Probiotics for improving intestinal health and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to probiotics for improving intestinal health and application thereof. Wherein the probiotics for improving the intestinal health are a Lactobacillus plantarum LV1000 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.30865, and the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2024; through unique activation culture, streak separation and bacterial colony identification steps, high activity and high purity of the strain are ensured; based on the application of the strain, two product forms of probiotic microcapsules and probiotic compositions are developed, the probiotic microcapsules adopt sodium alginate, calcium lactate and other raw materials, the intestinal colonization ability of the strain is effectively improved, and the synergistic interaction of oxidation resistance and immunoregulation is achieved by combining rosa roxburghii tratt freeze-dried powder; and the probiotic composition can comprehensively regulate the balance of intestinal flora and improve the digestive function and intestinal motility.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to a probiotic for improving gut health and its applications. Background Technology

[0002] As the largest digestive and immune organ in the human body, the gut microbiota balance is crucial for nutrient absorption, immune regulation, and disease prevention. Most people experience varying degrees of gut health problems, with functional constipation and chronic diarrhea being particularly prominent. Gut health management has become a pressing public health issue that needs to be addressed.

[0003] Probiotics, as a class of live microorganisms, can exert beneficial effects on host health when ingested in sufficient quantities, and their application in the field of gut health has attracted much attention. Currently, *Lactobacillus plantarum* has become a research hotspot due to its acid and bile salt resistance, antimicrobial peptide production, and immune regulation. However, existing probiotic strains still have significant limitations: First, the colonization efficiency of *Lactobacillus plantarum* traditionally isolated from fermented foods in the human gut is generally low. Clinical studies show that most strains are excreted in feces within 72 hours after oral administration, making it difficult to maintain their effects. Second, some strains have insufficient resistance to gastric acid and bile salts, resulting in a significant decrease in the number of live bacteria after passing through the upper digestive tract. For example, the survival rate of conventional *Lactobacillus plantarum* after exposure to a gastric acid environment at pH 2.0 for 2 hours is less than 10%. Third, their functions are often limited, with most strains targeting only a single intestinal symptom and lacking synergistic effects on gut microbiota, barrier function, and immune regulation. Therefore, directly screening new probiotic strains with natural adaptability from the human gut and developing probiotic resources with high colonization, strong tolerance, and multiple gut health improvement functions has become a pressing technical challenge in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a probiotic for improving gut health and its applications. The probiotic for improving gut health is *Lactobacillus plantarum* LV1000 strain (CGMCC No. 30865), which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2024. Through unique activation culture, streak plating, and colony identification procedures, the high activity and purity of the strain are ensured. Based on this strain, two product forms have been developed: probiotic microcapsules and probiotic compositions. The probiotic microcapsules are prepared using sodium alginate, calcium lactate, and other raw materials through specific emulsification and gelation processes, effectively enhancing the intestinal colonization ability of the strain. Combined with freeze-dried prickly pear powder, they achieve synergistic effects of antioxidant and immunomodulatory effects. The probiotic compositions are prepared by precisely proportioning freeze-dried *Lactobacillus plantarum* powder with prebiotics and excipients through a mixing and molding process, which can comprehensively regulate the balance of intestinal flora and improve digestive function and intestinal motility. This invention provides a complete technical solution from strain screening to product preparation, offering an effective solution for gut health.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a probiotic for improving gut health. The probiotic for improving gut health is obtained by culturing Lactobacillus plantarum strain LV1000 (hereinafter referred to as LV1000 strain), which was isolated from the intestine of a healthy human body and has the accession number CGMCC No. 30865. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2024.

[0007] The process of culturing and processing probiotics to improve gut health includes the following steps:

[0008] A1. Activation culture: Take the preserved LV1000 strain and inoculate it into a sterile test tube of MRS liquid medium. Place it in a constant temperature incubator at 30-37℃ and culture it in a facultative anaerobic manner for 18-24 h to restore the activity of the strain and obtain the activated bacteria.

[0009] A2, streak isolation: streak the activated bacteria on the surface of MRS agar medium in sections to ensure that the strains are evenly distributed on the surface of the medium. After streaking, invert the plate and place it in an incubator at 30-37℃ for facultative anaerobic culture for 48 hours.

[0010] A3. Colony identification: After the culture is completed, the colonies growing on the plate are observed morphologically to screen for target strains. The LV1000 strain grows well on MRS agar medium, forming medium-sized, milky-white colonies that are raised, moist, and have neat edges. They are easy to pick up with an inoculation loop. Further Gram staining identification of the strain showed a positive result, yielding a probiotic with good activity to improve intestinal health.

