A method for preparing a probiotic drop

Abstract

The present application relates to the technical field of microbial preparation processing, and discloses a preparation method of probiotic drops. The method comprises the following steps: activating and culturing probiotic freeze-dried powder to logarithmic growth phase, resuspending the probiotic freeze-dried powder in a protective agent solution after centrifugal washing to obtain a bacterial suspension; mixing the bacterial suspension with an aqueous solution of sodium alginate and gelatin, generating liquid drops through a microfluidic droplet generator, and dropping the liquid drops into a calcium chloride and chitosan coagulation bath to gelate into microspheres; low-temperature curing the microspheres, washing the microspheres, soaking the microspheres in a freeze-drying protective solution, and then vacuum freeze-drying the microspheres to obtain microcapsule dry powder; and mixing the microcapsule dry powder with medicinal-grade vegetable oil and natural antioxidants, homogenizing, and filling. The present application prepares composite gel microcapsules with uniform particle size through microfluidic technology, effectively maintains the activity of probiotics during processing and storage by combining freeze-drying protection and oil-phase antioxidant, realizes intestinal targeted release, and has good product stability, high safety, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation processing technology, specifically a method for preparing probiotic drops. Background Technology

[0002] Probiotics are a class of live microorganisms that are beneficial to the health of the host and are widely used in the food, health product, and pharmaceutical industries. With increasing consumer awareness of gut health, the market demand for probiotic products continues to grow. Currently, commercially available probiotic products mainly include powders, tablets, capsules, and drops. Among these, drops, due to their convenience, flexible dosage, and ease of swallowing, are particularly suitable for infants and the elderly, and have become a research hotspot in recent years.

[0003] The core technical challenge of probiotic drops lies in maintaining the long-term survival rate of bacterial strains in a liquid environment. Free-living probiotics are highly sensitive to environmental factors such as temperature, oxygen, moisture, and gastric acid and bile salts, and their activity rapidly declines during their shelf life. In existing technologies, some products use oil-based carriers to isolate oxygen and moisture, but the oil phase alone cannot effectively resist the damage from gastrointestinal digestive juices, resulting in insufficient numbers of live bacteria reaching the intestinal colonization site. Another approach uses microencapsulation technology to encapsulate probiotics within polymer wall materials to improve resilience. However, traditional microencapsulation preparation methods, such as extrusion, spray drying, or emulsification cross-linking, suffer from drawbacks such as wide particle size distribution, unstable encapsulation rates, and damage to bacterial activity caused by harsh process conditions (such as high temperature or strong shear force). Microcapsules prepared by extrusion are typically in the millimeter range in diameter, resulting in a rough texture and difficulty in uniform dispersion in drops; spray drying involves high-temperature airflow, significantly affecting the survival rate of heat-sensitive probiotics; and the organic solvents and surfactants used in emulsification may leave residues, posing safety risks.

[0004] Existing literature reports the use of a sodium alginate-calcium chloride system to prepare probiotic microcapsules, which offers mild conditions and good biocompatibility. However, the single calcium alginate gel network is loose, easily swelling and rupturing in a simulated gastric fluid environment, releasing the encapsulated material prematurely, and lacking sufficient mechanical strength, making it prone to sedimentation and clumping during drop storage. To improve gel density, researchers have attempted to add polycationic substances such as gelatin and chitosan to form composite gels, but these improvements often involve more complex preparation processes, such as requiring multiple cross-linking steps or long curing times, resulting in low production efficiency and difficulty in simultaneously achieving uniform microcapsule size and high bacterial loading.

[0005] On the other hand, the choice of the final carrier oil phase and the antioxidant system of probiotic drops directly affect product stability. Vegetable oils are rich in unsaturated fatty acids, which are prone to oxidation and rancidity during long-term storage, producing off-odors and inducing free radicals to attack bacterial cell membranes. Existing drop products often add synthetic antioxidants, which does not align with the development trend of natural health products. Furthermore, the redispersibility of lyophilized microcapsule powder in the oil phase is poor, leading to agglomeration and affecting the appearance of the drops and the uniformity of dosage per administration. Therefore, there is an urgent need to develop a preparation method that can simultaneously address the protection of probiotic encapsulation, controllable microcapsule size, antioxidant properties of the oil phase, and overall product stability to meet the industrial production needs of high-quality probiotic drops. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing probiotic drops to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides a method for preparing probiotic drops, the method comprising: Step A: After activating the probiotic freeze-dried powder, inoculate it into a liquid culture medium, culture it under anaerobic conditions until the logarithmic growth phase, collect the bacterial cells by centrifugation, wash them with sterile physiological saline, and resuspend them in a protective agent solution to obtain a bacterial suspension. Step B: The bacterial suspension is mixed with an aqueous solution containing sodium alginate and gelatin at a volume ratio of 1:2. The mixture is then used to generate uniformly sized droplets using a microfluidic droplet generator. The droplets fall into a coagulation bath containing calcium chloride and chitosan, where they gel to form microspheres. Step C: The microspheres are allowed to stand and mature at 4°C, then filtered and washed with deionized water, then soaked in a freeze-drying protective solution, and finally freeze-dried under vacuum to obtain probiotic microcapsule powder. Step D: The microcapsule powder is mixed with pharmaceutical-grade plant oil and natural antioxidants, homogenized, and then filled to obtain probiotic drops.

