Preparation method for high-density fermentation freeze-drying of high-activity lactobacillus paracasei KFY202407
By optimizing the fermentation medium and freeze-drying process, the viable count and survival rate of Lactobacillus paracasei KFY202407 were increased, solving the problems of low viable bacteria content and insufficient metabolites in existing technologies, and achieving a highly effective effect in relieving intrauterine adhesions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING UNIV OF EDUCATION
- Filing Date
- 2026-01-15
- Publication Date
- 2026-04-28
AI Technical Summary
The existing Lactobacillus paracasei KFY202407 fermentation freeze-drying technology has a low live bacteria content and insufficient synthesis of metabolites, resulting in poor efficacy in relieving intrauterine adhesions.
By employing a suitable specialized fermentation medium and a two-stage fermentation process, combined with a compound freeze-drying protectant and a gradient freeze-drying process, the metabolic characteristics and survival rate of the strain were optimized.
The number of viable bacteria in the fermentation broth was increased to over 2.0 × 10¹⁰ CFU/mL, enhancing the survival rate and functional stability of the strains and ensuring effective relief of reproductive health problems such as intrauterine adhesions.
Smart Images
Figure SMS_1
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial fermentation freeze-drying technology, and more specifically, to a method for preparing high-density fermented freeze-dried Lactobacillus paracasei KFY202407. Background Technology
[0002] Intrauterine adhesions (IUA), also known as Asherman's syndrome, are caused by damage to the basal layer of the endometrium and proliferation of fibrous tissue, resulting in partial or complete adhesions in the uterine cavity. They mainly cause serious reproductive health problems such as oligomenorrhea, amenorrhea, infertility, or recurrent miscarriage.
[0003] Existing technologies disclose a strain of *Lactobacillus paracasei* KFY202407 (CGMCC No. 30736) that can alleviate intrauterine adhesions, uterine edema, and fibrosis. However, its efficacy is highly dependent on the viable cell content, in vivo colonization ability, and functional stability of the formulation. Current freeze-drying fermentation techniques for *Lactobacillus paracasei* often employ traditional MRS media, using peptone and beef extract as the main nitrogen sources. These media are single-component and lack optimization tailored to the metabolic characteristics of *Lactobacillus paracasei* KFY202407, leading to rapid lactic acid accumulation during fermentation that inhibits cell proliferation, resulting in viable cell concentrations in the fermentation broth generally below 1.0 × 10⁻⁶. 9 The CFU / mL level is insufficient, and the strains using existing fermentation processes do not synthesize enough metabolites related to anti-inflammatory and anti-fibrotic functions.
[0004] Therefore, the present invention proposes a method for preparing highly active Lactobacillus paracasei KFY202407 through high-density fermentation freeze-drying, which has important practical significance. Summary of the Invention
[0005] In view of this, the present invention proposes a method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying, aiming to solve at least one of the problems in the above-mentioned background art.
[0006] This invention proposes a method for preparing highly active Lactobacillus paracasei KFY202407 through high-density fermentation freeze-drying, comprising the following preparation steps: Lactobacillus paracasei KFY202407 was inoculated into a dedicated culture medium and activated to obtain an activated bacterial solution. The activated bacterial solution was inoculated into a dedicated culture medium for large-scale cultivation to obtain a seed culture. The seed culture was inoculated into a fermentation medium for high-density anaerobic fermentation to obtain a fermentation broth. The fermentation broth was centrifuged and enriched to obtain bacterial sludge. The bacterial sludge was then mixed with a composite freeze-drying protectant in a gradient and subjected to gradient freeze-drying to obtain freeze-dried bacterial blocks. The freeze-dried bacterial blocks were post-processed to obtain high-density fermented freeze-dried Lactobacillus paracasei KFY202407 with high activity.
[0007] Furthermore, the specific culture medium comprises the following components in parts by weight: The ingredients included 12 parts plant peptone, 8 parts yeast extract, 25 parts glucose, 3 parts anhydrous sodium acetate, 2.5 parts diammonium hydrogen citrate, 0.6 parts MgSO4·7H2O, 0.3 parts MnSO4·H2O, 2.5 parts K2HPO4·3H2O, 1.2 parts Tween 80, 0.8 parts Astragalus polysaccharide, and 1.5 parts xylooligosaccharide.
[0008] Furthermore, the inoculum size for the activation culture is 1% (v / v), the culture temperature is 37°C, the culture environment is anaerobic, and the culture time is 18 hours.
[0009] Furthermore, the inoculum size for the expanded culture was 3% (v / v), the culture temperature was 37°C, the culture environment was anaerobic, the shaking speed was 50 rpm, and the culture time was 24 h.
[0010] Furthermore, the high-density anaerobic fermentation culture specifically includes: The seed culture was inoculated into the fermentation medium at an inoculum rate of 5% (v / v), and fermented under anaerobic conditions at 37°C with shaking at a speed of 50-100 rpm until the cell concentration in the fermentation broth reached 8.0 × 10⁻⁶. 9 CFU / mL, then adjust the temperature of the fermentation broth to 35℃, adjust the pH to between 6.0 and 6.2, add plant lactobacillus solution to the concentration of plant lactobacillus in the fermentation broth to 50μg / mL, and continue anaerobic fermentation for 20-28h; The fermentation medium comprises the following components in parts by weight: 15 parts plant peptone, 8 parts soybean protein peptide, 10 parts yeast extract, 35 parts glucose, 4 parts anhydrous sodium acetate, 3 parts diammonium hydrogen citrate, 0.8 parts MgSO4·7H2O, 0.4 parts MnSO4·H2O, 3 parts K2HPO4·3H2O, 1.5 parts Tween 80, 1.2 parts astragalus polysaccharide, 2.0 parts xylooligosaccharide, 1.0 part L-glutamic acid, and 0.1 parts vitamin B complex.
