Method for improving stability of lactobacillus plantarum

Abstract

The invention provides a method for improving the stability of lactobacillus plantarum, and relates to the field of lactobacillus plantarum preparation processes. The method for improving the stability of the lactobacillus plantarum comprises the following process steps: screening bacterial strains, separating candidate bacterial strains from traditional fermented food pickles, diluting a pickle sample, smearing the diluted pickle sample onto an MRS solid culture medium, putting the MRS solid culture medium into an anaerobic incubator, adjusting the temperature to 36 DEG C, carrying out anaerobic culture for 36 hours, and taking out the MRS solid culture medium; single colonies are selected for gram staining and catalase tests to identify lactic acid bacteria, bacterial liquid is placed in an MRS liquid culture medium with the pH value of 3.0, standing is conducted for 3 hours, and strains with the survival rate larger than 85% are screened. The pH response type microcapsule is constructed by utilizing a chitosan-sodium alginate electrostatic adsorption principle, a capsule shell rapidly swells to release a bacterial strain in an acid environment, and the structure is kept complete in an alkaline environment, so that the colonization rate of the bacterial strain in simulated gastrointestinal fluid is increased by 70%.

Description

Technical Field

[0001] The invention relates to the field of Lactobacillus plantarum preparation technology, and in particular to a method for improving the stability of Lactobacillus plantarum. Background Art

[0002] As an important member of the genus Lactobacillus, Lactobacillus plantarum has demonstrated irreplaceable value in many fields such as food, medicine, and feed, and is closely linked to people's lives and production practices. In the food field, Lactobacillus plantarum is a type of fermentation microorganism with extremely wide application. In the medical field, Lactobacillus plantarum has an important regulatory effect on human health. In the feed field, Lactobacillus plantarum, as a green and safe feed additive, can significantly promote animal growth. In the field of food fermentation, stability directly affects the quality and production efficiency of fermented food. In the field of medicine and health care, the stability of Lactobacillus plantarum is even more related to the realization of its prebiotic effects. In the field of feed applications, the stability of Lactobacillus plantarum cannot be ignored.

[0003] However, lactic acid bacteria are easily inactivated during processing, storage, transportation and use due to environmental conditions such as heat, acid, and oxygen. As the storage time increases, the number of live bacteria decreases and the vitality gradually weakens, affecting their physiological functions. Their poor stability is a bottleneck limiting their application. Plant lactic acid bacteria products require refrigerated storage and transportation due to their limitations in heat resistance, acid resistance and oxygen resistance. The products have a short shelf life and high cost, which hinders their application.

[0004] Therefore, the present invention proposes a method for improving the stability of Lactobacillus plantarum, thereby effectively solving the above-mentioned problems and difficulties. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In response to the deficiencies in the prior art, the present invention provides a method for improving the stability of Lactobacillus plantarum. By optimizing culture conditions, accurately controlling temperature and oxygen content, and formulating a precise temperature control scheme according to the requirements of different growth stages of Lactobacillus plantarum, a suitable anaerobic or microaerobic environment is created by using an anaerobic incubator or adding an oxygen scavenger, etc., thereby effectively reducing the adverse effects of environmental factors on the bacteria, maintaining the normal metabolism and physiological functions of the bacteria, and improving its stability. In addition, by utilizing the electrostatic adsorption principle of chitosan-sodium alginate, pH-responsive microcapsules are constructed. The capsule shell rapidly swells to release the strain in an acidic environment, while maintaining structural integrity in an alkaline environment, thereby increasing the colonization rate of the strain in simulated gastrointestinal fluid by 70%, significantly reducing losses compared to traditional encapsulation technology, and organically combining microencapsulation technology with processes such as freeze-drying, immobilization technology, and additive application, fully leveraging the advantages of each process and achieving synergistic efficiency.

[0007] (2) Technical solution

[0008] To achieve the above object, the present invention is achieved through the following technical solutions: a method for improving the stability of Lactobacillus plantarum, the process steps of improving the stability of Lactobacillus plantarum are as follows:

[0009] Step 1: Strain screening: Candidate strains were isolated from kimchi, a traditional fermented food. The kimchi sample was diluted and smeared onto MRS solid culture medium. The diluted kimchi sample was placed in an anaerobic incubator and incubated at 36 degrees Celsius for 36 hours. Single colonies were selected for Gram staining and catalase testing to identify lactic acid bacteria. The bacterial suspension was then placed in MRS liquid culture medium at pH 3.0 and allowed to stand for 3 hours to screen for strains with a survival rate greater than 85%.

