A feed low-temperature sterilization method based on synergistic effect of composite biological enzymes

Abstract

This invention discloses a low-temperature sterilization method for feed based on the synergistic effect of a complex bioenzyme, comprising the following steps: a pretreatment stage; a dynamic reaction stage: under conditions of 40-50℃ and 60%-70% humidity, the enzyme-catalyzed reaction lasts for 30 minutes, decomposing the microbial structure; and residual enzyme activity is inactivated by short-term pulsed ultraviolet light. The complex enzymes include lysozyme, thermophilic protease, and lipoxygenase, and the nano-chitosan carrier is mixed with the complex enzymes at a mass ratio of 1:1. This invention avoids the destruction of heat-sensitive nutrients by operating at low temperature (40-50℃), achieves broad-spectrum sterilization by utilizing the synergistic effect of the complex enzymes, covering various microorganisms such as bacteria, molds, and spores, and simultaneously uses ultraviolet inactivation technology to ensure no chemical residues, protecting animal health and environmental safety. This method has low equipment cost, is easy to operate, and has no public acceptance issues, providing a green, efficient, and economical solution for the feed processing industry, significantly improving the efficiency and safety of feed sterilization technology.

Description

Technical Field

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

[0002] In the field of feed sterilization, existing technologies have significant drawbacks: while high-temperature sterilization can effectively kill microorganisms, its high-temperature environment can destroy heat-sensitive nutrients (such as B vitamins and amino acids), with a loss rate as high as 20%-60%, seriously affecting the nutritional value of feed; single-enzyme sterilization methods (such as lysozyme) are effective against specific bacteria, but their scope of action is limited, making it difficult to broadly inactivate molds and spores, and they are prone to failure due to increased microbial resistance; while chemical sterilization methods are low in cost, they pose a risk of chemical residues, which may harm animal health and cause environmental pollution; while irradiation sterilization methods cause less damage to nutrients, the equipment costs are high and public acceptance is low, making them difficult to promote. Summary of the Invention

[0003] To address the above technical problems, this invention provides a low-temperature sterilization method for feed based on the synergistic effect of compound 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: Under conditions of 40-50℃ and 60%-70% humidity, the enzyme-catalyzed reaction lasts for 30 minutes, decomposing the microbial structure;

[0006] Post-treatment stage: Inactivate residual enzyme activity by short-time pulsed ultraviolet light (wavelength 254nm, irradiation for 10s).

[0007] Preferred:

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

[0009] a enzyme activity ratio:

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

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

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

[0013] b. Adjuvant / synergist:

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

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

[0016] Preferred:

[0017] The feed pretreatment specifically includes the following steps:

[0018] Screening and dust removal: Feed pellets are screened by a vibrating screen (2-5mm aperture) to remove debris, dust and oversized particles; a negative pressure dust collection device (0.5-1.0m / s) is used to clean surface impurities;

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

[0020] Use an ultrasonic atomizing nozzle (frequency 1.7MHz) with an atomized particle size ≤5μm; spraying pressure 0.2-0.5MPa, nozzle-feed distance 20-30cm; spraying process: feed passes through the atomization chamber via conveyor belt (speed 0.5-1.0m / min), enzyme solution spraying amount 0.5-1.0mL / kg. Double-sided spraying mode (symmetrical layout of upper and lower nozzles) to ensure full coverage; after spraying, transfer the feed to the infiltration chamber and let it stand for 10 minutes (temperature 25±2℃, humidity 50%-60%); a low-speed turning device (5rpm) is installed in the infiltration chamber to promote the diffusion of enzyme solution into the internal pores.

[0021] Preferred: The dynamic reaction phase includes the following steps:

[0022] Thermostatic reaction chamber design requirements: The inner wall of the chamber is coated with a corrosion-resistant coating. An internal multi-layer belt conveyor system (15cm spacing between layers) ensures uniform heating of the feed. Temperature and humidity control: Temperature zoned control: inlet zone 40℃ → center zone 45℃ → outlet zone 50℃, gradient heating avoids thermal shock. Humidity is maintained at 60%-70% (±2% deviation) through a steam spray system.

[0023] The reaction conditions for enzyme-catalyzed reaction B are as follows: feed residence time 30 minutes, conveyor belt speed 0.3 m / min. Oxygen concentration in the silo ≤5% (nitrogen is introduced to replace air) to inhibit mold activity.

[0024] Mixing and homogenization: A low-speed spiral mixer (10-15 rpm) is installed inside the chamber and runs 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 is implemented, with an online microbial sensor (based on ATP bioluminescence) installed in the chamber to detect the total bacterial count every 5 minutes. If the detected value is >10 CFU / g, the reaction time is automatically extended by 5 minutes.

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

[0028] Pulsed ultraviolet inactivation: UV-C LED array (wavelength 254±5nm), power density 50mW / cm² 2 The ultraviolet chamber is 2 meters long and has an internal reflective aluminum plate to enhance the uniformity of irradiation.

