A probiotic beadlet comprising lactobacillus plantarum and lactobacillus rhamnosus

Abstract

The present application belongs to the technical field of probiotic food processing, and particularly relates to a probiotic crystal ball containing plantarum and lactobacillus rhamnosus. The probiotic crystal ball comprises a shell and an inner core, wherein the inner core comprises plantarum with a preservation number of CCTCC NO: M2022173 and lactobacillus rhamnosus with a preservation number of CCTCC NO: M2021904; and the shell is prepared from gelatin, glycerol and water. The probiotic crystal ball can be used alone or in combination with prebiotics or other components to prepare probiotic solid beverages, and can be directly taken orally or added to various foods and beverages, such as various fruit and vegetable juices, yogurts and the like, to prepare probiotic composite beverages. The probiotic crystal ball can protect probiotics from reaching the intestinal tract smoothly and exerting a probiotic effect in the intestinal tract.

Description

Technical Field

[0001] This invention belongs to the field of probiotic food production technology, specifically relating to a probiotic sphere containing Lactobacillus plantarum and Lactobacillus rhamnosus. Background Technology

[0002] Lactic acid bacteria are a group of Gram-positive bacteria characterized by certain morphological, metabolic, and physiological features. They are non-spore-forming, anaerobic or facultative anaerobic cocci or bacilli that produce lactic acid, one of the main fermentation products of carbohydrates. Lactic acid bacteria are among the major types of probiotics, and their various beneficial effects, such as regulating intestinal health, skin microecology, and dental caries, have been reported in numerous articles. There are many types of lactic acid bacteria, mainly including *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, and *Lactobacillus paracasei*. However, the survival of lactic acid bacteria as intestinal probiotics is affected by many factors, such as temperature, pH, pressure, gastric acid, and bile salts. Most lactic acid bacteria cannot tolerate the digestion of gastric acid and bile salts, and upon reaching the intestines, they have very few viable bacteria, thus failing to effectively perform their intended physiological functions.

[0003] Many products on the market claim to be resistant to stomach acid, but after being treated with strong stomach acid, the amount of bacteria in the gut is practically zero, making it difficult to achieve any gut-regulating effect. Currently, microencapsulation technology is considered an important technique for protecting probiotics. Common microencapsulation techniques include spray drying, extrusion, and emulsification. Among these, multi-layer encapsulation technology using extrusion is a relatively new technology, requiring sophisticated equipment and wall materials. This technology encapsulates probiotics in multiple layers within a capsule shell or sphere shell, isolating the probiotics from contact with gastric juices, thus better protecting them.

[0004] The wall material and carrier used to encapsulate probiotics determine their survival rate and 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. Commonly used wall materials include polysaccharides such as sodium alginate, xanthan gum, gellan gum, carrageenan, agar, bacterial cellulose, and carbohydrates (including starch, starch syrup, dextrin, chitosan, and small-molecule sugars). These substances have low viscosity and good solubility. Except for starch, most of these materials lack lipophilic groups in their structure and therefore lack the emulsifying and film-forming interfacial properties required for efficient microencapsulation. They are usually used as auxiliary wall materials in combination with other wall materials with excellent emulsifying properties, such as proteins and colloids, to improve the density of the microcapsule membrane. Protein raw materials mainly include gelatin, whey protein, sodium caseinate, and soy protein. Proteins have excellent emulsifying and film-forming properties and are widely used, especially for encapsulating oils. Protein molecules have many amphiphilic groups. When in contact with oil droplets, their hydrophobic groups can adsorb onto the surface of the oil droplets, while their hydrophilic groups penetrate into the aqueous phase, forming a protective film around the oil droplets. This reduces the interfacial tension of the emulsion, which is beneficial to the formation and stability of the emulsion.

