Functional bacterium resource screening and raw material processing method

Abstract

The invention relates to the technical field of microorganisms and agriculture, in particular to a functional bacterium resource screening and raw material treatment method, which comprises the following steps: step 1, sample treatment: collecting a sample from a sampling environment, and pretreating to obtain a microbial suspension; step 2, separation and purification: carrying out five times of streak purification on the microbial suspension by adopting a streak separation method to obtain a purified strain; 3, primary screening of functional bacteria: further culturing the purified strains, and screening out bacterial colonies with large transparent circle diameters to obtain primary screening functional strains; step 4, re-screening the functional bacteria: re-screening the primarily screened functional bacteria to obtain re-screened functional bacteria; and step five, detecting functional bacteria: adding the functional bacteria subjected to secondary screening into a specific fermentation product to detect the activity of enzyme production of the functional bacteria. The functional bacterium resource screening method disclosed by the invention can be used for efficiently screening microbial strains with specific functions, and is beneficial to quickly positioning and obtaining valuable microbial resources.

Description

Technical Field

[0001] The invention relates to the field of microorganisms and agricultural technology, and in particular to a method for screening functional bacteria resources and processing raw materials. Background Art

[0002] Functional bacteria refer to microorganisms with specific biological activities or physiological functions, such as strains that can produce cellulase, laccase, protease and other enzymes, which are widely used in industry and environmental protection. In addition, functional bacteria also include strains with antibacterial, antioxidant, and immunomodulatory functions, which are also of great significance for improving human health. Therefore, screening functional bacteria resources with efficient biological activity has become one of the hot spots in current biotechnology research.

[0003] The shake flask culture method is one of the commonly used methods for screening functional bacteria. It is to inoculate single colonies of the strain to be tested into multiple triangular flasks of LB liquid culture medium, shake and culture them, and then analyze and measure the culture fluid. The conditions of the shake flask and the fermentation tank are relatively close, and the measured data are more practical. However, the shake flask culture method requires more labor, equipment and time, and is usually time-consuming and labor-intensive.

[0004] In summary, how to solve the problem that the shake flask culture method requires more manual labor, equipment and time has become a difficult problem that needs to be solved urgently in the current field, because it is necessary to propose a method for functional bacteria resource screening and raw material processing. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for screening functional bacteria resources and processing raw materials, which can significantly improve the culture efficiency and reduce manual labor through the automated operation of a shaker; the bacteria are constantly in contact with the surrounding culture medium during the shaking process, which not only obtains nutrients but also effectively absorbs dissolved oxygen, thereby reducing the culture time of the bacteria.

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

[0007] On the one hand, a method for screening functional bacteria resources is provided, comprising the following steps:

[0008] Step 1, sample processing: collect samples from the sampling environment, pre-treat the samples, and obtain a microbial suspension;

[0009] Step 2, separation and purification: using the streak separation method, inoculate the microbial suspension onto a sterile culture medium until visible colonies are formed; select a number of colonies with different morphological and color characteristics and perform streak purification five times to obtain purified strains;

[0010] Step 3, initial screening of functional bacteria: add the purified strain to sterile water and shake to mix, obtain a suspension, divide the suspension into several groups, add sterile water to dilute each group of suspension to 1 times concentration, 0.1 times concentration and 0.01 times concentration, take 100 μL of each diluted concentration suspension and spread it on the nutrient agar medium, then place the nutrient agar medium in a 30°C constant temperature box and culture it at a constant temperature for 2-3 days to obtain new colonies; pick a small amount of new colonies and inoculate them on different enzyme detection culture media, place the enzyme detection culture media in a 30-35°C constant temperature box and culture them until the colonies are abundant, take out the cultured enzyme culture media and observe the size of the transparent circle around the colonies, screen out the colonies with a transparent circle diameter greater than or equal to 20 mm, and obtain the initial screening functional strains;

[0011] Step 4, functional bacteria rescreening: Functional bacteria rescreening: inoculate the initially screened functional strains into LB liquid culture medium, put the LB liquid culture medium into a constant temperature box, set the constant temperature box temperature to 37°C, and culture for 12 hours to obtain a functional strain solution; after the culture is completed, inoculate the functional strain solution into a triangular flask LB liquid culture medium according to an inoculation volume of 5%, put the triangular flask LB liquid culture medium into a shaker, the shaker speed is 160r / min, put the shaker into a constant temperature box, set the constant temperature box temperature to 37°C, and culture for 48 hours. After the culture is completed, the rescreened functional strains are obtained;

