Compound microorganism freeze-drying protective agent as well as preparation method and use method thereof

Abstract

The invention discloses a compound microorganism freeze-drying protective agent, a preparation method of the compound microorganism freeze-drying protective agent and a use method of the compound microorganism freeze-drying protective agent. Comprising the following raw materials in parts by weight: 0.03 part of disodium ethylene diamine tetraacetate, 5 to 15 parts of trehalose, 2 to 5 parts of sorbitol, 0.5 to 2 parts of ascorbic acid, 0.05 to 0.2 part of N-acetyl-L-cysteine, 0.1 to 0.5 part of polylactic acid-glycolic acid copolymer, 0.01 to 0.05 part of alpha-tocopherol and 1 to 3 parts of bentonite. The PLGA, the alpha-tocopherol and the NAC construct a three-stage synergistic anti-oxidation system from outside to inside: the PLGA is externally blocked, so that rapid consumption of an antioxidant is avoided, and slow release is realized; the alpha-tocopherol realizes oxidative damage repair of cell membranes; nAC maintains the internal reduction environment of cells, reduces oxidative stress and can regenerate alpha-tocopherol at the same time, and reutilization of resources is achieved.

Description

Technical Field

[0001] This invention relates to a composite microbial freeze-drying protectant, its preparation method, and its application method, and more particularly to a composite microbial freeze-drying protectant based on natural products and novel synthetic materials, its preparation method, and its application method, belonging to the technical field of microbial freeze-drying protectants. Background Technology

[0002] Halogenated hydrocarbons, a class of pollutants with mutagenic, carcinogenic, and teratogenic effects, can infect the central nervous system or internal organs through skin and respiratory routes, leading to poisoning. They are widely found in various environmental media and organisms worldwide. After polluting the environment, they easily accumulate in soil, are difficult to degrade naturally, and cause persistent environmental pollution. Most halogenated hydrocarbons are poorly soluble in water and easily adsorbed by soil particles, further increasing the difficulty of their decomposition in soil.

[0003] Currently, remediation technologies for halogenated hydrocarbon-contaminated soils include physical, chemical, and biological methods. Among these, biological methods have attracted significant attention due to their environmental friendliness, high economic efficiency, and ease of operation. Specifically, the method of enhancing the native soil microbial community by adding microbial agents or nutrients to accelerate the adaptation and degradation rate of halogenated hydrocarbons is called bio-enhanced remediation, and it is an effective method for remediating halogenated hydrocarbon-contaminated soils.

[0004] Currently, the most effective way to store bacterial agents is to prepare them into dry powder form at low temperatures and then protect the microorganisms within them with a preservative. Therefore, the composition and ratio of the preservative are crucial. Commonly used preservative components include trehalose, skim milk powder, and mannitol. CN201911031744.9 discloses a method for preparing a preservative for anaerobic ammonia-oxidizing bacteria dry powder inoculum, using an aqueous solution of five agents—polyethylene glycol, mannitol, biotin, ethylenediaminetetraacetic acid (EDTA), and hydroxylamine—as the preservative, achieving a bacterial survival rate of over 80%.

[0005] Existing dry powder microbial agents are often affected by temperature and humidity fluctuations, oxidation, and other stresses during production, storage, transportation, and field use, leading to decreased agent viability, shortened shelf life, and unstable application. Traditional protection systems often rely on a single type of natural or synthetic material, making it difficult to simultaneously ensure stress resistance and long-term stability, thus failing to guarantee agent viability. Summary of the Invention

[0006] Objectives of the invention: The first objective of this invention is to provide a composite microbial freeze-drying protectant that simultaneously achieves both stress resistance and long-term stability; the second objective of this invention is to provide a method for preparing the composite microbial freeze-drying protectant; and the third objective of this invention is to provide a method for using the composite microbial freeze-drying protectant.

[0007] To achieve the first objective mentioned above, the technical solution of the composite microbial freeze-drying protectant provided by the present invention is as follows: The composite microbial freeze-drying protectant of the present invention comprises, by weight, the following raw materials: 0.03 parts disodium ethylenediaminetetraacetate, 5 to 15 parts trehalose, 2 to 5 parts sorbitol, 0.5 to 2 parts ascorbic acid, 0.05 to 0.2 parts N-acetyl-L-cysteine, 0.1 to 0.5 parts polylactic acid-glycolic acid copolymer, 0.01 to 0.05 parts α-tocopherol, and 1 to 3 parts bentonite.

