Method for improving high-temperature survival rate of lactobacillus paracasei
By adding stabilizers such as trehalose and gallic acid to the fermentation broth of Lactobacillus paracasei, the problem of the reduction in survival rate of Lactobacillus paracasei under high temperature conditions was solved, significantly improving its high temperature survival rate and broadening its application field.
Patent Information
- Application Number
- CN202411791656.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-02-18
AI Technical Summary
The survival rate of Lactobacillus paracasei is significantly reduced under high temperature conditions, which affects its application ability in the field of deodorizing livestock and poultry manure.
Add stabilizers to the bacterial solution after Lactobacillus paracasei fermentation, such as trehalose, 2-nitrihexylcyclone, gallic acid, glycerol, 3-phenylapropionic acid or sucrose to form a sterile aqueous solution, with an added mass concentration of 0.1- 8% to improve the high temperature survival rate of bacteria.
By optimizing stabilizer formulations, such as 0.5% trehalose and 0.1% gallic acid, the high temperature survival rate of Lactobacillus paracasei increased by 1.38 times, broadening its application areas in high temperature environments.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for improving the high-temperature survival rate of Lactobacillus paracasei and belongs to the field of biotechnology. Background Art
[0002] Lactobacillus paracasei is a Gram-positive, facultative anaerobic probiotic that is widely found in the human intestines, oral cavity, and some fermented foods. It has the functions of regulating intestinal flora, enhancing immunity, and improving digestive function. During the metabolism process, Lactobacillus paracasei produces organic acids (such as lactic acid), hydrogen peroxide, and other antibacterial substances, which can effectively inhibit and kill harmful bacteria that cause odor. By regulating the balance of microbial communities in the environment, Lactobacillus paracasei can reduce the number of malodorous bacteria, thereby achieving a deodorizing effect.
[0003] At present, Lactobacillus paracasei is widely used in agriculture and animal husbandry, and plays a role in the deodorization of livestock and poultry manure. It is expected to be used as a bacterial agent additive for deodorization of livestock and poultry manure. However, the survival rate of Lactobacillus paracasei is significantly reduced under high temperature conditions, and its metabolic activity is weakened. Therefore, when manure is centrally treated, the temperature rise will affect its fermentation ability and probiotic effect. Therefore, finding a method to improve the survival rate of Lactobacillus paracasei at high temperature can improve its application in deodorization of livestock and poultry manure. Summary of the invention
[0004] In view of the problems existing in the prior art, the present invention provides a method for improving the high temperature survival rate of Lactobacillus paracasei. The method of the present invention can reduce the activity loss of Lactobacillus paracasei at high temperature.
[0005] The method for improving the survival rate of Lactobacillus paracasei at high temperature is to add a stabilizer into the bacterial liquid after the Lactobacillus paracasei is fermented.
[0006] The stabilizer is a sterile aqueous solution prepared from one or more of trehalose, 2-azolopyrrolidone, gallic acid, glycerol, 3-phenylpropionic acid, and sucrose, and the mass concentration of the stabilizer added is 0.1-8%.
[0007] Furthermore, the stabilizer is trehalose, added at a concentration of 0.1-0.5%; or the stabilizer is 2-azolopyrrolidone, added at a concentration of 0.1-0.5%; or the stabilizer is gallic acid, added at a concentration of 0.1-0.5%; or the stabilizer is glycerol, added at a concentration of 3-8%; or the stabilizer is 3-phenylpropionic acid, added at a concentration of 0.1-0.5%; or the stabilizer is sucrose, added at a concentration of 3-8%.
[0008] Furthermore, the stabilizer is composed of trehalose and gallic acid.
[0009] The concentration of trehalose in the system is 0.1-0.5%, and the concentration of gallic acid is 0.1-0.5%.
[0010] Most preferably, the concentration of trehalose in the system is 0.5%, and the concentration of gallic acid is 0.1%.
