Freeze-drying protection method of paracoccus versicolor and application of freeze-drying protection method
By optimizing the freeze-drying protective agent and gradient freeze-drying technology, the problems of short shelf life and poor temperature resistance of Paracoccus mutans were solved, and efficient freeze-drying protection and long-term preservation effects were achieved.
Patent Information
- Application Number
- CN202510895000.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-09-12
AI Technical Summary
The existing storage methods for Paracoccus mutans have the disadvantages of short shelf life, rapid decline in strain activity, poor temperature resistance, complete inactivation due to spray drying at 60°C, and a lack of effective freeze-drying protection methods.
The freeze-drying process was optimized by using a vacuum freeze-drying protective agent consisting of 10-12% skim milk powder + 5-10% trehalose + 2-4% glycerol or 20% skim milk powder + 20% trehalose + 8% glycerol, combined with gradient precooling (-5 to -55°C, 3-6 h), gradient vacuum freeze-drying (-45 to 5°C, 16 to 24 h), and vacuum desorption drying (5-25°C, 8-17 h).
The freeze-dried survival rate and long-term storage effect of Paracoccus mutans were significantly improved. The survival rate reached 62.8% after 9 months of storage at 4°C, and the survival rate was 64.73% after 30 days of storage at room temperature, effectively extending the shelf life.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a freeze-drying protection method and application of Paracoccus mutans. Background Art
[0002] Paracoccus versutus belongs to the genus Paracoccus. It is Gram-negative, catalase-positive, oxidase-positive, and non-motile. Its colonies are 3-4 mm in diameter and can utilize glycerol, acetate, and pyruvate through respiratory metabolism, not fermentation. On LB medium, colonies grow singly, in pairs, or in clusters. It is light yellow in color, with a rounded, intact edge and a smooth, slightly convex surface. Individual bacteria measure 0.5–1.1 μm in diameter. Its optimal growth temperature is 30-32°C. It is not strictly aerobic and can grow in temperatures ranging from 10°C to 37°C.
[0003] Freeze-drying is by far the most common method for extending the shelf life of non-thermostable bacteria. The selection of appropriate freeze-drying protectants and the adjustment of storage method parameters are key factors in increasing the survival rate of bacteria during freeze-drying and subsequent storage.
[0004] There are many types of lyoprotectants, and their protective effects on different strains vary greatly. A study reported that the lyoprotectant survival rate of Lactobacillus plantarum M660 in 9.5% xylo-oligosaccharides, 9.5% whole milk powder, and 6% sodium glutamate was 88.39%, and the survival rate after 28 days of storage at -20°C was 82.5%. The lyoprotectant survival rate of Lactobacillus plantarum DMDL9010 in 12% skim milk powder, 7.95% bacteriological peptone, and 15.2% trehalose was 13.38%. Sun Juan's research showed that the optimal lyoprotectant formula for Lactobacillus plantarum 158 was 7% trehalose, 7% sucrose, 5% sodium glutamate, and 10% skim milk, with a lyoprotectant survival rate of 75.6%. The optimal lyoprotectant for Pediococcus acidilactici PP was 7% trehalose, 7% sucrose, 3% sodium glutamate, and 10% skim milk, with a lyoprotectant survival rate of 95.8%. Zeng Xiaoqun et al. found in their study on freeze-dried protective agents for Lactobacillus casei that, as sugar protective agents, the freeze-dried survival rate of Lactobacillus casei in 10% trehalose protective agent was 75%, while the freeze-dried survival rate in 10% lactose was only 15%; in the composite protective agent of trehalose, L-cysteine, sorbitol and sodium acetate, the freeze-dried protection rate of Lactobacillus casei in 10% trehalose, 2% L-cysteine, 2% sorbitol and 0.2% sodium acetate was 38%, and the protective effect after compounding was low. The protective effects of some of these components alone were significant (the freeze-dried protective rates for Lactobacillus casei were: 10% trehalose had a 75% protection rate, 2% sorbitol had a 67% protection rate, and 0.2% sodium acetate had a 57% protection rate). Furthermore, the freeze-dried protective rate for 8% trehalose, 2% L-cysteine, 2% sorbitol, and 0.25% sodium acetate was 25%, while the freeze-dried protective rate for 12% trehalose, 2% L-cysteine, 2% sorbitol, and 0.25% sodium acetate increased to 87%. Existing literature reports have demonstrated significant differences in freeze-dried protective agents for different strains. Even for the same strain, slight changes in the components or ratios of the protectant can lead to significant changes in the protective effect.
