A quick recovery liquid for frozen bacteria and its preparation method and application

Abstract

The application discloses a kind of quick recovery liquid of frozen bacteria and its preparation method and application, the recovery liquid includes 0.05-0.3 g / ml egg yolk lecithin, 1%-5% glucose and 1%-5% sucrose, dissolved in sterile water.It is mixed with frozen strain and then incubated at room temperature, to realize the quick recovery of strain activity.It is suitable for a variety of clinical bacteria, such as attenuated salmonella VNP20009, escherichia coli probiotic ECN, etc.The bacteria solution after recovery can be directly used for clinical treatment, including anti-tumor treatment, metabolic disease treatment, nervous system disease treatment and improvement of intestinal microecology, etc.The recovery liquid of the application is simple in preparation, low in cost, convenient to use, can significantly improve the biological activity and clinical efficacy of frozen bacteria, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a rapid resuscitation solution for cryopreserved bacterial strains, its preparation method, and its application. Background Technology

[0002] In recent years, live microbial therapy has attracted widespread attention in the medical field as a promising new treatment method. The core of this therapy lies in utilizing specific bacterial microorganisms, introducing them into the human body through various routes such as oral administration, intravenous injection, and intraperitoneal injection, allowing them to exert specific medicinal effects within the body. These bacterial microorganisms include attenuated Salmonella VNP20009, Escherichia coli probiotic ECN, attenuated Listeria, BCG, Streptococcus thermophilus (patent application number: 2024110944111), Enterococcus faecalis (patent application number: 2024110944516), Lactobacillus rhamnosus (patent application number: 2024110944520), and Bifidobacterium (patent application number: 2024110944126), among others. For example, in the field of anti-tumor therapy, numerous preclinical studies have confirmed that specific bacteria such as attenuated Salmonella can be highly enriched in the tumor site through various routes of administration. This is because the hypoxia and corresponding immunosuppressive microenvironment of the tumor site provide favorable conditions for bacterial growth and colonization. Once bacteria accumulate within a tumor, they can effectively treat the tumor through various strategies, such as activating the tumor's immune response and / or expressing and secreting engineered protein drugs. Furthermore, in vivo microbial therapy has shown broad application prospects in the treatment of metabolic diseases and the remodeling of the gut microbiota. For example, Bifidobacterium preparations have been effectively used to prevent and treat demyelinating diseases (patent application numbers: 2024110948095; 2024110948076), to prevent diabetes using Bifidobacterium longum and Streptococcus thermophilus preparations (patent application number: 2024110948108), to prevent and treat Parkinson's disease using Bifidobacterium longum preparations (patent application number: 2024110948080), and to improve ulcerative colitis using Bifidobacterium longum, Lactobacillus rhamnosus, and Enterococcus faecium preparations (patent application numbers: 2025102712456; 2025102712441; 2025102712437).

[0003] With the continuous advancements in synthetic biology and the rapid development of cell / bacterial therapy, the research and application of live microbial therapy are progressing rapidly, and numerous clinical trials are being conducted globally. However, in actual clinical applications, to save on costs related to use, transportation, and handling, while maintaining the stability of drug formulation and dosage, microbial drugs typically need to be cryopreserved with glycerol or freeze-dried into powder form after pilot-scale or large-scale production. Subsequently, these cryopreserved microbial drugs are transported via cold chain and, at the required time and place, thawed and administered to patients by healthcare professionals. While this cryopreservation and thaw process ensures drug stability and usability to a certain extent, it also introduces some problems.

[0004] Extensive evidence suggests that most bacteria enter a special dormant state under adverse environmental conditions (such as long-term cryopreservation in glycerol or lyophilized powder form). This dormancy is a self-protective mechanism employed by bacteria to resist adverse environments, but it is also accompanied by a significant reduction in cell activity to mitigate the impact of adverse conditions on the bacterial cells. However, this low-activity state cannot be quickly restored by simply removing the environmental stressors (such as removing them from the cryopreservation environment). When these low-activity microbial strains are used directly in clinical treatment, their insufficient activity may weaken or even eliminate their ultimate therapeutic effect. This undoubtedly presents a significant challenge to the clinical application of live microbial therapy.

[0005] Therefore, to maximize the clinical efficacy of microbial drugs and ensure their safety and effectiveness in clinical applications, it is necessary to find a method that can rapidly and efficiently restore the activity of frozen bacterial strains. This method must be able to significantly enhance the biological activity of frozen bacterial strains within a short period, restoring them to levels close to those of fresh strains, thereby guaranteeing their effectiveness in clinical treatment. Simultaneously, the method also needs to be simple to implement, low in cost, highly safe, and compatible with existing clinical procedures to ensure its widespread application in real-world clinical settings. Currently, although some studies have attempted to revive frozen bacterial strains by adding nutrients, these methods mostly suffer from problems such as complex operation, high cost, unsatisfactory revival effects, or insufficient safety, making it difficult to meet the needs of rapid revival and use in clinical practice.

[0006] Therefore, there is an urgent need to develop a simple and efficient rapid resuscitation solution for cryopreserved bacterial strains for clinical use, as well as its preparation method and application. Summary of the Invention

[0007] To address the problems of existing technologies, the purpose of this invention is to provide a simple and efficient rapid resuscitation solution for cryopreserved bacterial strains, its preparation method, and its application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In the first aspect, this application provides a rapid resuscitation solution for cryopreserved bacterial strains.

[0009] Secondly, this application provides a method for preparing a rapid resuscitation solution for cryopreserved bacterial strains.

[0010] Thirdly, this application provides the application of a rapid resuscitation solution for cryopreserved microorganisms in the preparation of drugs.

[0011] Fourthly, this application provides a combination of a rapid resuscitation solution for cryopreserved microorganisms with other conventional drugs or methods.

[0012] Fifthly, this application provides the application of a rapid resuscitation solution of cryopreserved bacterial strains in the preparation of antitumor drugs and other disease treatment drugs.

[0013] Sixthly, this application provides a combination of a cryopreserved bacterial strain rapid resuscitation solution with other conventional anti-tumor or disease treatment drugs or methods.