[0011] The present invention also provides an application of probiotics for improving gut health, wherein the application is to prepare probiotic microcapsules for improving gut health using probiotics for improving gut health;

[0012] Furthermore, the probiotic microcapsules comprise the following raw materials in parts by weight: 5-15 parts of *Lactobacillus plantarum* suspension, 2-6 parts of freeze-dried prickly pear powder, 1-3 parts of sodium alginate, 0.5-2 parts of calcium lactate, 20-40 parts of soybean oil, and 0.1-0.5 parts of Span80.

[0013] The processing procedure for the freeze-dried prickly pear powder is as follows: take prickly pears, wash and drain them, chop them, remove the seeds and impurities, and freeze them at -20℃. After freezing and pre-cooling, place them in a freeze dryer to dry for 72 hours. After the prickly pears are dried, take them out, grind them into powder, pass them through 100 mesh, and obtain freeze-dried prickly pear powder. Store it in a refrigerator at 4℃ for later use.

[0014] Preparation of the *Lactobacillus plantarum* suspension: Probiotics that improve intestinal health were inoculated into MRS medium and cultured facultatively at 35°C for 20 h. After the culture was completed, the bacterial cells were collected by centrifugation and washed with sterile physiological saline to prepare the *Lactobacillus plantarum* suspension.

[0015] The preparation method of the probiotic microcapsules includes the following steps:

[0016] (1) Thoroughly mix sodium alginate, calcium lactate and Bacillus plantarum suspension, and then...

[0017] Pour into a mixed solution containing soybean oil and Span80, stir magnetically to form a stable emulsion system;

[0018] (2) Add the freeze-dried prickly pear powder to the emulsion system and stir to evenly disperse the nutrients in the freeze-dried prickly pear powder in the emulsion to form a mixture;

[0019] (3) Slowly add glacial acetic acid to the mixture to adjust the pH of the system to 4.5-5.0, and carry out gelation for 35 minutes to form a microcapsule structure encapsulating the LV1000 strain;

[0020] (4) Add sterilized 1% Tween solution to the microcapsule structure. The mass ratio of Tween solution to microcapsule is 1:1.5-2. This promotes the precipitation of microcapsules. Let stand for 2 hours. After the emulsion forms a three-layer separation state, carefully discard the upper oil phase. Transfer the aqueous phase containing microcapsules to a centrifuge tube and centrifuge at 5000 rpm for 5 min. Discard the supernatant, wash, and remove residual emulsifier and impurities to obtain probiotic microcapsules.

[0021] The present invention also provides an application of probiotics for improving gut health, wherein the application is to prepare a probiotic composition for improving gut health using probiotics for improving gut health;

[0022] Further, the probiotic composition comprises the following raw materials in parts by weight: 0.2-2 parts of freeze-dried Lactobacillus plantarum powder, 5-15 parts of fructooligosaccharides, 3-10 parts of inulin, 2-8 parts of resistant dextrin, 1-5 parts of microcrystalline cellulose, and 0.1-0.5 parts of magnesium stearate.

[0023] The method for preparing the probiotic composition includes the following steps:

[0024] (a) Preparation of freeze-dried Bacillus plantarum powder: Probiotics that improve intestinal health were inoculated into MRS liquid medium and cultured in a facultative anaerobic incubator at 35℃ for 20 h. The bacterial solution was transferred to a sterile centrifuge tube and centrifuged at 4℃. The supernatant was discarded, the bacterial cells were collected, and the bacterial cells were washed with sterile physiological saline. Then, the cells were freeze-dried. The freeze dryer was pre-frozen at -40℃ for 2 hours and then dried at a vacuum of 10 Pa and a sublimation temperature of -20℃ for 12 h to obtain freeze-dried Bacillus plantarum powder.

[0025] (b) Weigh out fructooligosaccharides, inulin, resistant dextrin, microcrystalline cellulose and magnesium stearate, mix them initially to form a mixed excipient, add the freeze-dried Lactobacillus plantarum powder to the mixed excipient, transfer it to a three-dimensional mixer, stir and mix for 30 minutes to ensure that the strain and each excipient are in full contact and evenly dispersed. After mixing, directly package the material through an aseptic dispensing device to make a probiotic composition.

