Preparation method and application of solid-state microbial agent for fiber extraction

Abstract

The application discloses a kind of preparation method and application of solid bacterial agent for fiber extraction, comprising, after activation DCE-01 and BE-91 seed liquid is inoculated to fermentation culture solution and is expanded and is fermented, after obtaining mature fermentation liquor, it is concentrated, then protective agent is added to concentrated fermentation liquor and is mixed evenly to obtain wet bacterial powder, finally wet bacterial powder is spread and is dried at 25-30 DEG C, and the present application is obtained.The present application selects two kinds of excellent microorganisms to prepare composite microbial inoculant, enhances the effect of fiber extraction;Corn steep liquor dry powder and peanut cake powder are used to replace most of the protein peptone to prepare fermentation culture solution, not only reduce production cost, and have flocculation effect;Sterilized bran and cotton seed powder are selected as protective agent, and the adsorption capacity of bacterial body is large, the load of subsequent drying is reduced, and the protective agent can provide growth nutrients for bacterial agent in subsequent retting process;By using cold air normal pressure drying, DCE-01 and BE-91 strains are protected from harm, and energy is saved.

Description

Technical Field

[0001] This invention relates to the field of plant fiber raw material processing technology, specifically to a method for preparing and applying a solid microbial agent for fiber extraction. Background Technology

[0002] Plant-based raw materials are one of the world's most important sources of natural fiber. To obtain plant fibers that meet industrial requirements, a fiber extraction process is necessary to remove non-cellulose substances (commonly known as "colloids") such as pectin, hemicellulose, and lignin from the fiber raw materials. Traditional chemical extraction, which uses caustic soda as the core, has drawbacks such as high pollution and damage to fibers. In contrast, biological extraction, which uses microorganisms as the core, has the advantages of being "green, environmentally friendly, and highly efficient," and represents the future trend in fiber extraction.

[0003] The core of fiber bio-extraction is obtaining highly efficient microbial strains. The applicant's team previously bred a highly efficient degumming strain for bast fibers, *D. dadantii* DCE-01, capable of simultaneously and efficiently processing various plant fiber raw materials, including kenaf, jute, flax, and industrial hemp. This single strain, DCE-01, is suitable for pure cultivation in a fermentation workshop to complete the industrial-scale fiber extraction from plant raw materials. However, DCE-01 is a heat-sensitive Gram-negative bacterium, making its strain preparation technology relatively difficult, especially the technical issues of strain degradation and mutation, which restrict its further expanded application in jute / kenaf and flax retting. *B. subtilis* BE-91 is a strain previously screened by the team that can efficiently degrade hemicellulose, but it lacks pectinase and ligninase, and therefore cannot remove pectin and lignin from fiber raw materials; thus, it cannot be used alone for fiber extraction.

[0004] This invention solves the problems of low activity, incomplete enzyme system, and high cost of existing fiber bio-extraction agents by preparing a highly efficient mixed solid microbial agent by drying D. dadantiiDCE-01 and B. subtilis BE-91 under cold air conditions. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying a solid microbial agent for fiber extraction. The synergistic effect of DCE-01 and BE-91 is utilized to improve fiber extraction efficiency. Furthermore, by adjusting the preparation method, a microbial agent with a higher viable bacteria count is obtained.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing and applying a solid microbial agent for fiber extraction includes: mixing activated DCE-01 and BE-91 seed liquids and inoculating them into a fermentation culture medium for expansion and fermentation; concentrating the mature fermentation broth; adding a protectant to the concentrated fermentation broth and mixing it evenly to obtain a moist microbial powder; and finally spreading the moist microbial powder evenly and drying it at 25-30℃.

[0008] Specifically, the fermentation broth has the following mass ratio: 0.5-1.5% glucose + 1.0-2.0% corn steep liquor powder + 1.0-2.0% peanut cake powder + 0.5% peptone + 0.5% NaCl, with the remainder being water.