[0014] The first aspect of this application provides a rapid resuscitation solution for cryopreserved bacterial strains, which is composed of the following components: egg yolk lecithin: 0.05-0.3 g / ml; glucose: 0.01-0.05 g / ml; sucrose: 0.01-0.05 g / ml, and the remainder being sterile water.

[0015] Furthermore, the liquid volume range of the rapid resuscitation solution for cryopreserved bacterial strains is 100 μL-10000 μL.

[0016] The second aspect of this application provides a method for preparing a rapid resuscitation solution for cryopreserved bacterial strains, comprising the following steps: dissolving egg yolk lecithin, glucose and sucrose in sterile water, filtering for sterilization and then dispensing.

[0017] The third aspect of this application provides the application of a rapid resuscitation solution of cryopreserved bacterial strains in the preparation of pharmaceuticals.

[0018] Further, the rapid resuscitation solution of the cryopreserved bacterial strain is mixed with the cryopreserved bacterial strain and incubated statically at room temperature; the cryopreserved bacterial strain is a Gram-negative or Gram-positive bacterium, including any one of attenuated Salmonella VNP20009, Escherichia coli probiotic ECN, attenuated Listeria, BCG, Streptococcus thermophilus, Enterococcus faecalis, Lactobacillus rhamnosus, or Bifidobacterium.

[0019] Furthermore, the room temperature for standing is 16-37℃, and the standing time is 10-60 minutes; the bacterial concentration in the bacterial solution is not less than 10³ CFU / mL.

[0020] Furthermore, the revived cryopreserved bacterial solution, along with the resuscitation solution, can be used directly for subsequent treatment applications, including but not limited to intravenous infusion, intratumoral injection, or peritumoral injection after mixing with glucose injection solution, and oral administration.

[0021] The fourth aspect of this application provides a combination of a rapid resuscitation solution for cryopreserved microorganisms with other conventional drugs or methods.

[0022] The fifth aspect of this application provides the use of a rapid resuscitation solution of cryopreserved bacterial strains in the preparation of antitumor drugs and other disease treatment drugs.

[0023] The sixth aspect of this application provides a combination of a rapid resuscitation solution for cryopreserved microorganisms with other conventional antitumor or disease treatment drugs or methods.

[0024] Beneficial effects: The rapid resuscitation solution and its application method of this invention have significant innovation and practicality. They can effectively solve the problems of slow recovery of activity of frozen bacterial strains and insufficient clinical efficacy in existing technologies, and provide strong support for the clinical application of live microbial therapy. They have important clinical value and broad application prospects.

[0025] Compared with the prior art, the present invention has the following advantages: (1) The present invention can rapidly restore the activity of strains: The rapid recovery solution of the present invention can significantly enhance the biological activity of frozen strains in a short time, restoring them to a level close to that of fresh strains. By adding nutrients such as egg yolk lecithin, glucose and sucrose, the activity recovery of frozen strains can be effectively promoted, the recovery time can be shortened and the recovery efficiency can be improved.

[0026] (2) Improved clinical efficacy: The revived strains exhibit higher biological activity both in vivo and in vitro, enabling them to exert their therapeutic effects more effectively. For example, in anti-tumor therapy, the revived strains can accumulate and colonize more rapidly within tumors, effectively inhibiting tumors by activating immune responses or expressing drug proteins, thereby significantly improving the therapeutic effect.

[0027] Simple to operate: The resuscitation method of the present invention is simple and easy to implement. It only requires mixing the frozen strain with the rapid resuscitation solution and letting it stand at room temperature for a period of time. No complicated operation steps or special equipment are required, which makes it easy for the user to implement quickly. It is suitable for widespread application in clinical, laboratory and other application environments.

[0028] Low cost: Rapid resuscitation fluid is inexpensive to prepare, using common and affordable nutrients, and the production process is simple and easy to scale up. This makes the final cost of the resuscitation fluid controllable, which helps reduce the overall cost of clinical treatment and laboratory applications.

[0029] High safety: The selected nutrients (such as egg yolk lecithin, glucose and sucrose) have good biocompatibility and can be directly injected into animals or humans. After entering the body, they can be rapidly metabolized and absorbed without causing adverse effects on animals or humans, ensuring the safety of use.

[0030] Wide range of applications: This invention is applicable to a variety of Gram-negative or Gram-positive bacteria that can be used in clinical treatment, animal experiments, etc., including attenuated Salmonella VNP20009, Escherichia coli probiotic ECN, attenuated Listeria, BCG, Bifidobacterium, Streptococcus thermophilus, Enterococcus faecium, Lactobacillus rhamnosus, etc., as well as genetically modified strains based on these strains and synthetic biology engineered strains that can express drug proteins, and has broad application prospects.

[0031] Extended treatment window: By rapidly restoring the biological activity of the strain, this invention can shorten treatment time, extend the treatment window, improve treatment flexibility and adaptability, and provide a more timely and effective treatment plan.

[0032] Potential for combined applications: The rapid resuscitation solution of this invention can not only be used alone to prepare anti-tumor and other disease treatments, such as demyelinating diseases, Parkinson's disease, diabetes, ulcerative colitis, etc., but can also be used in combination with other conventional anti-tumor and other disease treatments (such as demyelinating diseases, Parkinson's disease, diabetes, ulcerative colitis, etc.) to exert a synergistic effect and further improve the treatment effect. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a flowchart illustrating the preparation and use of the rapid resuscitation solution for cryopreserved bacterial strains according to the present invention. The previously prepared cryopreserved glycerol strains or lyophilized powder strains are directly mixed with the aseptically treated rapid resuscitation solution in the required bacterial quantity and then thoroughly shaken. Subsequently, the mixture is allowed to stand at room temperature (25°C) for 20-60 minutes to obtain the reactivated bacterial strains, which can then be administered to the body via injection along with the resuscitation solution.