[0011] Furthermore, the centrifugal enrichment process specifically includes: Centrifuge the fermentation broth at 4℃ and 8000-12000 r / min for 10-20 min, discard the supernatant, collect the wet bacterial sludge, add 2 times the volume of isotonic protective washing solution pre-cooled to 4℃ to the wet bacterial sludge, stir and resuspend, centrifuge at 4℃ and 6000-10000 r / min for 5-15 min, discard the supernatant, and repeat the washing twice.
[0012] Furthermore, the composite lyophilization protectant comprises the following components in parts by weight: The ingredients are: 12 parts trehalose, 8 parts xylooligosaccharides, 6 parts glycerol, 1.5 parts glutathione, 2 parts astragalus polysaccharide, 0.8 parts vitamin C, 1.2 parts soybean lecithin, 10 parts plant protein hydrolysate, and 58.5 parts water.
[0013] Furthermore, the gradient mixing specifically involves: Add the bacterial sludge to one-third of the volume of water and stir to obtain a concentrated bacterial suspension. Add the composite freeze-drying protectant dropwise to the concentrated bacterial suspension at 4°C and 200-500 rpm for 15-20 minutes. After the addition is complete, let it stand at 4°C for 25-35 minutes to obtain a bacterial suspension.
[0014] Furthermore, the gradient freeze-drying specifically involves: The bacterial suspension was cooled from 4°C to -20°C at a rate of 2°C / min and kept at that temperature for 1-3 hours. Then, it was cooled from -20°C to -50°C at a rate of 5°C / min and kept at that temperature for 2-6 hours. Finally, it was cooled from -50°C to -70°C at a rate of 8°C / min and kept at that temperature for 8-12 hours to obtain pre-frozen bacterial blocks. The pre-frozen mycelium blocks were heated from -70°C to -40°C at a rate of 0.5°C / h under a vacuum of 1.5 Pa and held for 6-10 hours. Then, the temperature was heated from -40°C to -20°C at a rate of 1°C / h and held for 10-14 hours. Next, the temperature was heated from -20°C to 0°C at a rate of 2°C / h and held for 4-8 hours. Finally, while maintaining a vacuum of 1.5 Pa, the temperature was heated from 0°C to 28°C at a rate of 2°C / h.
[0015] Further, the post-processing involves crushing the freeze-dried bacterial blocks under nitrogen protection and then sieving them through an 80-mesh sieve.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses a specialized fermentation medium adapted to Lactobacillus paracasei KFY202407, employing a pure plant-derived, multi-stage nitrogen source blend (plant peptone, soybean protein peptides, yeast extract), and precisely adding synergistic components such as astragalus polysaccharides and xylooligosaccharides. This provides the strain with a balanced, sustained nutrient flow tailored to its metabolic characteristics. Combined with a two-stage fermentation process, it effectively alleviates metabolic product inhibition, ultimately achieving a stable viable cell count of 2.0 × 10⁻⁶ in the fermentation broth. 10 CFU / mL or higher.
[0017] 2. By using a composite freeze-drying protectant combined with a gradient freeze-drying process, this invention significantly reduces the damage to the strains caused by freeze-drying, thereby improving the survival rate of the strains. Detailed Implementation
[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.
[0019] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.
[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0023] This invention provides a method for preparing highly active Lactobacillus paracasei KFY202407 through high-density fermentation and freeze-drying, comprising the following preparation steps: Lactobacillus paracasei KFY202407 was inoculated into a dedicated culture medium and activated to obtain an activated bacterial solution. The activated bacterial solution was inoculated into a dedicated culture medium for large-scale cultivation to obtain a seed culture. The seed culture was inoculated into a fermentation medium for high-density anaerobic fermentation to obtain a fermentation broth. The fermentation broth was centrifuged and enriched to obtain bacterial sludge. The bacterial sludge was then mixed with a composite freeze-drying protectant in a gradient and subjected to gradient freeze-drying to obtain freeze-dried bacterial blocks. The freeze-dried bacterial blocks were post-processed to obtain high-density fermented freeze-dried Lactobacillus paracasei KFY202407 with high activity.
[0024] Specifically, the Lactobacillus paracasei KFY202407 has the accession number CGMCC NO.30736, is deposited at the China General Microbiological Culture Collection Center on May 22, 2024, and is classified as Lactobacillus paracasei.
[0025] Understandably, this invention employs a specialized fermentation medium adapted to Lactobacillus paracasei KFY202407, using a pure plant-derived, multi-stage nitrogen source blend (plant peptone, soybean protein peptides, yeast extract), and precisely adding synergistic components such as astragalus polysaccharides and xylooligosaccharides. This provides the strain with a balanced, sustained nutrient flow tailored to its metabolic characteristics. Combined with a two-stage fermentation process, this effectively alleviates metabolic product inhibition, ultimately resulting in a stable viable cell count of 2.0 × 10⁻⁶ in the fermentation broth. 10 CFU / mL or higher.
[0026] It is understood that by using a composite freeze-drying protectant combined with a gradient freeze-drying process, this invention significantly reduces the damage to the strains caused by freeze-drying, thereby improving the survival rate of the strains.
[0027] In this invention, the specific culture medium comprises the following components in parts by weight: The ingredients included 12 parts plant peptone, 8 parts yeast extract, 25 parts glucose, 3 parts anhydrous sodium acetate, 2.5 parts diammonium hydrogen citrate, 0.6 parts MgSO4·7H2O, 0.3 parts MnSO4·H2O, 2.5 parts K2HPO4·3H2O, 1.2 parts Tween 80, 0.8 parts Astragalus polysaccharide, and 1.5 parts xylooligosaccharide.