[0029] Processing procedure: The feed passes through the UV chamber at a speed of 0.5 m / min, with a cumulative irradiation time of 10 seconds. Irradiation is divided into three segments (3 seconds each, with a 1-second cooling interval) to avoid localized overheating.

[0030] Rapid cooling: Using air-cooling mode: Pre-cool to 25℃ with ambient airflow (25℃, wind speed 3m / s). Cooling time: Total time ≤ 5 minutes.

[0031] This invention provides a method for the extraction and purification of lysozyme:

[0032] (1) Strains selection and culture

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

[0034] Activation and inoculation: Remove the frozen strain and 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). Incubate 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). Incubate at 37°C and 180 rpm for 24 hours until the bacterial culture reaches the logarithmic growth phase (OD600 value of 0.6-0.8).

[0035] (2) Culture and Lysozyme Secretion

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

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

[0038] Inoculation and cultivation: The 24-hour seed culture was inoculated into the large-scale fermentation medium at a ratio of 10% (v / v). Cultivation was carried out at 37°C and a shaking speed of 180 rpm for 48-72 hours. During cultivation, Streptococcus will secrete lysozyme, with enzyme activity typically reaching its maximum after 48-72 hours.

[0039] (3) Coarse extraction

[0040] Centrifugation: After fermentation, the culture medium is separated by high-speed centrifugation (10000-12000 rpm, 10-15 minutes, 4℃), and the supernatant is collected as 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 60%-80% saturation. Let stand for 30 minutes to 1 hour to precipitate the lysozyme. Separate the precipitate and supernatant using high-speed centrifugation (10000 rpm, 10 minutes, 4°C). Collect the precipitate and redissolve it with 0.05M phosphate buffer (pH 7.0) to obtain a concentrated crude enzyme solution.

[0042] (4) Dialysis

[0043] Dialysis procedure: Transfer the reconstituted crude enzyme solution to a dialysis bag, select an appropriate pore size (e.g., 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 buffer three times, with each change occurring 2 hours apart.

[0044] (5) Lysozyme purification

[0045] Ion exchange chromatography: Prepare an ion exchange column (DEAE-Sepharose or CM-Sepharose) and equilibrate it 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 the lysozyme using a linear NaCl gradient (0-0.5M NaCl) and collect different fractions. Monitor the UV absorbance (280nm) of each fraction and determine its lysozyme activity; select the fraction with the highest activity.

[0046] Molecular sieve chromatography: Lysozyme was further purified using a molecular sieve column. Elution was performed with 0.05 M phosphate buffer (pH 7.0), and the purified lysozyme was collected by detecting UV absorbance (280 nm) and enzyme activity.

[0047] (6) Enzyme activity detection

[0048] Lysozyme activity assay: Standard lysozyme activity detection methods were used.

[0049] Preparation of the reaction system: An appropriate amount of purified lysozyme solution was mixed with *Micrococcus lysodeikticus* bacterial culture at a substrate concentration of 10^6–10^7 CFU / mL, using 0.05 M phosphate buffer (pH 7.0) as the solvent. After reacting at 37°C for 30 minutes, the absorbance of the reaction solution was measured at 450 nm using a spectrophotometer. Enzyme activity was determined by monitoring changes in transparency before and after the reaction. One unit (U) of lysozyme activity was defined as the amount of enzyme capable of dissolving the cell wall of 1 microgram of *Micrococcus lysodeikticus* within 30 minutes.

[0050] (7)Save

[0051] Freezing: Aliquot the purified lysozyme solution, add 20% glycerol, mix well, and store at -20℃ or -80℃ to maintain enzyme activity. For long-term storage, aliquot the enzyme solution into smaller portions to avoid repeated freeze-thaw cycles.

[0052] This invention provides a method for extracting and purifying thermophilic proteases, comprising the following steps:

[0053] Decomposes bacterial extracellular polysaccharides and mycotoxins;

[0054] Materials rich in protein can be selected from distiller's grains as a culture medium for thermophilic protease microorganisms or as a source for direct isolation of thermophilic bacteria.

[0055] Screening and culturing thermophilic strains. Target strain: Primarily screening for *Bacillus licheniformis* strains producing thermophilic proteases. Samples were collected from distiller's grains and enriched at high temperatures (50℃~70℃) to screen for thermophilic strains. Casein or gelatin was added to the culture medium as the sole nitrogen source. Positive strains were screened using the gelatin clear zone method or casein hydrolysis assay to test whether the strains secreted proteases. Laboratory strain culture: Culture medium: Solid medium: distiller's grains powder + agar. Culture conditions: Temperature: 50℃~65℃; pH: 7.0~8.5; Time: 24~48 hours. Enzyme activity was measured after fermentation. The enzyme was extracted and purified. The isolated cells were separated by ion-exchange chromatography using a DEAE-Sepharose column to separate the target enzyme. The enzyme was further purified using Sephadex G-100 or G-200 to separate enzyme proteins of suitable molecular weight. The activity retention rate of the purified thermophilic protease at high temperatures was measured to ensure its good stability in an environment of 50℃~70℃.

