High activity, long-term preservation and rapid revival method of microbial strains

Abstract

The present application relates to the technical field of microbial strain preservation, and discloses a high-activity, long-term preservation and rapid revival method for microbial strains. The method sequentially performs two-stage osmotic adaptation, and loads the microbial cells on a porous carrier having a rehydration buffer zone and a microbial cell loading zone; blank protection units without microbial cells are used to deduct the background conductivity of the carrier and the dissolved protective components. During revival, the first revival liquid enters the microbial cell loading zone after forming a transition osmotic environment in the rehydration buffer zone; according to the change in the corrected conductivity between the microbial rehydration system and the blank rehydration system, the first nutrient addition liquid for limiting proliferation and the second nutrient addition liquid for proliferation culture are added in sequence. The method associates rehydration transition, background correction and nutrition start, which is conducive to reducing osmotic disturbance in the preservation and revival processes, and improving the consistency of revival timing judgment of different preservation units.

Description

Technical Field

[0001] This invention relates to the field of microbial strain preservation and resuscitation technology, specifically to a method for achieving high activity, long-term preservation, and rapid resuscitation of microbial strains through stepwise permeation adaptation before preservation, porous carrier loading protection, low-temperature or freeze-drying preservation, and staged resuscitation based on changes in conductivity. Background Technology

[0002] Microbial strains are essential basic materials for microbial research, industrial fermentation, biopharmaceutical preparation, food processing, and environmental remediation. During preservation, strains need to have their metabolic activity reduced to minimize genetic drift and contamination risks, while also maintaining the integrity of cell membranes, proteins, and intracellular metabolic systems as much as possible so that they can regain their original growth capacity and target functions upon use.

[0003] Existing methods for preserving microbial strains mainly include periodic transplantation, liquid paraffin preservation, cryopreservation, liquid nitrogen preservation, and vacuum freeze-drying. Periodic transplantation requires repeated subculturing, which easily increases the risk of contamination and phenotypic drift; liquid paraffin preservation is limited to certain strains; although cryopreservation and liquid nitrogen preservation can extend the preservation period, ice crystal formation, solute concentration, and osmotic pressure changes during freezing and thawing can damage cell membranes; vacuum freeze-drying facilitates transportation, but pre-freezing, sublimation drying, and rehydration processes can also cause damage to membrane and protein structures.

[0004] To improve preservation survival rates, existing techniques typically add glycerol, dimethyl sulfoxide, trehalose, sucrose, sorbitol, skim milk, or high-molecular-weight protective components to the bacterial suspension. However, the preservation and resuscitation effects of existing methods are still easily affected by factors such as the type of bacterial strain, the culture stage, the composition of the protective solution, freezing or drying conditions, and resuscitation culture conditions. Some strains may experience a decrease in culturable bacterial count, a prolonged resuscitation lag period, or unstable recovery of target metabolic activity after preservation.

[0005] Furthermore, variations in the preservative solution, carrier material, and bacterial cell state between different batches can cause fluctuations in the test results during the resuscitation process. Fixed process parameters are also difficult to fully adapt to different bacterial strains and storage periods. Therefore, it remains necessary to improve the preservation and resuscitation procedures for bacterial strains to reduce the impact of process fluctuations on preservation survival and resuscitation consistency. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the high activity, long-term preservation, and rapid reactivation of microbial strains, in order to solve the problems in existing solutions where bacteria are directly transferred from the culture environment to the hyperosmotic protective environment, the permeability environment changes abruptly when the reactivation solution comes into contact with the bacteria, and the background conductivity caused by the dissolution of the carrier and protective components interferes with the determination of the reactivation timing.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: S1. Collect the bacterial cells when the microbial strain to be preserved is in the late logarithmic growth stage to the early stationary stage to obtain the bacterial cells to be preserved.

[0008] S2. Place the bacterial cells to be preserved in the first osmotic adaptation solution for the first adaptation treatment, and then transfer them to the second osmotic adaptation solution for the second adaptation treatment; the mass osmotic molar concentration of the first osmotic adaptation solution, the second osmotic adaptation solution and the preservation solution increases sequentially.

[0009] S3. The adapted bacterial cells are mixed with a preservation solution containing osmotic protective components and polymeric protective components and loaded onto a partitioned porous carrier. The partitioned porous carrier has a rehydration buffer zone and a bacterial cell loading zone sequentially along the infiltration direction of the first reactivation solution. A liquid-blocking coating layer is provided on the periphery of the carrier, and only the upstream end face of the rehydration buffer zone forms the liquid addition surface. The rehydration buffer zone is loaded with soluble buffer components that do not contain bacterial cells, and the bacterial cell loading zone is loaded with bacterial cells. A blank preservation unit is prepared with the same porous carrier partitions, buffer components, preservation solution composition, and loading amount of each component, except that it does not contain bacterial cells.

[0010] S4. The bacterial protection unit and the blank protection unit are stored at low temperature under the same conditions, or are pre-frozen and vacuum freeze-dried under the same conditions before storage.

