Preparation method of beef cattle straw feed based on synergistic fermentation of compound strains

Abstract

The invention discloses a beef cattle straw feed preparation method based on compound strain synergistic fermentation, and relates to the technical field of feed processing, and the beef cattle straw feed preparation method comprises the following steps: screening bacillus, lactic acid bacteria and the like to construct an initial compound strain, and screening a high-degradation-ability strain combination through a directed evolution system (30-45 DEG C, pH 4.0-7.0, and lignocellulose 5-15g / L); the straw lignocellulose structure is destroyed through microwave-assisted pretreatment (the power is 200-800 W, and the time is 1-5 min); high-efficiency fermentation is realized through double-stage fermentation (the pH is reduced at 30-35 DEG C for 12-24 hours in the first stage, and stubborn components are degraded at 35-40 DEG C for 36-60 hours in the second stage) and intelligent regulation and control. According to the method, the content of crude protein in the straw is increased to 15.2%, the content of crude fibers is reduced to 19.8%, the degradation rate of lignocellulose reaches 62%, the quality of the feed is remarkably improved, and a new path is provided for recycling of agricultural wastes.

Description

Technical Field

[0001] The invention relates to the technical field of feed processing, and in particular to a method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation. Background Art

[0002] In the field of beef cattle breeding, straw is an important source of roughage. Its stubborn lignocellulose structure leads to low digestibility and insufficient nutritional value. Traditional single-strain fermentation also has problems such as low efficiency, high risk of bacterial contamination, and poor raw material adaptability. In existing technologies, conventional microbial fermentation has difficulty breaking through the limitations of straw structure and lacks dynamic adaptation solutions for different raw material characteristics, resulting in unstable feed quality. In addition, traditional processes rely on manual control of fermentation parameters, which can easily affect the fermentation effect due to environmental fluctuations. There is an urgent need to develop efficient and precise straw feed preparation technology.

[0003] In view of this, this application is hereby filed. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing beef cattle straw feed based on the synergistic fermentation of composite strains, so as to solve the problems mentioned in the above background technology.

[0005] To solve the above technical problems, the present invention provides a method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation, comprising the following steps: S1 strain screening and evolution: Bacillus, lactic acid bacteria, yeast, and Aspergillus strains with lignocellulose degradation potential are screened from natural environments or bacterial strain libraries to construct an initial composite strain system. Using a directed evolution system that simulates natural evolution, the initial composite strains are cultured for multiple generations in a pressure environment with high lignocellulose concentrations, specific temperature, and pH values ​​to induce genetic mutations. Combined with high-throughput screening technology, composite strain combinations with significantly improved lignocellulose degradation capabilities are obtained. Through directed evolution and screening, the composite strain combination is optimized to significantly enhance its ability to degrade straw lignocellulose, laying the foundation for improving the nutritional value of feed. S2 Straw Pretreatment: Microwave-assisted technology is used to pretreat the straw, using microwave high-frequency vibration to destroy the straw lignocellulose structure and increase porosity. Microwave pretreatment effectively destroys the straw structure, increases the area of ​​action of subsequent strains, and improves fermentation efficiency and effect. S3 dual-stage fermentation: The pretreated straw is fed into a dual-stage fermentation system. In the first stage, a fast-growing and highly adaptable bacterial strain is introduced to rapidly consume the easily available sugars in the straw and lower the pH value. In the second stage, a strain with strong lignocellulose degradation ability after directed evolution is introduced to efficiently degrade stubborn components under the environment created in the early stage. The dual-stage fermentation fully utilizes the advantages of different bacterial strains, optimizes the fermentation environment in stages, and achieves efficient degradation of straw and nutrient conversion. S4 Fermentation Control: High-precision temperature, humidity, pH, and dissolved oxygen sensors are deployed in the fermentation equipment, and the data is fed back to the central control system in real time via wireless transmission. The system has a built-in intelligent control model based on fuzzy control theory and neural network algorithm. According to the preset optimal fermentation parameter range, it automatically adjusts the operating parameters of ventilation, heating, humidification, and stirring equipment; accurately monitors and intelligently controls fermentation conditions to ensure a stable and efficient fermentation process and improve the uniformity of feed quality.