[0028] Specifically, the method for preparing the specific culture medium is as follows (preparing 1L of specific culture medium): Prepare a 500 g / L glucose stock solution, sterilize it by filtration through a 0.22 μm filter membrane, seal it, and store it at 4°C for later use. Take 800ml of deionized water and, while stirring, add 12g of plant peptone, 8g of yeast extract, 3g of anhydrous sodium acetate, 2.5g of diammonium hydrogen citrate, 2.5g of K2HPO4·3H2O, 0.8g of astragalus polysaccharide, 1.5g of xylooligosaccharide, 1.2g of Tween 80, 0.6g of MgSO4·7H2O, and 0.3g of MnSO4·H2O. Ensure that each component is basically dissolved before adding the next. After the initial addition, use 1mol / L HCl or NaOH solution to precisely adjust the pH to 6.4±0.1. Add deionized water to bring the volume to 950ml. After bringing the volume to 950ml, steam sterilize at 115℃ and 0.11MPa for 15min. After sterilization, wait until the pressure drops to zero and the temperature drops to between 70-80℃, then remove the product and allow it to cool naturally to room temperature. Then add 50ml of the glucose stock solution and bring the volume to 1L using deionized water.
[0029] It is understandable that sterilizing glucose separately from other components can prevent glucose from reacting with other components at high temperatures to produce inhibitors through Maillard reactions.
[0030] Understandably, this dedicated culture medium provides a safe and balanced nitrogen source and growth factors with plant peptone and yeast extract. Glucose, combined with xylooligosaccharides and astragalus polysaccharides, forms a dual-function carbon source system that provides rapid energy supply and functional induction. This not only meets the nutritional needs for rapid bacterial proliferation but also enables the adaptive expression of anti-inflammatory and anti-fibrotic metabolic pathways in the strain in advance.
[0031] In this invention, the inoculum amount for the activation culture is 1% (v / v), the culture temperature is 37°C, the culture environment is an anaerobic environment, and the culture time is 18h.
[0032] Specifically, Lactobacillus paracasei KFY202407 was inoculated into the dedicated culture medium at an inoculation rate of 1% (v / v) and incubated statically for 18 hours at 37°C under an atmosphere of N2:CO2=85:15 (v / v).
[0033] In this invention, the inoculum amount for the expanded culture is 3% (v / v), the culture temperature is 37°C, the culture environment is an anaerobic environment, the shaking speed is 50 rpm, and the culture time is 24 h.
[0034] Specifically, the activated bacterial solution was inoculated into the dedicated culture medium at an inoculation rate of 3% (v / v) and cultured at 37°C with an atmosphere of N2:CO2=85:15 (v / v) and shaken at 50 rpm for 24 h.
[0035] In this invention, the high-density anaerobic fermentation culture specifically refers to: The seed culture was inoculated into the fermentation medium at an inoculum rate of 5% (v / v), and fermented under anaerobic conditions at 37°C with shaking at a speed of 50-100 rpm until the cell concentration in the fermentation broth reached 8.0 × 10⁻⁶. 9 CFU / mL, then adjust the temperature of the fermentation broth to 35℃, adjust the pH to between 6.0 and 6.2, add plant lactobacillus solution to the concentration of plant lactobacillus in the fermentation broth to 50μg / mL, and continue anaerobic fermentation for 20-28h; The fermentation medium comprises the following components in parts by weight: 15 parts plant peptone, 8 parts soybean protein peptide, 10 parts yeast extract, 35 parts glucose, 4 parts anhydrous sodium acetate, 3 parts diammonium hydrogen citrate, 0.8 parts MgSO4·7H2O, 0.4 parts MnSO4·H2O, 3 parts K2HPO4·3H2O, 1.5 parts Tween 80, 1.2 parts astragalus polysaccharide, 2.0 parts xylooligosaccharide, 1.0 part L-glutamic acid, and 0.1 parts vitamin B complex.
[0036] Specifically, a 50L stainless steel fermenter was used, filled with 30L of fermentation medium. An anaerobic environment was created by purging the tank with sterile gas (N2:CO2 = 85:15, v / v) to replace the air. The seed culture was inoculated into the fermentation medium at a rate of 5% (v / v). The fermentation was carried out at 37°C with shaking at 80 rpm. During this period, 200 g / L sterile glucose solution was added to maintain the glucose concentration in the fermentation broth at 8-10 g / L, and 1 mol / L sterile sodium lactate solution was added to maintain the pH between 6.2 and 6.5 until the cell concentration reached 8.0 × 10⁻⁶. 9 CFU / mL; after completion, adjust the temperature to 35℃, adjust the pH to between 6.0 and 6.2, reduce the amount of sterile glucose solution added, and maintain the residual sugar concentration at 2-3 g / L. At the same time, add sterile plant lactobacillus solution to the fermentation broth to a plant lactobacillus concentration of 50 μg / mL, and continue anaerobic culture for 24 h.
[0037] Specifically, the fermentation medium is prepared as follows (per 1L): Prepare a 500 g / L glucose stock solution, sterilize it by filtration through a 0.22 μm filter membrane, seal it, and store it at 4°C for later use. Weigh 0.1g of vitamin B complex powder (VB1:VB6:VB12 mass ratio = 1:2:0.5), dissolve it in 10ml of sterile deionized water, and then filter it through a 0.22μm filter membrane to obtain a vitamin B complex solution. Take 800ml of deionized water and add 15g of plant peptone, 8g of soybean protein peptide, 10g of yeast extract, 4g of sodium acetate aqueous solution, 3g of diammonium hydrogen citrate, 3g of K₂HPO₄·3H₂O, 1.2g of astragalus polysaccharide, 2.0g of xylooligosaccharide, 1.0g of L-glutamic acid, 1.5g of Tween 80, 0.8g of MgSO₄·7H₂O, and 0.4g of MnSO₄·H₂O. Ensure each ingredient is basically dissolved before adding the next. Afterward, use a 1mol / L solution. Adjust the pH of the HCl or NaOH solution precisely to 6.5±0.1, add deionized water to a final volume of 900 ml, and then steam sterilize at 115℃ and 0.11 MPa for 20 min. After sterilization, wait until the pressure drops to zero and the temperature drops to between 70-80℃, then remove the solution and allow it to cool naturally to room temperature. Then add 75 ml of the glucose stock solution and vitamin B complex solution, and bring the final volume to 1 L using deionized water.