[0010] Step 2: Strain activation: The selected strains were inoculated into MRS liquid medium containing 25% glycerol, frozen at -65°C for 24 hours, and then the freeze-dried strains were inoculated into 15 mL of MRS liquid medium. The culture was anaerobically cultured at 36°C for 48 hours, and then transferred to 100 mL of MRS liquid medium at a 5% inoculum volume and anaerobically cultured at 36°C for 24 hours.

[0011] Step 3: Stress pretreatment: add 0.5% sodium chloride solution to the strain seed solution and place it at 36 degrees Celsius for 3 hours to induce the expression of osmotic stress proteins and improve the cell osmotic pressure tolerance:

[0012] Step 4: High-density fermentation: Place the pretreated fermentation strain into the culture medium for lactobacillus culture. The culture conditions are set at 36 degrees Celsius and 50% humidity in an incubator. The incubator is set to a microaerobic environment with an oxygen concentration of less than 5% and an air flow rate of 0.2 vvm. Culture for 36 hours until the OD 600nm is greater than 2.5:

[0013] Step 5: Prepare the protective agent. The protective agent is formulated as 16.29 g / 100 mL skim milk, 10.85 g / 100 mL trehalose, and 0.07 g / 100 mL manganese sulfate. The preparation process is carried out at room temperature below 30 degrees Celsius. The protective agent and pre-strain fermentation broth are mixed in a ratio of 1.5:1 and placed in an incubator for 12 hours. The strain survival rate is tested to be greater than 85%;

[0014] Step 6: Concentrating the Lactobacillus, placing the fermentation liquid of the strain after being fully mixed with the protective agent into a centrifuge for centrifugation, removing the supernatant by centrifugation, retaining the bacterial suspension, and then placing the bacterial suspension into a reagent tube and freezing it at -70 degrees Celsius for 6 hours, and then drying the frozen bacterial suspension;

[0015] Step 7: Microcapsulation: The concentrated frozen strain powder was resuspended in sterile water to 15 CFU / mL. The inner wall of the microcapsule was prepared by using 2% sodium alginate solution and 1% gum arabic in a ratio of 1:2. After sterilization, it was cooled to 40 degrees Celsius and freeze-concentrated. The outer wall of the microcapsule was prepared by using 1% chitosan solution and 0.5% citric acid in a ratio of 1:1. The freeze-dried powder of the bacterial suspension was then mixed with the inner wall of the microcapsule in a ratio of 1:5.

[0016] Step 8: Storage of finished product: vacuum-pack the microcapsules in aluminum foil bags, place a desiccant in the vacuum bag, and store in a cool, dry place with a temperature ≤ 30 degrees Celsius and an air humidity ≤ 35% away from light to obtain highly stable Lactobacillus plantarum.

[0017] Preferably, in the strain screening step, the screened strains are cultured in MRS medium containing 0.5% bile salts, the OD 650nm value is measured, and strains with a growth rate greater than 0.25 OD / h are selected;

[0018] Through the above technical solution, the traditional fermented food kimchi can ferment natural high-stability strains, and the plant strains extracted from kimchi can adapt to more complex environments.

[0019] Preferably, the strain activation step requires that the OD 650nm value of the activated strain reaches 1.2-1.6;

[0020] Through the above technical solution, strains with substandard measurement values ​​are excluded through OD 650nm absorbance detection, and strains that can be used to cultivate highly stable Lactobacillus plantarum are retained.

[0021] Preferably, in the stress pretreatment step, the heat resistance of the strain needs to be additionally enhanced. Specifically, after standing, the bacterial solution is heated to 43-47°C for 60 minutes to activate heat shock proteins and cold shock proteins.

[0022] Through the above technical solution, the pressure-resistant osmotic properties of the cells are improved through the stress pretreatment step, and the heat resistance and cold resistance of the strain cells are improved by activating heat shock proteins.

[0023] Preferably, in the high-density fermentation step, the reconfiguration ratio of the culture medium is 26 g / L glucose, 26 g / L yeast extract, 16 g / L sodium glutamate, pH 6.2, and 0.1% to 0.5% tomato juice or soy peptone is additionally added;

[0024] Through the above technical solution, the rapid growth of the strain liquid and the secretion of ESP can be effectively promoted by adding tomato juice or soy peptone to the culture medium.

[0025] Preferably, in the bacterial cell concentration step, the drying treatment requires placing the frozen bacterial suspension in an incubator with a vacuum degree of less than 5 Pa, maintaining the temperature at -20 degrees Celsius for 36 hours, then raising the temperature to 30 degrees Celsius and letting it stand for 24 hours, so that the moisture content of the concentrated strain liquid is less than 3%, and then nitrogen-filled, sealed, and placed in a 4-degree Celsius cold room away from light;

[0026] Through the above technical solution, the cryoprotectant can effectively protect the bacteria from freezing damage during the freeze-drying process, thereby improving the survival rate and stability of the bacteria.

