Phytobacterium plantarum with bacteriostatic activity and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to plant lactobacillus with bacteriostatic activity and application of the plant lactobacillus. Wherein the plant lactobacillus with bacteriostatic activity is separated from the intestinal tract of a healthy human body, and the LV1000 fermentation filtrate freeze-dried powder is prepared through fermentation, centrifugation, filtration, ultrafiltration concentration and vacuum freeze-drying by taking inactivated staphylococcus epidermidis bacterial suspension as an inducer through a specific induction fermentation culture medium. Meanwhile, the freeze-dried powder is compounded with an embedding compound, galactooligosaccharide, beta-cyclodextrine, erythritol and the like, so that the fermented beverage capable of improving the intestinal microecology is prepared. The freeze-dried powder of the LV1000 fermentation filtrate can specifically inhibit enteropathogenic bacteria, the tea polyphenol in the embedded compound can further synergistically enhance the bacteriostatic action and exert the antioxidant effect, and the fermented beverage which is high in stability, fine and smooth in taste, free of refrigeration and capable of being directly drunk and improving the intestinal microecology is prepared.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to a plant lactobacillus with antibacterial activity and its applications. Background Technology

[0002] In recent years, the use of probiotics to regulate the gut microbiota has become a research hotspot. Among them, lactic acid bacteria are widely used in the development of functional foods due to their high safety and clear probiotic functions. However, traditional probiotic products mostly rely on live bacteria to exert their effects, and their activity is easily destroyed by processing, storage, and the gastrointestinal environment (such as gastric acid and bile), resulting in a significant decrease in the actual number of effective live bacteria reaching the intestines, making it difficult to stably exert a regulatory effect.

[0003] Furthermore, the antibacterial activity of existing probiotic fermentation products is often insufficient, and there is a lack of targeted functional enhancement methods, resulting in limited inhibitory effects against intestinal pathogens. Some products, in an effort to improve stability or palatability, add excessive additives, which may actually harm gut health or reduce consumer acceptance. Therefore, developing a strain with antibacterial activity and its derivative products holds significant promise. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a *Lactobacillus plantarum* strain with antibacterial activity and its applications. This strain is isolated from the intestines of healthy humans and, using a specific induced fermentation medium and an inactivated *Staphylococcus epidermidis* suspension as an inducer, undergoes fermentation, centrifugation, filtration, ultrafiltration concentration, and vacuum freeze-drying to obtain LV1000 fermentation filtrate freeze-dried powder. Simultaneously, this freeze-dried powder is compounded with an encapsulation complex, galactooligosaccharides, β-cyclodextrin, erythritol, etc., to prepare a fermented beverage that improves the intestinal microecology. This invention solves the problems of traditional probiotic products relying on live bacteria, requiring stringent storage conditions, and exhibiting unstable efficacy. By utilizing the high antibacterial activity of the LV1000 fermentation product, combined with the protective and synergistic effects of the encapsulation complex, and the synergistic effects of prebiotics and other components, the beverage effectively regulates the intestinal microecology, exhibits good stability, and is convenient to consume, providing a new approach for the development of functional foods for intestinal health.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a *Lactiplantibacillus plantarum* strain with antibacterial activity. This strain is *Lactiplantibacillus plantarum* LV1000, isolated from the intestines of healthy humans and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30865. It is classified as *Lactiplantibacillus plantarum*, deposited on June 5, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Furthermore, the *Lactobacillus plantarum* with antibacterial activity has the following characteristics: it is Gram-positive, and on MRS agar medium, the colonies are medium-sized, milky white, raised upwards, moist, with neat edges, and easy to pick up.

[0007] Furthermore, the antibacterial Lactobacillus plantarum is used in the preparation of fermented beverages that improve intestinal microecology in the form of LV1000 fermentation filtrate freeze-dried powder. Furthermore, the preparation method of the LV1000 fermentation filtrate freeze-dried powder includes the following steps: A1. Take Lactobacillus plantarum LV1000 strain, inoculate it into MRS liquid medium, and culture it at 37 ℃ for 18-24 h to complete the primary activation and form the primary bacterial culture. Transfer the primary bacterial culture to fresh MRS liquid medium, with the volume of the primary bacterial culture accounting for 1% of the total volume of the fresh MRS liquid medium. Culture it at 37 ℃ with shaking for 12 h to obtain the secondary seed culture. A2. The secondary seed culture obtained in step A1 is inoculated into the induction fermentation medium. The volume of the secondary seed culture accounts for 3% of the total volume of the induction fermentation medium. The fermentation conditions are controlled as follows: temperature 37 ℃, pH adjustment agent is added to control the pH value to 6, stirring speed is 150 rpm. When the fermentation has been going on for 12 h, an inducer with a volume fraction of 1% is added to the fermentation system and fermentation continues for 24 h. The total fermentation time is 36 h, and the fermentation broth is obtained. A3. Centrifuge the fermentation broth obtained in step A2 for 20 min, collect the supernatant, filter the supernatant to obtain sterile filtrate, use an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to perform tangential flow ultrafiltration on the sterile filtrate, concentrate it to one-tenth of the volume of sterile filtrate, and obtain concentrated liquid. A4. The concentrate obtained in step A3 is subjected to vacuum freeze drying under the following conditions: pre-freezing at -45 ℃ for 4 h, followed by primary drying at -20 ℃ and a vacuum degree below 0.2 mbar for 24 h, and finally desorption drying at 25 ℃ and a vacuum degree below 0.1 mbar for 8 h to obtain LV1000 fermentation filtrate freeze-dried powder.

