A method for improving the stability of probiotics by nitrogen sealing in a cellophane bottle

Abstract

The application relates to a method for improving the stability of probiotics by nitrogen sealing in a carboy, and relates to the technical field of probiotic sealing. Modified probiotic semi-permeable microspheres are obtained by using mercapto hyaluronic acid, polyethylene glycol glycidyl ether, baking soda, a solvent and probiotics; the carboy is cleaned by high-pressure sterile water and dried by sterile compressed air; the modified probiotic semi-permeable microspheres are filled into the carboy in a sterile nitrogen protection cover; nitrogen is injected through a double-path injection mode; butyl rubber plugs and aluminum covers are used to seal the carboy; the sealed carboy is embedded into a honeycomb plate slot hole, the honeycomb plate embedded with the carboy is loaded into a hard nest box, double-layer cover paper is covered, and heat sealing is fixed; the application can significantly improve the survival rate and production efficiency of probiotics, meets the demand of industrial large-scale production, prolongs the shelf life of the product due to long-acting fresh-keeping effect, and improves the storage safety and market competitiveness of the product.

Description

Technical Field

[0001] This invention relates to the field of probiotic encapsulation technology, and in particular to a method for nitrogen encapsulation of vials to improve the stability of probiotics. Background Technology

[0002] Probiotics are a class of live microorganisms that are beneficial to the host and are widely used in food, health products, and pharmaceuticals. Their physiological activity is the core of their probiotic effects. However, probiotics are extremely sensitive to environmental factors such as oxygen, moisture, and temperature. During production, packaging, storage, and transportation, their activity can be reduced or even inactivated due to oxidation or moisture, which seriously affects the quality and efficacy of the product.

[0003] Chinese Patent CN121249641A discloses a method for encapsulating single-cell probiotics with thiolated guluronic acid oligosaccharides and probiotic microcapsules, relating to the field of biomedical technology. The encapsulation method involves mixing a thiolated guluronic acid oligosaccharide solution and a probiotic suspension to form a probiotic-polysaccharide mixed solution; adding a calcium chloride solution to the probiotic-polysaccharide mixed solution and stirring to form single-cell microcapsules encapsulating the probiotics.

[0004] Chinese Patent CN121573283A discloses a method for controlling the stability of probiotic encapsulation based on deep learning, comprising the following steps: collecting multi-source data during the encapsulation process and preprocessing them separately; performing time-series feature analysis on the standardized structured dataset to extract multi-scale time-series features; performing target segmentation and multi-dimensional structural feature extraction on the standardized microscopic image dataset; performing feature fusion to construct a probiotic encapsulation stability feature space and generating a stable feature vector; inputting the data into a time-series prediction network for time-series modeling and prediction; adjusting control parameters and optimizing constraints based on the difference between the prediction results and stability reference data; executing and updating the encapsulation control adjustment scheme based on feedback to complete the encapsulation control closed loop.

[0005] Existing nitrogen encapsulation methods have the following drawbacks: they do not provide effective pre-protection for probiotics, which are directly exposed to the packaging environment. Even if nitrogen is replaced, the activity will still decrease rapidly due to trace amounts of oxygen and moisture residue, resulting in a low survival rate at room temperature for 12 months. Summary of the Invention

[0006] To address the above problems, this invention provides a method for nitrogen sealing of vials to improve the stability of probiotics, the operation steps of which are as follows: S1: Add 30-60 parts of thiolated hyaluronic acid, 5-10 parts of polyethylene glycol glycidyl ether, and baking soda to ensure the final pH is between 7.5 and 8.5, along with 200-350 parts of solvent, to the reactor. Purge with nitrogen to remove oxygen for 10-20 minutes, and stir at room temperature for 15-30 minutes to form a homogeneous solution. Add 10-20 parts of probiotics and continue stirring for 5-10 minutes. Send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres. S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 40-60% of its volume; inject nitrogen gas with a purity ≥99.99% through a dual-channel injection method, with a nitrogen flow rate of 5-10 L / min and a replacement time of 10-20 s, and control the pressure inside the vial to 0.10-0.12 MPa; S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0007] Optionally, the probiotic in S1 is one of Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus, Bacillus, or yeast.

[0008] Optionally, the solvent in S1 is sterile deionized water or physiological saline.

[0009] Optionally, the freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -40 to -35°C, keep warm for 3-5 hours; then at a vacuum of 10-30 Pa and a temperature of -10 to 0°C, keep warm for 12-18 hours; finally, at a vacuum of 5-15 Pa and a temperature of 15-20°C, keep warm for 4-6 hours.

