Method for preparing functional biological flora by coupling target biological flora and host original flora

Abstract

The invention discloses a method for preparing a functional biological flora in a targeted biological flora and host original flora coupling mode. The method comprises the following steps: screening a single strain with a function of degrading pesticides, antibiotics or heavy metals; purifying and amplifying the single strain, and compounding according to a ratio to obtain a compound flora; carrying out coupling symbiotic culture on the compound flora and a host original flora to obtain a coupled flora; carrying out thermal shock and gradient domestication on the coupled flora, and screening stress-tolerant sporulation strains; carrying out targeted delivery treatment on the stress-tolerant strain by utilizing microcapsule embedding, lipidosome or nanoparticles; the invention relates to the technical field of agriculture, animal husbandry and environmental restoration, and aims to realize the synergistic symbiosis of the flora and the host environment and improve the colonization capability and action effect of the microbial inoculum. Multiple functions of degrading pesticide residues, antibiotics and heavy metals are realized; high temperature resistance, acid and alkali resistance and adaptability to complex environments; the targeting property is strong, and the action efficiency is high.

Description

A method for preparing functional microbial communities by coupling targeted microorganisms with the host's original microbial community. Technical Field

[0001] This invention relates to the fields of agriculture, animal husbandry and environmental remediation, specifically to a method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbial community. Background Technology

[0002] With the development of modern agriculture and animal husbandry, the problems of pesticide, antibiotic, and heavy metal residues are becoming increasingly serious, posing a threat to food safety and human health. Although existing microbial agents have a certain degradation effect, they generally suffer from problems such as single function, poor environmental adaptability, insufficient targeting, and weak synergy with the host microbiota.

[0003] Most bacterial agents on the market are single-species or simple compound formulations, lacking a symbiotic mechanism with the host's original microbial community. This results in unstable and poor-lasting effects in practical applications. Furthermore, the low survival rate of these strains in extreme environments such as high temperatures and acidic / alkaline conditions limits their application scope.

[0004] Therefore, developing a method for preparing a composite microbial community that can coexist with the host microbial community, has strong environmental adaptability and targeting function, has important technical value and application prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for formulating functional microbial communities through a coupling of targeted microorganisms and the host's original microbial community. This method solves the problems of existing microbial preparations, such as limited functionality, poor environmental adaptability, insufficient targeting, weak synergy with the host microbial community, and lack of a symbiotic mechanism with the host's original microbial community.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: A method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbial community, comprising the following sequential steps: (1) constructing a targeted microbial strain resource library and screening single strains with the function of degrading pesticides, antibiotics or heavy metals; (2) purifying, amplifying and compounding the single strains in proportion to obtain compound microbial communities; (3) coupling and symbiotically culturing the compound microbial communities with the original host microbial community under anaerobic or microaerobic conditions at 25–40℃, pH 5.5–7.5 for 48–120 hours to obtain coupled microbial communities; (4) subjecting the coupled microbial communities to thermal shock at 60–80℃ and pH gradient acclimation at 4.0–9.0 to screen stress-resistant sporulation strains; (5) targeting and delivering the stress-resistant strains using at least one of microcapsule encapsulation, liposomes or nanoparticles; (6) obtaining a composite microbial community with the function of degrading pesticide residues, antibiotic residues and heavy metal residues by freeze drying or spray drying.

[0007] Preferably, the single strain is selected from at least one of Bacillus, Monosporus, Lactobacillus, Nitrifying Bacteria, and Yeast.

[0008] Preferably, the temperature for the coupled symbiotic culture in step (3) is 30–37°C, pH 6.0–7.0, and the culture time is 72–96 hours.

[0009] Preferably, the thermal shock temperature in step (4) is 65–75℃ and the duration is 5–15 min; the pH gradient acclimatization is carried out by first culturing at pH 4.0 for 12 h, and then culturing at pH 9.0 for 12 h, and the cycle is repeated twice.

[0010] Preferably, the microcapsule wall material in step (5) is sodium alginate-chitosan with a particle size of 50–500 μm and an encapsulation rate of ≥80%.

[0011] Preferably, in step (6), the inlet air temperature for spray drying is 170–180℃, the outlet air temperature is 70–80℃, the resulting bacterial powder has a moisture content of ≤5%, and a viable count of ≥1×10⁻⁶. 9 CFU·g⁻¹.

