CN-121991821-A - Complex bacterial enzyme preparation for efficiently degrading bush lignocellulose and application thereof

Abstract

The invention discloses a compound bacteria enzyme preparation for efficiently degrading bush lignocellulose and application thereof, and relates to the technical field of agricultural microorganisms. The preparation is prepared from high-efficiency lignocellulose degrading bacteria obtained by screening and a compound enzyme system (cellulase: xylanase: laccase=5:3:2) with a specific ratio by optimizing a fermentation process. The viable count of the product is more than or equal to 1 multiplied by 10 8 CFU/g, the neutral washing fiber and the acidic washing fiber of the shrubs can be respectively reduced by 6.4 percent and 8.27 percent, the digestibility of dry matters in vitro is improved by 13 percent, and the shrub neutral washing fiber and the acidic washing fiber can be stably stored for more than 6 months at room temperature. The preparation method comprises the steps of fermentation liquor preparation and shrub raw material treatment, is simple and convenient to use, can obviously improve the nutritive value of shrub feed, reduces the cultivation cost, and provides an economic and effective solution for the utilization of shrub resource feed in desertification areas.

Inventors

• XUE SHUYUAN
• LIU YANG
• TIAN FENG
• Li Jiuyue
• GAO YUAN
• LIANG JIANYONG
• ZHAO XIAOJUAN
• DE LEHEI

Assignees

• 内蒙古自治区农牧业科学院

Dates

Publication Date

20260508

Application Date

20251110

Priority Date

20250820

Claims (5)

1. 1. A compound bacterial enzyme preparation for efficiently degrading bush lignocellulose is characterized by comprising a microorganism strain and a compound enzyme system, wherein the microorganism strain is lactobacillus plantarum (Lactiplantibacillus Plantarum) with a preservation number of CGMCC 1.2437 and bacillus subtilis (Bacillus subtilis) with a preservation number of CGMCC 1.15792, and the compound enzyme system comprises cellulase, xylanase and laccase; The ratio of enzyme components in the compound enzyme system is cellulase, xylanase and laccase=5:3:2, the effective viable count of lactobacillus plantarum is more than 1.0 multiplied by 10 12 CFU/g, the effective viable count of bacillus subtilis is more than or equal to 1.0 multiplied by 10 12 CFU/g, the activity of the cellulase is more than 2 multiplied by 10 5 U/g, the activity of xylanase is more than 1 multiplied by 10 4 U/g, the activity of laccase is more than 6 multiplied by 10 3 U/g, the compound enzyme preparation can reduce the neutral washing fiber of shrubs by more than or equal to 6.4%, the acidic washing fiber is reduced by more than or equal to 8.27%, and the in-vitro dry matter digestibility is improved by more than or equal to 13%. .
2. 2. A method for preparing the compound bacterial enzyme preparation for efficiently degrading the lignocellulose of shrubs, which is characterized by comprising the steps of preparing a fermentation liquor and treating the raw materials of the shrubs, wherein the preparation of the fermentation liquor needs to use a high-density fermentation medium and an enlarged fermentation medium; 12-18 g of yeast extract, 10-15 g of glucose, 1.6-4.2 g of KH 2 PO 4 , 2.5-4.5 g of NaCl, 1-3 g of magnesium sulfate, 0.2-0.4 g of manganese sulfate, 0.5-2.5 mL of tween 80, 10-15 g of peptone, 80-121 mL/10-15 g of skim milk/skim milk powder, 0.8-1.5 g of beef extract, 8-15 mL of carrot juice and 10-15 mL of tomato juice are dissolved in 1-L distilled water, the pH is adjusted to 7.0-7.2, and sterilization is performed at 121 ℃ for 20-min; The composition and preparation of the expanded fermentation medium comprise 1200-1800 g of yeast extract, 4000-5200 mL/500-650 g of skim milk/skim milk powder, 1000-mL g of tomato juice, 2.6-4.2 g of magnesium sulfate, 1000-1500 g of glucose, g of MnSO 4 ·H 2 O 3 g、KH 2 PO 4 , 100-g of NaCl and 1500-2500 g of soybean meal powder, and the expanded fermentation medium is prepared by dissolving the expanded fermentation medium in 1L of distilled water, adjusting the pH to 7.0-7.2 and sterilizing at 121 ℃ to 20-min.
3. 3. The method according to claim 2, wherein the fermentation broth preparation comprises the steps of: (1) Inoculating bacillus subtilis into an LB liquid culture medium according to 5% of inoculum size, inoculating lactobacillus plantarum into an MRS liquid culture medium according to 5% of inoculum size, and respectively culturing until logarithmic growth phase is used as a first-stage strain; (2) Inoculating the first-level strain into a liquid culture medium corresponding to 300 mL, and culturing 24 h at 37 ℃ to serve as a second-level strain; (3) Inoculating the second-level strain into a liquid culture medium corresponding to 1500 mL, and culturing 24h at 37 ℃ to serve as a third-level strain; (4) Mixing the three-stage seed culture solution, inoculating the mixed solution into a 50L high-density fermentation culture medium, and culturing for 22-24 hours at the stirring rotation speed of 180R/min and the temperature of 34-37 ℃ to obtain a high-density fermentation bacteria solution; (5) Inoculating the high-density fermentation broth into a 700L expansion culture medium, culturing for 22-24 hours at the stirring rotation speed of 120-150R/min and the temperature of 34-37 ℃, controlling the pH value to be 6.0-6.5 in the whole process, and stopping adjusting the pH value after 18 hours to obtain an expansion culture broth; (6) And mixing cellulase, xylanase and laccase with the expansion culture solution according to the proportion of 50 g/ton to obtain the complex bacterial enzyme fermentation solution.
4. 4. The application of the compound bacteria enzyme preparation for efficiently degrading the wood cellulose of the shrubs, which is characterized in that the compound bacteria enzyme preparation is used for degrading the wood cellulose of the shrubs and realizing the forage utilization of shrubs resources.
5. 5. The method according to claim 4, comprising the steps of: (1) Harvesting shrubs in the last 10 months to the last 3 months of the next year, drying, removing dust and impurities, kneading, crushing, wherein the crushing length of cattle feed is 2 cm-3 cm, and the crushing length of sheep feed is 1 cm-2 cm; (2) Mixing the compound bacterial enzyme preparation with crushed shrub raw materials according to the proportion of 1.5-2.5%, adjusting the water content to 50-60%, and adjusting the content of soluble carbohydrates to be more than or equal to 3%; (3) And bundling and wrapping the mixture, and fermenting for 30-60 days at 15-25 ℃ or treating for 48-72 hours at 35-45 ℃.