[0011] S5. During reactivation, equal amounts of the first reactivation solution are added to the rehydration buffer zones of the bacterial protection unit and the blank protection unit, respectively, so that the first reactivation solution first forms a transitional permeation environment before entering the bacterial loading area; the conductivity of the bacterial rehydration system and the blank rehydration system are detected simultaneously, and the difference between the two is used as the correction conductivity, thereby deducting the background influence of the dissolution of the carrier and protective components.

[0012] S6. Nutrient initiation is controlled based on the unit-time change in corrected conductivity: When the unit-time change in corrected conductivity in two consecutive detection intervals is not greater than the first preset change, add the first nutrient additive solution to make the final concentrations of glucose and peptone in the system 0.2 g / L and 0.1 g / L, respectively; thereafter, when the change in the unit-time change in two consecutive detection intervals is not greater than the second preset change, and adjacent detection values ​​do not continuously increase beyond the repeatability limit of the conductivity detection method, add the second nutrient additive solution containing glucose, peptone, yeast extract, and inorganic salts to bring the system to the proliferation culture conditions. The first and second preset change values ​​are determined by adding twice the standard deviation to the average value of the corresponding reference stable phase, and the repeatability limit of the conductivity detection method is determined by 2.8 times the standard deviation of at least six repeated measurements of the same sample.

[0013] Preferably, the first osmosis adaptation solution and the second osmosis adaptation solution each contain at least one osmosis protection component selected from trehalose, glycerol, sorbitol, proline and glutamate, and the mass osmotic molar concentration of the first osmosis adaptation solution, the second osmosis adaptation solution and the preservation protection solution increases sequentially.

[0014] Preferably, the conductivity of the corresponding permeation adaptation solution is detected during the first and second adaptation processes, and the endpoint of the treatment is taken as the change in conductivity per unit time not being greater than the corresponding preset adaptation change; the mass permeation molar concentration difference between the second permeation adaptation solution and the preservation solution is less than the mass permeation molar concentration difference between the first and second permeation adaptation solutions.

[0015] Preferably, the permeation protection components in the preservation solution include trehalose and glycerol, and the polymer protection component includes γ-polyglutamic acid; the porous carrier has interconnected pores, the preservation solution carries the adapted bacteria into the pores, the γ-polyglutamic acid is distributed in the pores, and together with the bacteria, forms a polymer protection region.

[0016] Preferably, the rehydration buffer is loaded with trehalose and buffer salt, and its loading is adjusted according to the volume of the first rehydration solution, so that the mass permeation molar concentration of the first rehydration solution when it enters the cell loading zone is between the initial liquid phase formed after the second permeation adaptation solution and the preservation and protection solution are rehydrated according to the actual loading state of the cell-containing protection unit.

[0017] Preferably, the low-temperature preservation includes a first cooling stage, a constant-temperature nucleation stage, and a second cooling stage. The bacterial preservation unit and the blank preservation unit adopt the same cooling procedure: the first cooling stage lowers the temperature to -5 to -15°C at a rate of 0.5 to 2°C / min, maintains the temperature for 10 to 60 min, and uses ice crystal inoculation or mechanical contact for controlled nucleation; the second cooling stage lowers the temperature to below -20°C at a rate of 0.2 to 1.5°C / min to the final preservation temperature.

[0018] Preferably, the pre-freezing and vacuum freeze-drying sequentially include cooling pre-freezing, sublimation drying, and desorption drying; the bacterial protection unit and the blank protection unit adopt the same freeze-drying procedure. During sublimation drying, for crystalline systems, the product temperature is kept below the eutectic point temperature of the preservation solution; for amorphous systems, the product temperature is kept below its collapse temperature; desorption drying continues until the residual moisture in both protection units reaches the preset storage range.

[0019] Preferably, the blank protection unit, except for the absence of bacteria, has the same porous carrier partitioning, soluble buffer components, protection solution composition, component loading, preservation process, first reactivation solution volume, detection temperature, and detection time as the bacterial protection unit; the corrected conductivity is obtained by subtracting the conductivity of the blank rehydration system from the conductivity of the bacterial rehydration system.

[0020] Preferably, the unit time correction conductivity change, the first preset change, the second preset change, and the repeatability limit of the conductivity detection method for the i-th detection interval are calculated according to the following formulas:

[0021]

[0022]

[0023] In the formula, The detection interval number; and The first Corrected conductivity at each detection time point and the previous detection time point, in units of ; and These are the corresponding detection times, in units of ; For the first The change in corrected conductivity per unit time within each detection interval, in units of ; and These are the first preset change amount and the second preset change amount, respectively; and At least three reference batches of the same strain, using the same vector and under the same testing conditions, were tested during the first and second reference stability phases. The arithmetic mean; and They are respectively the corresponding The sample standard deviation; The repeatability limit of conductivity detection methods; The sample standard deviation is the conductivity measurement value obtained from at least six repeated measurements of the same sample under the same conditions.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects: First, the two-stage osmotic adaptation and rehydration buffer act on the pre-preservation and early-stage reactivation phases, respectively: the former allows the bacteria to gradually enter the preservation solution, while the latter, combined with the peripheral liquid-blocking coating, ensures that the first reactivation solution dissolves the soluble buffer components and forms a transitional osmotic environment before contacting the bacteria, thus reducing abrupt changes in the liquid phase in both directions. This effect stems from the control of the osmotic environment between the preceding and following phases, rather than from adding a single protective agent.

