Production process of bacteriostatic fermented feed based on dynamic regulation and phenolic acid precursor synergy
Through multi-stage dynamic regulation and synergistic fermentation process with phenolic acid precursors, the problems of low and unstable yield of antibacterial substances in existing fermented feeds have been solved, resulting in a significant improvement in antibacterial function and product stability, and promoting the development of antibiotic-free aquaculture.
Patent Information
- Application Number
- CN202511994999.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-02-27
AI Technical Summary
In current fermented feed production, the yield of antibacterial substances is low and unstable, the fermentation process is extensive, the added value of the products is low, and targeted production cannot be achieved.
The fermentation process employs multi-stage dynamic regulation and synergistic effects with phenolic acid precursors. By controlling parameters such as temperature, time, and dissolved oxygen in stages, and combining this with the addition of phenolic acid precursors, the process promotes the metabolism of probiotics and the generation of antibacterial substances.
It significantly enhances antibacterial function, improves product stability and repeatability, and has the potential to inhibit pathogenic bacteria in the animal gut, thus promoting the development of antibiotic-free aquaculture.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of feed fermentation technology, and in particular to a production process for antibacterial fermented feed based on dynamic regulation and synergistic effects of phenolic acid precursors. Background Technology
[0002] Fermented feed, especially fermented products using plant protein raw materials such as soybean meal as the main substrate, has become an important direction for improving the digestibility and utilization of raw materials and enhancing animal gut health in the feed industry. Current fermented feed production often uses single raw materials, single strains, or simple compound strains, and involves static fermentation at fixed temperatures and durations. This traditional process has the following significant drawbacks: 1) Low and unstable yield of antibacterial substances: Fixed environmental parameters cannot simultaneously meet the needs of rapid bacterial proliferation and efficient synthesis of antibacterial substances (such as bacteriocins and organic acids), resulting in limited antibacterial efficacy of the final product and significant batch-to-batch variations. 2) Inefficient fermentation process: It cannot precisely guide the metabolic flow of microorganisms; the fermentation endpoint relies heavily on experience, making it impossible to achieve targeted production for the specific function of "antibacterial inhibition." 3) Low added value: The resulting product is mainly used as a conventional protein raw material, and its potential in replacing antibiotics and improving gut health has not been fully explored.
[0003] Therefore, there is an urgent need in this field to develop a fermentation production process that can break through the framework of static fermentation, achieve stable, efficient and targeted production of antibacterial active substances through refined and dynamic control of the fermentation process and coordinated targeted biotransformation of functional precursor substances. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a production process for antibacterial fermented feed based on dynamic regulation and synergistic effects of phenolic acid precursors.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] A production process for antibacterial fermented feed based on dynamic regulation and synergistic effects of phenolic acid precursors is provided, comprising the following steps:
[0007] S1. Ingredients and Mixing: After crushing the main raw material, add agricultural by-products rich in phenolic acid precursors at a mass ratio of 10%-15% of the main raw material, and glucose or molasses at a mass ratio of 1%-3% of the main raw material, and mix evenly; then add purified water at a mass ratio of material to purified water of 1:0.4 to 1:0.5, stir evenly, and prepare a fermentation substrate with a water content of 50%-60%;
[0008] S2. Sterilization and cooling: The fermentation substrate is sterilized and then cooled.
[0009] S3. Enzymatic hydrolysis: Add a complex enzyme to the cooled fermentation substrate and hydrolyze it at 40-45℃ for 6-8 hours to obtain the enzymatically hydrolyzed fermentation substrate.
[0010] S4. Inoculation and Fermentation: The activated compound bacterial solution is inoculated into the enzymatic fermentation substrate at an inoculation rate of 3 v / w.%-5 v / w.%, stirred evenly, and then subjected to three stages of segmented fermentation:
[0011] First stage: Slowly stir at 35-40℃ and 80-120rpm, and aerate for 3-5 minutes per hour at an aeration rate of 0.1-0.3vvm, ferment for 20-24 hours or until the pH of the system drops below 4.5;
[0012] This stage is the period of bacterial density enrichment, which can rapidly increase the number of lactic acid bacteria, yeast and Bacillus, establish bacterial superiority, prevent contamination by other bacteria, and lay the foundation for the subsequent accumulation of antibacterial components.
[0013] Second stage: Reduce the temperature to 30-35℃, stop stirring and ventilation, and let it ferment for 20-30 hours.