[0012] A complex microbial community with a viable count ≥1×10⁻⁶ 9 CFU·g⁻¹, with a survival rate of ≥85% after 10 min at 80℃, and a degradation rate of ≥60% for pesticides, antibiotics, and heavy metals.

[0013] Preferably, the aforementioned complex microbial community is used in agriculture or animal husbandry for the degradation of pesticide, antibiotic, or heavy metal residues. Beneficial effects.

[0014] This invention provides a method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbial community, which has the following beneficial effects: achieving synergistic symbiosis between the microbial community and the host environment, enhancing the colonization ability and efficacy of the microbial agent; possessing multiple functions: degrading pesticide residues, antibiotics, and heavy metals; resistant to high temperatures and acids and alkalis, adapting to complex environments; highly targeted and efficient; and can be widely applied in green agriculture, organic farming, soil remediation, and other fields. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Embodiments

[0016] A method for preparing functional microbial communities by coupling targeted microorganisms with the original host microbial community includes the following sequential steps: (1) constructing a targeted microbial strain resource library and screening single strains with the function of degrading pesticides, antibiotics or heavy metals; (2) purifying, amplifying and compounding the single strains in proportion to obtain compound microbial communities; (3) coupling and symbiotically culturing the compound microbial communities with the original host microbial community under anaerobic or microaerobic conditions at 25–40℃, pH 5.5–7.5 for 48–120 hours to obtain coupled microbial communities; (4) subjecting the coupled microbial communities to thermal shock at 60–80℃ and pH gradient acclimation at 4.0–9.0 to screen stress-resistant sporulation strains; (5) targeting and delivering the stress-resistant strains using at least one of microcapsule encapsulation, liposomes or nanoparticles; (6) obtaining a composite microbial community with the function of degrading pesticide residues, antibiotic residues and heavy metal residues by freeze drying or spray drying.

[0017] In this embodiment, the single strain is further selected from at least one of Bacillus, Monosporus, Lactobacillus, Nitrifying Bacteria, and Yeast.

[0018] In this embodiment, the temperature for the coupled symbiotic culture in step (3) is 30–37°C, pH 6.0–7.0, and the culture time is 72–96 hours.

[0019] Table 1 Comparison of Coupling Symbiotic Conditions—Temperature & pH Three-Level Experiment Experimental Group Temperature (°C) pH Culture Time (h) Viable Bacteria Growth (log CFU·mL⁻¹) Spore Formation Rate (%) Degradation Rate* (%) A 255.5 480.9±0.14238 B 306.07 21.8±0.26855 C 377.09 62.4±0.38570 D 407.5 1201.5±0.27362 Table target: Chlorpyrifos 50 mg·L⁻¹, incubated at 30℃ for 72 h; preferred range B / C.

[0020] In this embodiment, the thermal shock temperature in step (4) is 65–75℃ and the duration is 5–15 min; the pH gradient acclimatization is carried out by first culturing at pH 4.0 for 12 h, and then culturing at pH 9.0 for 12 h, and the cycle is repeated twice.

[0021] Table 2. Comparison of Stress Tolerance Acclimation – Thermal Shock + pH Gradient Treatment Single Treatment Treatment Thermal Shock (°C·min) pH Gradient Cycle 80°C 10min Survival Rate (%) Spore Ratio (%) Degradation Rate* (%) No Acclimation – 45 35 42 Thermal Shock Only 70°C × 10 min 0 72 58 55 pH Gradient Only – 2 cycles 68 52 53 Thermal Shock + pH Gradient 70°C × 10 min 2 cycles 88 84 70 In this embodiment, the microcapsule wall material in step (5) is sodium alginate-chitosan with a particle size of 50–500 μm and an encapsulation rate of ≥80%.

[0022] Table 3 Comparison of Targeted Delivery Methods—Microcapsules vs. Liposomes vs. Carrier-Free Carrier Type Particle Size (μm) Encapsulation Efficiency (%) Survival Rate (%) after 30 days of storage at 25℃ Degradation Rate* (%) Claim 5 Compliance Carrier-Free — 42 45 — Liposomes 0.2–0.865±56 858 Compliance Sodium Alginate-Chitosan Microcapsules 200±50 82±38 570 ✓ Preferred Table target: Cd²⁺ 10 mg·L⁻¹; the microcapsule group was significantly superior to the liposome group (p<0.05).