[0008] Further, the induced fermentation medium comprises the following raw materials in the following mass ratio: peptone: yeast extract: beef extract: glucose: dipotassium hydrogen phosphate: triammonium citrate: magnesium sulfate: manganese sulfate: Tween 80: L-cysteine: L-valine = 2-3: 1-1.5: 1: 3: 0.4: 0.4: 0.04: 0.01: 0.2: 0.1: 0.1; the induced fermentation medium is prepared using distilled water, and the initial pH value of the induced fermentation medium is 6-7.

[0009] Furthermore, the inducer is an inactivated Staphylococcus epidermidis bacterial suspension with a concentration of 1×10⁻⁶. 9 CFU / mL.

[0010] Furthermore, the pH adjuster is a 5M NaOH solution.

[0011] The present invention also provides the application of *Lactobacillus plantarum* with antibacterial activity in the preparation of fermented beverages that improve intestinal microecology. The fermented beverages that improve intestinal microecology include the following raw materials in parts by weight: 1-3 parts of LV1000 fermentation filtrate freeze-dried powder, 5-15 parts of encapsulated complex, 40-60 parts of galactooligosaccharides, 0.5-2 parts of β-cyclodextrin, 20-40 parts of erythritol, and 1000 parts of purified water. The encapsulation complex comprises raw materials in the following mass ratio: resistant dextrin: tea polyphenols = 4:1; The method for preparing the embedding complex includes the following steps: i. Take pure water, heat it to 60 ℃, add resistant dextrin while stirring to obtain a uniform resistant dextrin solution with a mass fraction of 40%. Keep stirring and temperature, slowly add tea polyphenols to the resistant dextrin solution, and continue stirring for 60 min to form a mixture. ii. The mixture was homogenized twice under a high pressure homogenizer at 25 MPa, and then spray-dried at an inlet air temperature of 185 ℃ and an outlet air temperature of 85 ℃ to obtain the embedded complex.

[0012] The preparation method of the fermented beverage for improving gut microbiota includes the following steps: S1, add some purified water to the sugar dissolving pot, heat to 50 ℃, add galactooligosaccharide, erythritol and encapsulation complex in sequence while stirring, stir until completely dissolved to obtain liquid A; S2, pump liquid A into the main mixing tank, add the remaining purified water, start stirring, slowly add LV1000 fermentation filtrate freeze-dried powder and β-cyclodextrin, continue stirring for 30 min to ensure that all components are completely dissolved and dispersed to obtain liquid B; S3, under stirring, slowly add a 50% citric acid solution to solution B to obtain a mixture. Adjust the pH of the mixture to 3.8-4.2, and add purified water to the set total volume of 1000 parts to obtain solution C. S4. The liquid C is homogenized in a homogenizer at a pressure of 20-25 MPa to form liquid D. Liquid D is then sterilized using an ultra-high temperature instantaneous sterilization method. The sterilization conditions are set as follows: 135-140 ℃, held for 4-5 s. After sterilization, it is immediately cooled to below 25 ℃ and then bottled to obtain a fermented beverage that improves the intestinal microecology.

[0013] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The *Lactobacillus plantarum* LV1000 strain of this invention, isolated from the intestines of healthy humans, contains antibacterial active substances in itself and in its fermentation filtrate. These substances can specifically inhibit harmful intestinal bacteria while promoting the proliferation of beneficial bacteria, thus regulating the intestinal microecological balance from the source. Furthermore, it exhibits better human compatibility and is more easily adapted to the intestinal environment to exert its effects. Through targeted induction with inactivated *Staphylococcus epidermidis* suspension, the yield and activity of antibacterial active substances in the fermentation product are significantly increased. Combining ultrafiltration concentration and vacuum freeze-drying technology maximizes the preservation of the stability of active ingredients while avoiding the stringent dependence on storage conditions inherent in live bacteria preparations. This solves the technical pain point of traditional probiotics being easily inactivated, ensuring high antibacterial activity in the gastrointestinal environment and guaranteeing effective function.