[0025] Second, the rehydration buffer zone and the cell-loaded zone are set on the same porous carrier and arranged along the infiltration direction of the first revitalizing liquid. The peripheral liquid-blocking coating layer limits the liquid addition path to the length direction of the carrier, so that the release order of the buffer components is limited by the carrier structure. The polymer protective component is distributed in the pores of the cell-loaded zone and forms a protective area together with the cells, thereby reducing the probability that the first revitalizing liquid bypasses the buffer zone and directly contacts the cells.

[0026] Third, the blank protection unit and the bacterial protection unit undergo the same loading, preservation and rehydration process. The difference in conductivity between the two is used to form the corrected conductivity, which can deduct the background changes caused by the dissolution of porous carrier, buffer components and preservation components, so that the subsequent threshold mainly reflects the difference between the bacterial system and the blank system.

[0027] Fourth, the first and second nutrient addition solutions are not added at fixed times, but are constrained by the first preset change amount, the second preset change amount, and whether the corrected conductivity continues to increase, respectively. This creates a closed-loop control relationship between rehydration, basal metabolic recovery, and proliferation initiation, which helps to reduce the impact of different strains, storage periods, and carrier batches on the fixed resurrection time.

[0028] Fifth, the aforementioned partitioned rehydration, blank correction, and two threshold controls work together: the partitioned carrier regulates the actual rehydration process experienced by the bacteria, the blank unit eliminates the conductivity background generated by the carrier and buffer components themselves, and the two thresholds control nutrient addition accordingly. Therefore, all features work together to determine the resurrection state, improving the consistency and repeatability of the resurrection operation. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the overall process of the microbial strain preservation and reactivation method of the present invention.

[0030] Figure 2 This is a flowchart of the first adaptation process and the second adaptation process of the present invention.

[0031] Figure 3 This is a schematic diagram of the structure of the bacterial protection unit of the present invention.

[0032] Figure 4 This is a flowchart illustrating the staged cooling process for low-temperature preservation according to the present invention.

[0033] Figure 5 This is a flowchart of the vacuum freeze-drying preservation process of the present invention.

[0034] Figure 6 This is a flowchart of the phased reactivation process based on blank correction conductivity according to the present invention. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are used to illustrate the technical principles and possible implementation methods of the present invention, and are not intended to limit the scope of protection to a particular microorganism, a particular protective agent formulation, or a particular fixed storage temperature. Unless otherwise specified, operations such as cultivation, centrifugation, aseptic operation, conductivity measurement, mass permeate molar concentration measurement, freezing, and vacuum freeze-drying can all be performed using conventional equipment in the art. Operations involving microbial culture should be carried out in facilities and operating conditions appropriate to the biosafety level of the microbial strain.

[0036] I. Terminology and Testing Methods The "first osmotic adaptation solution" and "second osmotic adaptation solution" referred to in this invention are aqueous liquids that come into contact with the bacterial cells in a sequential manner before they enter the preservation solution, and are used to gradually change the osmolality of the liquid phase in which the bacterial cells are located. "First" and "second" are used only to distinguish the order of contact. The osmolality of the second osmotic adaptation solution is higher than that of the first osmotic adaptation solution but lower than that of the preservation solution. The osmolality can be measured using a freezing point permeameter and is expressed in milliosmols per kilogram (mOsm / kg).

[0037] The "preservative solution" referred to in this invention is a liquid that coexists with the bacterial cells during low-temperature preservation or vacuum freeze-drying preservation. A preferred preservative solution contains 10%–15% (w / v) trehalose, 5% (w / v) glycerol, and 0.5%–0.8% (w / v) γ-polyglutamic acid, where γ stands for the Greek letter gamma, representing the linkage of polyglutamic acid. Trehalose and glycerol act as osmotic protectants, while γ-polyglutamic acid acts as a polymeric protectant distributed within the pores of the porous carrier, forming a polymeric protective region together with the bacterial cells.

[0038] The "first revitalizing solution" referred to in this invention is a liquid used for rehydration, dilution of preservation and protective components, and osmotic equilibrium of the bacterial cells. It does not contain any available carbon or nitrogen sources. When the first revitalizing solution enters the bacterial loading zone through the rehydration buffer, its osmotic molar concentration is preferably between that of the second osmotic adaptation solution and the initial liquid phase formed after rehydration of the preservation and protective solution according to the actual loading state of the bacterial protection unit.

[0039] The "first nutrient additive solution" and "second nutrient additive solution" referred to in this invention are two nutrient solutions added sequentially to the bacterial rehydration system after rehydration. The first nutrient additive solution contains glucose and peptone, and its addition ensures that the final concentrations of glucose and peptone in the system are 0.2 g / L and 0.1 g / L, respectively. The second nutrient additive solution contains glucose, peptone, yeast extract, and inorganic salts suitable for the proliferation of the target bacterial strain, and its addition brings the system to the conditions for the proliferation and culture of the target bacterial strain.

