Freeze-drying protective agent and freeze-drying method for bifidobacterium breve
By using lyophilized protective agents composed of skim milk powder, trehalose, sodium ascorbate, etc. and an optimized freeze-drying method, the problem of low survival rate of Bifidobacter brevis during lyophilization is solved, and efficient bacterial protection and stability improvement is achieved.
Patent Information
- Application Number
- CN202510907832.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-08-22
AI Technical Summary
Existing lyophilized protective agents are difficult to effectively improve the survival rate of Bifidobacter brevis, especially during freeze-drying, which leads to poor bacterial stability.
Skimmed milk powder, trehalose, white sugar, sodium ascorbate, anhydrous glucose, glycerin and sodium glutamate are used as lyophilized protection agents, and specific freeze-drying methods, including bacterial sludge emulsification, prefreezing and 12-section freeze-drying processes, optimize the prefreezing temperature and protective agent ratio to form an efficient lyophilized protection system.
The number of live bacteria in lyophilized powder of Bifidobacterium brevis was increased to 9.0×1011CFU/g and the yield of bacterial sludge was increased to 1.3%, significantly improving the survival rate and stability of bacteria.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microorganisms, and in particular to a freeze-drying protectant and a freeze-drying method for Bifidobacterium breve. Background Art
[0002] Bifidobacterium breve ( Bifidobacterium breve Bifidobacterium breve is an important probiotic widely used in food, health supplements, and medicine. It regulates intestinal flora, enhances immunity, and alleviates inflammation. However, Bifidobacterium breve is sensitive to its environment and is easily inactivated during processing, storage, and transportation by factors such as temperature, oxygen, and humidity. This can lead to a significant decrease in viable bacteria count and compromise its efficacy.
[0003] Freeze-drying is currently one of the primary methods for preserving probiotics. Low-temperature dehydration causes the bacteria to enter a dormant state, thereby extending their shelf life. However, the freeze-drying process itself can cause damage to the bacteria through low temperatures, dehydration, and oxidation, resulting in a low survival rate. Therefore, improving the survival rate of Bifidobacterium breve, particularly its stability during the freeze-drying (lyophilization) process, has become a pressing technical challenge for the industry.
[0004] Generally, during freeze-drying, lyoprotectants are added to protect the probiotic bacteria. However, existing lyoprotectant technology is difficult to meet the efficient preservation requirements of Bifidobacterium breve. Summary of the Invention
[0005] In order to solve the above problems, the present invention provides a freeze-drying protectant and freeze-drying method for Bifidobacterium breve.
[0006] The specific scheme is as follows: a freeze-dried protective agent for Bifidobacterium breve, comprising the following components: skim milk powder, trehalose, white sugar, sodium ascorbate, anhydrous glucose, glycerol, sodium glutamate, and maltodextrin.
[0007] The above-mentioned Bifidobacterium breve freeze-drying protective agent includes the following components: trehalose, glycerol, sodium glutamate, and sodium ascorbate.
[0008] The above-mentioned Bifidobacterium breve freeze-dried protective agent comprises the following components in weight concentrations: trehalose 10-18%, glycerol 1.0-2.0%, sodium glutamate 1.0-1.5%, and sodium ascorbate 0.1-0.3%.
[0009] The above-mentioned Bifidobacterium breve freeze-dried protective agent comprises the following components in the following weight concentrations: 15% trehalose, 1.5% glycerol, 1.2% sodium glutamate, and 0.2% sodium ascorbate.
[0010] The above-mentioned Bifidobacterium breve freeze-dried protective agent is Bifidobacterium breve ( Bifidobacterium breve)RRJK1316, accession number is GDMCC No.64764.
[0011] A freeze-drying method for Bifidobacterium breve, the process is as follows: 1) Emulsification of bacterial sludge: Add the aforementioned Bifidobacterium breve freeze-drying protective agent to the Bifidobacterium breve bacterial sludge, stir and mix thoroughly to form a homogeneous bacterial sludge emulsion; 2) Prefreezing: Dispense the mixed emulsion into freeze-drying trays at a rate of 1.1 L per tray. Place the trays into the freeze-dryer and prefreeze for 2 hours. Stop prefreezing when the temperature reaches -40°C. 3) Freeze drying: After pre-freezing, the temperature is gradually raised and freeze drying is performed in 12 sections, with each section increasing by 5°C until it reaches 30°C. The freeze drying time is 52.5 hours.
[0012] In the above-mentioned freeze-drying method for Bifidobacterium breve, in step 1), the Bifidobacterium breve bacterial slurry is obtained by activating, expanding, fermenting in the primary stage, and fermenting in the secondary stage.
[0013] In the above-mentioned freeze-drying method for Bifidobacterium breve, in step 3), the freeze-drying temperature and time of the 12 sections are shown in the following table;
[0014] The above-mentioned freeze-drying method of Bifidobacterium breve further includes the following steps: collecting the material after freeze-drying, setting the environmental conditions to a temperature of 18-25° C. and a humidity of 30-35%, crushing the material after collection, and sealing and packaging.