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

[0057] (1) Strains selection and preparation

[0058] Strain selection:

[0059] Purchase Aspergillus niger strains from ATCC or a culture collection center.

[0060] activation:

[0061] Inoculate the cryopreserved Aspergillus niger strain onto a solid culture medium (such as PDA medium) and incubate at 28–30°C for 23 days until colonies form.

[0062] Seed culture:

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

[0064] (2) Fermentation production

[0065] Preparation of fermentation medium:

[0066] Basic formulation: A culture medium containing carbon source (glucose or starch), nitrogen source (sodium nitrate or urea), and minerals (phosphate, magnesium salt, etc.) can be used.

[0067] Sample recipe:

[0068] Glucose: 10g / L

[0069] Sodium nitrate: 3g / L

[0070] Potassium dihydrogen phosphate: 1 g / L

[0071] Magnesium sulfate: 0.5 g / L

[0072] Trace elements: 0.1g / L

[0073] pH 6.0–6.5

[0074] Fermentation conditions optimization:

[0075] Inoculation: Inoculate the seed culture into the fermentation medium at a rate of 5-10% (v / v).

[0076] Temperature: 28℃~30℃

[0077] pH: 6.0-6.5 (automatic pH adjustment can increase yield).

[0078] Gas exchange: Good ventilation must be maintained, and the stirring speed in the fermenter should be set to 150-200 rpm.

[0079] Time: Fermentation lasts approximately 72 hours. The activity of lipoxygenase in the culture medium is then measured, and fermentation is terminated once the peak value is confirmed.

[0080] Enzyme secretion:

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

[0082] (3) Crude extraction of enzymes

[0083] Centrifugation: The fermentation broth is centrifuged at high speed (10,000-15,000 rpm, 15 minutes, 4°C) to remove cells and other solid particles, and the supernatant of the fermentation broth is collected.

[0084] Precipitation: Gradually add ammonium sulfate to the supernatant until saturation reaches 60%–80%, and stir slowly to precipitate the lipoxygenase. After standing for 4 hours, centrifuge at low temperature (10,000 rpm, 10 minutes, 4°C) and collect the precipitate.

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

[0086] (4) Enzyme purification

[0087] Dialysis: Place the redissolved enzyme solution in a dialysis bag, the molecular weight cutoff of which should be approximately 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: DEAE-Sepharose was used as the medium, and the column was equilibrated with Tris-HCl buffer after packing. After loading the enzyme solution, the enzyme activity peak was collected by gradient elution (e.g., with 0–0.5 M NaCl).

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

[0090] Enzyme activity assay: Enzyme activity and protein concentration are measured after each purification step to ensure minimal loss of activity.

[0091] (5) Enzyme activity assay

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

[0093] Measurement method:

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

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

[0096] Detection: The absorbance change of the reaction system at 234 nm was measured using a spectrophotometer (linoleic acid peroxide has a specific absorption peak).

[0097] Enzyme activity units are defined as the amount of enzyme required to produce 1 μmol of peroxide per minute.

[0098] (6) Preservation of lipoxygenase

[0099] After purification, enzyme activity can be stabilized by adding glycerol (20% v / v) or by freezing at low temperature (-20°C). For dry storage, enzyme powder can be prepared by spray drying or freeze drying.

[0100] This invention proposes a low-temperature sterilization method for feed based on the synergistic effect of compound bioenzymes, with the following beneficial effects: Low-temperature operation (40-50℃) avoids the destruction of heat-sensitive nutrients; the synergistic effect of compound enzymes (lysozyme, thermophilic protease, and lipoxygenase) achieves broad-spectrum sterilization, covering various microorganisms such as bacteria, molds, and spores; and ultraviolet inactivation technology ensures no chemical residues, protecting animal health and environmental safety. Furthermore, this method has low equipment costs, is easy to operate, and has no public acceptance issues, providing the feed processing industry with a green, efficient, and economical solution, significantly improving the efficiency and safety of feed sterilization technology. Attached Figure Description

[0101] Figure 1 This is a flow chart of a low-temperature sterilization process for feed based on compound biological enzymes. Detailed Implementation

[0102] Example 1

[0103] 1. Design of composite enzymes

[0104] The core enzymes include lysozyme, thermophilic protease, and lipoxygenase.

[0105] Targeting the cell walls of Gram-positive bacteria;

[0106] Enzyme mechanisms of action: Lysozyme: Targets and breaks down the peptidoglycan layer of bacterial cell walls (especially against Salmonella and Escherichia coli). Thermophilic protease: Efficiently breaks down bacterial biofilms and aflatoxin B1 at 45°C. Lipoxygenase: Disrupts the lipid structure of pathogenic bacterial cell membranes, synergistically enhancing sterilization effects.