[0005] However, there are no reports on the preservation technology for Paracoccus variabilis. Conventional liquid storage methods have a short shelf life and rapid decline in strain activity. Furthermore, Paracoccus variabilis has poor temperature tolerance and is completely inactivated by spray drying at 60°C. To address these issues, the applicant has provided a freeze-drying protection method for Paracoccus variabilis. This method can improve the survival rate of Paracoccus variabilis during vacuum freeze-drying and extend the shelf life of freeze-dried samples at 4°C and room temperature. Summary of the Invention
[0006] The purpose of the present invention is to provide a freeze-drying protection method for Paracoccus mutans, which can achieve long-term storage and preservation of the activity of Paracoccus mutans at room temperature or 4°C, and provide theoretical and technical support for the promotion and application of Paracoccus mutans.
[0007] Another object of the present invention is to provide application of the above method in the preservation of Paracoccus mutans.
[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0009] A freeze-drying protection method for Paracoccus mutans comprises the following steps:
[0010] 1) Mixing Paracoccus mutans with a Paracoccus mutans vacuum freeze-drying protective agent, wherein the Paracoccus mutans is a microbial preparation containing live Paracoccus mutans in any dosage form, wherein the formula of the Paracoccus mutans vacuum freeze-drying protective agent is 10-12% skim milk powder + 5-10% trehalose + 2-4% glycerol, or 20% skim milk powder + 20% trehalose + 8% glycerol, and the solvent is water; after mixing, the effective bacterial concentration of Paracoccus mutans is: 10 9 ~10 11 CFU / ml;
[0011] 2) Gradient pre-cooling: The gradient pre-cooling temperature range is -5 to -55°C, and the total pre-cooling time is 3-6 hours;
[0012] 3) Gradient vacuum freeze drying: the gradient temperature span is -45 to 5°C, the total time is 16 to 24 hours, and the vacuum degree is 0.1 to 0.3 mbar;
[0013] 4). Vacuum desorption drying: drying temperature is 5-25℃, time is 8-17h, vacuum degree is 0.1~0.3mbar.
[0014] The method described above, preferably, in step 1), the vacuum freeze-drying protective agent for Paracoccus mutans is: 12% skim milk powder + 10% trehalose + 4% glycerol;
[0015] In the above method, preferably, in step 2), the parameters of the gradient precooling are preferably: the first gradient precooling temperature is -8°C, the time is 60 min, the second gradient precooling temperature is -20°C, the time is 60 min, and the third gradient precooling temperature is -50°C, the time is 180 min;
[0016] In the above method, preferably, in step 3), the parameters of the gradient vacuum freeze-drying are: the first stage is -30°C, 3 to 4 hours, preferably 3 hours; the second stage is -20°C, 8 to 15 hours, preferably 12 hours; the third stage is -10°C, 3 to 6 hours, preferably 5 hours;
[0017] In the above method, preferably, in step 4), the vacuum drying temperature is 25° C., the time is 10 h, and the vacuum degree is 0.1 mbar.
[0018] In the above method, after vacuum desorption and drying, the product continues to operate the vacuum pump to complete the vacuum sealing of the freeze-dried bacterial powder in the freeze-drying chamber, then the vacuum pump is turned off and the drying chamber is opened. The product seal is covered with an aluminum cap and the product is stored at 4-8°C. In the above method, the storage temperature of the product is preferably 4°C.
[0019] The protection method of the present invention also includes: application of the above method in the preservation of Paracoccus mutans.
[0020] Compared with the prior art, the present invention has the following advantages:
[0021] The present invention provides for the first time a freeze-drying protection method for Paracoccus mutans. The Paracoccus mutans preserved by this method can provide a good protection effect during long-term storage. After storage at 4°C for 9 months, the survival rate of Paracoccus mutans reaches 62.8%. After storage at room temperature of 15°C to 30°C for 30 days, the survival rate of Paracoccus mutans is 64.73%.
