A method for improving stability of lactobacillus plantarum

Abstract

The application provides a method for improving stability of lactobacillus plantarum, and relates to the field of lactobacillus plantarum preparation process. The process steps of the method for improving stability of lactobacillus plantarum are strain screening, candidate strain separation from traditional fermented food pickles, placing the pickle sample into a dilution tank, smearing the diluted pickle sample on MRS solid culture medium, placing the MRS solid culture medium into an anaerobic incubator, adjusting to 36 DEG C, anaerobic culture for 36 hours, picking single colonies for gram staining and peroxidase test to identify lactic acid bacteria, and placing the bacterial liquid in MRS liquid culture medium with pH 3.0 and standing for 3 hours to screen strains with a survival rate greater than 85%. The application utilizes the electrostatic adsorption principle of chitosan-sodium alginate to construct a pH-responsive microcapsule, the capsule shell is quickly swollen and releases the strain in an acidic environment, and the capsule shell remains intact in an alkaline environment, so that the colonization rate of the strain in simulated gastric and intestinal fluid is increased by 70%.

Description

Technical Field

[0001] This invention relates to the field of Lactobacillus plantarum preparation process, specifically a method for improving the stability of Lactobacillus plantarum. Background Technology

[0002] As an important member of the Lactobacillus genus, *Lactobacillus plantarum* has demonstrated irreplaceable value in multiple 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 widely used fermentation microorganism. In the pharmaceutical field, *Lactobacillus plantarum* plays an important regulatory role in human health. In the feed field, *Lactobacillus plantarum*, as a green and safe feed additive, can significantly promote animal growth. In the food fermentation field, stability directly affects the quality and production efficiency of fermented foods. In the pharmaceutical and health care field, the stability of *Lactobacillus plantarum* is even more related to the exertion of its probiotic effects. In the feed application field, the stability of *Lactobacillus plantarum* is equally important.

[0003] Lactic acid bacteria are easily deactivated during processing, storage, transportation, and use due to environmental conditions such as heat, acid, and oxygen. As storage time increases, the number of live bacteria decreases and their activity gradually weakens, affecting their physiological functions. Their poor stability is a bottleneck problem that limits their application. Plant lactic acid bacteria products, due to their limitations in heat resistance, acid resistance, and oxygen resistance, all require refrigerated storage and transportation. Their short shelf life and high cost hinder their application.

[0004] Therefore, this invention proposes a method to improve the stability of Lactobacillus plantarum, thereby effectively solving the aforementioned problems and difficulties. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a method for improving the stability of *Lactobacillus plantarum*. By optimizing culture conditions and precisely controlling temperature and oxygen content, a precise temperature control scheme is developed based on the needs of different growth stages of *Lactobacillus plantarum*. Furthermore, by using anaerobic incubators or adding oxygen absorbers to create suitable anaerobic or microaerobic environments, the adverse effects of environmental factors on the bacteria are effectively reduced, maintaining normal metabolism and physiological functions and improving stability. Moreover, by utilizing the chitosan-sodium alginate electrostatic adsorption principle to construct pH-responsive microcapsules, the capsule shell rapidly swells and releases the bacterial strain in an acidic environment, while maintaining structural integrity in an alkaline environment. This increases the colonization rate of the strain in simulated gastrointestinal fluid by 70%, significantly reducing losses compared to traditional encapsulation techniques. Furthermore, the microcapsule technology is organically combined with freeze-drying, immobilization, and additive applications, fully leveraging the advantages of each process to achieve synergistic effects.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for improving the stability of *Lactobacillus plantarum*, wherein the process steps for improving the stability of *Lactobacillus plantarum* are as follows:

[0009] Step 1: Strains screening. Candidate strains were isolated from traditional fermented food kimchi. The kimchi sample was diluted and spread onto MRS solid medium. The diluted kimchi sample was placed in an anaerobic incubator and anaerobically cultured at 36 degrees Celsius for 36 hours. Single colonies were picked for Gram staining and catalase test to identify lactic acid bacteria. The bacterial solution was placed in MRS liquid 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: Activation of the strain. The selected strain was inoculated into MRS liquid medium containing 25% glycerol and frozen at -65 degrees Celsius for 24 hours. The freeze-dried strain was then inoculated into 15 mL of MRS liquid medium and anaerobically cultured at 36 degrees Celsius for 48 hours. Then, it was transferred to 100 mL of MRS liquid medium at a 5% inoculation rate and anaerobically cultured at 36 degrees Celsius for 24 hours.

[0011] Step 3: Stress pretreatment. A 0.5% sodium chloride solution was added to the bacterial seed culture and incubated at 36°C for 3 hours to induce the expression of osmotic stress proteins and improve cell osmotic pressure tolerance.

[0012] Step 4: High-density fermentation. The pretreated fermentation strain is placed in a culture medium for Lactobacillus culture. The culture conditions are set in an incubator at 36 degrees Celsius and 50% humidity, with a micro-aerobic environment (oxygen concentration below 5%) and an air flow rate of 0.2 vvm. Culture for 36 hours until OD 600nm > 2.5.

[0013] Step 5: Preparation of the protectant. The protectant formulation consisted of 16.29 g / 100 mL skim milk, 10.85 g / 100 mL trehalose, and 0.07 g / 100 mL manganese sulfate. The preparation was carried out at room temperature below 30 degrees Celsius. The protectant and pre-strain fermentation broth were mixed evenly at a ratio of 1.5:1 and placed in an incubator for 12 hours. The survival rate of the strain was found to be greater than 85%.

[0014] Step 6: Lactobacillus concentration. The fermentation broth of the strain, which is thoroughly mixed with the protectant, is centrifuged in a centrifuge. The supernatant is removed by centrifugation, and the bacterial suspension is retained. The bacterial suspension is then placed in a reagent tube and frozen at -70 degrees Celsius for 6 hours. The frozen bacterial suspension is then dried.

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

[0016] Step 8: Finished product storage. The microcapsules are vacuum-packed in aluminum foil bags with a desiccant inside. They are then stored in a cool, dry place away from light at a temperature ≤30 degrees Celsius and an air humidity ≤35% to obtain highly stable Lactobacillus plantarum.

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

[0018] Through the above technical solutions, traditional fermented food kimchi can ferment to produce natural, highly stable bacterial strains, and the plant strains extracted from kimchi can adapt to more complex environments.

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

[0020] Using the above technical solution, strains with substandard absorbance values ​​are excluded by OD 650nm absorbance detection, while strains that can be cultured with high stability of Lactobacillus plantarum are retained.

[0021] Preferably, in the stress pretreatment step, it is necessary to further enhance the heat resistance of the strain. Specifically, after the bacterial solution has been allowed to stand, it is heated to 43-47°C for 60 minutes to activate heat shock proteins and cold shock proteins.

[0022] The above technical solution improves the cell's pressure resistance and osmotic properties through stress pretreatment, while simultaneously enhancing the heat resistance and cold resistance of the strain cells 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 monosodium glutamate, pH 6.2, with an additional addition of 0.1% to 0.5% tomato juice or soybean peptone;

[0024] The above technical solution demonstrates that adding tomato juice or soybean peptone to the culture medium can effectively promote the rapid growth of the bacterial culture and the secretion of ESP.

[0025] Preferably, in the bacterial cell concentration step, the drying process requires placing the frozen bacterial suspension in an incubator with a vacuum degree of <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 bacterial solution is less than 3%, and then sealing it with nitrogen and placing it in a refrigerator at 4 degrees Celsius away from light.

[0026] Through the above technical solutions, cryoprotectants can effectively protect 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 in using a peristaltic pump to form gel beads with a diameter of 3 mm to 5 mm. The gel beads are then immersed in a chitosan solution, stirred for 30 minutes, and the microcapsules are collected by centrifugation.