[0009] In the above technical solution, the expansion fermentation method is as follows: the mixed solution of DCE-01 and BE-91 seed liquids and the fermentation culture medium are placed in a sealed fermentation tank at a volume ratio of 1:40-50, and the filling volume is controlled to be 40-60% of the fermentation tank volume. Simultaneously, the pH value is controlled to be 6.5-7.0 by adding NH3·H2O, the temperature is 32-35℃, the stirring speed is 200-220 r / min, and the corresponding aeration rate is 0.3-0.5 m³ / L of seed liquid. 3 Fermentation is carried out at a rate of 1 / h for 12-14 hours until the pH value becomes constant or increases.

[0010] In the above technical solution, before being used for expansion fermentation, the fermentation culture medium is sterilized at 121°C for 20-30 minutes, and the pH value is adjusted to 7.0-7.5 with NH3·H2O before sterilization.

[0011] Furthermore, in the above technical solution, the concentration of DCE-01 bacteria in the mature fermentation broth is greater than 1×10⁻⁶. 9 CFU / mL, BE-91 concentration greater than 1×10⁻⁶ 10 cfu / mL.

[0012] In detail, in practical applications, the mature fermentation broth usually needs to undergo a degumming experiment to verify its bacterial activity. The specific method is as follows: Take 2 wt% of the mature fermentation broth and add it to 35°C preheated tap water to prepare a bacterial suspension. Soak the kenaf bast and bacterial suspension at a bath ratio of 1:10. Let it stand at 35°C for 10-12 hours to degumme. The kenaf bast softens and the fiber dispersibility is good, indicating that the DCE-01 and BE-01 mixed bacterial strains have high activity.

[0013] In the above technical solution, the concentrated fermentation broth is collected by centrifugation at a speed of 3000-5000 r / min for 20-30 min. In practical applications, 1 / 5-1 / 4 of the supernatant is usually retained, and after remixing, the concentrated fermentation broth is obtained.

[0014] In detail, the presence of corn steep liquor powder and peanut cake powder in the fermentation broth significantly increased its sedimentation coefficient, resulting in excellent precipitation. Furthermore, the supernatant after centrifugation was examined under a microscope (40×100x) with crystal violet staining to observe the bacterial count. The presence of fewer than 10 bacteria per field of view confirmed the effective centrifugation.

[0015] Furthermore, in the above technical solution, the protective agent is a mixture of sterilized wheat bran and cottonseed flour. Preferably, the mass ratio of the sterilized wheat bran to cottonseed flour is 1:1, and the mass ratio of the concentrated fermentation liquid to the mixture of sterilized wheat bran and peanut cake flour is 1:5-8.

[0016] In the above technical solution, the drying method of the moist bacterial powder is as follows: spread the moist bacterial powder evenly on a stainless steel mesh screen, control the spreading thickness to be 1-2cm, and dry it with cold air at 25-30℃ for 25-40 minutes until its moisture content is 6-8%.

[0017] Furthermore, in the above technical solution, the content of the pulverized and vacuum-packaged finished bacterial agent, DCE-01, is greater than 1×10⁻⁶ bacteria. 9 CFU / g, BE-91 bacteria content greater than 1×10 10 cfu / g.

[0018] Furthermore, in the above technical solution, the solid microbial agent obtained from the DCE-01 and BE-91 mixed strains can be directly used in the extraction of fibers such as jute / red hemp and hemp / flax retting.

[0019] Advantages of this invention:

[0020] (1) The preparation method and application of the solid microbial agent for fiber extraction provided by the present invention prepares a compound microbial agent by mixing and culturing DCE-01 and BE-01 strains, which enriches the gum degradation enzyme system of plant fiber raw materials, enhances the degradation ability of non-cellulose substances, and improves the fiber preparation efficiency.