[0015] The freeze-dried powder of Bifidobacterium breve obtained by the freeze-drying method of Bifidobacterium breve is prepared.
[0016] Beneficial effects of the present invention: The viable bacterial count of the freeze-dried powder of Bifidobacterium breve prepared by the freeze-drying method of the present invention can reach 9.0×10 11 CFU / g.
[0017] The yield of freeze-dried bacterial mud of Bifidobacterium breve prepared by the freeze-drying method of the present invention can reach more than 1.3%. DETAILED DESCRIPTION
[0018] Example 1 Fermentation of Bifidobacterium breve 1. Bacteria fermentation 1) Strain Activation: Bifidobacterium breve RRJK1316 was inoculated into a basal culture medium at a 10% inoculum rate, sealed, and incubated in an incubator at a constant temperature of 37±0.5°C for 17±0.5 hours. The Bifidobacterium breve RRJK1316 was deposited in the Guangdong Provincial Microbiological Culture Collection Center with the accession number GDMCC No. 64764.
[0019] 2) Strain expansion: Inoculate the activated bacterial suspension into the basal culture medium at a 5% inoculum volume, fill it to 80%, seal it, and incubate it in a 37±0.5℃ incubator for 17±0.5 hours.
[0020] 3) Primary Fermentation: Inoculate the fermenter with the optimized medium at 80% volume using the bacterial suspension from the expanded culture at a 5% inoculum volume. Incubate at 37.0±0.2°C with agitation, zero aeration, and a stirring speed of 60 rpm. At the start of fermentation, automatically feed food-grade NaOH to maintain the pH of the culture at 5.50±0.05. Calibrate the electrodes to ensure that the pH displayed by the system differs by less than 0.05 from the measured value. Starting at 5 h into fermentation, monitor the OD value of the culture every 0.5 h. Stop fermentation if the difference between two consecutive OD values is less than 0.6.
[0021] 4) Secondary Fermentation: Inoculate the bacterial suspension from the primary fermentation into a fermenter containing optimized culture medium at a 6% inoculum volume. Incubate at 37°C with constant agitation at 100 rpm and zero aeration. Maintain a constant pH of 5.50 ± 0.05 by feeding food-grade NaOH solution at the start of fermentation. Calibrate the electrodes to ensure that the pH displayed by the system differs from the measured value by less than 0.05. Starting from the 7th hour of fermentation, monitor the OD value of the bacterial suspension every 0.5 h. Fermentation is terminated when the difference between two consecutive OD values is less than 0.6.
[0022] 5) Bacteria collection: After the secondary fermentation is completed, the temperature of the fermentation tank is lowered. When the temperature of the fermentation liquid in the tank is below 20°C, centrifugation is performed. The centrifugal equipment uses a tubular centrifuge with a speed of 13,000 rpm. After the feeding is completed, the centrifuge is idle for 8 minutes. After the centrifugation is completed, the bacterial sludge is collected.
[0023] Basal medium: 30 g / L anhydrous glucose, 10 g / L yeast extract powder, 15 g / L yeast peptone, 2 g / L citric acid, 5 g / L sodium acetate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.01 g / L manganese sulfate, 0.6 g / L Tween-80, 5 g / L soy peptone. Adjust pH to 7.20. Fill to 80%.
[0024] Optimized culture medium: 30 g / L anhydrous glucose, 15 g / L yeast extract powder, 15 g / L yeast peptone, 2 g / L citric acid, 5 g / L sodium acetate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.01 g / L manganese sulfate, 0.6 g / L Tween-80, 10 g / L soy peptone, pH adjusted to 7.20. Filling volume 80%.
[0025] Example 2 Freeze-drying method for Bifidobacterium breve cells
[0026] 1) Cold stress pretreatment: Place the bacterial sludge at 4°C for 5 h.
[0027] 2) Emulsification of the Sludge: Add lyoprotectant to the sludge at a ratio of 1:2.5 (kg:L) and stir thoroughly to form a homogeneous emulsion. Lyoprotectant formulation: 15% trehalose, 1.5% glycerol, 1.2% sodium glutamate, and 0.2% sodium ascorbate, by weight.
[0028] 3) Prefreezing: Dispense the mixed emulsion into freeze dryer trays at a rate of 1.1 L per tray. Place the tray into the freeze dryer and prefreeze for 2 hours. Stop prefreezing when the temperature reaches -40°C.
[0029] 4) Freeze drying: After pre-freezing, the temperature was gradually raised to 12 sections for freeze drying. The temperature of each section was raised by 5°C until it reached 30°C. The freeze drying time was 52.5 hours. The operating time and end temperature of each section are shown in Table 1. Table 1
[0030] 5) Freeze-dried material collection and processing: After freeze-drying, the material was collected under environmental conditions set at 18-25°C and 30-35% humidity. After collection, the material was intermittently crushed to 100 mesh using a water-cooled universal grinder and packaged in double-layer sealed ziplock bags and aluminum foil bags. The viable bacterial count of the bacterial powder was tested to be 4.5-9.00×10 11 CFU / g.