[0012] Step 5, functional bacteria detection: weigh 3.0g of solid fermentation product, wash with water to remove impurities, and add it to a 100mL volumetric flask. Add the rescreened functional bacteria and water to the volumetric flask at a mass ratio of 1:100. After mixing evenly, incubate at room temperature for 20min, shake the volumetric flask once every 3min, then filter the mixed solution in the volumetric flask with filter paper, take 5ml of the filtrate and use HBS-1096A enzyme label analyzer to detect the activity of specific enzymes in the filtrate.

[0013] The technical principle of the above scheme is as follows: collect samples from the sampling environment, perform pretreatment, and obtain a microbial suspension; use the streak separation method to purify the microbial suspension 5 times to obtain a purified strain; further cultivate the purified strain and screen out colonies with a larger transparent circle diameter to obtain a primary screened functional strain; rescreen the primary screened functional strain to obtain a rescreened functional strain; add the rescreened functional strain to a specific fermentation product to detect its enzyme production activity.

[0014] The above scheme has the following beneficial effects:

[0015] 1. This scheme can shake the LB liquid culture medium in the triangular flask in all directions by using a shaker, so that the liquid surface of the culture medium is constantly in contact with the air above, thereby significantly improving the ventilation volume; sufficient ventilation volume ensures the supply of dissolved oxygen in the culture medium, which is beneficial to the growth and metabolic activities of microorganisms; the bacteria are constantly in contact with the surrounding culture medium during the oscillation process, which not only obtains nutrients but also effectively absorbs dissolved oxygen, reducing the culture time of the strain; at the same time, the automated operation of the shaker can also significantly improve the culture efficiency and reduce manual labor.

[0016] 2. This method can efficiently screen out microbial strains with efficient enzyme production through systematic sample processing, separation and purification, primary screening and secondary screening steps; this helps to quickly locate and obtain valuable microbial resources, providing an important basis for subsequent industrial applications and biopharmaceutical development.

[0017] 3. In this scheme, in the initial screening stage, the transparent circle on the enzyme detection culture medium can be observed to intuitively screen out strains with strong enzyme production ability; the size of the transparent circle directly reflects the strength of the strain's enzyme production ability, thereby improving the accuracy and reliability of screening; in the re-screening stage, by adjusting the culture temperature and the speed of the shaker, the growth environment of the functional bacteria is further optimized and the accumulation of its metabolites is promoted; it is helpful to obtain functional bacteria with higher yield and higher activity.

[0018] Furthermore, in step one, the sampling environment includes soil, water and plant roots.

[0019] Beneficial effects: Obtaining bacterial strains from soil, water and plant roots can efficiently obtain microbial resources with potential application value from complex sampling environments, providing a rich bacterial strain library for subsequent biotechnology research and industrial applications.

[0020] Further, in step one, the pretreatment includes filtering, grinding and diluting.

[0021] Beneficial effects: Impurities are removed by filtration, grinding increases the contact area between microorganisms and culture medium, and dilution facilitates the dispersion and counting of microorganisms, improving the efficiency and accuracy of subsequent separation and purification.

[0022] Furthermore, the different enzyme detection medium includes a cellulase detection medium, a laccase detection medium and a protease detection medium.

[0023] Beneficial effects: By screening different types of enzymes, functional bacteria with specific target enzyme production can be screened out. At the same time, other enzyme agents can be selected for screening. Through the selection of enzyme agents, functional bacteria corresponding to the enzyme product can be screened out, which is convenient for achieving subsequent effects.

