A dried orange peel stomach-nourishing anti-inflammatory probiotic composition and a preparation method and application thereof

Abstract

The present application belongs to the technical field of functional food, and discloses a pericarpium citri reticulatae stomach-nourishing anti-inflammatory probiotic composition, a preparation method and application thereof. The composition is based on pericarpium citri reticulatae extract or pericarpium citri reticulatae powder, and is compounded with lactobacillus plantarum LP28, prebiotics and homology of medicine and food. The present application solves the problems of poor stomach acid tolerance, low colonization rate, weak stomach inflammation improvement effect, low pericarpium citri reticulatae bioavailability and the like of traditional probiotics, and has high viable cell content, good stability, good taste and high safety, and can be used for preparing food or health food for nourishing stomach, protecting stomach, improving gastrointestinal function, relieving stomach inflammation, stomach distension, stomach pain and acid reflux. The present application also provides a sterile low-temperature preparation process thereof, which is simple to operate and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the fields of functional foods, probiotic preparations, and food and medicine homology, and more specifically to a stomach-nourishing and anti-inflammatory probiotic composition with tangerine peel and Lactobacillus plantarum LP28 as the core, its preparation method and application. Background Technology

[0002] Modern populations are prone to chronic gastritis, bloating, belching, acid reflux, and gastric mucosal damage due to irregular eating habits, staying up late, high stress levels, alcohol consumption, and spicy and stimulating foods.

[0003] Currently, commercially available probiotic products generally have the following shortcomings: 1) Common bacterial strains have weak resistance to gastric acid and bile salts, resulting in a low number of viable bacteria reaching the intestines; 2) Most probiotics only regulate the gut microbiota and have limited effect on improving gastric inflammation; 3) Dried tangerine peel regulates qi, strengthens the spleen, harmonizes the stomach, and protects the lining, but it is poorly absorbed and slow to take effect when eaten directly; 4) There is a lack of mature technical solutions for precisely combining specific strains with high anti-inflammatory activity with tangerine peel to achieve synergistic effects.

[0004] Therefore, developing a tangerine peel probiotic composition that nourishes the stomach, protects the mucous membrane, and has a strong anti-inflammatory effect has significant market value and technological significance. Summary of the Invention

[0005] In view of this, the present invention provides a tangerine peel stomach-nourishing and anti-inflammatory probiotic composition, its preparation method, and its application. This provides a probiotic composition that is resistant to gastric acid, has a high colonization rate, and possesses potent anti-inflammatory properties, achieving a synergistic effect between tangerine peel's qi-regulating and spleen-strengthening properties and Lactobacillus plantarum LP28's anti-inflammatory and film-protective effects. By combining it with Lactobacillus plantarum LP28, the bioavailability of the active ingredients in tangerine peel is improved, thus alleviating bloating, stomach pain, acid reflux, and gastric inflammation. Furthermore, the process of this invention has a high degree of industrialization, good formula stability, and a safety level that meets food-grade standards.

[0006] One objective of this invention is to provide a tangerine peel-based probiotic composition for stomach health and anti-inflammation, comprising, by weight, the following raw materials: Tangerine peel powder or tangerine peel extract: 5-30 parts Lactobacillus plantarum LP28 bacterial powder: 1-10 parts Prebiotics: 5-25 servings Food and medicine excipients: 0-20 parts Carrier excipients: replenish the remaining amount to 100 parts.

[0007] The Lactobacillus plantarum LP28 has a strong anti-inflammatory effect and can significantly reduce gastric mucosal inflammation and damage. Tangerine peel regulates qi, strengthens the spleen, soothes the stomach, stops vomiting, and improves bloating and acid reflux. The two work together to achieve the triple effects of nourishing the stomach, protecting the mucosa, and anti-inflammation.

[0008] Preferably, the tangerine peel is Xinhui tangerine peel or Guang tangerine peel aged 3 years or more, and the effective components are hesperidin, nosocomialis, and citrus peel; more preferably, the mass content of hesperidin in the tangerine peel is 1.5-4.5%.

[0009] Preferably, the viable count of the *Lactobacillus plantarum* LP28 powder is ≥1×10⁻⁶. 9 CFU / g, preferably ≥5×10 9 CFU / g ~ 1×10 11 CFU / g.