[0010] Optionally, the high-pressure sterile water pressure in S2 is 0.2-0.5 MPa, and the cleaning time is 5-10 seconds.

[0011] Optionally, the sterile compressed air drying temperature in S2 is 80-100℃, and the time is 3-10s.

[0012] Optionally, in S4, the inner diameter of the honeycomb plate slot is 0.5-1 mm larger than the outer diameter of the vial.

[0013] Optionally, in step S4, the heat sealing temperature is 95-115℃, the heat sealing pressure is 0.2-0.5MPa, and the time is 0.5-5s.

[0014] Reaction mechanism: The thiol groups on the thiolized hyaluronic acid molecular chain are strong nucleophilic groups. Under the mild catalysis of sodium bicarbonate, they undergo a highly efficient in-situ ring-opening addition reaction with the epoxy groups of polyethylene glycol glycidyl ether, forming stable thioether covalent bonds to construct a three-dimensional network cross-linked microsphere coating structure. This cross-linked structure is formed by freeze-drying and an anhydrous microenvironment, which not only ensures the compactness and structural stability of the coating layer, but also endows the coating layer with good flexibility and biocompatibility through covalent grafting. At the same time, the free radical scavenging properties of thiol groups can reduce oxidative damage to probiotics, achieving a dual effect of physical coating protection and chemical stress resistance for probiotics.

[0015] Technical effects: This invention provides a method for nitrogen sealing of vials to improve the stability of probiotics. Compared with existing technologies, this invention has the following significant advantages: 1. A dense oxygen- and moisture-controlling barrier is formed through a covalently cross-linked modified coating layer. Combined with the antioxidant properties of thiol groups, it effectively reduces the contact between probiotics and oxygen and moisture, reduces the impact of oxidation, miscellaneous bacteria and other factors on the activity of probiotics, and significantly improves the survival rate of probiotics stored at room temperature.

[0016] 2. The modified microsphere coating has both good film-forming properties and mechanical strength.

[0017] 3. The cross-linked microsphere coating has a stable structure and can withstand minor collisions and environmental fluctuations during storage and transportation. Combined with subsequent nitrogen encapsulation and sterilization processes, it further enhances the safety of product storage and transportation. At the same time, the modified coating has excellent biocompatibility and does not affect the probiotic efficacy, thus enhancing the overall application value of the product. Detailed Implementation

[0018] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description is provided in conjunction with embodiments and comparative examples: 1. Probiotic survival rate detection: The plate count method was used. An appropriate amount of microsphere sample was taken and serially diluted with sterile physiological saline. The bacterial solution with the appropriate dilution was spread on MRS medium and anaerobically cultured at 37℃ for 48h. The number of colonies was counted and the probiotic survival rate was calculated. The initial number of surviving bacteria and the survival rate after 12 months of storage at room temperature (25±2℃) were detected respectively.

[0019] 2. Production efficiency test: Record the number of qualified vials produced in one hour of continuous production and calculate the production efficiency (via vials / hour).

[0020] Example 1

[0021] A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 30g of thiolated hyaluronic acid, 5g of polyethylene glycol glycidyl ether, and baking soda to ensure the final pH is 7.5. Add 200g of solvent to the reaction vessel, purge with nitrogen to remove oxygen for 10 minutes, stir at room temperature for 15 minutes to form a homogeneous liquid, add 10g of probiotics, and continue stirring for 5 minutes. Send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres. S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 40% of its volume; inject nitrogen gas with a purity of ≥99.99% through a dual-channel injection method, with a nitrogen flow rate of 5L / min and a replacement time of 10s, and control the pressure inside the vial to 0.10MPa. S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0022] The probiotic in S1 is Bifidobacterium.

[0023] The solvent in S1 is sterile deionized water.

[0024] The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -40℃, keep warm for 3 hours; then at a vacuum of 10Pa and a temperature of -10℃, keep warm for 12 hours; finally, at a vacuum of 5Pa and a temperature of 15℃, keep warm for 4 hours.

[0025] The pressure of the high-pressure sterile water in S2 is 0.2 MPa, and the cleaning time is 5 seconds.

[0026] The sterile compressed air drying temperature in S2 is 80℃, and the time is 3s.

[0027] In the S4, the inner diameter of the honeycomb plate slot is 0.5 mm larger than the outer diameter of the vial.

[0028] In S4, the heat sealing temperature is 95℃, the heat sealing pressure is 0.2MPa, and the time is 0.5s.