[0040] The "change in conductivity per unit time", the first preset change, the second preset change, and the repeatability limit of the conductivity detection method, as referred to in this invention, are calculated according to the following formulas:

[0041]

[0042]

[0043] In the formula, The detection interval number; and The first Corrected conductivity at each detection time point and the previous detection time point, in units of ; and These are the corresponding detection times, in units of ; For the first The change in corrected conductivity per unit time within each detection interval, in units of ; and These are the first preset change amount and the second preset change amount, respectively; and At least three reference batches of the same strain, using the same vector and under the same testing conditions, were tested during the first and second reference stability phases. The arithmetic mean; and They are respectively the corresponding The sample standard deviation; The repeatability limit of conductivity detection methods; The sample standard deviation is the conductivity measurement value obtained from at least six repeated measurements of the same sample under the same conditions.

[0044] The first reference stabilization phase is confirmed by the plateauing of nucleic acid or protein leakage. The second reference stabilization phase is confirmed by the biomass change after the addition of the first nutrient supplement not exceeding the repeatability limit of the biomass detection method. Conductivity values ​​are uniformly converted to 25°C, or measured under the same isothermal conditions.

[0045] The "corrected conductivity" referred to in this invention is the conductivity of the bacterial rehydration system minus the conductivity of the blank rehydration system. Except for the absence of bacteria, the blank protection unit is identical to the bacterial protection unit in terms of its porous carrier partitioning, rehydration buffer zone, soluble buffer components, protective solution composition, component loading, preservation process, first rehydration solution volume, detection temperature, and detection time.

[0046] II. General Implementation Process This invention is applicable to bacteria, yeasts, actinomycetes, or fungi that are primarily preserved in vegetative cell form. For microorganisms that are primarily preserved and revived in spore or endospore form, the adaptation solution, revival solution, and nutrient initiation conditions should be determined separately based on their germination conditions, and the parameters in this embodiment should not be directly used.

[0047] During cultivation, the optical density, viable cell count, and biomass of the bacterial culture were measured periodically. When the slope of the bacterial growth curve began to decrease but before entering a significant decline phase, the bacteria were considered to be in the late logarithmic growth phase to the early stationary phase. The bacteria were collected by centrifugation or membrane filtration and washed with isotonic buffer containing no major carbon source to reduce the influence of residual original culture medium on subsequent conductivity measurements.

[0048] The mass permeation molar concentration of the first acclimation solution can be set to 300–600 mOsm / kg, the second acclimation solution to 500–900 mOsm / kg, and the preservation solution to 700–1200 mOsm / kg. The specific values ​​should simultaneously satisfy the relationship that the first acclimation solution < the second acclimation solution < the preservation solution. The increment of the mass permeation molar concentration between adjacent liquid phases should preferably not exceed 60% of that of the preceding liquid phase.

[0049] The first and second adaptation treatments can be carried out at 2–25℃ for 5–60 min, with a volume ratio of bacterial cells to the corresponding osmotic adaptation solution of 1:3 to 1:20. During the treatment, the supernatant is collected every 2–10 min to measure conductivity. The current adaptation stage ends when the change in conductivity per unit time between two consecutive detection intervals does not exceed the corresponding preset adaptation change. The preferred transfer method is centrifugation to collect the bacterial cells followed by resuspending, to avoid large amounts of the previous adaptation solution being carried into the subsequent adaptation solution. The first and second adaptation changes are determined by adding twice the standard deviation to the average of the changes in conductivity per unit time after at least three reference adaptation batches have entered the stable stage.

[0050] The porous carrier is a sterile cellulose porous sheet or alginate porous gel. For the *Lactobacillus plantarum* implementation, a sterile cellulose porous sheet measuring 30 mm long, 10 mm wide, and 3 mm thick with interconnected pores is used. The upstream third of the sheet serves as a rehydration buffer, and the remaining area is the cell loading zone. A biocompatible liquid-resistant membrane incompatible with the first reactivation solution is placed around the carrier, with only the upstream end of the rehydration buffer used as the liquid loading surface. The rehydration buffer is loaded with 0.05 mL of a buffer solution containing 6% (w / v) trehalose and phosphate, and the cell loading zone is loaded with 0.20 mL of a mixture of cells and preservation solution. The volumetric absorption and porosity of the carrier are maintained consistently within the same batch.

[0051] For low-temperature preservation, both the bacterial-containing and blank preservation units can be lowered to the preset nucleation temperature at a rate of 0.5–2 °C / min, maintained at this temperature for 10–60 min, and controlled nucleation should be triggered in both preservation units using the same ice crystal inoculation or mechanical contact method. Then, the temperature can be lowered to a final preservation temperature below -20 °C at a rate of 0.2–1.5 °C / min. For vacuum freeze-drying preservation, pre-freezing can be performed at -30 to -50 °C. For crystalline systems, sublimation drying is performed at a temperature below the eutectic point, and for amorphous systems, sublimation drying is performed at a temperature below the collapse temperature. Then, the temperature is raised for further desorption drying, reducing the residual moisture content to 1%–5%.

