Feed low-temperature sterilization method based on synergistic effect of composite biological enzyme

Abstract

The invention discloses a feed low-temperature sterilization method based on a composite biological enzyme synergistic effect. The feed low-temperature sterilization method comprises the following steps: a pretreatment stage; a dynamic reaction stage: under the conditions that the temperature is 40-50 DEG C and the humidity is 60%-70%, the enzyme catalytic reaction lasts for 30 minutes, and the microbial structure is decomposed; and inactivating the activity of the residual enzyme through short-time pulsed ultraviolet rays. The compound enzyme comprises lysozyme, thermophilic protease and lipoxygenase, and the nano chitosan carrier and the compound enzyme are mixed according to the mass ratio of 1: 1. According to the method, damage to heat-sensitive nutritional ingredients is avoided through low-temperature operation (40-50 DEG C), broad-spectrum sterilization is achieved through the synergistic effect of compound enzymes, multiple microorganisms such as bacteria, molds and spores are covered, meanwhile, it is guaranteed that no chemical residues exist through the ultraviolet inactivation technology, and animal health and environment safety are guaranteed. The method is low in equipment cost, easy and convenient to operate and free of public acceptance problems, a green, efficient and economical solution is provided for the feed processing industry, and the efficiency and safety of the feed sterilization technology are remarkably improved.

Description

Technical Field

[0001] It belongs to the field of feed processing technology, specifically involving bio-enzyme-assisted sterilization technology, and is suitable for scenarios with high hygiene requirements such as pellet feed and aquatic feed. Background Art

[0002] In the field of feed sterilization, existing technologies have significant defects: although high-temperature sterilization can effectively kill microorganisms, its high temperature environment will destroy heat-sensitive nutrients (such as B vitamins and amino acids), with a loss rate of up to 20%-60%, seriously affecting the nutritional value of feed; although single enzyme sterilization methods (such as lysozyme) are effective against specific bacteria, their range of action is limited, and it is difficult to inactivate broad-spectrum molds and spores, and they are easily ineffective due to enhanced microbial resistance; although chemical sterilization methods are low-cost, there is a risk of chemical residues, which may endanger animal health and pollute the environment; although irradiation sterilization methods have less damage to nutrients, the equipment cost is high and the public acceptance is low, making it difficult to promote. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes, comprising the following steps:

[0004] Pretreatment stage: enzyme atomization spraying technology (particle size ≤ 5μm) is used to make the compound enzyme evenly adhere to the surface of the feed and penetrate into the internal pores;

[0005] Dynamic reaction stage: At 40-50°C and 60%-70% humidity, the enzyme-catalyzed reaction lasts for 30 minutes to decompose the microbial structure;

[0006] Post-treatment stage: Residual enzyme activity was inactivated by short-time pulse ultraviolet light (wavelength 254nm, irradiation 10s).

[0007] Preferred:

[0008] The preparation of the complex enzyme solution includes the following steps:

[0009] a Enzyme activity ratio:

[0010] Lysozyme (activity ≥5000U / mg): 40% of the total enzyme activity;

[0011] Thermophilic protease (derived from Thermus aquaticus, activity ≥ 8000 U / g): 35%.

[0012] Lipoxygenase (activity ≥ 2000U / mg): 25%.

[0013] b. Auxiliary synergist:

[0014] Nano-chitosan carrier (particle size 50 - 100 nm): Mixed with the enzyme in a mass ratio of 1:1 to enhance enzyme stability.

[0015] Tea polyphenols (purity ≥ 98%): Addition amount 0.1% - 0.3% (w / w) to inhibit microbial regeneration.

[0016] Preferably:

[0017] The specific steps of the feed pretreatment are as follows:

[0018] Screening and dust removal: Feed particles pass through a vibrating screen (aperture 2 - 5 mm) to remove debris, dust and oversized particles; A negative pressure dust suction device (wind speed 0.5 - 1.0 m / s) is used to clean surface impurities;

[0019] Surface wetting: Use a micron-level water mist spraying system to increase the surface humidity of the feed to 10% - 15% (regulated in real-time by a humidity sensor); The wetting water is deionized water;

[0020] Use an ultrasonic atomizing nozzle (frequency 1.7 MHz), with an atomization particle size ≤ 5 μm; Spraying pressure 0.2 - 0.5 MPa, nozzle-to-feed distance 20 - 30 cm; Spraying process: The feed passes through the atomization chamber via a conveyor belt (speed 0.5 - 1.0 m / min), and the enzyme solution spraying amount is 0.5 - 1.0 mL / kg. Double-sided spraying mode (upper and lower nozzles symmetrically arranged) to ensure full coverage; The sprayed feed is transferred to the penetration chamber and left to stand for 10 minutes (temperature 25 ± 2 °C, humidity 50% - 60%); A low-speed turning device (rotation speed 5 rpm) is set inside the penetration chamber to promote the diffusion of the enzyme solution into the internal pores.

[0021] Preferably: The dynamic reaction stage includes the following steps:

[0022] a Design requirements for the constant temperature reaction chamber: The inner wall of the chamber is coated with a corrosion-resistant coating. An internal multi-layer belt conveyor system (layer spacing 15 cm) is installed to ensure uniform heating of the feed. Temperature and humidity control: Temperature zoning control: 40 °C in the inlet area → 45 °C in the central area → 50 °C in the outlet area, with gradient heating to avoid thermal shock. Humidity is maintained at 60% - 70% (± 2% deviation) through a steam spraying system.

[0023] b Reaction conditions for the enzyme-catalyzed reaction process: Feed residence time 30 minutes, conveyor belt speed 0.3 m / min. Oxygen concentration in the chamber ≤ 5% (nitrogen is introduced to displace air) to inhibit mold activity.

[0024] Stirring and homogenization: A low-speed spiral stirrer (rotation speed 10 - 15 rpm) is installed in the chamber and operates intermittently for 1 minute every 5 minutes.

[0025] The surface of the stirring blades is coated with polytetrafluoroethylene to reduce feed adhesion.

[0026] Real-time quality monitoring. An on-line microbial sensor (based on ATP bioluminescence method) is set in the warehouse, and the total number of colonies is detected every 5 minutes. If the detected value > 10 CFU / g, the reaction time is automatically extended by 5 minutes.