[0014] This stage is the period of efficient synthesis of antibacterial substances. During this period, lactic acid bacteria and Bacillus subtilis can be induced to synthesize a large amount of target antibacterial substances, such as organic acids, lactobacillusin, and subtilisin.
[0015] Third stage: Lower the temperature further to 25-30℃ and let it ferment for 10-14 hours.
[0016] This stage is the product stabilization and retention period, the purpose of which is to reduce the metabolic activity of the bacteria and stabilize and retain the synthesized antibacterial substances.
[0017] S5. Drying and Packaging: Dry the fermented material at a temperature not exceeding 60°C until the moisture content is below 12%, and then seal and package it after cooling.
[0018] Further, in step S1, the main raw material is one or a mixture of soybean meal, cottonseed meal, and rapeseed meal, and the particle size is 40-60 mesh.
[0019] Further, in step S1, the agricultural by-product rich in phenolic acid precursors is one or more of grape seed meal, apple pomace, and green tea residue, or a mixture thereof.
[0020] Further, in step S2, the fermentation substrate is sterilized at 80-100°C for 15-30 minutes, and then cooled to 40-45°C.
[0021] In a further step S3, the complex enzyme comprises an alkaline protease, a cellulase, and a pectinase, added in amounts of 0.3%-0.5% of the fermentation substrate mass, a cellulase, and a pectinase, respectively.
[0022] Further, in step S4, the compound bacterial solution is selected from two or three of lactic acid bacteria, Bacillus subtilis and yeast; the lactic acid bacteria are Lactobacillus acidophilus or Lactobacillus plantarum.
[0023] Furthermore, the activation method of the compound bacterial solution is as follows: dissolve the compound bacterial agent and glucose in water, and activate at 35-37℃ and 180-200 rpm for 40-60 minutes; wherein, the amount of compound bacterial agent added is 1-2 kg / (300-500L water), and the amount of glucose added is 500-1000 g / (300-500L water).
[0024] Furthermore, in step S2, the second stage also includes: starting anaerobic control by introducing nitrogen into the fermenter to maintain the dissolved oxygen (DO) value at 0.2% ± 0.1%.
[0025] Furthermore, in step S5, a low-temperature airflow drying method is used for drying.
[0026] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:
[0027] This invention achieves the following beneficial effects by dynamically controlling fermentation conditions in multiple stages and combining this with the addition of agricultural by-products rich in phenolic acid precursors:
[0028] 1) Significantly enhanced antibacterial function: Through phased dynamic regulation of the "bacterial proliferation phase—antibacterial substance synthesis phase—product stabilization phase" (including precise control of temperature, time, dissolved oxygen, and stirring), an optimal environment is created for probiotic metabolism and the synthesis of antibacterial substances. Combined with exogenously added phenolic acid precursors, the generation of antibacterial active substances is synergistically promoted. Experiments show that the obtained product has a significant inhibitory effect on Escherichia coli K99 and Staphylococcus aureus.
[0029] 2) Enhanced process controllability and product stability: The multi-stage dynamic control strategy allows for targeted adjustment of key fermentation parameters (such as pH, temperature, time, and dissolved oxygen) according to the fermentation process. This process route is clear and the conditions are well-defined, effectively reducing fluctuations associated with traditional static fermentation, improving production repeatability and product quality consistency, and facilitating industrial-scale promotion.
[0030] 3) Contributes to the development of antibiotic-free aquaculture: This invention provides a process for directly producing fermented feed with antibacterial properties. This product, through its contained antibacterial active substances, has the potential to regulate the intestinal flora of animals and inhibit specific pathogenic bacteria during the aquaculture process, providing a new solution for reducing antibiotic use. Attached Figure Description
[0031] Figure 1 The antibacterial properties of fermented soybean meal against Escherichia coli were tested using the perforation method; wherein, well 1 was the fermented soybean meal prepared in Example 1, well 2 was the fermented soybean meal prepared in Example 1, well 3 was the fermented soybean meal prepared in Example 1, well 4 was ordinary fermented soybean meal, well 5 was ordinary fermented soybean meal, and well 6 was ordinary fermented soybean meal.
[0032] Figure 2 The antibacterial properties of fermented soybean meal against Staphylococcus aureus were tested using the perforation method; wherein, well 7 was the fermented soybean meal prepared in Example 1, well 8 was the fermented soybean meal prepared in Example 1, well 9 was the fermented soybean meal prepared in Example 1, well 10 was ordinary fermented soybean meal, well 11 was ordinary fermented soybean meal, and well 12 was ordinary fermented soybean meal.