[0038] Understandably, the gradient release of nitrogen sources from plant peptone, soybean peptides, and yeast extract continuously meets the synthesis needs of high-density microbial cells. The precisely regulated carbon source (glucose) combined with exclusive prebiotics (astragalus polysaccharide and xylooligosaccharides) efficiently provides energy while directionally inducing anti-inflammatory and anti-fibrotic metabolic pathways in the strains. Furthermore, key metabolic cofactors such as L-glutamate and B vitamins comprehensively optimize cellular physiological states. This design not only effectively alleviates metabolic inhibition caused by lactic acid accumulation but also increases the viable cell count at the fermentation endpoint to ≥2.0 × 10⁻⁶. 10 The CFU / mL level ensures that the bacteria simultaneously accumulate functional active substances during rapid proliferation.
[0039] Understandably, the first stage of high-density anaerobic fermentation culture (37℃, pH 6.2-6.5, nutrient-rich) aims to drive rapid cell proliferation and quickly reach a high biomass plateau. The second stage (35℃, pH 6.0-6.2, carbon-limited with added inducing agents) shifts the metabolic focus from rapid proliferation to enhancing cellular stress tolerance, stabilizing membrane structure, and enriching anti-inflammatory and anti-fibrotic functional metabolites through gentle slow-release culture. This method not only increases the concentration of viable cells but also ensures that the harvested cells are in a robust and functionally active optimal physiological state.
[0040] In this invention, the centrifugal enrichment process specifically includes: Centrifuge the fermentation broth at 4℃ and 8000-12000 r / min for 10-20 min, discard the supernatant, collect the wet bacterial sludge, add 2 times the volume of isotonic protective washing solution pre-cooled to 4℃ to the wet bacterial sludge, stir and resuspend, centrifuge at 4℃ and 6000-10000 r / min for 5-15 min, discard the supernatant, and repeat the washing twice.
[0041] Specifically, the preparation method of the isotonic protective washing solution is as follows: Add 8.5g sodium chloride (NaCl) and 10.0g trehalose to 800ml of deionized water and stir until dissolved. Use 0.1mol / L HCl or NaOH solution to precisely adjust the pH of the solution to 6.8. Add deionized water to make up to 1L. Then autoclave at 121℃ for 20min. After that, equilibrate overnight in a refrigerator at 4℃.
[0042] Understandably, the isotonic protective washing solution can prevent plasmolysis or rupture damage caused by sudden changes in osmotic pressure when the bacterial sludge is resuspended after centrifugation by creating an isotonic environment (0.85% NaCl). The added trehalose can bind to cell membrane phospholipids in advance during the washing stage, initiating a dehydration protection mechanism and stabilizing the membrane structure. The entire operation is carried out at 4°C, which strongly inhibits bacterial metabolism and heat stress.
[0043] In this invention, the composite lyophilization protectant comprises the following components in parts by weight: The ingredients are: 12 parts trehalose, 8 parts xylooligosaccharides, 6 parts glycerol, 1.5 parts glutathione, 2 parts astragalus polysaccharide, 0.8 parts vitamin C, 1.2 parts soybean lecithin, 10 parts plant protein hydrolysate, and 58.5 parts water.
[0044] The gradient mixing specifically refers to: Add the bacterial sludge to one-third of the volume of water and stir to obtain a concentrated bacterial suspension. Add the composite freeze-drying protectant dropwise to the concentrated bacterial suspension at 4°C and 200-500 rpm for 15-20 minutes. After the addition is complete, let it stand at 4°C for 25-35 minutes to obtain a bacterial suspension.
[0045] Specifically, the preparation method of the composite freeze-drying protectant is as follows: take 12 parts of trehalose, 8 parts of xylooligosaccharide, 6 parts of glycerol, 1.5 parts of glutathione, 2 parts of astragalus polysaccharide, 0.8 parts of vitamin C, 1.2 parts of soybean lecithin, 10 parts of plant protein hydrolysate, and 58.5 parts of water, mix them, and stir in a water bath at 60°C until completely dissolved.
[0046] Specifically, the bacterial sludge is added to one-third of the volume of sterile deionized water at 4°C and stirred to obtain a concentrated bacterial suspension. The concentrated bacterial suspension is then added dropwise with a composite freeze-drying protectant at 4°C and 200-500 rpm for 15-20 minutes. After the addition is complete, the suspension is allowed to stand at 4°C for 25-35 minutes to obtain a bacterial suspension. The entire process is carried out under sterile nitrogen protection.
[0047] Understandably, pre-mixing and diluting the bacterial sludge first lowers the initial osmotic pressure. Then, under low temperature and low-speed stirring, the hypertonic protective agent solution is slowly added dropwise. This allows the bacterial cells sufficient time to gradually adapt to changes in the external environment through osmotic regulation, preventing cell membrane rupture or plasmolysis caused by sudden water imbalance. At the same time, the gentle mixing method preserves the integrity of the cell structure to the greatest extent and ensures that key protective components such as trehalose and glutathione are evenly and fully coated and penetrated to the bacterial surface and surrounding area.
[0048] In this invention, the gradient freeze-drying specifically refers to: The bacterial suspension was cooled from 4°C to -20°C at a rate of 2°C / min and kept at that temperature for 1-3 hours. Then, it was cooled from -20°C to -50°C at a rate of 5°C / min and kept at that temperature for 2-6 hours. Finally, it was cooled from -50°C to -70°C at a rate of 8°C / min and kept at that temperature for 8-12 hours to obtain pre-frozen bacterial blocks. The pre-frozen mycelium blocks were heated from -70°C to -40°C at a rate of 0.5°C / h under a vacuum of 1.5 Pa and held for 6-10 hours. Then, the temperature was heated from -40°C to -20°C at a rate of 1°C / h and held for 10-14 hours. Next, the temperature was heated from -20°C to 0°C at a rate of 2°C / h and held for 4-8 hours. Finally, while maintaining a vacuum of 1.5 Pa, the temperature was heated from 0°C to 28°C at a rate of 2°C / h.