[0026] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0027] The LV1000 strain in this invention originates from the gut of healthy humans, naturally adapted to the intestinal environment, exhibiting excellent resistance to gastric acid and bile salts, and a high survival rate. This high survival rate ensures that the strain reaches the gut in large quantities, fully exerting its functions in regulating gut microbiota and promoting digestion and absorption, laying a solid foundation for improving gut health. An optimized culture process, with each step from activation to identification working in close coordination, guarantees high activity and purity of the strain. The activation step allows the strain to quickly regain its growth capacity; streak plating achieves purification culture; morphological observation and Gram staining identification ensure the acquisition of the target strain, providing high-quality bacterial resources for subsequent applications and improving the stability and reliability of product efficacy. For the application of probiotics to improve gut health, the strain is encapsulated in a sodium alginate-calcium lactate gel system, combined with the functional enhancement of prickly pear freeze-dried powder, significantly improving product performance. The microcapsule structure effectively protects the bacterial strains from the harsh gastrointestinal environment. Simulated digestion experiments show that the colonization rate of microencapsulated strains in the intestine is significantly higher than that of unencapsulated strains. The antioxidant components in the freeze-dried prickly pear powder work synergistically with the bacterial strains to enhance intestinal antioxidant capacity and immune regulation, expanding the product's health benefits. Using a scientifically formulated probiotic composition, with the LV1000 strain as the core, combined with prebiotics and excipients, it achieves multi-dimensional improvement in intestinal health, improves bowel movements in patients with functional constipation, and effectively relieves discomfort symptoms such as bloating and abdominal pain. It has a significant comprehensive regulatory effect on intestinal flora balance, digestive function, and intestinal motility. Attached Figure Description

[0028] Figure 1 Image showing the successful cultivation of the LV1000 strain of this invention;

[0029] Figure 2 This is a diagram of a continuous simulated gastrointestinal digestion experiment of the LV1000 strain, probiotic microcapsules, and probiotic composition of the present invention.

[0030] Figure 3 This is a diagram illustrating the enteric coagulation experiment of the LV1000 strain, probiotic microcapsules, and probiotic composition of the present invention. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] Example 1: This example provides a probiotic for improving gut health. The probiotic for improving gut health is obtained by culturing and treating Lactobacillus plantarum LV1000 strain with accession number CGMCC No. 30865. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2024.

[0034] The process of culturing and processing probiotics to improve gut health includes the following steps:

[0035] A1, Activation culture: Take the preserved LV1000 strain and inoculate it into a sterile test tube of MRS liquid medium. Place it in a constant temperature incubator at 37℃ and culture it in a facultative anaerobic manner for 24 hours to restore the activity of the strain and obtain the activated bacteria.

[0036] A2, streak separation: streak the activated bacteria on the surface of MRS agar medium in sections to ensure that the strains are evenly distributed on the surface of the medium. After streaking, invert the plate and place it in a 37°C incubator for facultative anaerobic culture for 48 h.

[0037] A3. Colony identification: After the culture is completed, the colonies growing on the plate are observed morphologically to screen for target strains. The LV1000 strain grows well on MRS agar medium, forming medium-sized, milky-white colonies that are raised, moist, and have neat edges. They are easy to pick up with an inoculation loop. Further Gram staining identification of the strain showed a positive result, yielding a probiotic with good activity to improve intestinal health.

[0038] This embodiment also provides an application of probiotics for improving gut health, wherein the application is to prepare probiotic microcapsules for improving gut health using probiotics that improve gut health;

[0039] The probiotic microcapsules comprise the following raw materials in parts by weight: 5 parts of *Lactobacillus plantarum* suspension, 2 parts of freeze-dried prickly pear powder, 1 part of sodium alginate, 0.5 parts of calcium lactate, 20 parts of soybean oil, and 0.1 parts of Span80.

[0040] The processing procedure for the freeze-dried prickly pear powder is as follows: take prickly pears, wash and drain them, chop them, remove the seeds and impurities, and freeze them at -20℃. After freezing, place them in a freeze dryer to dry for 72 hours. After drying, take them out, grind them into powder, pass them through 100 mesh, and obtain freeze-dried prickly pear powder. Store them in a refrigerator at 4℃ for later use.

[0041] The preparation process of the *Lactobacillus plantarum* bacterial suspension is as follows: Probiotics for improving intestinal health are inoculated into MRS medium and cultured facultatively at 35 ℃ for 20 h. After culture, the bacterial cells are collected by centrifugation at 5000 rpm for 10 min at 4 ℃, washed twice with sterile physiological saline, and the bacterial suspension is prepared to a concentration of 1×10⁻⁶. 9 CFU / mL.

[0042] The preparation method of the probiotic microcapsules includes the following steps:

[0043] (1) Thoroughly mix sodium alginate, calcium lactate and plant lactobacillus suspension, then pour it into a mixed solution containing soybean oil and Span80, and stir magnetically at 350 rpm for 10 min to form a stable emulsion system.

[0044] (2) Add the freeze-dried prickly pear powder to the emulsion system and continue stirring at 350 rpm for 5 min to evenly disperse the nutrients in the freeze-dried prickly pear powder in the emulsion to form a mixture;

[0045] (3) Slowly add glacial acetic acid to the mixture to adjust the pH of the system to 4.5 and carry out gelation. This process lasts for 35 minutes to form microcapsules encapsulating the LV1000 strain.