[0052] During reactivation, the volume of the first reactivation solution added is preferably 5 to 30 times the volume of the adsorption protective solution in the bacterial protection unit. The first reactivation solution is added from the rehydration buffer side of both the bacterial protection unit and the blank protection unit. The conductivity of the bacterial rehydration system and the blank rehydration system is simultaneously measured every 1 to 10 minutes, and the corrected conductivity and its change per unit time are calculated. The first nutrient additive solution is added when two consecutive detection intervals do not exceed the first preset change amount; the second nutrient additive solution is added when two consecutive detection intervals do not exceed the second preset change amount, and adjacent detection values ​​do not continuously increase beyond the repeatability limit.

[0053] III. Example 1: Complete Preservation and Closed-Loop Reactivation of Lactobacillus plantarum Lactobacillus plantarum, primarily preserved in vegetative cell form, was selected. It was inoculated into MRS liquid medium (Derman-Rogoza-Sharp liquid medium, a medium for lactic acid bacteria culture) and incubated statically at 37°C. The late logarithmic growth phase was determined by changes in bacterial density and viable cell count. The bacterial culture was centrifuged at 4°C and 6000×g for 10 min, the supernatant was discarded, and the culture was washed twice with carbon-free phosphate buffer (pH 6.5) to obtain the bacterial cells to be preserved.

[0054] The first osmotic adaptation solution contained 3% (w / v, i.e., mass-volume percentage) trehalose and 1% (v / v, i.e., volume percentage) glycerol; the second osmotic adaptation solution contained 6% (w / v) trehalose and 4% (v / v) glycerol; and the preservation solution contained 10% (w / v) trehalose, 5% (v / v) glycerol, and 0.5% (w / v) γ-polyglutamic acid, where γ stands for gamma. The pH of all three solutions was adjusted to 6.5. The target ranges for the mass osmotic molar concentrations were set to 320±20 mOsm / kg, 720±20 mOsm / kg, and 960±20 mOsm / kg, respectively. The amount of buffer salt or water was adjusted according to the results of the freezing point permeameter measurements, ensuring that the concentrations increased sequentially, and that the difference between the second osmotic adaptation solution and the preservation solution was less than the difference between the first and second osmotic adaptation solutions.

[0055] The bacterial cells to be preserved were suspended in the first osmotic adaptation solution at a volume ratio of 1:10 and treated at 10℃ for 20 min. After centrifugation, the bacterial cells were collected and resuspended in the second osmotic adaptation solution at the same volume ratio, and treated at 10℃ for 20 min. During the treatment, the conductivity of the supernatant was measured every 5 min. The adaptation treatment was terminated when the change in conductivity per unit time between two consecutive detection intervals did not exceed the corresponding preset adaptation change.

[0056] In this embodiment, a sterile cellulose porous sheet with interconnected pores and a length of 30 mm, width of 10 mm, and thickness of 3 mm was selected. The upstream third of the sheet was designated as a rehydration buffer zone, and the remaining area as the cell loading zone. The periphery of the porous sheet was sealed with a biocompatible liquid-resistant membrane, leaving only the upstream end of the rehydration buffer zone as the liquid loading surface. The rehydration buffer zone was quantitatively loaded with 0.05 mL of a buffer solution containing 6% (w / v) trehalose and phosphate; the cell loading zone was quantitatively loaded with 0.20 mL of a mixture of adapted cells and preservation solution, forming a bacterial-containing protection unit. Blank protection units were prepared using the same porous sheet, with the same buffer solution loaded in the rehydration buffer zone and an equal volume of cell-free preservation solution loaded in the corresponding area of ​​the cell loading zone. The osmolar concentration of the first rehydration solution when it passed through the rehydration buffer zone and entered the cell loading zone was measured. The amount of buffer salt was adjusted to be between the initial liquid phase formed after rehydration of the second osmolarity adaptation solution and the preservation solution under the aforementioned loading conditions. Both protection units were cooled to -10℃ at a rate of 1℃ / min, held at that temperature for 30 min, and then cooled to -80℃ at a rate of 0.5℃ / min for preservation.

[0057] During revival, 2.0 mL of phosphate buffer solution with a pH of 6.5 and a concentration of 10 mmol / L was added to the liquid addition surface of each of the two protection units as the first revival solution. The first revival solution did not contain any available carbon or nitrogen sources. Conductivity was simultaneously measured every 5 min at 37°C, and the corrected conductivity was calculated. The first and second preset change amounts were predetermined according to the reference batch method described in this specification. When both consecutive detection intervals did not exceed the first preset change amount, the first nutrient additive solution was added to bring the final concentrations of glucose and peptone in the system to 0.2 g / L and 0.1 g / L, respectively. The change in optical density within 30 min at these concentrations was confirmed in a reference pre-experiment to not exceed the repeatability limit of the optical density detection method; the corrected conductivity was then measured again. When two consecutive detection intervals are both no greater than the second preset change amount, and adjacent detection values ​​do not continuously increase beyond the repeatability limit of the conductivity detection method, a second nutrient additive solution is added to bring the glucose, peptone, yeast extract, and inorganic salts in the system to the MRS proliferation culture conditions, and the culture continues to obtain the revived strain. Thus, this embodiment implements two-level adaptation, partitioned carrier, isomorphic blank unit, conductivity correction, and two-stage nutrient start-up in the same process.

