Microbial liquid inoculant, preparation method and application thereof

Abstract

The present application relates to a kind of microbial liquid inoculant and its preparation method and application, the preparation method of the inoculant includes the following steps: DCE-01 strain fermentation liquor is inoculated to the glucose bouillon medium containing protective agent and is cultivated, obtain microbial liquid inoculant;Wherein, the protective agent includes glycerol, D-sorbitol, mannitol, fructose and sucrose;In the glucose bouillon medium, glycerol volume is 8-15%, the molar concentration of D-sorbitol is 0.3-0.6M, the molar concentration of D-mannitol is 0.2-0.7M, the mass concentration of fructose is 0.15-0.4%, and the mass concentration of sucrose is 0.2-0.4%;By the preparation method of the present application, long-term storage, easy to transport, efficient microbial inoculant can be obtained to meet the use requirements of various complex fiber extraction scenes.

Description

Technical Field

[0001] This invention relates to the field of herbal fiber raw material processing technology, and more specifically, to a liquid microbial agent, its preparation method, and its application. Background Technology

[0002] Herbal fibers are materials made from fibers extracted from herbaceous plants such as hemp and grass straw. They are lightweight, environmentally friendly, biodegradable, and biocompatible, and have great potential applications in textiles, medicine, aerospace, automotive, construction, and packaging. A prerequisite for herbal fiber materials is obtaining high-quality herbal plant fibers. Typically, herbal plant fibers are coated with substances such as pectin, hemicellulose, and lignin. The extraction of herbal plant fibers involves the selective stripping of these substances. Industrially, chemical extraction methods are commonly used, which are costly, energy-intensive, and severely pollute the environment, resulting in low fiber yield and poor quality. Bio-extraction methods can save significant amounts of chemical raw materials and energy, greatly reduce environmental pollution, and yield higher-quality fibers.

[0003] Dickeya dadantii strain DCE-01 (preservation number: CGMCC5522, hereinafter referred to as strain DCE-01) is a highly efficient strain that can be used for the extraction of fibers from various herbal plants, including ramie, flax, and kenaf.

[0004] Currently, apart from large-scale factory production that can be equipped with complete microbial fermentation workshops to continuously supply DCE-01 strain fermentation broth for the extraction of herbal plant fibers, various complex fiber extraction scenarios, such as field-grown green degumming of flax, field-grown water-retting of kenaf, and farmer-style degumming of ramie, cannot be carried out on a large scale for microbial fermentation. This greatly limits the application scope of DCE-01 strain in the extraction of herbal plant fibers. Summary of the Invention

[0005] Based on the aforementioned technical problems in the existing technology, the present invention provides a method for preparing liquid microbial agents. The liquid microbial agents obtained by this method maintain high activity after two months of storage and have high extraction efficiency for herbal fibers, which can meet the needs of various complex fiber extraction scenarios.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for preparing a liquid microbial inoculant includes the following steps:

[0008] The fermentation broth of strain DCE-01 was inoculated into glucose broth medium containing a protectant and cultured to obtain liquid microbial inoculum.

[0009] The protective agent includes glycerol, D-sorbitol, mannitol, fructose, and sucrose; in the glucose broth medium, the volume content of glycerol is 8-15%, the molar concentration of D-sorbitol is 0.3-0.6M, the molar concentration of D-mannitol is 0.2-0.7M, the mass concentration of fructose is 0.15-0.4%, and the mass concentration of sucrose is 0.2-0.4%.

[0010] In some embodiments, the glucose broth culture medium contains 10% glycerol by volume, 0.4 M D-sorbitol by molar concentration, 0.6 M D-mannitol by molar concentration, 0.2% fructose by mass, and 0.3% sucrose by mass.

[0011] In some embodiments, after the fermentation broth of strain DCE-01 is inoculated into the glucose broth medium, it is incubated statically at 33-36°C for 12-16 hours.

[0012] In some embodiments, the fermentation broth of strain DCE-01 is inoculated into glucose broth medium containing a protectant at an inoculation rate of 5-15%.

[0013] In some embodiments, the basic formulation of the glucose broth culture medium includes: 5-15 g / L peptone, 2-10 g / L beef extract, 2-10 g / L sodium chloride, 5-15 g / L glucose, and pH 7.2-7.4.