[0024] Further, in step four, the shaking machine includes a bracket and a motor, a driving wheel is eccentrically fixedly connected to the motor output shaft, a slider is sleeved on the outer side of the driving wheel, and the slider and the driving wheel rotate in cooperation; a first slide groove is vertically symmetrically opened on the bracket, a pull rod is vertically slidably matched in the first slide groove, a plurality of fixed blocks are fixedly connected to the outer wall of the upper part of the bracket, and the side walls of the fixed blocks close to the bracket are fixedly connected to the support rod; a driving frame is vertically slidably matched on the inner side of the bracket, and a second slide groove is opened on the inner bottom wall and the inner top wall of the driving frame, and the slider and the second slide groove are horizontally slidably matched; a plurality of first connecting rods are hinged on the top of the driving frame, and the first connecting rod passes through the top wall of the bracket away from the driving frame and is hinged to the second connecting rod, and the side of the second connecting rod close to the support rod is hinged to the support rod, and the second connecting rod is hinged to the third connecting rod at one end away from the first connecting rod, and the third connecting rod is hinged to the pull rod at one end away from the second connecting rod; a shaking table assembly for omnidirectional self-rotation and oscillation of the LB liquid culture medium in the triangular flask is provided on the slider; a piston assembly for generating airflow is provided on the inner bottom wall of the bracket.

[0025] Beneficial effects: The use of a shaker can significantly improve culture efficiency and reduce manual labor; through the oscillation of the shaker, the growth rate and metabolic efficiency of functional bacteria are improved, thereby shortening the entire culture cycle.

[0026] Furthermore, the shaker assembly includes a shaker, one end of which is fixedly connected to the outer wall of the slider; a plurality of grooves are opened on the shaker, each of which is provided with a tray for placing the LB liquid culture medium in a triangular flask, a rotating rod is fixedly connected to the bottom of the tray, and the end of the rotating rod away from the tray penetrates the inner bottom wall of the groove and rotates with the inner bottom wall of the groove; the end of the rotating rod away from the tray is coaxially fixedly connected to a fan blade.

[0027] Beneficial effects: The shaker assembly can achieve all-round oscillation of the LB liquid culture medium in the triangular flask; this oscillation method helps to evenly mix the bacterial liquid and nutrients in the culture medium, ensuring that the functional bacteria can fully contact the required nutrients during the growth process, thereby promoting their uniform growth and reproduction; oscillation can also prevent the solid components in the culture medium from settling to the bottom of the bottle, ensuring that the bacteria can be in a uniform suspension state throughout the culture process, further improving the culture efficiency.

[0028] Furthermore, the piston assembly includes an air cylinder, the bottom of which is fixedly connected to the inner bottom wall of the bracket; a piston is vertically slidably fitted on the inner wall of the air cylinder, a one-way valve is connected to the piston, a piston rod is fixedly connected to the top of the piston, and the end of the piston rod away from the piston is fixedly connected to the pull rod; a first air pipe is connected to the bottom of the air cylinder, a plurality of second air pipes are connected to the first air pipe, and the ends of the second air pipes away from the first air pipe are all directly below adjacent fan blades.

[0029] Beneficial effect: The piston assembly generates airflow through the up and down movement of the piston in the air cylinder, and transports the airflow to the bottom of the fan blade through the first air pipe and the second air pipe. The airflow can drive the fan blade to rotate and drive the rotating rod to rotate, and then drive the tray at one end of the rotating rod away from the fan blade to rotate, so that the LB liquid culture medium in the triangular flask can achieve self-rotation oscillation during the all-round oscillation process, further allowing the bacteria in the LB liquid culture medium in the triangular flask to fully contact the required nutrients, and for some aerobic bacteria, the continuous self-rotation oscillation of the culture medium during the self-rotation oscillation process can increase the dissolved oxygen content in the culture medium, and sufficient dissolved oxygen is conducive to the rapid growth of the bacteria and the accumulation of metabolites.

[0030] Furthermore, in step five, the solid fermentation product includes one or more of corn straw fermentation product, distiller's grains, cassava fermentation product, soybean fermentation product and sugarcane bagasse fermentation product.

[0031] Beneficial effects: Corn straw fermentation products and distiller's grains can be converted into valuable bio-fertilizers or feed additives through solid-state fermentation. The products have rich application value. Using them as detection objects can detect strains suitable for fermenting corn straw fermentation products and distiller's grains; cassava fermentation products and soybean fermentation products are rich in cellulose, laccase and protease. Using them as detection objects can detect strains that can efficiently produce cellulose, laccase and protease. These enzymes have rich application value and economic value. This method can be used to perform detection quickly and in batches, which is suitable for large-scale screening and detection.

[0032] Furthermore, in step five, the specific enzyme includes one or more of cellulase, laccase and protease.