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

[0029] (III) Beneficial Effects

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

[0031] 1. This invention provides a method for improving the stability of Lactobacillus plantarum. By optimizing culture conditions and precisely controlling temperature and oxygen content, a precise temperature control scheme is formulated according to the needs of different growth stages of Lactobacillus plantarum. Furthermore, by using anaerobic incubators or adding oxygen absorbers to create a suitable anaerobic or micro-aerobic environment, the adverse effects of environmental factors on the bacteria are effectively reduced, maintaining the normal metabolism and physiological functions of the bacteria and improving their stability. At the same time, by optimizing parameters such as pre-freezing rate, vacuum degree, drying temperature, feeding speed, and atomization pressure, the damage to the activity of Lactobacillus plantarum during the drying process is reduced, and the survival rate of the bacteria is improved.

[0032] 2. This invention provides a method for improving the stability of Lactobacillus plantarum. By utilizing the principle of chitosan-sodium alginate electrostatic adsorption, pH-responsive microcapsules are constructed. In an acidic environment, the capsule shell rapidly swells and releases the strain, while in an alkaline environment, the structure remains intact. This increases the colonization rate of the strain in simulated gastrointestinal fluid by 70%, significantly reducing losses compared to traditional encapsulation techniques. Furthermore, the microcapsule technology is organically combined with processes such as freeze-drying, immobilization, and additive application, giving full play to the advantages of each process and achieving synergistic effects. Attached Figure Description

[0033] Figure 1 This is a process flow diagram for improving the stability of Lactobacillus plantarum according to the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the description of this application, it should be noted that the terminology used herein is only for describing specific implementations and is not intended to limit the exemplary implementations according to this application. For ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, 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 exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings indicate similar items, and therefore, once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0035] Example 1:

[0036] like Figure 1 As shown in the figure, this embodiment of the invention provides a method for improving the stability of Lactobacillus plantarum. The process steps for improving the stability of Lactobacillus plantarum are as follows:

[0037] Step 1: Strains screening. Candidate strains were isolated from traditional fermented food kimchi. The kimchi sample was diluted and spread onto MRS solid medium. The diluted kimchi sample was placed in an anaerobic incubator and anaerobically cultured at 36 degrees Celsius for 36 hours. Single colonies were picked for Gram staining and catalase test to identify lactic acid bacteria. The bacterial solution was placed in MRS liquid 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 and frozen at -65 degrees Celsius for 24 hours. The freeze-dried strains were then inoculated into 15 mL of MRS liquid medium and anaerobically cultured at 36 degrees Celsius for 48 hours. The strains were then transferred to 100 mL of MRS liquid medium at a 5% inoculation rate and anaerobically cultured at 36 degrees Celsius for 24 hours.

[0039] Step 3: Stress pretreatment. A 0.5% sodium chloride solution was added to the bacterial seed culture and incubated at 36°C for 3 hours to induce the expression of osmotic stress proteins and improve cell osmotic pressure tolerance.

[0040] Step 4: High-density fermentation. The pretreated fermentation strain is placed in a culture medium for Lactobacillus culture. The culture conditions are set in an incubator at 36 degrees Celsius and 50% humidity, with a micro-aerobic environment (oxygen concentration below 5%) and an air flow rate of 0.2 vvm. Culture for 36 hours until OD 600nm > 2.5.

[0041] Step 5: Preparation of the protectant. The protectant formulation consisted of 16.29 g / 100 mL skim milk, 10.85 g / 100 mL trehalose, and 0.07 g / 100 mL manganese sulfate. The preparation was carried out at room temperature below 30°C. The protectant and pre-strain fermentation broth were mixed evenly at a ratio of 1.5:1 and placed in an incubator for 12 hours. The survival rate of the strain was found to be greater than 85%.