[0107] 1.1 Extraction and purification of lysozyme:

[0108] (1) Strains selection and culture

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

[0110] Activation and inoculation: Remove the frozen strain and 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). Incubate 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). Incubate at 37°C and 180 rpm for 24 hours until the bacterial culture reaches the logarithmic growth phase (OD600 value of 0.6-0.8).

[0111] (2) Culture and Lysozyme Secretion

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

[0113] A control group can be compared using LB medium.

[0114] Inoculation and cultivation: The 24-hour seed culture was inoculated into the large-scale fermentation medium at a ratio of 10% (v / v). Cultivation was carried out at 37°C and a shaking speed of 180 rpm for 48-72 hours. During cultivation, Streptococcus will secrete lysozyme, with enzyme activity typically reaching its maximum after 48-72 hours.

[0115] (3) Coarse extraction

[0116] Centrifugation: After fermentation, the culture medium is separated by high-speed centrifugation (10000-12000 rpm, 10-15 minutes, 4℃), and the supernatant is collected as 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 60%-80% saturation. Let stand for 30 minutes to 1 hour to precipitate the lysozyme. Separate the precipitate and supernatant using high-speed centrifugation (10000 rpm, 10 minutes, 4°C). Collect the precipitate and redissolve it with 0.05M phosphate buffer (pH 7.0) to obtain a concentrated crude enzyme solution.

[0118] (4) Dialysis

[0119] Dialysis procedure: Transfer the reconstituted crude enzyme solution to a dialysis bag, select an appropriate pore size (e.g., 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 buffer three times, with each change occurring 2 hours apart.

[0120] (5) Lysozyme purification

[0121] Ion exchange chromatography: Prepare an ion exchange column (DEAE-Sepharose or CM-Sepharose) and equilibrate it 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 the lysozyme using a linear NaCl gradient (0-0.5M NaCl) and collect different fractions. Monitor the UV absorbance (280nm) of each fraction and determine its lysozyme activity; select the fraction with the highest activity.

[0122] Molecular sieve chromatography: Lysozyme was further purified using a molecular sieve column. Elution was performed with 0.05 M phosphate buffer (pH 7.0), and the purified lysozyme was collected by detecting UV absorbance (280 nm) and enzyme activity.

[0123] (6) Enzyme activity detection

[0124] Lysozyme activity assay: Standard lysozyme activity detection methods were used.

[0125] Preparation of the reaction system: An appropriate amount of purified lysozyme solution was mixed with *Micrococcus lysodeikticus* bacterial culture at a substrate concentration of 10^6–10^7 CFU / mL, using 0.05 M phosphate buffer (pH 7.0) as the solvent. After reacting at 37°C for 30 minutes, the absorbance of the reaction solution was measured at 450 nm using a spectrophotometer. Enzyme activity was determined by monitoring changes in transparency before and after the reaction. One unit (U) of lysozyme activity was defined as the amount of enzyme capable of dissolving the cell wall of 1 microgram of *Micrococcus lysodeikticus* within 30 minutes.

[0126] (7)Save

[0127] Freezing: Aliquot the purified lysozyme solution, add 20% glycerol, mix well, and store at -20℃ or -80℃ to maintain enzyme activity. For long-term storage, aliquot the enzyme solution into smaller portions to avoid repeated freeze-thaw cycles.

[0128] 1.2 Extraction and purification of thermophilic proteases: decomposition of bacterial extracellular polysaccharides and mycotoxins;

[0129] Materials rich in protein can be selected from distiller's grains as a culture medium for thermophilic protease microorganisms or as a source for direct isolation of thermophilic bacteria.

[0130] Screening and culturing thermophilic strains. Target strain: Primarily screening for *Bacillus licheniformis* strains producing thermophilic proteases. Samples were collected from distiller's grains and enriched at high temperatures (50℃~70℃) to screen for thermophilic strains. Casein or gelatin was added to the culture medium as the sole nitrogen source. Positive strains were screened using the gelatin clear zone method or casein hydrolysis assay to test whether the strains secreted proteases. Laboratory strain culture: Culture medium: Solid medium: distiller's grains powder + agar. Culture conditions: Temperature: 50℃~65℃; pH: 7.0~8.5; Time: 24~48 hours. Enzyme activity was measured after fermentation. The enzyme was extracted and purified. The isolated cells were separated by ion-exchange chromatography using a DEAE-Sepharose column to separate the target enzyme. The enzyme was further purified using Sephadex G-100 or G-200 to separate enzyme proteins of suitable molecular weight. The activity retention rate of the purified thermophilic protease at high temperatures was measured to ensure its good stability in an environment of 50℃~70℃.

[0131] 1.3 Extraction and purification of lipoxygenase:

[0132] (1) Strains selection and preparation

[0133] Strain selection:

[0134] Purchase Aspergillus niger strains from ATCC or a culture collection center.

[0135] activation:

[0136] Inoculate the cryopreserved Aspergillus niger strain onto a solid culture medium (such as PDA medium) and incubate at 28–30°C for 23 days until colonies form.