[0021] (2) The preparation method and application of the solid microbial agent for fiber extraction provided by the present invention greatly reduces the production cost by adjusting the formula of the fermentation culture medium and replacing most of the peptone with corn steep liquor powder and peanut cake powder; and these two new nitrogen sources can change the overall properties of the culture medium and promote the subsequent mixed bacterial growth and reproduction; the two coarse particle fermentation culture medium components also enhance the adsorption and sedimentation of bacteria in the mature fermentation broth, and play a role in immobilization and flocculation.

[0022] (3) The preparation method and application of the solid microbial agent for fiber extraction provided by the present invention, by selecting sterilized wheat bran and cottonseed powder as composite protective agents, makes the adsorption capacity of the microbial cells large, and the fluffy characteristics of sterilized wheat bran reduce the load of subsequent drying, resulting in excellent practical application effect; at the same time, the wheat bran and peanut cake powder contained in the microbial agent can provide the nutrients required for the growth of microbial strains for subsequent retting of hemp, which is convenient for direct feeding.

[0023] (4) The preparation method and application of the solid bacterial agent for fiber extraction provided by the present invention, by adopting the cold air normal pressure drying method, not only protects the DCE-01 and BE-91 strains from damage to the maximum extent, but also saves energy; the process is simple, the cost is low, and it is easy to promote and apply, with broad practical application prospects and great theoretical and practical significance. Attached Figure Description

[0024] Figure 1 This is a process flow diagram illustrating the preparation method and application of the solid microbial agent for fiber extraction provided in the embodiments of the present invention. Detailed Implementation

[0025] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples.

[0026] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

[0027] Unless otherwise specified, all experimental reagents and materials used in the embodiments of the present invention are commercially available. Unless otherwise specified, all technical means used in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0028] Example 1: Preparation method and application of a solid microbial agent for fiber extraction

[0029] One loopful of DCE-01 bacterial strain was selected from the slant culture preserved by the Plant Fiber Functional Materials Innovation Team of the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, and inoculated into 5 mL of modified broth medium (i.e., GR medium, with the following mass ratio: 1% glucose + 0.5% beef extract + 0.5% peptone + 0.5% NaCl, adjusted to pH 7.5). The culture was thoroughly resuspended and incubated at 35℃ for 6 h. After dilution, the culture was spread onto YQ nutrient agar plates and incubated at 35℃ for 20 h. Single colonies were isolated. Typical colonies were streaked onto fresh slant culture, incubated at 35℃ for 20 h, vacuum-packed, and stored at room temperature. One slant culture was poured into 1 mL of sterilized modified broth medium, and the surface bacterial growth was gently scraped with an inoculation loop to ensure that all bacterial growth was mixed into the culture medium.

[0030] One loopful of BE-91 bacterial strain was selected from the slant culture preserved by the Plant Fiber Functional Materials Innovation Team of the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, and inoculated into 5 mL of modified broth medium (i.e., GR medium, with the following mass ratio: 1% glucose + 0.5% beef extract + 0.5% peptone + 0.5% NaCl, adjusted to pH 7.5). The culture was thoroughly resuspended and incubated at 35℃ for 6 h. After dilution, the culture was spread onto YQ nutrient agar plates and incubated at 35℃ for 20 h to isolate single colonies. Typical colonies were selected and streaked onto fresh slant culture, incubated at 35℃ for 20 h, vacuum-packed, and stored at room temperature. One slant culture was poured into 1 mL of sterilized modified broth medium, and the surface bacterial growth was gently scraped with an inoculation loop to ensure that all bacterial growth was mixed into the culture medium.

[0031] The liquid containing bacterial growth was inoculated into 100mL Erlenmeyer flasks containing 40mL of GR culture medium, mixed thoroughly, and cultured at 35℃ and 180r / min in a water bath for 5h to obtain the primary seed culture. The primary seed culture was then inoculated into 3500mL Erlenmeyer flasks containing 1000mL of GR culture medium, mixed thoroughly, and cultured at 35℃ and 180r / min in a water bath for 6h to obtain the secondary seed culture.