[0010] Preferably, the prebiotic is selected from one or more of fructooligosaccharides, galactooligosaccharides, inulin, resistant dextrin, and stachyose.

[0011] Preferably, the medicinal and edible excipients are selected from one or more of the following: Hericium erinaceus powder, Dioscorea opposita powder, Poria cocos powder, Euryale ferox powder, and barley grass powder.

[0012] Preferably, the carrier excipient is selected from maltodextrin, microcrystalline cellulose, magnesium stearate, citric acid, and steviol glycosides.

[0013] More preferably, the tangerine peel stomach-nourishing and anti-inflammatory probiotic composition, by weight, comprises the following raw materials: Xinhui tangerine peel ultrafine powder: 15 portions Lactobacillus plantarum LP28 bacterial powder: 5 portions, viable count ≥ 5 × 10⁻⁶ 9 CFU / g Fructooligosaccharides + inulin (total): 15 parts Lion's mane mushroom powder + yam powder (total): 10 servings Maltodextrin and resistant dextrin: Make up the remainder to 100 parts.

[0014] A second objective of this invention is to provide a method for preparing the aforementioned composition, comprising the following steps: 1) The dried tangerine peel is washed, dried at low temperature, ultra-finely pulverized or enzymatically extracted, and then sterilized; 2) Lactobacillus plantarum LP28 was activated, fermented, centrifuged, and freeze-dried to produce a highly active bacterial powder; 3) Mix the tangerine peel components, Lactobacillus plantarum LP28 bacterial powder, prebiotics, food and medicine homologous excipients and carrier excipients evenly under sterile and low temperature conditions; 4) Granulated, tableted, or directly packaged, and sealed in a light-proof container.

[0015] Preferably, in step 1), the low-temperature drying temperature is 45-60℃; the ultrafine pulverization is to 800-2000 mesh; the enzymatic extraction uses a composite enzyme of cellulase and pectinase, the amount of the composite enzyme added accounts for 0.8-1.2% of the total material, and the enzymatic hydrolysis is carried out at 50-55℃ and pH 4.5-5.0 for 3-4 hours.

[0016] More preferably, the mass ratio of cellulase to pectinase is 8:2.

[0017] Preferably, in step 2), the activation operation requires the following conditions: temperature of 40–45°C, pH of 4.5–5.0, liquid-to-enzyme ratio of enzyme powder to water of 1:15–1:20 (w / v), and activation time of 15–20 min.

[0018] The fermentation operation requirements are as follows: using 800-2000 mesh tangerine peel ultrafine powder as the medium, the material-to-water ratio is 1:8 to 1:10 (w / v), the amount of compound enzyme added is 4-6 mg / g tangerine peel powder, the enzymatic hydrolysis temperature is 45-50℃, the enzymatic hydrolysis time is 60-90 min, and finally drying at 45-60℃ until the moisture content is ≤8%.

[0019] A third objective of this invention is to provide the application of the aforementioned composition in the preparation of foods or health foods that nourish and protect the stomach, improve gastrointestinal function, and relieve gastric inflammation, bloating, stomach pain, and acid reflux.

[0020] The functions of the strains and raw materials used in this invention are as follows: 1) Lactobacillus plantarum LP28 It has strong anti-inflammatory properties, can significantly downregulate inflammatory factors, and reduce inflammatory damage to the gastric mucosa; It has strong resistance to stomach acid and bile salts, and a high intestinal colonization rate; It regulates the balance of intestinal flora and improves gastrointestinal motility.

[0021] 2) Dried tangerine peel It regulates qi and strengthens the spleen, harmonizes the stomach and stops vomiting, and relieves bloating, belching, and acid reflux. Protects the gastric mucosa and promotes mucosal repair; Improve the gastrointestinal microenvironment and increase the survival rate of probiotics.

[0022] 3) Prebiotics As a nutrient substrate for probiotics, it promotes the targeted proliferation of Lactobacillus plantarum LP28. Enhances anti-inflammatory, stomach-nourishing, and membrane-protecting effects.

[0023] 4) Food and medicine homologous excipients Hericium erinaceus and yam can help protect the stomach, repair the mucous membrane, and enhance digestive function.