[0029] Example 2

[0030] A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 40g of thiolated hyaluronic acid, 6g of polyethylene glycol glycidyl ether, baking soda to ensure the final pH = 8, and 250g of solvent to the reaction vessel. Purge with nitrogen to remove oxygen for 15 minutes, stir at room temperature for 20 minutes to form a homogeneous liquid, add 14g of probiotics, and continue stirring for 6 minutes. Send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres. S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 45% of its volume; inject nitrogen gas with a purity ≥99.99% through a dual-channel injection method at a flow rate of 6L / min and a replacement time of 15s, and control the pressure inside the vial to 0.11MPa. S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0031] The probiotic in S1 is Lactobacillus.

[0032] The solvent in S1 is physiological saline.

[0033] The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -38°C, keep warm for 4 hours; then at a vacuum of 15Pa and a temperature of -8°C, keep warm for 14 hours; finally, at a vacuum of 8Pa and a temperature of 16°C, keep warm for 5 hours.

[0034] The high-pressure sterile water pressure in S2 is 0.3 MPa, and the cleaning time is 6 seconds.

[0035] The sterile compressed air drying temperature in S2 is 85℃, and the time is 4s.

[0036] In the S4, the inner diameter of the honeycomb plate slot is 0.6 mm larger than the outer diameter of the vial.

[0037] In S4, the heat sealing temperature is 100℃, the heat sealing pressure is 0.3MPa, and the time is 0.6s.

[0038] Example 3

[0039] A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 50g of thiolated hyaluronic acid, 8g of polyethylene glycol glycidyl ether, and baking soda to ensure the final pH is 8.5. Add 300g of solvent to the reaction vessel, purge with nitrogen to remove oxygen for 15 minutes, stir at room temperature for 25 minutes to form a homogeneous liquid, add 18g of probiotics, and continue stirring for 8 minutes. Send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres. S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 55% of its volume; inject nitrogen gas with a purity ≥99.99% through a dual-channel injection method, with a nitrogen flow rate of 8L / min and a replacement time of 15s, and control the pressure inside the vial to 0.11MPa. S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0040] The probiotic in S1 is streptococcus.

[0041] The solvent in S1 is sterile physiological saline.

[0042] The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -36°C, keep warm for 4 hours; then at a vacuum of 25Pa and a temperature of -2°C, keep warm for 16 hours; finally, at a vacuum of 13Pa and a temperature of 18°C, keep warm for 5 hours.

[0043] The pressure of the high-pressure sterile water in S2 is 0.3 MPa, and the cleaning time is 8 seconds.

[0044] The sterile compressed air drying temperature in S2 is 95℃, and the time is 4s.

[0045] In the S4, the inner diameter of the honeycomb plate slot is 0.8 mm larger than the outer diameter of the vial.

[0046] In S4, the heat sealing temperature is 110℃, the heat sealing pressure is 0.3MPa, and the time is 0.8s.

[0047] Example 4

[0048] A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 60g of thiolated hyaluronic acid, 10g of polyethylene glycol glycidyl ether, and baking soda to ensure the final pH is 8.5. Add 350g of solvent to the reaction vessel, purge with nitrogen to remove oxygen for 20 minutes, stir at room temperature for 30 minutes to form a homogeneous solution, add 20g of probiotics, and continue stirring for 10 minutes. Send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres. S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 60% of its volume; inject nitrogen gas with a purity ≥99.99% through a dual-channel injection method at a flow rate of 10L / min and a replacement time of 20s, and control the pressure inside the vial to 0.12MPa. S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0049] The probiotic in S1 is Enterococcus.

[0050] The solvent in S1 is sterile physiological saline.

[0051] The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -35°C, keep warm for 5 hours; then at a vacuum of 30Pa and a temperature of 0°C, keep warm for 18 hours; finally, at a vacuum of 15Pa and a temperature of 20°C, keep warm for 6 hours.

[0052] The high-pressure sterile water pressure in S2 is 0.5 MPa, and the cleaning time is 10 seconds.

[0053] The sterile compressed air drying temperature in S2 is 100℃, and the time is 10s.

[0054] In the S4, the inner diameter of the honeycomb plate slot is 1 mm larger than the outer diameter of the vial.

[0055] In S4, the heat sealing temperature is 115℃, the heat sealing pressure is 0.5MPa, and the time is 5s.