[0006] Furthermore, in the S1, the mass ratio of Bacillus, lactic acid bacteria, yeast, and Aspergillus strains in the composite strain combination is (2-5):(3-6):(1-3):(1-4); the reasonable strain mass ratio enables the composite strains to better synergize during the fermentation process, give full play to their respective functions, and improve the overall fermentation effect.

[0007] Furthermore, in S1, the temperature range of the pressure environment in the directed evolution system is 30-45°C, the pH range is 4.0-7.0, and the cellulose concentration is 5-15 g / L; the clear pressure environment parameter setting provides suitable conditions for the directed evolution of the strain, ensuring that the screened strain has stronger cellulose degradation ability.

[0008] Furthermore, in S2, the processing conditions of the microwave-assisted technology are: microwave power 200-800W, processing time 1-5min; the specific microwave processing parameters can effectively destroy the straw structure while avoiding nutritional loss due to excessive processing, thereby ensuring the pretreatment effect and feed quality.

[0009] Furthermore, in S3, the fermentation temperature of the first stage is controlled at 30-35°C, and the fermentation time is 12-24 hours; the fermentation temperature of the second stage is controlled at 35-40°C, and the fermentation time is 36-60 hours; the precise dual-stage fermentation temperature and time settings meet the growth and metabolic requirements of strains at different stages, promote the smooth progress of the fermentation process, and improve the quality of feed fermentation.

[0010] Furthermore, in S3, during the two-stage fermentation process, the microbial community of the fermentation system is monitored in real time. When the number of foreign bacteria exceeds a set threshold, a targeted antibacterial agent is added to the fermentation system; real-time monitoring and antibacterial measures effectively prevent foreign bacteria contamination, ensure the normal progress of the fermentation process, and improve the safety and quality stability of the feed.

[0011] Furthermore, the antibacterial agent is one of a specific plant extract and a bacteriophage; the specific antibacterial agent can accurately inhibit the growth of miscellaneous bacteria while avoiding adverse effects on the composite strain, ensuring that the fermentation process proceeds smoothly without introducing harmful substances.

[0012] Furthermore, in S4, the temperature error range detected by the high-precision sensor is ±0.5°C, the humidity error range is ±3%, the pH value error range is ±0.2, and the dissolved oxygen error range is ±0.5 mg / L; the high-precision sensor detection error range ensures that the obtained fermentation parameters are accurate and reliable, provides precise data support for intelligent regulation, and ensures stable fermentation conditions.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. Directed evolution of composite strains: By simulating natural stress environments to screen highly active strain combinations, the limitations of the degradation capacity of traditional single strains were broken through. The cellulose degradation rate was increased by more than 60% compared with the initial strain, significantly releasing the nutrients encapsulated in the straw.

[0014] 2. Microwave-assisted pretreatment: Using microwave high-frequency vibration to destroy the dense structure of straw, the specific surface area is increased by nearly 100%, providing more action sites for the strain, and increasing the subsequent fermentation efficiency by more than 30%.

[0015] 3. Two-stage precision fermentation: The temperature and strain composition are regulated in stages. The first stage quickly lowers the pH to inhibit bacteria, and the second stage degrades lignocellulose in a targeted manner. Combined with an intelligent control system (parameter fluctuation ≤±0.5℃), fermentation uniformity is ensured, and the crude protein content of the feed is increased by 80% compared with traditional processes.

