A probiotic bead containing lactobacillus plantarum and a method of preparing the same

Abstract

This invention relates to the field of functional microbial screening and application technology, specifically providing a probiotic crystal ball and its preparation method. The probiotic crystal ball consists of three parts: a crystal nucleus, a crystal mantle, and a crystal shell. The crystal nucleus contains *Lactobacillus plantarum* powder. The *Lactobacillus plantarum* strain is VHProbi R14, with accession number CCTCC NO: M2023721. This strain can effectively regulate the intestinal flora and significantly increase the abundance of beneficial *Akermansia* species, especially *Akermansia myxophilus* (Akk bacteria). The probiotic crystal ball provided by this invention has a two-layer protective structure, exhibiting excellent stability and heat resistance. It can effectively maintain the viable count of *Lactobacillus plantarum* VHProbi R14 during transportation, storage, and processing, significantly improving the applicability of *Lactobacillus plantarum* VHProbi R14, and can be widely used in food, health products, and other fields.

Description

Technical Field

[0001] This invention belongs to the field of functional probiotic screening and application technology, specifically relating to a probiotic pellet containing Lactobacillus plantarum and its preparation method. Background Technology

[0002] Probiotics are live microorganisms that, when ingested in sufficient quantities, can provide health benefits to the host. Currently, there are over a thousand clinical research papers on probiotics. Although these studies are heterogeneous due to differences in strains and study populations, the accumulated evidence from many different results supports the beneficial effects of probiotics on human health. Changes in the composition of the gut microbiota and its metabolites are associated with various metabolic abnormalities, including obesity, diabetes, neurological disorders, respiratory diseases, and cardiovascular diseases. Probiotics can improve these conditions by creating a healthy gut environment through balancing the number of microorganisms in the body and promoting good metabolism.

[0003] Lactobacillus plantarum, a type of beneficial bacteria, is widely distributed in nature, especially in various fermented foods such as fruits, vegetables, meat, dairy products, and wine, and is therefore widely used in human production activities. Simultaneously, Lactobacillus plantarum also exists in the human gastrointestinal tract, serving as a probiotic that greatly promotes human health. Currently, numerous studies have shown that Lactobacillus plantarum possesses various health benefits, including regulating immune function, regulating chronic metabolic diseases, antagonizing pathogenic bacterial infections, and regulating mental and neurological functions. For Lactobacillus plantarum to exert its probiotic effects, it must successfully navigate the acidic environment of the stomach and the high bile salt environment of the duodenum to reach the small and large intestines in a viable state, thereby exerting its microecological regulatory function. Therefore, increasing the stress of bile salts, osmotic pressure, and oxygen on Lactobacillus plantarum helps it survive and successfully colonize in the gastrointestinal tract.

[0004] In recent years, researchers both domestically and internationally have used various wall materials to encapsulate *Lactobacillus plantarum*. Probiotic pellets refer to non-dried, transparent spheres encapsulated with probiotics. Compared to ordinary microcapsules, probiotic pellets formed through secondary encapsulation can effectively improve the stability of probiotics, such as storage stability, gastric acid tolerance, and temperature tolerance. Currently, research on probiotic microcapsule pellets is limited; therefore, the development of novel probiotic pellet wall materials and their preparation processes has become a hot topic pursued by both academia and industry. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic pellet and its preparation method. The probiotic pellet contains *Lactobacillus plantarum*, which can significantly increase the abundance of Akk bacteria in the intestines and has broad application prospects.

[0006] One aspect of this invention is to provide probiotic pellets containing Lactobacillus plantarum powder.

[0007] The *Lactobacillus plantarum* mentioned is strain VHProbi R14, with accession number CCTCC NO: M2023721.

[0008] The viable count of the *Lactobacillus plantarum* powder is not less than 10. 10 CFU / g.

[0009] The probiotic crystals consist of three parts: a crystal nucleus, a crystal mantle, and a crystal shell.

[0010] The crystal nucleus is located in the innermost layer of the crystal sphere and is composed of plant lactobacillus powder, lecithin, and stearic palm oil.

[0011] The mass percentages of Bacillus plantarum powder, lecithin, and stearic palm oil in the crystal nucleus are 5-10%, 1.5-3%, and 88.5-92%, respectively.