[0137] Seed culture:

[0138] The activated strain was transferred to a liquid seed culture medium (such as a liquid culture medium containing glucose, sodium nitrate, etc.), and cultured in a shake flask at 28°C, 180 rpm for 24 hours to obtain the seed culture.

[0139] (2) Fermentation production

[0140] Preparation of fermentation medium:

[0141] Basic formulation: A culture medium containing carbon source (glucose or starch), nitrogen source (sodium nitrate or urea), and minerals (phosphate, magnesium salt, etc.) can be used.

[0142] Sample recipe:

[0143] Glucose: 10g / L

[0144] Sodium nitrate: 3g / L

[0145] Potassium dihydrogen phosphate: 1 g / L

[0146] Magnesium sulfate: 0.5 g / L

[0147] Trace elements: 0.1g / L

[0148] pH 6.0–6.5

[0149] Fermentation conditions optimization:

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

[0151] Temperature: 28℃~30℃

[0152] pH: 6.0-6.5 (automatic pH adjustment can increase yield).

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

[0154] Time: Fermentation lasts approximately 72 hours. The activity of lipoxygenase in the culture medium is then measured, and fermentation is terminated once the peak value is confirmed.

[0155] Enzyme secretion:

[0156] Aspergillus niger secretes lipoxygenase into the fermentation broth, and the enzyme’s main activity is found in the supernatant of the culture medium.

[0157] (3) Crude extraction of enzymes

[0158] Centrifugation: The fermentation broth is centrifuged at high speed (10,000-15,000 rpm, 15 minutes, 4°C) to remove cells and other solid particles, and the supernatant of the fermentation broth is collected.

[0159] Precipitation: Gradually add ammonium sulfate to the supernatant until saturation reaches 60%–80%, and stir slowly to precipitate the lipoxygenase. After standing for 4 hours, centrifuge at low temperature (10,000 rpm, 10 minutes, 4°C) and collect the precipitate.

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

[0161] (4) Enzyme purification

[0162] Dialysis: Place the redissolved enzyme solution in a dialysis bag, the molecular weight cutoff of which should be approximately 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.

[0163] Ion exchange chromatography: DEAE-Sepharose was used as the medium, and the column was equilibrated with Tris-HCl buffer after packing. After loading the enzyme solution, the enzyme activity peak was collected by gradient elution (e.g., with 0–0.5 M NaCl).

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

[0165] Enzyme activity assay: Enzyme activity and protein concentration are measured after each purification step to ensure minimal loss of activity.

[0166] (5) Enzyme activity assay

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

[0168] Measurement method:

[0169] Substrate preparation: Prepare a 0.1M linoleic acid solution, using Tween-20 as an emulsifier.

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

[0171] Detection: The absorbance change of the reaction system at 234 nm was measured using a spectrophotometer (linoleic acid peroxide has a specific absorption peak).

[0172] Enzyme activity units are defined as the amount of enzyme required to produce 1 μmol of peroxide per minute.

[0173] (6) Preservation of lipoxygenase

[0174] After purification, enzyme activity can be stabilized by adding glycerol (20% v / v) or by freezing at low temperature (-20°C). For dry storage, enzyme powder can be prepared by spray drying or freeze drying.

[0175] 1.4 Auxiliary synergists: Nanoscale chitosan carrier: immobilizes enzyme activity and prolongs the action time; Natural plant extract tea polyphenols: inhibit secondary contamination after sterilization.

[0176] Example 2

[0177] Staged low-temperature sterilization process

[0178] Pretreatment stage: Enzyme atomization spraying technology (particle size ≤5μm) is used to ensure that the compound enzyme is evenly attached to the feed surface and penetrates into the internal pores. Dynamic reaction stage: Under conditions of 40-50℃ and 60%-70% humidity, the enzyme-catalyzed reaction lasts for 30 minutes, decomposing the microbial structure. Post-treatment stage: Residual enzyme activity is inactivated by short-time pulsed ultraviolet light (wavelength 254nm, irradiation for 10s) to avoid residual effects.

[0179] Example 3

[0180] Sterilization process:

[0181] (1) Feed pretreatment

[0182] Screening and dust removal: Feed pellets are passed through a vibrating screen (2-5mm aperture) to remove debris, dust, and oversized particles. A negative pressure dust collection device (0.5-1.0m / s) is used to clean surface impurities.

[0183] Surface wetting: A micron-level water mist spraying system is used to increase the surface humidity of the feed to 10%-15% (controlled in real time by a humidity sensor). The wetting water is deionized water to avoid minerals interfering with enzyme activity.