[0016] 4. Dynamic control of foreign bacteria: Real-time monitoring of microbial communities and precise addition of plant extracts / bacteriophage antibacterial agents to control the foreign bacteria contamination rate below 5%, ensuring feed safety and storage stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a principle block diagram of a method for preparing beef cattle straw feed based on the synergistic fermentation of composite strains. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] See also Figure 1 The present invention provides a technical solution: a method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation, comprising: 1. Experimental Materials and Equipment Strains: Obtain Bacillus subtilis (CGMCC1.1), Lactobacillus plantarum (CGMCC1.2437), Saccharomyces cerevisiae (CGMCC2.1189), and Aspergillus niger (CGMCC3.4407), and activate and culture them in the corresponding culture medium before use.

[0020] Straw raw material: Select corn straw harvested in a certain area, remove the moldy parts, crush to a length of about 1-2 cm, and control the initial moisture content at 12%-15%.

[0021] Main equipment: microwave pretreatment instrument (model: MW-2000, power adjustment range 0-1000W), 50L fully automatic fermentation tank (equipped with temperature, humidity, pH, dissolved oxygen sensors and intelligent control system), constant temperature incubator, clean workbench, microbial culture shaker, high-speed refrigerated centrifuge, Kjeldahl nitrogen analyzer, dietary fiber meter, etc.

[0022] 2. Experimental methods and test indicators Directed evolution of composite strains: The initial composite strain was inoculated at 1% into a liquid culture medium containing 10 g / L lignocellulose. The culture was incubated at 38°C, pH 5.5, and shaken at 200 rpm. The strains were transferred to fresh culture medium every 24 hours for five consecutive passages. The strains' lignocellulose degradation activity was tested using a high-throughput screening instrument, and the strain combination with the largest degradation zone diameter was selected as the evolved composite strain.

[0023] Straw pretreatment method: spread the corn straw flatly in a microwave treatment container, set the microwave power and treatment time parameters, and then measure the change in the specific surface area of ​​the straw after treatment.

[0024] Two-stage fermentation process: In the first stage, the pretreated straw is loaded into the fermentation tank, and the composite strain is inoculated at a 2% inoculation rate. The temperature is controlled at 32°C, the humidity is 70%, and the dissolved oxygen concentration is 2-3 mg / L, and the fermentation is carried out for 18 hours. In the second stage, the temperature is raised to 38°C, and other conditions remain unchanged, and the fermentation is continued for 48 hours.

[0025] Detection indicators and methods Crude protein content: determined by Kjeldahl method (GB5009.5-2016).

[0026] Crude fiber content: determined according to GB / T 5009.10-2003.

[0027] Lignocellulose degradation rate: The degradation rate was calculated by measuring the contents of cellulose, hemicellulose and lignin in the straw before and after fermentation.

[0028] pH value: Use a precision pH meter to directly measure the fermentation material.

[0029] Sensory evaluation: Organize 5 professionals to score the color, smell and texture of the feed (maximum 10 points). 3. Specific embodiments Example 1 S1 strain screening and evolution: An initial composite strain system was constructed using a mixture of Bacillus, Lactobacillus, Saccharomyces, and Aspergillus strains in a mass ratio of 3:4:2:2. In a directed evolution system set at a pressure temperature of 38°C, a pH of 5.5, and a cellulose concentration of 10 g / L, the evolved composite strain was obtained after five generations of culture and screening. Its cellulose degradation rate was 62% higher than that of the initial strain.

[0031] S2 Straw pretreatment: Place corn straw in a microwave pretreatment instrument, set the power to 500W, and treat for 3 minutes. After treatment, the specific surface area of ​​the straw is reduced from 0.8m 2 / g increased to 1.5m 2 / g.

[0032] S3 two-stage fermentation: In the first stage, fermentation is carried out according to the above process, and the pH drops to 4.2 at the end of fermentation; in the second stage, after continued fermentation, the feed has a soft texture and a strong sour and fragrant smell.

[0033] S4 Fermentation Control: During the fermentation process, temperature fluctuations were controlled within ±0.3°C, humidity within ±2%, pH within ±0.1, and dissolved oxygen within ±0.3 mg / L. The final feed had a crude protein content of 15.2%, crude fiber content reduced to 19.8%, and a sensory score of 8.9.