[0035] Figure 2 This is a graph showing the detection of ATP content in the culture medium of cryopreserved bacterial strains after activation using a rapid resuscitation solution, as described in this invention. The cryopreserved strain of attenuated Salmonella VNP20009 was prepared according to... Figure 1The incubation process involved culture in nutrient solutions containing different nutrients, including 5% glucose solution, 5% sucrose solution, 0.1 g / mL egg yolk lecithin solution, and a mixture of the above three nutrients (i.e., the rapid recovery solution). Bacterial cells placed in normal physiological saline served as a control. Differences in ATP content in the supernatant were measured after incubation for 20 / 40 / 60 minutes to indirectly assess differences in bacterial bioactivity. au is an arbitrary unit. 1: 20 minutes; 2: 40 minutes; 3: 60 minutes. **P < 0.01.

[0036] Figure 3 This is a graph showing the detection of ATP content in the culture medium of different cryopreserved bacterial strains after activation using a rapid resuscitation solution, according to the present invention. Different cryopreserved bacterial strains (including *Escherichia coli* ECN, *Bifidobacterium longum*, selenium-enriched *Bifidobacterium longum*, attenuated *Listeria*, *BCG*, *Streptococcus thermophilus*, selenium-enriched *Streptococcus thermophilus*, *Enterococcus faecium*, selenium-enriched *Enterococcus faecium*, *Lactobacillus rhamnosus*, selenium-enriched *Lactobacillus rhamnosus*, etc.) were classified according to... Figure 1 The bacterial cells were incubated in a rapid resuscitation solution, with those placed in normal physiological saline serving as a control. *Escherichia coli* (ECN), attenuated *Listeria*, and *BCG* were cryopreserved at -80°C in glycerol; *Bifidobacterium longum*, selenium-enriched *Bifidobacterium longum*, *Streptococcus thermophilus*, selenium-enriched *Streptococcus thermophilus*, *Enterococcus faecium*, selenium-enriched *Enterococcus faecium*, *Lactobacillus rhamnosus*, and selenium-enriched *Lactobacillus rhamnosus* were lyophilized powders stored at 4°C. After 40 minutes of static incubation, the difference in ATP content in the supernatant was measured (expressed as relative bioluminescence intensity) to indirectly assess the differences in bacterial bioactivity. The results confirmed that the rapid resuscitation solution could effectively enhance the bioactivity of the aforementioned bacterial strains within 40 minutes. Figure 3 Only representative results for *Escherichia coli* ECN, *Bifidobacterium longum*, and selenium-enriched *Bifidobacterium longum* are provided. au represents any unit. 1: Saline group; 2: Rapid resuscitation solution group. NS indicates no significant difference; *P < 0.05; ****P < 0.0001.

[0037] Figure 4 This is a graph showing the difference in bacterial growth after the cryopreserved bacterial strains were activated using a rapid resuscitation solution according to the present invention. Figure 4 a represents the detection of changes in the bacterial growth curve of cryopreserved bacterial strains after activation with rapid resuscitation solution according to the present invention. The cryopreserved strain of attenuated Salmonella VNP20009 was processed according to... Figure 1 The bacterial cultures were incubated in nutrient solutions containing different nutrients, including 5% glucose solution, 5% sucrose solution, 0.1 g / mL egg yolk lecithin solution, and a mixture of the above three nutrients (i.e., the rapid recovery solution). Bacterial cells placed in normal physiological saline served as a control. After standing for 40 minutes, the bacterial cultures of different groups were diluted 200-fold and placed in bacterial LB agar to monitor changes in bacterial OD600 values ​​in real time. The culture conditions were 37°C with shaking. Figure 4 b is Figure 4 In section a, this invention compares the differences in OD600 values ​​of cryopreserved bacterial strains activated using a rapid resuscitation solution at the 5th hour of culture. 1: physiological saline; 2: glucose; 3: sucrose; 4: egg yolk lecithin; 5: rapid resuscitation solution. ****P < 0.0001.

[0038] Figure 5 This is a graph showing the intensity of red fluorescent protein (RFP) in cryopreserved bacteria after activation with a rapid resuscitation solution, as described in this invention. The cryopreserved strain of engineered attenuated Salmonella VNP20009 (abbreviated as VNP-RFP), expressing red fluorescent protein (RFP), was processed according to... Figure 1 The strains were incubated in nutrient solutions containing different nutrients, including 5% glucose solution, 5% sucrose solution, 0.1 g / mL egg yolk lecithin solution, and a mixture of the above three nutrients (i.e., the rapid recovery solution). Bacterial cells placed in normal physiological saline served as a control. After standing for 20 / 40 / 60 minutes, the differences in fluorescence intensity of the engineered strains after different treatments were detected to indirectly assess the differences in expression activity of the engineered strains. au is an arbitrary unit. 1: 20 minutes; 2: 40 minutes; 3: 60 minutes. ns = not significant, *P < 0.05.

[0039] Figure 6 This is a comparative graph showing the initial number of bacterial cells on plates after activation with rapid resuscitation solution for cryopreserved bacterial strains according to this invention. Cryopreserved attenuated Salmonella VNP20009 strains were incubated in rapid resuscitation solution (containing 5% glucose, 5% sucrose, and 0.1 g / mL egg yolk lecithin), with bacterial cells placed in normal physiological saline as a control. After incubation for 20 / 40 / 60 minutes, the strains were diluted and plated to detect differences in bacterial count at different treatment times. 1: 20 minutes; 2: 40 minutes; 3: 60 minutes. ns = not significant.

[0040] Figure 7 This is a comparative graph showing the difference in the activation ability (M1-type related gene expression) of macrophages by strains activated with rapid resuscitation solution after cryopreservation according to this invention. Cryopreserved attenuated Salmonella VNP20009 was incubated in rapid resuscitation solution (containing 5% glucose, 5% sucrose, and 0.1 g / mL egg yolk lecithin), with cells placed in normal physiological saline as a control. After 40 minutes of static incubation, the cells were collected and co-cultured in BMDM (bone marrow-derived macrophages) at MOI 10 for 3 hours. Cells were then collected for qPCR detection of M1-type macrophage-related genes (including IL1β, IL6, and TNFα). 1: Physiological saline; 2: Rapid resuscitation solution. ns = not significant, *P < 0.05, **P < 0.01.

