A probiotic beadlet comprising lactobacillus salivarius and bifidobacterium longum and a method of making the same

Abstract

The application provides a probiotic crystal ball preparation containing lactobacillus jensenii and bifidobacterium longum, which comprises a shell structure and a core structure, wherein the shell structure comprises gelatin, glycerol and water; and the core structure comprises lactobacillus jensenii, bifidobacterium longum and hydrogenated oil. The lactobacillus jensenii has a preservation number of CCTCC NO: M2022172; and the bifidobacterium longum has a preservation number of CCTCC NO: M2019780. The application adopts a double-layer seamless capsule technology with gelatin as the shell material, hydrogenated oil and bacterial powder as the core, so that the probiotics can not be digested by gastric juice and have a high survival rate in the intestinal tract. Moreover, the core of the application uses the screened lactobacillus jensenii VHProbi A17 strain which does not produce hemolysin and cannot dissolve blood cells, and has safety. The lactobacillus jensenii has scavenging effects on DPPH free radicals and hydroxyl radicals, and has a wide antibacterial spectrum.

Description

Technical Field

[0001] This invention relates to the field of probiotic application technology, specifically to a probiotic pellet containing Lactobacillus saliva-associated and Bifidobacterium longum and its preparation method. Background Technology

[0002] Probiotics are products made from live microorganisms. When ingested in sufficient quantities, they can provide benefits to the host by improving and maintaining the balance of the gut microbiota. They promote nutrient absorption and maintain gut health by regulating the host's mucosal and systemic immune functions or by modulating the balance of gut microbiota, thus producing single microorganisms or well-defined mixtures of microorganisms that contribute to health. The probiotic functions of probiotics are widely studied in the fields of medicine, food, and feed. Gut symbiotic microorganisms, through their metabolic activities and molecular interactions with host cells, significantly influence the physiology of the gastrointestinal tract, as well as extraintestinal organs and systems. An increasing number of strains have been confirmed as probiotics and are being used by humans, such as Lactobacillus and Bifidobacterium. However, the survival of gut probiotics is affected by many factors, such as temperature, pH, pressure, gastric acid, and bile salts. After oral administration, probiotics need to undergo digestion in the mouth and stomach before reaching the intestines to exert their active effects. The pH of gastric juice is generally between 0.8 and 3.0, which poses a significant challenge to the survival of probiotics. Probiotics easily lose their activity in the highly acidic environment of gastric juice, resulting in a low colonization and survival rate in the human body.

[0003] To exert their probiotic functions, probiotics need to improve their survival rate during storage and in the host's intestinal environment. Enabling these probiotics to successfully enter the intestines via oral administration and exert their effects is a challenging problem.

[0004] Microencapsulation technology has long been considered an important technique for protecting the viability of probiotics during processing, storage, and the acidic conditions of the stomach. Microencapsulation technology utilizes encapsulating materials to encapsulate a target core material (solid, liquid, or even gas), forming a microcapsule with a diameter ranging from 1 to 5000 μm (typically 5 to 400 μm) and possessing a semi-permeable or sealed membrane. Currently, the microcapsule structures used to protect probiotics mainly fall into three categories: matrix structures, cross-linked structures, and external coating structures. Matrix structures involve polymers forming a coating wall through hydrogen bonds, van der Waals forces, and hydrophobic forces; this is a typical structure for products prepared using spray drying, ultrasonic vacuum spray drying, spray cooling, electrospinning, and supercritical fluid dynamics. In cross-linked structures, polymer chains are typically cross-linked by ions or enzymes; this is a typical structure for samples prepared using techniques such as extrusion, emulsification, enzyme gelation, and impact aerosols. Both of these probiotic microcapsules expose a small number of probiotics to gastric juices, resulting in some bacterial loss, with a survival rate of 68%-78%. The outer encapsulation structure is mainly a capsule, which encapsulates the probiotics in multiple layers inside the capsule shell or crystal shell, isolating the probiotics from gastric juice and thus better protecting them. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic pellet preparation containing *Lagilactobacillus salivarius* and *Bifidobacterium longum*, and its applications. The *Lagilactobacillus salivarius* VHProbi A17 and *Bifidobacterium longum* VHProbi Y08 used are both screened from infant feces. *Bifidobacterium longum* VHProbi Y08 can effectively reduce the mutagenic effects of carcinogens on human cells, while *Lagilactobacillus salivarius* VHProbi A17 can inhibit the growth of oral pathogens. Both *Lagilactobacillus salivarius* VHProbi A17 and *Bifidobacterium longum* VHProbi Y08 exhibit strong tolerance to artificial gastric juices. The probiotic pellets have stronger resistance and better stability, and can be widely used in food, health products, and other fields.

