Preparation method and device of rumen fluid anaerobic bacteria culture medium additive

Abstract

The invention discloses a preparation method and device of a rumen fluid anaerobic bacteria culture medium additive, and the method comprises the following steps: injecting an anaerobic bacteria activity protective agent into a rumen fluid according to an initial flora activity value of a collected fresh rumen fluid, and carrying out pulse type oscillation treatment for 10-15 minutes in a low-temperature environment of 4-6 DEG C to obtain a pretreated rumen fluid; performing two-stage filtration according to particle distribution characteristics of the pretreated rumen fluid to obtain primary filtrate; performing staged variable-speed centrifugal treatment according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria; according to the microbial load capacity of the high-activity supernate, a pulse type high-temperature sterilization method is adopted, and sterilized supernate is obtained; according to the oxidation-reduction potential of the sterilized supernate, mixed inert gas is filled into the split charging bottle to replace residual oxygen, and the bottle opening is sealed by adopting a laser melting sealing technology. By utilizing the embodiment of the invention, efficient protection and culture of anaerobic bacteria in rumen fluid can be realized, the activity and the application effect of the anaerobic bacteria are improved, and a high-quality microbial culture medium is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of culture medium preparation, in particular to a method and a device for preparing a rumen fluid anaerobic bacteria culture medium additive. Background Art

[0002] In modern agriculture and animal husbandry, the breeding efficiency of ruminants such as cattle and sheep directly affects the yield and quality of meat and dairy products. Rumen fluid, as an important substance in the digestion process of ruminants, carries a rich microbial community, which plays a key role in food fermentation, nutrient conversion and animal health. Therefore, the development of an efficient rumen fluid processing and cultivation method can significantly improve the feed conversion rate and production performance of ruminants.

[0003] Traditional rumen fluid culture and treatment methods often have problems such as complex operation, low efficiency, and serious loss of microbial activity. In practical applications, how to effectively maintain and improve the activity of anaerobic bacteria in rumen fluid has become a technical problem that the industry urgently needs to solve. In addition, the existing culture medium lacks strict control of the anaerobic environment and is easily disturbed by oxygen, which leads to limited growth of anaerobic bacteria and affects the application effect of rumen fluid. Summary of the invention

[0004] The purpose of the present invention is to provide a method and device for preparing a rumen fluid anaerobic culture medium additive to solve the deficiencies in the prior art, to reasonably configure anaerobic bacteria active protective agents, to optimize operating conditions and to strengthen the control of the anaerobic environment, to achieve efficient protection and cultivation of anaerobic bacteria in rumen fluid, and to enhance their activity and application effect.

[0005] One embodiment of the present application provides a method for preparing a rumen fluid anaerobic culture medium additive, the method comprising: According to the initial bacterial flora activity value of the collected fresh rumen fluid, an anaerobic bacteria active protective agent is injected into the rumen fluid, and the rumen fluid is treated by pulse shaking for 10-15 minutes at a low temperature of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5Hz±0.5Hz; According to the particle distribution characteristics of the pretreated rumen fluid, a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter are used in turn for two-stage filtration, and a negative pressure of -0.05MPa or -0.1MPa is simultaneously applied in the ultrafine filtration stage to obtain the primary filtrate; wherein, the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; According to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria, a phased variable speed centrifugation treatment is adopted. In the first phase, large particles are removed by centrifugation at 5000-6000 rpm for 5 minutes, and in the second phase, the supernatant is collected by centrifugation at 10000-12000 rpm for 10 minutes. The centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain an anaerobic environment to obtain a highly active supernatant. According to the microbial load of the highly active supernatant, a pulse high temperature sterilization method is used to obtain a sterilized supernatant; According to the redox potential of the supernatant after sterilization, a mixed inert gas is filled into the sub-bottle to replace the residual oxygen, and the bottle mouth is sealed using laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

[0006] Optionally, according to the initial flora activity value of the collected fresh rumen fluid, an anaerobic bacteria activity protective agent is injected into the rumen fluid, and the rumen fluid is treated by pulse shaking for 10-15 minutes at a low temperature of 4°C-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5Hz±0.5Hz, including: According to the real-time ATP bioluminescence value, a portable bioluminescence detector was used to measure the initial bacterial colony activity value as the activity quantification data; According to the activity quantification data, 0.5 mL of protective agent was added for every 100 RLU, and a succinate buffer containing 10 mmol / L reduced glutathione was injected into the rumen fluid, and the pH value of the succinate buffer was 6.8±0.2 to obtain a protective agent mixture; The protective agent mixture was placed in a 4°C pulse oscillator, and the oscillation frequency was dynamically adjusted according to the initial bacterial flora activity value. When the activity value was >500RLU, the oscillation frequency was 5.5Hz, and when the activity value was ≤500RLU, the oscillation frequency was 4.5Hz. The oscillation treatment was performed for 12 minutes ± 30 seconds to obtain the pretreated rumen fluid.

[0007] Optionally, according to the particle distribution characteristics of the pretreated rumen fluid, a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter are sequentially used for two-stage filtration, and a negative pressure of -0.05MPa or -0.1MPa is simultaneously applied in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles, including: According to the test results of the laser scattering particle size analyzer, the median particle size D50 value of the particles was extracted; Select the coarse filter mesh number according to the D50 value, wherein, when D50>200μm, use 60 mesh for the coarse filter, and when D50≤200μm, use 100 mesh for the coarse filter, perform the first stage filtration, and obtain the coarse filtrate; According to the feedback of the coarse filtrate flow rate, the negative pressure value of the 200-300 mesh ultrafine filter is dynamically adjusted by the PID algorithm, wherein the negative pressure value is set to -0.1MPa when the flow rate is less than 10mL / min, and the negative pressure value is set to -0.05MPa when the flow rate is ≥10mL / min; According to the D50 value, the pore size of the ultrafine filter is adjusted in real time, wherein the pore size range is 1.3×D50×(1±5%), and the filter is cleaned with ultrasound every 2 minutes during the filtration process of the ultrafine filter to obtain an ultrafine filtrate; According to the bacterial activity loss rate of the ultrafine filtrate obtained by comparing the pretreated rumen fluid, if the bacterial activity loss rate is greater than 10%, the process returns to the step of dynamically adjusting the negative pressure value of the 200-300 mesh ultrafine filter through the PID algorithm according to the feedback of the coarse filtrate flow rate until the bacterial activity loss rate is ≤10%, and the primary filtrate is output.

[0008] Optionally, according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria, a staged variable speed centrifugation treatment is adopted, wherein the first stage is centrifuged at 5000-6000 rpm for 5 minutes to remove large particle residues, and the second stage is centrifuged at 10000-12000 rpm for 10 minutes to collect the supernatant; wherein the centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain an anaerobic environment, to obtain a highly active supernatant, comprising: According to the rotational viscometer test value η and the optical density value OD600, the viscosity-bacteria density relationship formula η=0.02×(OD600)²+1.5 was established; The first-stage centrifugal speed is set according to the η value, wherein, when η>20mPa·s, the first-stage centrifugal speed is 6000rpm, and when η≤20mPa·s, the first-stage centrifugal speed is 5000rpm, centrifuge at 8°C for 5 minutes, remove large particle residues, and obtain a first-stage supernatant; Nitrogen was introduced into the centrifuge chamber at a flow rate of 5 L / min to maintain the oxygen partial pressure < 0.5%, and an anaerobic primary supernatant was obtained; The secondary centrifugal speed was set according to the OD600 value, wherein the secondary centrifugal speed was 12000 rpm when OD600>1.0, and the secondary centrifugal speed was 10000 rpm when OD600≤1.0, and the mixture was centrifuged at 10°C for 10 minutes, and the supernatant was collected to obtain the secondary enrichment solution; According to the ATP value of the secondary enrichment solution compared with the primary filtrate, if the activity loss rate is greater than 8%, 0.1% cysteine-HCl solution is added to compensate, and finally a high-activity supernatant is output.