[0033] Figure 3 Growth curves showing the antibacterial effects of different fermented feed extracts on Escherichia coli K99. Detailed Implementation
[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.
[0035] Example 1
[0036] This embodiment provides a production process for antibacterial fermented feed based on dynamic regulation and synergistic effects of phenolic acid precursors. The specific implementation steps are as follows:
[0037] S1. Ingredients and Mixing:
[0038] 1000 kg of pulverized 46% protein soybean meal (as the main raw material) was mixed evenly with agricultural by-products rich in phenolic acid precursors (60 kg of grape seed meal, 60 kg of apple pomace, and 10 kg of green tea residue) and 15 kg of molasses. Then, 600 kg of purified water was added at a material-to-purified water mass ratio of approximately 1:0.5, and the mixture was stirred evenly (for 3 minutes) to prepare a fermentation substrate with a moisture content of approximately 50%.
[0039] S2. Sterilization and cooling:
[0040] The fermentation substrate was transferred to a fermentation tank, sterilized by introducing saturated steam, and maintained at 80°C for 30 minutes. Then, the material was cooled to 40°C using a cooling system.
[0041] S3, Enzymatic hydrolysis:
[0042] Add a complex enzyme, consisting of 0.3% alkaline protease, 0.05% cellulase, and 0.2% pectinase by weight of the fermentation substrate, to the cooled fermentation substrate. After stirring evenly, enzymatically hydrolyze the substrate at 40°C for 8 hours to obtain the enzymatically hydrolyzed fermentation substrate.
[0043] S4. Inoculation and Fermentation:
[0044] The activated compound bacterial solution (in which the live count ratio of Lactobacillus plantarum, Bacillus subtilis, and baker's yeast is 3:1:1) was inoculated into the enzymatic fermentation substrate at an inoculation rate of 4 v / w.%, stirred evenly, and then carried out staged fermentation:
[0045] First stage (cell proliferation period): Intermittent slow stirring at 37℃ and 100 rpm (stirring for 5 minutes and aerating at 0.2 vvm, let stand for 55 minutes, and circulate once per hour).
[0046] Fermentation time was approximately 20 hours. During this period, the online pH sensor showed that the pH dropped rapidly from the initial 6.8. When the pH dropped to 4.5, Phase Two began.
[0047] Second stage (antibacterial substance synthesis period): Reduce the temperature to 32℃, stop stirring and ventilation, start anaerobic control, introduce nitrogen into the fermenter, and maintain the dissolved oxygen (DO) value at 0.2% ± 0.1%;
[0048] This phase lasts for 24 hours, with online monitoring of pH changes. Once the pH changes slowly and approaches 4.0, phase three can begin.
[0049] The third stage (product stabilization period): The temperature is further reduced to 27°C, and fermentation is allowed to proceed for 12 hours until the pH value of the system stabilizes at 3.9.
[0050] S5. Drying and Packaging:
[0051] The fermented material is dried at 55°C using a low-temperature airflow drying method until the moisture content is below 12%, and then sealed and packaged after cooling.
[0052] The fermented soybean meal samples prepared above were extracted at a solid-liquid ratio of 1:3 for 30 min, and the supernatant was collected by centrifugation. The supernatant was sterilized at 121℃ for 20 min, and then centrifuged again to remove the precipitate, yielding the antibacterial extract of fermented soybean meal. This extract was then stored at 4℃ for later use. The antibacterial properties of the fermented soybean meal were tested using the perforation method (small holes were slowly punched in LB agar plates contaminated with Escherichia coli K99 and Staphylococcus aureus, the test liquid was added, the plate was stored at 4℃ for 3 h, then placed in a 37℃ incubator, and the size of the inhibition zone was observed after 12 h). The results were compared with those of ordinary fermented soybean meal, with three replicates per group. The results are shown in the figure below. Figure 1 and Figure 2 .
[0053] In the control example (ordinary fermented soybean meal): 1000 kg of pulverized 46% protein soybean meal was mixed evenly with 15 kg of molasses. Water was added at a material-to-water ratio of 1:0.5, and the mixture was stirred thoroughly. After sterilization and cooling, a compound bacterial solution of the same type and amount as in Example 1 (Lactobacillus plantarum: Bacillus subtilis: baker's yeast = 3:1:1) was inoculated, and static fermentation was carried out at 37°C for 56 hours (i.e., no segmented dynamic control was performed, maintaining a constant temperature, and no special stirring or dissolved oxygen regulation was applied). Subsequent drying and extraction methods were the same as in Example 1.