[0012] The crystal mantle is located in the middle layer of the crystal sphere and is composed of sodium alginate, gellan gum, carrageenan, and water.

[0013] The mass percentages of sodium alginate, gellan gum, carrageenan, and water in the crystal mantle are 3-10%, 1-2%, 0.5-1%, and 87.5-95.5%, respectively.

[0014] The crystal shell, located on the outermost layer of the crystal sphere, is composed of food coloring, gelatin, pectin, glycerin, and water.

[0015] The mass percentages of food coloring, gelatin, pectin, glycerin, and water in the crystal shell are 2%, 10-20%, 1-2%, 5-15%, and 62-77%, respectively.

[0016] The present invention also provides a method for preparing the probiotic pellets, comprising the following steps:

[0017] (1) Preparation of solutions containing crystal nuclei, crystal mantles, and crystal shells

[0018] Soft lecithin and stearic palm oil are heated and dissolved into a liquid state. The Bacterium plantarum powder, soft lecithin and stearic palm oil are thoroughly mixed at a mass percentage of 5-10%, 1.5-3% and 88.5-92% respectively to obtain a crystal nucleus solution, which is kept warm for later use.

[0019] Gellan gum and carrageenan are preheated and melted into a liquid state. Sodium alginate, gellan gum, carrageenan and water are thoroughly mixed at a mass percentage of 3-10%, 1-2%, 0.5-1%, and 87.5-95.5% respectively to obtain a crystal mantle solution, which is then kept warm for later use.

[0020] Preheat gelatin and pectin to melt them into a liquid state. Mix food coloring, gelatin, pectin, glycerin and water thoroughly at mass percentages of 2%, 10-20%, 1-2%, 5-15% and 62-77% to obtain a crystal shell solution, and keep it warm for later use.

[0021] (2) Preparation of crystal spheres

[0022] The solutions of crystal nuclei, crystal mantle, and crystal shell are added to a capsule machine with three concentric nozzles. The innermost nozzle sprays the crystal nuclei solution, the middle nozzle sprays the crystal mantle solution, and the outermost nozzle sprays the crystal shell solution. The capsule machine is started, and the appropriate nozzle speed is adjusted so that the three nozzles simultaneously spray the crystal ball components, which drip into rapeseed oil that has been cooled to 12°C and is flowing. The dripped crystal balls are dried at 16°C for 8 hours, and then freeze-dried under vacuum for 24 hours to reduce the moisture content to below 3%, thus obtaining probiotic crystal balls.

[0023] The probiotic pellets contain no less than 10 live bacteria. 8 CFU / g.

[0024] The present invention also provides the application of the probiotic crystals in food.

[0025] The probiotic crystals provided by this invention have a two-layer protective structure. The crystal shell isolates the probiotics from oxygen and moisture, while the crystal mantle is highly acid-resistant, effectively protecting the activity of the probiotics within the crystal nucleus and giving it excellent stability. This effectively maintains the viable count of *Lactobacillus plantarum* VHProbi R14 during transportation, storage, and processing, significantly expanding the applicability of *Lactobacillus plantarum* VHProbi R14. The probiotic crystals also exhibit strong heat resistance; after storage at 45°C and 75% humidity for 24 hours, they retain their morphology and color without change, making them suitable for wide application in food, health products, and other fields. Attached Figure Description

[0026] Figure 1 This is a colony morphology diagram of *Lactobacillus plantarum* VHProbi R14.

[0027] Figure 2 The species composition distribution of mouse fecal microbiota at the genus level;

[0028] Figure 3 Species differences in mouse fecal microbiota at the genus level;

[0029] Figure 4 To analyze species differences in mouse fecal microbiota at the species level;

[0030] Figure 5 The results are from the temperature resistance test of probiotic pellets. Detailed Implementation

[0031] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0032] Example 1: Isolation, screening and identification of Lactobacillus plantarum VHProbi R14

[0033] This invention screened a lactobacillus strain with strong acid resistance from fresh fermented sauerkraut samples. After identification by colony morphology, carbon source metabolism and molecular biology, the strain was identified as a new Lactiplantibacillus plantarum strain, which the applicant named Lactiplantibacillus plantarum VHProbi R14.