[0014] In some embodiments, the preparation of the DCE-01 strain fermentation broth includes the following steps:

[0015] S1. Take a loop of bacterial growth from the DCE-01 bacterial slant and place it in liquid glucose broth medium. Activate overnight to obtain seed culture.

[0016] S2. The activated seed culture was serially diluted on solid glucose broth plates and cultured.

[0017] S3. Pick a single colony from the plate and place it in liquid glucose broth medium for expansion culture, and then inoculate it into liquid broth medium to obtain the fermentation broth of strain DEC-01.

[0018] In some embodiments, in step S1, activation is performed overnight at 33-37°C and 160-180 rpm.

[0019] In some implementations, in step S2, the cells are incubated upside down at 33-37°C for 18-22 hours.

[0020] In some embodiments, in step S3, the culture is carried out at 34-37°C and 160-180 rpm for 12-16 hours; after inoculation, the culture is carried out at 34-37°C and 160-180 rpm for 6-8 hours.

[0021] In some embodiments, in step S3, the culture medium is placed in glucose broth medium at an inoculation rate of 1-10%.

[0022] The present invention also provides a liquid microbial agent obtained by the preparation method of any of the above embodiments.

[0023] The present invention also provides the application of the above-mentioned microbial liquid inoculant in the degumming of herbaceous fibers.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention extends the storage time of the microbial agent at room temperature by adding a specific protectant to a glucose broth culture medium and inoculating the activated DCE-01 strain fermentation broth into the broth culture medium containing the protectant. This allows the liquid microbial agent to maintain high activity and good extraction efficiency for herbal fibers even after being stored at room temperature for two months.

[0026] The microbial liquid inoculant of the present invention has a long storage time and is easy to transport. Compared with solid inoculants, the liquid inoculant has the advantages of high uniformity, easy control, easy scaling and high efficiency.

[0027] The method for preparing the liquid microbial agent of the present invention is simple, and the resulting liquid microbial agent is easy to store and transport. It can greatly promote the application of the DEC-01 strain in various complex scenarios where large-scale microbial fermentation cannot be carried out, such as field green degumming of herbaceous plants such as flax, field warm water retting of kenaf and other hemp, and farm-style degumming of ramie. Detailed Implementation

[0028] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] Example 1: Preparation of liquid microbial inoculant

[0031] (1) Prepare liquid glucose broth culture medium and solid glucose broth culture medium. The formula of the culture medium is: 10 g / L peptone, 5 g / L beef extract, 5 g / L sodium chloride, 10 g / L glucose, pH 7.2~7.4.

[0032] (2) Scrape a loop of bacterial growth from the DCE-01 bacterial slant and place it in liquid glucose broth medium, and activate overnight at 35 ℃ and 180 rpm;

[0033] (3) The activated seed culture was serially diluted and spread onto solid glucose broth agar plates and incubated upside down at 35 °C for 18-22 h.

[0034] (4) Pick a single colony from the plate and place it in liquid glucose broth medium. Incubate at 35 ℃ and 180 rpm for 12-16 h. Then, inoculate the culture medium with 2% inoculum and incubate at 35 ℃ and 180 rpm for 6-8 h to obtain the fermentation broth of strain DCE-01.

[0035] (5) The prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at an inoculation rate of 10%, and cultured statically at 35 °C for 12-16 h. After aseptic filling, the liquid microbial agent can be obtained. The specific protectant is composed of glycerol, D-sorbitol, D-mannitol, fructose and sucrose. The contents of each component of the protectant in the glucose broth medium are: 10% (v / v) glycerol, 0.4 M D-sorbitol, 0.6 M D-mannitol, 0.2% (w / v) fructose and 0.3% (w / v) sucrose.

[0036] Example 1: Optimization of Glycerol Addition in the Preparation of Liquid Microbial Agents

[0037] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0038] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 5% (v / v) glycerol, 10% (v / v) glycerol, 15% (v / v) glycerol, 20% (v / v) glycerol, and 25% (v / v) glycerol.

[0039] Example 2: Optimization of D-sorbitol dosage in the preparation of liquid microbial inoculants

[0040] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0041] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.2 M D-sorbitol, 0.4 M D-sorbitol, 0.6 M D-sorbitol, 0.8 M D-sorbitol, and 1.0 M D-sorbitol.