[0046] (4) Add sterilized 1% Tween solution to the microcapsules. The mass ratio of Tween solution to microcapsules is 1:1.5 to promote microcapsule precipitation. Let the emulsion stand for 2 hours. After the emulsion forms a three-layer separation state, carefully discard the upper oil phase. Transfer the aqueous phase containing microcapsules to a centrifuge tube and centrifuge at 5000 rpm for 5 minutes. Discard the supernatant. Wash the collected microcapsules twice with 1% Tween solution to remove residual emulsifier and impurities, and obtain probiotic microcapsules.

[0047] This embodiment also provides an application of probiotics for improving gut health, wherein the application is to prepare a probiotic composition for improving gut health using probiotics for improving gut health;

[0048] The probiotic composition comprises the following raw materials in parts by weight: 0.2 parts of freeze-dried Lactobacillus plantarum powder, 5 parts of fructooligosaccharides, 3 parts of inulin, 2 parts of resistant dextrin, 1 part of microcrystalline cellulose, and 0.1 parts of magnesium stearate.

[0049] (a) Preparation of freeze-dried *Lactobacillus plantarum* bacterial powder: LV1000 strain was inoculated into MRS liquid medium and cultured facultatively in a 35℃ constant temperature incubator for 20 h to obtain bacterial suspension. The bacterial suspension was transferred to sterile centrifuge tubes and centrifuged at 5000 rpm for 10 min at 4℃. The supernatant was discarded, and the bacterial cells were collected. The bacterial cells were washed 2-3 times with sterile physiological saline, followed by freeze-drying. The pre-freezing temperature of the freeze dryer was set to -40℃ for 2 h, and then dried under vacuum of 10 Pa and sublimation temperature of -20℃ for 12 h to obtain freeze-dried *Lactobacillus plantarum* bacterial powder. The viable cell content in the bacterial powder was determined by viable cell counting to ensure that the viable cell count of LV1000 strain per gram of bacterial powder met the requirement of 1×10⁻⁶. 10 Requirements for CFU / g;

[0050] (b) Weigh out fructooligosaccharides, inulin, resistant dextrin, microcrystalline cellulose and magnesium stearate, mix them to form a mixed excipient, add the freeze-dried Lactobacillus plantarum powder to the mixed excipient, transfer it to a three-dimensional mixer, set the speed of the three-dimensional mixer to 60 rpm and the mixing time to 30 min, so that the strain and each excipient can be fully contacted and evenly dispersed. After the mixing is completed, package it to obtain the probiotic composition.

[0051] Example 2: This example provides a probiotic for improving gut health. The probiotic for improving gut health is obtained by culturing and treating Lactobacillus plantarum LV1000 strain with accession number CGMCC No. 30865. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2024.

[0052] The process of culturing and processing probiotics to improve gut health includes the following steps:

[0053] A1, Activation culture: Take the preserved LV1000 strain and inoculate it into a sterile test tube of MRS liquid medium. Place it in a constant temperature incubator at 30℃ and culture it in a facultative anaerobic manner for 20 h to restore the activity of the strain and obtain the activated bacteria.

[0054] A2, streak isolation: streak the activated bacteria on the surface of MRS agar medium in sections to ensure that the strains are evenly distributed on the surface of the medium. After streaking, invert the plate and place it in a 32°C incubator for facultative anaerobic culture for 48 h.

[0055] A3. Colony identification: After the culture is completed, the colonies growing on the plate are observed morphologically to screen for target strains. The LV1000 strain grows well on MRS agar medium, forming medium-sized, milky-white colonies that are raised, moist, and have neat edges. They are easy to pick up with an inoculation loop. Further Gram staining identification of the strain showed a positive result, yielding a probiotic with good activity to improve intestinal health.

[0056] This embodiment also provides an application of probiotics for improving gut health, wherein the application is to prepare probiotic microcapsules for improving gut health using probiotics that improve gut health;

[0057] The probiotic microcapsules comprise the following raw materials in parts by weight: 10 parts of *Lactobacillus plantarum* suspension, 3 parts of freeze-dried prickly pear powder, 2 parts of sodium alginate, 1 part of calcium lactate, 30 parts of soybean oil, and 0.3 parts of Span80.

[0058] The processing procedure for the freeze-dried prickly pear powder is as follows: take prickly pears, wash and drain them, chop them, remove the seeds and impurities, and freeze them at -20℃. After freezing, place them in a freeze dryer to dry for 72 hours. After drying, take them out, grind them into powder, pass them through 100 mesh, and obtain freeze-dried prickly pear powder. Store them in a refrigerator at 4℃ for later use.