[0027] Preferably, in the microcapsule encapsulation step, a 3% CaCl2 solution is dripped into the microcapsule by a peristaltic pump to form gel beads with a diameter of 3MM-5MM, and the gel beads are immersed in the chitosan solution. After stirring for 30 minutes, the microcapsules are collected by centrifugation.

[0028] Through the above technical solution, the sodium alginate and chitosan composite wall material utilizes its good film-forming property and biocompatibility to wrap Lactobacillus plantarum inside the microcapsule, providing physical barrier protection for Lactobacillus plantarum.

[0029] (3) Beneficial effects

[0030] The present invention provides a method for improving the stability of Lactobacillus plantarum. It has the following beneficial effects:

[0031] 1. The present invention provides a method for improving the stability of Lactobacillus plantarum, by optimizing culture conditions, accurately controlling temperature and oxygen content, and according to the needs of different growth stages of Lactobacillus plantarum, formulating an accurate temperature control scheme, and creating a suitable anaerobic or microaerobic environment by using an anaerobic incubator or adding an oxygen scavenger, etc., effectively reducing the adverse effects of environmental factors on the bacterium, maintaining the normal metabolism and physiological function of the bacterium, and improving its stability. At the same time, by optimizing parameters such as pre-freezing rate, vacuum degree, drying temperature, feed rate, atomization pressure, etc., the damage to the activity of Lactobacillus plantarum during the drying process is reduced, and the survival rate of the bacterium is improved.

[0032] 2. The present invention provides a method for improving the stability of Lactobacillus plantarum. By utilizing the electrostatic adsorption principle of chitosan-sodium alginate, pH-responsive microcapsules are constructed. The capsule shell quickly swells and releases the strain in an acidic environment, while maintaining structural integrity in an alkaline environment, thereby increasing the colonization rate of the strain in simulated gastrointestinal fluid by 70%. Compared with traditional encapsulation technology, the loss is significantly reduced. In addition, the microencapsulation technology is organically combined with processes such as freeze-drying, immobilization technology and additive application, giving full play to the advantages of each process and achieving synergistic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a process flow chart for improving the stability of Lactobacillus plantarum. DETAILED DESCRIPTION

[0034] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. It should be noted that the described embodiments are only some of the embodiments of the present invention, and are not intended to be exhaustive. Based on the embodiments of the present invention, all other embodiments derived by persons of ordinary skill in the art without inventive effort are within the scope of protection of the present invention. It should be noted that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the exemplary embodiments of the present invention. For ease of description, the dimensions of the various parts shown in the drawings are not drawn to scale. Technologies, methods, and devices known to persons of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, such technologies, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely illustrative and not limiting. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar reference numerals and letters denote similar items in the following figures. Therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0035] Example 1:

[0036] like Figure 1 As shown, the embodiment of the present invention provides a method for improving the stability of Lactobacillus plantarum, and the process steps for improving the stability of Lactobacillus plantarum are as follows:

[0037] Step 1: Strain screening: Candidate strains were isolated from kimchi, a traditional fermented food. The kimchi sample was diluted and smeared onto MRS solid culture medium. The diluted kimchi sample was placed in an anaerobic incubator and incubated at 36 degrees Celsius for 36 hours. Single colonies were selected for Gram staining and catalase testing to identify lactic acid bacteria. The bacterial suspension was then placed in MRS liquid culture medium at pH 3.0 and allowed to stand for 3 hours to screen for strains with a survival rate greater than 85%.

[0038] Step 2: Strain activation: The selected strains were inoculated into MRS liquid medium containing 25% glycerol, frozen at -65°C for 24 hours, and then the freeze-dried strains were inoculated into 15 mL of MRS liquid medium. The culture was anaerobically cultured at 36°C for 48 hours, and then transferred to 100 mL of MRS liquid medium at a 5% inoculum volume and anaerobically cultured at 36°C for 24 hours.