[0022] The Paracoccus variabilis lyoprotectant and lyophilization method provided by the present invention are simple and easy to operate, highly practical, and have high preservation efficiency. The pre-cooling followed by freezing and gradient lyophilization methods adopted can effectively reduce freezing damage and drying stress loss, significantly reduce the loss of bacterial viability during freezing, lyophilization and storage, and show good effects on the long-term lyophilization preservation of Paracoccus variabilis. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with the examples. It should be understood that the examples described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The Paracoccus versutus used in the examples of the present invention is derived from Wuhan Keyuan Biological Development Co., Ltd., and the freeze-drying protection method of the present invention is also applicable to other Paracoccus versutus.
[0024] Example 1:
[0025] Preparation of Paracoccus versutus bacterial suspension
[0026] The Paracoccus mutans strain stored at -80°C was revived, streaked onto NA agar plates, and incubated at 30-32°C for 36-48 hours. After a single colony grew, a single colony was picked and inoculated into NB liquid medium. The culture was shaken at 30-32°C and 120 rpm for 18-24 hours. The liquid seed solution was inoculated into a 200 L fermentor at a 1% (v / v) inoculum. The fermentation medium formula was 20 g / L sucrose, 10 g / L peptone, 10 g / L yeast extract, 2 g / L sodium nitrate, 0.1 g / L magnesium sulfate, and 10 g / L calcium carbonate. The pH was 7.2-7.5, and the liquid filling volume was 60% of the tank volume. After inoculation, sterile air was introduced at an initial ventilation ratio of 0.5 vvm. The tank pressure was 0.05 MPa, the temperature was controlled at 32°C, and fermentation was initiated at the lowest agitation speed. According to the change in DO, the ventilation volume and stirring speed are increased (increasing ventilation is preferred) to maintain DO not less than 20%; in the later stage, when DO rises over 50%, the speed or ventilation is reduced (reducing the speed is preferred) to maintain 20%-50%. After 18-24 hours of fermentation, samples are taken for microscopic examination to observe the proliferation of bacterial counts. If there is no obvious increase, the fermentation broth can be collected in a tank, or the fermentation broth can be centrifuged and concentrated to collect the centrifuged concentrated fermentation broth. The fermentation broth or the centrifuged concentrated fermentation broth is the suspension of Paracoccus mutans, which is used in the following examples.
[0027] Example 2:
[0028] Shelf life of Paracoccus mutans in liquid
[0029] The fermentation broth of Paracoccus mutans prepared in Example 1 was stored at 4°C and room temperature of 15-30°C, respectively. Samples were taken on the 0th, 30th, 60th, and 90th days of storage for sample dilution and plate counting to determine the viable bacterial content of Paracoccus mutans under different storage conditions. Based on the viable bacterial count on day 0, the survival rate of Paracoccus mutans at different storage time points was calculated. The results are shown in Table 1. On day 0, the viable bacterial content was 1.48×10 10 CFU / ml, stored at 4℃ for 30 days, the survival rate was 89.9%, the survival rate was 13.34% at 60 days, and the survival rate dropped to 3.04% at 90 days; under room temperature conditions of 15-30℃, the survival rate dropped to 3.88% at 30 days, the survival rate dropped to 99.58% at 60 days, and the survival rate dropped to 100% at 90 days.
[0030] Table 1 Preservative activity of Paracoccus mutans liquid sample
[0031]
[0032] Example 3:
[0033] Selection of protective agent for vacuum freeze-drying of Paracoccus mutans:
[0034] The components and proportions of the different protective agents to be screened are shown in Table 2, where the percentages are by mass to volume ratios.
[0035] The preparation method of the freeze-drying protective agent comprises the following steps:
[0036] (1) dissolving skimmed milk powder, trehalose, sucrose, glycerol, maltodextrin, sodium glutamate, porous starch, etc. in sterile distilled water according to different protective agent formula ratios and sterilizing to obtain a sterile vacuum freeze-dried protective agent solution;
[0037] (2) For a protective agent formulation containing vitamin C, the other components are first sterilized at high temperature, and the vitamin C is sterilized by 0.22 μm filtration. After the sterilized components are cooled, they are mixed with the filtered and sterilized vitamin C solution in proportion to obtain a vacuum freeze-dried protective agent solution containing vitamin C.
[0038] In this embodiment, the sterilization temperature is 110° C. and the time is 15 minutes.
[0039] Table 2 Ratios of different protective agents for Paracoccus mutans
[0040]
[0041]
[0042] Example 4:
[0043] Freeze vacuum drying method
[0044] The Paracoccus mutans liquid prepared in Example 1 was mixed with different vacuum freeze-drying protective agents prepared in Example 3 and then dispensed into vials, 2 mL / vial.