[0011] The method for improving the high temperature survival rate of Lactobacillus paracasei of the present invention specifically comprises the following steps:
[0012] Step 1: Streak the strain of Lactobacillus paracasei glycerol storage tube on the MRS agar medium plate, culture it in an anaerobic incubator at 30°C for 24-48 hours, select a single colony and put it into a test tube containing 5 mL of MRS liquid culture medium, and culture it in an anaerobic incubator at 30°C for 24-48 hours to obtain the fermentation liquid.
[0013] Step 2: Inoculate the Lactobacillus paracasei test tube fermentation broth into 200 mL of MRS liquid culture medium at a volume ratio of 5%, and culture in an anaerobic incubator at 30° C. for 24-48 hours to obtain Lactobacillus paracasei fermentation broth.
[0014] Step 3: Centrifuge the fermentation liquid obtained in step 2 at 4°C and 10,000 rpm for 15 min to obtain Bacillus cereus fermentation cells; resuspend the obtained cells in sterile water and add a stabilizer.
[0015] The Lactobacillus paracasei can be obtained through conventional purchase.
[0016] The MRS medium comprises: 10.0 g / L casein digest, 10.0 g / L beef powder, 4.0 g / L yeast powder, 2.0 g / L ammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 20.0 g / L glucose, 2.0 g / L potassium dihydrogen phosphate, 1.0 g / L Tween 80, pH = 5.7 ± 0.2. When preparing the agar medium, add 2 g / L agar.
[0017] The beneficial effects of the present invention are embodied in:
[0018] The invention provides a method for improving the survival rate of Lactobacillus paracasei at high temperature, and optimizes the type and addition amount of a stabilizer. The optimized composite stabilizer formula comprises 0.5% of trehalose and 0.1% of gallic acid, and the high temperature survival rate of the bacteria is improved by 1.38 times.
[0019] The prior art has not paid attention to the high-temperature bacterial survival rate of Lactobacillus paracasei. The present invention improves the bacterial survival rate of Lactobacillus paracasei at high temperatures by adding a stabilizer, broadens the application field of Lactobacillus paracasei, and effectively solves the limitation of temperature on its application. DETAILED DESCRIPTION
[0020] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. In the following examples, various processes and methods not described in detail are conventional methods known in the art. The specific embodiments of the present invention described below are to supplement the purpose, technical solutions and effects of the present invention, but they are not intended to limit the scope of protection of the present invention. Within the scope of the present invention, simple changes, modifications or replacements can be made, which are all considered to be within the scope of protection of the present invention.
[0021] Embodiment 1: fermentation preparation of Lactobacillus paracasei
[0022] The strain of Lactobacillus paracasei glycerol preservation tube was streaked on an MRS agar medium plate, cultured in an anaerobic incubator at 30°C for 24 hours, and a single colony was selected and placed in a test tube containing 5 mL of MRS medium. After culturing in an anaerobic incubator at 30°C for 24 hours, a Lactobacillus paracasei test tube fermentation liquid was obtained. 1 mL of the test tube fermentation liquid was taken and inoculated into 200 mL of liquid MRS medium, and the Lactobacillus paracasei fermentation liquid was obtained after culturing in an anaerobic incubator at 30°C for 48 hours. The fermentation liquid was centrifuged at 4°C and 10,000 rpm for 15 minutes to obtain the bacterial cells.
[0023] Example 2: Selection of heat stabilizer
[0024] Single factor optimization of stabilizers: trehalose, 2-azolophenone, glycerol, gallic acid, 3-phenylpropionic acid, sucrose, and the protective effect of the added types and amounts on the activity of Lactobacillus paracasei under high temperature conditions. For the convenience of experimental operation, 10 mL of Lactobacillus paracasei fermentation broth was taken, and the cells were obtained by centrifugation at 4 ° C and 10000 rpm for 15 min. The cells were resuspended in 5 mL of sterile water. The types of stabilizers added and the final mass concentration of the system are shown in Table 1. Sterile water was added to make up to 10 mL to prepare a mixed solution. The control group was without stabilizer.