[0058] IV. Example 2: Vacuum freeze-drying preservation and reactivation of yeast Saccharomyces cerevisiae was selected as the example strain. Saccharomyces cerevisiae was inoculated into yeast culture medium and cultured with shaking at 30°C. The period from the late logarithmic growth phase to the early stationary phase was determined by the change in the optical density of the bacterial solution. The cells were collected by centrifugation and washed with carbon-free isotonic buffer.

[0059] The first osmosis adaptation solution contained 5% (w / v) trehalose and 2% (w / v) sorbitol; the second osmosis adaptation solution contained 10% (w / v) trehalose and 5% (w / v) sorbitol; the preservation solution contained 15% (w / v) trehalose, 5% (w / v) sorbitol, and 0.8% (w / v) γ-polyglutamic acid. The actual osmolar concentration of each solution was determined and adjusted so that the concentrations were increased sequentially.

[0060] The bacterial cells were sequentially treated in the first and second osmosis adaptation solutions at 15°C for 30 min. After centrifugation, they were mixed with the preservation solution and quantitatively loaded onto an alginate porous carrier with a rehydration buffer and a bacterial cell loading zone. Blank protection units with identical composition and loading amount except for the absence of bacterial cells were prepared. Both protection units were pre-frozen at -40°C until the temperature change of the product within 30 min was no more than 1°C, and then vacuum freeze-drying was initiated. During sublimation drying, based on the thermal analysis results of the protection solution, the product temperature was kept below its eutectic point temperature or collapse temperature, followed by desorption drying until the residual moisture content was 1%–5%. After sealing, the products were stored at 2–8°C in the dark.

[0061] During resurrection, equal volumes of glucose-free and nitrogen-containing organic-free first resurrection solution were added to the rehydration buffer side of the bacterial protection unit and the corresponding blank protection unit. Rehydration was carried out at 30°C while simultaneously measuring conductivity. The corrected conductivity was calculated, and according to the first and second preset change amounts determined by the reference batch of brewing yeast, the first nutrient supplement solution for limiting proliferation and the second nutrient supplement solution to achieve the yeast proliferation culture conditions were added sequentially. Culture continued to obtain the resurrected strain.

[0062] V. Example 3: Bacterial Protection Unit with Rehydration Buffer Zone In this embodiment, the porous carrier sequentially forms a rehydration buffer zone and a cell-loading zone along the infiltration direction of the first reactivation solution. A liquid-blocking coating layer is provided on its periphery, with only the upstream end face of the rehydration buffer zone serving as the liquid-adding surface. The rehydration buffer zone is loaded with soluble trehalose and buffer salts but not with cells; the cell-loading zone is loaded with adapted cells and a preservation solution. Figure 3As shown, the label "First Revitalizing Solution Addition Location" points to the center of the upstream end face of the rehydration buffer, indicating that the first revitalizing solution is added from this location; the arrow "First Revitalizing Solution Penetration Direction" points from the upstream end face to the cell loading area, indicating that the first revitalizing solution first enters the rehydration buffer, dissolves the soluble components, and then enters the cell loading area along the interconnecting pores of the porous carrier. Figure 3 The outer boundary represents the liquid-blocking coating layer, the dashed outline represents the protective region formed by γ-polyglutamic acid, and the solid ellipse represents the adapted bacteria.

[0063] The loading of soluble components in the rehydration buffer is determined based on the volume of the first rehydration solution, ensuring that the osmolar concentration of the first rehydration solution when it enters the cell-loading zone is between that of the second osmotic adaptation solution and the initial liquid phase formed after rehydration of the preservation solution according to the actual loading state of the cell-containing protection unit. Thus, the first rehydration solution first forms a transitional osmotic environment before contacting the cells, and then gradually reduces its osmolar concentration as the soluble components diffuse.

[0064] VI. Example 4: Blank Protection Unit Correction and Staged Nutrient Reactivation Blank protective units were prepared using the same porous carrier, rehydration buffer, soluble buffer component, preservation solution, and preservation process as the bacterial protective units. Except for the absence of bacterial cells, the structure, composition, and loading of each region were identical. During reactivation, an equal volume of the first reactivation solution from the same batch was added, and conductivity was measured at the same temperature and time point.

[0065] The corrected conductivity is obtained by subtracting the conductivity of the blank rehydrated system from the conductivity of the bacterial rehydrated system. The first preset change is determined by the change in corrected conductivity per unit time after the reference batch without added nutrients enters the stable phase. When two consecutive detection intervals are not greater than the first preset change, the first nutrient additive solution is added; its carbon and nitrogen source concentrations are limited by preliminary experiments to a level where the change in optical density during the first nutrient phase does not exceed the repeatability limit of the optical density detection method, in order to restore basal metabolism.