[0008] In the above technical solution, the functions of each raw material are as follows: Trehalose: Protects cell membranes and proteins during drying, reducing membrane changes and protein denaturation during cell dehydration; Sorbitol: Provides osmotic protection during drying, maintaining intracellular and extracellular osmotic pressure; Bentonite: Provides carrier support for bacteria, offering some physical protection, reducing damage to bacteria from drying and oxidation, while adsorbing excess moisture within the bacterial agent, maintaining the powdery structure of the agent, improving dispersibility, and preventing moisture absorption and clumping; Ascorbic acid: Slows down the oxidation reactions of intracellular lipids and proteins during microbial storage, counteracting oxidative stress during drying, storage, and transportation, and extending the shelf life of the agent; Disodium EDTA (2Na): A commonly used strong... The active ingredients include: chelating agents to stabilize the antioxidant components in the protective agent and prevent rapid consumption of antioxidants; N-acetyl-L-cysteine ​​(NAC): after cells absorb NAC, they convert it into cysteine, which combines with glutamic acid and glycine to form glutathione (GSH), which can scavenge free radicals and slow down the oxidative damage of the bacterial agent; α-tocopherol: a cell membrane antioxidant used to interrupt the lipid peroxidation chain reaction and reduce the peroxidation damage to unsaturated fatty acids in the cell membrane; and polylactic-co-glycolic acid copolymer (PLGA): which forms a film on the surface of microorganisms to protect cell structure, maintain cell structure, and achieve a sustained-release effect, prolonging the survival time of microorganisms in the bacterial agent.

[0009] Most importantly, PLGA, α-tocopherol, and NAC construct a three-tiered synergistic antioxidant system from the outside in: PLGA acts as an external barrier, preventing rapid consumption of antioxidants and achieving slow release; α-tocopherol repairs oxidative damage to cell membranes; and NAC maintains the reducing environment inside cells, reducing oxidative stress and potentially regenerating α-tocopherol, enabling resource reuse. Ultimately, this improves the long-term stability of the bacterial agent.

[0010] Preferably, the composite microbial freeze-drying protectant includes 0.03 parts disodium ethylenediaminetetraacetate, 5-10 parts trehalose, 2-3 parts sorbitol, 0.5-1 part ascorbic acid, 0.05-0.2 parts N-acetyl-L-cysteine, 0.1-0.5 parts polylactic acid-glycolic acid copolymer, 0.01-0.05 parts α-tocopherol, and 2 parts bentonite.

[0011] Preferably, the composite microbial freeze-drying protectant includes 0.03 parts disodium ethylenediaminetetraacetate, 10 parts trehalose, 3 parts sorbitol, 1 part ascorbic acid, 0.1 parts N-acetyl-L-cysteine, 0.3 parts polylactic acid-glycolic acid copolymer, 0.02 parts α-tocopherol, and 2 parts bentonite.

[0012] Preferably, the compound microbial freeze-drying protectant also includes phosphate buffer.

[0013] Preferably, the phosphate buffer concentration is 0.1 M.

[0014] To achieve the second objective mentioned above, the technical solution for preparing the composite microbial freeze-drying protectant provided by this invention is as follows: The preparation method of the composite microbial freeze-drying protectant of the present invention includes the following steps: Weigh the following raw materials according to their weight proportions: 0.03 parts disodium ethylenediaminetetraacetate, 5-15 parts trehalose, 2-5 parts sorbitol, 0.5-2 parts ascorbic acid, 0.05-0.2 parts N-acetyl-L-cysteine, 0.1-0.5 parts polylactic acid-glycolic acid copolymer, 0.01-0.05 parts α-tocopherol, and 1-3 parts bentonite. Place all raw materials in a buffer solution, mix and disperse evenly to obtain a composite microbial freeze-drying protectant.

[0015] Preferably, the composite microbial freeze-drying protectant contains 0.03 wt% disodium ethylenediaminetetraacetate, 5-15 wt% trehalose, 2-5 wt% sorbitol, 0.5-2 wt% ascorbic acid, 0.05-0.2 wt% N-acetyl-L-cysteine, 0.1-0.5 wt% polylactic acid-glycolic acid copolymer, 0.01-0.05 wt% α-tocopherol, and 1-3 wt% bentonite.

[0016] Preferably, the mixing and dispersion method is stirring or ultrasonication.