[0005] Probiotic capsules require an acid-resistant wall material that can dissolve in the intestines to exert their effects. To address this issue, 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 allows the probiotics to survive without being digested by gastric juices, resulting in a high survival rate in the intestines. Summary of the Invention

[0006] The purpose of this invention is to provide a probiotic crystal pellet preparation containing *Lactiplantibacillus plantarum* and *Lactobacillus rhamnosus* and its applications. *Lactiplantibacillus plantarum* is identified as *Lactiplantibacillus plantarum* VHProbi V38, obtained through screening from fermented sauerkraut, and can inhibit the growth of oral pathogens. *Lactobacillus rhamnosus* is identified as *Lactobacillus rhamnosus* VHPribo M15, selected from breast milk, and can effectively prevent and relieve constipation. Both *Lactobacillus rhamnosus* and *Lactobacillus plantarum* have strong tolerance to artificial gastric juice. After crystal pelleting, the prepared probiotic crystal pellets exhibit stronger resistance and better stability, and can be widely used in food, health products, and other fields.

[0007] The present invention first provides a probiotic crystal ball, which comprises an outer shell and an inner core, wherein the inner core contains *Lactobacillus plantarum* with accession number CCTCC NO: M2022173 and *Lactobacillus rhamnosus* with accession number CCTCC NO: M2021904.

[0008] The outer shell is made from gelatin, glycerin, and water.

[0009] As described in the example, the mass ratio of gelatin, glycerin and water is 16-25:6-8:67-78;

[0010] As a specific embodiment, the outer shell structure comprises the following components: 22 parts gelatin, 6 parts glycerin, and 72 parts water.

[0011] As a specific description of another embodiment, the outer shell structure includes the following components: 25 parts gelatin, 8 parts glycerin, and 67 parts water.

[0012] Furthermore, the inner core also contains hydrogenated oil with a melting point of 42°C.

[0013] In another aspect, the present invention also provides an application of the probiotic pellets in the preparation of probiotic products.

[0014] The product in question is a solid or liquid food.

[0015] The present invention also provides a probiotic solid or liquid product comprising the above-mentioned probiotic spheres.

[0016] The probiotic pellets provided by this invention can be used alone or in combination with prebiotics or other components to make probiotic solid beverages for direct oral consumption. They can also be added to various foods and beverages, such as fruit and vegetable juices and yogurt, to create probiotic compound beverages. The probiotic pellets help the probiotics reach the intestines smoothly and exert their beneficial effects within the gut. Detailed Implementation

[0017] Based on the re-screening of *Lactobacillus plantarum* and *Lactobacillus rhamnosus* with specific effects, this invention provides a probiotic sphere, thereby increasing the efficiency of probiotic strains reaching the intestines.

[0018] The present invention will now be described in detail with reference to the embodiments.

[0019] Example 1: Physicochemical properties and applications of Lactobacillus plantarum strain VHProbi V38

[0020] The *Lactiplantibacillus plantarum* strain VHProbiV38 obtained by screening in this invention has been identified as a novel *Lactobacillus* strain through polyphasic taxonomy. Its safety meets relevant regulatory requirements and it can be used as a food ingredient source without long-term side effects or the risk of overdose. This strain, *Lactiplantibacillus plantarum* VHProbiV38, was deposited on March 1, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M2022173.

[0021] The *Lactobacillus plantarum* VHProbi V38 strain exhibits good biocompatibility, resisting artificial gastric and intestinal fluids, and can exert probiotic effects in the gut. This strain possesses strong antioxidant capabilities, with scavenging rates of 20.9% and 36.3% against DPPH and hydroxyl radicals, respectively.

[0022] The inhibitory effects of *Lactobacillus plantarum* VHProbi V38 bacterial suspension and lysate on *Streptococcus mutans*, *Actinomyces villosa*, *Porphyromonas gingivalis*, *Actinomyces actinomycetes*, *Clostridium nucleatum*, *Escherichia coli*, and *Salmonella enteritidis* were determined using the Oxford cup method. The diameters of the inhibition zones are shown in Table 1.