[0033] Beneficial effects: Cellulase, laccase and protease are widely used in industry and environmental protection fields. For example, cellulase can be used for biomass degradation and feed processing, laccase can be used for dye decolorization and wastewater treatment, and protease can be used for food processing and leather manufacturing. Screening the functional bacteria of these enzymes has important application value.

[0034] On the other hand, a raw material processing method is provided, which is based on the functional bacteria obtained by the above-mentioned functional bacteria resource screening method, and comprises the following steps:

[0035] Step 1, raw material pretreatment: collecting agricultural waste, food processing by-products and plant raw materials, washing, crushing and drying them to obtain pretreated raw material particles;

[0036] Step 2, raw material sterilization: steam sterilize the pretreated raw material particles; wherein the sterilization temperature is 121-126° C. to obtain sterilized raw material particles;

[0037] Step 3, raw material inoculation and fermentation: under aseptic conditions, the functional bacteria are inoculated into the sterilized raw material particles, and fermentation treatment is performed to obtain a fermentation matrix;

[0038] Step 4, product extraction: extracting the fermentation substrate to obtain the target product, and purifying the extracted target product to obtain a purified product; wherein the extraction method includes one or more of filtration, centrifugation, solvent extraction and enzymatic hydrolysis; the purification method includes one or more of crystallization, distillation and chromatography;

[0039] Step 5, product drying and storage: drying the purified product, and storing the dried product in a cool, dry and ventilated place; wherein the drying method includes one or more of natural drying, hot air drying and vacuum drying.

[0040] Beneficial effects: Through systematic raw material pretreatment, sterilization, inoculation and fermentation, product extraction and drying and storage, the complete transformation process from raw materials to target products is realized, providing reliable technical support for the industrial application of functional bacteria resources; at the same time, this method also has the advantages of simple operation, low cost and environmentally friendly sampling.

[0041] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 4 is a flow chart of the method for screening functional bacteria resources in an embodiment of the present invention.

[0043] Figure 2 It is a method flow chart of the functional bacteria resource raw material processing method in an embodiment of the present invention.

[0044] Figure 3 It is an axonometric view of the shaking machine in the embodiment of the present invention.

[0045] Figure 4 It is a side sectional view of the rocking table assembly in an embodiment of the present invention.

[0046] The figure marks in the drawings of the specification include: 1, bracket; 2, driving wheel; 3, slider; 4, first slide groove; 5, pull rod; 6, fixed block; 7, support rod; 8, driving frame; 9, first connecting rod; 10, second connecting rod; 11, third connecting rod; 12, rocking table; 13, groove; 14, tray; 15, rotating rod; 16, fan blade; 17, air cylinder; 18, piston; 19, piston rod; 20, first air pipe; 21, second air pipe; 22, second slide groove. DETAILED DESCRIPTION

[0047] The following is further described in detail through specific implementation methods:

[0048] Embodiment 1:

[0049] like Figure 1 As shown: A method for screening functional bacteria resources comprises the following steps:

[0050] Step 1: Sample processing: Collect samples from soil, water and plant roots, filter, grind and dilute the samples to obtain a microbial suspension.

[0051] Step 2: Isolation and purification: Using the streak separation method, inoculate the microbial suspension onto a sterile culture medium until visible colonies are formed; select a number of colonies with different morphological and color characteristics and perform streak purification five times to obtain purified strains.

[0052] Step three, initial screening of functional bacteria: add the purified strain to sterile water, shake and mix, and obtain a suspension, divide the suspension into several groups, add sterile water to dilute each group of suspension to 1 times concentration, 0.1 times concentration and 0.01 times concentration, take 100 μL of each diluted concentration suspension and spread it on the nutrient agar medium, then place the nutrient agar medium in a 30°C constant temperature box and culture it at a constant temperature for 2-3 days to obtain new colonies; pick a small amount of new colonies and spot-inoculate them on cellulase detection medium, laccase detection medium and protease detection medium, place the cellulase detection medium, laccase detection medium and protease detection medium in a 30-35°C constant temperature box and culture them until the colonies are rich, take out the cultured cellulase detection medium, laccase detection medium and protease detection medium and observe the size of the transparent circle around the colonies, screen out colonies with a transparent circle diameter greater than or equal to 20 mm, and obtain the initial screening functional bacteria.