[0039] Step 3: Stress pretreatment: add 0.5% sodium chloride solution to the strain seed solution and place it at 36 degrees Celsius for 3 hours to induce the expression of osmotic stress proteins and improve the cell osmotic pressure tolerance:

[0040] Step 4: High-density fermentation: Place the pretreated fermentation strain into the culture medium for lactobacillus culture. The culture conditions are set at 36 degrees Celsius and 50% humidity in an incubator. The incubator is set to a microaerobic environment with an oxygen concentration of less than 5% and an air flow rate of 0.2 vvm. Culture for 36 hours until the OD 600nm is greater than 2.5:

[0041] Step 5: Prepare the protective agent. The protective agent formula is 16.29 g / 100 mL skim milk, 10.85 g / 100 mL trehalose, and 0.07 g / 100 mL manganese sulfate. The preparation process is carried out at room temperature below 30 degrees Celsius. The protective agent and pre-strain fermentation liquid are mixed in a ratio of 1.5:1 and placed in an incubator for 12 hours. The strain survival rate is tested to be greater than 85%;

[0042] Step 6: Concentrating the Lactobacillus, placing the fermentation liquid of the strain after being fully mixed with the protective agent into a centrifuge for centrifugation, removing the supernatant by centrifugation, retaining the bacterial suspension, and then placing the bacterial suspension into a reagent tube and freezing it at -70 degrees Celsius for 6 hours, and then drying the frozen bacterial suspension;

[0043] Step 7: Microcapsulation: The concentrated frozen strain powder was resuspended in sterile water to 15 CFU / mL. The inner wall of the microcapsule was prepared by using 2% sodium alginate solution and 1% gum arabic in a ratio of 1:2. After sterilization, it was cooled to 40 degrees Celsius and freeze-concentrated. The outer wall of the microcapsule was prepared by using 1% chitosan solution and 0.5% citric acid in a ratio of 1:1. The freeze-dried powder of the bacterial suspension was then mixed with the inner wall of the microcapsule in a ratio of 1:5.

[0044] Step 8: Storage of finished product: vacuum-pack the microcapsules in aluminum foil bags, place a desiccant in the vacuum bag, and store in a cool, dry place with a temperature ≤ 30 degrees Celsius and an air humidity ≤ 35% away from light to obtain highly stable Lactobacillus plantarum.

[0045] The strain activation step requires that the OD 650nm measurement value of the activated strain reach 1.2-1.6. The strains with substandard measurement values ​​are excluded through OD 650nm absorbance detection, and the strains that can be cultivated with high stability Lactobacillus plantarum are retained. In the stress pretreatment step, the heat resistance of the strain needs to be additionally enhanced. The specific steps are to heat the bacterial solution to 43-47°C for 60 minutes after standing to activate heat shock proteins and cold shock proteins. The stress pretreatment step improves the cell pressure osmotic properties and at the same time improves the heat resistance and cold resistance of the strain cells by activating heat shock proteins. In the high-density fermentation step, the reconfiguration ratio of the culture medium is 26 g / L glucose, 26 g / L yeast extract, 16 g / L sodium glutamate, pH 6.2, and an additional 0.1%-0.5% tomato juice or soy peptone is added to the culture medium. The addition of tomato juice or soy peptone to the culture medium can effectively promote the rapid growth of the strain solution and the secretion of ESP. In the bacterial concentration step, the drying process requires the frozen bacterial suspension to be placed in a vacuum degree of <5 Pa incubator, the temperature is kept at -20 degrees Celsius for 36 hours and then raised to 30 degrees Celsius and allowed to stand for 24 hours, so that the moisture content of the concentrated strain liquid is less than 3%, and then nitrogen is filled and sealed and placed in a 4-degree Celsius cold storage room away from light. During the freeze-drying process, the cryoprotectant can effectively protect the bacteria from freezing damage and improve the survival rate and stability of the bacteria. In the microencapsulation step, 3% CaCl2 solution needs to be dripped through a peristaltic pump to form gel beads with a diameter of 3MM-5MM. The gel beads are immersed in chitosan solution, stirred for 30 minutes, and then the microcapsules are collected by centrifugation. The sodium alginate and chitosan composite wall material uses its good film-forming property and biocompatibility to wrap Lactobacillus plantarum inside the microcapsule, providing physical barrier protection for Lactobacillus plantarum.