[0009] Optionally, the method of obtaining the sterilized supernatant by pulse high temperature sterilization according to the microbial load of the highly active supernatant comprises: Calculate the sterilization intensity coefficient based on the flow cytometry test results , where CFU is colony forming unit; Select the initial sterilization temperature according to the K value, wherein when K>3, the initial sterilization temperature is 115°C for 5 minutes, and when K≤3, the initial sterilization temperature is 110°C for 5 minutes, and the heating rate is 3°C / min to obtain a first-level sterilization solution; The primary sterilization solution was cooled to 80°C at a rate of 5°C / min and maintained for 2 minutes to activate the thermostable bacterial autolytic enzyme to obtain a resuscitation treatment solution; The resuscitation treatment solution was raised to 121°C at a rate of 3°C / min and maintained for 8 minutes. 0.2MPa overpressure protection was applied simultaneously, and the sterilized supernatant was output.

[0010] Optionally, according to the redox potential of the sterilized supernatant, a mixed inert gas is filled into the sub-filling bottle to replace the residual oxygen, and the bottle mouth is sealed by laser melting and sealing technology; wherein the oxygen content in the bottle after packaging is less than 0.1%, and an airtight packaging product is obtained, comprising: When the redox potential detection value ORP>-150mV, the mixed inert gas is injected into the sub-filling bottle for 3 cycles, wherein the mixed inert gas includes 85%, 10%, and 5% by volume of , , , after each replacement, stand for 10 seconds to obtain a hypoxic pretreatment bottle; Select the laser wavelength according to the bottle mouth material. Glass corresponds to a laser wavelength of 1064nm, and plastic corresponds to a laser wavelength of 980nm. Set the sealing energy range to 20x (1±5%) J / cm² for laser sealing. After laser sealing, anaerobic glue containing nano-silicon dioxide with a thickness range of 50x (1 ± 5) μm was sprayed on the bottle mouth seam and cured under ultraviolet light for 30 seconds to obtain a reinforced sealed bottle; A micro oxygen sensor is inserted into the bottle to detect the oxygen content <0.1%, then label it and output the airtight packaged product.

[0011] Another embodiment of the present application provides a device for preparing a rumen fluid anaerobic culture medium additive, the device comprising: A processing module is used to inject an anaerobic bacteria activity protective agent into the rumen fluid according to the initial bacterial flora activity value of the collected fresh rumen fluid, and treat it by pulse shaking for 10-15 minutes in a low temperature environment of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5Hz±0.5Hz; A filtration module is used to perform two-stage filtration using a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter in sequence according to the particle distribution characteristics of the pretreated rumen fluid, and a negative pressure of -0.05 MPa or -0.1 MPa is simultaneously applied in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; The centrifugal module is used to perform staged variable speed centrifugation according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria. In the first stage, the centrifugation is performed at 5000-6000 rpm for 5 minutes to remove large particle residues, and in the second stage, the centrifugation is performed at 10000-12000 rpm for 10 minutes to collect the supernatant. The centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain the anaerobic environment to obtain a highly active supernatant. A sterilization module, used for obtaining a sterilized supernatant by pulse high temperature sterilization according to the microbial load of the high-activity supernatant; The packaging module is used to fill the sub-packaging bottle with mixed inert gas to replace the residual oxygen according to the redox potential of the supernatant after sterilization, and seal the bottle mouth with laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

[0012] Yet another embodiment of the present application provides a storage medium, wherein the storage medium stores a computer program, wherein the computer program is configured to execute any of the above methods when running.

[0013] Yet another embodiment of the present application provides an electronic device, including a memory and a processor, wherein the memory stores a computer program, and the processor is configured to run the computer program to execute any of the methods described above.

[0014] Compared with the prior art, the invention provides a method for preparing an additive for anaerobic culture medium of rumen fluid. According to the initial bacterial flora activity value of the collected fresh rumen fluid, an anaerobic activity protective agent is injected into the rumen fluid, and the rumen fluid is treated by pulse shaking for 10-15 minutes in a low temperature environment of 4-6°C to obtain a pretreated rumen fluid; according to the particle distribution characteristics of the pretreated rumen fluid, two-stage filtration is performed to obtain a primary filtrate; according to the viscosity of the primary filtrate and the anaerobic bacteria density distribution, a phased variable speed centrifugation treatment is adopted; according to the microbial load of the high-activity supernatant, a pulse high-temperature sterilization method is adopted to obtain a sterilized supernatant; according to the redox potential of the sterilized supernatant, a mixed inert gas is filled into the sub-filling bottle to replace the residual oxygen, and the bottle mouth is sealed by laser melting sealing technology, so that the anaerobic activity protective agent can be reasonably configured, the operating conditions can be optimized, and the control of the anaerobic environment can be strengthened, so as to achieve efficient protection and cultivation of anaerobic bacteria in the rumen fluid and improve its activity and application effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A hardware structure block diagram of a computer terminal for a method for preparing a rumen fluid anaerobic culture medium additive provided by an embodiment of the present invention; Figure 2 A schematic flow chart of a method for preparing a rumen fluid anaerobic culture medium additive provided by an embodiment of the present invention; Figure 3 A schematic structural diagram of a device for preparing a rumen fluid anaerobic culture medium additive provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0016] The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, but should not be construed as limiting the present invention.

[0017] The embodiment of the present invention firstly provides a method for preparing a rumen fluid anaerobic culture medium additive, which can be applied to electronic devices such as computer terminals, specifically ordinary computers, etc.

[0018] The following describes it in detail by taking running on a computer terminal as an example. Figure 1 The hardware structure block diagram of a computer terminal for a method for preparing a rumen fluid anaerobic culture medium additive provided by an embodiment of the present invention. Figure 1 As shown, the computer device includes a processor, a memory, and a network interface connected via a device bus, wherein the memory may include a non-volatile storage medium and an internal memory.

[0019] The non-volatile storage medium can store an operating device and a computer program. The computer program includes program instructions, and when the program instructions are executed, the processor can execute any method for preparing a rumen fluid anaerobic culture medium additive.

[0020] The processor is used to provide computing and control capabilities and support the operation of the entire computer equipment.

[0021] The internal memory provides an environment for the operation of the computer program in the non-volatile storage medium. When the computer program is executed by the processor, the processor can execute any method for preparing a rumen fluid anaerobic culture medium additive.

[0022] The network interface is used for network communication, such as sending assigned tasks, etc. Those skilled in the art will understand that Figure 1 The structure shown in the figure is only a block diagram of a part of the structure related to the solution of the present application, and does not constitute a limitation on the computer device to which the solution of the present application is applied. The specific computer device may include more or fewer components than those shown in the figure, or combine certain components, or have a different arrangement of components.

[0023] It should be understood that the processor may be a central processing unit (CPU), and the processor may also be other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), field-programmable gate arrays (FPGA) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. Among them, the general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc.

[0024] See also Figure 2 The embodiment of the present invention provides a method for preparing a rumen fluid anaerobic culture medium additive, which may include the following steps: S201, according to the initial bacterial flora activity value of the collected fresh rumen fluid, injecting an anaerobic bacteria activity protective agent into the rumen fluid, and treating it with pulse shaking for 10-15 minutes at a low temperature of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5 Hz±0.5 Hz; Specifically, the initial bacterial flora activity value can be measured using a portable bioluminescence detector according to the real-time ATP bioluminescence value of the collected fresh rumen fluid as activity quantification data; Fresh rumen fluid needs to be tested for activity immediately after collection to prevent inactivation of the bacterial flora. Use a portable ATP bioluminescence detector (such as Hygiena SystemSURE Plus) for rapid determination. The detection principle is based on the firefly luciferase reaction: ATP releases photons after binding to luciferase, and the light intensity is positively correlated with the number of live bacteria. The operation process is as follows: Sample pretreatment: 1 mL of rumen fluid was added to lysis buffer (containing 0.1% Triton X-100) and vortexed for 30 seconds to release intracellular ATP; Reagent loading: inject 100 μL of lysate into the test tube (containing freeze-dried luciferase / luciferin complex) and immediately insert into the detector; Light signal collection: The instrument records relative light units (RLU) within 3 seconds, and the activity value range is usually 200-800 RLU; Data calibration: ATP standard ( ) Calibrate the instrument to ensure the error is <±5%.