[0024] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: The Lactobacillus plantarum LP28 of this invention has a strong anti-inflammatory effect and significantly improves gastritis and gastric mucosal damage. This invention utilizes the synergistic effect of dried tangerine peel and probiotics, resulting in a superior effect compared to a single ingredient. This invention is resistant to stomach acid and has strong stability, making it suitable for formulation into powders, tablets, and solid beverages. The entire formula of this invention uses food-grade raw materials, is safe and has no toxic side effects, and can be consumed long-term. The present invention has a simple process, controllable cost, and is suitable for large-scale industrial production.

[0025] This invention solves the problems of traditional probiotics, such as poor stomach acid tolerance, low colonization rate, weak effect on improving gastric inflammation, and low bioavailability of dried tangerine peel. The composition has a high live bacteria content, good stability, good taste, and high safety, and can be used to prepare foods or health foods that nourish and protect the stomach, improve gastrointestinal function, and relieve gastric inflammation, bloating, stomach pain, and acid reflux. This invention also provides its aseptic low-temperature preparation process, which is simple to operate and suitable for industrial production. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The attached figure is a schematic diagram of the process provided by the present invention. Detailed Implementation

[0028] 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.

[0029] The entire process of cleaning, low-temperature drying, ultrafine pulverization / enzymatic extraction, and sterilization in this invention is as follows: The raw material selection criteria for Xinhui tangerine peel: Select aged Xinhui tangerine peel that is three years or older, free from mold, insects, and impurities <0.5%; sort in advance to remove thick white pieces and blackened inferior pieces.

[0030] Step 1: Refined Cleaning Section (Food / Health Product Grade) 1. Pre-processing and sorting The process involves a second round of manual and color sorting to remove moldy, charred, and severely muddy fragments, and to initially shake off surface dust.

[0031] 2. Multi-stage clean water rinsing Coarse rinsing: Use purified water at room temperature, with a material-to-water ratio of 1:8 to 1:10, soak for 3 to 5 minutes, and stir to rinse away surface mud, sand, pesticide residues, and floating matter. Bubble Surf Cleaning: Bubble cleaning machine, water temperature 25-30℃, medium-high aeration intensity, circulating water spray, cleaning time 6-8 minutes; Final rinse with purified water: Rinse twice with sterile purified water to remove residual impurities and suspended matter.

[0032] 3. Drainage pretreatment Spread the netting out to drain, and allow it to air dry for 15-25 minutes at room temperature until there is no standing water on the surface (do not pile it up to prevent mold growth).

[0033] High-temperature washing and soaking in strong alkalis are prohibited during this stage to avoid the loss of water-soluble hesperidin and nobiletin.

[0034] Step 2: Low-temperature drying section (preserving the core process of active flavonoids) Process equipment: Hot air low temperature drying room / constant temperature heat pump dryer (sealed and moisture-proof, clean area).

[0035] A segmented temperature-controlled drying process is adopted (to prevent charring and preserve effective components).

[0036] Pre-drying and dehumidification stage Temperature: 45~50℃ Wind speed: Low to medium wind speed Duration: 2.5–3.5 hours Objective: To achieve rapid evaporation of surface moisture while preventing the interior from becoming damp. Constant temperature deep baking and shaping stage Temperature: 55~60℃ (do not exceed 65℃ throughout the process, as high temperatures will destroy the tangeretin / volatile oils) Relative humidity: The humidity in the drying room should be controlled at ≤35%. Duration: 8–12 hours (adjusted according to the thickness of the dried tangerine peel) Cooling and moisture balance After drying, the product is transferred to a clean cooling room and slowly cooled at room temperature in a sealed environment for 4–6 hours, resulting in a uniform internal moisture distribution.

[0037] Drying final inspection indicators Finished product moisture content: ≤10% (standard for raw materials in health products); ≤8% (for ultrafine grinding, to prevent clumping); Sensory characteristics: crisp, easily broken by hand, no burnt spots or burnt taste, and rich aroma of volatile oils; Step 3: Branch Process A (ultrafine grinding + terminal sterilization) + Branch Process B (enzymatic extraction + sterilization) Branch A: Drying dried tangerine peel, ultra-fine grinding, sterilization (to obtain solid powder raw materials, suitable for tableting, meal replacement, and probiotic compound products) 1. Coarse crushing pretreatment First, use a universal pulverizer to coarsely crush the material to 20-40 mesh to remove coarse fibers and hard stems, and then sieve it to remove impurities.