[0041] Figure 8This figure shows a comparison of the activation ability (ROS detection) of frozen bacterial strains activated with rapid resuscitation solution on macrophages. Frozen strains of attenuated Salmonella VNP20009 were incubated in rapid resuscitation solution (containing 5% glucose, 5% sucrose, and 0.1 g / mL egg yolk lecithin), with bacterial cells placed in normal physiological saline as a control. After 40 minutes of static incubation, bacterial cells were collected and co-cultured with BMDM (bone marrow-derived macrophages) at MOI 20 for 3 hours. Cells were then collected, labeled with a ROS detection fluorescent probe for 10 minutes, and ROS levels were detected by flow cytometry. The figure shows a representative flow cytometry plot (left) and a comparison of ROS detection differences between different groups (right). 1: Physiological saline; 2: Rapid resuscitation solution. *P < 0.05.

[0042] Figure 9 This is a comparative graph showing the differences in the initial enrichment of cryopreserved bacterial strains activated with rapid resuscitation solution against in vivo tumors (mouse in situ melanoma). Cryopreserved attenuated Salmonella VNP20009 strains were incubated in rapid resuscitation solution (containing 5% glucose, 5% sucrose, and 0.1 g / mL egg yolk lecithin), with bacterial cells placed in normal physiological saline as a control. After 40 minutes of static incubation, the bacterial cells were collected and administered via intraperitoneal injection to an in situ melanoma mouse model. Twenty hours after administration, mouse tumors were obtained, and the bacterial titers within the tumors were detected by dilution and plate testing in different groups. 1: Physiological saline group; 2: Rapid resuscitation solution group. ****P < 0.0001.

[0043] Figure 10 This is a comparative graph showing the differences in the initial enrichment of cryopreserved bacterial strains activated with rapid resuscitation solution on in vivo tumors (orthotopic breast cancer in mice). Cryopreserved attenuated Salmonella VNP20009 strains were incubated in rapid resuscitation solution (containing 5% glucose, 5% sucrose, and 0.1 g / mL egg yolk lecithin), with bacteria placed in normal physiological saline as a control. After 40 minutes of static incubation, the bacteria were collected and administered via intraperitoneal injection to an orthotopic breast cancer mouse model. Twenty hours after administration, mouse tumors were obtained, and the bacterial titers within the tumors were detected by dilution and plate testing in different groups. 1: Physiological saline group; 2: Rapid resuscitation solution group. ****P < 0.0001. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0046] In this application, "~ one less" means one or more, and "more than" means two or more. "~ one less item (item) below" or similar expressions refer to any combination of these items, including any combination of single items (items) or multiple items (items). For example, "~ one less item (item) in a, b, or c", or "~ one less item (item) in a, b, and c", can all mean: a, b, c, a~b (i.e., a and b), a~c, b~c, or a~b~c, where a, b, and c can be single or multiple.

[0047] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0048] The first aspect of this application provides a rapid resuscitation solution for cryopreserved bacterial strains, which is composed of the following components: egg yolk lecithin: 0.05-0.3 g / ml; glucose: 0.01-0.05 g / ml; sucrose: 0.01-0.05 g / ml, and the remainder being sterile water.

[0049] In some embodiments, the liquid volume of the rapid resuscitation solution for cryopreserved microorganisms ranges from 100 μL to 10000 μL.

[0050] The second aspect of this application provides a method for preparing a rapid resuscitation solution for cryopreserved bacterial strains, comprising the following steps: dissolving egg yolk lecithin, glucose and sucrose in sterile water, filtering for sterilization and then dispensing.

[0051] The third aspect of this application provides the application of a rapid resuscitation solution of cryopreserved bacterial strains in the preparation of drugs.

[0052] In some embodiments, a rapid resuscitation solution for cryopreserved bacterial strains is mixed with the cryopreserved bacterial strains and cultured statically at room temperature; the cryopreserved bacterial strains are Gram-negative or Gram-positive bacteria, including any one of attenuated Salmonella VNP20009, Escherichia coli probiotic ECN, attenuated Listeria, BCG, Streptococcus thermophilus, Enterococcus faecalis, Lactobacillus rhamnosus, or Bifidobacterium.

[0053] In some embodiments, the room temperature is 16-37°C and the standing time is 10-60 minutes; the bacterial concentration in the bacterial solution is not less than 10³ CFU / mL.

[0054] In some embodiments, the revived cryopreserved bacterial solution can be used directly with the resuscitation solution for subsequent treatment applications, including but not limited to intravenous infusion, intratumoral injection or peritumoral injection after mixing with glucose injection solution, and oral administration.

[0055] The fourth aspect of this application provides a combination of a rapid resuscitation solution for cryopreserved microorganisms with other conventional drugs or methods.

[0056] The fifth aspect of this application provides the application of a rapid resuscitation solution of cryopreserved bacterial strains in the preparation of antitumor drugs and other disease treatment drugs.

[0057] The sixth aspect of this application provides a combination of a rapid resuscitation solution for cryopreserved microorganisms with other conventional antitumor or disease treatment drugs or methods. Example 1

[0058] The present invention provides a rapid resuscitation solution for cryopreserved bacterial strains for clinical use. The rapid resuscitation solution for cryopreserved bacterial strains consists of the following components: egg yolk lecithin: 0.05 g / ml; glucose: 0.05 g / ml; sucrose: 0.01 g / ml, and the remainder is sterile water.

[0059] The liquid volume range for rapid resuscitation solution of cryopreserved bacterial strains is 100 μL.

[0060] The resulting rapid resuscitation solution containing the bacterial strain, after undergoing the resuscitation process described above, can be directly used in downstream clinical treatment. Options include: 1) Centrifuging at 6000-10000 rpm for 3-10 minutes, removing the supernatant, resuspending the bacteria in the appropriate injection solution, and then injecting in vivo; 2) More recommended option: Directly mixing the injection solution with the bacterial rapid resuscitation solution and then injecting in vivo.