[0034] Example 2 (Changing the mass ratio of composite strains) S1 strain screening and evolution: The mass ratio of the composite strain was adjusted to Bacillus: Lactobacillus: Yeast: Aspergillus = 2:5:1:3, and other evolution conditions were the same as in Example 1. After screening, the lignocellulose degradation rate of the strain increased by 55%.

[0035] Steps S2-S4 were the same as those in Example 1. The final feed had a crude protein content of 14.5%, a crude fiber content of 21.2%, and a sensory score of 8.2 points.

[0036] Example 3 (Changing the Directed Evolution Temperature) S1 strain screening and evolution: The directed evolution temperature was adjusted to 30°C, and other conditions were the same as in Example 1. After screening, the lignocellulose degradation rate of the strain increased by 48%.

[0037] Steps S2-S4 were the same as those in Example 1. The final feed had a crude protein content of 13.8%, a crude fiber content of 22.5%, and a sensory score of 7.8.

[0038] Example 4 (Changing Microwave Treatment Power) Step S1-S2: The microwave power was adjusted to 200W and the treatment time was 3 min. Other aspects were the same as in Example 1. After treatment, the specific surface area of ​​the straw was increased to 1.2 m 2 / g.

[0039] Steps S3-S4 were the same as those in Example 1. The final feed had a crude protein content of 14.0%, a crude fiber content of 22.0%, and a sensory score of 8.0.

[0040] Example 5 (Changing the first stage fermentation time) Step S1-S2: The fermentation time of the first stage was extended to 24 hours, and the rest was the same as in Example 1. At the end of the first stage, the pH dropped to 4.0.

[0041] Steps S3-S4 were the same as those in Example 1. The final feed had a crude protein content of 15.0%, a crude fiber content of 20.0%, and a sensory score of 8.5.

[0042] Example 6 (Changing the type of antibacterial agent) Steps S1-S3: In the double-stage fermentation, when the number of bacteria exceeds 10 5 CFU / g, rosemary extract (0.1%) was added as an antibacterial agent, and the rest was the same as in Example 1.

[0043] Step S4: The same as Example 1. The final feed crude protein content was 15.1%, crude fiber content was 19.9%, sensory score was 8.8 points, and the number of bacteria was controlled at 10 3 CFU / g or less.

[0044] Example 7 (Changing the Dissolved Oxygen Control Range) Steps S1-S3: Adjust the dissolved oxygen control range to 3-4 mg / L, and the rest are the same as in Example 1.

[0045] Step S4: The same as Example 1. The final feed had a crude protein content of 14.3%, a crude fiber content of 21.8%, and a sensory score of 8.1 points.

[0046] Example 8 (Changing the pH Detection Error Range) Steps S1-S3: relax the detection error range of the pH sensor to ±0.5, and the rest are the same as in Example 1.

[0047] Step S4: The same as Example 1. The pH fluctuated greatly during the fermentation process. The final feed had a crude protein content of 14.0%, a crude fiber content of 22.2%, and a sensory score of 7.9.

[0048] Example 9 (Changing the Particle Size of Straw Crushing) Step S1-S2: Crush the straw to a length of about 0.5-1 cm, with the rest being the same as in Example 1.

[0049] Steps S3-S4 were the same as those in Example 1. The final feed had a crude protein content of 15.5%, a crude fiber content of 19.5%, and a sensory score of 9.0.

[0050] Example 10 (Changing the activation time of bacteria) Step S1: Extend the bacterial activation time from 24 h to 36 h. Other steps are the same as those in Example 1.

[0051] Steps S2-S4 were the same as those in Example 1. The final feed had a crude protein content of 15.3%, a crude fiber content of 19.7%, and a sensory score of 8.8.

[0052] 4. Comparative analysis of experimental data: The experimental data are shown in Table 1 below: Table 1: Experimental data comparison table.