[0014] In the application of *Lactobacillus* with antibacterial activity, the LV1000 fermentation filtrate freeze-dried powder can specifically inhibit pathogenic bacteria in the intestines. The tea polyphenols in the encapsulated complex further synergistically enhance the antibacterial effect and exert antioxidant effects. At the same time, resistant dextrin, as a prebiotic, selectively promotes the proliferation of beneficial bacteria in the intestines. Galacto-oligosaccharides and resistant dextrin synergistically enhance each other, continuously nourishing the intestinal flora. β-cyclodextrin effectively encapsulates and masks undesirable flavors, while erythritol provides a palatable sweetness. Finally, through precise acidification, homogenization, and sterilization processes, a fermented beverage with high stability, delicate taste, no refrigeration required, and direct consumption that improves the intestinal microecology is obtained. Attached Figure Description

[0015] Figure 1 Stability diagram of the fermented beverage for improving gut microbiota prepared according to the present invention; Figure 2 The figure shows the experimental results of the fermented beverage prepared in this invention to improve intestinal microecology and resist artificial gastric juice. Detailed Implementation

[0016] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0018] Example 1: This example provides a plant lactobacillus strain with antibacterial activity. The plant lactobacillus strain with antibacterial activity is strain LV1000 of Lactiplantibacillus plantarum, which was isolated from the intestine of a healthy human and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30865 and deposit date of June 5, 2024.

[0019] The *Lactobacillus plantarum* with antibacterial activity has the following characteristics: it is Gram-positive, and on MRS agar medium, the colonies are medium-sized, milky white, raised, moist, with neat edges, and easy to pick up.

[0020] The plant lactobacillus with antibacterial activity is used in the preparation of fermented beverages that improve intestinal microecology in the form of LV1000 fermentation filtrate freeze-dried powder. The method for preparing the LV1000 fermentation filtrate freeze-dried powder includes the following steps: A1. Take Lactobacillus plantarum LV1000 strain, inoculate it into MRS liquid medium, and culture it at 37 ℃ for 18 h to complete the primary activation and form the primary bacterial culture. Transfer the primary bacterial culture to fresh MRS liquid medium, with the volume of the primary bacterial culture accounting for 1% of the total volume of the fresh MRS liquid medium. Shake culture at 37 ℃ and 150 rpm for 12 h to obtain the secondary seed culture. A2. The secondary seed culture obtained in step A1 is inoculated into the induction fermentation medium. The volume of the secondary seed culture accounts for 3% of the total volume of the induction fermentation medium. The fermentation conditions are controlled as follows: temperature 37 ℃, pH value controlled by adding 5M NaOH solution, stirring speed 150 rpm. When the fermentation has been going on for 12 h, 1% of the inducer is added to the fermentation system and fermentation continues for 24 h. The total fermentation time is 36 h, and the fermentation broth is obtained. A3. The fermentation broth obtained in step A2 is centrifuged at 4 ℃ and 10000 for 20 min, and the supernatant is collected. The supernatant is filtered through 0.45 μm and 0.22 μm microporous membranes to obtain sterile filtrate. The sterile filtrate is then subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to concentrate it to one-tenth of the volume of sterile filtrate to obtain concentrated liquid. A4. The concentrate obtained in step A3 is subjected to vacuum freeze drying under the following conditions: pre-freezing at -45 ℃ for 4 h, followed by primary drying at -20 ℃ and a vacuum of 0.1 mbar for 24 h, and finally desorption drying at 25 ℃ and a vacuum of 0.05 mbar for 8 h to obtain LV1000 fermentation filtrate freeze-dried powder.

[0021] The induced fermentation medium comprises the following raw materials in the following mass ratio: peptone: yeast extract: beef extract: glucose: dipotassium hydrogen phosphate: triammonium citrate: magnesium sulfate: manganese sulfate: Tween 80: L-cysteine: L-valine = 2:1:1:3:0.4:0.4:0.04:0.01:0.2:0.1:0.1; the induced fermentation medium is prepared with distilled water, and the initial pH of the induced fermentation medium is 6; The inducer is an inactivated Staphylococcus epidermidis bacterial suspension with a concentration of approximately 1 × 10⁻⁶. 9 CFU / mL.

[0022] This embodiment also provides the application of *Lactobacillus plantarum* with antibacterial activity in the preparation of fermented beverages that improve intestinal microecology. The fermented beverages that improve intestinal microecology include the following raw materials in parts by weight: 1 part of LV1000 fermentation filtrate freeze-dried powder, 5 parts of encapsulated complex, 40 parts of galactooligosaccharides, 0.5 parts of β-cyclodextrin, 20 parts of erythritol, and 1000 parts of purified water. The encapsulation complex comprises raw materials in the following mass ratio: resistant dextrin: tea polyphenols = 4:1; The method for preparing the embedding complex includes the following steps: i. Take pure water, heat it to 60 ℃, add resistant dextrin while stirring to obtain a uniform resistant dextrin solution with a mass fraction of 40%. Keep stirring and temperature, slowly add tea polyphenols to the resistant dextrin solution, and continue stirring for 60 min to form a mixture. ii. The mixture was homogenized twice under a high pressure homogenizer at 25 MPa, and then spray-dried at an inlet air temperature of 185 ℃ and an outlet air temperature of 85 ℃ to obtain the embedded complex.