[0006] The present invention first provides a probiotic crystal ball, which comprises an outer shell structure and an inner core structure, wherein the outer shell structure comprises gelatin, glycerin and water; and the inner core structure comprises Lactobacillus salivarius, Bifidobacterium longum and hydrogenated oil.

[0007] Preferably, the mass fractions of the gelatin, glycerin and water are as follows: 18-25 parts gelatin, 6-8 parts glycerin, and 69-76 parts water.

[0008] As a specific example, the mass ratio of gelatin, glycerin and water is 18 parts gelatin, 6 parts glycerin and 76 parts water; or 20 parts gelatin, 8 parts glycerin and 72 parts water; or 25 parts gelatin, 6 parts glycerin and 69 parts water.

[0009] The hydrogenated oil mentioned is a hydrogenated oil with a melting point of 42°C.

[0010] As a preferred embodiment, the Lactobacillus salivarii has the accession number CCTCC NO: M2022172.

[0011] The aforementioned Bifidobacterium longum has the preservation number CCTCC NO: M2019780.

[0012] The probiotic pellets are prepared by the following steps:

[0013] Preparation of the core solution: Mix and disperse the Lactobacillus saliva-associated and Bifidobacterium longum bacterial powders in hydrogenated vegetable oil and stir until homogeneous;

[0014] Preparation of the outer shell solution: Dissolve gelatin in water, then add glycerin and continue to dissolve, then stir evenly and pour into the outer shell solution storage tank of the capsule making machine;

[0015] Preparation of probiotic crystals: The outer shell solution tank and the inner core solution tank of the capsule machine have concentric nozzles. The pump speed is controlled at 650 mL / h, and the pressure in the inner core tank is 5 kPa. During titration, the outer shell solution completely surrounds the inner core solution, which is then dripped into a tank containing a liquid paraffin solution at 10°C, causing gelation and forming continuous core-shell spheres. The resulting spheres are then dried at room temperature (25°C) until the moisture content is below 3.0%, yielding composite probiotic crystals with a diameter of 1.6 mm.

[0016] This invention employs a double-layer seamless capsule technology with gelatin as the main outer shell and hydrogenated oil and bacterial powder as the inner core. This technology allows the probiotics to be undigested by gastric juices and maintain a high survival rate in the intestines. Furthermore, the inner core uses a screened strain of *Lactobacillus salivarius* VHProbi A17, which does not produce hemolysin and cannot lyse blood cells, ensuring safety. It exhibits scavenging effects against DPPH and hydroxyl radicals; it also shows significant inhibitory effects against five oral pathogens: *Streptococcus mutans*, *Porphyromonas gingivalis*, *Actinomyces viscosus*, *Actinomyces actinomycetes*, and *Clostridium nucleatum*. Additionally, it demonstrates significant antibacterial effects against intestinal pathogens such as *Escherichia coli* and *Salmonella enteritidis*, exhibiting a broad antibacterial spectrum. Detailed Implementation

[0017] Probiotic microspheres are a type of encapsulation structure, where probiotics are encapsulated by a wall material. Different wall materials determine the survival rate of probiotics and their resistance to adverse environmental conditions. Choosing a suitable wall material has a decisive impact on the survival rate, pH adaptability, temperature tolerance, and storage stability of probiotics after microencapsulation. Choosing a suitable wall material is the first step in preparing probiotic microcapsules. An ideal probiotic microcapsule wall material should meet the following conditions: (1) good biocompatibility and film-forming properties; (2) able to protect and improve the activity of the core material; (3) not react with the core material; (4) no toxic side effects; (5) easy to process and with good rheological properties; (6) viscosity, permeability, hydrophilicity, and solubility meet the requirements.

[0018] This invention primarily encapsulates functional bacterial strains that regulate the gut. Therefore, the wall material must be resistant to stomach acid and able to dissolve and release probiotics rapidly within the intestines, allowing them to quickly colonize and exert their beneficial effects. This invention employs a double-layer seamless capsule technology using gelatin as the outer shell and hydrogenated oil and bacterial powder as the inner core. This technology ensures that the probiotics are not digested by gastric juices and have a high survival rate within the intestines.

