Method for preparing freeze-dried bacterial powder and freeze-dried bacterial powder
Through the activation of rejuvenating bacterial strains, solid culture and skim milk powder embedding technology, the problems of low gene mutation and survival rates are solved, the survival rate and stability of lyophilized bacteria powder are improved, and better strain preservation effect is achieved.
Patent Information
- Application Number
- CN202411715619.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-01-10
AI Technical Summary
In the prior art, modified strains after gene editing are prone to problems such as gene mutation, plasmid loss, and strain degeneration, resulting in insufficient survival rate of lyophilized bacterial powder and it is difficult to effectively maintain the original traits and stability of the strain.
The activated rejuvenating bacteria were used to carry out solid culture and purification and enrichment, and the skimmed milk powder was used as the protective liquid for embedding, and the lyophilized bacteria powder was prepared through freeze-drying technology to increase the bacterial density and reduce the impact of the nutrient solution on the bacteria.
It significantly improves the survival rate of lyophilized bacteria powder, maintains the traits and stability of the strain, and is suitable for long-term storage and transportation.
Smart Images
Figure BDA0005156819510000081
Abstract
Description
Technical Field
[0001] The invention belongs to the field of freeze-dried bacterial powder preparation, and particularly relates to a method for preparing freeze-dried bacterial powder and the freeze-dried bacterial powder. Background Art
[0002] Existing organic acid strains are basically improved strains through gene editing. The genetically modified strains are prone to gene mutation, plasmid loss, strain degeneration and other phenomena. Therefore, in order to better maintain the original strain performance, freeze-dried powder needs to be produced and preserved. The dry powder is stored at -80°C and in liquid nitrogen, which can better maintain the original properties and stability of the strain.
[0003] At present, the bacterial strains are mostly preserved by drying the bacterial liquid into dry powder. However, the survival rate of this method of preserving the strains is insufficient and still needs to be improved. Therefore, a method for preparing freeze-dried bacterial powder is proposed. Summary of the invention
[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing freeze-dried bacterial powder and freeze-dried bacterial powder, which solves the problems in the prior art.
[0005] The purpose of the present invention can be achieved through the following technical solutions:
[0006] A method for preparing freeze-dried bacterial powder comprises the following steps:
[0007] S1, activate the desired strains for rejuvenation and prepare a suspension;
[0008] S2, purifying and culturing the activated suspension on a solid medium, and selecting a single colony for subsequent cultivation;
[0009] S3, picking up the single colony on the solid culture medium to the slant for solid purification and enrichment culture to form colony mud;
[0010] S4, scrape all the colony mud on the slope into the skimmed milk powder protective solution, mix thoroughly, and embed in the protective solution;
[0011] S5, pre-freezing the mixed solution of the colony mud and the skimmed milk powder protective solution in a refrigerator, then freezing it in a refrigerator, and then freeze-drying it in a freeze dryer to obtain a freeze-dried sample;
[0012] S6, after drying, the freeze-dried sample is ground to obtain a powdery form, namely, freeze-dried bacterial powder.
[0013] Further, in S1, the step of preparing the suspension is:
[0014] Place the bacteria in a sterile culture dish and add MRS medium;
[0015] The culture medium containing the bacterial strain was placed in a constant temperature oscillator and cultured under shaking at 37° C. for 24 hours to obtain a suspension.
[0016] Furthermore, the bacterial species is Lactobacillus acidophilus.
[0017] Further, in S2, the purification and culturing steps are:
[0018] The activated bacterial suspension is evenly applied on the surface of the solid culture medium;
[0019] The culture dish was placed in a constant temperature incubator at 37°C for 48 hours.
[0020] Furthermore, the solid culture medium is: MRS agar culture medium.
[0021] Further, in S3, the preparation process of the colony mud is:
[0022] Pick a single colony from the solid culture medium and transfer it to a slant test tube containing MRS agar medium;
[0023] The slant test tube was placed in a 37°C constant temperature incubator for 48 hours and the colony morphology was observed;
[0024] After purification, a stable single strain is obtained and cultured to form colony mud.
[0025] Further, in S4, the step of embedding with protective liquid is:
[0026] Scrape all the colony mud in the inclined test tube into the prepared skim milk powder protective solution;
[0027] Use a magnetic stirrer to stir the colony mud and the skimmed milk powder protective solution to form an embedding effect.