[0027] Preferably, the post-treatment stage includes the following steps:

[0028] Pulsed ultraviolet inactivation: Use a UV-C LED array (wavelength 254 ± 5 nm), and the power density is 50 mW / cm 2 . The length of the ultraviolet warehouse is 2 m, and an internal reflective aluminum plate is provided to enhance the irradiation uniformity.

[0029] Processing flow: The feed passes through the ultraviolet warehouse at a speed of 0.5 m / min, and the cumulative irradiation time is 10 seconds. It is irradiated in three segments (3 seconds for each segment, with a 1-second interval for cooling) to avoid local overheating.

[0030] Rapid cooling: Use the air-cooling mode: pre-cool to 25 °C with normal-temperature air flow (25 °C, wind speed 3 m / s). Cooling time: The total duration ≤ 5 minutes.

[0031] The present invention provides a method for extracting and purifying lysozyme:

[0032] (1) Strain selection and culture

[0033] Strain selection: Select streptococcal strains with high lysozyme activity, such as Streptococcus pyogenes, Streptococcus thermophilus,

[0034] Activation and inoculation: Take out the cryopreserved strain, inoculate it into a petri dish containing LB solid medium (10 g / L tryptone, 5 g / L yeast extract, 1 g / L sodium chloride, 15 g / L agar), and culture at 37 °C for 24 - 48 hours until colonies form. Subsequently, pick a single colony and inoculate it into a liquid seed medium (LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 1 g / L sodium chloride), and culture on a shaker at 37 °C and 180 rpm for 24 hours until the bacterial liquid reaches the logarithmic growth phase (OD600 value is 0.6 - 0.8).

[0035] (2) Culture and lysozyme secretion

[0036] Fermentation medium preparation: Prepare a liquid fermentation medium, and the common formula is: glucose (20 g / L), tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (1 g / L), potassium dihydrogen phosphate (1 g / L), magnesium sulfate (0.1 g / L), and the pH is adjusted to 6.8 - 7.0.

[0037] The control group can be compared using LB medium.

[0038] Inoculation and culture: Inoculate the 24-hour seed solution into the large-scale fermentation medium at a ratio of 10% (v / v). Incubate at 37 °C with a shaker speed of 180 rpm for 48 - 72 hours. During the culture process, streptococcus will secrete lysozyme, and the enzyme activity usually reaches its maximum value at 48 - 72 hours.

[0039] (3) Crude extraction

[0040] Centrifugal separation: After the fermentation is completed, separate the culture broth by high-speed centrifugation (10000 - 12000 rpm, 10 - 15 minutes, 4 °C), and collect the supernatant as the crude enzyme solution. The supernatant contains lysozyme and other soluble substances.

[0041] Ammonium sulfate precipitation: Slowly add ammonium sulfate to the collected supernatant, gradually increasing the concentration to a saturation of 60% - 80%, let it stand for 30 minutes to 1 hour to precipitate lysozyme. Separate the precipitate and the supernatant by high-speed centrifugation (10000 rpm, 10 minutes, 4 °C). Collect the precipitate and redissolve it in 0.05 M phosphate buffer (pH 7.0) to obtain a concentrated crude enzyme solution.

[0042] (4) Dialysis

[0043] Dialysis operation: Transfer the redissolved crude enzyme solution into a dialysis bag, select an appropriate pore size (such as 3500 Da) for dialysis, and dialyze overnight at 4 °C using 0.05 M phosphate buffer (pH 7.0) to remove low-molecular-weight impurities and salts. After dialysis, change the solution 3 times, with an interval of 2 hours each time.

[0044] (5) Lysozyme purification

[0045] Ion exchange chromatography: Prepare an ion exchange chromatography column DEAE-Sepharose or CM-Sepharose, and equilibrate the column with 0.05 M phosphate buffer (pH 7.0). Load the dialyzed crude enzyme solution onto the ion exchange column at a uniform flow rate. Elute lysozyme using a linear NaCl gradient (0 - 0.5 M NaCl), and collect different fractions. Monitor the ultraviolet absorbance (280 nm) of each fraction and detect its lysozyme activity, and select the fraction with higher activity.

[0046] Size exclusion chromatography: Further purify lysozyme using a size exclusion chromatography column. Elute with 0.05 M phosphate buffer (pH 7.0), and collect the purified lysozyme by detecting the ultraviolet absorbance (280 nm) and enzyme activity.

[0047] (6) Enzyme activity detection

[0048] Lysozyme activity assay: Use a standard lysozyme activity detection method.

[0049] Prepare the reaction system: Mix an appropriate amount of purified lysozyme solution with Micrococcus lysodeikticus bacterial solution. The substrate concentration is 10^6 - 10^7 CFU / mL, and 0.05M phosphate buffer (pH 7.0) is used as the solvent. After reacting at 37°C for 30 minutes, measure the absorbance change of the reaction solution at 450 nm using a spectrophotometer. Enzyme activity is measured by monitoring the change in transparency before and after the start of the reaction. 1 unit (U) of lysozyme activity is defined as the amount of enzyme that can dissolve 1 microgram of Micrococcus lysodeikticus cell wall within 30 minutes.

[0050] (7) Preservation

[0051] Cryopreservation: Aliquot the purified lysozyme solution, add 20% glycerol, mix well, and store it at -20°C or -80°C to maintain enzyme activity. For long-term storage, the enzyme solution can be aliquoted into small portions to avoid repeated freezing and thawing.

[0052] The present invention provides a method for extracting and purifying a thermophilic protease, comprising the following steps:

[0053] Decompose extracellular polysaccharides and mycotoxins of bacteria;

[0054] Select materials rich in protein from distiller's grains as the culture medium for thermophilic protease microorganisms or directly as the source for isolating thermophilic bacteria.