[0023] In this embodiment, the inlet air temperature of the spray drying step (6) is 170–180℃, the outlet air temperature is 70–80℃, the moisture content of the obtained bacterial powder is ≤5%, and the viable count is ≥1×10⁻⁶. 9 CFU·g⁻¹.

[0024] Table 4. Comparison of Spray Drying and Freeze Drying | Drying Method | Inlet Air Temperature (°C) | Outlet Air Temperature (°C) | Moisture (%) | Viable Bacteria Count (CFU·g⁻¹) | Survival Rate at 80°C (%) | Compliance with Claim 6 | Freeze Drying | 3.1 ± 0.3 | 2.8 × 10⁻⁶ 9 90% conforms to spray drying 175754.2±0.21.2×10 9 88✓ Meets Both met the requirements, but spray drying is more suitable for continuous production.

[0025] A complex microbial community with a viable count ≥1×10⁻⁶ 9 CFU·g⁻¹, survival rate ≥85% at 80℃ for 10 min, degradation rate of pesticides, antibiotics, and heavy metals ≥60% Table 5: Comprehensive Performance Verification Test Items, Test Conditions, Results, Indicators, Thresholds, Conclusions Viable Bacteria Count, Plate Count 1.2×10⁻¹ 9 CFU·g⁻¹≥1×10 9 CFU·g⁻¹✓ Resistant to 80℃ 10 min water bath + plate: 88% ≥ 85%✓ Pesticide degradation: Chlorpyrifos 50 mg·L⁻¹, 72 h: 70% ≥ 60%✓ Antibiotic degradation: Enrofloxacin 20 mg·L⁻¹, 72 h: 68% ≥ 60%✓ Heavy metal removal: Cd²⁺ 10 mg·L⁻¹, 72 h: 70% ≥ 60%✓ All indicators in the table meet the requirements at once.

[0026] This embodiment is further configured such that the composite microbial community is used in agriculture or animal husbandry to degrade pesticide, antibiotic, or heavy metal residues.

[0027] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, the phrase "comprising an element defined as..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0028] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing functional microbial communities by coupling targeted microorganisms with the host's original microbial community, characterized in that, The following sequential steps are included: (1) constructing a targeted biological strain resource library and screening single strains with the function of degrading pesticides, antibiotics or heavy metals; (2) purifying, amplifying and compounding the single strains in proportion to obtain compound bacterial groups; (3) coupling and symbiotically culturing the compound bacterial groups with the host original bacterial groups at 25–40℃, pH 5.5–7.5, anaerobic or microaerobic conditions for 48–120 hours to obtain coupled bacterial groups; (4) subjecting the coupled bacterial groups to thermal shock at 60–80℃ and pH gradient acclimatization at 4.0–9.0 to screen stress-resistant sporulation strains; (5) targeting and delivering the stress-resistant strains using at least one of microcapsule encapsulation, liposomes or nanoparticles; (6) obtaining a composite biological group with the function of degrading pesticide residues, antibiotic residues and heavy metal residues by freeze drying or spray drying.

2. The method according to claim 1, characterized in that, The single strain is selected from at least one of the genera Bacillus, Monosporus, Lactobacillus, Nitrifying Bacteria, and Yeast.

3. The method according to claim 1, characterized in that, Step (3) The temperature for coupled symbiotic culture is 30–37℃, pH 6.0–7.0, and the culture time is 72–96 hours.

4. The method according to claim 1, characterized in that, Step (4) The thermal shock temperature is 65–75℃ and the duration is 5–15 min; pH gradient acclimatization is carried out by first culturing at pH 4.0 for 12 h, and then culturing at pH 9.0 for 12 h, and the cycle is repeated twice.

5. The method according to claim 1, characterized in that, Step (5) The microcapsule wall material is sodium alginate-chitosan with a particle size of 50–500 μm and an encapsulation rate of ≥80%.

6. The method according to claim 1, characterized in that, Step (6): Spray drying inlet air temperature 170–180℃, outlet air temperature 70–80℃, resulting bacterial powder moisture ≤5%, viable bacteria count ≥1×10⁻⁶. 9 CFU·g⁻¹.

7. A composite microbial community prepared by any one of claims 1-6, characterized in that, viable count ≥1×10 9 CFU·g⁻¹, with a survival rate of ≥85% after 10 min at 80℃, and a degradation rate of ≥60% for pesticides, antibiotics, and heavy metals.

8. The use of the composite microbial community according to claim 7 in agriculture or animal husbandry for degrading pesticide, antibiotic or heavy metal residues.