[0053] Step 4, re-screening of functional bacteria: inoculate the initially screened functional strains into LB liquid culture medium, place the LB liquid culture medium in an incubator, set the incubator temperature to 37°C, and culture for 12 hours to obtain a functional strain solution; after the culture is completed, inoculate the functional strain solution into a triangular flask LB liquid culture medium according to an inoculation volume of 5%, place the triangular flask LB liquid culture medium in a shaker, the shaker speed is 160r / min, place the shaker in an incubator, set the incubator temperature to 37°C, and culture for 48 hours. After the culture is completed, the re-screened functional strains are obtained.

[0054] Step 5, functional bacteria detection: weigh 3.0g of soybean fermentation product and sugarcane bagasse fermentation product, wash with water to remove impurities, and add them to a 100mL volumetric flask, add the re-screened functional bacteria and water to the volumetric flask at a mass ratio of 1:100, mix well, incubate at room temperature for 20min, shake the volumetric flask once every 3min, then filter the mixed solution in the volumetric flask with filter paper, take 5ml of the filtrate, and use HBS-1096A enzyme labeled analyzer to detect the activity of protease and cellulase in the filtrate.

[0055] The present invention can shake the LB liquid culture medium in a triangular flask in all directions by using a shaking machine, so that the liquid surface of the culture medium is further continuously in contact with the air above, thereby significantly improving the ventilation volume; sufficient ventilation volume ensures the supply of dissolved oxygen in the culture medium, which is beneficial to the growth and metabolic activities of microorganisms; the bacteria are continuously in contact with the surrounding culture medium during the oscillation process, not only to obtain nutrition, but also to effectively absorb dissolved oxygen, thereby reducing the culture time of the strains; at the same time, the automatic operation of the shaking machine can also significantly improve the culture efficiency and reduce manual labor; through systematic sample processing, separation and purification, primary screening and secondary screening steps, the method can efficiently screen out microbial strains with efficient enzyme production function; this helps to quickly locate and obtain valuable microbial resources, and provides an important basis for subsequent industrial applications and biological preparation development.

[0056] Embodiment 2:

[0057] like Figure 3 and Figure 4 As shown, the difference from the above embodiment is that the shaking machine includes a bracket 1 and a motor, a driving wheel 2 is eccentrically fixedly connected to the motor output shaft by bolts, a slider 3 is sleeved on the outer side of the driving wheel 2, and the slider 3 and the driving wheel 2 are rotatably matched; a first sliding groove 4 is vertically symmetrically opened on the bracket 1, and a pull rod 5 is vertically slidably matched in the first sliding groove 4.

[0058] The upper outer wall of the bracket 1 is fixedly connected with a plurality of fixing blocks 6 by bolts, and the side walls of the fixing blocks 6 close to the bracket 1 are integrally formed with support rods 7; a driving frame 8 is vertically slidably matched on the inner side of the bracket 1, and a second slide groove 22 is provided on the inner bottom wall and the inner top wall of the driving frame 8, and the slider 3 and the second slide groove 22 are horizontally slidably matched; a plurality of first connecting rods 9 are hinged on the top of the driving frame 8, and the ends of the first connecting rods 9 away from the driving frame 8 all penetrate the top wall of the bracket 1 and are hinged with the second connecting rod 10, and the side of the second connecting rod 10 close to the support rod 7 is hinged to the support rod 7, and the ends of the second connecting rod 10 away from the first connecting rod 9 are hinged to the third connecting rod 11, and the ends of the third connecting rod 11 away from the second connecting rod 10 are hinged to the pull rod 5; a shaking table assembly for performing all-round self-rotation and oscillation of the LB liquid culture medium in the triangular flask is provided on the slider 3; a piston assembly for generating airflow is provided on the inner bottom wall of the bracket 1.

[0059] The shaking table assembly includes a shaking table 12, one end of which is integrally formed with the outer wall of the slider 3; a plurality of grooves 13 are provided on the shaking table 12, each of which is provided with a tray 14 for placing a triangular flask of LB liquid culture medium, and a rotating rod 15 is integrally formed at the bottom of the tray 14, and the end of the rotating rod 15 away from the tray 14 penetrates the inner bottom wall of the groove 13 and rotates with the inner bottom wall of the groove 13; the end of the rotating rod 15 away from the tray 14 is coaxially fixedly connected to a fan blade 16 by bolts.