[0028] Further, in S5, the preparation process of the freeze-dried sample is:
[0029] The mixed solution of skimmed milk powder protection solution and colony mud was placed in a -20℃ refrigerator for pre-freezing for 1.5h;
[0030] After prefreezing, the samples were transferred to a -80°C freezer for another 10 hours;
[0031] The sample was taken out from the -80°C refrigerator and placed in a freeze dryer for freeze drying to obtain a freeze-dried sample; the temperature of the freeze dryer was -50°C, the pressure was 50Pa, and the drying time was 24h.
[0032] Furthermore, in S6, a planetary grinder is used for grinding, the rotation speed of the grinder is 3000 r / min, the grinding time is 20 min, and after grinding, a 100-mesh sieve is used for screening.
[0033] A freeze-dried bacterial powder is prepared using the above method for preparing freeze-dried bacterial powder.
[0034] Beneficial effects of the present invention:
[0035] 1. When preparing freeze-dried bacterial powder, the present invention replaces the traditional centrifuged bacterial liquid with a large amount of pure bacterial mud, which greatly increases the density of the bacterial population and significantly improves the survival rate.
[0036] 2. The present invention uses skimmed milk powder as a suspension of bacterial mud, which can better maintain the properties and stability of the strain and reduce the influence of substances in the nutrient solution on the bacteria. DETAILED DESCRIPTION
[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0038] Example
[0039] A method for preparing freeze-dried bacterial powder comprises the following steps:
[0040] S1, activate the desired bacteria (Lactobacillus acidophilus) for rejuvenation and prepare a suspension;
[0041] The specific steps are:
[0042] S11, taking an appropriate amount of Lactobacillus acidophilus seeds, placing them in a sterile culture dish, and adding an appropriate amount of MRS culture medium to ensure that the strains are evenly distributed;
[0043] S12, placing the culture medium containing the bacterial strain in a constant temperature oscillator, and culturing at 37° C. for 24 hours with shaking until the bacterial cells reach a logarithmic growth phase, thereby obtaining a suspension;
[0044] S13, taking the suspension and observing it under a microscope to ensure the activity of the strain.
[0045] S2, purifying and culturing the activated suspension on solid culture medium;
[0046] The specific steps are:
[0047] S21, evenly coating the activated bacterial suspension on the surface of MRS agar medium (solid medium);
[0048] S22, place the culture dish in a constant temperature incubator at 37°C for 48 hours and observe the growth of the colonies;
[0049] S23, pick a representative single colony for subsequent culture;
[0050] The selection is based on the morphological characteristics of the colonies, such as their size, shape, color, edge, smoothness, etc., as well as the uniformity of their growth; the selected colonies should show a typical and stable morphology, indicating that they are pure and uncontaminated single species.
[0051] S3, picking up the single colony on the solid culture medium to the slant for solid purification and enrichment culture to form colony mud;
[0052] The specific steps are:
[0053] S31, pick a single colony from the solid culture medium and transfer it to a slant test tube containing MRS agar medium;
[0054] S32, place the slant test tube in a 37°C constant temperature incubator for 48 hours and observe the colony morphology;
[0055] S33, after several rounds of purification, a stable single strain was obtained and cultured to form colony slurry.
[0056] S4, adding 5 g of skim milk powder to 100 ml of the protective solution to prepare a skim milk powder protective solution, and then scraping all the colony mud on the slope into the skim milk powder protective solution for thorough mixing to produce an embedding effect;
[0057] The specific steps are:
[0058] S41, dissolve 5g skim milk powder in 100ml sterile water, making sure it is completely dissolved, to prepare skim milk powder protective solution:
[0059] S42, scrape all the colony mud in the inclined test tube into the prepared skim milk powder protective solution;
[0060] S43, using a magnetic stirrer to stir at a low speed to fully and evenly mix the colony mud with the protective liquid to ensure a good embedding effect; the stirring speed is 100 r / min and the stirring time is 15 min.