Description

Complex bacterial enzyme preparation for efficiently degrading bush lignocellulose and application thereof Technical Field The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a compound bacteria enzyme preparation for efficiently degrading bush lignocellulose and application thereof. Background The shrub woodland in China is rich in resources, and the annual stumping amount of sand shrubs such as caragana microphylla, salix psammophila and the like in arid and semiarid regions such as inner mongolia, gansu and the like exceeds ten millions of tons. The traditional treatment mode not only occupies land, but also has the natural degradation period of lignin as long as 10-14 months, and the resource circulation efficiency is low. Although shrubs have feeding value, with prolonged growth time, the increased degree of lignification results in reduced nutritional content and palatability. The existing commercial strains are mostly aimed at crop straws, the degradation efficiency of special lignin monomers of shrubs is insufficient, and the product has the stability problems of short storage period, rapid spore attenuation, rapid enzyme activity reduction and the like, so that the forage application of shrubs resources is restricted. Disclosure of Invention The invention aims to provide a compound bacteria enzyme preparation capable of efficiently degrading the lignocellulose of shrubs and a preparation method thereof, and the compound bacteria enzyme preparation is low in cost, pollution-free and convenient to popularize. The invention aims to solve the technical problems of low lignocellulose degradation efficiency, high cost, large environmental pollution and the like of the traditional treatment method in the feed utilization process of the current shrub plant resources, and develops a biological agent capable of efficiently decomposing the shrub lignocellulose structure by screening the optimized combination of the microorganism strain with a specific function and the compound enzyme system, thereby remarkably improving the nutritional value and the digestion utilization rate of the shrub unconventional feed raw materials, simultaneously reducing the energy consumption and the environmental pollution in the treatment process and providing an economic and effective technical solution for the feed utilization of the shrub resources in desertification areas. Firstly, screening a shrub lignin sensitive strain in a strain library, carrying out enrichment culture on the shrub lignin sensitive strain by using a selective culture medium with alkaline lignin as a unique carbon source, initially screening the strain with lignin degradation capability by using a Congo red decolorization method, a aniline blue decolorization method and a guaiacol color development method, and carrying out re-screening by measuring activities of laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP), thereby finally obtaining 3-5 high-efficiency lignocellulose degrading bacteria; simultaneously screening cellulase (from Trichoderma reesei), xylanase (from Aspergillus niger) and laccase (from Bacillus subtilis) from commercial enzyme preparations to form a compound enzyme system, determining the optimal ratio of enzyme components (cellulase: xylanase: laccase=5:3:2) through orthogonal experiments, adopting Plackett-Burman design to screen key influencing factors (inoculum size, temperature, pH, rotating speed and the like) in the aspect of fermentation process optimization, establishing a mathematical model by a Box-Behnken response surface method to optimize fermentation conditions, performing amplification culture in a 50L fermentation tank, determining the optimal harvest time (72-96 h) by monitoring Dissolved Oxygen (DO), reducing sugar content and enzyme activity change, wherein the final product comprises the viable count of 1X 10 8 CFU/g, the cellulase activity of > 2X 10 5 U/g, the xylanase activity of > 1X 10 4 U/g, respectively reducing neutral washing fiber and acid washing fiber of shrubs by 6.4% and 8.27%, improving the dry matter digestibility by 13%, the preparation can be stably stored for more than 6 months at room temperature, and has the innovation points that 1) the optimized proportion of the synergism of the bacteria and the enzymes and 2) the special formula suitable for the shrub raw materials are provided, the preparation is simple and convenient to use, the preparation is only mixed with the crushed shrub raw materials according to the proportion of 1.5-2.5%, the water content is regulated to 50-60%, the feed quality can be obviously improved after the preparation is treated for 48-72 hours at the temperature of 35-45 ℃, and a brand new biotechnology solution is provided for the development and the utilization of shrub resources in desertification areas. In order to solve the problems of the background technology, the invention provides a compound