[0049] Specifically, the preferred method for gradient freeze-drying is as follows: Pre-freezing stage: Spread the bacterial suspension evenly on a freeze-drying tray (1.5 cm thick) and immediately place it in a vacuum freeze dryer pre-cooled to 4°C. Procedure: Depress from 4°C to -20°C at a rate of 2°C / min and hold for 2 hours; then depress from -20°C to -50°C at a rate of 5°C / min and hold for 4 hours; finally, depress from -50°C to -70°C at a rate of 8°C / min and hold at this temperature for 10 hours to ensure complete solidification and formation of fine ice crystals, resulting in pre-frozen bacterial blocks. Sublimation drying stage (segmented heating): The vacuum system is activated to stabilize the vacuum level in the chamber to 1.5 Pa, and the cold trap temperature is maintained at -65℃. First stage: The sample temperature is raised from -70℃ to -40℃ at a rate of 0.5℃ / h and held for 8 hours. Second stage: The temperature is raised from -40℃ to -20℃ at a rate of 1℃ / h and held for 12 hours. Third stage: The temperature is raised from -20℃ to 0℃ at a rate of 2℃ / h and held for 6 hours. This stage removes most of the free water. Drying stage: Maintain a vacuum of 1.5 Pa and raise the sample temperature from 0°C to 28°C at a rate of 2°C / h. Drying is complete when the sample moisture probe shows that the moisture content is consistently ≤3% for 1 hour.
[0050] Understandably, gradient freeze-drying manages the water crystal morphology, ice crystal sublimation interface, and final moisture content by controlling the temperature, vacuum, and time parameters of the three stages: pre-freezing, sublimation, and desorption. This minimizes structural and functional damage to bacterial cells during the freeze-drying process. The step-by-step, slow-drying pre-freezing procedure guides the formation of fine, uniform microcrystals, effectively preventing ice crystals from puncturing the cell membrane. The segmented, slow-heating sublimation drying ensures stable sublimation of ice crystals, preventing sample collapse and cell structure tearing caused by localized overheating or drying stress. Finally, the desorption drying based on real-time moisture monitoring completely removes bound water while avoiding excessive heat treatment.
[0051] In this invention, the post-processing specifically involves: pulverizing the freeze-dried bacterial blocks under nitrogen protection and then sieving them using an 80-mesh sieve.
[0052] Specifically, the freeze-dried bacterial blocks are transferred to a sterile ultra-micro pulverizer and pulverized to a particle size ≤80μm under continuous nitrogen protection, and then sieved through an 80-mesh sterile sieve.
[0053] Example 1 Reagent preparation: The specific culture medium, fermentation culture medium, isotonic protective washing solution, and composite lyophilization protectant were all prepared using the methods disclosed above.
[0054] Preparation method: S1. Inoculate Lactobacillus paracasei KFY202407 into the dedicated culture medium at an inoculation rate of 1% (v / v) and incubate statically for 18 hours at 37℃ under an atmosphere of N2:CO2=85:15 (v / v) to obtain the bacterial culture solution. S2. Inoculate the activated bacterial solution into the dedicated culture medium at an inoculation rate of 3% (v / v), and culture at 37℃ and N2:CO2=85:15 (v / v) with shaking at 50 rpm for 24 h to obtain the seed culture. S3. A 50L stainless steel fermenter was used, filled with 30L of fermentation medium. A sterile gas mixture of N2:CO2 = 85:15 (v / v) was introduced to replace the air in the fermenter, creating an anaerobic environment. The seed culture was inoculated into the fermentation medium at a rate of 5% (v / v). The fermentation was carried out at 37℃ with shaking at 50 rpm. During this period, 200 g / L sterile glucose solution was added to maintain the glucose concentration in the fermentation broth at 8-10 g / L, and 1 mol / L sterile sodium lactate solution was added to maintain the pH between 6.2 and 6.5 until the cell concentration reached 8.0 × 10⁻⁶. 9CFU / mL; after completion, adjust the temperature to 35℃, adjust the pH to between 6.0 and 6.2, reduce the amount of sterile glucose solution added, and maintain the residual sugar concentration at 2-3 g / L. At the same time, add sterile plant lactobacillus solution to make the plant lactobacillus concentration in the fermentation broth 50 μg / mL, and continue anaerobic culture for 24 h to obtain the fermentation broth; S4. Centrifuge the fermentation broth at 4℃ and 8000r / min for 10min, discard the supernatant, collect the wet bacterial sludge, add 2 times the volume of isotonic protective washing solution pre-cooled to 4℃ to the wet bacterial sludge, stir and resuspend, centrifuge at 4℃ and 6000r / min for 5min, discard the supernatant, repeat the washing twice to obtain bacterial sludge. S5. Add one-third volume of sterile deionized water at 4℃ to the bacterial sludge and stir to obtain a concentrated bacterial suspension. Add the composite freeze-drying protectant dropwise to the concentrated bacterial suspension at 4℃ and 200 rpm for 15 minutes. After the addition is completed, let it stand at 4℃ for 25 minutes to obtain the bacterial suspension. The whole process is carried out under sterile nitrogen protection. S6. Spread the bacterial suspension evenly on a freeze-drying tray (1.5 cm thick), and immediately place it in a vacuum freeze dryer pre-cooled to 4°C. Execute the following program: reduce the temperature from 4°C to -20°C at a rate of 2°C / min and hold for 1 hour; then reduce the temperature from -20°C to -50°C at a rate of 5°C / min and hold for 2 hours; finally, reduce the temperature from -50°C to -70°C at a rate of 8°C / min and hold at this temperature for 8 hours to obtain pre-frozen bacterial blocks; start the vacuum system and stabilize the vacuum level in the chamber to 1.5. The cold trap temperature was maintained at -65℃. The sample temperature was increased from -70℃ to -40℃ at a rate of 0.5℃ / h and held for 6 hours. Then, the temperature was increased from -40℃ to -20℃ at a rate of 1℃ / h and held for 10 hours. The temperature was then increased from -20℃ to 0℃ at a rate of 2℃ / h and held for 4 hours. The vacuum degree was maintained at 1.5Pa, and the sample temperature was increased from 0℃ to 28℃ at a rate of 2℃ / h. When the sample moisture probe showed that the moisture content was stable at ≤3% for 1 hour, the drying was completed, and freeze-dried bacterial blocks were obtained. S7. Transfer the freeze-dried bacterial blocks into a sterile ultra-micro pulverizer and pulverize them to a particle size ≤80μm under continuous nitrogen protection. Then, sieve them through an 80-mesh sterile sieve to obtain high-activity Lactobacillus paracasei KFY202407 high-density fermented freeze-dried bacteria.