[0023] Table 1: Inhibitory effect of Lactobacillus plantarum VHProbi V38 on pathogenic bacteria

[0024] Streptococcus mutans 27.5mm 25mm Porphyromonas gingivalis 21mm 18mm Sticky Actinomycetes 15.5mm 13mm Aggregates actinomycetes 12mm 11mm Clostridium nucleatum 10.5mm 10mm E. coli >30mm >30mm Salmonella enteritidis >30mm >30mm

[0025] As shown in Table 1, *Lactobacillus plantarum* VHProbi V38 not only has a significant inhibitory effect on five oral pathogens, namely *Streptococcus mutans*, *Porphyromonas gingivalis*, *Actinomyces viscerata*, *Actinomyces actinomyces*, and *Clostridium nucleatum*, but also has a significant antibacterial effect on intestinal pathogens, namely *Escherichia coli* and *Salmonella enteritidis*, exhibiting a broad spectrum of antibacterial activity.

[0026] Lactobacillus plantarum VHProbi V38 can effectively bind to common oral pathogens, significantly inhibiting their adhesion to teeth or gums. Among these, this strain exhibits the best co-agglutination effect against Streptococcus mutans, Clostridium nucleatum, and Aggregates hemiactophorae, with co-agglutination rates reaching 60.04%-76.99% after 6 hours. This strain can also effectively colonize the oral cavity, achieving a self-agglutination rate of 68.40% after 6 hours.

[0027] Lactobacillus plantarum VHProbi V38 effectively removes biofilms formed by Streptococcus mutans, thus preventing and treating dental caries. The fermentation broth and lysate of this bacterium showed the best biofilm removal efficiency, reaching up to 100%, significantly higher than the bacterial suspension. This strain also significantly inhibits the proliferation of Streptococcus mutans. In the control group, Streptococcus mutans began to grow rapidly 10 hours after inoculation, reaching a stationary phase after approximately 20 hours; while in the experimental group supplemented with the supernatant of Lactobacillus plantarum VHProbi V38 lysate, Streptococcus mutans showed almost no growth within 24 hours.

[0028] Cellular experiments further confirmed that *Lactobacillus plantarum* VHProbi V38 is non-toxic to normal human gingival epithelial cells and can adhere to the cells, effectively colonizing the oral cavity and balancing the oral flora. This strain can significantly inhibit the adhesion and growth of periodontal pathogens (*Porphyromonas gingivalis*, *Clostridium nucleatum*, and *Aggregatibacter hemiactiformis*) on normal human gingival epithelial cells, effectively preventing and improving symptoms such as periodontitis and gingival bleeding caused by these pathogens.

[0029] Example 2: Physicochemical properties and applications of Lactobacillus rhamnosus strain VHPribo M15

[0030] The Lactobacillus rhamnosus VHPribo M15 strain used in this invention was obtained by screening from breast milk and was deposited on July 19, 2021, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2021904.

[0031] Lactobacillus rhamnosus strain VHPribo M15 exhibits strong tolerance to simulated gastrointestinal fluid. After 3 hours of digestion in simulated gastric fluid, its survival rate reaches 99.8%, successfully traversing the gastrointestinal tract and colonizing in the colon to exert its probiotic functions. This strain is sensitive to common antibiotics such as ampicillin and tetracycline, does not produce hemolysin, and does not lyse blood cells, demonstrating good biocompatibility. Furthermore, Lactobacillus rhamnosus VHPribo M15 possesses certain antioxidant activity, achieving a DPPH free radical scavenging rate of 46.19%, and its supernatant exhibits a lipid peroxidation inhibition rate of 43.37%. This strain can also effectively degrade cholesterol, demonstrating probiotic properties that lower serum cholesterol.

[0032] Lactobacillus rhamnosus VHPribo M15 can significantly increase the water content of mouse feces and improve the transit capacity of the small intestine in constipated mice, thereby promoting defecation and improving constipation symptoms.

[0033] Lactobacillus rhamnosus M15 can also significantly increase the content of acetic acid and total organic acids in mouse feces. Acetic acid is the main product of colonic fermentation. Increasing the concentration of acetic acid in the intestine can lead to an increase in intestinal osmotic pressure and an increase in the water content of intestinal contents, thereby stimulating the intestinal wall, increasing intestinal peristalsis, and thus relieving constipation.

