Microbial liquid inoculant as well as preparation method and application thereof

Abstract

The invention relates to a microbial liquid inoculant as well as a preparation method and application thereof, and the preparation method of the inoculant comprises the following steps: inoculating DCE-01 strain fermentation liquor into a glucose broth culture medium containing a protective agent, and culturing to obtain the microbial liquid inoculant, wherein the protective agent comprises glycerol, D-sorbitol, mannitol, fructose and cane sugar; in the glucose broth culture medium, the volume content of glycerol is 8-15%, the molar concentration of D-sorbitol is 0.3-0.6 M, the molar concentration of D-mannitol is 0.2-0.7 M, the mass concentration of fructose is 0.15-0.4%, and the mass concentration of cane sugar is 0.2-0.4%; the efficient microbial agent which can be stored for a long time and is easy to transport can be obtained through the preparation method, so that the use requirements of various complex fiber extraction scenes can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of herbal fiber raw material processing, and more specifically, to a liquid microbial agent and a preparation method and application thereof. Background Art

[0002] Herbal fiber refers to a material made from fibers extracted from herbaceous plants such as hemp and grass straw. It is lightweight, environmentally friendly, degradable, and biocompatible, and has great application potential in the fields of textiles, medical care, aerospace, automobiles, construction, and packaging. The necessary prerequisite for herbal fiber materials is to obtain high-quality herbal fiber. Usually, the surface of herbal fiber is coated with pectin, hemicellulose, lignin and other substances. The extraction of herbal fiber is the process of selective stripping of pectin, hemicellulose, lignin and other substances. Chemical extraction is often used in industry, which is costly, energy-intensive, and seriously pollutes the environment. In addition, the fiber production rate is low and the quality is poor. The biological extraction method saves a lot of chemical raw materials and energy, greatly reduces environmental pollution, and can obtain better quality fibers.

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

[0004] At present, except for large-scale factory production that can be equipped with a complete microbial fermentation workshop to continuously supply DCE-01 strain fermentation liquid for the extraction of herbal plant fibers, various complex fiber extraction scenarios such as field green degumming of industrial hemp and flax, field water retting of ramie, and farmer-style degumming of ramie cannot be carried out for large-scale microbial fermentation, which greatly limits the application scope of DCE-01 strain in herbal plant fiber extraction. Summary of the invention

[0005] Based on the above-mentioned technical problems existing in the prior art, the present invention provides a method for preparing a microbial liquid agent. The microbial liquid agent obtained by the method still maintains high activity after being stored for two months and has a high extraction efficiency for herbal fiber, which can meet the use of a variety of complex fiber extraction scenarios.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for preparing a liquid microbial agent comprises the following steps:

[0008] The fermentation liquid of the DCE-01 strain is inoculated into a glucose broth medium containing a protective agent to obtain a liquid microbial agent;

[0009] The protective agent comprises glycerol, D-sorbitol, 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%.

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

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

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

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

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

[0015] S1. Take a loop of the DCE-01 bacterial lawn on the slant and place it in liquid glucose broth medium, activate it overnight, and obtain seed solution;

[0016] S2, gradient dilution of the activated seed solution on solid glucose broth medium plates for culture;

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

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

[0019] In some embodiments, in step S2, the culture is inverted at 33-37° C. for 18-22 h.

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

[0021] In some embodiments, in step S3, the culture solution is placed in a glucose broth medium at an inoculum size 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 application of the liquid microbial agent in herbal fiber degumming.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention can extend the storage time of the inoculum under normal temperature conditions by adding a specific protective agent to a glucose broth culture medium and inoculating the cremated DCE-01 strain fermentation liquid into the broth culture medium containing the protective agent, so that the liquid microbial inoculum can still maintain a high activity after being stored at room temperature for two months and still have a good extraction efficiency for the herbal fiber.

[0026] The liquid microbial agent of the present invention has a long storage time and is easy to transport. Compared with solid microbial agents, the liquid microbial agent has the advantages of high uniformity, easy control, easy scale-up and high efficiency.

[0027] The preparation method of the microbial liquid inoculant of the present invention is simple, and the obtained liquid inoculant is easy to store and transport, which 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 industrial hemp and flax, field warm water retting of kenaf, and farmer-based workshop degumming of ramie. DETAILED DESCRIPTION

[0028] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] Example 1 Preparation of liquid microbial agent

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

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

[0033] (3) The activated seed solution was diluted in gradients and spread on solid glucose broth medium plates, and incubated inverted at 35°C for 18 to 22 hours;

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

[0035] (5) The prepared DCE-01 fermentation broth is inoculated with a 10% inoculum into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent is obtained; the specific protective agent is composed of glycerol, D-sorbitol, D-mannitol, fructose and sucrose, wherein the content of each component of the protective agent in the glucose broth medium is: 10% (v / v) glycerol, 0.4M D-sorbitol, 0.6M D-mannitol, 0.2% (w / v) fructose, and 0.3% (w / v) sucrose.