[0042] Step 6: Lactobacillus concentration. The fermentation broth of the strain, which is thoroughly mixed with the protectant, is centrifuged in a centrifuge. The supernatant is removed by centrifugation, and the bacterial suspension is retained. The bacterial suspension is then placed in a reagent tube and frozen at -70 degrees Celsius for 6 hours. The frozen bacterial suspension is then dried.

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

[0044] Step 8: Finished product storage. The microcapsules are vacuum-packed in aluminum foil bags with a desiccant inside. They are then stored in a cool, dry place away from light at a temperature ≤30 degrees Celsius and an air humidity ≤35% to obtain highly stable Lactobacillus plantarum.

[0045] The strain activation step requires the activated strain to achieve an OD 650nm absorbance value of 1.2-1.6. Strains failing to meet this standard are excluded by OD 650nm absorbance detection, retaining only those strains capable of culturing highly stable *Lactobacillus plantarum*. In the stress pretreatment step, additional enhancement of the strain's heat resistance is required. Specifically, after settling, the bacterial suspension is heated to 43-47℃ for 60 minutes to activate heat shock and cold shock proteins. This stress pretreatment improves the cell's pressure and osmotic properties, while activating heat shock proteins enhances the strain's heat and cold resistance. In the high-density fermentation step, the culture medium is prepared with 26 g / L glucose, 26 g / L yeast extract, and 16 g / L monosodium glutamate at pH 6.2, with the addition of 0.1%–0.5% tomato juice or soy peptone. Adding tomato juice or soy peptone effectively promotes rapid growth of the strain and ESP secretion. In the cell concentration step, the drying process requires placing the frozen bacterial suspension under a vacuum of <5°C. In a Pa incubator, the temperature was maintained at -20°C for 36 hours, then raised to 30°C and left to stand for 24 hours to ensure that the moisture content of the concentrated bacterial solution was less than 3%. The solution was then sealed with nitrogen and placed in a 4°C refrigerator away from light. During the freeze-drying process, the cryoprotectant effectively protected the bacteria from freezing damage, improving their survival rate and stability. In the microcapsule encapsulation step, a 3% CaCl2 solution was dripped in using a peristaltic pump to form gel beads with a diameter of 3-5 mm. These gel beads were then immersed in a chitosan solution and stirred for 30 minutes before being collected by centrifugation. The sodium alginate and chitosan composite wall material, utilizing its excellent film-forming properties and biocompatibility, encapsulated *Lactobacillus plantarum* within the microcapsules, providing a physical barrier for its protection.

