in a simple linear relation is explained.
8. Determination of optimal solid fermentation process
Solving a fitting equation by adopting Design Expert 13.0 software to obtain the optimal technological parameters when the solid fermentation sporulation number prediction of beauveria bassiana is maximum: the sample loading amount is 85g, the feed-water ratio is 2:1, the inoculation amount is 25%, and the predicted solid fermentation spore yield is 2.89 multiplied by 10 9/g.
9. Verification of optimal solid fermentation process
And carrying out verification experiments according to the optimal solid fermentation conditions obtained by the design of the response surface curve, and repeating the experiments three times, wherein the experimental results are shown in table 23. The average value of the spore production number is 3.30X10 9/g (RSD is 0.39%), which is relatively close to the predicted spore production number of 2.89X 10 9/g. Therefore, the model has accurate predictability and reliability, and can also prove the feasibility of optimizing the beauveria bassiana solid fermentation process by using the response surface. Under the optimal condition, the spore yield is slightly higher than the predicted value of the regression equation, and the heat generated by spore yield in the solid fermentation process possibly causes the temperature of the solid culture material to slightly rise, so that the spore yield is increased and the difference is generated between the spore yield and the predicted value.
Table 23 verifies the results of the experiment
3. Liquid-solid biphasic fermentation process pilot scale test of beauveria bassiana
1. Test in strain production Process
1.1 Determination of the transfer of the liquid fermentation to the second liquid fermentation
The fermentation broth obtained by primary shake flask culture in the liquid fermentation step needs to be subjected to secondary conversion to expand liquid fermentation culture, and the transfer amount can directly influence the fermentation time and yield of secondary fermentation. The results of the experiments using the concentration of blastospores and the dry weight of mycelia obtained by the secondary liquid fermentation as the detection index are shown in FIG. 6 and Table 24. The spore concentration and mycelium dry weight of the bud spores under different transfer amounts reach the maximum value at 60h, respectively reduced slightly to 72h, and when the transfer amount is 5%, the spore concentration and mycelium dry weight of the bud spores are obviously higher than those of the rest treatment groups, so that the secondary expansion fermentation culture is carried out by transferring 5% of primary shake flask fermentation liquor for 60 hours.
TABLE 24 two-stage liquid fermentation of mycelium dry weight at different transfer rates
1.2 Pilot plant test production process flow of strains
The strain J-2 is used as a pilot plant strain, and pilot plant production is carried out in Sichuan Naruhito source agricultural science and technology Co-Ltd on the basis of the best liquid-solid double-phase fermentation process of beauveria bassiana which is preferably obtained by laboratory experiments. The preparation and the debugging are carried out before the production, in the solid fermentation link, secondary fermentation bacteria liquid is used as inoculation bacteria liquid in pilot plant test, a large stainless steel tray (24 cm multiplied by 18cm multiplied by 3 cm) is selected as a fermentation container, the sample conversion amount is adjusted to 170g, and the fresh-keeping film is used for covering during sealing culture. About 50g of beauveria spores powder is produced in each batch in a pilot-scale mode. The specific process flow is shown in figure 7.
1.3 Quality detection of pilot plant spore powder
Measuring appearance characters, drying decrement, spore content, viable spore number and mixed bacterial rate of the pilot plant spore powder; the results are shown in Table 25.
The spore powder cultured by liquid-solid double-phase fermentation has biological components of culture medium during fermentation, and is dried to separate spore powder from rice solid culture medium, and the spore powder has white or yellowish white powder with special bacterial fragrance and rice fragrance, as shown in figure 8
The drying decrement of the pilot-plant spore powder for three times is 5.66% -6.53%, the spore content is 9.36×10 10 spores/g-9.56×10 10 spores/g, and the viable count is 8.81×10 10 spores/g-8.95×10 10 spores/g. The drying decrement ZS-1 is obviously higher than ZS-2 and ZS-3, but the key indexes of the three batches of spore powder have no obvious difference in spore content and viable spore number, and the mixed bacteria test is shown in figure 9, and the mixed bacteria are not detected.
The quality index of the spore powder in Table 25±s，n=3）
Numbering device Drying decrement (%) Spore content (10 10 spores/g) Living spore number (10 10 spores/g) Rate of mixed bacteria (%)
ZS-1 6.53±0.22a 9.36±0.75a 8.81±0.67a Not detected
ZS-2 5.66±0.23b 9.56±0.32a 8.98±0.20a Not detected
ZS-3 5.71±0.05b 9.53±0.23a 8.95±0.13a Not detected
EXAMPLE 4 application of the Bombyx Batryticatus in the production of the present invention
1. Instrument and reagent
BS-200S-WEI precision electronic balance (thousandth, beijing cerdolis instrument systems limited); BP-121s precision electronic balance (thousandth, zhejiang precision instruments Co., ltd.); box-type resistance furnace (Shanghai electric furnace Co., ltd.); electronic thermostat water bath (Beijing Zhongxing Wei industry instruments Co., ltd.); siemens UltiMate high performance liquid chromatograph (Thermo Fisher Co., USA); ULUP-IV-10T Utility model ultra-pure water device (Chengdu ultra-pure technology Co., ltd.); intelligent electrothermal blast drying oven (Shanghai Lang laboratory equipment Co., ltd.); KQ-500DE type numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.).