[0025] For example, if a test shows an RLU of 650, it indicates that the bacterial activity is high and the amount of protective agent needs to be reduced to avoid over-inhibition. This step should be completed within 10 minutes after collection to prevent ATP degradation from underestimating activity.

[0026] According to the activity quantification data, 0.5 mL of protective agent was added for every 100 RLU, and a succinate buffer containing 10 mmol / L reduced glutathione was injected into the rumen fluid, and the pH value of the succinate buffer was 6.8±0.2 to obtain a protective agent mixture; The preparation and addition of protective agents must strictly follow the proportion to balance bacterial flora protection and metabolic inhibition: Buffer preparation: Dissolve 5.4 g succinic acid (C4H6O4) in 800 mL ultrapure water, adjust to pH 6.8 with 1 mol / L NaOH, and make up to 1 L. After autoclaving (121°C, 15 minutes), cool to 4°C, add reduced glutathione (GSH) to a final concentration of 10 mmol / L (i.e., 3.07 g / L), and store in the dark; Addition amount calculation: If the detected activity value is 600RLU, the amount of protective agent added = 600 / 100×0.5mL = 3mL / 100mL rumen fluid; For example, 15 mL of protective agent should be added to 500 mL of rumen fluid; Mixed Operations: Precool the protective agent to 4°C and slowly inject the rumen fluid along the container wall to avoid violent foaming; Magnetic stirring (200 rpm) was performed for 5 min to ensure uniform mixing, during which the temperature was monitored to be ≤6 °C.

[0027] Critical Control Points: pH stability: pH after mixing needs to be maintained at 6.6-7.0. If it is out of range, fine-tune with 0.1mol / L HCl / NaOH; GSH activity protection: Reduced glutathione can scavenge free radicals (such as ) Maintain the anaerobic redox potential (ORP≤-250mV).

[0028] The protective agent mixture was placed in a 4°C pulse oscillator, and the oscillation frequency was dynamically adjusted according to the initial bacterial flora activity value. When the activity value was >500RLU, the oscillation frequency was 5.5Hz, and when the activity value was ≤500RLU, the oscillation frequency was 4.5Hz. The oscillation treatment was performed for 12 minutes ± 30 seconds to obtain the pretreated rumen fluid.

[0029] Pulse oscillation promotes the penetration of protective agents through mechanical stress while avoiding shear damage to bacteria: Device Setup: Use a programmable pulse oscillator (such as IKA HS 501 digital) with an amplitude of 5 mm; The temperature control module maintains the cavity temperature at 4-6°C; Frequency dynamic adjustment: High activity samples (RLU>500): oscillation frequency 5.5 Hz (330 times / min), pulse width 50 ms; Low activity samples (RLU ≤ 500): oscillation frequency 4.5 Hz (270 times / min), pulse width 70 ms; Process monitoring: Take samples every 2 minutes to test the ORP value and ensure that it is ≤-200mV (if the ORP rises, stop the oscillation and add 0.1% cysteine); After shaking, the mixture was immediately cooled to 2°C in an ice water bath to inhibit residual metabolic activity.

[0030] Effect verification: Bacterial colony survival rate: assessed by viable bacteria count (such as MPN method), with a target of ≥90%; Retention of metabolic activity: short-chain fatty acid (SCFA) production was measured, with a target of ≥85% of initial value.

[0031] For example, the number of viable bacteria in a batch of rumen fluid after treatment changed from CFU / mL decreased to CFU / mL (survival rate 92%), and propionic acid production was maintained from 45mol / L to 39mol / L (retention 87%), meeting the subsequent processing requirements.

[0032] S202, according to the particle distribution characteristics of the pretreated rumen fluid, a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter are sequentially used for two-stage filtration, and a negative pressure of -0.05 MPa or -0.1 MPa is simultaneously applied in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; Specifically, the median particle size D50 value of the particles can be extracted according to the test results of the laser scattering particle size analyzer of the pretreated rumen fluid; Laser scattering particle size analyzer (such as Malvern Mastersizer 3000) is used to determine the particle distribution of pre-treated rumen fluid. The instrument irradiates the sample with a red and blue dual-wavelength laser (red light 632.8nm, blue light 470nm) and detects the distribution of particle scattered light intensity. The extraction process of D50 value (i.e., the particle size of 50% of the volume is smaller than this value) is as follows: Sample preparation: Take 5 mL of pretreated rumen fluid and dilute it with deionized water to an occlusion of 10%-15% to avoid multiple scattering interference.

[0033] Instrument calibration: The instrument was calibrated using standard polystyrene latex particles (nominal particle size 100x (1±5%) nm) to ensure an error of <±1%.

[0034] Measurement parameter settings: Pump speed: 2000rpm, to ensure uniform suspension; Measurement time: 30 seconds, repeated 3 times for each sample; Refractive index settings: particle refractive index 1.52 (similar to cellulose particles), dispersion medium (water) refractive index 1.33.

[0035] Data analysis: The built-in software of the instrument (such as Mastersizer 3000 software) automatically fits the Mie scattering theory model and outputs the D10, D50, and D90 values. For example, in a certain measurement, D50=180μm and D90=250μm were obtained, indicating that most of the particles are concentrated below 180μm.

[0036] Select the coarse filter mesh number according to the D50 value, wherein, when D50>200μm, use 60 mesh for the coarse filter, and when D50≤200μm, use 100 mesh for the coarse filter, perform the first stage filtration, and obtain the coarse filtrate; The corresponding relationship between the number of coarse filter mesh and the pore size is: 60 mesh: pore size 250μm (in accordance with ISO 3310-1 standard); 100 mesh: pore size 150μm.

[0037] Dynamically select the filter based on the D50 value: Filter installation: Install a nylon filter of the selected mesh size (such as Millipore NY6004700) into a stainless steel filter holder (100 mm in diameter) and seal it with a silicone gasket.

[0038] Filter operation: When D50=220μm (>200μm), select 60 mesh filter; When D50=180μm (≤200μm), select 100 mesh filter.

[0039] Filtration control: Gravity filtration is the main method, and the initial flow rate is controlled at 15-20 mL / min. For example, it takes about 25 minutes for 500 mL of pretreated rumen fluid to pass through a 60-mesh filter, and about 480 mL of coarse filtrate is collected, with a loss rate of 4%.

[0040] According to the feedback of the coarse filtrate flow rate, the negative pressure value of the 200-300 mesh ultrafine filter is dynamically adjusted by the PID algorithm, wherein the negative pressure value is set to -0.1MPa when the flow rate is less than 10mL / min, and the negative pressure value is set to -0.05MPa when the flow rate is ≥10mL / min; PID (Proportional-Integral-Derivative) control algorithm is used to maintain a stable filtration flow rate: Sensor Configuration: A flow meter (such as Cole-Parmer EW-32707-10) monitors flow rate in real time; The vacuum gauge (range -0.1~0MPa, accuracy ±0.5%) monitors the negative pressure.

[0041] PID parameter settings: Proportional coefficient Kp = 2.5, integral time Ti = 30 seconds, differential time Td = 5 seconds; Set the flow rate target value to 10 mL / min.

[0042] Control logic: When the measured flow rate = 8mL / min (<10mL / min), the PID output increases the negative pressure to -0.1MPa; When the flow rate increases to 12 mL / min (≥10 mL / min), PID reduces the negative pressure to -0.05 MPa.

[0043] Actuator: Rapid response to pressure changes through electric vacuum control valve (such as SMC ITV2050-212BL), response time <1 second.

[0044] According to the D50 value, the pore size of the ultrafine filter is adjusted in real time, wherein the pore size range is 1.3×D50×(1±5%), and the filter is cleaned with ultrasound every 2 minutes during the filtration process of the ultrafine filter to obtain an ultrafine filtrate; The ultra-fine filter uses an adjustable pore size ceramic membrane (such as Whatman Anodisc 47), and the pore size is adjusted by a mechanical knob: Aperture calculation: If D50 = 180 μm, then the target aperture = 1.3 × 180 = 234 μm, with an allowable error of ±5% (222.3 ~ 245.7 μm); Select the closest standard pore size of 250 μm (corresponding to 200 mesh).