[0055] Screen and culture thermophilic strains. Target strains: Mainly screen Bacillus licheniformis strains producing thermophilic protease. Sample from distiller's grains and enrich and culture at high temperature (50°C - 70°C) to screen for thermophilic strains resistant to high temperature. Add casein or gelatin as the sole nitrogen source to the culture medium, and screen positive strains by the gelatin clear zone method or casein hydrolysis experiment to test whether the strains secrete protease. Laboratory strain culture: Culture medium: Solid medium: Distiller's grains dry powder + agar. Culture conditions: Temperature: 50°C - 65°C; pH value: 7.0 - 8.5; Time: 24 - 48 hours. After fermentation, measure the enzyme activity, extract and purify the enzyme, separate the bacterial cells by ion exchange chromatography, use a DEAE-Sepharose packing column to separate the target enzyme. Further purify the enzyme using Sephadex G-100 or G-200 to separate the enzyme protein with a suitable molecular weight. Measure the activity retention rate of the purified thermophilic protease at high temperature to ensure its good stability in the environment of 50°C - 70°C.

[0056] The present invention provides a method for extracting and purifying a lipoxygenase, comprising the following steps:

[0057] (1) Strain Selection and Preparation

[0058] Strain Selection:

[0059] Purchase the strain Aspergillus niger from ATCC or a culture collection center

[0060] Activation:

[0061] Inoculate the cryopreserved Aspergillus niger strain onto a solid medium (such as PDA medium) and culture at 28 - 30°C for 2 - 3 days until colonies form

[0062] Seed Culture:

[0063] Transfer the activated strain to a liquid seed medium (such as a liquid medium containing glucose, sodium nitrate, etc.) and culture in a shaking flask at 28°C, 180 rpm for 24 hours to obtain a seed solution

[0064] (2) Fermentation Production

[0065] Fermentation Medium Preparation:

[0066] Basic Formula: A medium containing a carbon source (glucose or starch), a nitrogen source (sodium nitrate or urea), and minerals (phosphates, magnesium salts, etc.) can be used

[0067] Sample Formula:

[0068] Glucose: 10 g / L

[0069] Sodium Nitrate: 3 g / L

[0070] Potassium Dihydrogen Phosphate: 1 g / L

[0071] Magnesium Sulfate: 0.5 g / L

[0072] Trace Elements: 0.1 g / L

[0073] pH 6.0 - 6.5

[0074] Fermentation Condition Optimization:

[0075] Inoculation: Inoculate the seed solution into the fermentation medium at 5 - 10% (v / v)

[0076] Temperature: 28°C - 30°C

[0077] pH: 6.0 - 6.5 (Automatically adjusting the pH can increase the yield)

[0078] Gas Exchange: Good ventilation needs to be maintained, and the stirring speed in the fermenter is set to 150 - 200 rpm

[0079] Time: Ferment for about 72 hours, detect the activity of lipoxygenase in the culture medium, and terminate fermentation after confirming that it reaches the peak value.

[0080] Enzyme secretion:

[0081] Aspergillus niger secretes lipoxygenase into the fermentation broth, and the main activity of the enzyme exists in the supernatant of the culture medium.

[0082] (3) Crude extraction of the enzyme

[0083] Centrifugation: Centrifuge the fermentation broth at high speed (10000 - 15000 rpm, 15 minutes, 4°C) to remove cells and other solid particles, and collect the supernatant of the fermentation broth.

[0084] Precipitation: Gradually add ammonium sulfate to the supernatant to a saturation of 60% - 80%, and precipitate lipoxygenase by slowly stirring. After standing for 4 hours, centrifuge at low temperature (10000 rpm, 10 minutes, 4°C) to collect the precipitate.

[0085] Redissolution: Redissolve the precipitate with an appropriate amount of Tris-HCl buffer (0.05 M, pH 7.5 - 8.0).

[0086] (4) Purification of the enzyme

[0087] Dialysis: Place the redissolved enzyme solution in a dialysis bag with a molecular weight cut-off of about 10 kDa. Dialyze overnight at 4°C with Tris-HCl buffer (0.05 M, pH 7.5 - 8.0) to remove small molecule impurities and salts.

[0088] Ion exchange chromatography: Use DEAE-Sepharose as the medium, equilibrate the column with Tris-HCl buffer after packing. After loading the enzyme solution, collect the activity peak of the enzyme by gradient elution (such as using 0 - 0.5 M NaCl).

[0089] Size exclusion chromatography: Use Sephadex G-100 or Superdex 200 for size exclusion separation to remove high molecular weight impurities.

[0090] Enzyme activity detection: Detect the enzyme activity and protein concentration after each step of purification to ensure the minimum loss of activity.

[0091] (5) Determination of enzyme activity

[0092] Lipoxygenase activity is usually detected by generating peroxides after oxidizing substrates (such as linoleic acid or linolenic acid).

[0093] Determination method:

[0094] Substrate preparation: Prepare a 0.1M linoleic acid solution, using Tween-20 as an emulsifier.

[0095] Reaction system: Mix an appropriate amount of buffer (such as 0.05M Tris-HCl, pH 7.5), linoleic acid solution, and enzyme solution. React at 25 °C for 5 - 10 minutes.

[0096] Detection: Use a spectrophotometer to measure the change in absorbance of the reaction system at 234 nm (linoleic acid peroxide has a specific absorption peak).

[0097] The enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of peroxide per minute.

[0098] (6) Preservation of lipoxygenase

[0099] After enzyme purification, the enzyme activity can be stabilized by adding glycerol (20% v / v) or storing at low temperature (-20 °C). Dry preservation can be achieved by spray drying or freeze drying to prepare enzyme powder.

[0100] The present invention proposes a low-temperature sterilization method for feed based on the synergistic effect of composite bioenzymes, and the beneficial effects are as follows: By operating at low temperature (40 - 50 °C), the destruction of heat-sensitive nutritional components is avoided. The synergistic effect of composite enzymes (lysozyme, thermophilic protease, lipoxygenase) is utilized to achieve broad-spectrum sterilization, covering various microorganisms such as bacteria, molds, and spores. At the same time, ultraviolet inactivation technology is adopted to ensure no chemical residues, protecting animal health and environmental safety. In addition, this method has low equipment cost, simple operation, and no public acceptance problems, providing a green, efficient, and economical solution for the feed processing industry, and significantly improving the efficiency and safety of feed sterilization technology. Description of the drawings

[0101] Figure 1 It is a process flow chart of low-temperature sterilization of feed based on composite bioenzymes. Detailed implementation manners

[0102] Example 1

[0103] 1. Composite enzyme design

[0104] The core enzyme species include lysozyme, thermophilic protease, and lipoxygenase

[0105] For the cell wall of Gram-positive bacteria;

[0106] Enzyme action mechanism: Lysozyme: Targetedly decomposes the peptidoglycan layer of the bacterial cell wall (especially for Salmonella and Escherichia coli). Thermophilic protease: Efficiently decomposes the bacterial biofilm and aflatoxin B1 at 45 °C. Lipoxygenase: Destroys the lipid structure of the pathogen cell membrane, synergistically enhancing the sterilization effect.