Control: beauverine (lot number: 419469, beijing Baoling technology Co., ltd., purity: 99%) water is ultrapure water, and methanol, ethanol and acetonitrile are all chromatographic purities.
2. Test method
2.1 Feeding of young silkworms
And (3) selecting qualified silkworm seeds from factory, and breeding the silkworm seeds under a proper environment by referring to the Sichuan province local standard (mulberry silkworm small silkworm co-breeding technical procedure) (DB 51T 849-2008).
2.2 Inoculation and cultivation of silkworm
The silkworms grow to the early five-age period, are fed for 1 time and then are inoculated.
Three batches of pilot spore powder are taken for standby, and the inoculating bacterial liquid is beauveria bassiana working liquid prepared by sterile water (containing 0.05% of Tween-80 and 23-28 ℃). The inoculation concentration is 2X 10 7 spores/mL, and the mixture is uniformly sprayed for three times, and the temperature and humidity environment with 90% RH and 26 ℃ is maintained for 2 hours; one sheet of paper for each group, three replicates.
2.3 Harvesting and processing
And when the silkworm is normally cultivated to start to dead, harvesting is completed in batches. After harvesting, spreading the stiff silkworm in a tray, removing impurities such as mulberry leaves, silkworm excrement and the like, and curing under the conditions that the temperature is 24-26 ℃ and the relative humidity is more than 90%, so that the silkworm body grows with beauveria bassiana spores. And (3) after curing for 48-60 h, drying until the moisture content is not higher than 13%.
2.4 Appearance and cross-section property of stiff silkworm medicinal material
And combining with the characteristic description index under the stiff silkworm item of the Chinese pharmacopoeia 2020 edition, photographing and observing and recording the appearance characteristics of the stiff silkworm, including length, diameter and weight. Cutting between 1 st to 2 nd pairs of feet of the abdomen of stiff silkworm, and dividing into 4, 3, 2, 0 silk gland rings and white silk gland rings according to the condition of the silk gland ring of the section. Sampling by using a quartering method, wherein each batch of medicinal materials is sampled by 50 g, the ratio is calculated according to the number of each group, and each group is repeated three times.
2.5 Sample grade determination
The classification of the medicinal materials is divided into first class, second class and third class according to the stiff silkworm medicinal material class standard established in the earlier stage of the subject group, and the stiff silkworm class standard classification index is shown in a table 26.
Table 26 standard grading index for stiff silkworm
2.6 Determination of intrinsic Mass of samples
The method is used for measuring the content of water, total ash and extract and the content of beauvericin under the condition of stiff silkworm item in Chinese pharmacopoeia of 2020 edition.
3. Results and analysis
3.1 Influence of the spore powder on the death rate of Bombyx Batryticatus and the yield of Bombyx Batryticatus
As shown in Table 27, the three pilot spore powders showed no significant difference (P > 0.05) in the dead rate of silkworm, which reached 95% or more; the output of the stiff silkworm medicinal materials reaches 15.77 Kg/paper to 16.29 Kg/paper, and no significant difference (P is more than 0.05).
The test spore powder in Table 27 shows the death rate of silkworm and the yield of medicinal materials±s，n=3）
3.2 Influence of the pilot spore powder on the characteristics of the Bombyx Batryticatus
3.2.1 Appearance characters of the Bombyx Batryticatus medicinal material produced by pilot plant spore powder
As can be seen from Table 28, the appearance of the stiff silkworm medicinal materials produced by the three batches of spore powder is stronger and full, and no significant difference exists.
Table 28 appearance traits of Bombyx Batryticatus obtained by inoculating different beauveria strains
3.2.2 Medium test spore powder production of Bombyx Batryticatus medicinal material silk gland ring character
The results of silk gland ring proportion of stiff silkworm medicine produced by pilot plant spore powder are shown in Table 29. The results show that the stiff silkworm medicinal materials produced by the three batches of pilot-scale spore powder are mainly 4 silk gland rings, the ratio of the stiff silkworm medicinal materials is more than 70%, and the three batches of pilot-scale spore powder have no significant difference (P is more than 0.05).
The test result of the silk gland ring character of the stiff silkworm medicine produced by the spore powder in the table 29 is [ (]±s，n=3，%）
3.3 Grading of Bombyx Batryticatus medicinal materials produced by pilot plant spore powder
The grading results of the stiff silkworm medicinal materials produced by the pilot plant spore powder are shown in Table 30. The results show that the first grade products of the stiff silkworm medicinal materials produced by the three batches of pilot plant spore powder are all more than 50%; 1. the sum of the two medicinal materials exceeds 90 percent, and the two medicinal materials have no obvious difference (P is more than 0.05).