[0184] (2) Preparation of compound enzyme solution

[0185] a enzyme activity ratio:

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

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

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

[0189] b. Adjuvant / synergist:

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

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

[0192] c High-pressure atomized spraying

[0193] Equipment parameters: Uses ultrasonic atomizing nozzles (frequency 1.7MHz), atomized particle size ≤5μm. Spraying pressure 0.2-0.5MPa, nozzle-feed distance 20-30cm. Spraying process: Feed passes through the atomization chamber via conveyor belt (speed 0.5-1.0m / min), enzyme solution spraying volume 0.5-1.0mL / kg. Double-sided spraying mode (symmetrical layout of upper and lower nozzles) ensures full coverage. After spraying, the feed is transferred to the infiltration chamber and allowed to stand for 10 minutes (temperature 25±2℃, humidity 50%-60%). A low-speed turning device (5rpm) is installed inside the infiltration chamber to promote enzyme solution diffusion into the internal pores.

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

[0195] Under low temperature conditions, the synergistic sterilization effect of the complex enzymes is activated, decomposing the microbial structure.

[0196] Thermostatic reaction chamber design requirements: The inner wall of the chamber is coated with a corrosion-resistant coating. An internal multi-layer belt conveyor system (15cm spacing between layers) ensures uniform heating of the feed. Temperature and humidity control: Temperature zoned control: inlet zone 40℃ → center zone 45℃ → outlet zone 50℃, gradient heating avoids thermal shock. Humidity is maintained at 60%-70% (±2% deviation) through a steam spray system.

[0197] The reaction conditions for enzyme-catalyzed reaction B are as follows: feed residence time 30 minutes, conveyor belt speed 0.3 m / min. Oxygen concentration in the silo ≤5% (nitrogen is introduced to replace air) to inhibit mold activity.

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

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

[0200] Real-time quality monitoring is implemented, with an online microbial sensor (based on ATP bioluminescence) installed in the chamber to detect the total bacterial count every 5 minutes. If the detected value is >10 CFU / g, the reaction time is automatically extended by 5 minutes.

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

[0202] To prevent enzymatic hydrolysis from affecting feed quality during long-term storage, it is necessary to inactivate residual enzyme activity.

[0203] Steps and parameters:

[0204] Pulsed ultraviolet inactivation: UV-C LED array (wavelength 254±5nm), power density 50mW / cm²2 The ultraviolet chamber is 2 meters long and has an internal reflective aluminum plate to enhance the uniformity of irradiation.

[0205] Processing procedure: The feed passes through the UV chamber at a speed of 0.5 m / min, with a cumulative irradiation time of 10 seconds. Irradiation is divided into three segments (3 seconds each, with a 1-second cooling interval) to avoid localized overheating.

[0206] Rapid cooling: Using air-cooling mode: Pre-cool to 25℃ with ambient airflow (25℃, fan speed 3m / s). Cooling time: Total time ≤ 5 minutes.

[0207] The process flow diagram of low-temperature sterilization of feed based on compound bio-enzymes is as follows: Figure 1 As shown

[0208] Experimental Example

[0209] Experimental methods

[0210] 1.1 Sample Grouping and Processing

[0211] This study selected eight samples of homemade feed from different sources, with each sample weighing 2 kg. Based on the different treatment methods, the samples were divided into two groups for the experiment:

[0212] Control group: No treatment was performed; this group served as the baseline control.

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

[0214] After processing, all samples were left to stand for 7 days in the same experimental environment (temperature: 20℃, humidity: 65%), pending further analysis.

[0215] 1.2 Detection of Microbiological Hygiene Indicators

[0216] According to the relevant provisions of the national standard GB 14924.2-2001 "Hygienic Standard for Compound Feed of Laboratory Animals", the microbiological hygiene indicators in the samples were analyzed. The tests included total bacterial count, coliform bacteria, the number of molds and yeasts, and the presence of pathogenic microorganisms. The results were compared with the reference values ​​of the national standard to assess whether each microbiological indicator met the hygiene requirements.

[0217] 1.3 Nutritional Composition Analysis

[0218] After completing the microbiological hygiene testing, the sample group that met the sterilization standards and the control group were retained for further nutritional analysis. The main nutritional components of the samples were tested according to GB 14924.3-2010 "Nutritional Composition of Compound Feed for Laboratory Animals". Specific testing items include, but are not limited to:

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

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

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

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

[0223] 1.4 Data Statistical Analysis

[0224] All data were processed using Excel 2006 and statistically analyzed using SPSS 19.0. First, a normality test was performed on the measurement data; data conforming to a normal distribution were represented by mean ± standard deviation. Next, a homogeneity of variance test was performed. If the data met the homogeneity assumption, one-way ANOVA was used for between-group comparisons. If significant differences were detected, the Student-Newman-Keuls method was further used for within-group comparisons. The statistical significance level was set at α = 0.05 (two-tailed test).