[0061] The direct administration of the bacterial rapid resuscitation solution includes at least one of the following: intravenous injection preparation, intratumoral injection preparation, intraperitoneal injection preparation, or oral administration preparation.

[0062] The various nutrients in the rapid resuscitation solution, such as egg yolk lecithin and / or glucose and / or sucrose, along with sufficient resuscitation time, are key factors in optimizing the effectiveness of this type of resuscitation solution. Similarly, rapid reactivation of cryopreserved bacterial strains is crucial for these strains to exert their in vivo biological therapeutic effects.

[0063] Cryopreserved bacterial strains that can be used clinically include, but are not limited to, various types of Gram-negative / positive bacteria, including attenuated Salmonella VNP20009, Escherichia coli probiotic ECN, attenuated Listeria, BCG, Streptococcus thermophilus (patent application number: 2024110944111), Enterococcus faecalis (patent application number: 2024110944516), Lactobacillus rhamnosus (patent application number: 2024110944520), Bifidobacterium (patent application number: 2024110944126), etc., as well as genetically modified strains based on the above strains and synthetic biology engineered strains that can express drug proteins, etc., all of which are suitable for the activation and application of this rapid resuscitation solution. The activation and application scheme of this rapid resuscitation solution is applicable to the treatment of diseases such as tumors (e.g., patents: 2025102714625; 2025102714610; 2025102714606), demyelinating diseases (e.g., patent: 2024110948076), Parkinson's disease (e.g., patent: 2024110948080), diabetes (e.g., patents: 2024110948095; 2024110948108), and ulcerative colitis (e.g., patents: 2025102712456, 2025102712441; 2025102712437) based on the above-mentioned strains. Since other bacteria, such as Escherichia coli probiotic ECN, attenuated Listeria, BCG, Streptococcus thermophilus, Enterococcus faecalis, Lactobacillus rhamnosus, and Bifidobacterium, have all obtained similar results to attenuated Salmonella VNP20009 in the experiment, the following examples only use attenuated Salmonella VNP20009 as a specific and representative research object for description. Example 2

[0064] This invention provides a rapid resuscitation solution for cryopreserved bacterial strains for clinical use. The rapid resuscitation solution comprises the following components: egg yolk lecithin: 0.3 g / ml; glucose: 0.01 g / ml; sucrose: 0.05 g / ml; and the remainder being sterile water. The liquid volume of the rapid resuscitation solution ranges from 10,000 μL. Example 3

[0065] The present invention provides a rapid resuscitation solution for cryopreserved bacterial strains for clinical use. The rapid resuscitation solution for cryopreserved bacterial strains consists of the following components: egg yolk lecithin: 0.25 g / ml; glucose: 0.04 g / ml; sucrose: 0.04 g / ml, with the remainder being sterile water.

[0066] The liquid volume range of the rapid resuscitation solution for cryopreserved bacterial strains is 9000 μL. Example 4

[0067] The present invention provides a method for preparing a rapid resuscitation solution for cryopreserved bacterial strains for clinical use, comprising the following steps: dissolving egg yolk lecithin, glucose and sucrose in sterile water, filtering for sterilization and then dispensing. Example 5

[0068] The present invention provides a method for reviving frozen bacterial strains using a resuscitation solution, comprising the following steps: mixing the frozen bacterial strain with a rapid resuscitation solution and incubating it statically at room temperature.

[0069] The frozen bacterial strains were either Gram-negative or Gram-positive bacteria, and the Gram-negative or Gram-positive bacteria were attenuated Salmonella VNP20009.

[0070] The room temperature range for standing is 16°C, and the standing time is 60 minutes; the bacterial concentration in the bacterial solution is not less than 10³ CFU / mL.

[0071] The revived cryopreserved bacterial solution, along with the resuscitation solution, can be used directly for subsequent treatment applications, including but not limited to intratumoral injection after being mixed with glucose injection.

[0072] Taking the rapid resuscitation of attenuated Salmonella VNP20009 cryopreserved in glycerol as an example, considering the efficiency of bioactivity optimization, the difference in bacterial titer changes over time during resuscitation, and the convenience of treatment use, a room temperature static resuscitation time of no more than 60 minutes, preferably 20-40 minutes, is recommended. Under the 40-minute condition, compared to static incubation in physiological saline, the bioactivity of the strain in the rapid resuscitation solution was significantly improved both in vitro and in vivo. Example 6

[0073] The difference between Example 6 and Example 5 is that: a method for reviving frozen strains using a resuscitation solution according to the present invention includes the following steps: mixing the rapid resuscitation solution of the frozen strain with the frozen strain and incubating it statically at room temperature.

[0074] The frozen strains are Gram-positive bacteria that can be used for disease treatment / prevention.

[0075] The room temperature range for standing is 37°C, and the standing time is 10 minutes; the bacterial concentration in the bacterial solution is not less than 10³ CFU / mL.

[0076] The revived cryopreserved bacterial solution, along with the resuscitation solution, can be used directly for subsequent treatment applications, including but not limited to intratumoral injection after being mixed with glucose injection solution. Example 7

[0077] The difference between Example 7 and Example 5 is that: a method for reviving frozen strains using a resuscitation solution according to the present invention includes the following steps: mixing the rapid resuscitation solution of the frozen strain with the frozen strain and incubating it statically at room temperature.

[0078] The frozen strains are Gram-negative bacteria that can be used for disease treatment. The Gram-negative bacteria or Gram-positive bacteria are attenuated Listeria.

[0079] The room temperature range for standing is 27°C, and the standing time is 50 minutes; the bacterial concentration in the bacterial solution is not less than 10³ CFU / mL.

[0080] The revived cryopreserved bacterial solution, along with the resuscitation fluid, can be used directly for subsequent treatment applications, including but not limited to intravenous infusion after being mixed with glucose injection. Example 8

[0081] The difference between Example 8 and Example 5 is that: a method for reviving frozen strains using a resuscitation solution according to the present invention includes the following steps: mixing the rapid resuscitation solution of the frozen strain with the frozen strain and incubating it statically at room temperature.