[0034] On May 10, 2023, the applicant deposited strain VHProbi R14 of Lactobacillus plantarum at the China Center for Type Culture Collection (CCTCC) of Wuhan University in Wuhan, China, with accession number CCTCC NO: M2023721.

[0035] The colony morphology of *Lactobacillus plantarum* VHProbi R14 is as follows: Figure 1 As shown, the colonies are smooth, moist, milky white, round, with neat edges, and the surface of the colonies is raised. The diameter of the colonies is about 1.5 to 2 mm.

[0036] The 16S rDNA sequence of *Lactobacillus plantarum* VHProbi R14 is SEQ ID NO: 1, and the specific sequence is as follows:

[0037]

[0038] The maximum salt tolerance of *Lactobacillus plantarum* VHProbi R14 is 6%; it ferments glucose to produce acid but not gas; it can grow normally at 15-45℃; it has good biocompatibility, does not produce hemolysin, and cannot lyse blood cells; it is sensitive to common antibiotics such as erythromycin, clindamycin, and ampicillin; and its cell surface hydrophobicity is 8.02%.

[0039] Example 2: Tolerance of *Lactobacillus plantarum* VHProbi R14 to artificial gastric and intestinal fluids

[0040] 1. Preparation of bacterial culture:

[0041] The cryopreserved *Lactobacillus plantarum* strain VHProbi R14 was streaked onto MRS solid medium and cultured at 37°C for 24–48 h. After one subculture on MRS liquid medium, *Lactobacillus plantarum* strain VHProbi R14 was inoculated into fresh MRS liquid medium at a 5% inoculum and cultured at 40°C with shaking for 24–48 h to obtain fresh bacterial culture.

[0042] 2. Preparation of artificial gastric juice

[0043] 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 3.0 with dilute hydrochloric acid, and then sterilize at 115℃ for 20min. 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.

[0044] 3. Preparation of artificial intestinal fluid

[0045] 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.

[0046] 4. Test methods

[0047] Take 2 mL of fresh bacterial culture, centrifuge at 5000 rpm for 5 min to collect bacterial cells, wash the cells three times with physiological saline, and then resuspend them in 2 mL of physiological saline as the inoculum. Take 1 mL of the inoculum and add it to 9 mL of artificial gastric fluid that has been warmed for 1 h. Place the mixture in a 37℃ water bath shaker at 200 rpm for 2 h. Take 1 mL samples at 0 h and 2 h to detect the viable bacterial count. Then take 1 mL of artificial gastric fluid after 2 h of digestion and add it to 24 mL of artificial intestinal fluid. Place the mixture in a 37℃ water bath shaker (200 rpm) for 3 h. Take 1 mL samples to detect the viable bacterial count.

[0048] The viable count method was determined according to the national standard GB4789.35-2016-Food Microbiology Examination - Lactic Acid Bacteria Examination. The LOG (CFU / mL) of the viable count of this strain after artificial gastric fluid and artificial intestinal fluid are shown in Table 1.

[0049] Table 1. Viable bacterial count after digestion in artificial gastrointestinal tract

[0050]

[0051] As shown in Table 1, the viable count of *Lactobacillus plantarum* VHProbi R14 decreased only slightly after digestion with artificial gastric and intestinal fluids, indicating that this strain has strong tolerance to artificial gastric and intestinal fluids.

[0052] Example 3: In vitro cholesterol degradation test of Lactobacillus plantarum VHProbi R14

[0053] 1. Preparation of cholesterol micelle solution: Accurately weigh 1g of cholesterol, dissolve it in anhydrous ethanol, and make up to 100mL. Filter the solution under sterile conditions using a 0.22μm microporous membrane.

[0054] 2. Weigh out 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 2.0g diammonium citrate, 20.0g glucose, 1.0mL Tween 80, 5.0g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 2.0g dipotassium hydrogen phosphate, and 1000ml distilled water. Adjust the pH to 7.3, sterilize at 115℃ for 30min, then add cholesterol solution to bring the final cholesterol concentration to 0.1%. Inoculate fresh bacterial culture at a rate of 0.1%, incubate at 37℃ for 48 hours, then take 0.2ml of bacterial culture, add 1.8ml anhydrous ethanol, mix well, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and use the supernatant to determine the cholesterol content. The cholesterol determination method follows GB / T5009.128-2003 "Determination of Cholesterol in Food".