[0019] The functional strain encapsulated in this invention is *Lagilactobacillus salivarius* VHProbi A17, which was screened and deposited on March 1, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M2022172. This strain exhibits significant inhibitory effects on *Streptococcus mutans* and *Actinomyces viscosus*, which cause dental caries, and on *Porphyromonas gingivalis*, *Clostridium nucleatum*, and *Gastromyxobolus hemiactiformis*, which cause periodontal disease. Furthermore, it can remove biofilms formed by *Streptococcus mutans* and inhibit the growth and reproduction of *Streptococcus mutans*, *Actinomyces viscosus*, *Porphyromonas gingivalis*, *Clostridium nucleatum*, and *Gastromyxobolus hemiactiformis*.

[0020] The Bifidobacterium longum used in this embodiment of the invention is Bifidobacterium longum VHProbi Y08, which was deposited on October 8, 2019 at the China Center for Type Culture Collection, Wuhan University, China, with accession number CCTCC NO: M2019780.

[0021] The present invention will now be described in detail with reference to the embodiments.

[0022] Example 1: Physicochemical properties of Lactobacillus salivarius VHProbi A17

[0023] The strain VHProbi A17 of *Lactobacillus salivarius* was obtained by screening from the feces of healthy infants. Blood cell plate assays showed that VHProbi A17 does not produce hemolysin and cannot lyse blood cells, indicating its safety. Its scavenging rate against DPPH free radicals was 20.9%, and against hydroxyl free radicals was 10.6%.

[0024] The Oxford cup method was used to determine the antibacterial effects of salivary Lactobacillus VHProbi A17 bacterial suspension and lysis buffer against Streptococcus mutans, Actinomyces viscerata, Porphyromonas gingivalis, Aggregobacter actinomycetes, Clostridium nucleatum, Escherichia coli, and Salmonella enteritidis. The diameter of the inhibition zone is shown in Table 1.

[0025] Table 1: Inhibitory effect of Lactobacillus saliva-associated with pathogenic bacteria VHProbi A17

[0026] Oral pathogens A17 fermentation broth inhibition zone diameter A17 lysis buffer inhibition zone diameter Streptococcus mutans 27mm 26mm Porphyromonas gingivalis 14.5mm 10mm Sticky Actinomycetes 23.5mm 22mm Aggregates actinomycetes 11mm 10mm Clostridium nucleatum 12.5mm 11mm E. coli >30mm >30mm Salmonella enteritidis >30mm >30mm

[0027] As shown in Table 1, Lactobacillus salivarius VHProbi A17 not only has a significant inhibitory effect on five oral pathogens, namely Streptococcus mutans, Porphyromonas gingivalis, Actinomyces coliformis, Aggregobacter actinomycetes, and Clostridium nucleatum, but also has a significant antibacterial effect on intestinal pathogens Escherichia coli and Salmonella enteritidis, exhibiting a broad spectrum of antibacterial activity.

[0028] Furthermore, *Lactobacillus salivarius* VHProbi A17 can effectively bind to common oral pathogens, significantly inhibiting their adhesion to teeth or gums. Moreover, *Lactobacillus salivarius* VHProbi A17 has no significant effect on the proliferative activity of normal human gingival epithelial cells, demonstrating good safety and no cytotoxicity.

[0029] Example 2: Physicochemical properties of Bifidobacterium longum strain VHProbi Y08

[0030] The *Bifidobacterium longum* VHProbi Y08 strain provided by this invention was screened from infant feces and is sensitive to common antibiotics such as erythromycin, exhibiting good biosafety. This strain possesses strong antioxidant properties, with scavenging rates of 23.8% and 74.5% against DPPH and HRS free radicals, respectively. Its supernatant showed an anti-lipid peroxidation inhibition rate of 41.99%, while the bacterial cell showed an inhibition rate of 43.17%. This strain can also effectively degrade cholesterol, achieving a degradation rate of 20%. Simultaneously, *Bifidobacterium longum* VHProbi Y08 can effectively reduce the mutagenic toxicity of carcinogens such as 4NQO and IQ on human cells, demonstrating an effective tumor prevention mechanism and possessing certain value in cancer prevention and treatment.

[0031] Example 3: Preparation of bacterial powder

[0032] (1) The activated Lactobacillus salivans VHProbi A17 and Bifidobacterium longum VHProbi Y08 were inoculated into MRS broth medium and cultured at 37℃ for 24h to obtain seed culture.

[0033] (2) Inoculate the seed liquid into MRS broth medium at a volume ratio of 5%, and after culturing at 37°C for 24 hours, stop fermentation to obtain fermentation broth;

[0034] (3) Centrifuge the fermentation broth at 5000 rpm for 10 minutes and collect the bacterial sludge.