[0055] Example 2 Reagent preparation: The specific culture medium, fermentation culture medium, isotonic protective washing solution, and composite lyophilization protectant were all prepared using the methods disclosed above.
[0056] Preparation method: S1. Inoculate Lactobacillus paracasei KFY202407 into the dedicated culture medium at an inoculation rate of 1% (v / v) and incubate statically for 18 hours at 37℃ under an atmosphere of N2:CO2=85:15 (v / v) to obtain the bacterial culture solution. S2. Inoculate the activated bacterial solution into the dedicated culture medium at an inoculation rate of 3% (v / v), and culture at 37℃ and N2:CO2=85:15 (v / v) with shaking at 50 rpm for 24 h to obtain the seed culture. S3. A 50L stainless steel fermenter was used, filled with 30L of fermentation medium. Sterile gas (N2:CO2 = 85:15, v / v) was introduced to replace the air in the fermenter, creating an anaerobic environment. The seed culture was inoculated into the fermentation medium at a rate of 5% (v / v). The fermentation was carried out at 37℃ with shaking at 80 rpm. During this period, 200 g / L sterile glucose solution was added to maintain the glucose concentration in the fermentation broth at 8-10 g / L, and 1 mol / L sterile sodium lactate solution was added to maintain the pH between 6.2 and 6.5 until the cell concentration reached 8.0 × 10⁻⁶. 9 CFU / mL; after completion, adjust the temperature to 35℃, adjust the pH to between 6.0 and 6.2, reduce the amount of sterile glucose solution added, and maintain the residual sugar concentration at 2-3 g / L. At the same time, add sterile plant lactobacillus solution to make the plant lactobacillus concentration in the fermentation broth 50 μg / mL, and continue anaerobic culture for 24 h to obtain the fermentation broth; S4. Centrifuge the fermentation broth at 4℃ and 10000r / min for 15min, discard the supernatant, collect the wet bacterial sludge, add 2 times the volume of isotonic protective washing solution pre-cooled to 4℃ to the wet bacterial sludge, stir and resuspend, centrifuge at 4℃ and 8000r / min for 10min, discard the supernatant, repeat the washing twice to obtain bacterial sludge. S5. Add one-third volume of sterile deionized water at 4℃ to the bacterial sludge and stir to obtain a concentrated bacterial suspension. Add the composite freeze-drying protectant dropwise to the concentrated bacterial suspension at 4℃ and 400rpm with stirring for 18 minutes. After the addition is completed, let it stand at 4℃ for 30 minutes to obtain the bacterial suspension. The whole process is carried out under sterile nitrogen protection. S6. Spread the bacterial suspension evenly on a freeze-drying tray (1.5 cm thick), and immediately place it in a vacuum freeze dryer pre-cooled to 4°C. Execute the following program: reduce the temperature from 4°C to -20°C at a rate of 2°C / min and hold for 2 hours; then reduce the temperature from -20°C to -50°C at a rate of 5°C / min and hold for 4 hours; finally, reduce the temperature from -50°C to -70°C at a rate of 8°C / min and hold at this temperature for 10 hours to obtain pre-frozen bacterial blocks; start the vacuum system and stabilize the vacuum level in the chamber to 1.5. The cold trap temperature was maintained at -65℃. The sample temperature was increased from -70℃ to -40℃ at a rate of 0.5℃ / h and held for 8 hours. Then, the temperature was increased from -40℃ to -20℃ at a rate of 1℃ / h and held for 12 hours. The temperature was then increased from -20℃ to 0℃ at a rate of 2℃ / h and held for 6 hours. The vacuum degree was maintained at 1.5Pa, and the sample temperature was increased from 0℃ to 28℃ at a rate of 2℃ / h. When the sample moisture probe showed that the moisture content was stable at ≤3% for 1 hour, the drying was completed, and freeze-dried bacterial blocks were obtained. S7. Transfer the freeze-dried bacterial blocks into a sterile ultra-micro pulverizer and pulverize them to a particle size ≤80μm under continuous nitrogen protection. Then, sieve them through an 80-mesh sterile sieve to obtain high-activity Lactobacillus paracasei KFY202407 high-density fermented freeze-dried bacteria.
[0057] Example 3 Reagent preparation: The specific culture medium, fermentation culture medium, isotonic protective washing solution, and composite lyophilization protectant were all prepared using the methods disclosed above.