[0045] Ultrasonic cleaning: Cleaning parameters: frequency 40kHz, power 50W; Cleaning cycle: automatically starts every 2 minutes of filtration and lasts for 10 seconds; Cleaning effect: filter flux recovery rate>95%.

[0046] Quality control: Check the integrity of the filter after each batch of filtration (such as bubble point test, no bubbles generated under a pressure of 0.15MPa).

[0047] According to the bacterial activity loss rate of the ultrafine filtrate obtained by comparing the pretreated rumen fluid, if the bacterial activity loss rate is greater than 10%, the process returns to the step of dynamically adjusting the negative pressure value of the 200-300 mesh ultrafine filter through the PID algorithm according to the feedback of the coarse filtrate flow rate until the bacterial activity loss rate is ≤10%, and the primary filtrate is output.

[0048] Calculation formula for bacterial flora activity loss rate (L):

[0049] ATP detection: Promega BacTiter-Glo kit was used to detect fluorescence intensity (RLU) and convert it into ATP (adenosine triphosphate) concentration.

[0050] Feedback Adjustment: For example, at the 0th cycle (i.e. at the beginning), the current coarse filtrate flow rate is assumed to be 8 mL / min, the negative pressure value is adjusted to -0.1 MPa, and L is calculated to be 18% (>10%), then return to execute the first cycle, and the current coarse filtrate flow rate feedback is measured and assumed to be 12 mL / min, then the negative pressure value is adjusted to -0.05 MPa, and after re-filtration, L is calculated to be 9% (≤10%), and the cycle is terminated.

[0051] Data recording: Record the flow rate, negative pressure, and activity loss rate for each cycle to form an optimized parameter table (see Table 1).

[0052] Table 1 Filter parameter optimization example

[0053] By dynamically adjusting the filter mesh aperture and negative pressure gradient, combined with ultrasonic cleaning and activity feedback control, the bacterial activity loss rate of the primary filtrate is reduced from 20%-25% in traditional methods to ≤10%, the particle removal efficiency is increased by 30%, and the filtration time is shortened to less than 40 minutes, significantly improving preparation efficiency and product quality.

[0054] S203, according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria, a staged variable speed centrifugation treatment is performed, wherein the first stage is centrifuged at 5000-6000 rpm for 5 minutes to remove large particle residues, and the second stage is centrifuged at 10000-12000 rpm for 10 minutes to collect the supernatant; wherein the centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain an anaerobic environment, so as to obtain a highly active supernatant; Specifically, the viscosity-bacteria density relationship formula η=0.02×(OD600)²+1.5 can be established based on the rotational viscometer detection value η and the optical density value OD600 of the primary filtrate; The viscosity (η) of the primary filtrate is measured by a rotational viscometer (such as Brookfield DV2T) at 8°C, with a LV-3 rotor, a speed of 60 rpm, a measurement time of 30 seconds, and three averages are taken. The optical density (OD600) is measured by a spectrophotometer (such as Thermo Scientific NanoDrop 2000), and the filtrate is diluted to an OD600 value in the range of 0.1 to 1.0 to ensure a linear response.

[0055] The viscosity-bacteria density relationship modeling is based on historical experimental data fitting. For example, when OD600=0.5, η=0.02×(0.5)²+1.5=1.505mPa·s; when OD600=1.0, η=0.02×1.0+1.5=1.52mPa·s. This formula fits 100 sets of sample data (R²=0.93) by the least squares method, which can reflect the nonlinear effect of bacterial concentration on filtrate viscosity. In actual operation, if OD600=1.2 is detected, η=0.02×1.44+1.5≈1.53mPa·s, and the system automatically matches the subsequent centrifugation parameters.

[0056] The first-stage centrifugal speed is set according to the η value, wherein, when η>20mPa·s, the first-stage centrifugal speed is 6000rpm, and when η≤20mPa·s, the first-stage centrifugal speed is 5000rpm, centrifuge at 8°C for 5 minutes, remove large particle residues, and obtain a first-stage supernatant; Precool the centrifuge (such as Eppendorf 5910R) to 8°C and load 50mL centrifuge tubes with 40mL primary filtrate per tube. Dynamically set the speed according to the η value: High viscosity mode (η>20mPa·s): 6000rpm, centrifugal force about 4020×g, suitable for viscous filtrate containing fiber residue; Low viscosity mode (η≤20mPa·s): 5000rpm, centrifugal force about 2800×g, suitable for thin filtrates with uniform bacterial dispersion.

[0057] During the centrifugation process, the chamber temperature is monitored in real time and maintained at 8-10°C by compressor cooling. After centrifugation, the supernatant is carefully aspirated with a sterile pipette to avoid disturbing the bottom sediment (large particles such as plant fiber and undigested feed fragments). For example, after a certain centrifugation, the sediment volume accounts for about 15%, and the transmittance of the supernatant is increased to 85%.

[0058] Nitrogen was introduced into the centrifuge chamber at a flow rate of 5 L / min to maintain the oxygen partial pressure < 0.5%, and an anaerobic primary supernatant was obtained; The nitrogen inlet system is controlled by a mass flow controller (MFC, such as Alicat MC-5SLPM), and high-purity nitrogen (purity ≥ 99.999%) is injected at a flow rate of 5L / min through a dedicated interface on the centrifuge cover. The oxygen partial pressure is monitored in real time using a fiber optic oxygen sensor (such as PreSens Fibox 4), and the probe is inserted into the cavity exhaust port.

[0059] Anaerobic environment maintenance strategy: Pre-rinsing: Pass nitrogen for 2 minutes before centrifugation to replace the air in the chamber; Dynamic adjustment: If the oxygen partial pressure is > 0.5%, an alarm is triggered and the nitrogen flow rate is increased to 8L / min for 30 seconds; Seal verification: After centrifugation, test the redox potential (ORP) of the supernatant, which is required to be ORP < -200mV (typical anaerobic conditions).

[0060] For example, in one run, the initial oxygen partial pressure was 21% (air), which dropped to 0.3% after nitrogen flushing, and maintained at 0.2%~0.4% during centrifugation. The final supernatant ORP = -250mV, meeting the anaerobic requirements.

[0061] The secondary centrifugal speed was set according to the OD600 value, wherein the secondary centrifugal speed was 12000 rpm when OD600>1.0, and the secondary centrifugal speed was 10000 rpm when OD600≤1.0, and the mixture was centrifuged at 10°C for 10 minutes, and the supernatant was collected to obtain the secondary enrichment solution; Before the second stage centrifugation, transfer the first stage supernatant to a new centrifuge tube and precool it to 10°C. The speed selection depends on the bacterial concentration: High bacterial density (OD600>1.0): 12000rpm (centrifugal force about 13500×g), suitable for concentrating Firmicutes and other easy-to-sediment bacteria; Low bacterial density (OD600 ≤ 1.0): 10000rpm (centrifugal force about 9400×g), suitable for dispersed Bacteroidetes flora.

[0062] After centrifugation, discard the supernatant and keep the bacterial precipitate at the bottom (about 2~5mL). For example, when OD600=1.5, the precipitate after centrifugation at 12000rpm is dense milky white, with a wet weight of about 0.5g / mL; when OD600=0.8, the precipitate after centrifugation at 10000rpm is loose, with a wet weight of about 0.3g / mL.

[0063] According to the ATP value of the secondary enrichment solution compared with the primary filtrate, if the activity loss rate is greater than 8%, 0.1% cysteine-HCl solution is added to compensate, and finally a high-activity supernatant is output.

[0064] ATP activity is detected by bioluminescence (such as Promega BacTiter-Glo). Take 100 μL of sample and mix it with an equal volume of detection reagent, and measure the relative light unit (RLU) using a luminometer (such as Tecan Infinite 200 Pro). The activity loss rate is calculated by:

[0065] Compensatory measures: Loss rate>8%: add 0.1% cysteine-HCl solution (prepared immediately, pH 6.0), mix at a ratio of 1 mL solution / 10 mL enrichment solution, and vortex for 30 seconds; Loss rate ≤ 8%: directly divide and store.