[0023] The preparation method of the fermented beverage for improving gut microbiota includes the following steps: S1, add 200 parts of purified water to the sugar dissolving pot, heat to 50 ℃, add galacto-oligosaccharide, erythritol and encapsulation complex in sequence while stirring, and stir until completely dissolved to obtain liquid A; S2, pump liquid A into the main mixing tank, add the remaining purified water, start stirring, slowly add LV1000 fermentation filtrate freeze-dried powder and β-cyclodextrin, continue stirring for 30 min to ensure that all components are completely dissolved and dispersed to obtain liquid B; S3, under stirring, slowly add a 50% citric acid solution to the feed solution B to obtain a mixture. Monitor the pH value online and adjust the pH of the mixture to 3. Add purified water to the set total volume of 1000 parts to obtain feed solution C. S4. The liquid C is homogenized in a homogenizer at a pressure of 20 MPa to form liquid D. Liquid D is then sterilized using an ultra-high temperature instantaneous sterilization method. The sterilization conditions are set as follows: 135 ℃, held for 4 s. After sterilization, it is immediately cooled to below 25 ℃ and then bottled to obtain a fermented beverage that improves the intestinal microecology.

[0024] Example 2: This example provides a plant lactobacillus strain with antibacterial activity. The plant lactobacillus strain with antibacterial activity is strain LV1000 of Lactiplantibacillus plantarum, which was isolated from the intestine of a healthy human and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30865 and deposit date of June 5, 2024.

[0025] The *Lactobacillus plantarum* with antibacterial activity has the following characteristics: it is Gram-positive, and on MRS agar medium, the colonies are medium-sized, milky white, raised, moist, with neat edges, and easy to pick up.

[0026] The plant lactobacillus with antibacterial activity is used in the preparation of fermented beverages that improve intestinal microecology in the form of LV1000 fermentation filtrate freeze-dried powder. The method for preparing the LV1000 fermentation filtrate freeze-dried powder includes the following steps: A1. Take Lactobacillus plantarum LV1000 strain, inoculate it into MRS liquid medium, and culture it at 37 ℃ for 24 h to complete the primary activation and form the primary bacterial culture. Transfer the primary bacterial culture to fresh MRS liquid medium, with the volume of the primary bacterial culture accounting for 1% of the total volume of the fresh MRS liquid medium. Culture it at 37 ℃ and 150 rpm for 12 h with shaking to obtain the secondary seed culture. A2. The secondary seed culture obtained in step A1 is inoculated into the induction fermentation medium. The volume of the secondary seed culture accounts for 3% of the total volume of the induction fermentation medium. The fermentation conditions are controlled as follows: temperature 37 ℃, pH value controlled by adding 5M NaOH solution, stirring speed 150 rpm. When the fermentation has been going on for 12 h, 1% of the inducer is added to the fermentation system and fermentation continues for 24 h. The total fermentation time is 36 h, and the fermentation broth is obtained. A3. The fermentation broth obtained in step A2 is centrifuged at 4 ℃ and 10000 for 20 min, and the supernatant is collected. The supernatant is filtered through 0.45 μm and 0.22 μm microporous membranes to obtain sterile filtrate. The sterile filtrate is then subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to concentrate it to one-tenth of the volume of sterile filtrate to obtain concentrated liquid. A4. The concentrate obtained in step A3 is subjected to vacuum freeze drying under the following conditions: pre-freezing at -45 ℃ for 4 h, followed by primary drying at -20 ℃ and a vacuum degree below 0.2 mbar for 24 h, and finally desorption drying at 25 ℃ and a vacuum degree of 0.08 mbar for 8 h to obtain LV1000 fermentation filtrate freeze-dried powder.

[0027] The preparation of the LV1000 fermentation filtrate freeze-dried powder includes: Lactobacillus plantarum LV1000 strain, MRS liquid culture medium, induced fermentation medium, inducer, buffer, and pH adjuster. The induced fermentation medium comprises the following raw materials in the following mass ratio: peptone: yeast extract: beef extract: glucose: dipotassium hydrogen phosphate: triammonium citrate: magnesium sulfate: manganese sulfate: Tween 80: L-cysteine: L-valine = 2.5:1:1:3:0.4:0.4:0.04:0.01:0.2:0.1:0.1; the induced fermentation medium is prepared with distilled water, and the initial pH value of the induced fermentation medium is 6.8; The inducer is an inactivated Staphylococcus epidermidis bacterial suspension with a concentration of approximately 1 × 10⁻⁶. 9 CFU / mL.