[0017] To achieve the third objective mentioned above, the technical solution for the use of the composite microbial freeze-drying protectant provided by this invention is as follows: The method of using the composite microbial freeze-drying protectant of the present invention includes the following steps: The compound microbial freeze-drying protectant is mixed with concentrated bacterial mud, then packaged and frozen. After freezing, the mixture is freeze-dried to obtain freeze-dried blocks, which are then crushed to obtain dry powder.

[0018] Preferred method for preparing concentrated bacterial sludge: Cultivate the target bacteria to the logarithmic phase; harvest the bacterial cells by centrifugation, discard the supernatant, and replenish the volume with physiological saline to the volume before centrifugation to obtain a mixture; shake the mixture to form a bacterial suspension, and collect the bacterial sludge at the bottom.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The natural product extract components and novel synthetic material components form a synergistic system, balancing storage stability and stress-induced slow release, and are compatible with target strains to form highly active dry powder products. After using the composite microbial freeze-drying protectant of this invention, the survival rate decline curve of the strain becomes smoother, indicating that the protectant can improve the long-term stability of the bacterial agent. The advantages of the natural product extract components are as follows: They are selected from one or more of polysaccharides, oligosaccharides, amino acids / peptides, antioxidant active substances, and plant-derived small molecules, providing comprehensive effects such as permeation protection, membrane stabilization, and free radical scavenging; Trehalose has been proven to have good protective effects, but it is prone to crystallization during drying. Sorbitol can effectively inhibit the crystallization process of trehalose, maintaining its glassy state during drying; NAC, as a GSH precursor, can not only be converted into GSH in vivo but also has stronger stability, and its molecular structure contains thiol groups, which can effectively scavenge free radicals and provide intracellular antioxidant protection; α-Tocopherol, as a form of vitamin E, is inexpensive and can effectively protect cell membranes from peroxidation, playing an important role in maintaining cell structural integrity. Advantages of the novel synthetic material components: They are selected from biocompatible polymers, biodegradable polymers, surface functionalized materials or their complexes, and are used to form physical barriers, slow-release microenvironments or directional adsorption protection; PLGA can isolate the bacteria from contact with oxygen and water during the drying process of the bacterial agent, so as to achieve slow release. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the preparation and use of the composite microbial freeze-drying protectant of the present invention.

[0021] Figure 2 This is a comparison chart of the survival rates of various embodiments of the present invention.

[0022] Figure 3 This is a survival rate decay graph for Embodiment 1 and various comparative examples of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] This invention utilizes a strain previously screened and cultured by the applicant that exhibits good degradation effects on halogenated hydrocarbons in soil. The full-length 16S rRNA gene of the pure culture strain was amplified using universal primer pairs: 27F: AGAGTTTGATCMTGGCTCAG (forward), 1492R: TACGGYTACCTTGTTACGACTT (reverse). The amplified product was approximately 1450 bp. The full 16S rRNA gene sequence was obtained by Sanger sequencing and BLAST alignment using the NCBI database. Its 16S sequence showed 100% identity with *Dehalogenimonas formicexedens* (RefSeq accession number KX274280.1, NSZ-14 type strain), thus identifying it as *Dehalogenimonas formicexedens*.

[0025] This strain uses aqueous solutions of trehalose, sorbitol, bentonite, polylactic-co-glycolic acid copolymer (PLGA), ascorbic acid, and glutathione as protectants, with a volume ratio of protectant to culture medium of 1:1. The strain used in this invention was derived from soil samples from typical halogenated hydrocarbon contaminated sites.

[0026] Example 1

[0027] The formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 10 wt%; Sorbitol: 3 wt%; Ascorbic acid: 1 wt%; N-acetyl-L-cysteine ​​(NAC): 0.1 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.3 wt%; α-Tocopherol: 0.02 wt%; Bentonite: 2 wt%.

[0028] like Figure 1 As shown, the preparation and use method of the compound microbial freeze-dried product in this embodiment specifically includes the following steps: (1) Cell pretreatment: The screened degrading bacteria were cultured in LB medium to the logarithmic phase, and the cells were harvested by centrifugation at 10,000 g for 15 min at 4℃. After discarding the supernatant, the volume was replenished to the pre-centrifugation volume with pre-cooled 0.9% physiological saline. (2) Resuspension solution: Use a vortex shaker to shake at low speed to ensure that the bacterial sludge is completely and uniformly resuspended to form a bacterial suspension. Repeat the above centrifugation and resuspension steps 1-2 times and collect the bottom bacterial sludge.