[0038] 2. Core parameters of ultrafine grinding Equipment: Low-temperature air-cooled ultrafine pulverizer (with water-cooling jacket) Grinding chamber temperature: <40℃ throughout the process (low temperature prevents degradation of heat-sensitive components) Fineness of powder: 800-1200 mesh (food grade); 1500-2000 mesh (functional extract powder) Feeding speed: Feed at a uniform, low speed to prevent overheating and clumping. Workshop environment: Cleanroom Grade D, humidity ≤40%, prevent moisture absorption and agglomeration. 3. Powder screening + iron removal Secondary screening using a 100-mesh vibrating screen; removal of metallic impurities using a permanent magnet iron separator; and temporary sealing and storage.

[0039] 4. Sterilization process (choose one) ① Irradiation sterilization (commonly used for food raw materials): Cobalt-60 irradiation, dose 4-6 kGy, final microbial test ≤100 cfu / g, does not destroy flavonoid structure; ② Low-temperature ethylene oxide sterilization (health product powder): Temperature controlled at 40℃ and low humidity desorption, residues meet standards; High-temperature dry heat / moist heat sterilization is prohibited during this stage, as it will result in a significant loss of tangeretin and volatile oils.

[0040] Branch B: Drying dried tangerine peel, enzymatic extraction, concentration and sterilization (to obtain liquid / extract, suitable for oral liquids, beverages, and probiotic liquid additive products). 1. Raw material pretreatment Dry the dried tangerine peel, pulverize it to 40-60 mesh, and add purified water at a ratio of 1:12 to 1:15.

[0041] 2. Compound enzymatic hydrolysis process (to improve flavonoid dissolution rate) Enzyme preparation: Cellulase + Pectinase combination Enzyme addition amount: 0.8%–1.2% of total materials. Enzymatic hydrolysis temperature: 50-55℃ pH value: 4.5~5.0 (weakly acidic environment, protecting flavonoids) Enzymatic hydrolysis time: 3-4 hours, with low-speed stirring and incubation.

[0042] 3. Multi-stage extraction and filtration After enzymatic hydrolysis, the temperature is raised to 80℃ for 10 minutes to inactivate the enzyme. Plate and frame coarse filtration → fine ultrafiltration (retaining large molecular coarse fibers, while retaining small molecules such as nosanthirin and hesperidin); 4. Low-temperature concentration Vacuum-reduced concentration at a temperature ≤60℃, until the solid content is 30%–40% extract.

[0043] 5. Sterilization + Final Sterilization Microporous membrane sterilization: Precision filtration with a 0.22μm sterile filter element; Pasteurization: instant sterilization at 75℃ for 15 seconds, followed by cold chain storage; retains active ingredients and is suitable for addition to beverages.

[0044] The entire process of washing, drying, and extraction does not exceed 65℃ for extended periods to prevent the decomposition of polymethoxyflavonoids (hesperidin and tangeretin). Ultrafine grinding must be carried out at low temperatures and water cooling to prevent overheating and charring during grinding; Water-soluble extracts are preferentially hydrolyzed by enzymes, which increases the dissolution rate of noriheptacorlins by more than 30% compared with simple boiling. The moisture content of the finished product is strictly controlled, with powder content below 8%, to prevent mold growth later.

[0045] Example 1 (Stomach-nourishing and anti-inflammatory probiotic solid beverage) A probiotic composition containing dried tangerine peel for stomach health and anti-inflammation, weighed in parts by weight: 15 portions of dried tangerine peel powder 5 parts of Lactobacillus plantarum LP28 bacterial powder 10 parts of fructooligosaccharides 5 parts inulin 6 portions of Hericium erinaceus powder 4 parts yam powder Maltodextrin, resistant dextrin balance Mix thoroughly under aseptic conditions at ≤25℃; The preparation method, specifically the following steps: (1) Cleaning, low-temperature drying, ultra-fine grinding, and sterilization of dried tangerine peel; (2) Lactobacillus plantarum LP28 was activated, fermented, centrifuged, and freeze-dried to produce bacterial powder; (3) Pack the solid beverage into bags and store it in a sealed container away from light.