[0035] (4) Add freeze-drying protectant to the fungal mud at a mass ratio of 10%, stir well, and then freeze-dry to obtain fungal powder.

[0036] The viable bacteria count in the above-mentioned bacterial powder was determined according to the national standard GB4789.35-2016-Food Microbiology Examination - Lactic Acid Bacteria Examination.

[0037] The results showed that the viable bacterial counts of Lactobacillus salivae A17 and Lactobacillus longum Y08 prepared by this invention both exceeded 200 billion cfu / g.

[0038] Example 4: Preparation of compound probiotic pellets

[0039] The probiotic pellets provided in this embodiment include an outer shell structure and an inner core structure. The components and their mass fractions in the outer shell structure are as follows: 18 parts gelatin, 6 parts glycerin, and 76 parts water.

[0040] The core structure contains the following components and their mass fractions: 3 parts of Lactobacillus saliva-associated powder, 2 parts of Lactobacillus longum powder, and 95 parts of hydrogenated oil with a melting point of 42℃.

[0041] The preparation method is as follows: A standard capsule making machine is used to prepare the capsules. The outer shell solution and inner core solution are prepared separately. The inner core solution is prepared as follows: hydrogenated vegetable oil is dissolved at 45°C, bacterial powder is added, and the mixture is stirred at 200 rpm for 15 minutes to ensure the bacterial powder is evenly dispersed in the hydrogenated vegetable oil. This solution is then poured into the inner core solution tank of the capsule making machine. The stirring speed of the inner core solution tank is set to 10 rpm, and the temperature is set to 45°C. The outer shell solution is prepared as follows: gelatin is dissolved at 70°C, then glycerin is added, and the mixture is dissolved again at 70°C. The mixture is then stirred evenly and poured into the outer shell solution tank of the capsule making machine. The temperature of the outer shell solution tank is set to 70°C. The outer shell solution tank and the inner core solution tank of the capsule making machine have concentric nozzles. Then, the pump speed was controlled at 650 mL / h and the inner core tank pressure at 5 kPa, so that the outer shell solution completely enveloped the inner core solution during the titration process. The solution was then dropped into a tank containing a liquid paraffin solution at a temperature controlled at 10°C, causing it to gel and form continuous core-shell spheres. The resulting spheres were then dried in an ventilated environment at room temperature (25°C) until the moisture content was below 3.0%, yielding composite probiotic spheres with a diameter of 1.6 mm.

[0042] Example 5: Preparation of compound probiotic crystals

[0043] The probiotic pellets provided in this embodiment include an outer shell structure and an inner core structure, wherein the components and their mass fractions included in the outer shell structure are as follows: 20 parts gelatin, 8 parts glycerin, and 72 parts water.

[0044] The core structure contains the following components and their mass fractions: 3 parts of Lactobacillus salivae powder, 2 parts of Bifidobacterium longum powder, and 95 parts of hydrogenated oil with a melting point of 42℃.

[0045] The preparation methods for the outer shell solution and the inner core solution are the same as above. Then, the pump speed is controlled at 650 mL / h and the pressure in the inner core tank is 8 kPa, so that the outer shell solution completely surrounds the inner core solution during the titration process. The solution is then dropped into a tank containing a liquid paraffin solution at a temperature controlled at 10°C to gel it, thereby forming continuous core-shell spheres. The obtained spheres are then dried in a ventilated environment at room temperature (25°C) until the moisture content is below 3.0%, resulting in composite probiotic spheres with a diameter of 2.2 mm.

[0046] Example 6: Preparation of composite bacterial crystals

[0047] The probiotic pellets provided in this embodiment include an outer shell structure and an inner core structure. The components and their mass fractions in the outer shell structure are as follows: 25 parts gelatin, 6 parts glycerin, and 69 parts water.

[0048] The components and their mass fractions contained in the inner core structure are as follows: 5 parts of Lactobacillus saliva-associated powder, 2 parts of Lactobacillus longum powder, and 93 parts of hydrogenated oil with a melting point of 42℃.

[0049] The preparation methods for the outer shell solution and the inner core solution are the same as above. Then, the pump speed is controlled at 600 mL / h and the pressure in the inner core tank is 8 kPa, so that the outer shell solution completely surrounds the inner core solution during the titration process. The solution is then dropped into a tank containing a liquid paraffin solution at a temperature controlled at 10°C to gel it, thereby forming continuous core-shell spheres. The obtained spheres are then dried in a ventilated environment at room temperature (25°C) until the moisture content is below 3.0%, resulting in composite probiotic spheres with a diameter of 2.5 mm.