[0059] The preparation process of the *Lactobacillus plantarum* bacterial suspension is as follows: Probiotics that improve intestinal health are inoculated into MRS medium and cultured facultatively at 35℃ for 20 h. After culture, the bacterial cells are collected by centrifugation at 5000 rpm for 10 min at 4℃. The cells are washed twice with sterile physiological saline to prepare a bacterial suspension with a concentration of 1×10⁻⁶. 9 CFU / mL.

[0060] The preparation method of the probiotic microcapsules includes the following steps:

[0061] (1) Thoroughly mix sodium alginate, calcium lactate and plant lactobacillus suspension, then pour it into a mixed solution containing soybean oil and Span80, stir magnetically at 350 rpm for 10 min to form a stable emulsion system;

[0062] (2) Add the freeze-dried prickly pear powder to the emulsion system and continue stirring at 350 rpm for 5 min to evenly disperse the nutrients in the freeze-dried prickly pear powder in the emulsion to form a mixture;

[0063] (3) Slowly add glacial acetic acid to the mixture to adjust the pH of the system to 5.0 and carry out gelation. This process lasts for 35 minutes to form microcapsules encapsulating the LV1000 strain.

[0064] (4) Add sterilized 1% Tween solution to the microcapsules. The mass ratio of Tween solution to microcapsules is 1:1.5 to promote the precipitation of microcapsules. Let the emulsion stand for 2 hours. After the emulsion forms a three-layer separation state, carefully discard the upper oil phase. Transfer the aqueous phase containing microcapsules to a centrifuge tube and centrifuge at 5000 rpm for 5 minutes. Discard the supernatant. Wash the collected microcapsules twice with 1% Tween solution to remove residual emulsifier and impurities to obtain probiotic microcapsules.

[0065] This embodiment also provides an application of probiotics for improving gut health, wherein the application is to prepare a probiotic composition for improving gut health using probiotics for improving gut health;

[0066] The probiotic composition comprises the following raw materials in parts by weight: 1.5 parts of freeze-dried Lactobacillus plantarum powder, 10 parts of fructooligosaccharides, 8 parts of inulin, 6 parts of resistant dextrin, 3 parts of microcrystalline cellulose, and 0.3 parts of magnesium stearate.

[0067] (a) Processing of *Lactobacillus plantarum* freeze-dried bacterial powder: LV1000 strain was inoculated into MRS liquid medium and cultured facultatively in a 35℃ constant temperature incubator for 20 h to obtain bacterial suspension. The bacterial suspension was transferred to sterile centrifuge tubes and centrifuged at 5000 rpm for 10 min at 4℃. The supernatant was discarded, and the bacterial cells were collected. The bacterial cells were washed three times with sterile physiological saline, followed by freeze-drying. The pre-freezing temperature of the freeze dryer was set to -40℃ for 2 h, and then dried for 12 h under a vacuum of 10 Pa and a sublimation temperature of -20℃ to obtain *Lactobacillus plantarum* freeze-dried bacterial powder. The viable cell content in the bacterial powder was determined by viable cell counting to ensure that the viable cell count of LV1000 strain per gram of bacterial powder met the requirement of 5 × 10⁻⁶. 10 Requirements for CFU / g;

[0068] (b) Weigh out fructooligosaccharides, inulin, resistant dextrin, microcrystalline cellulose and magnesium stearate, mix them to form a mixed excipient, add the freeze-dried Lactobacillus plantarum powder to the mixed excipient, transfer it to a three-dimensional mixer, set the speed of the three-dimensional mixer to 60 rpm and the mixing time to 30 min, so that the strain and each excipient can be fully contacted and evenly dispersed. After the mixing is completed, package it to obtain the probiotic composition.

[0069] Example 3: This example provides a probiotic for improving gut health. The probiotic for improving gut health is obtained by culturing and treating Lactobacillus plantarum LV1000 strain with accession number CGMCC No. 30865. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2024.

[0070] The process of culturing and processing probiotics to improve gut health includes the following steps:

[0071] A1. Activation culture: Take the preserved LV1000 strain and inoculate it into a sterile test tube of MRS liquid medium. Place it in a 30 ℃ constant temperature incubator and culture it in a facultative anaerobic manner for 18 h to restore the activity of the strain and obtain the activated bacteria.

[0072] A2, streak isolation: streak the activated bacteria on the surface of MRS agar medium in sections to ensure that the strains are evenly distributed on the surface of the medium. After streaking, invert the plate and place it in a 30 ℃ incubator for facultative anaerobic culture for 48 h.

[0073] A3. Colony identification: After the culture is completed, the colonies growing on the plate are observed morphologically to screen for target strains. The LV1000 strain grows well on MRS agar medium, forming medium-sized, milky-white colonies that are raised, moist, and have neat edges. They are easy to pick up with an inoculation loop. Further Gram staining identification of the strain showed a positive result, yielding a probiotic with good activity to improve intestinal health.