[0056] Comparative Example 1 A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 10g of probiotics and 200g of solvent to the reaction vessel, purge with nitrogen to remove oxygen for 10 minutes, and stir for 5 minutes; send the mixture to a freeze dryer for freeze drying to obtain probiotic semi-permeable microspheres; S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with semi-permeable probiotic microspheres to 40% of its volume; inject nitrogen gas with a purity of ≥99.99% through a dual-channel injection method at a flow rate of 5L / min and a replacement time of 10s, and control the pressure inside the vial to 0.10MPa. S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0057] The probiotic in S1 is Bifidobacterium.

[0058] The solvent in S1 is sterile deionized water.

[0059] The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -40℃, keep warm for 3 hours; then at a vacuum of 10Pa and a temperature of -10℃, keep warm for 12 hours; finally, at a vacuum of 5Pa and a temperature of 15℃, keep warm for 4 hours.

[0060] The pressure of the high-pressure sterile water in S2 is 0.2 MPa, and the cleaning time is 5 seconds.

[0061] The sterile compressed air drying temperature in S2 is 80℃, and the time is 3s.

[0062] In the S4, the inner diameter of the honeycomb plate slot is 0.5 mm larger than the outer diameter of the vial.

[0063] In S4, the heat sealing temperature is 95℃, the heat sealing pressure is 0.2MPa, and the time is 0.5s.

[0064] Comparative Example 2 A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 30g of thiolated hyaluronic acid and baking soda to ensure the final pH is 7.5. Add 200g of solvent to the reaction vessel, purge with nitrogen to remove oxygen for 10 minutes, stir at room temperature for 15 minutes to form a homogeneous liquid, add 10g of probiotics, and continue stirring for 5 minutes; send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres; S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 40% of its volume; inject nitrogen gas with a purity of ≥99.99% through a dual-channel injection method, with a nitrogen flow rate of 5L / min and a replacement time of 10s, and control the pressure inside the vial to 0.10MPa. S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0065] The probiotic in S1 is Bifidobacterium.

[0066] The solvent in S1 is sterile deionized water.

[0067] The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -40℃, keep warm for 3 hours; then at a vacuum of 10Pa and a temperature of -10℃, keep warm for 12 hours; finally, at a vacuum of 5Pa and a temperature of 15℃, keep warm for 4 hours.

[0068] The pressure of the high-pressure sterile water in S2 is 0.2 MPa, and the cleaning time is 5 seconds.

[0069] The sterile compressed air drying temperature in S2 is 80℃, and the time is 3s.

[0070] In the S4, the inner diameter of the honeycomb plate slot is 0.5 mm larger than the outer diameter of the vial.

[0071] In S4, the heat sealing temperature is 95℃, the heat sealing pressure is 0.2MPa, and the time is 0.5s.

[0072] Comparative Example 3 A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 5g of polyethylene glycol glycidyl ether and 200g of solvent to the reactor, add baking soda to ensure the final pH is 7.5, purge with nitrogen to remove oxygen for 10 minutes, stir at room temperature for 15 minutes to form a homogeneous liquid, add 10g of probiotics, and continue stirring for 5 minutes; send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres; S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 40% of its volume; inject nitrogen gas with a purity of ≥99.99% through a dual-channel injection method, with a nitrogen flow rate of 5L / min and a replacement time of 10s, and control the pressure inside the vial to 0.10MPa. S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

[0073] The probiotic in S1 is Bifidobacterium.

[0074] The solvent in S1 is sterile deionized water.

[0075] The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -40℃, keep warm for 3 hours; then at a vacuum of 10Pa and a temperature of -10℃, keep warm for 12 hours; finally, at a vacuum of 5Pa and a temperature of 15℃, keep warm for 4 hours.

[0076] The pressure of the high-pressure sterile water in S2 is 0.2 MPa, and the cleaning time is 5 seconds.

[0077] The sterile compressed air drying temperature in S2 is 80℃, and the time is 3s.

[0078] In the S4, the inner diameter of the honeycomb plate slot is 0.5 mm larger than the outer diameter of the vial.

[0079] In S4, the heat sealing temperature is 95℃, the heat sealing pressure is 0.2MPa, and the time is 0.5s.

[0080] Comparative Example 4: Nitrogen gas was not introduced for deoxygenation in step S1 for 10 minutes, and the rest was the same as in Example 1.