[0050] Example 7: Preparation of composite bacterial crystal spheres

[0051] The probiotic pellets provided in this embodiment include an outer shell structure and an inner core structure. The components and their mass fractions in the outer shell structure are as follows: 25 parts gelatin, 8 parts glycerin, and 67 parts water.

[0052] The core structure contains the following components and their mass fractions: 5 parts of Lactobacillus saliva-associated powder, 5 parts of Lactobacillus longum powder, and 90 parts of hydrogenated oil with a melting point of 42℃.

[0053] The preparation methods for the outer shell solution and the inner core solution are the same as above. Then, the pump speed is controlled at 600 mL / h and the pressure in the inner core tank is 10 kPa, so that the outer shell solution completely surrounds the inner core solution during the titration process. The solution is then dropped into a tank containing a liquid paraffin solution at a temperature controlled at 10°C to gel and form continuous core-shell spheres. The obtained spheres are then dried in a ventilated environment at room temperature (25°C) until the moisture content is below 3.0%, resulting in composite probiotic spheres with a diameter of 3.0 mm.

[0054] Example 8: Test for resistance to artificial gastric juice

[0055] 1. Preparation of artificial gastric juice

[0056] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, and 2g of NaCl, add them to 1000ml of distilled water, adjust the pH to 1.2 with dilute hydrochloric acid, and then sterilize at 121℃ for 15min. Before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath shaker for 1h to simulate human body temperature.

[0057] 2. Probiotic pellet artificial gastric juice tolerance test

[0058] 2.1 Determination of the amount of crystalloid bacteria

[0059] 38 ml of anaerobic diluent was kept in a 45°C water bath for 7 min. 2 g of probiotic pellets from Examples 4, 5, 6 and 7 were added to each, and the mixture was kept in a 5 min water bath. The mixture was then homogenized at 45°C for 5 min using a homogenizer. The amount of pellets was then determined according to GB4789.35-2016 "Food Microbiology Examination - Lactic Acid Bacteria Examination".

[0060] 2.2 Artificial gastric juice digestion test

[0061] Weigh 2 grams of probiotic pellets from Examples 4, 5, 6, and 7, and add them to 8 ml of artificial gastric fluid that has been kept at a constant temperature (37°C for 10 min). Shake well by hand, and then place in a 37°C water bath for digestion for 2 hours. Filter the artificial gastric fluid away with sterile filter paper, then place the filtered pellets in a sterile container and soak in 18 ml of physiological saline for 10 min. Filter again with sterile filter paper and rinse twice with sterile physiological saline. Add anaerobic diluent to make the total weight of the pellets and anaerobic diluent approximately 40 grams, and record the actual mass. Then determine the bacterial count according to the method for determining the bacterial count of the pellets. The survival rate of the probiotic pellets after digestion in artificial gastric fluid is obtained by comparing the data before and after digestion.

[0062] 2.3 Experiment on tolerance of bacterial powder to artificial gastric juice

[0063] 0.04 g of Lactobacillus saliva-associated salivaryis VHProbi A17 and Bifidobacterium longum VHProbi Y08 bacterial powder were added to 9.96 ml of artificial gastric juice that had been kept at a constant temperature (37℃ for 10 min), shaken manually to mix, and then placed in a 37℃ water bath for 2 h for digestion. The number of viable bacteria before and after digestion in the artificial gastric juice was measured. The survival rate of the bacterial powder after digestion in the artificial gastric juice was obtained by comparing the data before and after digestion.

[0064] Table 2: Survival Rate of Probiotic Spheroids After Digestion with Artificial Gastric Juice

[0065] sample Probiotic survival rate Example 4: Probiotic Crystals 91.11％±2.11％ Example 5: Probiotic Crystals 90.67％±3.04％ Example 6: Probiotic Crystals 91.25％±2.35％ Example 7 Probiotic Crystals 92.02％±4.06％ VHProbi A17 Lactobacillus salivans powder 1.00％±0.01％ Bifidobacterium longum VHProbi Y08 bacterial powder 0.80％±0.01％

[0066] After digestion with artificial gastric juice, the probiotic pellets prepared in Examples 4, 5, and 6 were all able to withstand artificial gastric juice at pH 1.2, with a bacterial survival rate of over 90%. Among them, the probiotic pellets in Example 7 showed the best resistance to artificial gastric acid. The results indicate that the prepared pellets can effectively protect the probiotics from digestion by artificial gastric juice and allow them to successfully reach the intestines.