[0074] This embodiment also provides an application of probiotics for improving gut health, wherein the application is to prepare probiotic microcapsules for improving gut health using probiotics that improve gut health;

[0075] The probiotic microcapsules comprise the following raw materials in parts by weight: 15 parts of *Lactobacillus plantarum* suspension, 6 parts of freeze-dried prickly pear powder, 3 parts of sodium alginate, 2 parts of calcium lactate, 40 parts of soybean oil, and 0.5 parts of Span80.

[0076] The processing procedure for the freeze-dried prickly pear powder is as follows: take prickly pears, wash and drain them, chop them, remove the seeds and impurities, and freeze them at -20℃. After freezing, place them in a freeze dryer to dry for 72 hours. After drying, take them out, grind them into powder, pass them through 100 mesh, and obtain freeze-dried prickly pear powder. Store them in a refrigerator at 4℃ for later use.

[0077] The preparation process of the *Lactobacillus plantarum* bacterial suspension is as follows: Probiotics for improving intestinal health are inoculated into MRS medium and cultured facultatively at 35 ℃ for 20 h. After culture, the bacterial cells are collected by centrifugation at 5000 rpm for 10 min at 4 ℃, washed three times with sterile physiological saline, and the bacterial suspension is prepared to a concentration of 1×10⁻⁶. 9 CFU / mL.

[0078] The preparation method of the probiotic microcapsules includes the following steps:

[0079] (1) Thoroughly mix sodium alginate, calcium lactate and plant lactobacillus suspension, then pour it into a mixed solution containing soybean oil and Span80, and stir magnetically at 350 rpm for 10 min to form a stable emulsion system.

[0080] (2) Add the freeze-dried prickly pear powder to the emulsion system and continue stirring at 350 rpm for 5 min to evenly disperse the nutrients in the freeze-dried prickly pear powder in the emulsion to form a mixture;

[0081] (3) Slowly add glacial acetic acid to the mixture to adjust the pH of the system to 5.0 and carry out gelation. This process lasts for 35 minutes to form microcapsules encapsulating the LV1000 strain.

[0082] (4) Add sterilized 1% Tween solution to the microcapsules. The mass ratio of Tween solution to microcapsules is 1:2 to promote the precipitation of microcapsules. Let the emulsion stand for 2 hours. After the emulsion forms a three-layer separation state, carefully discard the upper oil phase. Transfer the aqueous phase containing microcapsules to a centrifuge tube and centrifuge at 5000 rpm for 5 minutes. Discard the supernatant. Wash the collected microcapsules twice with 1% Tween solution to remove residual emulsifier and impurities, and obtain probiotic microcapsules.

[0083] This embodiment also provides an application of probiotics for improving gut health, wherein a probiotic composition for improving gut health is prepared using probiotics for improving gut health;

[0084] The probiotic composition comprises the following raw materials in parts by weight: 2 parts of freeze-dried Lactobacillus plantarum powder, 15 parts of fructooligosaccharides, 10 parts of inulin, 8 parts of resistant dextrin, 5 parts of microcrystalline cellulose, and 0.5 parts of magnesium stearate.

[0085] (a) Preparation of freeze-dried *Lactobacillus plantarum* powder: Probiotics for improving intestinal health were inoculated into MRS liquid medium and cultured facultatively in a 35℃ constant temperature incubator for 20 h to obtain bacterial suspension. The bacterial suspension was transferred to sterile centrifuge tubes and centrifuged at 5000 rpm for 10 min at 4℃. The supernatant was discarded, and the bacterial cells were collected. The bacterial cells were washed three times with sterile physiological saline and then freeze-dried. The pre-freezing temperature of the freeze dryer was set to -40℃ for 2 h, and then dried for 12 h under a vacuum of 10 Pa and a sublimation temperature of -20℃ to obtain freeze-dried *Lactobacillus plantarum* powder. The viable cell content in the powder was determined by viable cell counting to ensure that the viable cell count of LV1000 strain per gram of powder met the requirement of 5 × 10⁻⁶. 10 Requirements for CFU / g;

[0086] (b) Weigh out fructooligosaccharides, inulin, resistant dextrin, microcrystalline cellulose and magnesium stearate, mix them to form a mixed excipient, add the freeze-dried Lactobacillus plantarum powder to the mixed excipient, transfer it to a three-dimensional mixer, set the speed of the three-dimensional mixer to 60 rpm and the mixing time to 30 min, so that the strain and each excipient can be fully contacted and evenly dispersed. After the mixing is completed, package it to obtain the probiotic composition.

[0087] The difference between Comparative Example 1 and Example 1 is that the LV1000 strain was replaced with common Lactobacillus plantarum, and the rest is exactly the same as Example 1.