[0042] Example 3: Optimization of D-mannitol addition in the preparation of liquid microbial inoculants

[0043] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0044] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.2 M D-mannitol, 0.4 M D-mannitol, 0.6 M D-mannitol, 0.8 M D-mannitol, and 1.0 M D-mannitol.

[0045] Example 4: Optimization of Fructose Addition in the Preparation of Liquid Microbial Agents

[0046] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0047] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.1% (w / v) fructose, 0.2% (w / v) fructose, 0.3% (w / v) fructose, 0.4% (w / v) fructose, and 0.5% (w / v) fructose.

[0048] Example 5: Optimization of sucrose addition in the preparation of liquid microbial inoculants

[0049] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0050] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.1% (w / v) sucrose, 0.2% (w / v) sucrose, 0.3% (w / v) sucrose, 0.4% (w / v) sucrose, and 0.5% (w / v) sucrose.

[0051] Comparative Example 1: Protective Agent Combination in the Preparation of Liquid Microbial Agents

[0052] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0053] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol and 0.4 M D-sorbitol.

[0054] Comparative Example 2: Protectant Combination Two in the Preparation of Microbial Liquid Inoculants

[0055] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0056] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol and 0.6 M D-mannitol.

[0057] Comparative Example 3: Protectant Combination Three in the Preparation of Microbial Liquid Inoculants

[0058] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0059] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol and 0.2% (w / v) fructose.

[0060] Comparative Example 4: Protective Agent Combination Four in the Preparation of Microbial Liquid Inoculants

[0061] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0062] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol and 0.3% (w / v) sucrose.

[0063] Comparative Example 5: Five combinations of protective agents in the preparation of liquid microbial inoculants.

[0064] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0065] Subsequently, the prepared D. dadantii DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size. The mixture was then incubated statically at 35 °C for 12–16 h. After aseptic filling, the liquid microbial inoculum was obtained. The specific protectant consisted of 0.4 M D-sorbitol and 0.6 M D-mannitol.

[0066] Comparative Example 6: Six combinations of protective agents in the preparation of liquid microbial inoculants.

[0067] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0068] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculation rate, and incubated statically at 35 ℃ for 12-16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.4 M D-sorbitol and 0.2% (w / v) fructose.

[0069] Comparative Example 7: Seven Protective Agent Combinations in the Preparation of Liquid Microbial Agents

[0070] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0071] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculation rate, and incubated statically at 35 ℃ for 12-16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.4 M D-sorbitol and 0.3% (w / v) sucrose.

[0072] Comparative Example 8: Combination of Protective Agents in the Preparation of Liquid Microbial Agents

[0073] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0074] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12-16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.6 M D-mannitol and 0.2% (w / v) fructose.

[0075] Comparative Example 9: Protective Agent Combination Nine in the Preparation of Microbial Liquid Inoculants

[0076] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0077] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12-16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.6 M D-mannitol and 0.3% (w / v) sucrose.

[0078] Comparative Example 10: Combination of Protective Agents in the Preparation of Liquid Microbial Agents

[0079] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0080] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculation rate, and incubated statically at 35 ℃ for 12-16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.2% (w / v) fructose and 0.3% (w / v) sucrose.

[0081] Comparative Example 11: Eleven Protective Agent Combinations in the Preparation of Microbial Liquid Inoculants

[0082] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0083] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol, 0.4 M D-sorbitol, and 0.6 M D-mannitol.

[0084] Comparative Example 12: Twelve Protectant Combinations in the Preparation of Microbial Liquid Inoculants

[0085] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0086] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol, 0.4 M D-sorbitol, and 0.2% (w / v) fructose.

[0087] Comparative Example 13: Thirteen Protective Agent Combinations in the Preparation of Microbial Liquid Inoculants

[0088] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0089] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol, 0.4 M D-sorbitol, and 0.3% (w / v) sucrose.

[0090] Comparative Example 14: Fourteen Protective Agent Combinations in the Preparation of Microbial Liquid Inoculants

[0091] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0092] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol, 0.6 M D-mannitol, and 0.2% (w / v) fructose.

[0093] Comparative Example 15: Fifteen Protective Agent Combinations in the Preparation of Microbial Liquid Inoculants

[0094] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0095] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol, 0.6 M D-mannitol, and 0.3% (w / v) sucrose.