[0060] The piston assembly includes an air cylinder 17, the bottom of which is fixedly connected to the inner bottom wall of the bracket 1 by bolts; a piston 18 is vertically slidably fitted on the inner wall of the air cylinder 17, a one-way valve is connected to the piston 18, a piston rod 19 is integrally formed on the top of the piston, and the end of the piston rod 19 away from the piston 18 is fixedly connected to the pull rod 5 by bolts; a first air pipe 20 is connected to the bottom of the air cylinder 17, a plurality of second air pipes 21 are connected to the first air pipe 20, and the ends of the second air pipes 21 away from the first air pipe 20 are all directly below the adjacent fan blades 16.

[0061] The specific implementation process is as follows: put the LB liquid culture medium in the triangular bottle containing functional bacteria after initial screening and inoculation into the tray 14 on the shaking table 12 and fix it, start the shaking table machine, and set the motor speed to 160r / min; when the motor is started, its output shaft starts to rotate; since the driving wheel 2 is eccentrically fixedly connected to the motor output shaft by bolts, the driving wheel 2 will rotate with the rotation of the motor; the slider 3 is sleeved on the outside of the driving wheel 2 and rotates with the driving wheel 2, and the slider 3 and the shaking table 12 are integrally formed. Since the driving wheel 2 is eccentrically fixed, when the driving wheel 2 rotates, the slider 3 will reciprocate up and down and left and right along the contour of the driving wheel 2. Since the slider 3 and the second slide groove 22 in the driving frame 8 are slidably matched, and the driving frame 8 and the inner wall of the bracket 1 are vertically slidably matched, when the slider 3 moves up and down, it can drive the driving frame 8 to slide up and down in the bracket 1, and at the same time drive the shaking table 12 to move up and down, and the driving frame 8 can slide Block 3 performs a rotational limiting function to prevent the slider 3 from rotating as the driving wheel 2 rotates; when the slider 3 moves left and right, the slider 3 also drives the shaker 12 to move left and right, thereby driving the LB liquid culture medium in the triangular bottle in the tray 14 on the shaker 12 to oscillate in all directions up and down and left and right; when the driving frame 8 slides up and down in the bracket 1, the first connecting rod 9 hinged on the driving frame 8 is driven by the driving frame 8 to move up and down. At the same time, since the second connecting rod 10 is hinged on the support rod 7, due to the lever principle, the up and down movement of the first connecting rod 9 drives the second connecting rod 10 to move up and down away from one end of the first connecting rod 9. Since the third connecting rod 11 is hinged to one end of the second connecting rod 10 away from the first connecting rod 9, the second connecting rod 10 moves up and down away from one end of the first connecting rod 9 and then drives the third connecting rod 11 to move up and down. Since the third connecting rod 11 is hinged to one end of the second connecting rod 10 away from the second connecting rod 10 and the pull rod 5, the third connecting rod 11 can drive the pull rod 5 to slide up and down on the first slide groove 4 in the bracket 1.

[0062] The air cylinder 17 is fixed to the inner bottom wall of the bracket 1, and the piston 18 is vertically slidably matched in the air cylinder 17; the top of the piston 18 is fixedly connected to the pull rod 5 through the piston rod 19, so when the pull rod 5 moves up and down, it will drive the piston 18 to slide up and down in the air cylinder 17; because the piston 18 is connected with a one-way valve, it can ensure that the gas can only enter the air cylinder 17 from the outside; when the piston 18 moves upward, the external air enters the air cylinder 17 through the one-way valve, and when the piston 18 moves downward, the gas is compressed and enters the first air pipe 20 and is blown out by the second air pipe 21, and the second air pipe 21 blows The outgoing airflow can drive the adjacent fan blades 16 to rotate, and at the same time drive the rotating rod 15 to rotate, and then drive the tray 14 at one end of the rotating rod 15 away from the fan blades 16 to rotate, so that the LB liquid culture medium in the triangular flask can realize self-rotation oscillation during the all-round oscillation process, and further enable the bacteria in the LB liquid culture medium in the triangular flask to fully contact the required nutrients. At the same time, for some aerobic bacteria, the continuous self-rotation oscillation of the culture medium during the self-rotation oscillation process can increase the dissolved oxygen content in the culture medium. Adequate dissolved oxygen is conducive to the rapid growth of the bacteria and the accumulation of metabolites.