[0061] S5, first pre-freeze in a refrigerator, then freeze in a refrigerator, and then freeze-dry in a freeze dryer to obtain a freeze-dried sample;
[0062] The specific steps are:
[0063] S51, placing the mixed solution of skimmed milk powder protective solution and colony mud into a -20°C refrigerator for pre-freezing for 1.5 hours;
[0064] S52, after prefreezing, transfer the sample to a -80°C freezer and place it for another 10 hours; further lower the temperature to allow the cells to better form a solid structure;
[0065] S53, taking the sample out of the -80°C freezer and immediately placing it in a freeze dryer for freeze drying to obtain a freeze-dried sample;
[0066] The freeze dryer temperature was set at -50°C, the pressure was 50Pa, and the drying time was 24h; and during the freeze drying process, the temperature and pressure of the equipment were monitored to ensure the stability of the freeze drying process.
[0067] S6, after drying, the freeze-dried sample is ground into a fine powder form.
[0068] The grinding was carried out using a planetary grinder with a speed of 3000 r / min and a grinding time of 20 min. After grinding, a 100-mesh sieve was used for sieving to ensure the fineness of the bacterial powder particles.
[0069] In addition, it should be noted that when the freeze-dried bacterial powder is stored and packaged into sterile bags, a vacuum sealer is used to seal the container to ensure that it is sealed under a vacuum state to prevent the bacterial powder from contacting the air and ensure its long-term stability.
[0070] Comparative Example
[0071] The method for preparing freeze-dried bacterial powder using centrifuged bacterial liquid (traditional centrifuged bacterial liquid dry powder method) comprises the following steps:
[0072] Step 1, activating the desired bacteria (Lactobacillus acidophilus) for rejuvenation and preparing a suspension;
[0073] The specific steps of step 1 are consistent with the steps of S1 in the embodiment.
[0074] Step 2, the activated suspension is separated from the bacteria by a centrifuge, the centrifuge speed is 3000r / min, and the centrifugation time is 10min;
[0075] Step 3: Use a vacuum concentrator to concentrate the suspension after centrifugation to remove excess solvent (such as water) and increase the concentration of the bacterial solution.
[0076] The parameters of the vacuum concentrator are: reduce the pressure to 30 mbar to reduce the heat during solvent evaporation; during the concentration process, the temperature should be maintained at 25°C to avoid thermal damage to the cells caused by excessive temperature; the concentration time is 2 hours.
[0077] Step 4, adding skimmed milk powder protective solution to the concentrated bacterial solution and stirring thoroughly;
[0078] The skimmed milk powder protective liquid is configured in the same manner as the skimmed milk powder protective liquid in Example 1.
[0079] Step 5: pre-freeze and freeze-dry the mixed solution of the bacterial solution and the skimmed milk powder protective solution.
[0080] The step parameters of pre-freezing and freeze-drying are consistent with the step parameters in S5 of the embodiment, and the specific steps are:
[0081] 1) Place the mixed solution of bacterial liquid and skimmed milk powder protective solution in a -20°C refrigerator for pre-freezing for 1.5 hours;
[0082] 2) After prefreezing, transfer the sample to a -80°C freezer and place it for another 10 hours; further lower the temperature to allow the cells to form a solid structure better;
[0083] 3) Take the sample out of the -80°C freezer and immediately put it into a freeze dryer for freeze drying to obtain a freeze-dried sample;
[0084] The freeze dryer temperature was set at -50°C, the pressure was 50Pa, and the drying time was 24h; and during the freeze drying process, the temperature and pressure of the equipment were monitored to ensure the stability of the freeze drying process.
[0085] Step 6: After drying, the freeze-dried sample is ground into a fine powder.
[0086] The grinding parameters are consistent with those of S6 in Example 1.
[0087] Comparative analysis shows that in the comparative example, the traditional centrifuged bacterial liquid dry powder method omits the step of "colony purification (S2-S3)" compared to the freeze-dried bacterial powder method in Example 1.
[0088] Experimental testing;
[0089] The survival rate experiment was conducted on the freeze-dried bacterial powder prepared in the embodiment and the comparative example, and the experimental steps were as follows:
[0090] 1. Resuscitation experiment
[0091] Take appropriate amounts of freeze-dried bacterial powder samples (prepared in the examples and comparative examples) and add them to MRS liquid culture medium respectively; place them in a 37° C. constant temperature incubator, let them stand for 1 hour, and observe the recovery.
[0092] 2. Colony count:
[0093] Samples were taken from the resuscitation solution and spread onto MRS agar medium for plate counting. Each set of plates was cultured for 48 hours and the number of visible colonies was counted.