[0107] 1.1 Extraction and purification of lysozyme:

[0108] (1) Strain selection and cultivation

[0109] Strain selection: Select streptococcus strains with high lysozyme activity, such as Streptococcus pyogenes, Streptococcus thermophilus,

[0110] Activation and inoculation: Take out the cryopreserved strains and inoculate them into petri dishes containing LB solid medium (10 g / L tryptone, 5 g / L yeast extract, 1 g / L sodium chloride, 15 g / L agar), and culture at 37 °C for 24 - 48 hours until colonies form. Then pick a single colony and inoculate it into liquid seed medium (LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 1 g / L sodium chloride), and culture on a shaker at 37 °C and 180 rpm for 24 hours until the bacterial liquid reaches the logarithmic growth phase (OD600 value is 0.6 - 0.8).

[0111] (2) Cultivation and lysozyme secretion

[0112] Fermentation medium preparation: Prepare liquid fermentation medium, and the common formula is: glucose (20 g / L), tryptone (10 g / L), yeast extract (5 g / L), sodium chloride (1 g / L), potassium dihydrogen phosphate (1 g / L), magnesium sulfate (0.1 g / L), and adjust the pH to 6.8 - 7.0.

[0113] The control group can use LB medium for comparison.

[0114] Inoculation and cultivation: Inoculate the 24-hour seed liquid into the large-scale fermentation medium at a ratio of 10% (v / v). Cultivate at 37 °C and a shaker speed of 180 rpm for 48 - 72 hours. During the cultivation process, streptococcus will secrete lysozyme, and the enzyme activity usually reaches the maximum value at 48 - 72 hours.

[0115] (3) Crude extraction

[0116] Centrifugal separation: After the fermentation is completed, separate the culture solution by high-speed centrifugation (10000 - 12000 rpm, 10 - 15 minutes, 4 °C), and collect the supernatant as the crude enzyme solution. The supernatant contains lysozyme and other soluble substances.

[0117] Ammonium sulfate precipitation: Slowly add ammonium sulfate to the collected supernatant, gradually increasing the concentration to a saturation of 60%-80%, let stand for 30 minutes to 1 hour to precipitate lysozyme. Separate the precipitate and supernatant by high-speed centrifugation (10000 rpm, 10 minutes, 4°C). Collect the precipitate and redissolve it in 0.05M phosphate buffer (pH 7.0) to obtain a concentrated crude enzyme solution.

[0118] (4) Dialysis

[0119] Dialysis operation: Transfer the redissolved crude enzyme solution into a dialysis bag, select an appropriate pore size (such as 3500 Da) for dialysis, and dialyze overnight at 4°C using 0.05M phosphate buffer (pH 7.0) to remove low-molecular impurities and salts. After dialysis, change the solution 3 times, with a 2-hour interval each time.

[0120] (5) Lysozyme purification

[0121] Ion exchange chromatography: Prepare an ion exchange chromatography column DEAE-Sepharose or CM-Sepharose, and equilibrate the column with 0.05M phosphate buffer (pH 7.0). Load the dialyzed crude enzyme solution onto the ion exchange column at a uniform flow rate. Elute lysozyme using a linear NaCl gradient (0-0.5M NaCl), and collect different fractions. Monitor the ultraviolet absorbance (280 nm) of each fraction and detect its lysozyme activity, and select the fraction with higher activity.

[0122] Molecular sieve chromatography: Further purify lysozyme using a molecular sieve chromatography column. Elute with 0.05M phosphate buffer (pH 7.0), and collect the purified lysozyme by detecting ultraviolet absorbance (280 nm) and enzyme activity.

[0123] (6) Enzyme activity detection

[0124] Lysozyme activity assay: Use a standard lysozyme activity detection method.

[0125] Prepare the reaction system: Mix an appropriate amount of purified lysozyme solution with Micrococcus lysodeikticus bacterial solution, with a substrate concentration of 10^6-10^7 CFU / mL, and use 0.05M phosphate buffer (pH 7.0) as the solvent. After reacting at 37°C for 30 minutes, measure the change in absorbance of the reaction solution at 450 nm using a spectrophotometer. Enzyme activity is determined by monitoring the change in transparency before and after the start of the reaction. 1 unit (U) of lysozyme activity is defined as the amount of enzyme that can dissolve 1 microgram of Micrococcus lysodeikticus cell wall within 30 minutes.

[0126] (7) Preservation

[0127] Cryopreservation: Aliquot the purified lysozyme solution and add 20% glycerol. After mixing, it can be stored at -20°C or -80°C to maintain enzyme activity. For long-term storage, the enzyme solution can be aliquoted into small portions to avoid repeated freezing and thawing.

[0128] 1.2 Extraction and purification of thermophilic protease: Degrade extracellular polysaccharides and mycotoxins of bacteria;

[0129] Select materials rich in protein from distiller's grains as the culture medium for thermophilic protease microorganisms or directly as the source for isolating thermophilic bacteria.