[0045] After mixing, a gradient precooling method is first adopted, with the first gradient precooling temperature being -8°C and the time being 60 min, the second gradient precooling temperature being -20°C and the time being 60 min, and the third gradient precooling temperature being -50°C and the time being 180 min.
[0046] Then vacuum freeze drying was used: the first stage was -30°C, 3 hours; the second stage was -20°C, 12 hours; the third stage was -10°C, 5 hours; the vacuum degree throughout the process was 0.1 mbar.
[0047] The product was dried under vacuum at 25°C for 10 h at a vacuum degree of 0.1 mbar.
[0048] After lyophilization is complete, continue running the vacuum pump and slowly tighten the latex cap on the vial using an external knob to complete the vacuum seal of the lyophilized bacterial powder within the lyophilization chamber. Then, turn off the vacuum pump, open the drying chamber, remove the sealed vial, and seal the latex cap with an aluminum cap. This will yield a lyophilized sample of Paracoccus mutans, which will be used in the following examples.
[0049] Example 5:
[0050] Freeze-drying protection efficiency of different lyoprotectants on Paracoccus mutans
[0051] The lyophilized sample of Paracoccus mutans prepared in Example 4 was reconstituted with sterile water to 2 ml, and the viable bacteria content was counted. The lyophilization protection efficiency of the lyoprotectant for Paracoccus mutans was calculated by comparing the viable bacteria content with that before lyophilization.
[0052] The formula for calculating the freeze-drying protection rate is: live bacteria concentration after freeze-drying / live bacteria concentration before freeze-drying * 100%
[0053] The results are shown in Table 3. In 20% skim milk powder (protectant 10), the freeze-drying protection rate of Paracoccus variabilis was the highest, at 94.29%; in the freeze-drying protectant of 12% skim milk powder + 10% trehalose + 4% glycerol (protectant 6), the freeze-drying protection rate of Paracoccus variabilis was 80.82%; the highest protection rate of commercial freeze-drying protectants did not exceed 53.34% (protectant 11).
[0054] While optimizing the freeze-drying protectant for Paracoccus mutans, Streptococcus suis was used to explore the protective efficiency of different protectants against different species of microorganisms. The steps were the same as above. The results showed that the freeze-drying protection rate of a commercially available heat-resistant bacterial protectant (protectant 13) for Streptococcus suis was 90.5%, but the freeze-drying protection rate for the Paracoccus mutans of the present invention was only 35.56%.
[0055] Table 3 Freeze-drying protection efficiency of different protective agents
[0056]
[0057] Example 6:
[0058] Evaluation of storage stability of different lyoprotectants
[0059] The freeze-dried samples prepared in Example 4 were stored at 4° C. and room temperature (15-30° C.), respectively, and the survival of Paracoccus mutans at different storage time points and temperatures was monitored. The viable bacterial concentration was calculated in the same manner as in Example 5.
[0060] The formula for calculating the survival rate is: viable bacteria concentration after freeze-drying / initial viable bacteria concentration after freeze-drying*100%.
[0061] That is, the ratio of the viable bacterial concentration at different storage times and storage temperatures in Tables 4 and 5 to the viable bacterial concentration after lyophilization of each protective agent in Table 3.
[0062] The results, shown in Tables 4 and 5, show that after storage at 4°C in a lyoprotectant of 12% skim milk powder, 10% trehalose, and 4% glycerol for one, six, and nine months, the survival rates reached 92.07%, 74.13%, and 62.80%, respectively, compared to those immediately following lyophilization. After storage at room temperature of 15°C-30°C for 30 days, the survival rate reached 64.73%, demonstrating good heat stability.
[0063] When evaluating the storage stability of different protective agents on Paracoccus mutans, the protective effect of water-in-water colloid emulsions with good storage stability for Lactobacillus plantarum (Construction of water-in-water (W_W) Pickering emulsion and its encapsulation of lactic acid bacteria, Liu Shilin, 2022) on Paracoccus mutans was compared. The results showed that the colloid that can improve the stress resistance and storage stability of Lactobacillus plantarum did not enhance the storage stability of Paracoccus mutans. The results are shown in Table 6.