[0028] This embodiment also provides the application of *Lactobacillus plantarum* with antibacterial activity in the preparation of fermented beverages that improve intestinal microecology. The fermented beverages that improve intestinal microecology include the following raw materials in parts by weight: 2 parts of LV1000 fermentation filtrate freeze-dried powder, 10 parts of encapsulated complex, 50 parts of galactooligosaccharides, 1.5 parts of β-cyclodextrin, 30 parts of erythritol, and 1000 parts of purified water. The encapsulation complex comprises raw materials in the following mass ratio: resistant dextrin: tea polyphenols = 4:1; The method for preparing the embedding complex includes the following steps: i. Take pure water, heat it to 60 ℃, add resistant dextrin while stirring to obtain a uniform resistant dextrin solution with a mass fraction of 40%. Keep stirring and temperature, slowly add tea polyphenols to the resistant dextrin solution, and continue stirring for 60 min to form a mixture. ii. The mixture was homogenized twice under a high pressure homogenizer at 25 MPa, and then spray-dried at an inlet air temperature of 185 ℃ and an outlet air temperature of 85 ℃ to obtain the embedded complex.

[0029] The preparation method of the fermented beverage for improving gut microbiota includes the following steps: S1. Add 300 parts of purified water to the sugar dissolving pot and heat to 50 ℃. Add galacto-oligosaccharide, erythritol and the encapsulation complex in sequence while stirring. Stir until completely dissolved to obtain liquid A. S2, pump liquid A into the main mixing tank, add the remaining purified water, start stirring, slowly add LV1000 fermentation filtrate freeze-dried powder and β-cyclodextrin, continue stirring for 30 min to ensure that all components are completely dissolved and dispersed to obtain liquid B; S3, under stirring, slowly add a 50% citric acid solution to the feed solution B to obtain a mixture. Monitor the pH value online, adjust the pH of the mixture to 4, and add purified water to the set total amount of 1000 parts to obtain feed solution C. S4. The liquid C is homogenized in a homogenizer at a pressure of 25 MPa to form liquid D. Liquid D is then sterilized using an ultra-high temperature instantaneous sterilization method. The sterilization conditions are set as follows: 137 ℃, held for 5 s. After sterilization, it is immediately cooled to below 25 ℃ and then bottled to obtain a fermented beverage that improves the intestinal microecology.

[0030] Example 3: This example provides a plant lactobacillus strain with antibacterial activity. The plant lactobacillus strain with antibacterial activity is strain LV1000 of Lactiplantibacillus plantarum, which was isolated from the intestine of a healthy human and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30865 and deposit date of June 5, 2024.

[0031] The *Lactobacillus plantarum* with antibacterial activity has the following characteristics: it is Gram-positive, and on MRS agar medium, the colonies are medium-sized, milky white, raised, moist, with neat edges, and easy to pick up.

[0032] The plant lactobacillus with antibacterial activity is used in the preparation of fermented beverages that improve intestinal microecology in the form of LV1000 fermentation filtrate freeze-dried powder. The method for preparing the LV1000 fermentation filtrate freeze-dried powder includes the following steps: A1. Take Lactobacillus plantarum LV1000 strain, inoculate it into MRS liquid medium, and culture it at 37 ℃ for 24 h to complete the primary activation and form the primary bacterial culture. Transfer the primary bacterial culture to fresh MRS liquid medium, with the volume of the primary bacterial culture accounting for 1% of the total volume of the fresh MRS liquid medium. Culture it at 37 ℃ and 150 rpm for 12 h with shaking to obtain the secondary seed culture. A2. The secondary seed culture obtained in step A1 is inoculated into the induction fermentation medium. The volume of the secondary seed culture accounts for 3% of the total volume of the induction fermentation medium. The fermentation conditions are controlled as follows: temperature 37 ℃, pH value controlled by adding 5M NaOH solution, stirring speed 150 rpm. When the fermentation has been going on for 12 h, 1% of the inducer is added to the fermentation system and fermentation continues for 24 h. The total fermentation time is 36 h, and the fermentation broth is obtained. A3. The fermentation broth obtained in step A2 is centrifuged at 4 °C and 10,000 rpm for 20 min. The supernatant is collected and filtered sequentially through microporous membranes of 0.45 μm and 0.22 μm to obtain sterile filtrate. The sterile filtrate is then subjected to tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to concentrate it to one-tenth of the volume of the sterile filtrate, thus obtaining a concentrated solution. A4. The concentrate obtained in step A3 is subjected to vacuum freeze drying under the following conditions: pre-freezing at -45 ℃ for 4 h, followed by primary drying at -20 ℃ and a vacuum of 0.1 mbar for 24 h, and finally desorption drying at 25 ℃ and a vacuum of 0.05 mbar for 8 h to obtain LV1000 fermentation filtrate freeze-dried powder.