[0031] Example 3 Screening of Freeze-Drying Protectants for Bifidobacterium breve
[0032] The microbial cell freeze-drying method described in Example 2 was followed, and some different freeze-drying protectant formulations were listed in Table 2. The survival rate and stability results are shown in Table 3. The optimal freeze-drying protectant formulation was 15% trehalose, 1.5% glycerol, 1.2% sodium glutamate, and 0.2% sodium ascorbate.
[0033] Count viable bacteria before freeze-drying: dilute the bacterial slurry before freeze-drying in 10-fold series with sterile physiological saline, take 1000 μL of the bacterial solution of appropriate dilution for viable bacteria counting operation, repeat 3 times for each dilution, and culture anaerobically in MRS+L solid medium at 39±0.5℃ for 48-72h before counting.
[0034] Viable bacteria count after freeze-drying: Add a certain amount of sterile water to the freeze-dried sample to rehydrate it to the mass before freeze-drying, and then count the viable bacteria.
[0035]
[0036] Table 2 Screening scheme for protective agents (weight concentration)
[0037] Table 3 Screening results of protective agents
[0038] Example 4 Screening of the ratio of bacterial mud to freeze-drying protective agent The viable bacterial count and survival rate were determined using the freeze-drying method described in Example 2, using different ratios of bacterial sludge to freeze-drying protectant as shown in Table 4. The results showed that the highest survival rate was achieved when the bacterial sludge to freeze-drying protectant ratio (kg:L) was 1:2.
[0039] Table 4 Screening results of the ratio of bacterial sludge to freeze-drying protective agent
[0040] Example 5 Optimization of the freeze-drying method of Bifidobacterium breve 1. Pre-freezing process optimization The freeze-drying survival rate was investigated using the freeze-drying method described in Example 2 and the different process parameters shown in Table 5. The results showed that the survival rate was highest when the pre-freezing temperature was -40°C.
[0041] Table 5 Pre-freezing process parameter screening results
Claims
1. A Bifidobacterium breve freeze-drying protective agent, characterized in that The invention comprises the following components: trehalose, glycerol, sodium glutamate and sodium ascorbate.
2. A Bifidobacterium breve freeze-drying protective agent according to claim 1, characterized in that The invention comprises the following components in weight concentrations: 10-18% of trehalose, 1.0-2.0% of glycerol, 1.0-1.5% of sodium glutamate and 0.1-0.3% of sodium ascorbate.
3. A Bifidobacterium breve freeze-drying protective agent according to claim 2, characterized in that The invention comprises the following components in weight concentrations: trehalose 15%, glycerol 1.5%, sodium glutamate 1.2%, and sodium ascorbate 0.2%.
4. A Bifidobacterium breve freeze-drying protective agent according to claim 1, characterized in that The invention comprises the following components: skimmed milk powder, trehalose, white sugar, sodium ascorbate, anhydrous glucose, glycerol, sodium glutamate and maltodextrin.
5. The Bifidobacterium breve freeze-drying protectant according to any one of claims 1 to 4, characterized in that The Bifidobacterium breve is Bifidobacterium breve ( Bifidobacterium breve )RRJK1316, accession number is GDMCC No.64764.
6. A freeze-drying method for Bifidobacterium breve, characterized in that: The process is as follows, 1) Cold stress pretreatment: Place the bacterial sludge at 4°C for 5 h; 2) Emulsifying the bacterial sludge: adding the Bifidobacterium breve freeze-drying protective agent according to any one of claims 1 to 5 to the Bifidobacterium breve bacterial sludge, stirring and mixing thoroughly to form a homogeneous bacterial sludge emulsion; 3) Pre-freezing: Dispense the mixed emulsion into freeze-drying trays at a rate of 1.0 L / tray. Place the trays into the freeze-dryer and pre-freeze for 2 hours. Stop pre-freezing when the temperature reaches -40°C. 4) Freeze drying: After pre-freezing, the temperature is gradually raised and freeze drying is performed in 12 sections, with each section increasing by 5°C until it reaches 30°C. The freeze drying time is 52.5 hours.
7. The freeze-drying method for Bifidobacterium breve according to claim 6, characterized in that: In step 1), the Bifidobacterium breve bacterial sludge is obtained by activating, expanding, fermenting in the primary stage and fermenting in the secondary stage.
8. The freeze-drying method for Bifidobacterium breve according to claim 6, characterized in that: In step 3), the freeze-drying temperature and time of the 12 sections are shown in the following table; 。 9. The freeze-drying method for Bifidobacterium breve according to claim 4, characterized in that: The following steps are also included: collecting the materials after freeze drying, setting the environmental conditions to a temperature of 18-25°C and a humidity of 30-35%, crushing and sealing the materials after collection.
10. Freeze-dried Bifidobacterium breve powder obtained according to the freeze-drying method for Bifidobacterium breve according to any one of claims 6 to 9.