[0034] Lactobacillus rhamnosus VHPribo M15 can effectively regulate the secretion of gastrointestinal regulatory peptides. By promoting the secretion of excitatory neurotransmitters motilin, gastrin, and substance P, and reducing the secretion of inhibitory neurotransmitters endothelin, somatostatin-1, and vasoactive intestinal peptide, it effectively regulates gastrointestinal motility and gastric acid secretion, thus relieving constipation symptoms.

[0035] Lactobacillus rhamnosus VHPribo M15 can prevent and alleviate intestinal barrier damage caused by constipation, reduce inflammatory response, and restore the integrity of the intestinal mucosal barrier.

[0036] Lactobacillus rhamnosus strain VHPribo M15 can increase the richness and evenness of fecal flora in constipated mice, making the abundance of dominant species at the phylum and genus levels tend to be similar to the flora characteristics of the control group mice, reducing the differences in flora composition, and making the community structure composition similar to the control group, tending to the level of normal mice.

[0037] Example 3: Preparation of bacterial powder

[0038] (1) Inoculate activated Lactobacillus plantarum VHProbi V38 or Lactobacillus rhamnosus VHPribo M15 into MRS broth medium and culture at 37°C for 24 h to obtain seed culture.

[0039] (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;

[0040] (3) Centrifuge the fermentation broth at 5000 rpm for 10 minutes and collect the bacterial sludge.

[0041] (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.

[0042] 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.

[0043] The results showed that the viable bacterial counts of *Lactobacillus plantarum* VHProbi V38 and *Lactobacillus rhamnosus* VHPriboM15 prepared by this invention both exceeded 200 billion CFU / g.

[0044] Example 4: Preparation of compound probiotic crystals

[0045] 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: 16 parts gelatin, 6 parts glycerin, and 78 parts water.

[0046] The core structure contains the following components and their mass fractions: 3 parts of Lactobacillus plantarum VHProbi V38 bacterial powder, 2 parts of Lactobacillus rhamnosus VHPribo M15 bacterial powder, and 95 parts of hydrogenated oil with a melting point of 42℃.

[0047] 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.

[0048] Example 5: Preparation of compound probiotic crystals

[0049] The probiotic pellets provided in this embodiment include an outer shell structure and an inner core structure. The components and their mass fractions contained in the outer shell structure are as follows: 18 parts gelatin, 8 parts glycerin, and 74 parts water.

[0050] The core structure contains the following components and their mass fractions: 2 parts of Lactobacillus plantarum VHProbi V38 bacterial powder, 3 parts of Lactobacillus rhamnosus VHPribo M15 bacterial powder, and 95 parts of hydrogenated oil with a melting point of 42℃.

[0051] 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.

[0052] Example 6: Preparation of composite bacterial crystals

[0053] The probiotic pellets provided in this embodiment include an outer shell structure and an inner core structure. The components and their mass fractions contained in the outer shell structure are as follows: 22 parts gelatin, 6 parts glycerin, and 72 parts water.

[0054] The core structure contains the following components and their mass fractions: 3 parts of Lactobacillus plantarum VHProbi V38 bacterial powder, 4 parts of Lactobacillus rhamnosus VHPribo M15 bacterial powder, and 93 parts of hydrogenated oil with a melting point of 42℃.

[0055] The preparation methods for the outer shell solution and the inner core solution are the same as in Example 4. Then, the pump speed is controlled and adjusted to 600 mL / h, and the pressure of the inner core tank is 8 kPa, so that the outer shell solution just covers 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.

[0056] Example 7: Preparation of composite bacterial crystal spheres

[0057] 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.

[0058] The core structure contains the following components and their mass fractions: 5 parts of Lactobacillus plantarum VHProbi V38 bacterial powder, 5 parts of Lactobacillus rhamnosus VHPribo M15 bacterial powder, and 90 parts of hydrogenated oil with a melting point of 42℃.

[0059] 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.

[0060] Example 8: Test for resistance to artificial gastric juice

[0061] 1. Preparation of artificial gastric juice

[0062] 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.

[0063] 2. Probiotic pellet artificial gastric juice tolerance test

[0064] 2.1 Determination of the amount of crystalloid bacteria

[0065] 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".

[0066] 2.2 Artificial gastric juice digestion test

[0067] 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.