[0082] The frozen bacterial strains are either Gram-negative or Gram-positive bacteria, and the Gram-negative or Gram-positive bacteria are any one of attenuated Listeria, BCG, or Bifidobacterium. The Gram-negative or Gram-positive bacteria are Escherichia coli probiotics (ECN). Example 9

[0083] The difference between Example 9 and Example 5 is that the frozen strain is a Gram-negative or Gram-positive bacterium, and the Gram-negative or Gram-positive bacterium is an attenuated Listeria. Example 10

[0084] The difference between Example 10 and Example 5 is that the frozen strain is a Gram-negative bacterium or a Gram-positive bacterium, and the Gram-negative bacterium or the Gram-positive bacterium is BCG. Example 11

[0085] The difference between Example 11 and Example 5 is that the frozen strain is a Gram-negative bacterium or a Gram-positive bacterium, and the Gram-negative bacterium or the Gram-positive bacterium is a thermophilic streptococcus. Example 12

[0086] The difference between Example 12 and Example 5 is that the frozen strain is a Gram-negative bacterium or a Gram-positive bacterium, and the Gram-negative bacterium or the Gram-positive bacterium is Enterococcus faecalis. Example 13

[0087] The difference between Example 13 and Example 5 is that the frozen strain is a Gram-negative bacterium or a Gram-positive bacterium, and the Gram-negative bacterium or the Gram-positive bacterium is Lactobacillus rhamnosus.

[0088] Example 14

[0089] The difference between Example 14 and Example 5 is that the frozen strain is a Gram-negative bacterium or a Gram-positive bacterium, and the Gram-negative bacterium or the Gram-positive bacterium is Bifidobacterium. Example 15

[0090] The present invention relates to the application of a rapid resuscitation solution of a cryopreserved bacterial strain in the preparation of drugs.

[0091] The present invention relates to the combined use of a rapid resuscitation solution for cryopreserved bacterial strains with other conventional drugs or methods.

[0092] The present invention relates to the application of a rapid resuscitation solution of a cryopreserved bacterial strain in the preparation of antitumor drugs and other disease treatment drugs.

[0093] The present invention relates to the combined use of a rapid resuscitation solution for cryopreserved bacterial strains with other conventional anti-tumor or disease treatment drugs or methods. Example 16

[0095] To prepare a rapid resuscitation solution for cryopreserved microorganisms, several nutrients with good human or animal compatibility, absorbability, and promoting effects on microbial survival and activity recovery were selected, including egg yolk lecithin, glucose, and sucrose. 0.1 g / mL of egg yolk lecithin and / or 0.05 g / mL of glucose and / or 0.05 g / mL of sucrose were thoroughly dissolved in sterile water (this can be achieved by heating at 42°C and sonicating for 30-60 seconds for rapid dissolution). The solution was then filtered through a 0.22-micron filter in a sterile operating table for sterilization. The resuscitation solution was then aliquoted into sterile EP tubes (1000-1500 μL per tube) and refrigerated for later use. Figure 1 ).

[0096] Experimental Example 1 The frozen attenuated Salmonella VNP20009, activated overnight in LB liquid medium, and the engineered attenuated Salmonella VNP20009-RFP strain expressing red fluorescent protein (RFP), were centrifuged at 9000 rpm for 5 minutes. The supernatant was then discarded, and the culture was resuspended in a solution containing 20% ​​sucrose (prepared with sterile water), with the OD600 adjusted to 1.5. An equal volume of 50% glycerol was added and thoroughly mixed by pipetting, resulting in a final concentration of 10% sucrose and 25% glycerol in the cryopreservation solution. The cryopreserved bacteria were then transferred to -80°C for storage.

[0097] Experimental Example 2

[0098] Effects of rapid resuscitation solution pretreatment on the biological and expression activities of the strain

[0099] To investigate the effects of resuscitation solutions with different nutrient additions on the bioactivity of cryopreserved bacterial strains, strains frozen for more than one week were retrieved and rapidly thawed at 37°C. Subsequently, 50 μL of the cryopreserved bacterial culture was added to 1000 μL of either physiological saline, sterile water with 5% glucose, sterile water with 0.05 g / mL sucrose, sterile water with 0.1 g / mL egg yolk lecithin, or sterile water with 0.05 g / mL glucose, 0.05 g / mL sucrose, and 0.1 g / mL egg yolk lecithin (i.e., the rapid resuscitation solution group), resulting in five groups. After thorough mixing, the cultures were incubated at room temperature (25°C) for 20, 40, and 60 minutes, respectively. Then, the cultures were centrifuged at 9000 rpm for 5 minutes, and the supernatant was collected. Following the instructions of the microbial cell viability assay kit (i.e., the ATP assay kit, Beyotime, C0052S), a luminescent assay reagent was added to the supernatant. Differences in ATP content in the supernatant were detected using an ELISA reader. Generally, ATP content in the supernatant is positively correlated with bacterial bioactivity and quantity. The results showed that the addition of glucose / sucrose / egg yolk lecithin effectively enhanced bacterial bioactivity, with the mixture of the three (i.e., rapid resuscitation solution) achieving the most significant increase in bacterial bioactivity. Compared to the saline group, glucose group, sucrose group, and egg yolk lecithin group, the relative ATP content in the rapid resuscitation solution group increased by 2.46 times, 3.12 times, 2.93 times, and 4.29 times, respectively, at 40 minutes. Figure 2 Further testing was conducted on the resuscitation effects of the rapid resuscitation solution on other bacterial species (including Gram-negative and Gram-positive bacteria), including *Escherichia coli* ECN, *Bifidobacterium longum*, selenium-enriched *Bifidobacterium longum*, attenuated *Listeria*, *BCG*, *Streptococcus thermophilus*, selenium-enriched *Streptococcus thermophilus*, *Enterococcus faecium*, selenium-enriched *Enterococcus faecium*, *Lactobacillus rhamnosus*, and selenium-enriched *Lactobacillus rhamnosus*. The results confirmed that the rapid resuscitation solution could also effectively enhance the bioactivity of the above-mentioned strains within 40 minutes. Only representative strains such as *Escherichia coli* ECN, *Bifidobacterium longum*, and selenium-enriched *Bifidobacterium longum* are presented here. Figure 3 ).