[0054] like Figure 1 As shown: wells 1, 2 and 3 have inhibition zones, indicating a significant antibacterial effect; wells 4, 5 and 6 do not have inhibition zones, indicating no antibacterial effect; it can be seen that the fermented soybean meal produced by this invention has an antibacterial effect on Escherichia coli.
[0055] like Figure 2 As shown: wells 7, 8 and 9 have inhibition zones, indicating a significant antibacterial effect; wells 10, 11 and 12 do not have inhibition zones, indicating no antibacterial effect; it can be seen that the fermented soybean meal produced by this invention has an antibacterial effect against Staphylococcus aureus.
[0056] Example 2
[0057] This embodiment provides a production process for antibacterial fermented feed based on dynamic regulation and synergistic effects of phenolic acid precursors. The specific implementation steps are as follows:
[0058] S1. Ingredients and Mixing:
[0059] 700 kg of crushed 46% protein soybean meal and 300 kg of cottonseed meal (as the main raw materials) were mixed evenly with agricultural by-products rich in phenolic acid precursors (50 kg of grape seed meal and 70 kg of apple pomace) and 10 kg of molasses. Then, 565 kg of purified water was added at a material-to-purified water mass ratio of approximately 1:0.5, and the mixture was stirred evenly (for 3 minutes) to prepare a fermentation substrate with a moisture content of approximately 56%.
[0060] S2. Sterilization and cooling:
[0061] The fermentation substrate was transferred to a fermentation tank, sterilized by introducing saturated steam, and maintained at 90°C for 20 minutes. Then, the material was cooled to 40°C using a cooling system.
[0062] S3, Enzymatic hydrolysis:
[0063] Add a complex enzyme, consisting of 0.4% alkaline protease, 0.05% cellulase, and 0.1% pectinase by weight of the fermentation substrate, to the cooled fermentation substrate. After stirring evenly, enzymatically hydrolyze the substrate at 40°C for 6 hours to obtain the enzymatically hydrolyzed fermentation substrate.
[0064] S4. Inoculation and Fermentation:
[0065] The activated compound bacterial solution (in which the live count ratio of Lactobacillus acidophilus to Bacillus subtilis is 3:1) was inoculated into the enzymatic fermentation substrate at an inoculation rate of 5 v / w.%, stirred evenly, and then carried out staged fermentation:
[0066] First stage (cell proliferation period): Intermittent slow stirring at 40℃ and 80 rpm (stir for 10 minutes and aerate at 0.2 vvm, let stand for 50 minutes, and circulate once per hour);
[0067] Fermentation took approximately 21 hours, during which time the online pH sensor showed a rapid decrease in pH from the initial 7.0; when the pH dropped to 4.5, Phase Two commenced.
[0068] Second stage (antibacterial substance synthesis period): Reduce the temperature to 34℃, stop stirring and ventilation; this stage lasts for 20 hours, monitor pH changes online, and when the pH reaches 4.0, stage three can begin.
[0069] The third stage (product stabilization period): The temperature is further reduced to 28℃, and fermentation is allowed to proceed for 10 hours until the pH value of the system stabilizes at 4.0.
[0070] S5. Drying and Packaging:
[0071] The fermented material is dried at 60°C using a low-temperature airflow drying method until the moisture content is below 12%, and then sealed and packaged after cooling.
[0072] The fermented feed sample prepared above was extracted at a solid-liquid ratio of 1:3 for 30 min, and the supernatant was collected by centrifugation. The supernatant was sterilized at 121℃ for 20 min, and then centrifuged again to remove the precipitate, yielding the fermented soybean meal antibacterial extract. This extract was stored at 4℃ for later use. 5 ml of the extract was added to 45 mL of LB medium, followed by 50 μL of logarithmic-phase *E. coli* K99. The mixture was incubated at 38℃ and 200 rpm for 12 h on a shaker. OD600 absorbance was measured hourly. A growth curve was plotted with incubation time on the x-axis and absorbance on the y-axis, and compared with that of ordinary fermented feed. The results are as follows: Figure 3 As shown:
[0073] The control group showed good growth of Escherichia coli K99 and Staphylococcus aureus, indicating that the results of this experiment are accurate and reliable. The growth curve of the ordinary fermented feed was similar to that of the control group, and it had no antibacterial properties. The growth curve of the fermented feed group of this invention showed no significant changes within 12 hours, and there were no signs of Escherichia coli growth, indicating a significant antibacterial effect.