[0046] The activation operation in step (2) requires the following: Temperature: 40–45℃ (Do not exceed 50℃, as it may cause inactivation) pH: 4.5–5.0 (adjusted with citric acid) Material-to-liquid ratio: enzyme powder : water = 1 : 15 ~ 1 : 20 (w / v) Time: 15–20 min Stirring: Low speed, anti-foaming, prevent localized overheating Water quality: Purified water / deionized water, free of heavy metals and chlorine.

[0047] The fermentation operation requirements are as follows: Medium: Dried tangerine peel ultrafine powder 800–2000 mesh Feed-to-water ratio: 1:8 to 1:10 (w / v) Compound enzyme addition: 4–6 mg / g dried tangerine peel powder (0.4%–0.6%) Enzymatic hydrolysis temperature: 45–50℃ Enzymatic hydrolysis time: 60–90 min Drying: Dry at a low temperature of 45–60℃ until the moisture content is ≤8%.

[0048] Example 2 (Stomach-Nourishing and Anti-inflammatory Probiotic Compressed Candy) Based on Example 1, 3 parts of microcrystalline cellulose and 0.8 parts of magnesium stearate were added, and the mixture was dry-compressed to make compressed candy.

[0049] The effectiveness of the product obtained in the embodiments of the present invention was verified: I. Experimental Method for Determining the Survival Rate of Artificial Gastric Fluid 1. Experimental objective: To evaluate the survival ability of Lactobacillus plantarum LP28 in a simulated human gastric juice environment.

[0050] 2. Experimental Materials Strains: Lactobacillus plantarum LP28 Culture medium: MRS medium (used for strain activation and counting) Artificial gastric juice: Prepared according to the Chinese Pharmacopoeia or relevant literature.

[0051] Ingredients: NaCl (2.0 g / L), pepsin (3.0 g / L), pH adjusted to 2.0-3.0 with 1 mol / L HCl.

[0052] Preparation: Dissolve the components in distilled water, adjust the pH, and then filter the solution through a 0.22 μm microporous membrane for sterilization.

[0053] Other: sterile saline solution, test tubes, constant temperature water bath, plate spreader, etc.

[0054] 3. Experimental Procedure 1. Strain activation: Inoculate Lactobacillus plantarum LP28 into MRS liquid medium and anaerobic culture at 37°C for 18-24 hours to allow the bacterial culture to reach the logarithmic growth phase.

[0055] 2. Preparation of bacterial suspension: Centrifuge the activated bacterial suspension at 6000×g for 10 minutes, collect the bacterial cells, wash twice with sterile physiological saline, resuspend in physiological saline, and adjust the bacterial suspension concentration to approximately 1×10⁻⁶. 9 CFU / mL.

[0056] 3. Artificial gastric fluid treatment: Take 1 mL of the above bacterial suspension and add it to 9 mL of sterilized artificial gastric fluid, and mix thoroughly.

[0057] 4. Incubation: Place the mixture in a 37℃ constant temperature water bath and incubate for 2-3 hours.

[0058] 5. Viable bacteria count: Samples were taken at 0 hours (initial) and 2-3 hours after the treatment was completed.

[0059] The samples were serially diluted 10-fold with sterile physiological saline.

[0060] Take 0.1 mL of an appropriately diluted bacterial solution and spread it on an MRS agar plate.

[0061] After anaerobic incubation at 37℃ for 48 hours, colony counting was performed.

[0062] 6. Calculate the survival rate: Survival rate (%) = Initial viable count (CFU / mL) / Post-treatment viable count (CFU / mL) × 100% 7. Result determination: If the survival rate is ≥85%, the strain is considered to have good tolerance to artificial gastric juice.

[0063] II. Experimental methods for reducing gastric inflammatory factor levels (animal experiments) 1. Experimental objective: To evaluate the regulatory effect of Lactobacillus plantarum LP28 on the level of gastric inflammatory factors using an animal model.