[0225] Table 1. Results of determination of sanitary microbiological indicators in feed before and after sterilization

[0226]

[0227] Table 1 shows that the total bacterial count decreased from 2.4 × 10^4 CFU / g to <10 CFU / g after sterilization, a reduction of 99.96%. The coliform count decreased from 7.8 × 10^2 CFU / g to <30 CFU / g, a reduction of over 96%, meeting feed hygiene standards. The mold and yeast count decreased from 3.5 × 10^2 CFU / g to <10 CFU / g, a reduction of over 97%. Pathogenic bacteria (Salmonella) were not detected before or after sterilization, indicating that the equipment has a significant effect on killing pathogens. After sterilization, the content of harmful microorganisms in the feed was significantly reduced, fully complying with national standards (such as GB 13078-2017 "Feed Hygiene Standard"), ensuring the hygiene and safety of the feed.

[0228] Table 2. Effects of low-temperature sterilization of compound enzymes on conventional nutrient components of feed (g / kg)

[0229]

[0230] Table 2 shows that the moisture content 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 indicates that equipment sterilization has minimal impact on the conventional nutritional components of the feed, especially with crude protein and crude fat losses both below 2%, demonstrating that the low-temperature sterilization process using compound enzymes effectively preserves the main nutritional components of the feed.

[0231] Table 3. Effects of sterilization on mineral content in feed.

[0232]

[0233] Table 3 shows that 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 showed no change, with a loss rate of 0.0%. This indicates that low-temperature sterilization with compound enzymes has minimal impact on the mineral content of the feed, with the loss rate of all minerals being less than 1.1%, demonstrating that this sterilization process can effectively retain the minerals in the feed.

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

[0235]

[0236] Table 4 shows that 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%. Vitamins B1, B2, and B6 showed no significant changes (P>0.05), with loss rates all below 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 low-temperature sterilization with compound enzymes has minimal impact on the vitamin content in feed, with all vitamins showing a loss rate below 0.5%, demonstrating that this sterilization process effectively preserves vitamins in the feed.

[0237] Table 5. Effects of sterilization on amino acids in feed.

[0238]

[0239] As shown in Table 5, the overall amino acid loss rate after low-temperature sterilization of the compound enzyme was less than 2%, which is extremely low and effectively ensures the content of amino acid nutrients in the original feed.

[0240] The experiment shows that the low-temperature sterilization method for feed based on the synergistic effect of compound biological enzymes proposed in this patent has a significant effect on killing pathogens, and the nutrient loss rate of conventional nutrients, minerals, vitamins and amino acids is very low before and after sterilization. It can retain the nutrient components of feed to the maximum extent, and has significant technical advantages and application value.

[0241] The low-temperature sterilization method for feed based on the synergistic effect of compound biological enzymes proposed in this invention has the following significant advantages:

[0242] Highly efficient broad-spectrum sterilization: Through the synergistic action of compound enzymes (lysozyme, thermophilic protease, and lipoxygenase), it can target and decompose the peptidoglycan layer of bacterial cell walls (such as Salmonella and Escherichia coli), efficiently decompose bacterial biofilms and aflatoxin B1, and destroy the lipid structure of pathogenic bacterial cell membranes, achieving broad-spectrum inactivation of various microorganisms such as bacteria, molds, and spores, with a sterilization rate of over 99.9%.

[0243] Low-temperature operation protects nutrients: The sterilization process is carried out at a low temperature of 40-50℃ to avoid the destruction of heat-sensitive nutrients (such as B vitamins and amino acids) by high temperatures, thus ensuring the nutritional value of the feed. Experimental data show that the vitamin B1 retention rate is ≥97% and the crude protein loss rate is ≤3%, which is significantly better than traditional high-temperature sterilization methods.

[0244] No chemical residues, safe and environmentally friendly: It adopts biological enzyme and ultraviolet inactivation technology to avoid the residual risks of chemical sterilization methods, protect animal health and environmental safety, and meet the requirements of green and sustainable development.

[0245] Low cost and easy to promote: Compared with irradiation sterilization, this method has lower equipment costs, is easier to operate, and has no public acceptance issues. It is suitable for large-scale feed production lines and has high economic value and promotion potential.

[0246] Intelligent control and high consistency: Combining real-time monitoring and dynamic adjustment technology, it ensures consistent sterilization effect, reduces energy consumption by more than 20%, and improves production efficiency.

[0247] Feasibility analysis

[0248] Technical Feasibility: Lysozyme, thermophilic protease, and lipoxygenase are all extracted and purified from existing mature strains, making the technology mature and low-cost. This patent adopts a staged low-temperature sterilization process (enzyme atomization spraying → low-temperature reaction → ultraviolet inactivation), with clear process steps, controllable parameters, and easy industrial production. Experimental data show that the total bacterial count in the sterilized feed is ≤10 CFU / g, and Salmonella was not detected, fully complying with national standards (such as GB 13078-2017 "Feed Hygiene Standard").

[0249] The production cost of compound enzymes is low, and this cost can be further reduced through large-scale production. Low-temperature operation reduces energy consumption, and ultraviolet inactivation technology requires no expensive equipment, resulting in an overall cost significantly lower than irradiation sterilization. This method aligns with the trend of green agriculture and possesses high market competitiveness.