[0130] Screen and culture thermophilic strains. Target strain: Mainly screen Bacillus licheniformis strains producing thermophilic protease. Sample from distiller's grains and enrich culture at high temperature (50°C - 70°C) to screen thermophilic strains resistant to high temperature. Add casein or gelatin as the sole nitrogen source to the culture medium, and screen positive strains by the gelatin clear zone method or casein hydrolysis experiment to test whether the strain secretes protease. Laboratory strain culture: Culture medium: Solid medium: Distiller's grains dry powder + agar. Culture conditions: Temperature: 50°C - 65°C; pH value: 7.0 - 8.5; Time: 24 - 48 hours. After fermentation, measure the enzyme activity, extract and purify the enzyme, separate the bacterial cells by ion exchange chromatography, use a DEAE-Sepharose packing column to separate the target enzyme. Further purify the enzyme using Sephadex G-100 or G-200 to separate the enzyme protein with a suitable molecular weight. Measure the activity retention rate of the purified thermophilic protease at high temperature to ensure its good stability in the environment of 50°C - 70°C.

[0131] 1.3 Extraction and purification of lipoxygenase:

[0132] (1) Strain selection and preparation

[0133] Strain selection:

[0134] Purchase the Aspergillus niger strain from ATCC or the culture collection center

[0135] Activation:

[0136] Inoculate the cryopreserved Aspergillus niger strain onto a solid medium (such as PDA medium), culture at 28 - 30°C for 2 - 3 days until colonies are formed.

[0137] Seed culture:

[0138] Transfer the activated strain to a liquid seed culture medium (such as a liquid medium containing glucose, sodium nitrate, etc.), culture in a shaking flask at 28°C, 180 rpm for 24 hours to obtain a seed solution.

[0139] (2) Fermentation production

[0140] Preparation of fermentation medium:

[0141] Basic formula: A medium containing a carbon source (glucose or starch), a nitrogen source (sodium nitrate or urea), and minerals (phosphates, magnesium salts, etc.) can be used.

[0142] Sample formula:

[0143] Glucose: 10 g / L

[0144] Sodium nitrate: 3 g / L

[0145] Potassium dihydrogen phosphate: 1 g / L

[0146] Magnesium sulfate: 0.5 g / L

[0147] Trace elements: 0.1 g / L

[0148] pH 6.0 - 6.5

[0149] Optimization of fermentation conditions:

[0150] Inoculation: Inoculate the seed solution into the fermentation medium at 5 - 10% (v / v).

[0151] Temperature: 28°C - 30°C

[0152] pH: 6.0 - 6.5 (Automatically adjusting the pH can increase the yield).

[0153] Gas exchange: Good ventilation needs to be maintained, and the stirring speed in the fermenter is set at 150 - 200 rpm.

[0154] Time: Ferment for about 72 hours, detect the lipoxygenase activity in the culture solution, and terminate the fermentation after confirming that it reaches the peak.

[0155] Enzyme secretion:

[0156] Aspergillus niger secretes lipoxygenase into the fermentation broth, and the main activity of the enzyme exists in the supernatant of the culture solution.

[0157] (3) Crude extraction of the enzyme

[0158] Centrifugation: Remove cells and other solid particles from the fermentation broth by high-speed centrifugation (10000 - 15000 rpm, 15 minutes, 4°C), and collect the supernatant of the fermentation broth.

[0159] Precipitation: Gradually add ammonium sulfate to the supernatant to a saturation of 60% - 80%, and precipitate the lipoxygenase by slowly stirring. After standing for 4 hours, centrifuge at low temperature (10000 rpm, 10 minutes, 4°C) and collect the precipitate.

[0160] Redissolution: Redissolve the precipitate with an appropriate amount of Tris-HCl buffer (0.05 M, pH 7.5 - 8.0).

[0161] (4) Purification of the enzyme

[0162] Dialysis: Place the redissolved enzyme solution in a dialysis bag with a molecular weight cut-off of approximately 10 kDa. Dialyze overnight at 4°C against Tris-HCl buffer (0.05 M, pH 7.5 - 8.0) to remove small molecule impurities and salts.

[0163] Ion exchange chromatography: Use DEAE-Sepharose as the medium. After packing the column, equilibrate it with Tris-HCl buffer. After loading the enzyme solution, collect the enzyme activity peak by gradient elution (such as using 0 - 0.5 M NaCl).

[0164] Size exclusion chromatography: Use Sephadex G-100 or Superdex 200 for size exclusion separation to remove high molecular weight impurities.

[0165] Enzyme activity detection: Detect the enzyme activity and protein concentration after each step of purification to ensure minimal loss of activity.

[0166] (5) Determination of enzyme activity

[0167] Lipoxygenase activity is usually detected by the formation of peroxides after oxidizing substrates such as linoleic acid or linolenic acid.

[0168] Determination method:

[0169] Substrate preparation: Prepare a 0.1 M linoleic acid solution with Tween-20 as the emulsifier.

[0170] Reaction system: Mix an appropriate amount of buffer (such as 0.05 M Tris-HCl, pH 7.5), linoleic acid solution, and enzyme solution. React at 25°C for 5 - 10 minutes.

[0171] Detection: Use a spectrophotometer to measure the change in absorbance of the reaction system at 234 nm (linoleic acid peroxide has a specific absorption peak).

[0172] The enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of peroxide per minute.

[0173] (6) Preservation of lipoxygenase

[0174] After enzyme purification, the enzyme activity can be stabilized by adding glycerol (20% v / v) or storing at low temperature (-20°C). Dry preservation can be achieved by spray drying or freeze drying to prepare enzyme powder.

[0175] 1.4 Auxiliary synergist: Nano chitosan carrier: Immobilize enzyme activity and extend the action time; Natural plant extract tea polyphenols: Inhibit secondary pollution after sterilization.

[0176] Example 2

[0177] Staged low-temperature sterilization process

[0178] Pretreatment stage: Adopt enzyme atomization spraying technology (particle size ≤ 5μm) to make the composite enzyme evenly adhere to the surface of the feed and penetrate into the internal pores. Dynamic reaction stage: Under the conditions of 40 - 50°C and humidity of 60% - 70%, the enzyme-catalyzed reaction lasts for 30 minutes to decompose the microbial structure. Post-treatment stage: Inactivate the residual enzyme activity through short-time pulsed ultraviolet light (wavelength 254nm, irradiation for 10s) to avoid the influence of residues.

[0179] Example 3

[0180] Sterilization process:

[0181] (1) Feed pretreatment

[0182] Screening and dust removal: Feed particles pass through a vibrating screen (aperture 2 - 5mm) to remove debris, dust, and oversized particles. Use a negative pressure dust suction device (wind speed 0.5 - 1.0m / s) to clean the surface impurities.