[0055] The results showed that the *Lactobacillus plantarum* VHProbi R14 provided by this invention had a cholesterol degradation rate as high as 21.24%.

[0056] Example 4: Determination of the antioxidant function of Lactobacillus plantarum VHProbi R14

[0057] 1. Determination of the strain's ability to scavenge DPPH and hydroxyl radicals (HRS)

[0058] 1) Preparation of bacterial suspension

[0059] A single colony of *Lactobacillus plantarum* VHProbi R14 in excellent growth condition was inoculated into 3 ml of MRS liquid medium and cultured at 37°C for 18-20 h. Using this culture as the inoculum, 2% of the colony was inoculated into 50 ml of MRS liquid medium and incubated statically for 18 h to obtain *Lactobacillus plantarum* VHProbi R14 bacterial suspension. 1 mL of the bacterial suspension was collected by centrifugation, and the bacterial cells were washed twice with 1 mL of PBS buffer, then resuspended in 2 mL of PBS solution for later use.

[0060] 2) Determination of the strain's ability to scavenge DPPH free radicals

[0061] Take 1 mL of *Lactobacillus plantarum* VHProbi R14 bacterial suspension, add 1 mL of 0.4 mM freshly prepared DPPH free radical solution, mix well, and then incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the sample at a wavelength of 517 nm. 样本 The test was performed in triplicate. The control group sample was prepared with an equal volume of PBS solution and DPPH-ethanol mixture, and the blank was zeroed with an equal volume of Lactobacillus plantarum VHProbi R14 bacterial suspension and ethanol mixture.

[0062] The clearance rate is calculated using the following formula:

[0063] Clearance rate % = [1-(A 样品 -A 空白 ) / A 对照 ×100%.

[0064] The results showed that the *Lactobacillus plantarum* VHProbi R14 provided by this invention had a DPPH free radical scavenging rate of up to 19.52% with a standard deviation of 0.91%.

[0065] 3) Determination of the strain's ability to scavenge hydroxyl radicals (HRS)

[0066] Mix 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water, and 200 μL of *Lactobacillus plantarum* VHProbi R14 suspension, then add 100 μL of 3 mM hydrogen peroxide solution. After incubating in a water bath at 37°C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm.

[0067] The hydroxyl radical scavenging rate is calculated using the following formula:

[0068] Clearance rate (%) = (A 样品 -A 控制 ) / (A 空白 -A 控制 )×100%.

[0069] Where A 控制 A was used as a substitute for deionized water in the sample. 空白 Deionized water was used to replace the sample and H2O2.

[0070] The results showed that the supernatant of *Lactobacillus plantarum* VHProbi R14 fermentation provided by this invention had a scavenging rate of up to 83.89% for HRS free radicals, with a standard deviation of 0.42%; and its bacterial suspension had a scavenging rate of up to 25.36% for HRS free radicals, with a standard deviation of 3.06%.

[0071] 2. Determination of the strain's resistance to lipid peroxidation

[0072] 1) Preparation of bacterial strain culture and fermentation supernatant, bacterial cells, and intracellular extracts:

[0073] The strain was cultured in MRS liquid medium at 37°C for 24 h, and after 3 passages, it was centrifuged at 6000 rpm for 10 min at 4°C, and the supernatant was collected as the fermentation supernatant. The collected bacterial cells were washed three times by centrifugation at 6000 rpm for 10 min in PBS buffer (pH 7.4). The bacterial cells were then resuspended in PBS buffer to adjust the bacterial concentration to 1.0 × 10⁻⁶ cells / mL. 9 The bacterial suspension was obtained by measuring cells / mL.

[0074] 2) Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL LTween 20, 19.7 mL deionized water.

[0075] 3) Add 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample. Incubate at 37°C for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture, incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 r / min for 15 min, and collect the supernatant. 532 nm The absorbance measured is A; the control group is represented by 0.5 mL of distilled water instead of the sample, which is A0. Inhibition rate / % = (A0 - A) / A0 × 100.