[0053] In summary, in the present invention, through the synergistic technical means of composite strain directed evolution, microwave-assisted pretreatment, two-stage fermentation process and intelligent control system, the present invention breaks through the efficiency bottleneck and quality fluctuation problems of traditional straw fermentation. Among them, the strain combination screened by directed evolution has a non-obvious synergistic advantage for lignocellulose degradation, the coupling design of microwave pretreatment and two-stage fermentation forms a complete technical chain from structural destruction to component conversion, and the intelligent control system realizes dynamic and precise regulation of fermentation parameters. Experimental data show that this method increases the crude protein content of straw feed to more than 15%, and the crude fiber degradation rate exceeds 40%, and can adapt to different raw material characteristics through multi-parameter adjustment, showing significant technical creativity and practical application value, and providing an innovative solution for the efficient conversion of agricultural waste and cost reduction and efficiency improvement of beef cattle breeding.

Claims

1. A method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation, characterized in that: The following steps are involved: S1 Strain Screening and Evolution: Bacillus, lactic acid bacteria, yeast, and Aspergillus strains with lignocellulose degradation potential were screened from the strain library to construct an initial composite strain system. Using a directed evolution system that simulates natural evolution, the initial composite strains were cultured for multiple generations in a stress environment with high lignocellulose concentrations, specific temperature, and pH values ​​to induce genetic mutations. Combined with high-throughput screening technology, a composite strain combination with significantly improved lignocellulose degradation capabilities was obtained. S2 straw pretreatment: microwave-assisted technology is used to pretreat the straw, using microwave high-frequency vibration to destroy the straw lignocellulose structure and increase the porosity; S3 two-stage fermentation: The pretreated straw is put into a two-stage fermentation system. In the first stage, a fast-growing and highly adaptable bacterial strain is introduced to quickly consume the easily available sugars in the straw and reduce the pH value. In the second phase, strains with strong lignocellulose degradation capabilities that have undergone directed evolution are used to efficiently degrade stubborn components under the environment created in the early stages. S4 Fermentation Control: High-precision temperature, humidity, pH, and dissolved oxygen sensors are deployed in the fermentation equipment, and the data is fed back to the central control system in real time via wireless transmission. The operating parameters of the ventilation, heating, humidification, and stirring equipment are automatically adjusted according to the preset optimal fermentation parameter range.

2. The method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation according to claim 1, characterized in that: In the S1, the mass ratio of Bacillus, lactic acid bacteria, yeast and Aspergillus strains in the composite strain combination is (2-5):(3-6):(1-3):(1-4).

3. The method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation according to claim 1, characterized in that: In S1, the temperature range of the pressure environment in the directed evolution system is 30-45° C., the pH range is 4.0-7.0, and the lignocellulose concentration is 5-15 g / L.

4. The method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation according to claim 1, characterized in that: In S2, the processing conditions of the microwave-assisted technology are: microwave power 200-800W, and processing time 1-5 minutes.

5. The method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation according to claim 1, characterized in that: In S3, the fermentation temperature in the first stage is controlled at 30-35° C., and the fermentation time is 12-24 hours; the fermentation temperature in the second stage is controlled at 35-40° C., and the fermentation time is 36-60 hours.

6. The method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation according to claim 1, characterized in that: In S3, during the dual-stage fermentation process, the microbial community of the fermentation system is monitored in real time, and when the number of miscellaneous bacteria exceeds a set threshold, a targeted antibacterial agent is added to the fermentation system.

7. The method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation according to claim 6, characterized in that: The antibacterial agent is one of a specific plant extract and a bacteriophage.

8. The method for preparing beef cattle straw feed based on composite bacterial strain synergistic fermentation according to claim 1, characterized in that: In S4, the temperature error range detected by the high-precision sensor is ±0.5°C, the humidity error range is ±3%, the pH value error range is ±0.2, and the dissolved oxygen error range is ±0.5 mg / L.

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