[0036] Experimental Example 1 Optimization of the amount of glycerol added in the preparation of liquid microbial inoculants

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

[0038] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and the liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 5% (v / v) glycerol, 10% (v / v) glycerol, 15% (v / v) glycerol, 20% (v / v) glycerol, 25% (v / v) glycerol.

[0039] Experimental Example 2 Optimization of the amount of D-sorbitol added in the preparation of liquid microbial inoculants

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

[0041] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.2M D-sorbitol, 0.4M D-sorbitol, 0.6M D-sorbitol, 0.8M D-sorbitol, 1.0M D-sorbitol.

[0042] Experimental Example 3 Optimization of the amount of D-mannitol added in the preparation of liquid microbial inoculants

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

[0044] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.2M D-mannitol, 0.4M D-mannitol, 0.6M D-mannitol, 0.8M D-mannitol, 1.0M D-mannitol.

[0045] Experimental Example 4 Optimization of fructose addition in the preparation of liquid microbial inoculant

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

[0047] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.1% (w / v) fructose, 0.2% (w / v) fructose, 0.3% (w / v) fructose, 0.4% (w / v) fructose, 0.5% (w / v) fructose.

[0048] Experimental Example 5 Optimization of the amount of sucrose added in the preparation of liquid microbial inoculants

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

[0050] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.1% (w / v) sucrose, 0.2% (w / v) sucrose, 0.3% (w / v) sucrose, 0.4% (w / v) sucrose, 0.5% (w / v) sucrose.

[0051] Comparative Example 1: Protective agent combination in the preparation of liquid microbial agent

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

[0053] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 10% (v / v) glycerol, 0.4M D-sorbitol.

[0054] Comparative Example 2: Protective Agent Combination 2 in the Preparation of Microbial Liquid Inoculant

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

[0056] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 10% (v / v) glycerol, 0.6M D-mannitol.

[0057] Comparative Example 3: Protective agent combination 3 in the preparation of liquid microbial agent

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

[0059] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 10% (v / v) glycerol, 0.2% (w / v) fructose.

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

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

[0062] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 10% (v / v) glycerol, 0.3% (w / v) sucrose.

[0063] Comparative Example 5: Protective agent combination 5 in the preparation of liquid microbial agent

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

[0065] Subsequently, the prepared D.dadantii DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.4M D-sorbitol, 0.6M D-mannitol.

[0066] Comparative Example 6: Protective Agent Combination 6 in the Preparation of Microbial Liquid Inoculant

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

[0068] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.4M D-sorbitol, 0.2% (w / v) fructose.

[0069] Comparative Example 7: Protective agent combination 7 in the preparation of liquid microbial agent

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

[0071] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.4M D-sorbitol, 0.3% (w / v) sucrose.

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

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

[0074] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.6M D-mannitol, 0.2% (w / v) fructose.

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

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

[0077] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.6M D-mannitol, 0.3% (w / v) sucrose.

[0078] Comparative Example 10: Protective agent combination in the preparation of liquid microbial agent

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

[0080] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 0.2% (w / v) fructose, 0.3% (w / v) sucrose.

[0081] Comparative Example 11 Protective Agent Combination XI in the Preparation of Microbial Liquid Bacteria Agent

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

[0083] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 10% (v / v) glycerol, 0.4M D-sorbitol, 0.6M D-mannitol.

[0084] Comparative Example 12 Protective Agent Combination Twelve in the Preparation of Microbial Liquid Bacteria Agent

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

[0086] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 10% (v / v) glycerol, 0.4M D-sorbitol, 0.2% (w / v) fructose.

[0087] Comparative Example 13 Protective Agent Combination Thirteen in the Preparation of Microbial Liquid Bacteria Agent

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

[0089] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 10% (v / v) glycerol, 0.4M D-sorbitol, 0.3% (w / v) sucrose.

[0090] Comparative Example 14 Protective Agent Combination 14 in the Preparation of Microbial Liquid Inoculant

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

[0092] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 10% (v / v) glycerol, 0.6M D-mannitol, 0.2% (w / v) fructose.

[0093] Comparative Example 15: Protective agent combination 15 in the preparation of liquid microbial agent

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

[0095] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 10% (v / v) glycerol, 0.6M D-mannitol, 0.3% (w / v) sucrose.

[0096] Comparative Example 16 Protective Agent Combination 16 in the Preparation of Microbial Liquid Inoculant

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

[0098] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 10% (v / v) glycerol, 0.2% (w / v) fructose, 0.3% (w / v) sucrose.