[0076] The results showed that the supernatant of *Lactobacillus plantarum* VHProbi R14 provided by this invention had an anti-lipid peroxidation inhibition rate of 35.81% with a standard deviation of 0.18%; the bacterial cell anti-lipid peroxidation inhibition rate was 32.47% with a standard deviation of 0.21%; and the intracellular extract had an anti-lipid peroxidation inhibition rate of 27.43% with a standard deviation of 0.29%.

[0077] Example 5: Effect of Lactobacillus plantarum VHProbi R14 on increasing beneficial bacteria in the mouse gut.

[0078] 1. Laboratory animals

[0079] C57 mice, SPF grade, male, 7 weeks old, 20 mice, weighing 19–25g. Environmental conditions for animal husbandry: room temperature 20–26℃, daily temperature difference ≤4℃, relative humidity 40–70%, light / dark cycle 12 / 12h. Animals were housed in standard mouse cages, 10 mice per cage. Feed and water: ad libitum. Feed consisted of SPF grade rat and mouse growth and breeding feed. Drinking water was sterilized municipal tap water.

[0080] 2. Experimental Methods

[0081] After 7 days of acclimatization, mice were randomly divided into a blank group and a probiotic group, with 10 mice in each group. The probiotic group was administered *Lactobacillus plantarum* VHProbi R14 bacterial suspension (0.2 mL / 10 g) via gavage. 9 Mice in the control group were administered an equal volume of physiological saline by gavage for 60 days. Mice were placed in clean, empty cages, and after a period of time, 3-5g of feces were collected using sterile forceps, placed in a 1.5mL tube, flash-frozen in liquid nitrogen, and stored at -80°C.

[0082] 3. 16S amplicon sequencing protocol for fecal samples

[0083] 30 ng of qualified fecal DNA sample and corresponding fusion primers were used to prepare the PCR reaction system. PCR reaction parameters were set for PCR amplification. The PCR amplification product was purified using Agencourt AMPure XP magnetic beads and dissolved in Elution Buffer. Labeling was then performed to complete library construction. The fragment range and concentration of the library were detected using an Agilent 2100 Bioanalyzer. Libraries that passed the detection were sequenced using the HiSeq platform according to the insert fragment size.

[0084] After the data is filtered, the remaining high-quality clean data is used for later analysis; the reads are spliced ​​into tags by the overlap relationship between the reads; the tags are clustered into OTUs and compared with the database, and species are annotated; sample species analysis is performed based on OTUs and annotation results, including intergroup species difference analysis at the genus and species levels, and species composition histogram analysis.

[0085] 4. Sequencing results

[0086] like Figure 2 As shown, at the genus level, the dominant genera in the feces of the blank group mice included *Lactobacillus*, *Bacteroides*, *Alloprevotella*, *Barnesiella*, *Clostridium XlVb*, and *Prevotella*. The dominant genera in the feces of the probiotics group mice were *Lactobacillus*, *Alloprevotella*, *Barnesiella*, *Akkermansia*, *Clostridium XlVb*, and *Prevotella*. Figure 5 The area framed in the image represents Akkermansia. It can be seen that, at the genus level, the abundance of Akkermansia in the fecal microbiota of mice in the probiotic group that were administered Lactobacillus plantarum VHProbi R14 via gavage was significantly higher than that in the control group.

[0087] like Figure 3 As shown, compared with the blank group mice, at the genus level, the levels of Prevotella, Lachnospiraceae incertae sedis, Mucispirillum, and Clostridium were downregulated in the feces of mice in the probiotics group (which were administered Lactobacillus plantarum VHProbiR14 via gavage), while Akkermansia was significantly upregulated.

[0088] like Figure 4 As shown, at the species level, the abundance of Akkermansia muciniphila in the feces of mice in the probiotics group, which were administered Lactobacillus plantarum VHProbiR14 via gavage, was significantly upregulated compared with the blank group mice.