[0066] For example, in a certain test, the primary filtrate RLU = 5000, the secondary enrichment solution RLU = 4300, the loss rate = (5000-4300) / 5000 = 14%, and 0.5mL of cysteine ​​solution needs to be added to 5mL of enrichment solution. After the addition, the RLU is restored to 4800 (the loss rate is reduced to 4%). The final supernatant is stored at -80℃, and the activity retention rate is >90% (within 3 months).

[0067] S204, according to the microbial load of the high-activity supernatant, a pulse high-temperature sterilization method is used to obtain a sterilized supernatant; Specifically, the sterilization intensity coefficient can be calculated based on the flow cytometry test results of the high-activity supernatant. , where CFU is colony forming unit; The microbial load of the highly active supernatant was quantitatively detected by flow cytometry (FCM). First, 1 mL of the supernatant sample was stained with SYBR Green I fluorescent dye for 15 minutes (concentration 1:1000) to allow bacterial DNA to bind to the fluorescent dye. The fluorescence signal was detected by a flow cytometer (such as BD Accuri C6), with an excitation wavelength of 488 nm, an emission wavelength of 530 nm, and a threshold of 5000 particles / μL to exclude background noise. The instrument automatically counted the number of fluorescent positive particles (i.e., the number of viable bacteria) and recorded it as CFU (Colony Forming Units).

[0068] Calculation of sterilization intensity coefficient K: Data normalization: Divide the detected CFU value by (i.e., the number of colonies per milliliter is converted to a value in millions). For example, if , then it is 50 after standardization.

[0069] Logarithmic transformation: Take the base 10 logarithm of the standardized value, that is, .

[0070] Threshold determination: Sterilization intensity levels are divided according to K value: K>3: indicates extremely high microbial load , high-intensity sterilization is required; K≤3: indicates medium microbial load , using standard sterilization strength.

[0071] For example, if the CFU of a batch of supernatant is ,but , which belongs to the category of K≤3, and the initial sterilization temperature of 110℃ is selected.

[0072] Select the initial sterilization temperature according to the K value, wherein when K>3, the initial sterilization temperature is 115°C for 5 minutes, and when K≤3, the initial sterilization temperature is 110°C for 5 minutes, and the heating rate is 3°C / min to obtain a first-level sterilization solution; The sterilization process is carried out in a program-controlled high-pressure steam autoclave (such as Hirayama HV-110), and the specific operations are as follows: Temperature setting: K>3: Set the initial sterilization temperature to 115℃ and the holding time to 5 minutes; K≤3: Set the initial sterilization temperature to 110°C and the holding time to 5 minutes.

[0073] Temperature control: The autoclave heats up from room temperature (25°C) to the target temperature at a rate of 3°C / min. For example, it takes 30 minutes to heat from 25°C to 115°C ((115-25) / 3=30). The heating power is adjusted in real time using the PID (proportional-integral-differential) algorithm.

[0074] Insulation stage: After reaching the target temperature, the timer starts, and the temperature is recorded every 30 seconds to ensure stability. For example, if the temperature is maintained at 115°C for 5 minutes, the temperature fluctuation error during the insulation period is ±0.3°C, that is, it is maintained at 114.7°C-115.3°C.

[0075] Sterilization effect verification: Take a sample after sterilization for plate count verification. For example, if the initial CFU is / mL (K=2.698), after sterilization at 110℃, the CFU decreased to / mL, the sterilization efficiency reaches 99.9998%.

[0076] The primary sterilization solution was cooled to 80°C at a rate of 5°C / min and maintained for 2 minutes to activate the thermostable bacterial autolytic enzyme to obtain a resuscitation treatment solution; The cooling process is achieved through the rapid cooling system of the sterilizer. The specific steps are as follows: Cooling rate control: After sterilization, start the water cooling system immediately and cool down at a rate of 5℃ / min. For example, it takes 7 minutes to cool down from 115℃ to 80℃ ((115-80) / 5=7).

[0077] The temperature gradient is monitored in real time during the cooling process to avoid the glass container from breaking due to excessive speed.

[0078] Temperature maintenance stage: After reaching 80℃, maintain it for 2 minutes. This stage activates the autolysin of heat-resistant bacteria (such as Bacillus). Autolysin is most active at 80℃ and can decompose the cell wall of bacteria and promote the lysis of residual bacteria.

[0079] During the maintenance period, nitrogen gas (flow rate 2 L / min) was introduced to prevent oxidation and ensure an anaerobic environment.

[0080] Biological Mechanisms: Activation of autolytic enzymes: The spores of heat-resistant bacteria may survive high-temperature sterilization, but the autolytic enzymes of their vegetative cells are activated at 80°C, causing autolysis. For example, the activity of the autolytic enzyme Lyta of Bacillus subtilis increases 3 times at 80°C, accelerating the rupture of the bacteria.

[0081] The resuscitation treatment solution was raised to 121°C at a rate of 3°C / min and maintained for 8 minutes. 0.2MPa overpressure protection was applied simultaneously, and the sterilized supernatant was output.

[0082] The final sterilization stage uses overpressure steam sterilization technology to ensure that residual microorganisms are completely inactivated: Secondary temperature rise control: It took 13.67 minutes to increase the temperature from 80°C to 121°C at a rate of 3°C / min ((121-80) / 3≈13.67).

[0083] The pressure increases simultaneously during the heating process to prevent the liquid from boiling (the boiling point increases with increasing pressure).

[0084] Overvoltage protection: At 121°C, an overpressure of 0.2MPa (absolute pressure 0.3MPa) is applied to prevent the container from bursting. The pressure is dynamically adjusted by the mechanical pressure relief valve of the sterilizer, with a fluctuation range of ±0.02MPa.

[0085] Insulation stage: Maintaining at 121°C for 8 minutes can completely inactivate all microorganisms (including heat-resistant spores). For example, the D121 value (90% inactivation time) of Clostridium botulinum spores is 0.2 minutes, and 8 minutes of sterilization can achieve a 40-log inactivation effect.

[0086] Quality Control: Biological indicator verification: Place a biological indicator containing thermophilic Bacillus stearothermophilus (ATCC 7953) in the autoclave. If no growth is observed after 48 hours of incubation, sterilization is confirmed to be qualified.

[0087] Physical monitoring: Sterilization process data (temperature, pressure, time) are automatically recorded and reports are generated to meet GMP (Good Manufacturing Practice) requirements.

[0088] S205, according to the redox potential of the supernatant after sterilization, a mixed inert gas is filled into the sub-filling bottle to replace the residual oxygen, and the bottle mouth is sealed by laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

[0089] Specifically, when the redox potential detection value ORP of the sterilized supernatant is greater than -150mV, a mixed inert gas can be injected into the sub-filling bottle for 3 cycles of replacement, wherein the mixed inert gas includes 85%, 10%, and 5% by volume of , , , after each replacement, stand for 10 seconds to obtain a hypoxic pretreatment bottle; Oxidation reduction potential (ORP) detection is a key indicator for measuring the residual oxygen content in liquids. Use a high-precision ORP electrode (range ±2000mV, accuracy ±1mV) to insert into the sterilized supernatant and monitor its potential value in real time. When the ORP value is greater than -150mV (indicating the presence of significant residual oxygen), start the inert gas replacement procedure.

[0090] Mixed inert gas configuration: Gas ratio: Nitrogen ( ) 85% as the main inert gas, carbon dioxide ( ) 10% maintains the slightly acidic environment required for anaerobic metabolism, hydrogen ( ) 5% as a reducing agent to further eliminate trace oxygen; Gas flow control: Precisely adjusted by mass flow meter (MFC), the total flow rate is set to 5L / min to ensure replacement efficiency.