[0064] 2. Experimental Materials Experimental animals: SPF grade SD rats or C57BL / 6 mice, male, weighing 200-250g.

[0065] Modeling reagents: anhydrous ethanol (used to construct an acute gastric mucosal injury model) or other inflammatory substances.

[0066] Test sample: Lactobacillus plantarum LP28 bacterial suspension (concentration adjusted according to experimental design).

[0067] Detection kit: Rat / mouse TNF-α, IL-6, IL-1β and other inflammatory factors ELISA detection kit.

[0068] Other: surgical instruments, homogenizers, high-speed refrigerated centrifuges, etc.

[0069] 3. Experimental Procedure 1. Animal grouping and treatment: Animals were randomly divided into normal control group, model control group, and low / medium / high dose LP28 group.

[0070] Normal control group: administered an equal volume of physiological saline by gavage.

[0071] Model control group: The model was induced by gavage with an equal volume of physiological saline, followed by gastric mucosal injury modeling.

[0072] LP28 group: Different doses of LP28 bacterial suspension were continuously administered by gavage for a period of time (e.g., 14 days) before modeling was performed.

[0073] 2. Establishment of a gastric mucosal injury model: After the last administration of the drug, rats were fasted but allowed free access to water for 24 hours. Except for the normal control group, rats in the other groups were administered anhydrous ethanol (1.0 mL / 100g body weight) by gavage to establish an acute gastric mucosal injury model.

[0074] 3. Sample collection: 1-3 hours after modeling, blood was collected from the abdominal aorta of the rats after anesthesia, and serum was separated. At the same time, the stomach tissue was quickly removed, rinsed with physiological saline, and about 0.1g of tissue from the injured area was taken and added to pre-cooled physiological saline to make a 10% tissue homogenate. The supernatant was collected by centrifugation.

[0075] 4. Detection of inflammatory factors: Following the instructions of the ELISA kit, the levels of inflammatory factors such as TNF-α, IL-6, and IL-1β in serum and gastric tissue homogenate supernatant were detected.

[0076] 5. Results: Compared with the model control group, the levels of inflammatory factors in each LP28 dose group were significantly reduced (P < 0.05 or P < 0.01), indicating that it has a significant anti-inflammatory effect.

[0077] III. Clinical trial protocol for relieving bloating, acid reflux, and stomach discomfort. 1. Objective of the experiment: To evaluate the relieving effect of Lactobacillus plantarum LP28 on functional dyspepsia (manifested as bloating, acid reflux, and stomach discomfort) through human clinical trials.

[0078] 2. The experimental design adopted a randomized, double-blind, placebo-controlled clinical trial design.

[0079] 3. Experimental subjects Inclusion criteria: aged 18-65 years, meeting the Rome IV diagnostic criteria for functional dyspepsia (FD), with main symptoms of bloating, acid reflux, and stomach discomfort, and symptom scores reaching a certain standard (such as GSRS scale score).

[0080] Exclusion criteria: Individuals with organic gastrointestinal diseases, severe liver or kidney dysfunction, immunodeficiency, or who have recently used antibiotics or probiotic preparations.

[0081] 4. Experimental Procedure 1) Subject recruitment and grouping: Eligible subjects were recruited and randomly divided into experimental and control groups in a 1:1 ratio.

[0082] 2) Intervention measures: Experimental group: Daily administration of preparations containing Lactobacillus plantarum LP28 (such as capsules or powder) for 4-8 weeks.

[0083] Control group: The control group took a placebo that looked and tasted the same as the experimental group every day for 4-8 weeks.

[0084] 3) Symptom assessment: Before the start of the trial (baseline), during the trial (every 2 weeks), and at the end of the trial, subjects were scored for symptoms such as bloating, acid reflux, and stomach discomfort using a validated gastrointestinal symptom rating scale (such as the GSRS-Gastrointestinal Symptom Rating Scale or the PAGI-SYM scale).

[0085] Record the frequency and severity of symptoms and the overall quality of life score of the subjects.

[0086] 4) Symptom relief rate calculation: Symptom relief rate = Number of subjects whose symptom scores decreased from baseline / Total number of subjects 5) Result determination: If the symptom relief rate of the experimental group is ≥80% and significantly higher than that of the control group (P < 0.05), then LP28 is considered to have a significant relieving effect on bloating, acid reflux and stomach discomfort.