[0081] Table 1. Results of initial probiotic survival count, 12-month survival rate at room temperature, and production efficiency in the specific implementation plan. This technical solution uses thiolated hyaluronic acid as the core polymer substrate, combined with polyethylene glycol glycidyl ether as a crosslinking agent and sodium bicarbonate as a food-grade catalyst. Through in-situ covalent crosslinking reactions of epoxy groups and thiol groups, the structure of the probiotic microsphere coating layer is modified and its function enhanced. The thiol active groups of thiolated hyaluronic acid provide reaction sites for covalent crosslinking, constructing a dense and stable coating barrier. Its antioxidant properties also actively protect probiotics from oxidative damage. The crosslinking effect of polyethylene glycol glycidyl ether creates a three-dimensional network structure in the coating layer, improving the mechanical strength and oxygen and moisture control capabilities of the microspheres. Sodium bicarbonate, while gently catalyzing the crosslinking reaction, is non-toxic and does not inhibit the activity of probiotics, ensuring their initial activity. The synergistic effect of this adjuvant system makes the modified microspheres significantly more effective in protecting probiotics than the original process. It improves the survival rate of probiotics during storage while balancing production efficiency and product storage and transportation stability, achieving a dual optimization of probiotic protection and industrial production requirements.

[0082] In this invention, the nitrogen replacement step significantly improves the long-term stability of probiotics. Compared with Example 1, the survival rate of Comparative Example 4 (without nitrogen deoxygenation) after 12 months of storage at room temperature decreased from 85.6% to 74.0%, an increase of 11.6%, indicating that nitrogen deoxygenation and nitrogen protection inside the bottle can significantly reduce oxidative stress and further enhance the long-term stability of probiotics, with significant effects.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for nitrogen sealing of vials to improve the stability of probiotics, comprising the following steps: S1: Add 30-60 parts of thiolated hyaluronic acid, 5-10 parts of polyethylene glycol glycidyl ether, and baking soda to ensure the final pH is between 7.5 and 8.5, along with 200-350 parts of solvent, to the reactor. Purge with nitrogen to remove oxygen for 10-20 minutes, and stir at room temperature for 15-30 minutes to form a homogeneous solution. Add 10-20 parts of probiotics and continue stirring for 5-10 minutes. Send the mixture to a freeze dryer for freeze drying to obtain modified probiotic semi-permeable microspheres. S2: Wash the vial with sterile water under high pressure with the bottle opening facing down, and then dry it with sterile compressed air with the bottle opening facing up. S3: In a sterile nitrogen protective hood, fill the vial with modified probiotic semi-permeable microspheres to 40-60% of its volume; inject nitrogen gas with a purity ≥99.99% through a dual-channel injection method, with a nitrogen flow rate of 5-10 L / min and a replacement time of 10-20 s, and control the pressure inside the vial to 0.10-0.12 MPa; S4: Within 3 seconds after nitrogen purging, use butyl rubber stoppers and aluminum caps to press and seal the vials; embed the sealed vials into the honeycomb plate slots, then put the honeycomb plate embedded in the vials into a rigid honeycomb box, cover with double layers of cover paper, and heat seal to fix.

2. The method for nitrogen sealing of vials to improve the stability of probiotics according to claim 1, characterized in that: The probiotics in S1 are one of Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus, Bacillus, and yeast.

3. The method for nitrogen sealing of vials to improve the stability of probiotics according to claim 1, characterized in that: The solvent in S1 is sterile deionized water or physiological saline.

4. The method for nitrogen sealing of vials to improve the stability of probiotics according to claim 1, characterized in that: The freeze-drying operation steps in S1 are as follows: under nitrogen atmosphere, at a current temperature of -40 to -35°C, keep warm for 3-5 hours; then at a vacuum of 10-30 Pa and a temperature of -10 to 0°C, keep warm for 12-18 hours; finally, at a vacuum of 5-15 Pa and a temperature of 15-20°C, keep warm for 4-6 hours.

5. The method for nitrogen sealing of vials to improve the stability of probiotics according to claim 1, characterized in that: The pressure of the high-pressure sterile water in S2 is 0.2-0.5 MPa, and the cleaning time is 5-10 seconds.

6. The method for nitrogen sealing of vials to improve the stability of probiotics according to claim 1, characterized in that: The sterile compressed air drying temperature in S2 is 80-100℃, and the time is 3-10s.

7. The method for nitrogen sealing of vials to improve the stability of probiotics according to claim 1, characterized in that: In the S4, the inner diameter of the honeycomb plate slot is 0.5-1 mm larger than the outer diameter of the vial.

8. The method for nitrogen sealing of vials to improve the stability of probiotics according to claim 1, characterized in that: In S4, the heat sealing temperature is 95-115℃, the heat sealing pressure is 0.2-0.5MPa, and the time is 0.5-5s.

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