[0091] Replacement process: First replacement: inject the mixed gas into the sub-bottle for 5 seconds, then let it stand for 10 seconds to reduce the oxygen concentration in the bottle through gas diffusion; Second replacement: Repeat the gas injection, but increase the flow rate to 6L / min to accelerate the replacement kinetics; The third replacement: the flow rate is adjusted back to 5L / min and the standing time is extended to 15 seconds to ensure uniform gas distribution.

[0092] Quality Control: After each replacement, the target oxygen content is less than 0.5% by random inspection using a micro oxygen sensor (limit detection concentration 0.01%); If the oxygen content is still greater than 0.3% after three replacements, perform an additional replacement and let it stand for 20 seconds.

[0093] For example, after a certain replacement, the oxygen content in the bottle dropped from the initial 1.2% to 0.08%, meeting the hypoxia pretreatment requirements.

[0094] Select the laser wavelength according to the bottle mouth material. Glass corresponds to a laser wavelength of 1064nm, and plastic corresponds to a laser wavelength of 980nm. Set the sealing energy range to 20x (1±5%) J / cm² for laser sealing. Laser sealing technology uses a high-energy laser beam to instantly heat the bottle mouth material to achieve sealing. The wavelength and energy parameters need to be optimized according to the material characteristics: Material adaptation strategy: Glass bottle mouth: Using Nd:YAG solid laser (wavelength 1064nm), its photon energy (1.17eV) can effectively penetrate the glass surface and induce deep melting; Plastic bottle mouth: semiconductor laser (wavelength 980nm) is used because plastic (such as PET) has a higher absorption rate in the near-infrared band, avoiding surface carbonization.

[0095] Energy parameter setting: Energy density: 20J / cm² is the basic value, which is adjusted dynamically by ±5% according to the thickness of the bottle mouth. For example, when the thickness of the glass bottle mouth is 2mm, 21J / cm² is used, and when it is 1.5mm, 19J / cm² is used; Pulse frequency: 200Hz, single pulse width 10ns, ensuring concentrated heat transfer to avoid thermal damage.

[0096] Sealing process control: Pre-alignment: The bottle mouth seam is positioned by CCD visual system with an accuracy of ±0.1mm; Laser scanning path: The spiral track shrinks from outside to inside, covering a width of 3mm to ensure the continuity of the sealing line; Real-time monitoring: Infrared thermal imager monitors the temperature of the sealing area. The target temperature of the glass bottle mouth is 850℃±20℃, and the target temperature of the plastic bottle mouth is 220℃±10℃.

[0097] For example, after a glass bottle mouth is sealed by 1064nm laser, the sealing line width is 0.5mm, and the helium mass spectrometer leak rate is , reaching ultra-high airtightness standards.

[0098] After laser sealing, anaerobic glue containing nano-silicon dioxide with a thickness range of 50x (1 ± 5) μm was sprayed on the bottle mouth seam and cured under ultraviolet light for 30 seconds to obtain a reinforced sealed bottle; Nano-silica modified anaerobic adhesive (model Anaerlok-S30) forms a high-strength sealing layer through free radical polymerization initiated by ultraviolet light: Spraying process parameters: Colloid ingredients: 30% nano (particle size 50nm), 60% methacrylate monomer, 10% photoinitiator (TPO); Spraying thickness: The thickness of the adhesive layer is controlled to be 50μm±5 by a piezoelectric nozzle (aperture 100μm), and the spraying speed is 10cm / s; Spraying path: Spray parallel to the outer side of the sealing line 0.2mm, with a coverage width of 1.2mm.

[0099] UV curing process: Light intensity: 365nm UV-LED array, radiation intensity 200mW / cm²; Exposure time: 30 seconds, two-stage curing - high intensity (300mW / cm²) for the first 10 seconds to initiate polymerization, and then reduced to 150mW / cm² for the last 20 seconds to complete crosslinking; Temperature control: The air cooling system maintains the temperature of the curing area below 40°C to prevent micro cracks caused by thermal stress.

[0100] Performance Verification: After curing, the hardness of the adhesive layer reaches Shore D 85, and the shear strength is >15MPa; Salt spray test (5% NaCl, 35℃) for 240 hours without corrosion penetration.

[0101] For example, after a batch of treatments, the bottle mouths were tested for leakage and the rubber layer did not fall off after pressure testing (0.3MPa for 1 hour).

[0102] A micro oxygen sensor is inserted into the bottle to detect the oxygen content <0.1%, then label it and output the airtight packaged product.

[0103] Oxygen content detection is the final verification link of packaging quality. Fiber optic oxygen sensor (FOXY-OR125) is used for non-destructive testing: Testing process: Sensor calibration: Use standard gas (0% , 0.1% , 0.5% )Three-point calibration to ensure the range 0-1% accuracy ±0.01%; Sampling method: insert the sensor probe into the bottle through the reserved detection hole (aperture 0.5mm) to a depth of 1cm above the liquid surface; Data collection: Monitor continuously for 30 seconds, and take the average value of the last 10 seconds after stabilization as the final oxygen content.

[0104] Qualification determination and labeling: Qualification standard: oxygen content <0.1% (volume fraction); Abnormal handling: If the oxygen content is ≥0.1%, start the rework process - re-inflate, replace and re-seal; Automatic labeling: Use thermal transfer labeling machine to print batch number and expiration date. The attachment position is 5mm from the bottle mouth, and the positioning accuracy is ±0.2mm.

[0105] Data traceability system: Each bottle generates a unique QR code, which is associated with the test data (ORP value, oxygen content, sealing parameters, etc.); The database storage period is ≥ 5 years, supporting reverse tracing of production batches and process parameters.

[0106] For example, 2,000 bottles of finished products are produced on a certain day, the sampling rate is 5% (100 bottles), the average oxygen content is 0.06%, the maximum value is 0.09%, and the qualified rate is 100%.

[0107] It can be seen that according to the initial bacterial flora activity value of the collected fresh rumen fluid, the anaerobic activity protective agent is injected into the rumen fluid, and the pretreated rumen fluid is treated with pulsed oscillation for 10-15 minutes at a low temperature of 4-6°C to obtain the pretreated rumen fluid; according to the particle distribution characteristics of the pretreated rumen fluid, two-stage filtration is performed to obtain the primary filtrate; according to the viscosity of the primary filtrate and the anaerobic density distribution, a staged variable speed centrifugation treatment is adopted; according to the microbial load of the highly active supernatant, a pulsed high temperature sterilization method is adopted to obtain the sterilized supernatant; according to the redox potential of the sterilized supernatant, a mixed inert gas is filled into the sub-bottle to replace the residual oxygen, and the bottle mouth is sealed with laser melting technology, so that the anaerobic activity protective agent can be reasonably configured, the operating conditions can be optimized, and the control of the anaerobic environment can be strengthened, so as to achieve efficient protection and cultivation of anaerobic bacteria in rumen fluid and improve its activity and application effect.

[0108] Another embodiment of the present invention provides a device for preparing a rumen fluid anaerobic culture medium additive, see Figure 3 , the device may include: The processing module 301 is used to inject an anaerobic bacteria activity protective agent into the rumen fluid according to the initial bacterial flora activity value of the collected fresh rumen fluid, and treat it by pulse shaking for 10-15 minutes in a low temperature environment of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5Hz±0.5Hz; The filtering module 302 is used to perform two-stage filtration using a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter in sequence according to the particle distribution characteristics of the pre-treated rumen fluid, and simultaneously apply a negative pressure of -0.05 MPa or -0.1 MPa in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; The centrifugal module 303 is used to perform staged variable speed centrifugation according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria. In the first stage, the centrifugation is performed at 5000-6000 rpm for 5 minutes to remove large particle residues, and in the second stage, the centrifugation is performed at 10000-12000 rpm for 10 minutes to collect the supernatant. The centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain the anaerobic environment to obtain a highly active supernatant. The sterilization module 304 is used to obtain a sterilized supernatant by using a pulse high temperature sterilization method according to the microbial load of the high-activity supernatant; The packaging module 305 is used to fill the sub-packaging bottle with mixed inert gas to replace the residual oxygen according to the redox potential of the supernatant after sterilization, and seal the bottle mouth using laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

[0109] It can be seen that according to the initial bacterial flora activity value of the collected fresh rumen fluid, the anaerobic activity protective agent is injected into the rumen fluid, and the pretreated rumen fluid is treated with pulsed oscillation for 10-15 minutes at a low temperature of 4-6°C to obtain the pretreated rumen fluid; according to the particle distribution characteristics of the pretreated rumen fluid, two-stage filtration is performed to obtain the primary filtrate; according to the viscosity of the primary filtrate and the anaerobic density distribution, a staged variable speed centrifugation treatment is adopted; according to the microbial load of the highly active supernatant, a pulsed high temperature sterilization method is adopted to obtain the sterilized supernatant; according to the redox potential of the sterilized supernatant, a mixed inert gas is filled into the sub-bottle to replace the residual oxygen, and the bottle mouth is sealed with laser melting technology, so that the anaerobic activity protective agent can be reasonably configured, the operating conditions can be optimized, and the control of the anaerobic environment can be strengthened, so as to achieve efficient protection and cultivation of anaerobic bacteria in rumen fluid and improve its activity and application effect.