[0066] After adding the first nutrient additive, the conductivity is continuously monitored and corrected. The second preset change is determined by the change in conductivity per unit time of the reference batch after it enters the stable phase in the first nutrient stage. When two consecutive detection intervals are not greater than the second preset change, and adjacent detection values ​​do not continuously increase beyond the repeatability limit of the conductivity detection method, the second nutrient additive is added; if adjacent detection values ​​continuously increase beyond the repeatability limit, the first nutrient stage is extended until the above conditions are met again.

[0067] VII. Comparative Evaluation Methods To evaluate the effects of each technical feature, the following groups can be set up under the same bacterial species, the same initial bacterial age, and the same number of viable cells loaded: Control group A, where the bacteria are directly introduced into the preservation solution without osmotic adaptation, and are directly added to the intact culture medium upon reactivation; Control group B, where the bacteria undergo two stages of osmotic adaptation, but are directly added to the intact culture medium upon reactivation; Control group C, where the bacteria undergo two stages of osmotic adaptation and a partitioned porous carrier, but nutrient addition is initiated based on uncorrected conductivity; and Experimental group D, which uses a two-stage osmotic adaptation, a partitioned porous carrier, blank correction, and two threshold controls for staged nutrient reactivation.

[0068] Each group should have at least three independent preservation batches, with at least three replicates per batch. Pre- and post-preservation assays should include viable cell count, survival rate, lag phase, maximum specific growth rate, membrane integrity, and target functional indicators. For lactic acid bacteria, the target functional indicator can be acid production rate or the amount of a specific metabolite produced; for yeast, it can be fermentation rate or the yield of the target product. If genetic stability is emphasized, the bacteria should be continuously passaged after revival, and genetic or phenotypic indicators representing the species identity and target trait should be tested.

[0069] Viable cell count can be determined using the plate count method; survival rate is the ratio of culturable viable cells after preservation to the number of viable cells loaded before preservation; reactivation lag period can be obtained by fitting a growth curve; membrane integrity can be characterized by nucleic acid leakage, protein leakage, or membrane integrity fluorescence staining results. These indicators are used to evaluate the relative performance of different schemes, and this invention does not use specific improvement margins that have not been actually tested as essential technical features.

[0070] VIII. Other Optional Implementation Methods The first and second osmotic adaptation solutions can use the same or different biocompatible osmotic protection components, as long as the measured mass osmotic molar concentration of the second osmotic adaptation solution is higher than that of the first osmotic adaptation solution and lower than that of the preservation protection solution, and does not produce unacceptable toxicity to the microbial cells to be preserved.

[0071] The polymeric protective component is γ-polyglutamic acid, with a concentration of 0.5%–0.8% by mass / volume. The porous carrier is a sterile cellulose porous sheet or alginate porous gel with interconnected pores, and the consistency of the protection units in the same batch is controlled by carrier size, liquid absorption per unit volume, and cell recovery rate.

[0072] The permeamolar concentration of the first resurrection solution upon entering the cell-loaded zone is determined jointly by the initial formulation of the first resurrection solution and the release of soluble components in the rehydration buffer. Nucleic acid or protein leakage can serve as an auxiliary evaluation indicator for the stabilization phase of a reference batch; however, the addition of the first and second nutrient supplements is still directly judged based on the change in corrected conductivity per unit time, the repeatability limit of the conductivity detection method, and the trend of adjacent detection values.

[0073] The above description is merely a preferred embodiment of the present invention. Those skilled in the art can make equivalent substitutions or conventional adjustments to the strain type, protective components, carrier material, storage temperature, and reactivation culture medium without departing from the technical concept of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for the preservation and reactivation of a microbial strain, wherein the microbial strain is primarily preserved in vegetative cells, characterized in that... Includes the following steps: S1. Collect the bacterial cells when the strain to be preserved is in the late logarithmic growth stage to the early stationary stage. S2. The bacterial cells are placed in a first osmotic adaptation solution for a first adaptation treatment, and then transferred to a second osmotic adaptation solution for a second adaptation treatment. The mass osmotic molar concentration of the first osmotic adaptation solution, the second osmotic adaptation solution, and the preservation solution increases sequentially. S3. Load the second-adapted bacterial cells and preservation solution onto the bacterial cell loading area of ​​a porous carrier with interconnected pores to form a bacterial-containing protection unit; the preservation solution contains trehalose, glycerol, and γ-polyglutamic acid; the porous carrier has a rehydration buffer zone upstream of the bacterial cell loading area along the penetration direction of the first reactivation solution; the rehydration buffer zone is loaded with trehalose and buffer salts; the porous carrier has a liquid-blocking coating layer on its periphery; and only the upstream end face of the rehydration buffer zone is the liquid-adding surface; prepare a blank protection unit that is identical to the bacterial-containing protection unit in terms of porous carrier structure, component composition, and loading amount, except that it does not contain bacteria. S4. The bacterial-containing protective unit and the blank protective unit are stored at low temperature under the same conditions, or are stored after pre-freezing and vacuum freeze-drying under the same conditions. S5. Add equal amounts of the first reactivation solution to the liquid filling surface of the two protection units respectively, and simultaneously detect the conductivity of the bacterial rehydration system and the blank rehydration system, and use the difference between the two as the correction conductivity. S6. Calculate the ratio of the absolute value of the difference in corrected conductivity between adjacent detection time points to the time interval to obtain the change in corrected conductivity per unit time. When the change in corrected conductivity per unit time in two consecutive detection intervals is not greater than the first preset change, add the first nutrient additive solution to make the final concentrations of glucose and peptone in the system 0.2 g / L and 0.1 g / L, respectively. When the change in corrected conductivity in two consecutive detection intervals after adding the first nutrient additive solution is not greater than the second preset change, and the corrected conductivity does not continuously increase beyond the repeatability limit of the conductivity detection method, add the second nutrient additive solution containing glucose, peptone, yeast extract, and inorganic salts to make the system meet the proliferation and culture conditions of the microbial strain to obtain the revived strain. The first preset change and the second preset change are the sum of the average value and twice the standard deviation of the change in corrected conductivity per unit time in the corresponding reference stable phase, respectively. The repeatability limit of the conductivity detection method is 2.8 times the standard deviation of at least six repeated measurements of the same sample.