[0063] Embodiment 3:

[0064] like Figure 2 As shown, the difference from the above embodiment is that a raw material processing method, based on the functional bacteria obtained in Example 1, comprises the following steps:

[0065] Step 1, raw material pretreatment: collect agricultural waste, food processing by-products and plant raw materials, wash, crush and dry them to obtain pretreated raw material particles; the raw material used in this embodiment is corn stalks among the plant raw materials.

[0066] Step 2, sterilizing the raw materials: sterilizing the pretreated corn stalk particles with steam at 122° C. to obtain sterilized corn stalk particles;

[0067] Step three, raw material inoculation and fermentation: under aseptic conditions, the functional bacteria are inoculated into the sterilized corn stalk particles, and fermentation treatment is performed to obtain a fermentation matrix.

[0068] Step 4, product extraction: filtering the fermentation substrate to obtain the target product; the target product in this embodiment is cellulase corn straw fermented feed.

[0069] Step 5, product drying and storage: vacuum drying the filtered cellulase corn straw fermented feed, and storing the dried cellulase corn straw fermented feed in a cool, dry and ventilated place.

[0070] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for screening functional bacteria resources, characterized in that: The following steps are involved: Step 1, sample processing: collect samples from the sampling environment, pre-treat the samples, and obtain a microbial suspension; Step 2, separation and purification: using the streak separation method, inoculate the microbial suspension onto a sterile culture medium until visible colonies are formed; select a number of colonies with different morphological and color characteristics and perform streak purification five times to obtain purified strains; Step 3, initial screening of functional bacteria: add the purified strain to sterile water and shake to mix, obtain a suspension, divide the suspension into several groups, add sterile water to dilute each group of suspension to 1 times concentration, 0.1 times concentration and 0.01 times concentration, take 100 μL of each diluted concentration suspension and spread it on the nutrient agar medium, then place the nutrient agar medium in a 30°C constant temperature box and culture it at a constant temperature for 2-3 days to obtain new colonies; pick a small amount of new colonies and inoculate them on different enzyme detection culture media, place the enzyme detection culture media in a 30-35°C constant temperature box and culture them until the colonies are abundant, take out the cultured enzyme culture media and observe the size of the transparent circle around the colonies, screen out the colonies with a transparent circle diameter greater than or equal to 20 mm, and obtain the initial screening functional strains; Step 4, functional bacteria rescreening: inoculate the initially screened functional strains into LB liquid culture medium, place the LB liquid culture medium in a constant temperature box, set the constant temperature box temperature to 37°C, and culture for 12 hours to obtain a functional strain solution; after the culture is completed, inoculate the functional strain solution into a triangular flask LB liquid culture medium according to an inoculation amount of 5%, place the triangular flask LB liquid culture medium in a shaker, the shaker speed is 160r / min, place the shaker in a constant temperature box, set the constant temperature box temperature to 37°C, and culture for 48 hours. After the culture is completed, the rescreened functional strains are obtained; Step 5, functional bacteria detection: weigh 3.0g of solid fermentation product, wash with water to remove impurities, and add it to a 100mL volumetric flask. Add the rescreened functional bacteria and water to the volumetric flask at a mass ratio of 1:

100. After mixing evenly, incubate at room temperature for 20min, shake the volumetric flask once every 3min, then filter the mixed solution in the volumetric flask with filter paper, take 5ml of the filtrate and use HBS-1096A enzyme label analyzer to detect the activity of specific enzymes in the filtrate.

2. The method for screening functional bacteria resources according to claim 1, characterized in that: In step one, the sampling environment includes soil, water, and plant roots.

3. The method for screening functional bacteria resources according to claim 2, characterized in that: In step one, pretreatment includes filtering, grinding and dilution.

4. The method for screening functional bacteria resources according to claim 3, characterized in that: In step three, different enzyme detection culture media include cellulase detection culture media, laccase detection culture media and protease detection culture media.