[0032] Mix the secondary seed cultures of DCE-01 and BE-91 at a ratio of 1:40-50. Take 0.6-0.8L of the mixture and inoculate it into a 50L fermenter (Bio, Switzerland, Germany) containing 30L of fermentation broth. Continuously add NH3·H2O automatically to maintain the pH at 6.7 and the aeration rate at 0.7m³ / h. 3 Culture at 34℃ and 180r / min for 10 hours, until the pH rises slightly, then stop fermentation and harvest the mature fermentation broth.

[0033] Specifically, the mass ratio formula of the above fermentation culture medium is: 0.5-1.5% glucose + 1.0-2.0% corn steep liquor powder + 1.0-2.0% peanut cake powder + 0.5% peptone + 0.5% NaCl, with the remainder being water.

[0034] Referring to the standard "Test Methods for Lactic Acid Bacteria in Imported and Exported Foods" (SN / T 1941.1-2017), the viable bacteria DCE-01 and BE-91 in the mature fermentation broth were isolated and counted using the serial dilution method. The viable bacterial counts were 1.2 × 10⁻⁶. 9 cfu / mL and 2.2×10 10 cfu / mL.

[0035] A bacterial suspension was prepared by adding 2% of DCE-01 mature fermentation broth to preheated tap water at 35℃. 20g of kenaf was soaked in 200ml of the bacterial suspension. Degumming was performed at 35℃. The kenaf bast began to soften after 10 hours and was completely softened after 11.5 hours, exhibiting good fiber dispersibility. This verified the high activity of the mixed DCE-01 and BE-01 bacterial strains.

[0036] The cells in the mature fermentation broth were collected by centrifugation at 5000 rpm for 20 min. The supernatant was then examined under an oil immersion microscope (40×100x) after staining with crystal violet. The number of cells was observed in each field of view, with 5-8 cells per field. The centrifugation was effective.

[0037] Take 30L of mature fermentation broth, concentrate it to 5-6L, add 25kg of sterilized wheat bran and cottonseed powder, mix thoroughly, and then spread the wet inoculum powder evenly on a stainless steel mesh sieve to a thickness of 1cm. Dry at 28℃ for about 30 minutes under cold air blowing conditions in a tea dryer (mixing and spreading every 10 minutes). Grind the inoculum powder through a 100-mesh sieve using a pulverizer, vacuum package it, and the resulting solid inoculum agent is the finished product. The moisture content of the finished solid inoculum agent is approximately 8-10% as measured by a moisture analyzer. The viable cell counts of DCE-01 and BE-91, determined by the gradient dilution method, are 8.1 × 10⁻⁶. 9 cfu / g and 9.09×10 10 cfu / g.

[0038] According to calculations, the total recovery rate of live bacteria DCE-01 and BE-91 prepared by the solid bacterial powder in the example was 24.5%.

[0039] Comparative Example 1

[0040] The difference from Example 1 is as follows:

[0041] The fermentation broth is formulated with the following mass ratio: 0.5-1.5% glucose + 1.0-2.0% corn steep liquor powder + 1.0-2.0% soybean meal + 0.5% peptone + 0.5% NaCl, with the remainder being water.

[0042] The remaining preparation methods are the same as in Example 1.

[0043] Referring to the standard "Test Methods for Lactic Acid Bacteria in Imported and Exported Foods" (SN / T 1941.1-2017), the viable bacteria DCE-01 and BE-91 in the mature fermentation broth were isolated and counted using the serial dilution method. The viable bacteria counts were 5.2 × 10⁻⁶. 8 cfu / mL and 1.4×10 9 cfu / mL.

[0044] The activity of the fermentation broth was verified by the degumming test of kenaf. The kenaf began to soften after 14 hours and the fiber dispersibility was poor, which verified that the activity of the mixed strains DCE-01 and BE-01 was basically qualified.