[0088] The difference between Comparative Example 2 and Example 1 is that the LV1000 strain was replaced with Lactobacillus acidophilus, and the rest is exactly the same as Example 1.

[0089] Experiment Example 1: Tests on tolerance to gastrointestinal fluids and bile salts

[0090] Solution: The survival rate of LV1000 strains in Examples 1-3 and strains in Comparative Examples 1-2 after being stored in environments containing artificial gastric fluid, artificial intestinal fluid, and artificial bile for a certain period of time was tested.

[0091] Artificial gastric juice containing 0.3% pepsin was adjusted to pH=3, and the survival rate of LV1000 after incubation at 37℃ for 1 h was counted. Artificial simulated intestinal juice containing 0.3% pancreatic enzyme was adjusted to pH=6.8, and the survival rate of LV1000 after incubation at 37℃ for 3 h was counted. Artificial simulated bile containing 0.3% bile salts was adjusted to pH=7, and the survival rate of LV1000 after incubation at 37℃ for 3 h was counted. The experimental results are recorded in Table 1.

[0092] Experiment Example 2: Test on the ability to inhibit common pathogenic bacteria

[0093] Experimental protocol: Single colonies of Listeria and Salmonella were obtained by streaking. These colonies were then picked and placed in LB broth and incubated at 37°C with shaking for 12 hours. The bacterial suspension was adjusted to a suitable concentration and spread onto LB agar plates. After the surface dried, the plates were placed in sterile Oxford cups, and supernatants of strains LV1000 from Examples 1-3 and strains from Comparative Examples 1-2 (with MRS medium as a control) and bacterial suspensions (with PBS as a control) were added, respectively. The plates were incubated overnight at 4°C for diffusion, followed by incubation at 37°C for 12 hours. The size of the inhibition zone was observed and measured, and the experiment was repeated three times. The results are recorded in Table 1.

[0094] Table 1

[0095]

[0096] - indicates no antibacterial activity; + indicates an inhibition zone diameter of 11-16 mm; ++ indicates an inhibition zone diameter of 17-22 mm; +++ indicates an inhibition zone diameter ≥23 mm.

[0097] Table 1 shows that the LV1000 strains cultured in Examples 1-3 all had high survival rates after being stored at 37°C for one hour in artificial gastric juice, intestinal juice, and bile, demonstrating the bacterium's good resistance to acid, bile salts, and gastrointestinal environment. The LV1000 strain of this invention has a good inhibitory effect on the activity and growth of common pathogenic bacteria Listeria and Salmonella, with inhibition zone diameters all above 23 cm, indicating good antibacterial properties.

[0098] Experiment Example 3: Cell Adhesion Experiment

[0099] Experimental protocol: Caco-2 (intestinal cells) were seeded into 24-well plates and cultured in DMEM (containing 10% fetal bovine serum) for 18-24 h until 90%-100% polymerization. The culture medium was aspirated, and after washing with PBS, LV1000 bacterial suspension (1×10^8 CFU / mL) was added. A control group without bacterial suspension was included. Cells were incubated at 37°C and 5% CO2 for 6-12 h. After washing to remove unadhered bacteria, cells were scraped to prepare a suspension, diluted, and plated onto MRS plates. Cells were incubated at 37°C for 48 h for counting. Adhesion rate was calculated as (number of adhered bacteria / number of added bacteria × 100%).

[0100] Table 2

[0101]

[0102] Table 2 shows that the average adhesion rate of *Lactobacillus plantarum* in Examples 1-3 of this invention was 70.5%, indicating that the LV1000 strain has a good adhesion ability to Caco-2 cells.

[0103] Figure 1 Image showing the successful cultivation of the LV1000 strain of this invention; Figure 2 The results showed that the number of live bacteria digested under acidic gastric conditions was affected to some extent, but the number of live bacteria in the prepared probiotic microcapsules and probiotic compositions remained stable over time, indicating that the prepared probiotic microcapsules and probiotic compositions could provide good protection for the LV1000 strain. Figure 3 As shown, the number of live bacteria did not decrease significantly in the enteric coagulation experiment. The slightly alkaline environment of the intestine can act as a suitable environment to slowly dissolve the microcapsules and release probiotics. The slightly alkaline environment of the intestine can neutralize gastric acid, which is conducive to maintaining the number of live bacteria.