5. The method for screening functional bacteria resources according to claim 4, characterized in that: In step 4, the shaking bed machine includes a bracket (1) and a motor, a driving wheel (2) is eccentrically fixedly connected to the motor output shaft, a slider (3) is sleeved on the outer side of the driving wheel (2), and the slider (3) and the driving wheel (2) are rotatably matched; a first slide groove (4) is vertically symmetrically opened on the bracket (1), a pull rod (5) is vertically slidably matched in the first slide groove (4), a plurality of fixed blocks (6) are fixedly connected to the outer side wall of the upper part of the bracket (1), and the side walls of the fixed blocks (6) close to the bracket (1) are fixedly connected to support rods (7); a driving frame (8) is vertically slidably matched on the inner side of the bracket (1), and the inner bottom wall and the inner top wall of the driving frame (8) are both opened with a second slide groove (22), and the slider (3) and the second slide groove (22) are all laterally slidably matched; a plurality of first connecting rods (9) are hinged on the top of the driving frame (8); the ends of the first connecting rods (9) away from the driving frame (8) all penetrate the top wall of the bracket (1) and are hinged with the second connecting rods (10); the second connecting rods (10) are hinged to the support rod (7) on the side close to the support rod (7); the ends of the second connecting rods (10) away from the first connecting rods (9) are hinged with the third connecting rods (11); the ends of the third connecting rods (11) away from the second connecting rods (10) are hinged with the pull rod (5); a shaking table assembly for performing all-round self-rotational oscillation on the LB liquid culture medium in the triangular flask is provided on the slider (3); a piston assembly for generating airflow is provided on the inner bottom wall of the bracket (1).

6. The method for screening functional bacteria resources according to claim 5, characterized in that: The shaking table assembly comprises a shaking table (12), one end of which is fixedly connected to the outer wall of the slider (3); a plurality of grooves (13) are provided on the shaking table (12), each of which is provided with a tray (14) for placing a triangular flask LB liquid culture medium; a rotating rod (15) is fixedly connected to the bottom of the tray (14); an end of the rotating rod (15) away from the tray (14) penetrates the inner bottom wall of the groove (13) and is rotatably matched with the inner bottom wall of the groove (13); and a fan blade (16) is coaxially fixedly connected to the end of the rotating rod (15) away from the tray (14).

7. The method for screening functional bacteria resources according to claim 6, characterized in that: The piston assembly comprises an air cylinder (17), the bottom of which is fixedly connected to the inner bottom wall of the bracket (1); a piston (18) is vertically slidably fitted on the inner wall of the air cylinder (17), a one-way valve is connected to the piston (18), a piston rod (19) is fixedly connected to the top of the piston (18), and the end of the piston rod (19) away from the piston (18) is fixedly connected to the pull rod (5); the bottom of the air cylinder (17) is connected to a first air pipe (20), the first air pipe (20) is connected to a plurality of second air pipes (21), and the ends of the second air pipes (21) away from the first air pipe (20) are all located directly below adjacent fan blades (16).

8. The method for screening functional bacteria resources according to claim 7, characterized in that: In step five, the solid fermentation product includes one or more of corn straw fermentation product, distiller's grains, cassava fermentation product, soybean fermentation product and sugarcane bagasse fermentation product.

9. The method for screening functional bacteria resources according to claim 8, characterized in that: In step five, the specific enzyme includes one or more of cellulase, laccase and protease.

10. A raw material processing method, based on the functional bacteria obtained by the functional bacteria resource screening method according to any one of claims 1 to 9, characterized in that: The following steps are involved: Step 1, raw material pretreatment: collecting agricultural waste, food processing by-products and plant raw materials, washing, crushing and drying them to obtain pretreated raw material particles; Step 2, raw material sterilization: steam sterilize the pretreated raw material particles; wherein the sterilization temperature is 121-126° C. to obtain sterilized raw material particles; Step 3, raw material inoculation and fermentation: under aseptic conditions, the functional bacteria are inoculated into the sterilized raw material particles, and fermentation treatment is performed to obtain a fermentation matrix; Step 4, product extraction: extracting the fermentation substrate to obtain the target product, and purifying the extracted target product to obtain a purified product; wherein the extraction method includes one or more of filtration, centrifugation, solvent extraction and enzymatic hydrolysis; the purification method includes one or more of crystallization, distillation and chromatography; Step 5, product drying and storage: drying the purified product, and storing the dried product in a cool, dry and ventilated place; wherein the drying method includes one or more of natural drying, hot air drying and vacuum drying.

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