[0094] 3. Calculation of survival rate;
[0095] The calculation formula is:
[0096] Survival rate (%) = (number of colonies after recovery / total amount of initial bacterial powder) × 100%
[0097] The experimental test results are shown in Table 1 below:
[0098] Table 1 Survival rate test results of freeze-dried bacterial powder prepared in Examples and Comparative Examples
[0099]
[0100] According to the experimental data shown in Table 1, it can be seen that the survival rate of the freeze-dried bacterial powder prepared by the method of the present invention is significantly higher than that of the traditional bacterial liquid centrifugal powder method. In this experiment, the survival rate of the freeze-dried bacterial powder prepared by the traditional method was only 18%, while the survival rate of the freeze-dried bacterial powder prepared by the method of the present invention was 29%, and the survival rate was increased by more than 50%. This result shows that the freeze-dried bacterial powder preparation method of the present invention has a higher strain survival rate, can better maintain the activity of the strain, and is suitable for long-term storage and transportation.
[0101] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0102] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.
Claims
1. A method for preparing freeze-dried bacterial powder, characterized in that: The following steps are involved: S1, activate the desired strains for rejuvenation and prepare a suspension; S2, purifying the suspension on solid culture medium and selecting single colonies for subsequent culture; S3, picking up the single colony on the solid culture medium to the slant for solid purification and enrichment culture to form colony mud; S4, scrape all the colony mud on the slope into the skimmed milk powder protective solution, mix thoroughly, and embed in the protective solution; S5, pre-freezing the mixed solution of the colony mud and the skimmed milk powder protective solution in a refrigerator, then freezing it in a refrigerator, and then freeze-drying it in a freeze dryer to obtain a freeze-dried sample; S6, after drying, the freeze-dried sample is ground to obtain a powdery form, namely, freeze-dried bacterial powder.
2. The method for preparing freeze-dried bacterial powder according to claim 1, characterized in that: In S1, the steps of preparing the suspension are: Place the bacteria in a sterile culture dish and add MRS medium; The culture medium containing the bacterial strain was placed in a constant temperature oscillator and cultured under shaking at 37° C. for 24 hours to obtain a suspension.
3. A method for preparing freeze-dried bacterial powder according to claim 1 or 2, characterized in that: In S1, the bacterial species is Lactobacillus acidophilus.
4. The method for preparing freeze-dried bacterial powder according to claim 1, characterized in that: In S2, the steps of purification and cultivation are: The activated bacterial suspension is evenly applied on the surface of the solid culture medium; The culture dish was placed in a constant temperature incubator at 37°C for 48 hours.
5. The method for preparing freeze-dried bacterial powder according to claim 1, characterized in that: The solid culture medium is: MRS agar culture medium.
6. The method for preparing freeze-dried bacterial powder according to claim 1, characterized in that: In S3, the preparation process of the colony mud is as follows: Pick a single colony from the solid culture medium and transfer it to a slant test tube containing MRS agar medium; The slant test tube was placed in a 37°C constant temperature incubator for 48 hours and the colony morphology was observed; After purification, a stable single strain is obtained and cultured to form colony mud.
7. The method for preparing freeze-dried bacterial powder according to claim 1, characterized in that: In S4, the step of embedding with protective solution is: Scrape all the colony mud in the inclined test tube into the prepared skim milk powder protective solution; Use a magnetic stirrer to stir the colony mud and the skimmed milk powder protective solution to form an embedding effect.
8. The method for preparing freeze-dried bacterial powder according to claim 1, characterized in that: In S5, the preparation process of the freeze-dried sample is: The mixed solution of skimmed milk powder protection solution and colony mud was placed in a -20℃ refrigerator for pre-freezing for 1.5h; After prefreezing, the samples were transferred to a -80°C freezer for another 10 hours; The sample was taken out from the -80°C refrigerator and placed in a freeze dryer for freeze drying to obtain a freeze-dried sample; the temperature of the freeze dryer was -50°C, the pressure was 50Pa, and the drying time was 24h.
9. The method for preparing freeze-dried bacterial powder according to claim 1, characterized in that: In S6, a planetary grinder is used for grinding, the speed of the grinder is 3000 r / min, the grinding time is 20 min, and after grinding, a 100-mesh screen is used for screening.
10. A freeze-dried bacterial powder, characterized in that: The freeze-dried bacterial powder is prepared using the method for preparing freeze-dried bacterial powder according to any one of claims 1 to 9.