[0096] Comparative Example 16: Sixteen Protective Agent Combinations in the Preparation of Microbial Liquid Inoculants

[0097] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0098] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 10% (v / v) glycerol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0099] Comparative Example 17: Seventeen Protective Agent Combinations in the Preparation of Microbial Liquid Inoculants

[0100] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0101] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.4 M D-sorbitol, 0.6 M D-mannitol, and 0.2% (w / v) fructose.

[0102] Comparative Example 18: Eighteen Combinations of Protective Agents in the Preparation of Liquid Microbial Agents

[0103] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0104] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.4 M D-sorbitol, 0.6 M D-mannitol, and 0.3% (w / v) sucrose.

[0105] Comparative Example 19: Nineteen Protective Agent Combinations in the Preparation of Microbial Liquid Inoculants

[0106] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0107] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculation rate, and incubated statically at 35 ℃ for 12-16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.4 M D-sorbitol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0108] Comparative Example 20: Twenty Combinations of Protective Agents in the Preparation of Microbial Liquid Inoculants

[0109] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0110] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculation rate, and incubated statically at 35 ℃ for 12-16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of 0.6 M D-mannitol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0111] Comparative Example 21: Combination of Protective Agents in the Preparation of Liquid Microbial Agents (21)

[0112] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0113] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of: 10% (v / v) glycerol, 0.4 M D-sorbitol, 0.6 M D-mannitol, and 0.2% (w / v) fructose.

[0114] Comparative Example 22: Twenty-two combinations of protective agents in the preparation of liquid microbial inoculants.

[0115] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0116] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of: 10% (v / v) glycerol, 0.4 M D-sorbitol, 0.6 M D-mannitol, and 0.3% (w / v) sucrose.

[0117] Comparative Example 23: Combination of Protective Agents in the Preparation of Liquid Microbial Agents (23)

[0118] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0119] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of: 10% (v / v) glycerol, 0.6 M D-mannitol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0120] Comparative Example 24: Twenty-four Combinations of Protectants in the Preparation of Liquid Microbial Agents

[0121] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0122] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of: 0.4 M D-sorbitol, 0.6 M D-mannitol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0123] Comparative Example 25: Combination of Protective Agents in the Preparation of Liquid Microbial Agents (25)

[0124] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0125] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium containing a specific protectant at a 10% inoculum size, and incubated statically at 35 ℃ for 12–16 h. After aseptic filling, the liquid microbial agent was obtained. The specific protectant consisted of: 10% (v / v) glycerol, 0.4 M D-sorbitol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0126] Comparative Example 26: Liquid microbial inoculant without added specific protective agents

[0127] The fermentation broth of strain DCE-01 was prepared according to steps (1) to (4) in Example 1.

[0128] Subsequently, the prepared DCE-01 fermentation broth was inoculated into glucose broth medium at an inoculation rate of 10%, and cultured statically at 35°C for 12-16 hours. After aseptic filling, the liquid microbial agent was obtained.

[0129] Efficiency evaluation of microbial liquid inoculant extraction of herbal fiber

[0130] The liquid microbial agents prepared in all the above examples and comparative examples were stored for 2 months and then used for the extraction of ramie fiber. The specific methods are briefly described below:

[0131] (1) Prepare a ramie degumming system by adding tap water and ramie bast (undegummed dry ramie bast) to a 500 mL beaker at a bath ratio of 1:10.

[0132] (2) Add 2% of microbial liquid inoculant to the ramie degumming system and culture at 35 ℃ and 180 rpm for 8 h with shaking. Here, DCE-01 fermentation broth was set as control group 1 to detect whether the preparation process of microbial liquid inoculant affects the extraction efficiency of herbaceous plant fiber. The liquid microbial inoculant newly prepared in Example 1 was set as control group 2 to detect the protective effect of specific protectant on DCE-01 inoculant during long-term storage.

[0133] (3) After degumming, the ramie bast fibers were rinsed and then dried at 60 ℃ to constant weight. The weight loss rate of the ramie bast fibers was used as the evaluation index to assess the extraction efficiency of the microbial liquid inoculant on herbaceous plant fibers. The weight loss rate of ramie bast fibers was calculated as follows:

[0134] V1=(M 0- M1) / M0×100 %

[0135] Wherein, V1 is the weight loss rate of ramie bast fiber; M0 is the weight of dry ramie bast before degumming; and M1 is the weight of ramie fiber after degumming and drying to constant weight.

[0136] Table 1. Optimization of glycerol addition amount in the preparation of liquid microbial inoculants.