[0068] 2.3 Tolerance test of bacterial powder to artificial gastric juice

[0069] 0.04 g of *Lactobacillus plantarum* VHProbi V38 powder and *Lactobacillus rhamnosus* VHPribo M15 powder were weighed and added to 9.96 ml of artificial gastric juice that had been kept at a constant temperature (37℃ for 10 min). The mixture was shaken by hand and then placed in a 37℃ water bath for 2 hours 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.

[0070] Table 2: Survival Rate of Probiotic Spheroids After Digestion with Artificial Gastric Juice

[0071] Example 4: Probiotic Crystals 91.21％±2.01％ Example 5: Probiotic Crystals 91.67％±2.04％ Example 6: Probiotic Crystals 92.05％±2.75％ Example 7 Probiotic Crystals 92.00％±3.06％ Lactobacillus plantarum VHProbi V38 powder 0.92％±0.02％ Lactobacillus rhamnosus VHPribo M15 bacterial powder 1.01％±0.01％

[0072] The results of artificial gastric juice digestion showed that the probiotic pellets prepared in Examples 4, 5, 6, and 7 could all tolerate artificial gastric juice at pH 1.2, with a bacterial survival rate of over 91%. Among them, the probiotic pellets in Examples 4 and 5 showed the best resistance to artificial gastric acid. Meanwhile, the survival rates of the two bacterial powders in artificial gastric juice were 0.92% and 1.01%, respectively, indicating that the pellets effectively protected the probiotics from digestion in artificial gastric juice and enabled them to successfully reach the intestines.

[0073] Example 9: Probiotic sphere disintegration test

[0074] 1. Preparation of artificial gastric juice

[0075] The artificial gastric juice is prepared as described above.

[0076] 2. Preparation of artificial intestinal fluid

[0077] 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.

[0078] 3. Disintegration test

[0079] 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 simulated gastric fluid without baffles for 2 hours. No pellets showed 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 repeated in simulated intestinal fluid, and the complete dissolution time of the pellets was recorded. The results showed that the disintegration times of the pellets described in Examples 4, 5, 6, and 7 were 5, 6, 8, and 8 minutes, respectively, and all were completely dissolved in intestinal fluid within 1 hour.

[0080] Example 10: Accelerated Stability Test

[0081] 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.

[0082] 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 testing was conducted continuously for one month. The stability of the probiotic pellets and bacterial powder was observed.

[0083] Table 3: Stability test results of bacterial count at 37℃ (LOG CFU / g)

[0084]

[0085] 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 0.99 and 0.90 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 plantarum* VHProbi V38 not only inhibits oral pathogens but also intestinal pathogens such as *Escherichia coli* and *Salmonella*. *Lactobacillus rhamnosus* VHPribo M15 can prevent and alleviate intestinal barrier damage caused by constipation, reduce inflammatory responses, and restore the integrity of the intestinal mucosal barrier. The combined use of these two strains can better regulate the gut and improve the body's immunity.

[0086] 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 and an inner core, wherein the inner core contains *Lactiplantibacillus plantarum* VHProbiV38 (accession number CCTCC NO: M2022173) and *Lactobacillus rhamnosus* VHPribo M15 (accession number CCTCC NO: M2021904). The outer shell is prepared from gelatin, glycerin and water, and the mass ratio of gelatin, glycerin and water in the outer shell is 16-25:6-8:67-78. The inner core also contains hydrogenated oil with a melting point of 42°C.

2. The probiotic pellets as described in claim 1, characterized in that, The mass parts of gelatin, glycerin and water in the outer shell are as follows: 22 parts gelatin, 6 parts glycerin and 72 parts water.

3. The probiotic pellets as described in claim 1, characterized in that, The mass parts of gelatin, glycerin and water are as follows: 25 parts gelatin, 8 parts glycerin and 67 parts water.

4. The application of the probiotic pellets as described in claim 1 in the preparation of probiotic products.

5. The application as described in claim 4, characterized in that, The product in question is a solid or liquid food.

6. A probiotic solid or liquid product, characterized in that, The product contains the probiotic pellets as described in claim 1.

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