[0089] Combination Figure 2 , 3 As shown in section 4, gavage administration of the *Lactobacillus plantarum* VHProbi R14 bacterial solution provided by this invention to normal mice significantly increased the abundance of *Akkermansia* spp. in their feces, particularly *Akkermansia myxophilus*. *Akkermansia myxophilus* plays an important role in regulating intestinal and host health and has been shown to improve metabolic diseases such as obesity, diabetes, and cardiovascular disease. Numerous studies have demonstrated that *Akkermansia myxophilus* has positive effects in reducing fat accumulation, alleviating inflammation, improving insulin resistance, and improving glucose homeostasis, and is considered a promising "second-generation probiotic." Therefore, the *Lactobacillus plantarum* VHProbi R14 provided by this invention can significantly increase the abundance of *Akkermansia* spp. in the mouse intestine, which is of great significance for maintaining intestinal flora health and host health.

[0090] Example 6: A crystal ball containing *Lactobacillus plantarum* and its preparation method

[0091] The probiotic crystals prepared in this embodiment consist of three parts: a crystal nucleus, a crystal mantle, and a crystal shell. The innermost crystal nucleus is composed of *Lactobacillus plantarum* VHProbi R14 bacterial powder, lecithin, and stearic palm oil. The middle crystal mantle is composed of sodium alginate, gellan gum, carrageenan, and water. The outermost crystal shell is composed of water, food coloring, gelatin, pectin, and glycerin.

[0092] Nucleus preparation method: Lecithin and stearic palm oil are heated and dissolved into a liquid state. The bacterial powder, lecithin, and stearic palm oil are thoroughly mixed at a mass fraction ratio of 5%, 3%, and 92%, and then kept at a warm temperature for later use. Mantle preparation method: Gellan gum and carrageenan are preheated and melted into a liquid state. Sodium alginate, gellan gum, carrageenan, and water are thoroughly mixed at a mass fraction ratio of 3%, 1%, 0.5%, and 95.5%, and then kept at a warm temperature for later use. Shell preparation method: Gelatin and pectin are preheated and melted into a liquid state. Pigment, gelatin, pectin, glycerin, and water are thoroughly mixed at a mass fraction ratio of 2%, 15%, 1%, 5%, and 77%, and then kept at a warm temperature for later use.

[0093] The liquid formulations of probiotic crystal pellets were added to a capsule forming machine equipped with three concentric nozzles. The innermost nozzle sprayed the nucleus solution, the middle nozzle sprayed the mantle solution, and the outermost nozzle sprayed the shell solution. After starting the capsule forming machine, the nozzle speed was adjusted to simultaneously spray the crystal pellet components from all three nozzles, which then dripped into flowing rapeseed oil cooled to 12°C, thus producing three-layered crystal pellets with a diameter of 2.5 mm. The dripped crystal pellets were dried at 16°C for 8 hours, followed by vacuum freeze-drying for 24 hours to reduce the moisture content to below 3%, yielding the finished probiotic crystal pellet product.

[0094] Example 7: A crystal ball containing *Lactobacillus plantarum* and its preparation method

[0095] The probiotic crystals prepared in this embodiment consist of three parts: a crystal nucleus, a crystal mantle, and a crystal shell. The innermost crystal nucleus is composed of *Lactobacillus plantarum* VHProbi R14 bacterial powder, lecithin, and stearic palm oil. The middle crystal mantle is composed of sodium alginate, gellan gum, carrageenan, and water. The outermost crystal shell is composed of water, food coloring, gelatin, pectin, and glycerin.

[0096] Nucleus preparation method: Lecithin and stearic palm oil are heated and dissolved into a liquid state. The bacterial powder, lecithin, and stearic palm oil are thoroughly mixed at a mass fraction ratio of 8%, 2%, and 90%, and then kept at a warm temperature for later use. Mantle preparation method: Gellan gum and carrageenan are preheated and melted into a liquid state. Sodium alginate, gellan gum, carrageenan, and water are thoroughly mixed at a mass fraction ratio of 5%, 2%, 1%, and 92%, and then kept at a warm temperature for later use. Shell preparation method: Gelatin and pectin are preheated and melted into a liquid state. Edible coloring, gelatin, pectin, glycerin, and water are thoroughly mixed at a mass fraction ratio of 2%, 20%, 2%, 10%, and 66%, and then kept at a warm temperature for later use.