[0110] An embodiment of the present invention further provides a storage medium, in which a computer program is stored, wherein the computer program is configured to execute the steps of any of the above method embodiments when running.

[0111] Specifically, in this embodiment, the above storage medium may be configured to store a computer program for performing the following steps: S201, according to the initial bacterial flora activity value of the collected fresh rumen fluid, injecting an anaerobic bacteria activity protective agent into the rumen fluid, and treating it with pulse shaking for 10-15 minutes at a low temperature of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5 Hz±0.5 Hz; S202, according to the particle distribution characteristics of the pretreated rumen fluid, a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter are sequentially used for two-stage filtration, and a negative pressure of -0.05 MPa or -0.1 MPa is simultaneously applied in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; S203, according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria, a staged variable speed centrifugation treatment is performed, wherein the first stage is centrifuged at 5000-6000 rpm for 5 minutes to remove large particle residues, and the second stage is centrifuged at 10000-12000 rpm for 10 minutes to collect the supernatant; wherein the centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain an anaerobic environment, so as to obtain a highly active supernatant; S204, according to the microbial load of the high-activity supernatant, a pulse high-temperature sterilization method is used to obtain a sterilized supernatant; S205, according to the redox potential of the supernatant after sterilization, a mixed inert gas is filled into the sub-filling bottle to replace the residual oxygen, and the bottle mouth is sealed by laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

[0112] It can be seen that according to the initial bacterial flora activity value of the collected fresh rumen fluid, the anaerobic activity protective agent is injected into the rumen fluid, and the pretreated rumen fluid is treated with pulsed oscillation for 10-15 minutes at a low temperature of 4-6°C to obtain the pretreated rumen fluid; according to the particle distribution characteristics of the pretreated rumen fluid, two-stage filtration is performed to obtain the primary filtrate; according to the viscosity of the primary filtrate and the anaerobic density distribution, a staged variable speed centrifugation treatment is adopted; according to the microbial load of the highly active supernatant, a pulsed high temperature sterilization method is adopted to obtain the sterilized supernatant; according to the redox potential of the sterilized supernatant, a mixed inert gas is filled into the sub-bottle to replace the residual oxygen, and the bottle mouth is sealed with laser melting technology, so that the anaerobic activity protective agent can be reasonably configured, the operating conditions can be optimized, and the control of the anaerobic environment can be strengthened, so as to achieve efficient protection and cultivation of anaerobic bacteria in rumen fluid and improve its activity and application effect.

[0113] An embodiment of the present invention further provides an electronic device, including a memory and a processor, wherein the memory stores a computer program, and the processor is configured to run the computer program to execute the steps in any one of the above method embodiments.

[0114] Specifically, the electronic device may further include a transmission device and an input / output device, wherein the transmission device is connected to the processor, and the input / output device is connected to the processor.

[0115] Specifically, in this embodiment, the processor may be configured to perform the following steps through a computer program: S201, according to the initial bacterial flora activity value of the collected fresh rumen fluid, injecting an anaerobic bacteria activity protective agent into the rumen fluid, and treating it with pulse shaking for 10-15 minutes at a low temperature of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5 Hz±0.5 Hz; S202, according to the particle distribution characteristics of the pretreated rumen fluid, a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter are sequentially used for two-stage filtration, and a negative pressure of -0.05 MPa or -0.1 MPa is simultaneously applied in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; S203, according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria, a staged variable speed centrifugation treatment is performed, wherein the first stage is centrifuged at 5000-6000 rpm for 5 minutes to remove large particle residues, and the second stage is centrifuged at 10000-12000 rpm for 10 minutes to collect the supernatant; wherein the centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain an anaerobic environment, so as to obtain a highly active supernatant; S204, according to the microbial load of the high-activity supernatant, a pulse high-temperature sterilization method is used to obtain a sterilized supernatant; S205, according to the redox potential of the supernatant after sterilization, a mixed inert gas is filled into the sub-filling bottle to replace the residual oxygen, and the bottle mouth is sealed by laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

[0116] It can be seen that according to the initial bacterial flora activity value of the collected fresh rumen fluid, the anaerobic activity protective agent is injected into the rumen fluid, and the pretreated rumen fluid is treated with pulsed oscillation for 10-15 minutes at a low temperature of 4-6°C to obtain the pretreated rumen fluid; according to the particle distribution characteristics of the pretreated rumen fluid, two-stage filtration is performed to obtain the primary filtrate; according to the viscosity of the primary filtrate and the anaerobic density distribution, a staged variable speed centrifugation treatment is adopted; according to the microbial load of the highly active supernatant, a pulsed high temperature sterilization method is adopted to obtain the sterilized supernatant; according to the redox potential of the sterilized supernatant, a mixed inert gas is filled into the sub-bottle to replace the residual oxygen, and the bottle mouth is sealed with laser melting technology, so that the anaerobic activity protective agent can be reasonably configured, the operating conditions can be optimized, and the control of the anaerobic environment can be strengthened, so as to achieve efficient protection and cultivation of anaerobic bacteria in rumen fluid and improve its activity and application effect.

[0117] The above describes in detail the structure, features and effects of the present invention based on the embodiments shown in the drawings. The above is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown in the drawings. Any changes made according to the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the description and drawings, should be within the protection scope of the present invention.

Claims

1. A method for preparing a rumen fluid anaerobic culture medium additive, characterized in that: The method comprises: According to the initial bacterial flora activity value of the collected fresh rumen fluid, an anaerobic bacteria active protective agent is injected into the rumen fluid, and the rumen fluid is treated by pulse shaking for 10-15 minutes at a low temperature of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5Hz±0.5Hz; According to the particle distribution characteristics of the pretreated rumen fluid, a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter are used in turn for two-stage filtration, and a negative pressure of -0.05MPa or -0.1MPa is simultaneously applied in the ultrafine filtration stage to obtain the primary filtrate; wherein, the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; According to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria, a phased variable speed centrifugation treatment is adopted. In the first phase, large particles are removed by centrifugation at 5000-6000 rpm for 5 minutes, and in the second phase, the supernatant is collected by centrifugation at 10000-12000 rpm for 10 minutes. The centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain an anaerobic environment to obtain a highly active supernatant. According to the microbial load of the highly active supernatant, a pulse high temperature sterilization method is used to obtain a sterilized supernatant; According to the redox potential of the supernatant after sterilization, a mixed inert gas is filled into the sub-bottle to replace the residual oxygen, and the bottle mouth is sealed using laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

2. The method according to claim 1, characterized in that According to the initial bacterial flora activity value of the collected fresh rumen fluid, an anaerobic bacteria active protective agent is injected into the rumen fluid, and the rumen fluid is treated by pulse shaking for 10-15 minutes at a low temperature of 4°C-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5Hz±0.5Hz, including: According to the real-time ATP bioluminescence value, a portable bioluminescence detector was used to measure the initial bacterial colony activity value as the activity quantification data; According to the activity quantification data, 0.5 mL of protective agent was added for every 100 RLU, and a succinate buffer containing 10 mmol / L reduced glutathione was injected into the rumen fluid, and the pH value of the succinate buffer was 6.8±0.2 to obtain a protective agent mixture; The protective agent mixture was placed in a 4°C pulse oscillator, and the oscillation frequency was dynamically adjusted according to the initial bacterial flora activity value. When the activity value was >500RLU, the oscillation frequency was 5.5Hz, and when the activity value was ≤500RLU, the oscillation frequency was 4.5Hz. The oscillation treatment was performed for 12 minutes ± 30 seconds to obtain the pretreated rumen fluid.