[0029] (3) Preparation of protective agent solution: Prepare the protective agent in the following order. Phosphate-buffered saline (PBS): pH 7.4, 0.1 M Disodium ethylenediaminetetraacetate (EDTA-2Na): 0.03 wt% Trehalose: 10 wt%, dissolved at 50°C Sorbitol: 3 wt% Ascorbic acid: 1 wt% N-acetyl-L-cysteine ​​(NAC): 0.1 wt% Polylactic acid-glycolic acid copolymer (PLGA): 0.3 wt% α-Tocopherol: 0.02 wt% Bentonite: 2 wt%, forming an emulsion under high-speed stirring or ultrasound.

[0030] (4) Freeze-drying and powder preparation: After sterilization of the preservative, it was mixed with the collected concentrated bacterial sludge in a sterile environment at a ratio of 1:1, and dispensed into freeze-drying trays, controlling the liquid level to be less than 1 cm. The samples were then quickly placed in an environment of -80℃ to freeze, ensuring that the samples were completely frozen solid. The frozen samples were then transferred to a pre-cooled freeze dryer. The vacuum pump was started, and the pressure inside the drying chamber was kept less than 10 Pa. The shelf temperature was set to -20℃, and drying was continued for 20 h. Then, the temperature was increased to 25℃ at a rate of 1℃ per minute, and drying was continued for 8 h to obtain freeze-dried blocks. The freeze-dried blocks were crushed in a clean environment and screened through an 80-mesh sieve to obtain uniform and fine dry powder.

[0031] Example 2

[0032] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution, while the usage and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.1 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.3 wt%; α-Tocopherol: 0.02 wt%; Bentonite: 1 wt%.

[0033] Example 3

[0034] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution. The application and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 15 wt%; Sorbitol: 5 wt%; Ascorbic acid: 2 wt%; N-acetyl-L-cysteine ​​(NAC): 0.1 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.3 wt%; α-Tocopherol: 0.02 wt%; Bentonite: 3 wt%.

[0035] Example 4

[0036] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution, while the usage and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 10 wt%; Sorbitol: 3 wt%; Ascorbic acid: 1 wt%; N-acetyl-L-cysteine ​​(NAC): 0.05 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.1 wt%; α-Tocopherol: 0.01 wt%; Bentonite: 2 wt%.

[0037] Example 5

[0038] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution, while the usage and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.05 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.3 wt%; α-Tocopherol: 0.02 wt%; Bentonite: 2 wt%.

[0039] Example 6

[0040] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution, while the usage and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.05 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.5% wt%; α-Tocopherol: 0.05 wt%; Bentonite: 2 wt%.

[0041] Example 7

[0042] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution, while the usage and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.1 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.1% wt%; α-Tocopherol: 0.05 wt%; Bentonite: 2 wt%.

[0043] Example 8

[0044] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution. The application and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.1 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.5% wt%; α-Tocopherol: 0.01 wt%; Bentonite: 2 wt%.

[0045] Example 9

[0046] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution. The application and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.2 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.1% wt%; α-Tocopherol: 0.05 wt%; Bentonite: 2 wt%.

[0047] Example 10

[0048] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution. The application and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.2 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.3% wt%; α-Tocopherol: 0.01 wt%; Bentonite: 2 wt%.

[0049] Example 11

[0050] The difference between this embodiment and Example 1 lies in the formulation of the composite microbial lyophilization protectant solution, while the usage and preparation methods of the protectant are the same as in Example 1. Specifically, the formulation of the composite microbial lyophilization protectant solution in this embodiment is as follows: Phosphate-buffered saline (PBS): pH 7.4, 0.1 M; Disodium EDTA-2Na: 0.03 wt%; Trehalose: 5 wt%; Sorbitol: 2 wt%; Ascorbic acid: 0.5 wt%; N-acetyl-L-cysteine ​​(NAC): 0.2 wt%; Polylactic-co-glycolic acid copolymer (PLGA): 0.5% wt%; α-Tocopherol: 0.02 wt%; Bentonite: 2 wt%.

[0051] Comparative Example 1 The difference between this comparative example and Example 1 is that the formulation of the composite microbial freeze-drying protectant solution is different: polylactic acid-glycolic acid copolymer (PLGA), N-acetyl-L-cysteine ​​(NAC) and α-tocopherol are removed from the original example. The method of using and preparing the protectant is the same as in Example 1.

[0052] Comparative Example 2 The difference between this comparative example and Example 1 is that the formulation of the composite microbial freeze-drying protectant solution is different: N-acetyl-L-cysteine ​​(NAC) and α-tocopherol are removed from the original example, while the method of using and preparing the protectant is the same as in Example 1.