[0183] Surface wetting: Use a micron-sized water mist spraying system to increase the surface humidity of the feed to 10% - 15% (regulated by real-time feedback of a humidity sensor). The wetting water is deionized water to avoid interference of minerals with enzyme activity.

[0184] (2) Preparation of composite enzyme solution

[0185] a Enzyme activity ratio:

[0186] Lysozyme (activity ≥ 5000U / mg): Accounts for 40% of the total enzyme activity.

[0187] Thermophilic protease (derived from Thermus aquaticus, activity ≥ 8000U / g): Accounts for 35%.

[0188] Lipoxygenase (activity ≥ 2000U / mg): Accounts for 25%.

[0189] b Auxiliary synergist:

[0190] Nano chitosan carrier (particle size 50 - 100nm): Mixed with the enzyme in a mass ratio of 1:1 to improve enzyme stability.

[0191] Tea polyphenols (purity ≥ 98%): Addition amount 0.1% - 0.3% (w / w) to inhibit microbial regeneration.

[0192] c High-pressure atomization spraying

[0193] Equipment parameters: Use an ultrasonic atomization nozzle (frequency 1.7 MHz), with an atomization particle size ≤ 5 μm. Spraying pressure is 0.2 - 0.5 MPa, and the distance between the nozzle and the feed is 20 - 30 cm. Spraying process: The feed passes through the atomization chamber via a conveyor belt (speed 0.5 - 1.0 m / min), and the spraying amount of the enzyme solution is 0.5 - 1.0 mL / kg. Double-sided spraying mode (symmetrical layout of upper and lower nozzles) to ensure full coverage. Penetration and static setting: The sprayed feed is transferred to the penetration chamber and left to stand for 10 minutes (temperature 25 ± 2 °C, humidity 50% - 60%). A low-speed turning device (rotation speed 5 rpm) is set inside the penetration chamber to promote the diffusion of the enzyme solution into the internal pores.

[0194] (3) Low-temperature enzyme-catalyzed sterilization

[0195] Activate the synergistic sterilization effect of the composite enzyme under low-temperature conditions to decompose the microbial structure.

[0196] a Design requirements for the constant-temperature reaction chamber: The inner wall of the chamber is coated with a corrosion-resistant coating. A multi-layer belt conveyor system (layer spacing 15 cm) is installed inside to ensure uniform heating of the feed. Temperature and humidity control: Temperature zoning control: Inlet area 40 °C → Central area 45 °C → Outlet area 50 °C, with gradient heating to avoid thermal shock. Humidity is maintained at 60% - 70% (± 2% deviation) through a steam spraying system.

[0197] b Reaction conditions during the enzyme-catalyzed reaction: Feed residence time is 30 minutes, and the conveyor belt speed is 0.3 m / min. The oxygen concentration inside the chamber ≤ 5% (nitrogen is introduced to displace air) to inhibit the activity of molds.

[0198] Stirring and homogenization: A low-speed spiral stirrer (rotation speed 10 - 15 rpm) is installed inside the chamber and operates intermittently for 1 minute every 5 minutes.

[0199] The surface of the stirring blades is coated with a polytetrafluoroethylene coating to reduce feed adhesion.

[0200] Real-time quality monitoring: An online microbial sensor (based on the ATP bioluminescence method) is set inside the chamber to detect the total number of colonies every 5 minutes. If the detected value > 10 CFU / g, the reaction time is automatically extended by 5 minutes.

[0201] (4) Post-treatment stage: Inactivation of enzyme activity and cooling

[0202] To avoid the influence of enzymatic hydrolysis on the feed quality during long-term storage, it is necessary to inactivate the residual enzyme activity.

[0203] Steps and parameters:

[0204] Pulse ultraviolet inactivation: Use a UV-C LED array (wavelength 254 ± 5 nm), power density 50 mW / cm2 The length of the ultraviolet chamber is 2m, and the built-in reflective aluminum plate enhances the irradiation uniformity.

[0205] Processing flow: The feed passes through the ultraviolet chamber at a speed of 0.5m / min, and the cumulative irradiation time is 10 seconds. It is irradiated in three sections (3 seconds for each section, with a 1-second interval for cooling) to avoid local overheating.

[0206] Rapid cooling: Use the air-cooling mode: Pre-cool to 25°C with normal-temperature air flow (25°C, wind speed 3m / s). Cooling time: The total duration ≤ 5 minutes

[0207] The process flow chart of the low-temperature sterilization of feed based on composite bio-enzymes is as Figure 1 shown

[0208] Experimental examples

[0209] Experimental methods

[0210] 1.1 Sample grouping and treatment

[0211] In this study, 8 self-made feed samples from different sources were selected, and the sampling amount of each sample was 2kg. According to the differences in treatment methods, the samples were divided into two groups for experiments:

[0212] Control group: Without any treatment, used as a benchmark control group.

[0213] Sterilization treatment group: The samples were sterilized using the composite enzyme low-temperature sterilization process.

[0214] After the treatment was completed, all samples were left standing for 7 days in the same experimental environment (temperature: 20°C, humidity: 65%) for subsequent analysis.

[0215] 1.2 Detection of microbial hygiene indicators

[0216] According to the relevant regulations in the national standard GB 14924.2-2001 "Hygiene Standard for Formula Feed for Laboratory Animals", the microbial hygiene indicators in the samples were analyzed. The detection contents included the total number of colonies, coliform bacteria, the number of molds and yeasts, and the presence of pathogenic microorganisms. By comparing with the reference values of the national standard, it was evaluated whether each microbial indicator met the hygiene requirements.

[0217] 1.3 Nutritional component analysis

[0218] After the microbial hygiene detection was completed, the sample groups and the control group that met the sterilization standards were retained for further nutritional component analysis. According to the standard of GB 14924.3-2010 "Nutritional Components of Formula Feed for Laboratory Animals", the main nutritional components of the samples were detected. The specific detection items included but were not limited to:

[0219] Conventional components: moisture, crude protein, crude fat, crude fiber, ash, calcium, total phosphorus, etc.