[0033] The preparation of the LV1000 fermentation filtrate freeze-dried powder includes: Lactobacillus plantarum LV1000 strain, MRS liquid culture medium, induced fermentation medium, inducer, buffer, and pH adjuster. The induced fermentation medium comprises the following raw materials in the following mass ratio: peptone: yeast extract: beef extract: glucose: dipotassium hydrogen phosphate: triammonium citrate: magnesium sulfate: manganese sulfate: Tween 80: L-cysteine: L-valine = 3: 1.5: 1: 3: 0.4: 0.4: 0.04: 0.01: 0.2: 0.1: 0.1; the induced fermentation medium is prepared with distilled water, and the initial pH of the induced fermentation medium is 7; The inducer is an inactivated Staphylococcus epidermidis bacterial suspension with a concentration of approximately 1 × 10⁻⁶.9 CFU / mL.

[0034] This embodiment also provides the application of *Lactobacillus plantarum* with antibacterial activity in the preparation of fermented beverages that improve intestinal microecology. The fermented beverages that improve intestinal microecology include the following raw materials in parts by weight: 3 parts of LV1000 fermentation filtrate freeze-dried powder, 15 parts of encapsulated complex, 60 parts of galactooligosaccharides, 2 parts of β-cyclodextrin, 40 parts of erythritol, and 1000 parts of purified water. The encapsulation complex comprises raw materials in the following mass ratio: resistant dextrin: tea polyphenols = 4:1; The method for preparing the embedding complex includes the following steps: i. Take pure water, heat it to 60 ℃, add resistant dextrin while stirring to obtain a uniform resistant dextrin solution with a mass fraction of 40%. Keep stirring and temperature, slowly add tea polyphenols to the resistant dextrin solution, and continue stirring for 60 min to form a mixture. ii. The mixture was homogenized twice under a high pressure homogenizer at 25 MPa, and then spray-dried at an inlet air temperature of 185 ℃ and an outlet air temperature of 85 ℃ to obtain the embedded complex.

[0035] The preparation method of the fermented beverage for improving gut microbiota includes the following steps: S1. Add 300 parts of purified water to the sugar dissolving pot and heat to 50 ℃. Add galacto-oligosaccharide, erythritol and the encapsulation complex in sequence while stirring. Stir until completely dissolved to obtain liquid A. S2, pump liquid A into the main mixing tank, add the remaining purified water, start stirring, slowly add LV1000 fermentation filtrate freeze-dried powder and β-cyclodextrin, continue stirring for 30 min to ensure that all components are completely dissolved and dispersed to obtain liquid B; S3, under stirring, slowly add 50% citric acid solution to solution B to obtain a mixture. Monitor the pH value online, adjust the pH of the mixture to 4.2, and add purified water to the set total volume of 1000 parts to obtain solution C. S4. The liquid C is homogenized in a homogenizer at a pressure of 25 MPa to form liquid D. Liquid D is then sterilized using an ultra-high temperature instantaneous sterilization method. The sterilization conditions are set as follows: 140 ℃, held for 5 s. After sterilization, it is immediately cooled to below 25 ℃ and then bottled to obtain a fermented beverage that improves the intestinal microecology.

[0036] The difference between Comparative Example 1 and Example 2 is that the Lactobacillus plantarum LV1000 was not cultured using an induced fermentation medium, while the rest of the process was exactly the same as in Example 2.

[0037] The difference between Comparative Example 2 and Example 2 is that no inducing agent was added; otherwise, they are exactly the same as Example 2.

[0038] The difference between Comparative Example 3 and Example 2 is that no embedding complex was added; otherwise, they are exactly the same as Example 2.

[0039] Experimental example: 1. Cell compatibility: Cell compatibility was tested using the CCK-8 assay. Caco-2 cells were seeded in 96-well plates and cultured until the cell confluence reached 70%-80%. Different concentrations of LV1000 bacterial suspension (diluted with sterile PBS) and LV1000 fermentation filtrate lyophilized powder solutions (reconstituted and diluted with cell culture medium) were prepared. The LV1000 fermentation filtrate lyophilized powders prepared in Examples 1-3 and Comparative Examples 1-3 were used as samples, and cell culture medium without samples was used as a blank control group. Different concentrations of samples were added to the cell wells, with 3 replicates per group, and incubated at 37°C and 5% CO2 for 24 h. After incubation, CCK-8 reagent was added to each well, and the cells were cultured for another 2-4 h. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated as: absorbance of sample group A / absorbance of control group A × 100%. Cytotoxicity was assessed, and the results are recorded in Table 1.

[0040] 2. Gastrointestinal tolerance: Artificial gastric juice with pH 3.0 and containing 0.3% pepsin, artificial intestinal juice with pH 6.8 and containing 0.3% pancreatin, and artificial bile with pH 7.0 and containing 0.3% bile salts were prepared respectively. The changes in the number of colonies of the LV1000 fermentation filtrate freeze-dried powder prepared in Examples 1-3 and Comparative Examples 1-3 before and after treatment with artificial gastric juice, artificial intestinal juice, and artificial bile were investigated by plate count method. The antibacterial activity retention rate was calculated and the results are recorded in Table 1.