[0053] Comparative Example 3 The difference between this comparative example and Example 1 is that the formulation of the composite microbial freeze-drying protectant solution is different: polylactic acid-glycolic acid copolymer (PLGA) and α-tocopherol are removed from the original example, while the method of using and preparing the protectant is the same as in Example 1.

[0054] Comparative Example 4 The difference between this comparative example and Example 1 is that the formulation of the composite microbial freeze-drying protectant solution is different: polylactic acid-glycolic acid copolymer (PLGA) and N-acetyl-L-cysteine ​​(NAC) are removed from the original example of Example 1, while the method of using and preparing the protectant is the same as in Example 1.

[0055] Comparative Example 5 The difference between this comparative example and Example 1 is that the formulation of the composite microbial freeze-drying protectant solution is different: the polylactic acid-glycolic acid copolymer (PLGA) is removed from the original example, while the method of using and preparing the protectant is the same as in Example 1.

[0056] Comparative Example 6 The difference between this comparative example and Example 1 is that the formulation of the composite microbial freeze-drying protectant solution is different: N-acetyl-L-cysteine ​​(NAC) is removed from the solution of Example 1, while the method of using and preparing the protectant is the same as in Example 1.

[0057] Comparative Example 7 The difference between this comparative example and Example 1 is that the formulation of the composite microbial freeze-drying protectant solution is different: α-tocopherol is removed from the solution of Example 1, while the method of using and preparing the protectant is the same as in Example 1.

[0058] The survival rate of the strains in the freeze-dried powder containing concentrated bacterial mud obtained in the above examples and comparative examples was tested. The test method is as follows: add physiological saline (0.9%) of the same volume as before freeze-drying to the freeze-dried bacterial powder for rehydration. Dilute the bacterial solution of the same volume before and after freeze-drying and spread it for viable count. The calculation formula is as follows. Each group was tested in parallel for 3 times.

[0059] Freeze-dried survival rate = Number of colonies on plates coated with the pre-freeze-drying bacterial agent / Number of colonies on plates coated with the post-freeze-drying rehydration bacterial agent Survival rates of examples using different ratios of protective agents are as follows: Figure 2 As shown in the figures, Examples 1, 2, and 3 were designed to determine the optimal ratio of the protective agents, excluding PLGA, α-tocopherol, and NAC. The remaining examples were used to compare the optimal ratio of the key components PLGA, α-tocopherol, and NAC. Results showed that the survival rate of the bacterial agent obtained from Example 1 after freeze-drying reached 80%, significantly higher than that of Examples 2 and 3. Range analysis revealed the following ranges (R) for the key components: R_PLGA = 9.43, R_NAC = 8.57, and R_α-tocopherol = 6.10. The results indicate that PLGA concentration is the most critical factor affecting the survival rate of the bacterial agent, with its level causing the greatest change in effect; the influence of NAC concentration is second; while the α-tocopherol concentration, within the range set in this experiment (0.01%~0.05%), has a relatively small direct impact on the final survival rate. The order of importance of the factors is: PLGA > NAC > α-tocopherol.

[0060] Calculations of the average survival rates of each factor at different levels showed that NAC had the highest average survival rate (80.5%) at 0.10 wt%; PLGA performed best at 0.30 wt% (82.2%); and α-tocopherol performed best at 0.02 wt% (81.1%). Considering both economic efficiency and trend of effect, the optimal combination of levels was determined to be Example 1, namely NAC 0.10%, PLGA 0.30%, and α-tocopherol 0.02%.

[0061] Analysis of variance (ANOVA) of the data was expected to show that the main effects of PLGA and NAC were extremely significant (P<0.05). The measured value (85.5%) of the optimal combination (Example 1) was much higher than the theoretical summation prediction based solely on the main effects. Furthermore, when both NAC and PLGA were at optimal levels (Examples 1, 5, 10), the survival rate generally remained at a high level (78.5%-85.5%). However, when one of them was at a low level, even with a high concentration of the other component, the effect was significantly limited (Examples 7, 9). This indicates that NAC, PLGA, and α-tocopherol constitute a synergistic protective network, where PLGA provides a physical barrier, α-tocopherol protects the membrane system, and NAC maintains intracellular redox homeostasis, collectively enhancing the overall upper limit of the protective effect.