[0097] The liquid formulations of probiotic crystal pellets were added to a capsule forming machine equipped with three concentric nozzles. The innermost nozzle sprayed the nucleus solution, the middle nozzle sprayed the mantle solution, and the outermost nozzle sprayed the shell solution. After starting the capsule forming machine, the nozzle speed was adjusted to simultaneously spray the crystal pellet components from all three nozzles, which then dripped into flowing rapeseed oil cooled to 12°C, thus producing three-layered crystal pellets with a diameter of 1.5 mm. The formed crystal pellets were dried at 16°C for 20 hours, followed by vacuum freeze-drying for 36 hours to reduce the moisture content to below 2%, yielding the finished probiotic crystal pellet product.

[0098] Example 8: A crystal ball containing *Lactobacillus plantarum* and its preparation method

[0099] The probiotic crystals prepared in this embodiment consist of three parts: a crystal nucleus, a crystal mantle, and a crystal shell. The innermost crystal nucleus is composed of *Lactobacillus plantarum* VHProbi R14 bacterial powder, lecithin, and stearic palm oil. The middle crystal mantle is composed of sodium alginate, gellan gum, carrageenan, and water. The outermost crystal shell is composed of food coloring, water, gelatin, pectin, and glycerin.

[0100] Nucleus preparation method: Lecithin and stearic palm oil are heated and dissolved into a liquid state. The bacterial powder, lecithin, and stearic palm oil are thoroughly mixed at a mass fraction ratio of 10%, 1.5%, and 88.5%, and then kept at a warm temperature for later use. Mantle preparation method: Gellan gum and carrageenan are preheated and melted into a liquid state. Sodium alginate, gellan gum, carrageenan, and water are thoroughly mixed at a mass fraction ratio of 10%, 2%, 0.5%, and 87.5%, and then kept at a warm temperature for later use. Shell preparation method: Gelatin and pectin are preheated and melted into a liquid state. Edible coloring, gelatin, pectin, glycerin, and water are thoroughly mixed at a mass fraction ratio of 2%, 10%, 1%, 15%, and 62%, and then kept at a warm temperature for later use.

[0101] The liquid formulations of probiotic crystal pellets were added to a capsule forming machine equipped with three concentric nozzles. The innermost nozzle sprayed the nucleus solution, the middle nozzle sprayed the mantle solution, and the outermost nozzle sprayed the shell solution. After starting the capsule forming machine, the nozzle speed was adjusted to simultaneously spray the crystal pellet components from all three nozzles, which then dripped into flowing rapeseed oil cooled to 12°C, thus producing three-layered crystal pellets with a diameter of 3mm. The dripped and formed crystal pellets were dried at 16°C for 10 hours, followed by vacuum freeze-drying for 48 hours to reduce the moisture content to below 1.5%, yielding the finished probiotic crystal pellet product.

[0102] Example 9: Heat resistance test of probiotic pellets

[0103] The three types of probiotic pellets prepared in Examples 6-8 were subjected to temperature resistance tests.

[0104] After weighing a certain amount of probiotic pellets, place them in an environment with a temperature of 35℃ and a humidity of 75% for 24 hours and in an environment with a temperature of 45℃ and a humidity of 75% for 24 hours respectively. Observe whether the probiotic pellets remain intact and whether there is any leakage.

[0105] The results are as follows Figure 5 As shown, after 24 hours at an ambient temperature of 35°C and an ambient humidity of 75%, the morphology of the three types of probiotic spheres prepared in Examples 6-8 remained intact, the color of the spheres did not change, and there was no leakage.

[0106] After 24 hours at an ambient temperature of 45°C and an ambient humidity of 75%, the probiotic spheres prepared in Example 6 remained intact, and their color did not change. The probiotic spheres prepared in Examples 7 and 8 showed signs of cracking, color change, and leakage.

[0107] The above results indicate that the probiotic pellets provided by the present invention have strong heat resistance, and the pellets prepared in Example 6 have the strongest heat resistance.

[0108] Example 10: Stability Test of Probiotic Spheres

[0109] Accelerated stability testing was conducted on the three types of probiotic pellets prepared in Examples 6-8. A certain amount of pellets was weighed and sealed in an aluminum foil bag, and placed in an incubator at 37°C and 75% humidity for 90 days (equivalent to 2 years of storage at room temperature). The viable count was measured every 30 days to analyze the stability of the probiotic pellet viable count. The results are shown in Table 2.