[0045] The moisture content of the finished solid microbial agent was measured to be approximately 8-10% using a moisture analyzer. The viable cell count of DCE-01, determined using a gradient dilution method, was 2.2 × 10⁻⁶. 8 cfu / g and 8.4×10 8 cfu / g.

[0046] After calculation, the total recovery rate of live bacteria DCE-01 and BE-91 in the solid bacterial powder prepared by the preparation method of Comparative Example 1 was 15.1%.

[0047] Comparative Example 2

[0048] The fermentation broth has the following mass ratio: 1% glucose + 2.0% beef extract + 0.5% peptone + 0.5% NaCl, with the remainder being water.

[0049] The remaining preparation methods are the same as in Example 1.

[0050] Referring to the standard "Test Methods for Lactic Acid Bacteria in Imported and Exported Foods" (SN / T 1941.1-2017), the viable bacteria DCE-01 and BE-91 in the mature fermentation broth were isolated and counted using the serial dilution method. The viable bacterial counts were 1.04 × 10⁻⁶. 8 cfu / mL and 1.38×10 9 cfu / mL.

[0051] The activity of the fermentation broth was verified by the degumming test of kenaf. The kenaf softened after 14 hours and the fiber dispersion was good, which verified that the activity of the mixed strains DCE-01 and BE-01 was basically qualified.

[0052] The moisture content of the finished solid microbial agent was measured to be approximately 8-10% using a moisture analyzer. The viable cell count of DCE-01, determined using a gradient dilution method, was 8.02 × 10⁻⁶. 7 cfu / g and 9.4×10 8 cfu / g.

[0053] After calculation, the total recovery rate of live bacteria DCE-01 and BE-91 in the solid bacterial powder prepared by the preparation method of Comparative Example 2 was 8.1%.

[0054] Comparative Example 3

[0055] The rest is the same as in Example 1.

[0056] Take 30L of mature fermentation broth, concentrate it to 5-6L, add 25kg of sterilized cottonseed powder, mix thoroughly, and then spread the wet inoculum powder evenly on a stainless steel mesh sieve to a thickness of 1cm. Dry at 28℃ for approximately 30 minutes under cold air blowing conditions in a tea dryer (mixing and spreading every 10 minutes). Grind the inoculum powder through a 100-mesh sieve using a pulverizer, vacuum package, and the resulting solid inoculum agent is ready. The moisture content of the finished solid inoculum agent is approximately 12% as measured by a moisture analyzer. The viable cell counts of DCE-01 and BE-91, determined by the gradient dilution method, are 3.7 × 10⁻⁶. 8 cfu / g and 9.09×10 7 cfu / g.

[0057] After calculation, the total recovery rate of live bacteria DCE-01 and BE-91 in the solid bacterial powder prepared by the preparation method of Comparative Example 3 was 1.4%.

[0058] Comparative Example 4

[0059] The rest is the same as in Example 1.

[0060] Take 30L of mature fermentation broth, concentrate it to 5-6L, add 25kg of sterilized wheat bran, mix thoroughly, and then spread the wet inoculum powder evenly on a stainless steel mesh sieve to a thickness of 1cm. Dry at 28℃ for approximately 30 minutes under cold air blowing conditions in a tea dryer (mixing and spreading every 10 minutes). Grind the inoculum powder through a 100-mesh sieve using a pulverizer, vacuum package, and the resulting solid inoculum agent is ready. The moisture content of the finished solid inoculum agent is approximately 8-9% as determined by a moisture analyzer. The viable cell counts of DCE-01 and BE-91, measured using a gradient dilution method, are 2.37 × 10⁻⁶. 8 cfu / g and 3.09×10 8 cfu / g.

[0061] After calculation, the total recovery rate of live bacteria DCE-01 and BE-91 prepared by solid bacterial powder in Comparative Example 4 was 4.5%.