[0074] The control example (ordinary fermented soybean meal) was prepared by mixing 700 kg of pulverized 46% protein soybean meal and 300 kg of cottonseed meal with 10 kg of molasses. Water was added at a material-to-water ratio of 1:0.5, and the mixture was stirred thoroughly. After sterilization and cooling, a compound bacterial solution of the same type and amount as in Example 2 was inoculated, and static fermentation was carried out at 37°C for 51 hours (i.e., no segmented dynamic control was performed, maintaining a constant temperature, and no special stirring or dissolved oxygen regulation was applied). Subsequent drying and extraction methods were the same as in Example 2.
[0075] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.
Claims
1. A production process for antibacterial fermented feed based on dynamic regulation and synergistic effects of phenolic acid precursors, characterized in that, Includes the following steps: S1. Ingredients and Mixing: After crushing the main raw material, add agricultural by-products rich in phenolic acid precursors at a mass ratio of 10%-15% of the main raw material, and glucose or molasses at a mass ratio of 1%-3% of the main raw material, and mix evenly; then add purified water at a mass ratio of material to purified water of 1:0.4 to 1:0.5, stir evenly, and prepare a fermentation substrate with a water content of 50%-60%; S2. Sterilization and cooling: The fermentation substrate is sterilized and then cooled. S3. Enzymatic hydrolysis: Add a complex enzyme to the cooled fermentation substrate and hydrolyze it at 40-45℃ for 6-8 hours to obtain the enzymatically hydrolyzed fermentation substrate. S4. Inoculation and Fermentation: The activated compound bacterial solution is inoculated into the enzymatic fermentation substrate at an inoculation rate of 3 v / w.%-5 v / w.%, stirred evenly, and then subjected to three stages of segmented fermentation: First stage: Slowly stir at 35-40℃ and 80-120rpm, and aerate for 3-5 minutes per hour at an aeration rate of 0.1-0.3vvm, ferment for 20-24 hours or until the pH of the system drops below 4.5; Second stage: Reduce the temperature to 30-35℃, stop stirring and ventilation, and let it ferment for 20-30 hours. Third stage: Lower the temperature further to 25-30℃ and let it ferment for 10-14 hours. S5. Drying and Packaging: Dry the fermented material at a temperature not exceeding 60°C until the moisture content is below 12%, and then seal and package it after cooling.
2. The production process according to claim 1, characterized in that, In step S1, the main raw material is one or a mixture of soybean meal, cottonseed meal, and rapeseed meal, and the particle size is 40-60 mesh.
3. The production process according to claim 1, characterized in that, In step S1, the agricultural by-product rich in phenolic acid precursors is one or more of grape seed meal, apple pomace, and green tea residue, or a mixture thereof.
4. The production process according to claim 1, characterized in that, In step S2, the fermentation substrate is sterilized at 80-100°C for 15-30 minutes, and then cooled to 40-45°C.
5. The production process according to claim 1, characterized in that, In step S3, the complex enzyme includes alkaline protease, cellulase, and pectinase added at an amount of 0.3%-0.5% of the mass of the fermentation substrate.
6. The production process according to claim 1, characterized in that, In step S4, the compound bacterial solution is selected from two or three of lactic acid bacteria, Bacillus subtilis and yeast; the lactic acid bacteria are Lactobacillus acidophilus or Lactobacillus plantarum.
7. The production process according to claim 6, characterized in that, The activation method of the compound bacterial solution is as follows: dissolve the compound bacterial agent and glucose in water, and activate at 35-37℃ and 180-200 rpm for 40-60 minutes; wherein, the amount of the compound bacterial agent added is 1-2 kg / (300-500 L water), and the amount of glucose added is 500-1000 g / (300-500 L water).
8. The production process according to claim 1, characterized in that, In step S2, the second stage further includes: starting anaerobic control by introducing nitrogen into the fermenter to maintain the dissolved oxygen (DO) value at 0.2% ± 0.1%.
9. The production process according to claim 1, characterized in that, In step S5, a low-temperature airflow drying method is used for drying.