[0110] Table 2 Comparison of live bacteria count in probiotic pellets (Log CFU / g)

[0111] Grouping 0 days 30 days 60 days 90 days Example 6 10.17±0.22 9.98±0.17 9.96±0.23 9.87±0.19 Example 7 9.96±0.14 9.83±0.23 9.51±0.12 9.01±0.32 Example 8 10.99±0.11 9.17±0.19 9.79±0.15 8.69±0.25

[0112] After 90 days of accelerated stability testing, the number of live bacteria in the probiotic pellets provided by this invention decreased only slightly. Among them, the number of live bacteria in the probiotic pellets prepared in Example 6 decreased by only 0.3 Log CFU / g, showing the strongest stability and achieving unexpected technical results.

[0113] The *Lactobacillus plantarum* probiotic crystals provided by this invention have a two-layer protective structure. The crystal shell structure isolates oxygen and moisture, while the crystal mantle structure provides acid resistance, effectively protecting the activity of probiotics in the crystal nucleus and giving it excellent stability. This effectively maintains the viable count of *Lactobacillus plantarum* VHProbi R14 during transportation, storage, and processing, significantly improving the applicability of *Lactobacillus plantarum* VHProbi R14. Therefore, it can be widely used in food, health products, and other fields.

Claims

1. A probiotic crystal ball, characterized in that, The probiotic crystal spheres consist of three parts: a crystal nucleus, a crystal mantle, and a crystal shell. The crystal nucleus is located in the innermost layer of the crystal sphere and is composed of *Lactobacillus plantarum* powder, lecithin, and stearic palm oil. The crystal mantle is located in the middle layer of the crystal sphere and is composed of sodium alginate, gellan gum, carrageenan, and water. The crystal shell is located in the outermost layer of the crystal sphere and is composed of food coloring, gelatin, pectin, glycerin, and water. The preservation number of *Lactobacillus plantarum* is CCTCC NO: M2023721.

2. The probiotic pellets as described in claim 1, characterized in that, The mass percentages of Bacillus plantarum powder, lecithin, and stearic palm oil in the crystal nucleus are 5-10%, 1.5-3%, and 88.5-92%, respectively.

3. The probiotic pellets as described in claim 2, characterized in that, The mass percentages of sodium alginate, gellan gum, carrageenan, and water in the crystal mantle are 3-10%, 1-2%, 0.5-1%, and 87.5-95.5%, respectively.

4. The probiotic pellets as described in claim 3, characterized in that, The mass percentages of food coloring, gelatin, pectin, glycerin, and water in the crystal shell are 2%, 10-20%, 1-2%, 5-15%, and 62-77%, respectively.

5. The probiotic pellets as described in claim 1, characterized in that, The preparation method of the probiotic crystals includes the following steps: (1) Preparation of solutions containing crystal nuclei, crystal mantles, and crystal shells Soft lecithin and stearic palm oil are heated and dissolved into a liquid state. The plant lactobacillus powder, soft lecithin and stearic palm oil are mixed thoroughly in proportion to obtain a crystal nucleus solution, which is then kept warm for later use. Gellan gum and carrageenan are preheated and melted into a liquid state. Sodium alginate, gellan gum, carrageenan and water are mixed thoroughly in proportion to obtain a crystal mantle solution, which is then kept warm for later use. Melt the gelatin and pectin in advance until they are in a liquid state. Mix the food coloring, gelatin, pectin, glycerin and water in proportion to obtain a crystal shell solution and keep it warm for later use. (2) Preparation of crystal spheres The solutions for crystal nuclei, crystal mantle, and crystal shell are added to a capsule machine with three concentric nozzles. The innermost nozzle sprays the crystal nuclei solution, the middle nozzle sprays the crystal mantle solution, and the outermost nozzle sprays the crystal shell solution. Start the capsule machine, adjust the appropriate nozzle speed so that the three nozzles spray out the crystal ball components at the same time and drip them into the rapeseed oil that has been cooled to 12°C and is flowing. After the crystal balls are dried at 16°C for 8 hours, they are then vacuum freeze-dried for 24 hours to reduce the moisture content to below 3%, thus obtaining probiotic crystal balls.

6. The application of the probiotic crystals according to claim 1 in the preparation of food.

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