[0062] Comparative Example 5

[0063] Take 0.6-0.8L of DCE-01 secondary seed and inoculate it into a 50L fermenter (Bio-Switzerland, Germany) containing 30L of fermentation broth. Continuously add NH3·H2O automatically to maintain the pH at 6.7 and maintain an aeration rate of 0.7m³ / h. 3 Culture at 34℃ and 180r / min for 10 hours, until the pH rises slightly, then stop fermentation and harvest the mature fermentation broth.

[0064] The rest is the same as in Example 1.

[0065] Referring to the standard "Test Methods for Lactic Acid Bacteria in Imported and Exported Foods" (SN / T 1941.1-2017), the viable DCE-01 bacteria in the mature fermentation broth were isolated and counted using the serial dilution method. The viable count was 6.74 × 10⁻⁶. 9 cfu / mL.

[0066] The activity of the fermentation broth was verified by a kenaf degumming test. The kenaf began to soften after 14 hours and the fiber dispersibility was poor, which verified that the activity of the DCE-01 strain was insufficient.

[0067] The moisture content of the finished solid microbial agent was measured to be approximately 8-10% using a moisture analyzer. The viable count of DCE-01, determined using a gradient dilution method, was 3.02 × 10⁻⁶. 9 cfu / g.

[0068] After calculation, the total recovery rate of live bacteria of DCE-01 prepared by solid bacterial powder in Comparative Example 5 was 10.2%.

[0069] Comparative Example 6

[0070] Take 0.6-0.8 L of BE-91 secondary seed and inoculate it into a 50 L fermenter (Bio-Switzerland, Germany) containing 30 L of fermentation broth. Continuously add NH3·H2O automatically to maintain the pH at 6.7 and maintain an aeration rate of 0.7 m³ / h. 3 Culture at 34℃ and 180r / min for 10 hours, until the pH rises slightly, then stop fermentation and harvest the mature fermentation broth.

[0071] The rest is the same as in Example 1.

[0072] Referring to the standard "Test Methods for Lactic Acid Bacteria in Imported and Exported Foods" (SN / T 1941.1-2017), the viable BE-91 bacteria in the mature fermentation broth were isolated and counted using the serial dilution method. The viable bacterial count was 1.38 × 10⁻⁶. 9 cfu / mL.

[0073] The activity of the fermentation liquid was verified by a kenaf degumming test. The kenaf did not soften and the fibers did not disperse, which verified that the BE-01 strain basically had no independent degumming ability.

[0074] The moisture content of the finished solid microbial agent was measured to be approximately 8-10% using a moisture analyzer. The viable cell count of BE-01, determined using a gradient dilution method, was 3.4 × 10⁻⁶. 9 cfu / g.

[0075] After calculation, the total recovery rate of BE-91 live bacteria prepared by solid bacterial powder in Comparative Example 6 was 6.2%.

[0076] Therefore, when using the kenaf retting effect to measure the viability of the microbial strain, the kenaf retting effect of the microbial strains in the mature fermentation broths prepared in Examples and Comparative Examples 1-6, from strongest to weakest, is as follows: Example 1 > Comparative Example 2 > Comparative Example 1 > Comparative Example 3 = Comparative Example 4 > Comparative Example 5 > Comparative Example 6; the effect of the microbial agents prepared in Examples and Comparative Examples 1-6, from strongest to weakest, is as follows: Example 1 > Comparative Example 1 > Comparative Example 5 > Comparative Example 2 > Comparative Example 6 > Comparative Example 4 > Comparative Example 3. Comparative Examples 5 and 6 show that although BE-91 does not have independent degumming ability, its combination with DCE-01 can significantly improve the degumming effect, indicating that DCE-01 and BE-91 in this invention have a synergistic effect. Comparative Examples 1 and 2 show that using soybean meal or beef extract as a nitrogen source in the fermentation culture medium will affect the bacterial count and recovery rate in the microbial agent. Comparative Examples 3 and 4 show that when cottonseed flour and sterilized wheat bran are used alone as protectants, the viable count and recovery rate of the microbial agent are significantly reduced; indicating that the use of cottonseed flour and sterilized wheat bran together as protectants in this application can improve the viable count and recovery rate of the microbial agent in this application.