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

Claims

1. A method for improving the stability of Lactobacillus plantarum, characterized by: The process steps for improving the stability of Lactobacillus plantarum are as follows: Step 1: Strain screening, candidate strains are isolated from traditional fermented food pickles, pickle samples are placed for dilution, the diluted pickle sample is smeared on MRS solid medium and placed in an anaerobic incubator, adjusted to 36 degrees Celsius, anaerobic culture for 36 hours, single colonies are picked for Gram staining and peroxidase test to identify lactic acid bacteria, the bacterial solution is placed in MRS liquid medium with pH 3.0 and left for 3 hours to screen strains with a survival rate greater than 85%; Step 2: Strain activation, the screened strain is inoculated into MRS liquid medium containing 25% glycerol, frozen at -65 degrees Celsius for 24 hours, then inoculated into 15 mL MRS liquid medium, cultured anaerobically at 36 degrees Celsius for 48 hours, then inoculated into 100 mL MRS liquid medium at a 5% inoculation amount, cultured anaerobically at 36 degrees Celsius for 24 hours; Step 3: Stress pretreatment, add 0.5% sodium chloride solution to the strain seed liquid, place it in a 36 degree Celsius temperature environment for 3 hours to induce the expression of osmotic stress proteins and improve cell osmotic pressure tolerance; Step 4: High-density fermentation, the pretreated fermentation strain is placed in the culture medium for Lactobacillus culture, the culture conditions are set to 36 degrees Celsius and 50% humidity in the incubator, the incubator is set to a micro-aerobic environment with oxygen concentration less than 5%, air flow is 0.2 vvm, and culture is carried out for 36 hours to OD 600nm>2.5; Step 5: Preparation of protective agent, the protective agent configuration formula is 16.29 g / 100mL skim milk, 10.85 g / 100mL trehalose, and 0.07 g / 100mL manganese sulfate, the configuration process is carried out at room temperature below 30 degrees Celsius, the protective agent pre-strain fermentation broth is mixed uniformly at a ratio of 1.5:1 and placed in the incubator for 12 hours, and the strain survival rate is greater than 85%; Step 6: Lactobacillus body concentration, the strain fermentation broth mixed with the protective agent is placed in a centrifuge for centrifugation, the supernatant is removed, the bacterial suspension is retained, and the bacterial suspension is placed in a reagent tube for freezing at -70 degrees Celsius for 6 hours, then the frozen bacterial suspension is dried; Step 7: Microcapsule encapsulation, the concentrated frozen strain dry powder is resuspended to 15 CFU / mL with sterile water, the inner wall of the microcapsule is prepared by configuring 2% sodium alginate solution and 1% gum arabic at a ratio of 1:2, sterilized and cooled to 40 degrees Celsius for freezing and concentration, the outer wall of the microcapsule is prepared by configuring 1% chitosan solution and 0.5% citric acid at a ratio of 1:1, and then the bacterial suspension freeze-dried powder is mixed with the inner wall of the microcapsule at a ratio of 1:5; Step 8: Product storage, the microcapsules are vacuum stored using aluminum foil bag vacuum packaging, and a desiccant is placed in the vacuum bag, stored in a cool and dry place with temperature ≤30 degrees Celsius and air humidity ≤35%, and stored in the dark to obtain high-stability Lactobacillus plantarum.

2. The method of claim 1, wherein the Lactobacillus plantarum is stabilized by adding a stabilizer to the Lactobacillus plantarum. In the strain screening step, the screened strain is placed in MRS medium containing 0.5% bile salt for culture, the OD 650nm value is determined, and the strain with a growth rate > 0.25 OD / h is selected.

3. The method of claim 1, wherein the Lactobacillus plantarum is stabilized by adding a stabilizer. In the strain activation step, the activated strain needs to reach an OD 650nm value of 1.2-1.

6.

4. The method of claim 1, wherein the Lactobacillus plantarum is stabilized by adding a stabilizer. In the stress pretreatment step, the strain needs to be additionally enhanced for heat resistance, and the specific steps are to warm the bacterial liquid to 43-47℃ for 60 minutes after standing to activate heat shock proteins and cold shock proteins.

5. The method of claim 1, wherein the Lactobacillus plantarum is stabilized by adding a stabilizer. In the high-density fermentation step, the reconfiguration ratio of the culture medium is glucose 26 g / L, yeast extract 26 g / L, and sodium glutamate 16 g / L, pH 6.2, and additionally adding 0.1%-0.5% tomato juice or soybean peptone.

6. The method of claim 1, wherein the Lactobacillus plantarum is stabilized by adding a stabilizer. In the bacterial cell concentration step, the dried bacteria suspension after freezing is placed in a vacuum incubator with a vacuum degree < 5 Pa, the temperature is kept at -20℃ for 36 hours, then warmed to 30℃ for 24 hours, so that the moisture content of the concentrated bacterial liquid is less than 3%, and then sealed with nitrogen and placed in a 4℃ refrigerator in the dark.

7. The method of claim 1, wherein the Lactobacillus plantarum is stabilized by adding a stabilizer. In the microcapsule packaging step, 3% CaCl2 solution is dropped by peristaltic pump to form gel beads with a diameter of 3-5 mm, the gel beads are soaked in chitosan solution, stirred for 30 minutes, and then microcapsules are collected by centrifugation.

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