[0046] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the stability of plant lactobacillus, characterized in that: The described processing step of improving plant lactobacillus stability is as follows: Step 1: Strain screening: Candidate strains were isolated from kimchi, a traditional fermented food. The kimchi sample was diluted and smeared onto MRS solid culture medium. The diluted kimchi sample was placed in an anaerobic incubator and incubated at 36 degrees Celsius for 36 hours. Single colonies were selected for Gram staining and catalase testing to identify lactic acid bacteria. The bacterial suspension was then placed in MRS liquid culture medium at pH 3.0 and allowed to stand for 3 hours to screen for strains with a survival rate greater than 85%. Step 2: Strain activation: The selected strains were inoculated into MRS liquid medium containing 25% glycerol, frozen at -65°C for 24 hours, and then the freeze-dried strains were inoculated into 15 mL of MRS liquid medium. The culture was anaerobically cultured at 36°C for 48 hours, and then transferred to 100 mL of MRS liquid medium at a 5% inoculum volume and anaerobically cultured at 36°C for 24 hours. Step 3: Stress pretreatment: add 0.5% sodium chloride solution to the strain seed solution and place it at 36 degrees Celsius for 3 hours to induce the expression of osmotic stress proteins and improve the cell osmotic pressure tolerance: Step 4: High-density fermentation: Place the pretreated fermentation strain into the culture medium for lactobacillus cultivation. The culture conditions are set at 36 degrees Celsius and 50% humidity in an incubator. The incubator is set to a microaerobic environment with an oxygen concentration of less than 5% and an air flow rate of 0.2 vvm. Cultivate for 36 hours until the OD 600nm is greater than 2.

5. Step 5: Prepare the protective agent. The protective agent is formulated as 16.29 g / 100 mL skim milk, 10.85 g / 100 mL trehalose, and 0.07 g / 100 mL manganese sulfate. The preparation process is carried out at room temperature below 30 degrees Celsius. The protective agent and pre-strain fermentation broth are mixed in a ratio of 1.5:1 and placed in an incubator for 12 hours. The strain survival rate is tested to be greater than 85%; Step 6: Concentrating the Lactobacillus, placing the fermentation liquid of the strain after being fully mixed with the protective agent into a centrifuge for centrifugation, removing the supernatant by centrifugation, retaining the bacterial suspension, and then placing the bacterial suspension into a reagent tube and freezing it at -70 degrees Celsius for 6 hours, and then drying the frozen bacterial suspension; Step 7: Microcapsulation: The concentrated frozen strain powder was resuspended in sterile water to 15 CFU / mL. The inner wall of the microcapsule was prepared by using 2% sodium alginate solution and 1% gum arabic in a ratio of 1:

2. After sterilization, it was cooled to 40 degrees Celsius and freeze-concentrated. The outer wall of the microcapsule was prepared by using 1% chitosan solution and 0.5% citric acid in a ratio of 1:

1. The freeze-dried powder of the bacterial suspension was then mixed with the inner wall of the microcapsule in a ratio of 1:

5. Step 8: Storage of finished product: vacuum-pack the microcapsules in aluminum foil bags, place a desiccant in the vacuum bag, and store in a cool, dry place with a temperature ≤ 30 degrees Celsius and an air humidity ≤ 35% away from light to obtain highly stable Lactobacillus plantarum.

2. a method for improving the stability of plant lactobacillus according to claim 1, is characterized in that: In the strain screening step, the screened strains need to be cultured in an MRS medium containing 0.5% bile salts, the OD 650nm value is measured, and strains with a growth rate greater than 0.25 OD / h are selected.

3. a method for improving the stability of plant lactobacillus according to claim 1, is characterized in that: The strain activation step requires that the OD 650nm value of the activated strain be 1.2-1.

6.

4. A method for improving the stability of plant lactobacillus according to claim 1, characterized in that: In the stress pretreatment step, the heat resistance of the strain needs to be additionally enhanced. The specific steps are to heat the bacterial solution to 43-47° C. for 60 minutes after standing to activate heat shock proteins and cold shock proteins.

5. A method for improving the stability of plant lactobacillus according to claim 1, characterized in that: In the high-density fermentation step, the culture medium is reconfigured to a ratio of 26 g / L glucose, 26 g / L yeast extract, 16 g / L sodium glutamate, pH 6.2, and 0.1% to 0.5% tomato juice or soy peptone is additionally added.

6. A method for improving the stability of plant lactobacillus according to claim 1, characterized in that: In the bacterial concentration step, the drying process requires that the frozen bacterial suspension be placed in an incubator with a vacuum degree of <5 Pa, maintained at -20 degrees Celsius for 36 hours, and then raised to 30 degrees Celsius and left to stand for 24 hours, so that the moisture content of the concentrated strain liquid is less than 3%, and then nitrogen-filled, sealed, and placed in a 4-degree Celsius cold storage room away from light.

7. A method for improving the stability of Lactobacillus plantarum according to claim 1, characterized in that: In the microcapsule encapsulation step, a 3% CaCl2 solution is dripped into the microcapsule through a peristaltic pump to form gel beads with a diameter of 3MM-5MM. The gel beads are immersed in the chitosan solution and stirred for 30 minutes before collecting the microcapsules by centrifugation.

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