[0062] like Figure 3As shown, under accelerated storage conditions at 37°C, the survival rate of the microbial agents in all formulations decreased over time, but at different rates. By day 7, all formulations maintained a survival rate close to or higher than 90%. By day 28, the protective efficacy of each group began to differentiate, with the survival rate of the complete composite protectant (Example 1) (95.9%) significantly higher than that of Comparative Example 1 (73.5%) and any single-component addition group (Comparative Examples 2-4, 80.1-85.7%) (P<0.05). By day 56, this differentiation was greatly amplified, with the survival rate of Example 1 (89.0%) almost twice that of Comparative Example 1 (53.4%), and significantly better than all binary combinations (Comparative Examples 5-7, 74.4-85.1%) (P<0.01). The data indicate that the synergistic protective effect of PLGA, α-tocopherol, and NAC becomes more pronounced with prolonged storage time and accumulated stress, and its advantage over simple protective strategies becomes increasingly significant.

Claims

1. A composite microbial lyophilizate, characterized in that By weight, it includes the following ingredients: 0.03 parts disodium ethylenediaminetetraacetate, 5-15 parts trehalose, 2-5 parts sorbitol, 0.5-2 parts ascorbic acid, 0.05-0.2 parts N-acetyl-L-cysteine, 0.1-0.5 parts polylactic acid-glycolic acid copolymer, 0.01-0.05 parts α-tocopherol, and 1-3 parts bentonite.

2. The composite microbial lyophilization protectant according to claim 1, characterized in that, It includes 0.03 parts disodium ethylenediaminetetraacetate, 5-10 parts trehalose, 2-3 parts sorbitol, 0.5-1 part ascorbic acid, 0.05-0.2 parts N-acetyl-L-cysteine, 0.1-0.5 parts polylactic acid-glycolic acid copolymer, 0.01-0.05 parts α-tocopherol, and 2 parts bentonite.

3. The composite microbial lyophilization protectant of claim 1, wherein, It includes 0.03 parts disodium EDTA, 10 parts trehalose, 3 parts sorbitol, 1 part ascorbic acid, 0.1 parts N-acetyl-L-cysteine, 0.3 parts polylactic acid-glycolic acid copolymer, 0.02 parts α-tocopherol, and 2 parts bentonite.

4. The composite microbial lyophilization protectant of claim 1, wherein, It also includes phosphate buffer.

5. The composite microbial lyophilizate according to claim 1, characterized in that The phosphate buffer concentration is 0.1M.

6. A method of preparing a composite microbial lyophilization protectant, characterized by, Weigh the following raw materials according to their weight proportions: 0.03 parts disodium ethylenediaminetetraacetate, 5-15 parts trehalose, 2-5 parts sorbitol, 0.5-2 parts ascorbic acid, 0.05-0.2 parts N-acetyl-L-cysteine, 0.1-0.5 parts polylactic acid-glycolic acid copolymer, 0.01-0.05 parts α-tocopherol, and 1-3 parts bentonite. Place all raw materials in a buffer solution, mix and disperse evenly to obtain a composite microbial freeze-drying protectant.

7. The method of claim 6, wherein the freeze-dried protective agent for a complex microorganism is prepared by adding a protective agent for a complex microorganism to a culture medium for a complex microorganism, and then culturing the complex microorganism in the culture medium. The compound microbial freeze-drying protectant contains 0.03 wt% disodium ethylenediaminetetraacetate, 5-15 wt% trehalose, 2 ~ 5 wt% sorbitol, 0.5 ~ 2 wt% ascorbic acid, 0.05 ~ 0.2 wt% N-acetyl-L-cysteine, 0.1 ~ 0.5 wt% polylactic acid-glycolic acid copolymer, 0.01 ~ 0.05 wt% α-tocopherol, 1 ~ 3 wt% bentonite.

8. The method for preparing the composite microbial freeze-drying protectant according to claim 6, characterized in that, The mixing and dispersion methods are stirring or ultrasonication.

9. A method of using the lyophilized protective agent of complex microorganisms according to any one of claims 1 to 5, characterized in that, The compound microbial freeze-drying protectant is mixed with concentrated bacterial mud, then packaged and frozen. After freezing, the mixture is freeze-dried to obtain freeze-dried blocks, which are then crushed to obtain dry powder.

10. The method of using a composite microbial lyophilizate according to claim 9, characterized in that Preparation method of concentrated bacterial sludge: Cultivate the target bacteria to the logarithmic phase; harvest the bacterial cells by centrifugation, discard the supernatant, and replenish the volume with physiological saline to the volume before centrifugation to obtain a mixture; shake the mixture to form a bacterial suspension, and collect the bacterial sludge at the bottom.

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