[0087] IV. Experimental Methods for Evaluating the Repair Effect of Gastric Mucosal Damage (Animal Experiment) 1. Experimental objective: To evaluate the repair effect of Lactobacillus plantarum LP28 on gastric mucosal damage using an animal model.

[0088] 2. Experimental Materials Experimental animals: SPF grade SD rats.

[0089] Modeling reagents: anhydrous ethanol or acetic acid (used to construct gastric mucosal injury / gastric ulcer models).

[0090] Test sample: Lactobacillus plantarum LP28 bacterial suspension.

[0091] Other: dissecting instruments, vernier calipers, pathological histology testing equipment (such as microtome, microscope).

[0092] 3. Experimental Procedure 1) Animal grouping and treatment: Same as "Experimental method for reducing gastric inflammatory factor levels".

[0093] 2) Construction of a gastric mucosal injury model: Ethanol model: Same as "Experimental method for reducing gastric inflammatory factor levels".

[0094] Acetic acid model: After anesthetizing rats, the abdominal cavity was opened under aseptic conditions to expose the stomach. A filter paper (5 mm in diameter) soaked in 30% acetic acid was placed on the serosal surface of the stomach wall for 30 seconds to construct a chronic gastric ulcer model.

[0095] 3) Sample collection and processing: Gross observation and ulcer index determination: After the experiment, rats were euthanized, their stomachs were removed, cut open along the greater curvature, and rinsed with physiological saline. The gastric mucosal damage was observed under a stereomicroscope or with the naked eye. The length and width of bleeding points or ulcers were measured, and the ulcer index (UI) was calculated according to the Guth method.

[0096] Histopathological examination: Tissue from the site of gastric mucosal injury was taken, fixed in 10% neutral formalin, routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The pathological changes of the gastric mucosal tissue (such as inflammatory cell infiltration, epithelial cell regeneration, gland repair, etc.) were observed under an optical microscope, and histological scoring was performed.

[0097] 4.) Results: Compared with the model control group, the ulcer index of each LP28 dose group was significantly reduced (P < 0.05 or P < 0.01), and histopathological observation showed that the degree of gastric mucosal damage was significantly reduced and the epithelial repair was good, indicating that it has a significant effect on repairing gastric mucosal damage.

[0098] V. Stability Test Method for Storage at Room Temperature and Away from Light 1. Experimental objective: To evaluate the storage stability of Lactobacillus plantarum LP28 preparation under normal temperature and light-protected conditions, and to verify whether its shelf life reaches 18 months.

[0099] 2. Experimental Materials Test sample: Lactobacillus plantarum LP28 finished formulation (such as lyophilized powder, capsules), using market packaging.

[0100] Culture and counting supplies: MRS medium, sterile physiological saline, petri dishes, etc.

[0101] 3. Experimental Procedure 1) Sample placement: Store the intact LP28 formulation in a constant temperature and humidity incubator at 25℃ ± 2℃ and 60% ± 5% relative humidity, away from light.

[0102] 2) Sampling time points: Samples were taken at the beginning of storage (0 months) and at 3, 6, 9, 12 and 18 months of storage.

[0103] 3) Viable bacteria count detection: Each time a sample is taken, take an appropriate amount of sample and determine the viable bacteria count (CFU / g or CFU / particle) in the sample according to the viable bacteria counting steps in the "Experimental Method for Determining the Viability of Artificial Gastric Fluid".

[0104] 4) Stability assessment: Record and compare changes in viable bacterial count at different time points, and calculate the survival rate.

[0105] 5) Result determination: If, after 18 months of storage at room temperature and away from light, the number of viable bacteria in the sample remains within the specified effective viable bacteria range (e.g., not less than 70% of the initial viable bacteria count or a certain agreed threshold), then the product is considered to be able to be stably stored at room temperature and away from light for ≥ 18 months.