[0041] Table 1: Results of Cell Compatibility and Gastrointestinal Tolerance Tests

[0042] Table 1 shows that the lyophilized powder of LV1000 fermentation filtrate prepared in Examples 1-3 had a higher cell survival rate than the suspension of Bacillus plantarum LV1000, indicating that induced culture can enhance cell compatibility and reduce potential adverse effects on cells. In the gastrointestinal tolerance test, the retention rate of antibacterial activity after induced culture in Examples 1-3 was significantly higher than that of the suspension of Bacillus plantarum LV1000, indicating that the lyophilized powder of LV1000 fermentation filtrate after induced culture can better exert its antibacterial effect.

[0043] 3. Listeria, Salmonella, Escherichia coli, and Staphylococcus aureus were streaked onto LB agar medium and incubated at 37 °C for 18-24 hours to obtain single colonies. Single colonies were then picked and inoculated onto LB liquid medium and incubated at 37 °C with shaking. Listeria, Salmonella, and Escherichia coli were incubated for 12 hours, and Staphylococcus aureus for 16 hours. The concentration of each bacterial culture was adjusted to 1×10⁻⁶. 6 -1×10 7 CFU / mL; the adjusted bacterial suspension was evenly spread on LB plates, and after the surface dried, sterile Oxford cups were placed in them. *Lactobacillus plantarum* LV1000 bacterial suspension, blank PBS control group, and lyophilized LV1000 fermentation filtrate powder prepared in Examples 1-3 and Comparative Examples 1-3 were added to the Oxford cups respectively; the plates were first diffused overnight at 4 ℃, and then incubated at 37 ℃ for 12-16 h. The diameter of the inhibition zone was observed and measured. All experiments were repeated 3 times, and the results are recorded in Table 2.

[0044] Table 2: Results of antibacterial performance

[0045] Table 2 shows that the diameter of the inhibition zone in Comparative Examples 1-2 was significantly smaller than that in Examples 1-3, indicating that the antibacterial ability of the LV1000 fermentation filtrate freeze-dried powder was effectively improved after induced fermentation culture, and it could better inhibit the growth of these common pathogenic bacteria.

[0046] 4. Sensory testing: Sensory and microbiological indicators were tested on the fermented beverages prepared in Examples 1-3 and Comparative Examples 1-3 to improve the intestinal microecology. The results are shown in Table 3.

[0047] Table 3: Results of Sensory and Microbiological Indicators Tests

[0048] Table 3 shows that the sensory and microbiological indicators of the fermented beverages prepared in Examples 1-3 that improve gut microbiota all meet the standards. However, adding the LV1000 fermentation filtrate freeze-dried powder directly to the beverage without induced fermentation culture will affect the taste. This indicates that the encapsulation complex added in this invention masks the unpleasant flavor that the LV1000 fermentation filtrate freeze-dried powder may bring. The fermentation culture of the induced fermentation medium not only improves the activity, but also has a positive effect on improving the flavor and color of the final beverage.

[0049] Figure 1 The results show that the antibacterial activity retention rate of the fermented beverage for improving gut microbiota prepared in Example 2 decreased slowly over time within 3 months, and remained at a high level after 3 months, indicating that the raw materials and preparation process of the present invention can maintain long-term antibacterial activity. Figure 2This describes the survival rate of *Lactobacillus plantarum* LV1000 in simulated gastric juice of the fermented beverage prepared in Example 2 of this invention to improve intestinal microecology. This indicates that the live bacteria in the fermented beverage can successfully pass through the acidic environment of the stomach and reach the intestines to exert their probiotic function.

[0050] In summary, this invention uses *Lactobacillus plantarum* LV1000 isolated from the intestines of healthy humans as its core. Through induced fermentation and precise separation and purification techniques, a freeze-dried powder of LV1000 fermentation filtrate rich in highly active antibacterial components is prepared. This powder not only retains excellent antibacterial activity but also exhibits improved cell compatibility after processing, enabling it to function stably in the gastrointestinal environment. Based on this, the freeze-dried powder is scientifically compounded with a specially formulated resistant dextrin-tea polyphenol encapsulation complex, galactooligosaccharides, and other functional components. Through an optimized preparation process, a fermented beverage that improves the intestinal microecology is developed. Specifically, the LV1000 fermentation filtrate freeze-dried powder specifically inhibits harmful intestinal bacteria; the encapsulation complex provides targeted intestinal protection for the active ingredients and synergistically enhances the effects of the antioxidant properties of tea polyphenols and the prebiotic properties of resistant dextrin; while galactooligosaccharides and other components further promote the proliferation of beneficial bacteria and optimize the palatability of the beverage. The entire technical solution achieves a dual improvement in functionality and practicality through the deep integration of strain characteristics, fermentation process, encapsulation technology and formulation design, providing innovative ideas and practical paths for the development of functional foods that regulate intestinal microecology.