[0077] Finally, the above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a solid microbial agent for fiber extraction, characterized in that, Activated Dickeya dandantii DCE-01 and Bacillus subtilis BE-91 secondary seed culture is inoculated into fermentation broth and mixed for expansion and fermentation. After obtaining mature fermentation broth, it is concentrated, and then a protective agent is added to the concentrated fermentation broth and mixed evenly to obtain moist bacterial powder. Finally, the moist bacterial powder is spread evenly and dried at 25-30℃. DCE-01 and BE-91 secondary seed cultures are mixed at a volume ratio of 1:40-50, and 0.6-0.8L of the mixture is inoculated into a fermenter containing 30L of fermentation broth. The filling volume of fermentation broth is controlled to be 40-60% of the fermenter volume. The protective agent is a mixture of sterilized wheat bran and cottonseed powder; The fermentation broth contains the following mass ratio: 0.5-1.5% glucose + 1.0-2.0% corn steep liquor powder + 1.0-2.0% peanut cake powder + 0.5% peptone + 0.5% NaCl, with the remainder being water.

2. The preparation method according to claim 1, characterized in that, The propagation fermentation method is as follows: the pH is controlled to 6.5-7.0 by adding NH3·H2O, the temperature is 32-35℃, the stirring speed is 200-220 r / min, corresponding to an aeration rate of 0.3-0.5 m³ / L for each liter of seed culture. 3 / h, fermentation culture for 12-14 h until its pH value is constant or rises; The final concentration of the culture medium in the DCE-01 inoculated fermenter is 1-5 × 10⁻⁵. 5 cfu / ml, the final concentration of BE-91 inoculated was 1×10⁻⁶. 4 cfu / ml.

3. The preparation method according to claim 1, characterized in that, Before being used for expansion fermentation, the fermentation broth is sterilized at 121°C for 20-30 min, and the pH is adjusted to 7.0-7.5 with NH3·H2O before sterilization.

4. The preparation method according to any one of claims 1-3, characterized in that, The concentration of DCE-01 bacteria in the mature fermentation broth is greater than 1×10⁻⁶. 9 CFU / mL, BE-91 concentration greater than 1×10⁻⁶ 10 cfu / mL.

5. The preparation method according to claim 1, characterized in that, The concentrated fermentation broth is collected by centrifugation, with a centrifugation speed of 3000-5000 r / min and a centrifugation time of 20-30 min.

6. The preparation method according to claim 1, characterized in that, The mass ratio of sterilized wheat bran to peanut cake powder is 1:1, and the mass ratio of concentrated fermentation liquid to the mixture of sterilized wheat bran and cottonseed powder is 1:5-8.

7. The preparation method according to claim 1, characterized in that, The drying method for the moistened bacterial powder is as follows: The moistened bacterial powder is thoroughly mixed, spread evenly on a stainless steel mesh sieve, controlling the spreading thickness to 1-2 cm, and dried with cold air at 25-30℃ for 25-40 minutes until its moisture content is less than 10%. After pulverizing, it is vacuum-packed to obtain the finished bacterial agent, wherein the content of DCE-01 bacteria is greater than 1×10⁻⁶. 9 CFU / g, BE-91 bacteria content greater than 1×10 10 cfu / g.

8. The solid microbial agent for fiber extraction prepared by the preparation method according to any one of claims 1-7.

9. The application of the solid microbial agent for fiber extraction as described in claim 8 in the retting process of jute, kenaf, hemp, ramie or flax.

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