[0220] Vitamin content: vitamin A, D, E, B1, B2, B6, niacin, etc.

[0221] Amino acids: lysine, arginine, histidine, phenylalanine + tyrosine, threonine, leucine, isoleucine, etc.

[0222] Minerals: magnesium, potassium, sodium, iron, manganese, copper, zinc, iodine, selenium, etc.

[0223] 1.4 Data statistical analysis

[0224] All data were sorted using Excel 2006 software and statistically analyzed using SPSS 19.0. First, a normality test was performed on the measurement data. For data that conformed to the normal distribution, the mean ± standard deviation was used to represent. Then, a homogeneity of variance test was conducted. If the data met the homogeneity assumption, one-way analysis of variance (ANOVA) was used for between-group comparison. In the case of detecting significant differences, the Student-Newman-Keuls method was further used for within-group comparison. The statistical significance level was set at α = 0.05 (two-tailed test).

[0225] Table 1 Determination results of hygienic microbial indicators in feed before and after sterilization

[0226]

[0227] As can be seen from Table 1, for the total number of colonies: after sterilization, it decreased from 2.4×10^4 cfu / g to <10 cfu / g, with a decrease of 99.96%. The number of coliforms decreased from 7.8×10^2 cfu / g to <30 cfu / g, with a decrease of more than 96%, meeting the feed hygiene standard. The number of molds and yeasts decreased from 3.5×10^2 cfu / g to <10 cfu / g, with a decrease of more than 97%. Pathogenic bacteria (Salmonella): were not detected before and after sterilization, indicating that the equipment had a significant killing effect on pathogenic bacteria. After the equipment was sterilized, the content of harmful microorganisms in the feed decreased significantly, fully meeting the national standards (such as GB 13078-2017 "Feed Hygiene Standard"), ensuring the hygienic safety of the feed.

[0228] Table 2 Effects of compound enzyme low-temperature sterilization on the conventional nutritional components of feed (g / kg)

[0229]

[0230] As can be seen from Table 2, moisture: decreased from 71.5 ± 5.0 g / kg to 70.8 ± 4.0 g / kg, with a loss rate of 1.0%. Crude protein: decreased from 16.5 ± 4.2 g / kg to 16.2 ± 4.1 g / kg, with a loss rate of 1.8%. Crude fat: decreased from 31.0 ± 2.1 g / kg to 30.8 ± 2.0 g / kg, with a loss rate of 0.6%. Crude fiber: decreased from 12.9 ± 6.8 g / kg to 12.7 ± 6.7 g / kg, with a loss rate of 1.6%. The loss rates of crude ash, calcium, and total phosphorus were all less than 1%. Table 2 shows that the equipment sterilization has minimal impact on the conventional nutritional components of the feed. In particular, the loss rates of crude protein and crude fat are both less than 2%, indicating that the main nutritional components of the feed can be effectively retained during the low-temperature sterilization process with complex enzymes.

[0231] Table 3 Effects of sterilization on mineral content in feed before and after

[0232]

[0233] As can be seen from Table 3, iron decreased from 455 ± 14 mg / kg to 450 ± 14 mg / kg, with a loss rate of 1.1%. The changes in manganese, sodium, magnesium, potassium, copper, zinc, and selenium were not significant (P > 0.05), and the loss rates were all less than 1%. Selenium: no change, with a loss rate of 0.0%. This indicates that the low-temperature sterilization with complex enzymes has minimal impact on the mineral content in the feed, and the loss rates of all minerals are less than 1.1%, suggesting that this sterilization process can better retain the minerals in the feed.

[0234] Table 4 Effects of sterilization on vitamin content in feed before and after (x ± s)

[0235]

[0236] As can be seen from Table 4, vitamin A: decreased from 18500 ± 130 IU / kg to 18400 ± 129 IU / kg, with a loss rate of 0.5%. Niacin: decreased from 113.0 ± 4.5 mg / kg to 112.5 ± 4.4 mg / kg, with a loss rate of 0.4%. The changes in vitamin B1, B2, and B6 were not significant (P > 0.05), and the loss rates were all less than 0.5%. Vitamin D: decreased from 1700 ± 14 IU / kg to 1695 ± 13 IU / kg, with a loss rate of 0.3%. This indicates that the low-temperature sterilization with complex enzymes has minimal impact on the vitamin content in the feed, and the loss rates of all vitamins are less than 0.5%, suggesting that this sterilization process can better retain the vitamins in the feed.

[0237] Table 5 Effects of sterilization on amino acids in feed before and after

[0238]

[0239] As can be seen from Table 5, the overall amino acid loss rate after low-temperature sterilization of the compound enzyme is below 2%, and the loss rate is extremely low, which well guarantees the content of amino acid nutrients in the original feed.

[0240] Through this experiment, it can be known that the low-temperature sterilization method of feed based on the synergistic effect of compound bioenzymes proposed in this patent has a significant effect on killing pathogenic bacteria, and the nutritional loss rate before and after sterilization of conventional nutrients, minerals, vitamins and amino acids is very low, which can maximize the retention of feed nutrients and has significant technical advantages and application value.

[0241] The low-temperature sterilization method of feed based on the synergistic effect of compound bioenzymes proposed by the present invention has the following significant advantages:

[0242] High-efficiency broad-spectrum sterilization: Through the synergistic effect of compound enzymes (lysozyme, thermophilic protease, lipoxidase), it can targetedly decompose the peptidoglycan layer of the bacterial cell wall (such as Salmonella, Escherichia coli), efficiently decompose the bacterial biofilm and aflatoxin B1, and destroy the lipid structure of the pathogenic bacteria cell membrane, realizing the broad-spectrum inactivation of various microorganisms such as bacteria, molds and spores, and the sterilization rate is over 99.9%.

[0243] Low-temperature operation to protect nutrients: The sterilization process is carried out under low-temperature conditions of 40 - 50 °C to avoid the destruction of heat-sensitive nutrients (such as vitamin B group, amino acids) by high temperature and ensure the nutritional value of the feed. Experimental data shows that the retention rate of vitamin B1 ≥ 97%, and the loss rate of crude protein ≤ 3%, which is significantly better than the traditional high-temperature sterilization method.