[0099] Comparative Example 17: Protective Agent Combination 17 in the Preparation of Liquid Microbial Agent

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

[0101] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 0.4M D-sorbitol, 0.6M D-mannitol, 0.2% (w / v) fructose.

[0102] Comparative Example 18 Protective Agent Combination 18 in the Preparation of Microbial Liquid Inoculant

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

[0104] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 0.4M D-sorbitol, 0.6M D-mannitol, 0.3% (w / v) sucrose.

[0105] Comparative Example 19: Protective Agent Combination 19 in the Preparation of Microbial Liquid Inoculant

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

[0107] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.4M D-sorbitol, 0.2% (w / v) fructose, 0.3% (w / v) sucrose.

[0108] Comparative Example 20: Preparation of liquid microbial agent with protective agent combination 20

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

[0110] Subsequently, the prepared DCE-01 fermentation liquid was inoculated into a glucose broth medium containing a specific protective agent at a 10% inoculation rate, and cultured at 35°C for 12 to 16 hours. After aseptic filling, a liquid microbial agent was obtained. The composition of the specific protective agent: 0.6M D-mannitol, 0.2% (w / v) fructose, 0.3% (w / v) sucrose.

[0111] Comparative Example 21 Protective Agent Combination 21 in the Preparation of Microbial Liquid Bacteria Agent

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

[0113] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 10% (v / v) glycerol, 0.4M D-sorbitol, 0.6M D-mannitol, 0.2% (w / v) fructose.

[0114] Comparative Example 22: Protective Agent Combination 22 in the Preparation of Microbial Liquid Bacteria Agent

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

[0116] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 10% (v / v) glycerol, 0.4M D-sorbitol, 0.6M D-mannitol, 0.3% (w / v) sucrose.

[0117] Comparative Example 23: Protective Agent Combination 23 in the Preparation of Microbial Liquid Bacteria Agent

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

[0119] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 10% (v / v) glycerol, 0.6M D-mannitol, 0.2% (w / v) fructose, 0.3% (w / v) sucrose.

[0120] Comparative Example 24: Protective Agent Combination 24 in the Preparation of Microbial Liquid Bacteria Agent

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

[0122] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 0.4M D-sorbitol, 0.6M D-mannitol, 0.2% (w / v) fructose, 0.3% (w / v) sucrose.

[0123] Comparative Example 25: Protective agent combination 25 in the preparation of liquid microbial agent

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

[0125] Subsequently, the prepared DCE-01 fermentation broth was inoculated with a 10% inoculation amount into a glucose broth medium containing a specific protective agent, and cultured at 35°C for 12 to 16 hours, and a liquid microbial agent was obtained after aseptic filling. The composition of the specific protective agent: 10% (v / v) glycerol, 0.4M D-sorbitol, 0.2% (w / v) fructose, 0.3% (w / v) sucrose.

[0126] Comparative Example 26: Liquid microbial agent without adding a specific protective agent

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

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

[0129] Evaluation of the efficiency of extracting herbal fiber with liquid microbial inoculants

[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 method is briefly described as follows:

[0131] (1) preparing a ramie degumming system, namely, adding tap water and ramie bast (undegummed dry ramie skin) in a 500 mL beaker at a bath ratio of 1:10;

[0132] (2) Adding a liquid microbial agent at an inoculation rate of 2% to the ramie degumming system, and culturing at 35°C and 180 rpm for 8 h; Here, the DCE-01 fermentation broth was set as the control group 1 to detect whether the preparation process of the liquid microbial agent has an effect on the extraction efficiency of the herbaceous plant fiber; the liquid microbial agent newly prepared in Example 1 was set as the control group 2 to detect the protective effect of the specific protective agent on the DCE-01 agent during long-term storage.

[0133] (3) After degumming, the ramie bast fiber was washed and then dried at 60°C to a constant weight. The weight loss rate of the ramie bast fiber was used as an evaluation index to evaluate the extraction efficiency of the microbial liquid inoculant on the herbaceous plant fiber. The weight loss rate of the ramie bast fiber was calculated as follows:

[0134] V1=(M0-M1) / M0×100%

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

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

[0137] Glycerol addition 5％ 10％ 15％ 20％ 25％ Weight loss rate 14.64％ 17.13％ 15.23％ 14.60％ 13.71％

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

[0139] D-sorbitol addition 0.2M 0.4M 0.6M 0.8M 1.0M Weight loss rate 11.60％ 15.53％ 13.92％ 12.12％ 11.43％

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

[0141] D-Mannitol Addition 0.2M 0.4M 0.6M 0.8M 1.0M Weight loss rate 13.28％ 13.70％ 14.10％ 10.53％ 10.21％