[0064] Table 4 Comparison of survival rates of different lyophilized protective agents stored at 4°C
[0065]
[0066] Table 5 Comparison of survival rates of different lyophilized protective agents stored at room temperature 15-30°C
[0067]
[0068]
[0069] Table 6 Comparison of storage stability of water-in-water colloidal emulsions against Lactobacillus plantarum and Paracoccus mutans (30°C)
[0070]
[0071] Example 7:
[0072] Protective effect of different ratios of protective agent 6 on freeze-dried Paracoccus mutans
[0073] Through the evaluation of the effects of different protective agents on the freeze-drying protection rate and storage stability in Examples 5 and 6, it can be seen that protective agent 6 has a freeze-drying protection rate of 80.82%, and still has a survival rate as high as 62.80% after storage at 4°C for 9 months; protective agent 10 has a freeze-drying protection rate of 94.29%, but after storage at 4°C for 9 months, the survival rate drops to 26%. Therefore, protective agent 6 was selected for further testing, and the proportions of each component are shown in Table 7.
[0074] Protective agents were prepared using the ratios and components of the protective agents in Table 7. Freeze-dried samples of Paracoccus mutans were prepared in the same manner as in Example 4. The freeze-dried protection rates of different freeze-dried protective agents on Paracoccus mutans were determined in the same manner as in Example 5.
[0075] The results are shown in Table 8. Except for the ratio of protective agent 16 and protective agent 17, the freeze-drying protection effect of the protective agent composed of skimmed milk powder, trehalose and glycerol is better than that of other component protective agents.
[0076] Table 7 Ratios and components of different protective agents
[0077]
[0078] Table 8 Freeze-drying protection rate of different protective agents
[0079]
Claims
1. A freeze-drying protection method for Paracoccus mutans, comprising the following steps: 1) Mix Paracoccus mutans with a Paracoccus mutans vacuum freeze-drying protective agent. The Paracoccus mutans is a microbial preparation containing live Paracoccus mutans in any dosage form. The Paracoccus mutans vacuum freeze-drying protective agent has a formula of 10-12% skim milk powder + 5-10% trehalose + 2-4% glycerol, or 20% skim milk powder + 20% trehalose + 8% glycerol, with water as the solvent. After mixing, the effective bacterial concentration of Paracoccus mutans is: 10 9 ~10 11 CFU / ml; 2) Gradient pre-cooling: The gradient pre-cooling temperature range is -5 to -55°C, and the total pre-cooling time is 3 to 6 hours; 3) Gradient vacuum freeze drying: the gradient temperature span is -45 to 5°C, the total time is 16 to 24 hours, and the vacuum degree is 0.1 to 0.3 mbar; 4). Vacuum desorption drying: drying temperature is 5-25℃, time is 8-17h, vacuum degree is 0.1~0.3mbar.
2. The method according to claim 1, wherein: In step 1), the protective agent for vacuum freeze-drying of Paracoccus mutans is: 12% skim milk powder + 10% trehalose + 4% glycerol.
3. The method according to claim 1, wherein: In step 2), the parameters of the gradient precooling are: the first gradient precooling temperature is -8°C, the time is 60 minutes, the second gradient precooling temperature is -20°C, the time is 60 minutes, and the third gradient precooling temperature is -50°C, the time is 180 minutes.
4. The method according to claim 1, wherein: In step 3), the parameters of gradient vacuum freeze drying are: first stage at -30°C, 3 to 4 h; second stage at -20°C, 8 to 15 h, preferably 12 h; third stage at -10°C, 3 to 6 h, preferably 5 h.
5. The method according to claim 4, characterized in that: In step 3), the first stage is -30°C for 3 hours; the second stage is -20°C for 12 hours; and the third stage is -10°C for 5 hours.
6. The method according to claim 1, wherein: In step 4), the vacuum drying temperature is 25° C., the time is 10 h, and the vacuum degree is 0.1 mbar.
7. The method according to claim 1, wherein: After vacuum desorption and drying, the product continues to operate the vacuum pump to complete the vacuum sealing of the freeze-dried bacterial powder in the freeze-drying chamber, then turns off the vacuum pump and opens the drying chamber, covers the product seal with an aluminum cap, and stores the product at 4-8°C.
8. The method according to claim 7, wherein: Store the product at 4°C.
9. Use of the method according to claim 1 in the preservation of Paracoccus mutans.