[0244] No chemical residues, safe and environmentally friendly: Using bioenzyme and ultraviolet inactivation technology to avoid the residual risk of chemical sterilization method, ensuring animal health and environmental safety, and meeting the requirements of green sustainable development.

[0245] Low cost and easy to promote: Compared with the irradiation sterilization method, this method has low equipment cost, simple operation, and no public acceptance problem, is suitable for large-scale feed production lines, and has high economic efficiency and promotion value.

[0246] Intelligent regulation with high consistency: Combining real-time monitoring and dynamic adjustment technology to ensure the consistency of sterilization effect, reducing energy consumption by more than 20% and improving production efficiency.

[0247] Feasibility analysis

[0248] Technical feasibility: Lysozyme, thermophilic protease, and lipoxygenase are all extracted and purified from existing mature strains. The technology is mature and the cost is low. This patent adopts a staged low-temperature sterilization process (enzyme atomization spraying → low-temperature reaction → ultraviolet inactivation). The process steps are clear, the parameters are controllable, and it is easy to realize industrial production. Experimental data shows that the total number of colonies in the feed after sterilization is ≤ 10 CFU / g, and Salmonella is not detected, fully meeting the national standards (such as GB 13078-2017 "Feed Hygiene Standard").

[0249] The production cost of the composite enzyme is low, and the cost can be further reduced through large-scale production. Low-temperature operation reduces energy consumption, and the ultraviolet inactivation technology does not require expensive equipment. The overall cost is significantly lower than that of irradiation sterilization. This method conforms to the development trend of green agriculture and has high market competitiveness.

[0250] Through the synergistic effect of the composite enzyme and the innovative design of the low-temperature sterilization process, the present invention solves the problems in the prior art such as high-temperature damage to nutritional components, limited action range of single enzymes, high risk of chemical residues, and high cost of irradiation. It has multiple advantages such as high-efficiency sterilization, nutritional retention, safety and environmental protection, and low cost. Its technology is mature, the process is feasible, the economy is high, and the market prospect is broad. It has significant industrial feasibility and provides a new sterilization solution for the feed processing industry.

Claims

1. A method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes, characterized in that: The following steps are involved: Pretreatment stage: enzyme atomization spraying technology is used to make the compound enzyme evenly adhere to the surface of the feed and penetrate into the internal pores; Dynamic reaction stage: At 40-50°C and 60%-70% humidity, the enzyme-catalyzed reaction lasts for 30 minutes to decompose the microbial structure; Post-treatment stage: Residual enzyme activity is inactivated by short-duration pulses of UV light.

2. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 1, characterized in that: The preparation of complex enzyme solution includes: a. Enzyme activity ratio: Lysozyme: 40% of total enzyme activity; Thermophilic protease: 35% of total enzyme activity; Lipoxygenase: 25% of total enzyme activity; b. Auxiliary synergists: Nano chitosan carrier: mixed with enzyme in a mass ratio of 1:1; Tea polyphenols: added amount 0.1%-0.3% (w / w).

3. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 1, characterized in that: The feed pretreatment specifically comprises the following steps: a. Screening and dust removal b. Surface wetting c. Use ultrasonic atomization nozzle with atomization particle size ≤ 5μm; pass the feed through the atomization chamber via a conveyor belt, and spray the enzyme solution at a rate of 0.5-1.0mL / kg; transfer the sprayed feed to the osmosis chamber and let it stand for 10 minutes (temperature 25±2℃, humidity 50%-60%); stir the chamber at a low speed.

4. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 1, characterized in that: The dynamic reaction phase includes: a. The temperature in the constant temperature reaction chamber is controlled by different zones: entrance zone 40℃→center zone 45℃→exit zone 50℃; humidity is maintained at 60%-70%; b. Reaction conditions of enzyme catalysis reaction process: feed residence time 30 minutes, conveyor belt speed 0.3m / min. Oxygen concentration in the bin ≤5%; c. Stirring and homogenizing.

5. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 4, characterized in that: An online microbial sensor is installed in the constant temperature reaction chamber to detect the total colony count every 5 minutes; if the detection value is greater than 10 CFU / g, the reaction time is automatically extended by 5 minutes.

6. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 1, characterized in that: The post-processing stage includes: Pulse UV inactivation: UV-CLED array (wavelength 254±5nm), power density 50mW / cm 2 ; The feed passes through the UV chamber at a speed of 0.5m / min, with a cumulative exposure time of 10 seconds; the exposure is divided into three sections, each section is 3 seconds, and the interval is 1 second for cooling; Rapid cooling: Use air cooling mode: pre-cool to 25°C with normal temperature airflow (25°C, wind speed 3m / s); cooling time: total time ≤5 minutes.

7. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 1, characterized in that: The extraction and purification of lysozyme includes the following steps: (1) Strain selection and cultivation Strain selection: Select streptococcal strains with higher lysozyme activity; Activation and inoculation; (2) Culture and lysozyme secretion (3) Rough extraction (4) Dialysis (5) Lysozyme purification (6) Enzyme activity detection (7) Storage: The purified lysozyme solution was divided into aliquots, and 20% glycerol was added. After mixing, the aliquots were stored at -20°C or -80°C.

8. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 1, characterized in that: Thermophilic protease extraction and purification includes: Decompose bacterial exopolysaccharides and mycotoxins; Select protein-rich materials from wine lees as culture medium for thermophilic protease microorganisms or as a source for directly isolating thermophilic bacteria.

9. The method for low-temperature sterilization of feed based on the synergistic effect of composite biological enzymes according to claim 1, characterized in that: The extraction and purification of lipoxygenase includes the following steps: (1) Strain selection and preparation Strain selection: Purchase Aspergillus niger strains from ATCC or culture collection centers; Activation: Inoculate the frozen Aspergillus niger strain onto solid culture medium and culture at 28-30°C for 23 days until colonies are formed; Seed cultivation; (2) Fermentation production (3) Crude extraction of enzymes (4) Enzyme purification (5) Enzyme activity determination (6) Storage of lipoxygenase: The enzyme activity was stabilized by adding glycerol (20% v / v) or freezing at low temperature (-20°C).

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