[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A plant lactobacillus with antibacterial activity, characterized in that, The *Lactobacillus plantarum* strain with antibacterial activity is *Lactobacillus plantarum* LV1000, with accession number CGMCC No.30865. The *Lactobacillus plantarum* with antibacterial activity has the following characteristics: it is Gram-positive, and on MRS agar medium, the colonies are medium-sized, milky white, raised, moist, with neat edges, and easy to pick up.

2. The application of *Lactobacillus plantarum* with antibacterial activity according to claim 1, characterized in that, The *Lactobacillus plantarum* with antibacterial activity is used to prepare fermented beverages that improve the intestinal microecology; The *Lactobacillus plantarum* with antibacterial activity is in the form of LV1000 fermentation filtrate freeze-dried powder in the preparation of fermented beverages that improve intestinal microecology. The method for preparing the LV1000 fermentation filtrate freeze-dried powder includes the following steps: A1. Inoculate Lactobacillus plantarum LV1000 strain into MRS liquid medium and culture to complete the first-stage activation, forming the first-stage bacterial solution. Culture the first-stage bacterial solution again to obtain the second-stage seed solution. A2. The secondary seed liquid obtained in step A1 is inoculated into the induction fermentation medium for induction fermentation culture, and then an inducer is added to continue fermentation to obtain the fermentation broth. A3. Centrifuge the fermentation broth obtained in step A2, collect the supernatant, filter the supernatant to obtain a sterile filtrate, ultrafilter, concentrate, and obtain a concentrated solution. A4. The concentrate obtained in step A3 is subjected to vacuum freeze-drying to obtain LV1000 fermentation filtrate freeze-dried powder.

3. The application of *Lactobacillus plantarum* with antibacterial activity according to claim 2, characterized in that, In step A2, the induced fermentation medium comprises the following raw materials in the following mass ratio: peptone: yeast extract: beef extract: glucose: dipotassium hydrogen phosphate: triammonium citrate: magnesium sulfate: manganese sulfate: Tween 80: L-cysteine: L-valine = 2-3: 1-1.5: 1: 3: 0.4: 0.4: 0.04: 0.01: 0.2: 0.1: 0.1; the induced fermentation medium is prepared with distilled water, and the initial pH value of the induced fermentation medium is 6-7.

4. The application of *Lactobacillus plantarum* with antibacterial activity according to claim 2, characterized in that, In step A2, the inducer is an inactivated Staphylococcus epidermidis bacterial suspension with a concentration of 1×10⁻⁶. 9 CFU / mL.

5. The application of *Lactobacillus plantarum* with antibacterial activity according to claim 2, characterized in that, In step A2, the conditions for induced fermentation are controlled as follows: temperature 37 ℃, pH value controlled at 6, and after 12 h of fermentation, an inducer is added and fermentation continues for another 24 h, with a total fermentation time of 36 h.

6. The application of *Lactobacillus plantarum* with antibacterial activity according to claim 2, characterized in that, The fermented beverage for improving gut microbiota includes the following ingredients in parts by weight: 1-3 parts of LV1000 fermentation filtrate freeze-dried powder, 5-15 parts of encapsulated complex, 40-60 parts of galactooligosaccharides, 0.5-2 parts of β-cyclodextrin, 20-40 parts of erythritol, and 1000 parts of purified water. The encapsulation complex comprises raw materials in the following mass ratio: resistant dextrin: tea polyphenols = 4:1; The method for preparing the embedding complex includes the following steps: i. Take pure water, heat it, add resistant dextrin while stirring to obtain a resistant dextrin solution, keep stirring and temperature, slowly add tea polyphenols, and continue stirring to form a mixture; ii. Homogenize the mixture and spray dry to obtain the encapsulated complex.

7. The application of *Lactobacillus plantarum* with antibacterial activity according to claim 2, characterized in that, The preparation method of the fermented beverage for improving gut microbiota includes the following steps: S1, take a portion of pure water, heat it, and add galacto-oligosaccharide, erythritol and the encapsulation complex in sequence while stirring. Stir until completely dissolved to obtain solution A; S2, pump liquid A into the main mixing tank, add the remaining purified water, start stirring, slowly add LV1000 fermentation filtrate freeze-dried powder and β-cyclodextrin, and continue stirring to obtain liquid B; S3, under stirring, slowly add citric acid solution to solution B to obtain a mixture. Adjust the pH of the mixture to 3.8-4.2 to obtain solution C. S4. Homogenize liquid C to form liquid D. Sterilize liquid D and fill it to obtain a fermented beverage that improves the intestinal microecology.

Images