[0137]

[0138] Table 2 Optimization of D-sorbitol addition in the preparation of liquid microbial inoculants

[0139]

[0140] Table 3 Optimization of D-mannitol addition in the preparation of liquid microbial inoculants

[0141]

[0142] Table 4 Optimization of fructose addition in the preparation of liquid microbial inoculants

[0143]

[0144] Table 5 Optimization of sucrose addition in the preparation of liquid microbial inoculants

[0145]

[0146] Table 6. Effects of different combinations of protective agents on the efficiency of liquid bacterial agent extraction of herbaceous plant fiber

[0147]

[0148] As shown in Tables 1, 2, 3, 4, and 5, under single-factor optimization conditions, the optimal addition amounts are as follows: 10% (v / v) glycerol, 0.4 M D-sorbitol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0149] The following information can be obtained from Table 6:

[0150] Compared with the specific protective agent ratios in Comparative Examples 1 to 25, it can be seen that adding the specific protective agent of the present invention can store the microbial agent for a longer period of time and maintain a high herbal fiber extraction efficiency.

[0151] Compared with Comparative Example 26, Example 1 shows that after two months of storage under the same conditions, the herbal fiber extraction efficiency of the microbial liquid inoculant with the added specific protective agent of the present invention is significantly higher than that of the microbial liquid inoculant without any added specific protective agent.

[0152] Compared with control group 1, after storage for up to 2 months, the herbal fiber extraction efficiency of the microbial liquid inoculant with the added specific protective agent of the present invention was still higher than that of the fresh fermentation broth of DCE-01.

[0153] Compared with control group 2, the microbial liquid inoculant with added specific protective agents of the present invention showed no significant decrease in herbal fiber extraction efficiency after 2 months of storage.

[0154] Compared with control group 2, the addition of the specific protective agent of the present invention to the culture medium in control group 1 promoted the herbal fiber extraction efficiency of strain DCE-01.

[0155] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0156] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a microbial liquid inoculant, characterized by, The method comprises the following steps: The DCE-01 strain fermentation liquor is inoculated into the glucose broth culture medium containing the protective agent at an inoculation amount of 5-15%, and is incubated at 33-36°C for 12-16h to obtain the microbial liquid inoculum; The protective agent is composed of glycerol, D-sorbitol, D-mannitol, fructose and sucrose; in the glucose broth culture medium, the volume content of glycerol is 8-15%, the molar concentration of D-sorbitol is 0.3-0.6M, the molar concentration of D-mannitol is 0.2-0.7M, the mass concentration of fructose is 0.15-0.4%, and the mass concentration of sucrose is 0.2-0.4%; The preservation number of the DCE-01 strain is CGMCC No.5522; The basic formula of the glucose broth culture medium is as follows: 5-15g / L of proteose peptone, 2-10g / L of beef extract, 2-10g / L of sodium chloride and 5-15g / L of glucose, and the pH value is 7.2-7.

4.

2. The production method of the microbial liquid inoculant according to claim 1, characterized by, The preparation of the DCE-01 strain fermentation liquor comprises the following steps: S1, a ring of bacterial moss is taken from a DCE-01 strain slant, is placed in a liquid glucose broth culture medium, is activated overnight, and seed liquor is obtained; S2, the activated seed liquor is gradiently diluted in a solid glucose broth culture medium flat plate culture; S3, a single colony is picked from the flat plate, is placed in a liquid glucose broth culture medium for expansion culture, and then is inoculated into a liquid broth culture medium to obtain the DCE-01 strain fermentation liquor.

3. The method for preparing a microbial liquid inoculant according to claim 2, characterized by, In step S1, the activation is performed overnight at 33-37°C and 160-180rpm; and / or, in step S2, the inversion culture is performed at 33-37°C for 18-22h; and / or, in step S3, the expansion culture is performed at 34-37°C and 160-180rpm for 12-16h; and after inoculation, the culture is performed at 34-37°C and 160-180rpm for 6-8h.

4. The production method of the microbial liquid culture agent according to claim 2, characterized by, In step S3, the culture liquor is placed in the glucose broth culture medium at an inoculation amount of 1-10%.

5. The microbial liquid inoculum obtained by the preparation method in any one of claims 1-4.

6. The application of the microbial liquid inoculum in claim 5 in herbaceous fiber degumming.

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