The classification result of the stiff silkworm medicinal materials produced by the spore powder in table 30±s，n=3，%）
3.4 Inherent quality of stiff silkworm medicine produced by pilot plant spore powder
The results of the silk gland ring proportion of the stiff silkworm medicinal materials produced by the pilot spore powder are shown in Table 31. The results show that the water content, the total ash content and the extract of the stiff silkworm medicinal materials produced by three batches of pilot spore powder meet the specification under stiff silkworm item in Chinese pharmacopoeia of 2020 edition, the total ash content is between 6.36% and 6.40%, the extract is between 27.72% and 28.08%, the content of beauvericin is between 0.030% and 0.032%, certain differences but no significant differences exist in the intrinsic quality of the medicinal materials, and the stability of the beauveria bassiana spore powder produced by the liquid-solid biphasic fermentation method established by the research and the stability of the beauveria bassiana spore powder applied to stiff silkworm cultivation production are verified from the side.
The inherent quality of the stiff silkworm medicinal material produced by the spore powder in the table 31 is [ ]±s，n=3，%）
4. Knot (S)
The fermentation of the microorganism is a complex metabolic process, and the fermentation production of the beauveria bassiana by adopting a liquid-solid double-phase fermentation method combines the dual advantages of liquid-phase fermentation and solid-phase fermentation, and is the main production process of the beauveria bassiana at present. At present, the beauveria bassiana production process research is applied to the aspects of biological control, and early-stage interview investigation shows that the beauveria bassiana culture base adopts spore powder eluted from the body surface of the beauveria bassiana as a strain source, so that the purity and quality of the beauveria bassiana cannot be ensured.
The invention determines key experimental parameters of two links of the beauveria bassiana liquid fermentation and the solid fermentation by a response surface method, establishes a liquid-solid double-phase fermentation method, and utilizes the method to produce three batches of beauveria bassiana spore powder, wherein the inherent quality (spore content, living spore rate and mixed fungus rate) of the spore powder has no significant difference. The spore powder is applied to the stiff silkworm breeding production, the obtained medicinal materials have small quality difference, and the method does not need large-scale fermentation tank equipment, is simple and convenient to operate, is more suitable for large-scale production of stiff silkworm breeding enterprises, and reduces the production cost.

Claims (4)

1. A fermentation process of beauveria bassiana spore powder of medicinal stiff silkworm is characterized in that: the method comprises two fermentation links of liquid fermentation and solid fermentation, and comprises the following steps:
a. Activating the strain; inoculating purified slant strain to SDY solid culture medium by streaking method, standing in constant temperature incubator under semi-illumination to culture, and collecting when the culture medium grows light yellow white conidium powder; the temperature of the constant temperature incubator is 26 ℃; the semi-illumination conditions were: l: d=12:12; culturing for 14d;
b. preparing a sterilized liquid culture medium:
the liquid medium is SDY medium with agar removed: 40g of glucose, 10g of yeast extract powder, 10g of peptone, 1000mL of distilled water and 0.05% tween-80, weighing the raw materials according to a formula, mixing, dissolving, packaging and sterilizing;
c. preparation of primary fermentation liquor:
Under the aseptic condition, preparing spore powder in the step a into spore suspension by using 0.05% Tween-80 aseptic aqueous solution, sucking the spore suspension to ensure that the initial inoculation concentration is 2.4X10 6 spores/mL, and culturing for 72 hours on a shaking table with the temperature of 26.2 ℃ and the shaking table rotating speed of 174r/min to obtain beauveria bassiana primary fermentation liquor;
d. Preparation of a secondary inoculation liquid: transferring 5% of the primary fermentation liquid in the step c under the aseptic condition, and culturing for 60 hours on a shaking table with the rotation speed of 174r/min at the temperature of 26.2 ℃ to obtain a beauveria bassiana secondary inoculation liquid;
e. Preparation of solid medium: loading rice into a stainless steel tray, adding 0.4% glucose and potassium nitrate, adding water with a material-water ratio of 2:1W:V, sterilizing with high-pressure steam at 121deg.C for 15min, taking out, and cooling to room temperature;
f. Solid spore production treatment: under the aseptic condition, adding 25% of the secondary inoculation liquid in the step d into a solid culture medium, uniformly mixing the bacterial liquid and rice, sealing with a preservative film, and culturing for 10d under the condition that the temperature is 26 ℃ and the humidity RH=85%; the inoculation amount is 25% v/w based on the total rice; culturing for 10d, sealing the front 3d for dark culture, and opening the rear 7d for illumination culture;
g. drying and sieving: and (5) drying and sieving after the culture is finished, and separating rice matrixes to obtain spore powder.
2. The fermentation process of the beauveria bassiana spore powder of the medicinal stiff silkworm according to claim 1, which is characterized in that: the size of the stainless steel shallow tray in the step e is as follows: 24 cm. Times.18 cm. Times.3 cm.
3. The fermentation process of the beauveria bassiana spore powder of the medicinal stiff silkworm according to claim 1, which is characterized in that: the drying conditions in the step g are as follows: drying at 35 ℃ for 2d; sieving with 100 mesh sieve.
4. Use of beauveria bassiana spore powder prepared by fermentation of the beauveria bassiana spore powder of medicinal stiff silkworm by a fermentation process of any one of claims 1-3 in the preparation of stiff silkworm.
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