[0100] Meanwhile, the growth curves of the strains under the above different conditions were examined. Bioscreen C software (OYGrowth 175 Curves Ab Ltd., Finland) was used to detect the growth curves of the strains in different groups on LB medium. Specifically, the strains under the above different resuscitation conditions were diluted 100-fold after 40 minutes of resuscitation treatment. Subsequently, 10 μL of the suspension was added to 1 mL of LB medium, and 300 μL of the solution was added to each well of a Bioscreen C multi-well plate. The multi-well plates were incubated at 37°C for 15 hours. OD values ​​were measured every 10-30 minutes using a 600 nm wavelength brown filter. The results confirmed that the strains treated with the rapid resuscitation solution were able to undergo logarithmic growth earlier and reach the plateau phase earlier. Figure 4 a). The OD600 of the strain in the rapid resuscitation solution group reached 0.53 within 5 hours after inoculation, significantly higher than that in the saline group (0.29), glucose group (0.44), sucrose group (0.40), and egg yolk lecithin group (0.37). Figure 4 b).

[0101] Subsequently, the differences in the expression of the target protein in the engineered strains under the above-mentioned different conditions were detected. An engineered plasmid containing the constitutive promoter J23100 and the red fluorescent protein (RFP) gene sequence was electroporated into the VNP20009 strain to obtain an engineered strain that continuously expresses red fluorescence (denoted as the VNP-RFP strain). The VNP-RFP strain was frozen under the same method but different conditions. Strains frozen for more than one week were removed and rapidly thawed at 37°C. Then, 50 μL of the frozen bacterial culture was added to 1000 μL of physiological saline, sterile water supplemented with 0.05 g / mL glucose, sterile water supplemented with 0.05 g / mL sucrose, sterile water supplemented with 0.1 g / mL egg yolk lecithin, or sterile water supplemented with 0.05 g / mL glucose, 0.05 g / mL sucrose, and 0.1 g / mL egg yolk lecithin (i.e., the rapid thawing solution group), for a total of five groups. After thorough mixing, the samples were allowed to stand at room temperature (25℃) for 20, 40, and 60 minutes, respectively. Then, 100 μL of the bacterial resuscitation solution was taken, and the differences in red fluorescence intensity among the internal strains were detected using a microplate reader (excitation light 550 nm, emission light 585 nm). The results showed that the strains treated with the rapid resuscitation solution for 40 minutes produced stronger red fluorescence than other groups, further confirming the effective restoration of the strain's biological expression activity by the rapid resuscitation solution. Figure 5 ).

[0102] To confirm whether the differences in ATP content and RFP intensity among the above-mentioned bacterial strains might be related to differences in bacterial count, 50 μL of frozen bacterial culture was added to 1000 μL of physiological saline or sterile water supplemented with 0.05 g / mL glucose, 0.05 g / mL sucrose, and 0.1 g / mL egg yolk lecithin (i.e., rapid resuscitation solution group). After thorough mixing, the mixtures were incubated at room temperature (25℃) for 20, 40, and 60 minutes, respectively. Then, the strains at different time points were serially diluted and plated onto LB agar plates of the corresponding antibiotic resistance (Kana), and incubated overnight upside down at 37℃. The results showed no significant difference in the number of strains between the two groups within 60 minutes. Figure 6 These results emphasize that the rapid resuscitation solution did indeed restore the bacteria's biological activity, rather than simply increasing various indicators due to differences in bacterial numbers. At 60 minutes, the rapid resuscitation solution group did induce a certain increase in the number of strains, but this was not significantly different from the 40-minute group. To avoid differences in biodosage due to subsequent bacterial self-replication, it is recommended to treat frozen strains with the rapid resuscitation solution for no more than 60 minutes (40 minutes is preferred).

[0103] Experimental Example 3 Evaluation of the effect of strains pretreated with rapid resuscitation fluid on immune cell activation

[0104] Intratumoral bacteria can typically indirectly control tumors by activating immune cells, especially macrophages. Therefore, the effects of different treatments on macrophage immune activation were evaluated. 50 μL of frozen bacterial culture was added to 1000 μL of physiological saline or sterile water supplemented with 0.05 g / mL glucose, 0.05 g / mL sucrose, and 0.1 g / mL egg yolk lecithin (i.e., rapid resuscitation solution). After thorough mixing, the solutions were incubated at room temperature (25°C) for 40 minutes. Next, mouse bone marrow-derived macrophages (BMDM) (2.0 × 10⁻⁶ cells) were... 5The cells were inoculated into 12-well plates and cultured adherently for 6-8 hours. Following an MOI of 10, different groups of strains were co-incubated with mouse bone marrow-derived macrophages. After 3 hours, cells were collected, total RNA was extracted, and cDNA was obtained using a reverse transcription kit. Changes in macrophage M1 marker genes, including IL1β (F: 5-GCA ACT GTT CCT GAA CTC AAC T-3; R: 5-TCT TTT GGG GTCCGT CAA CT-3), IL6 (F: 5-TAG TCC TTC CTA CCC CAA TTT CC-3; R: 5-TTG GTC CTT AGCCAC TCC TTC-3), and TNFα (F: 5-CCC TCA CAC TCA GAT CAT CTT CT-3; R: 5-GCT AC GAC GTGGGC TAC AG-3), were detected using qPCR. The results showed that M1-related genes in the rapidly resuscitated strains were slightly upregulated to varying degrees. Figure 7 Subsequently, the ROS production of macrophages in different groups was detected using a ROS probe. Macrophages obtained under the above conditions were labeled with a ROS probe (MCE) for 10-15 minutes, washed 2-3 times with PBS solution, and then fluorescence intensity changes (FITC channel) were detected by flow cytometry. The results showed that, compared with saline-treated strains, rapidly resuscitated strains significantly induced ROS production in macrophages (increasing it by 1.43 times). Figure 8 These results confirm that strains treated with the rapid resuscitation solution possess higher biological activity and more significant immune cell activation properties. This also further demonstrates the great potential of the rapid resuscitation solution of this invention to assist in the clinical translation of different frozen strains.