[0025] The mixed solutions of each group were placed in incubators at 30°C, 40°C and 45°C respectively, incubated for 12-24 hours, and then taken out and the OD was measured using an enzyme-labeled instrument. 600 The value was used to calculate the survival rate of Lactobacillus paracasei under high temperature conditions. The formula is as follows:
[0026] A%=(A1-A0) / A0×100%
[0027] Among them, A% is the survival rate of Lactobacillus paracasei at high temperature, A0 is the number of live bacteria in the control group after the culture is completed, and A1 is the number of live bacteria in the sample after the culture is completed at different high temperatures, that is, the increase rate of the number of live bacteria in each experimental group compared with the control group is compared.
[0028]
[0029] The survival rate of Lactobacillus paracasei at different temperatures under different stabilizers and different addition amounts are shown in Table 2. The addition of stabilizers can significantly improve the survival rate of Lactobacillus paracasei in high temperature environment. The survival rates of trehalose at 0.5%, gallic acid at 0.1%, glycerol at 3%, and 3-phenylpropionic acid at 0.1% are all improved. Compared with the group without protective agent, the high temperature survival rate of the bacteria is increased by up to 1.34 times.
[0030]
[0031] Example 3: Combination of Stabilizers
[0032] Optimize the compound addition of stabilizers, select additives and concentrations with the highest relative improvement rate of bacterial survival: trehalose (system mass concentration 0.5%), gallic acid (system mass concentration 0.1%), and glycerol (system mass concentration 3%), and make a combined compound, and carry out high-temperature culture at 30℃, 40℃, and 45℃, respectively.
[0033] Take 10 mL of Lactobacillus paracasei fermentation broth, centrifuge at 4°C and 10,000 rpm for 15 min to obtain the bacterial cells, resuspend the bacterial cells in 5 mL of sterile water, add the stabilizer compound as shown in Table 3, and make up to 10 mL with sterile water to prepare a mixed solution. The control group was a control group without stabilizer.
[0034] The mixture was placed in an incubator at 30°C, 40°C and 45°C, incubated for 12-24 hours, and then the OD was measured using an enzyme-labeled instrument. 600 The values were calculated and the survival rate of Lactobacillus paracasei under high temperature conditions was increased.
[0035] The results are shown in Table 3. In the combination of different protective agents, when 0.5% trehalose and 0.1% gallic acid were added, at 45°C, the high temperature survival rate of the bacteria was increased by up to 1.38 times compared with the group without protective agent, which improved the survival rate of Lactobacillus paracasei.
[0036]
Claims
1. A method for improving the high temperature survival rate of Lactobacillus paracasei, characterized in that: adding a stabilizer to the bacterial solution after fermentation of Lactobacillus paracasei; The stabilizer is a sterile aqueous solution prepared from one or more of trehalose, 2-azolopyrrolidone, gallic acid, glycerol, 3-phenylpropionic acid, and sucrose, and the mass concentration of the stabilizer added is 0.1-8%.
2. The method according to claim 1, characterized in that: The stabilizer is trehalose, and the added concentration is 0.1-0.5%.
3. The method according to claim 1, characterized in that: The stabilizer is 2-azocetone, and the added concentration is 0.1-0.5%.
4. The method according to claim 1, characterized in that: The stabilizer is gallic acid, and the added concentration is 0.1-0.5%.
5. The method according to claim 1, characterized in that: The stabilizer is glycerol, and the added concentration is 3-8%.
6. The method according to claim 1, characterized in that: The stabilizer is 3-phenylpropionic acid, and the added concentration is 0.1-0.5%.
7. The method according to claim 1, characterized in that: The stabilizer is sucrose, and the added concentration is 3-8%.
8. The method according to claim 1, characterized in that: The stabilizer is composed of trehalose and gallic acid.
9. The method according to claim 8, characterized in that: The concentration of trehalose in the system is 0.1-0.5%, and the concentration of gallic acid is 0.1-0.5%.
10. The method according to claim 9, characterized in that: The concentration of trehalose in the system is 0.5%, and the concentration of gallic acid is 0.1%.