[0058] Preparation method: S1. Inoculate Lactobacillus paracasei KFY202407 into the dedicated culture medium at an inoculation rate of 1% (v / v) and incubate statically for 18 hours at 37℃ under an atmosphere of N2:CO2=85:15 (v / v) to obtain the bacterial culture solution. S2. Inoculate the activated bacterial solution into the dedicated culture medium at an inoculation rate of 3% (v / v), and culture at 37℃ and N2:CO2=85:15 (v / v) with shaking at 50 rpm for 24 h to obtain the seed culture. S3. A 50L stainless steel fermenter was used, filled with 30L of fermentation medium. A sterile gas mixture of N2:CO2 = 85:15 (v / v) was introduced to replace the air in the fermenter, creating an anaerobic environment. The seed culture was inoculated into the fermentation medium at a rate of 5% (v / v). The fermentation was carried out at 37℃ with shaking at 100 rpm. During the incubation, 200 g / L sterile glucose solution was added to maintain the glucose concentration in the fermentation broth at 8-10 g / L, and 1 mol / L sterile sodium lactate solution was added to maintain the pH between 6.2 and 6.5 until the cell concentration reached 8.0 × 10⁻⁶. 9CFU / mL; after completion, adjust the temperature to 35℃, adjust the pH to between 6.0 and 6.2, reduce the amount of sterile glucose solution added, and maintain the residual sugar concentration at 2-3 g / L. At the same time, add sterile plant lactobacillus solution to make the plant lactobacillus concentration in the fermentation broth 50 μg / mL, and continue anaerobic culture for 24 h to obtain the fermentation broth; S4. Centrifuge the fermentation broth at 4℃ and 12000r / min for 20min, discard the supernatant, collect the wet bacterial sludge, add 2 times the volume of isotonic protective washing solution pre-cooled to 4℃ to the wet bacterial sludge, stir and resuspend, centrifuge at 4℃ and 10000r / min for 15min, discard the supernatant, repeat the washing twice to obtain bacterial sludge. S5. Add one-third volume of sterile deionized water at 4℃ to the bacterial sludge and stir to obtain a concentrated bacterial suspension. Add the composite freeze-drying protectant dropwise to the concentrated bacterial suspension at 4℃ and 500rpm for 20 minutes. After the addition is completed, let it stand at 4℃ for 35 minutes to obtain the bacterial suspension. The whole process is carried out under sterile nitrogen protection. S6. Spread the bacterial suspension evenly on a freeze-drying tray (1.5 cm thick), and immediately place it in a vacuum freeze dryer pre-cooled to 4°C. Execute the following program: reduce the temperature from 4°C to -20°C at a rate of 2°C / min and hold for 3 hours; then reduce the temperature from -20°C to -50°C at a rate of 5°C / min and hold for 6 hours; finally, reduce the temperature from -50°C to -70°C at a rate of 8°C / min and hold at this temperature for 12 hours to obtain pre-frozen bacterial blocks; start the vacuum system and stabilize the vacuum level in the chamber to 1.5. The cold trap temperature was maintained at -65℃. The sample temperature was increased from -70℃ to -40℃ at a rate of 0.5℃ / h and held for 10 hours. Then, the temperature was increased from -40℃ to -20℃ at a rate of 1℃ / h and held for 14 hours. The temperature was then increased from -20℃ to 0℃ at a rate of 2℃ / h and held for 8 hours. The vacuum degree was maintained at 1.5Pa, and the sample temperature was increased from 0℃ to 28℃ at a rate of 2℃ / h. When the sample moisture probe showed that the moisture content was stable at ≤3% for 1 hour, the drying was completed, and freeze-dried bacterial blocks were obtained. S7. Transfer the freeze-dried bacterial blocks into a sterile ultra-micro pulverizer and pulverize them to a particle size ≤80μm under continuous nitrogen protection. Then, sieve them through an 80-mesh sterile sieve to obtain high-activity Lactobacillus paracasei KFY202407 high-density fermented freeze-dried bacteria.
[0059] Compare with Example 1 The only difference from Example 2 is that step S3 is as follows: a 50L stainless steel fermenter is used, 30L of fermentation medium is added, and sterile gas with N2:CO2=85:15 (v / v) is introduced to replace the air in the fermenter to create an anaerobic environment. The seed liquid is inoculated into the fermentation medium in the fermenter at an inoculation rate of 5% (v / v). The fermenter is cultured at 37°C and shaken at 100 rpm for 60 hours. During the process, 200g / L sterile glucose solution is added to maintain the glucose concentration of the fermentation broth at 8-10g / L, and 1mol / L sterile sodium lactate solution is added to maintain the pH between 6.2 and 6.5 to obtain the fermentation broth. Compare with Example 2 The only difference from Control Example 1 was that the exclusive culture medium and fermentation medium used in Control Example 2 were replaced with traditional MRS medium (prepared according to the standard formula: peptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, glucose 20 g / L, Tween 80 1 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.05 g / L, sodium acetate 5 g / L, pH 6.5).
[0060] Test Example 1 (High-Density Fermentation Effect Test) Experimental groups: experimental group (Example 2), control group 1 (control example 1) and control group 2 (control example 2); Test method: Samples of the fermentation broth were taken and tested during the fermentation process of Example 2, Control Example 1, and Control Example 2. Sampling points were: 0, 12, 24, 36, 48, and 60 hours. Viable cell count: Aseptic sampling was performed, and the samples were serially diluted 10-fold with 0.85% physiological saline, spread on MRS agar plates, and anaerobically incubated at 37°C for 48 hours. The counts were then expressed as CFU / mL.
[0061] pH value: measured directly using a pH meter.
[0062] Glucose and lactate concentrations: Take 1 mL of sample, centrifuge at 12,000 rpm for 5 minutes to remove bacterial cells, and filter the supernatant through a 0.22 μm filter membrane. Determine glucose concentration using high-performance liquid chromatography (HPLC) equipped with a differential refractive index detector and a carbohydrate column (or a dedicated biosensor analyzer); determine lactate concentration using HPLC equipped with a UV detector and a C18 column (or a dedicated lactate kit), in g / L.
[0063] The test data is shown in Table 1: Table 1. Test results of fermentation broth
[0064] As shown in Table 1, the viable cell count of the experimental group prepared by the present invention reached 2.3 × 10⁻⁶ at the fermentation endpoint. 10 The CFU / mL concentration was 2.7 times and 3.4 times that of control groups 1 and 2, respectively, representing a significant order-of-magnitude improvement. Secondly, key metabolic parameters showed that the experimental group effectively alleviated metabolic inhibition, with a higher fermentation endpoint pH (6.05) and lower lactic acid concentration (45.2 g / L), indicating that the cells were in a healthier physiological state. In contrast, both control groups prematurely entered the growth plateau phase 48 hours prior due to rapid lactic acid accumulation and a sharp drop in pH.