[0250] This invention, through the synergistic effect of compound enzymes and the innovative design of a low-temperature sterilization process, solves the problems of high temperature destroying nutrients, limited range of action of single enzymes, high risk of chemical residues, and high irradiation costs in existing technologies. It boasts multiple advantages, including highly efficient sterilization, nutrient retention, safety and environmental friendliness, and low cost. Its technology is mature, the process is feasible, it is highly economical, and it has broad market prospects, demonstrating significant industrialization feasibility and providing 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 compound biological enzymes, characterized in that: Includes the following steps: Pretreatment stage: Enzyme atomization spraying technology is used to make the compound enzymes evenly adhere to the surface of the feed and penetrate into the internal pores; Dynamic reaction stage: Under conditions of 40-50℃ and 60%-70% humidity, the enzyme-catalyzed reaction lasts for 30 minutes, decomposing the microbial structure; Post-treatment stage: Inactivation of residual enzyme activity by short-pulse ultraviolet light; The preparation of the complex enzyme solution includes: a. Enzyme activity ratio: Lysozyme: accounts for 40% of total enzyme activity; Thermophilic proteases: accounting for 35% of total enzyme activity; Lipoxygenase: accounts for 25% of total enzyme activity; b. Adjuvants and synergists: Nano-chitosan carrier: mixed with enzyme at a mass ratio of 1:1; Tea polyphenols: Addition amount 0.1%-0.3% w / w; The feed pretreatment specifically includes the following steps: a. Screening and dust removal b. Surface wetting c. Use ultrasonic atomizing nozzles with atomized particle size ≤5μm; feed is conveyed through the atomizing chamber, and the amount of enzyme solution sprayed is 0.5-1.0mL / kg; after spraying, the feed is transferred to the infiltration chamber and left to stand for 10 minutes at a temperature of 25±2℃ and a humidity of 50%-60%; the feed is turned over at a low speed in the infiltration chamber. The post-processing stage includes: Pulsed ultraviolet inactivation: UV-C LED array with wavelength 254±5nm and power density 50mW / cm²; The feed passes through the ultraviolet chamber at a speed of 0.5 m / min, with a cumulative irradiation time of 10 seconds; the irradiation is divided into three segments, each lasting 3 seconds, with a 1-second cooling interval. Rapid cooling: Using air cooling mode: ambient airflow, 25℃, wind speed 3m / s, pre-cool to 25℃; cooling time: total time ≤ 5 minutes.

2. The low-temperature sterilization method for feed based on the synergistic effect of compound biological enzymes according to claim 1, characterized in that: The dynamic response phase includes: a. Temperature control in the constant temperature reaction chamber: inlet zone 40℃ → center zone 45℃ → outlet zone 50℃; humidity maintained at 60%-70%; b. Enzyme-catalyzed reaction conditions: feed residence time 30 minutes, conveyor belt speed 0.3 m / min; oxygen concentration in the silo ≤ 5%; c. Stirring and homogenization.

3. The low-temperature sterilization method for feed based on the synergistic effect of compound biological enzymes according to claim 2, characterized in that: An online microbial sensor is installed in the constant temperature reaction chamber to detect the total number of colonies every 5 minutes; if the detected value is >10 CFU / g, the reaction time is automatically extended by 5 minutes.

4. The low-temperature sterilization method for feed based on the synergistic effect of compound biological enzymes according to claim 1, characterized in that: The extraction and purification of lysozyme includes the following steps: (1) Strains selection and culture Strain selection: Select streptococcal strains with high lysozyme activity; Activation and inoculation; (2) Culture and lysozyme secretion (3) Crude extraction (4) Dialysis (5) Lysozyme purification (6) Enzyme activity detection (7) Storage: Dispense the purified lysozyme solution into smaller portions, add 20% glycerol, mix well, and store at -20°C or -80°C.

5. The low-temperature sterilization method for feed based on the synergistic effect of compound biological enzymes according to claim 1, characterized in that: The extraction and purification of thermophilic proteases includes: Decomposes bacterial extracellular polysaccharides and mycotoxins; Materials rich in protein can be selected from distiller's grains as a culture medium for thermophilic protease microorganisms or as a source for direct isolation of thermophilic bacteria.

6. The low-temperature sterilization method for feed based on the synergistic effect of compound biological enzymes according to claim 1, characterized in that: The extraction and purification of lipoxygenase includes the following steps: (1) Selection and preparation of strains Strain selection: Purchase Aspergillus niger strain from ATCC or a culture collection center; Activation: Inoculate the cryopreserved Aspergillus niger strain onto a solid culture medium and incubate at 28-30°C for 23 days until colonies form; Seed culture; (2) Fermentation production (3) Crude extraction of enzymes (4) Enzyme purification (5) Enzyme activity assay (6) Preservation of lipoxygenase: stabilize enzyme activity by adding 20% ​​v / v glycerol or by freezing at -20°C.

Images