[0104] In summary, this invention constructs a complete and efficient technical system centered on probiotics for improving gut health and their applications. From the perspective of strain screening, the LV1000 strain, derived from the gut of healthy humans, is naturally adapted to the intestinal environment, exhibiting excellent resistance to gastric acid and bile salts, laying the foundation for its survival and efficacy in the complex gastrointestinal environment. In the cultivation and processing stage, a scientifically optimized process ensures the high activity and purity of the strain, providing high-quality and stable bacterial resources for subsequent applications. Regarding application development, the probiotic microcapsule preparation utilizes a unique formula combining a sodium alginate-calcium lactate gel system with freeze-dried prickly pear powder. This not only significantly enhances the intestinal colonization ability of the strain but also strengthens the intestinal antioxidant and immune-regulating functions through the synergistic effect of antioxidant components and the strain. The probiotic composition, through precise proportioning of the LV1000 strain with prebiotics and excipients and a scientific preparation process, achieves a synergistic improvement in gut microbiota balance, digestive function, and intestinal motility. Meanwhile, antibacterial experiments showed that the LV1000 strain had a strong inhibitory effect on common pathogens such as Listeria and Salmonella, while cell adhesion experiments further confirmed its good adhesion ability to intestinal cells, enabling it to effectively colonize the intestines and exert its effect in regulating intestinal health. Overall, the technical solution of this invention achieves comprehensive improvement of intestinal health from multiple dimensions, providing a highly innovative and practical solution for the field of intestinal health.

[0105] 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 probiotic for improving gut health, characterized in that, The probiotics that improve gut health were obtained by culturing and treating Lactobacillus plantarum LV1000 strain with accession number CGMCC No.30865; The process of culturing and processing probiotics to improve gut health includes the following steps: A1, Activation culture: The preserved Lactobacillus plantarum LV1000 strain was cultured at a constant temperature to obtain activated bacteria; A2, streak separation, streak the activated bacteria into sections, and culture in a constant temperature facultative anaerobic culture; A3. Colony identification involves morphological observation to screen target strains, followed by Gram staining of the target strains to obtain probiotics that improve gut health.

2. The probiotic for improving gut health according to claim 1, characterized in that, In step A3, during morphological observation, strains with good growth status, medium-sized colonies, milky white color, upward convexity, moist surface, and neat colony edges are selected as target strains.

3. The application of a probiotic for improving gut health according to any one of claims 1-2, characterized in that, Probiotic microcapsules that improve gut health were prepared using probiotics that improve gut health.

4. The application of the probiotic for improving intestinal health according to claim 3, characterized in that, The probiotic microcapsules comprise the following raw materials in parts by weight: 5-15 parts of *Lactobacillus plantarum* suspension, 2-6 parts of freeze-dried prickly pear powder, 1-3 parts of sodium alginate, 0.5-2 parts of calcium lactate, 20-40 parts of soybean oil, and 0.1-0.5 parts of Span80. The processing procedure for the freeze-dried prickly pear powder is as follows: take prickly pear, wash and chop it, freeze-dry it, take it out and grind it into powder, sieve it to obtain freeze-dried prickly pear powder; The preparation process of the plant lactobacillus suspension is as follows: take probiotics that improve intestinal health and culture them in a facultative anaerobic manner, collect the bacterial cells by centrifugation, and prepare plant lactobacillus suspension.

5. The application of a probiotic for improving gut health according to claim 4, characterized in that, The preparation method of the probiotic microcapsules includes the following steps: (1) Thoroughly mix sodium alginate, calcium lactate and plant lactobacillus suspension, then pour it into a mixed solution containing soybean oil and Span80, and stir magnetically to form an emulsion system; (2) Add the lyophilized prickly pear powder to the emulsion system and stir to form a mixture; (3) Add glacial acetic acid to the mixture, adjust the pH, and gel to form microcapsules; (4) Add Tween solution to the microcapsules, let stand, discard the upper oil phase, centrifuge, discard the supernatant, wash, and obtain probiotic microcapsules.

6. The application of the probiotic for improving gut health according to claim 3, characterized in that, A probiotic composition for improving gut health was prepared using probiotics that improve gut health.

7. The application of a probiotic for improving gut health according to claim 6, characterized in that, The probiotic composition comprises the following raw materials in parts by weight: 0.2-2 parts of freeze-dried Lactobacillus plantarum powder, 5-15 parts of fructooligosaccharides, 3-10 parts of inulin, 2-8 parts of resistant dextrin, 1-5 parts of microcrystalline cellulose, and 0.1-0.5 parts of magnesium stearate. The method for preparing the probiotic composition includes the following steps: (a) Preparation of freeze-dried Bacillus plantarum powder: Probiotics that improve intestinal health are cultured at a constant temperature to obtain bacterial solution. After centrifugation, the supernatant is discarded, the bacterial cells are collected, washed, and then freeze-dried to obtain freeze-dried Bacillus plantarum powder. (b) Weigh out fructooligosaccharides, inulin, resistant dextrin, microcrystalline cellulose and magnesium stearate, mix them to form a mixed excipient, add freeze-dried Lactobacillus plantarum powder to the mixed excipient, stir and mix, and package to obtain a probiotic composition.

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