[0067] Example 9: Probiotic sphere disintegration test

[0068] 7.1 Preparation of artificial gastric juice

[0069] The artificial gastric juice is prepared as described above.

[0070] 7.2 Preparation of artificial intestinal fluid

[0071] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH₂PO₄, and 3.0g of ox bile salts, respectively. Add them to 77mL of 0.2mol / L NaOH solution, and bring the volume to 1000mL. Adjust the pH to 6.8±0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 115℃ for 20min. Before use, add 1g of trypsin, shake well to dissolve, and incubate in a 37℃ water bath for 1h to simulate human body temperature.

[0072] 7.3 Disintegration Test

[0073] Following the method and apparatus described in the "Disintegration Time Test" of the Chinese Pharmacopoeia, six probiotic pellets from Examples 4, 5, 6, and 7 were taken respectively and tested in artificial gastric fluid without baffles for 2 hours. Each pellet showed no disintegration or cracking. The basket was then removed, washed with a small amount of physiological water, and baffles were added to each tube. The test was then conducted in artificial intestinal fluid using the same method, and the complete dissolution time of the pellets was recorded. The disintegration times were found to be 7 min, 6 min, 8 min, and 6 min, respectively, all of which could be completely dissolved in intestinal fluid within 1 hour.

[0074] Example 10: Accelerated Stability Test

[0075] Accelerated probiotic testing refers to accelerating the decomposition of probiotic products by intensifying temperature conditions without altering the product's failure mechanism. This allows for the achievement of necessary probiotic stability within a shorter timeframe, thus assessing the reliability or lifespan of probiotics under normal conditions. Because probiotic pellets have a relatively long shelf life, we determined their stability under extreme temperature conditions of 37°C.

[0076] The compound probiotic pellets prepared in Examples 4, 5, 6 and 7 were sealed and placed in an incubator at 37°C. Simultaneously, bacterial powder was taken for comparison. Samples were taken every 5 days to determine the bacterial count, and the test was conducted continuously for one month.

[0077] Table 2: Stability test results of bacterial count at 37℃ (LOG CFU / g)

[0078]

[0079] The results show that the number of live bacteria in the probiotic powder gradually decreased over time. After 30 days of storage, the number of bacteria in the probiotic pellets did not decrease, while the number of live bacteria in the freeze-dried probiotic powder decreased by 0.89 and 1.2 orders of magnitude, respectively. Therefore, compared with the freeze-dried probiotic powder alone, the bacteria in the probiotic pellets showed better stability and a slower decline. *Lactobacillus salivarius* not only inhibits oral pathogens but also intestinal pathogens such as *Escherichia coli* and *Salmonella*, while *Bifidobacterium longum* can reduce the effects of carcinogens on human cell gene mutations. The combined use of these two strains can better regulate the gut and improve the body's immunity.

[0080] Probiotic pellets can be combined alone with prebiotics or other ingredients to make probiotic solid beverages for direct oral consumption, or they can be added to various foods and beverages, such as fruit and vegetable juices and yogurt, to create probiotic complex beverages. Probiotic pellets help protect probiotics as they reach the intestines and exert their beneficial effects.

Claims

1. A probiotic crystal ball, characterized in that, The probiotic pellets comprise an outer shell structure and an inner core structure, wherein the outer shell structure comprises gelatin, glycerin, and water; the inner core structure comprises Lactobacillus salivarius, Bifidobacterium longum, and hydrogenated oil; the mass fractions of the gelatin, glycerin, and water are as follows: 18-25 parts gelatin, 6-8 parts glycerin, and 69-76 parts water. The aforementioned Lactobacillus salivae ( Lagilactobacillus salivarius The accession number for this is CCTCC NO: M2022172; The aforementioned Bifidobacterium longum ( Bifidobacterium longum The accession number of the object is CCTCC NO: M2019780.

2. The probiotic pellets as described in claim 1, characterized in that, The mass proportions of gelatin, glycerin, and water are as follows: 18 parts gelatin, 6 parts glycerin, and 76 parts water.

3. The probiotic pellets as described in claim 1, characterized in that, The mass proportions of gelatin, glycerin, and water are as follows: 25 parts gelatin, 6 parts glycerin, and 69 parts water.

4. The probiotic pellets as described in claim 1, characterized in that, The hydrogenated oil mentioned is a hydrogenated oil with a melting point of 42°C.

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