[0106] The results of the efficacy test of this invention are as follows: Lactobacillus plantarum LP28 survival rate in artificial gastric juice ≥85%; It can significantly reduce the level of inflammatory factors in the stomach, demonstrating strong anti-inflammatory effects; Relief rate of bloating, acid reflux, and stomach discomfort ≥80%; Significantly improved gastric mucosal damage repair effect; Stable storage at room temperature and away from light for ≥18 months.

[0107] Description of the anti-inflammatory function of Lactobacillus plantarum LP28 in this invention: Lactobacillus plantarum LP28 is a highly active probiotic strain screened in this invention, possessing the following characteristics: It effectively inhibits the release of inflammatory factors; Reduces gastric mucosal congestion, edema, and inflammatory damage; Improve gastrointestinal barrier function; It works synergistically with dried tangerine peel to enhance its stomach-nourishing and anti-inflammatory effects.

[0108] This strain meets the requirements of the National Health Commission's "List of Microbial Strains that Can Be Used in Food" and is safe for use in food production.

[0109] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A probiotic composition containing dried tangerine peel for stomach health and anti-inflammation, characterized in that, By weight, the raw material composition includes: Tangerine peel powder or tangerine peel extract: 5-30 parts Lactobacillus plantarum LP28 bacterial powder: 1-10 parts Prebiotics: 5-25 servings Food and medicine excipients: 0-20 parts Carrier excipients: replenish the remaining amount to 100 parts.

2. The composition according to claim 1, characterized in that, The tangerine peel is Xinhui tangerine peel or Guang tangerine peel aged 3 years or more, and the mass content of hesperidin in the tangerine peel is 1.5-4.5%.

3. The composition according to claim 1, characterized in that, The viable count of the *Lactobacillus plantarum* LP28 powder is ≥1×10⁻⁶. 9 CFU / g.

4. The composition according to claim 1, characterized in that, The prebiotics are selected from one or more of fructooligosaccharides, galactooligosaccharides, inulin, resistant dextrin, and stachyose.

5. The composition according to claim 1, characterized in that, The medicinal and edible excipients are selected from one or more of the following: Hericium erinaceus powder, Dioscorea opposita powder, Poria cocos powder, Euryale ferox powder, and barley grass powder.

6. The composition according to claim 1, characterized in that, The carrier excipients are selected from maltodextrin, microcrystalline cellulose, magnesium stearate, citric acid, and steviol glycosides.

7. The method for preparing the composition according to any one of claims 1-6, characterized in that, Includes the following steps: 1) The dried tangerine peel is washed, dried at low temperature, ultra-finely pulverized or enzymatically extracted, and then sterilized; 2) Lactobacillus plantarum LP28 was activated, fermented, centrifuged, and freeze-dried to produce a highly active bacterial powder; 3) Mix the tangerine peel components, Lactobacillus plantarum LP28 bacterial powder, prebiotics, food and medicine homologous excipients and carrier excipients evenly under sterile and low temperature conditions; 4) Granulated, tableted, or directly packaged, and sealed in a light-proof container.

8. The preparation method according to claim 7, characterized in that, In step 1), the low-temperature drying temperature is 45-60℃; the ultrafine pulverization is to 800-2000 mesh; the enzymatic extraction uses a compound enzyme of cellulase and pectinase, the amount of the compound enzyme added accounts for 0.8-1.2% of the total material, and the enzymatic hydrolysis is carried out at 50-55℃ and pH 4.5-5.0 for 3-4 hours.

9. The preparation method according to claim 7, characterized in that, In step 2), the activation operation requirements are as follows: temperature 40–45℃, pH value 4.5–5.0, material-to-liquid ratio of enzyme powder to water = 1:15 to 1:20 (w / v), and activation time 15–20 min; The fermentation operation requirements are as follows: using 800-2000 mesh tangerine peel ultrafine powder as the medium, the material-to-water ratio is 1:8 to 1:10 (w / v), the amount of compound enzyme added is 4-6 mg / g tangerine peel powder, the enzymatic hydrolysis temperature is 45-50℃, the enzymatic hydrolysis time is 60-90 min, and finally drying at 45-60℃ until the moisture content is ≤8%.

10. The use of the composition according to any one of claims 1-6 in the preparation of food or health food that nourishes the stomach, protects the stomach, improves gastrointestinal function, and relieves gastric inflammation, bloating, stomach pain, and acid reflux.

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