3. The method according to claim 2, characterized in that According to the particle distribution characteristics of the pretreated rumen fluid, a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter are sequentially used for two-stage filtration, and a negative pressure of -0.05 MPa or -0.1 MPa is simultaneously applied in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles, including: According to the test results of the laser scattering particle size analyzer, the median particle size D50 value of the particles was extracted; Select the coarse filter mesh number according to the D50 value, wherein, when D50>200μm, use 60 mesh for the coarse filter, and when D50≤200μm, use 100 mesh for the coarse filter, perform the first stage filtration, and obtain the coarse filtrate; According to the feedback of the coarse filtrate flow rate, the negative pressure value of the 200-300 mesh ultrafine filter is dynamically adjusted by the PID algorithm, wherein the negative pressure value is set to -0.1MPa when the flow rate is less than 10mL / min, and the negative pressure value is set to -0.05MPa when the flow rate is ≥10mL / min; According to the D50 value, the pore size of the ultrafine filter is adjusted in real time, wherein the pore size range is 1.3×D50×(1±5%), and the filter is cleaned with ultrasound every 2 minutes during the filtration process of the ultrafine filter to obtain an ultrafine filtrate; According to the bacterial activity loss rate of the ultrafine filtrate obtained by comparing the pretreated rumen fluid, if the bacterial activity loss rate is greater than 10%, the process returns to the step of dynamically adjusting the negative pressure value of the 200-300 mesh ultrafine filter through the PID algorithm according to the feedback of the coarse filtrate flow rate until the bacterial activity loss rate is ≤10%, and the primary filtrate is output.

4. The method according to claim 3, characterized in that According to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria, a staged variable speed centrifugation treatment is adopted. In the first stage, large particles are removed by centrifugation at 5000-6000 rpm for 5 minutes, and in the second stage, the supernatant is collected by centrifugation at 10000-12000 rpm for 10 minutes; wherein the centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain an anaerobic environment, so as to obtain a highly active supernatant, including: According to the rotational viscometer test value η and the optical density value OD600, the viscosity-bacteria density relationship formula η=0.02×(OD600)²+1.5 was established; The first-stage centrifugal speed is set according to the η value, wherein, when η>20mPa·s, the first-stage centrifugal speed is 6000rpm, and when η≤20mPa·s, the first-stage centrifugal speed is 5000rpm, centrifuge at 8°C for 5 minutes, remove large particle residues, and obtain a first-stage supernatant; Nitrogen was introduced into the centrifuge chamber at a flow rate of 5 L / min to maintain the oxygen partial pressure < 0.5%, and an anaerobic primary supernatant was obtained; The secondary centrifugal speed was set according to the OD600 value, wherein the secondary centrifugal speed was 12000 rpm when OD600>1.0, and the secondary centrifugal speed was 10000 rpm when OD600≤1.0, and the mixture was centrifuged at 10°C for 10 minutes, and the supernatant was collected to obtain the secondary enrichment solution; According to the ATP value of the secondary enrichment solution compared with the primary filtrate, if the activity loss rate is greater than 8%, 0.1% cysteine-HCl solution is added to compensate, and finally a high-activity supernatant is output.

5. The method according to claim 4, characterized in that The method of obtaining the sterilized supernatant by pulse high temperature sterilization according to the microbial load of the highly active supernatant comprises: Calculate the sterilization intensity coefficient based on the flow cytometry test results , where CFU is colony forming unit; Select the initial sterilization temperature according to the K value, wherein when K>3, the initial sterilization temperature is 115°C for 5 minutes, and when K≤3, the initial sterilization temperature is 110°C for 5 minutes, and the heating rate is 3°C / min to obtain a first-level sterilization solution; The primary sterilization solution was cooled to 80°C at a rate of 5°C / min and maintained for 2 minutes to activate the thermostable bacterial autolytic enzyme to obtain a resuscitation treatment solution; The resuscitation treatment solution was raised to 121°C at a rate of 3°C / min and maintained for 8 minutes. 0.2MPa overpressure protection was applied simultaneously, and the sterilized supernatant was output.

6. The method according to claim 5, characterized in that According to the redox potential of the sterilized supernatant, a mixed inert gas is filled into the sub-filling bottle to replace the residual oxygen, and the bottle mouth is sealed by laser melting and sealing technology; wherein the oxygen content in the bottle after packaging is less than 0.1%, and an airtight packaging product is obtained, including: When the redox potential detection value ORP>-150mV, the mixed inert gas is injected into the sub-filling bottle for 3 cycles, wherein the mixed inert gas includes 85%, 10%, and 5% by volume of , , , after each replacement, stand for 10 seconds to obtain a hypoxic pretreatment bottle; Select the laser wavelength according to the bottle mouth material. Glass corresponds to a laser wavelength of 1064nm, and plastic corresponds to a laser wavelength of 980nm. Set the sealing energy range to 20x (1±5%) J / cm² for laser sealing. After laser sealing, anaerobic glue containing nano-silicon dioxide with a thickness range of 50x (1 ± 5) μm was sprayed on the seam of the bottle mouth and cured under ultraviolet light for 30 seconds to obtain a reinforced sealed bottle; A micro oxygen sensor is inserted into the bottle to detect the oxygen content <0.1%, then label it and output the airtight packaged product.

7. A device for preparing an additive for rumen fluid anaerobic culture medium, characterized in that: The device comprises: A processing module is used to inject an anaerobic bacteria activity protective agent into the rumen fluid according to the initial bacterial flora activity value of the collected fresh rumen fluid, and treat it by pulse shaking for 10-15 minutes in a low temperature environment of 4-6°C to obtain a pretreated rumen fluid; wherein the protective agent comprises reduced glutathione and succinate buffer, and the pulse shaking frequency is 5Hz±0.5Hz; A filtration module is used to perform two-stage filtration using a 60-100 mesh coarse filter and a 200-300 mesh ultrafine filter in sequence according to the particle distribution characteristics of the pretreated rumen fluid, and a negative pressure of -0.05 MPa or -0.1 MPa is simultaneously applied in the ultrafine filtration stage to obtain a primary filtrate; wherein the pore size of the ultrafine filter is dynamically adapted to 1.2-1.5 times the median particle size of the particles; The centrifugal module is used to perform staged variable speed centrifugation according to the viscosity of the primary filtrate and the density distribution of anaerobic bacteria. In the first stage, the centrifugation is performed at 5000-6000 rpm for 5 minutes to remove large particle residues, and in the second stage, the centrifugation is performed at 10000-12000 rpm for 10 minutes to collect the supernatant. The centrifugation temperature is controlled at 8-10°C and nitrogen is introduced to maintain the anaerobic environment to obtain a highly active supernatant. A sterilization module, used for obtaining a sterilized supernatant by pulse high temperature sterilization according to the microbial load of the high-activity supernatant; The packaging module is used to fill the sub-packaging bottle with mixed inert gas to replace the residual oxygen according to the redox potential of the supernatant after sterilization, and seal the bottle mouth with laser melting technology; wherein, after packaging, the oxygen content in the bottle is less than 0.1%, and an airtight packaging product is obtained.

8. A storage medium, characterized in that: The storage medium stores a computer program, wherein the computer program is configured to execute the method according to any one of claims 1 to 6 when executed.

9. An electronic device, comprising a memory and a processor, characterized in that: A computer program is stored in the memory, and the processor is configured to run the computer program to perform the method according to any one of claims 1 to 6.

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