[0105] Test Example 4

[0106] B16F10 (mouse melanoma cells) / 4T1 (mouse breast cancer cells) were grown in DMEM cell culture medium until they reached the exponential growth phase. Cells were then collected and resuspended in PBS to adjust the final cell concentration to 1 × 10⁻⁶. 7 Cells / mL. For B16F10 cells, 100 μL was injected into the axillary fat pad of each C57BL / 6 mouse, i.e., 1 × 10⁻⁶ cells / mL. 6 100 μL per mouse. For 4T1 cells, 100 μL was injected into the mammary fat pad of each Balb / c mouse, i.e., 1 × 10⁶ cells / mouse. 6 Each mouse was inoculated with one tumor. Mice were housed in a clean-grade animal facility after inoculation, and the tumors were allowed to grow to approximately 80-100 mm in size. 3 Subsequent experiments will be conducted at that time.

[0107] Following the same method described above, 50 μL of attenuated Salmonella VNP20009 cryopreserved bacterial suspension was added to 1000 μL of physiological saline or sterile water supplemented with 0.05 g / mL glucose, 0.05 g / mL sucrose, and 0.1 g / mL egg yolk lecithin (i.e., the rapid resuscitation solution group). After thorough mixing, the solutions were incubated at room temperature (25°C) for 40 minutes. Then, the bacterial concentration was adjusted to 1×10⁻⁶ as needed. 7 CFU / mL was administered intraperitoneally to either an orthotopic melanoma mouse model or an orthotopic breast cancer mouse model (100 μL per mouse). Twenty hours after administration of the bacterial drug to the tumor-bearing mice, the mice were randomly sacrificed, and tumors were harvested under sterile conditions. The tumors were weighed and homogenized in 2 mL PBS using a tissue homogenizer (frequency: 60 Hz; time: 100 s). The tumor tissue was serially diluted, plated on LB agar plates, and incubated upside down in a 37°C bacterial incubator for 12 h. Colony counting was performed, and the differences in the number of VNP20009 strains in mouse tumors between different groups were compared and analyzed. The results showed that effective colonization of bacteria within the tumor was detectable in both groups. However, compared to the saline-treated bacterial group, the group treated with rapid resuscitation fluid showed more rapid enrichment of the bacterial strain within the tumor. In an orthotopic melanoma mouse model, the intratumoral bacterial titer in the saline-treated bacterial group was 43.1 CFU / mg, while the intratumoral bacterial titer in the rapid resuscitation fluid-treated bacterial group was 115.4 CFU / mg. Pretreatment with rapid resuscitation fluid increased the intratumoral bacterial titer by 2.68 times. Figure 9 In an orthotopic breast cancer mouse model, the intratumoral bacterial titer in the saline-treated bacterial group was 9.2 CFU / mg, while the intratumoral bacterial titer in the rapid resuscitation fluid-treated bacterial group was 74.8 CFU / mg. Pretreatment with rapid resuscitation fluid increased the intratumoral bacterial titer by 8.15 times. Figure 10 These results confirm that the rapid resuscitation solution for frozen bacterial strains described in this invention can effectively enhance the in vivo biological activity of the strains after pretreatment, and indirectly enhance their tumor-targeting and colonization activities. Therefore, this invention has a significant enhancing effect on the clinical efficacy of such frozen bacterial strains and has great application value.

[0108] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above-described experimental examples. The experimental examples and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A rapid resuscitation solution for cryopreserved bacterial strains, characterized in that: The rapid resuscitation solution for frozen bacterial strains consists of the following components Composition: Egg yolk lecithin: 0.05-0.3 g / ml; Glucose: 0.01-0.05 g / ml; Sucrose: 0.01-0.05g / ml, the remainder is sterile water.

2. The rapid resuscitation solution for cryopreserved bacterial strains according to claim 1, characterized in that: The liquid volume range of the rapid resuscitation solution for frozen bacterial strains is 100 μL-10000 μL.

3. A method for preparing the rapid resuscitation solution of cryopreserved bacterial strains as described in claim 1, characterized in that... The process includes the following steps: dissolving the egg yolk lecithin, glucose, and sucrose described in claim 1 in sterile water, filtering to remove bacteria, and then dispensing.

4. The use of the rapid resuscitation solution of the frozen bacterial strain according to claim 1 in the preparation of drugs.

5. The application according to claim 4, characterized in that: The rapid resuscitation solution of the cryopreserved bacterial strain described in claim 1 is mixed with the cryopreserved bacterial strain and cultured statically at room temperature; the cryopreserved bacterial strain is a Gram-negative bacterium or a Gram-positive bacterium, including any one of attenuated Salmonella VNP20009, Escherichia coli probiotic ECN, attenuated Listeria, BCG, Streptococcus thermophilus, Enterococcus faecalis, Lactobacillus rhamnosus, or Bifidobacterium.

6. The application according to claim 5, characterized in that: The room temperature for standing is 16-37℃, and the standing time is 10-60 minutes; the bacterial concentration in the bacterial solution is not less than 10³ CFU / mL.

7. The application according to claim 4, characterized in that: The revived cryopreserved bacterial solution can be used directly with the resuscitation solution for subsequent treatment applications, including but not limited to intravenous infusion, intratumoral injection or peritumoral injection after mixing with glucose injection solution, and oral administration.

8. The combined use of the rapid resuscitation solution for cryopreserved microorganisms as described in claim 1 with other conventional drugs or methods.

9. The use of the cryopreserved bacterial strain rapid resuscitation solution according to claim 1 in the preparation of antitumor drugs and other disease treatment drugs.

10. The combined use of the cryopreserved bacterial strain rapid resuscitation solution according to claim 1 with other conventional antitumor or disease treatment drugs or methods.

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