2. The method for preservation and reactivation of microbial strains according to claim 1, characterized in that, The first osmosis adaptation solution and the second osmosis adaptation solution each contain at least one osmosis protection component selected from trehalose, glycerin, sorbitol, proline and glutamate.

3. The method for preserving and reactivating microbial strains according to claim 2, characterized in that, During the first adaptation process of placing the bacterial cells in the first osmotic adaptation solution, the conductivity of the first osmotic adaptation solution is detected at preset time intervals. When the change in conductivity of the first osmotic adaptation solution per unit time is not greater than the change in conductivity during the first adaptation process, the bacterial cells are transferred to the second osmotic adaptation solution. During the second adaptation process, in which the bacterial cells are placed in the second osmotic adaptation solution, the conductivity of the second osmotic adaptation solution is detected at the preset time interval. When the change in conductivity of the second osmotic adaptation solution per unit time is not greater than the change in conductivity during the second adaptation process, the second adaptation process is terminated.

4. The method for preserving and reactivating microbial strains according to claim 3, characterized in that, The difference in molality between the second osmotic adaptation solution and the preservation solution is less than the difference in molality between the first osmotic adaptation solution and the second osmotic adaptation solution.

5. The method for preserving and reactivating microbial strains according to claim 1, characterized in that, The preservation solution contains 10%–15% by volume trehalose, 5% by volume glycerol, and 0.5%–0.8% by volume γ-polyglutamic acid. The porous carrier is a sterile cellulose porous sheet or alginate porous gel with interconnected pores. The preservation solution carries the bacteria into the pores, and the γ-polyglutamic acid is distributed within the pores, forming a polymeric protective region together with the bacteria located within the pores.

6. The method for preserving and reactivating microbial strains according to claim 5, characterized in that, The soluble buffer components in the rehydration buffer include trehalose and buffer salts; the loading of the soluble buffer components is adjusted according to the volume of the first rehydration solution so that the mass permeation molar concentration of the first rehydration solution when it enters the cell loading zone is between that of the second permeation adaptation solution and the initial liquid phase formed after the preservation and protection solution is rehydrated according to the actual loading state of the cell-containing protection unit.

7. The method for preserving and reactivating microbial strains according to claim 1, characterized in that, The low-temperature preservation process includes a first cooling stage, a constant-temperature nucleation stage, and a second cooling stage. In the first cooling stage, the bacterial-containing protective unit and the blank protective unit are cooled to a nucleation temperature of -5 to -15°C at a rate of 0.5 to 2°C / min. In the constant-temperature nucleation stage, the temperature is maintained for 10 to 60 minutes, and controlled nucleation is triggered on both protective units using the same ice crystal inoculation or mechanical contact method. In the second cooling stage, the two protective units are cooled to a final preservation temperature below -20°C at a rate of 0.2 to 1.5°C / min.

8. The method for preservation and reactivation of microbial strains according to claim 1, characterized in that, The pre-freezing and vacuum freeze-drying include: pre-freezing the bacterial protection unit and the blank protection unit by cooling until the temperature change of the product within a preset time is not greater than the preset temperature change; for crystalline systems, sublimation drying is performed under the condition that the product temperature is lower than the eutectic point temperature of the preservation solution, and for amorphous systems, sublimation drying is performed under the condition that the product temperature is lower than the collapse temperature of the preservation solution; and increasing the product temperature and performing desorption drying until the residual moisture reaches the preset storage range.

9. The method for preserving and reactivating microbial strains according to claim 1, characterized in that, The first revitalizing solution does not contain any available carbon or nitrogen sources; the conductivity values ​​of the bacterial rehydration system and the blank rehydration system are both converted to 25°C or measured under the same constant temperature conditions.

10. The method for preserving and reactivating microbial strains according to claim 9, characterized in that, After adding the first nutrient additive solution, if the adjacent detection values ​​of the corrected conductivity increase in two consecutive detection intervals and the increase exceeds the repeatability limit of the conductivity detection method, the first nutrient stage is extended until the change in corrected conductivity per unit time in two consecutive detection intervals is not greater than the second preset change and the adjacent detection values ​​do not increase continuously beyond the repeatability limit of the conductivity detection method, and then the second nutrient additive solution is added.

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