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

[0143] Fructose added 0.1％ 0.2％ 0.3％ 0.4％ 0.5％ Weight loss rate 12.13％ 14.52％ 13.22％ 12.32％ 11.26％

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

[0145] Sucrose added 0.1％ 0.2％ 0.3％ 0.4％ 0.5％ Weight loss rate 10.98％ 12.15％ 14.26％ 12.68％ 11.29％

[0146] Table 6 Effects of different protective agent combinations on the efficiency of extracting herbal fiber by liquid bacterial agent

[0147] Group Name Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Weight loss rate 20.75％ 17.46％ 17.23％ 17.42％ 17.33％ Group Name Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 Comparative Example 9 Weight loss rate 15.84％ 15.98％ 15.55％ 14.65％ 14.38％ Group Name Comparative Example 10 Comparative Example 11 Comparative Example 12 Comparative Example 13 Comparative Example 14 Weight loss rate 14.57％ 17.85％ 17.63％ 17.55％ 17.48％ Group Name Comparative Example 15 Comparative Example 16 Comparative Example 17 Comparative Example 18 Comparative Example 19 Weight loss rate 17.87％ 17.93％ 16.72％ 16.65％ 16.28％ Group Name Comparative Example 20 Comparative Example 21 Comparative Example 22 Comparative Example 23 Comparative Example 24 Weight loss rate 15.23％ 18.86％ 18.66％ 18.47％ 17.98％ Group Name Comparative Example 25 Comparative Example 26 Control group 1 Control group 2 Weight loss rate 19.35％ 9.98％ 16.56％ 20.96％

[0148] It can be seen from Tables 1, 2, 3, 4 and 5 that under the 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 Example 1 and Comparative Examples 1 to 25, it can be seen that the addition of the specific protective agent in the present invention can store the microbial agent for a longer period of time and maintain a higher herbal fiber extraction efficiency;

[0151] It can be seen from the comparison between Example 1 and Comparative Example 26 that, after being stored for two months under the same conditions, the herbal fiber extraction efficiency of the microbial liquid inoculant to which the specific protective agent of the present invention is added is significantly higher than that of the microbial liquid inoculant to which no specific protective agent is added;

[0152] Compared with the control group 1, after 2 months of storage, the herbal fiber extraction efficiency of the liquid microbial inoculum added with the specific protective agent of the present invention is still higher than that of the fresh fermentation liquid of DCE-01;

[0153] Compared with the control group 2, the herbal fiber extraction efficiency of the liquid microbial inoculant added with the specific protective agent of the present invention in Example 1 did not decrease significantly after 2 months of storage;

[0154] Compared with the control group 2, the addition of the specific protective agent of the present invention into the culture medium of the control group 1 has a promoting effect on the herbal fiber extraction efficiency of the DCE-01 strain.

[0155] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a liquid microbial agent, characterized in that: The following steps are involved: The fermentation liquid of the DCE-01 strain is inoculated into a glucose broth medium containing a protective agent to obtain a liquid microbial agent; The protective agent comprises glycerol, D-sorbitol, 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%.

2. The method for preparing a liquid microbial agent according to claim 1, characterized in that: In the glucose broth culture medium, the volume content of glycerol is 10%, the molar concentration of D-sorbitol is 0.4M, the molar concentration of D-mannitol is 0.6M, the mass concentration of fructose is 0.2%, and the mass concentration of sucrose is 0.3%.

3. The method for preparing a liquid microbial agent according to claim 1, characterized in that: After the DCE-01 strain fermentation liquid is inoculated into the glucose broth medium, the medium is statically cultured at 33-36° C. for 12-16 hours.

4. The method for preparing a liquid microbial agent according to claim 1, characterized in that: The fermentation culture fluid of DCE-01 strain was inoculated into glucose broth medium containing protective agent at an inoculation rate of 5-15%.

5. The method for preparing a liquid microbial agent according to claim 1, characterized in that: The basic formula of the glucose broth culture medium is: 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.

6. The method for preparing a liquid microbial agent according to claim 1, characterized in that: The preparation of the DCE-01 strain fermentation broth comprises the following steps: S1. Take a loop of the DCE-01 bacterial lawn on the slant and place it in liquid glucose broth medium, activate it overnight, and obtain seed solution; S2, gradient dilution of the activated seed solution on solid glucose broth medium plates for culture; S3. Pick a single colony from the plate and place it in a liquid glucose broth medium for expansion, and then inoculate it into the liquid broth medium to obtain a fermentation broth of the DEC-01 strain.

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

8. The method for preparing a liquid microbial agent according to claim 6, characterized in that: In step S3, the culture solution is placed in a glucose broth medium at an inoculum size of 1-10%.

9. The liquid microbial agent obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the liquid microbial agent according to claim 9 in herbal fiber degumming.