[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A method for preparing highly active Lactobacillus paracasei KFY202407 through high-density fermentation freeze-drying, characterized in that, The preparation steps include the following: Lactobacillus paracasei KFY202407 was inoculated into a dedicated culture medium for activation culture to obtain activated bacterial solution; The activated bacterial solution was inoculated into a dedicated culture medium for large-scale cultivation to obtain a seed culture. The seed culture was inoculated into a fermentation medium for high-density anaerobic fermentation to obtain a fermentation broth. The fermentation broth was centrifuged and enriched to obtain bacterial sludge. The bacterial sludge was then mixed with a composite freeze-drying protectant in a gradient and subjected to gradient freeze-drying to obtain freeze-dried bacterial blocks. The freeze-dried bacterial blocks were post-processed to obtain high-density fermented freeze-dried Lactobacillus paracasei KFY202407 with high activity.
2. The method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 1, characterized in that, The specific culture medium comprises the following components in parts by weight: The ingredients included 12 parts plant peptone, 8 parts yeast extract, 25 parts glucose, 3 parts anhydrous sodium acetate, 2.5 parts diammonium hydrogen citrate, 0.6 parts MgSO4·7H2O, 0.3 parts MnSO4·H2O, 2.5 parts K2HPO4·3H2O, 1.2 parts Tween 80, 0.8 parts Astragalus polysaccharide, and 1.5 parts xylooligosaccharide.
3. The method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 2, characterized in that, The inoculum amount for the activation culture was 1% (v / v), the culture temperature was 37℃, the culture environment was anaerobic, and the culture time was 18h.
4. The method for preparing high-activity Lactobacillus paracasei KFY202407 high-density fermentation freeze-dried according to claim 3, characterized in that, The inoculum size for the expanded culture was 3% (v / v), the culture temperature was 37°C, the culture environment was anaerobic, the shaking speed was 50 rpm, and the culture time was 24 h.
5. The method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 4, characterized in that, The high-density anaerobic fermentation culture specifically refers to: The seed culture was inoculated into the fermentation medium at an inoculum rate of 5% (v / v), and fermented under anaerobic conditions at 37°C with shaking at a speed of 50-100 rpm until the cell concentration in the fermentation broth reached 8.0 × 10⁻⁶. 9 CFU / mL, then adjust the temperature of the fermentation broth to 35℃, adjust the pH to between 6.0 and 6.2, add plant lactobacillus solution to the concentration of plant lactobacillus in the fermentation broth to 50μg / mL, and continue anaerobic fermentation for 20-28h; The fermentation medium comprises the following components in parts by weight: 15 parts plant peptone, 8 parts soybean protein peptide, 10 parts yeast extract, 35 parts glucose, 4 parts anhydrous sodium acetate, 3 parts diammonium hydrogen citrate, 0.8 parts MgSO4·7H2O, 0.4 parts MnSO4·H2O, 3 parts K2HPO4·3H2O, 1.5 parts Tween 80, 1.2 parts astragalus polysaccharide, 2.0 parts xylooligosaccharide, 1.0 part L-glutamic acid, and 0.1 parts vitamin B complex.
6. The method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 5, characterized in that, The centrifugal enrichment process specifically involves: Centrifuge the fermentation broth at 4℃ and 8000-12000 r / min for 10-20 min, discard the supernatant, collect the wet bacterial sludge, add 2 times the volume of isotonic protective washing solution pre-cooled to 4℃ to the wet bacterial sludge, stir and resuspend, centrifuge at 4℃ and 6000-10000 r / min for 5-15 min, discard the supernatant, and repeat the washing twice.
7. The method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 6, characterized in that, The composite lyophilization protectant comprises the following components in parts by weight: The ingredients are: 12 parts trehalose, 8 parts xylooligosaccharides, 6 parts glycerol, 1.5 parts glutathione, 2 parts astragalus polysaccharide, 0.8 parts vitamin C, 1.2 parts soybean lecithin, 10 parts plant protein hydrolysate, and 58.5 parts water.
8. The method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 7, characterized in that, The gradient mixing specifically refers to: Add the bacterial sludge to one-third of the volume of water and stir to obtain a concentrated bacterial suspension. Add the composite freeze-drying protectant dropwise to the concentrated bacterial suspension at 4°C and 200-500 rpm for 15-20 minutes. After the addition is complete, let it stand at 4°C for 25-35 minutes to obtain a bacterial suspension.
9. The method for preparing high-activity Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 8, characterized in that, The gradient freeze-drying specifically refers to: The bacterial suspension was cooled from 4°C to -20°C at a rate of 2°C / min and kept at that temperature for 1-3 hours. Then, it was cooled from -20°C to -50°C at a rate of 5°C / min and kept at that temperature for 2-6 hours. Finally, it was cooled from -50°C to -70°C at a rate of 8°C / min and kept at that temperature for 8-12 hours to obtain pre-frozen bacterial blocks. The pre-frozen mycelium blocks were heated from -70°C to -40°C at a rate of 0.5°C / h under a vacuum of 1.5 Pa and held for 6-10 hours. Then, the temperature was heated from -40°C to -20°C at a rate of 1°C / h and held for 10-14 hours. Next, the temperature was heated from -20°C to 0°C at a rate of 2°C / h and held for 4-8 hours. Finally, while maintaining a vacuum of 1.5 Pa, the temperature was heated from 0°C to 28°C at a rate of 2°C / h.
10. The method for preparing highly active Lactobacillus paracasei KFY202407 by high-density fermentation freeze-drying according to claim 9, characterized in that, The post-processing involves crushing the freeze-dried bacterial blocks under nitrogen protection and then sieving them through an 80-mesh sieve.