[0008] Furthermore, the protective agent solution in step A comprises skim milk powder, glycerin and monosodium glutamate in a mass ratio of 10:5:1; Furthermore, the OD600 value of the bacterial suspension is adjusted to 2.0-3.0.

[0009] Furthermore, the anaerobic culture temperature in step A is 37±1℃, and the culture time is 12-18 hours; The centrifugation speed is 4000-6000 rpm, and the centrifugation time is 10-15 minutes; Wash 2-3 times, and resuspend in sterile saline after each wash.

[0010] Furthermore, in step B, the mass fraction of sodium alginate in the aqueous solution is 1.5%-2.5%, and the mass fraction of gelatin is 0.5%-1.0%. Furthermore, the aqueous solution is degassed under vacuum for 10-20 minutes and cooled to 10-15°C before mixing.

[0011] Furthermore, in step B, the dispersed phase flow rate of the microfluidic droplet generator is 0.5-2.0 mL / h, and the continuous phase flow rate is 5-15 mL / h; The droplet diameter is controlled between 50-200 μm; The continuous phase is liquid paraffin or vegetable oil.

[0012] Furthermore, in step B, the concentration of calcium chloride in the coagulation bath is 0.1-0.3 mol / L, and the mass fraction of chitosan is 0.2%-0.8%. The temperature of the coagulation bath is 25-35℃, and the gelation time is 10-30 minutes; During the gelation process, keep stirring slowly at a speed of 100-200 rpm.

[0013] Furthermore, the temperature of the ripening step in step C is 4-8°C, and the standing time is 2-6 hours; During the maturation process, stir for 1 minute at a speed of 50-100 rpm every 30 minutes.

[0014] Furthermore, the freeze-drying protective solution in step C contains trehalose, sucrose, and vitamin C in a mass ratio of 3:2:1; Furthermore, the osmotic pressure of the freeze-drying protective solution was adjusted to 600-800 mOsm / kg, and the soaking time was 30-60 minutes.

[0015] Furthermore, the vacuum freeze-drying described in step C includes a pre-freezing stage, a primary drying stage, and a secondary drying stage; The pre-freezing temperature is -40℃ to -50℃, and the pre-freezing time is 2-4 hours; The shelf temperature for one-time drying is -10℃ to 0℃, the vacuum degree is 10-20 Pa, and the drying time is 8-12 hours; The shelf temperature for secondary drying is 20-30℃, the vacuum degree is 5-10 Pa, and the drying time is 4-6 hours.

[0016] Furthermore, the pharmaceutical-grade plant oil mentioned in step D is a medium-chain triglyceride oil, and the natural antioxidant is rosemary extract or tocopherol; The mass ratio of vegetable oil to microcapsule powder is 3:1 to 5:1, and the amount of antioxidant added is 0.05%-0.2% of the mass of vegetable oil. The homogenization is performed using a high-pressure homogenizer with a homogenization pressure of 20-40 MPa and a homogenization cycle of 2-4 times. After homogenization, the particle size D90 of the drops is no greater than 10 μm.

[0017] Compared with the prior art, the beneficial effects of the present invention are: The probiotic drop preparation method provided by this invention significantly improves product performance at multiple levels through the synergistic coordination of process steps. Firstly, in the pretreatment stage, the lyophilized probiotic powder is activated and cultured to the logarithmic growth phase. At this stage, the bacteria exhibit vigorous metabolism and high cell membrane integrity, resulting in superior tolerance to subsequent encapsulation and drying processes. Resuspension with a specific ratio of skim milk powder, glycerol, and monosodium glutamate protectant, while controlling the OD600 value of the bacterial suspension within an appropriate range, provides osmotic pressure buffering and membrane stabilization for the bacteria, reducing damage caused by mechanical forces during centrifugation and washing. Simultaneously, it ensures a highly consistent initial bacterial load for each batch, facilitating stable control of subsequent encapsulation operations.

[0018] In the microcapsule preparation stage, a microfluidic droplet generator is used to precisely drop the aqueous bacterial suspension and sodium alginate-gelatin mixed solution. Coordinated regulation of the flow rates of the dispersed and continuous phases generates tiny droplets with a narrow particle size distribution. Compared to traditional stirring emulsification or extrusion methods, this method avoids the shear damage to the bacteria caused by severe turbulence. Simultaneously, the resulting microspheres are uniform in size and can maintain a relatively stable suspension in the droplet oil phase, making them less prone to gravity stratification. The coagulation bath contains both calcium ions and chitosan. After the droplets fall in, sodium alginate and calcium ions rapidly cross-link to form a gel framework. Chitosan molecules, due to their polycationic properties, electrostatically complex with the carboxyl groups of sodium alginate, filling the gaps in the gel network. This dual cross-linking mechanism forms a dense structure the instant the droplets solidify, effectively limiting the leakage of bacteria from the core material. Furthermore, the antibacterial properties of chitosan are only effective against harmful microorganisms, with no negative impact on the target probiotics due to the physical isolation caused by encapsulation. In addition, the addition of gelatin improves the film-forming properties and flexibility of the sodium alginate solution, making the surface of the gelled microspheres smooth and moderately elastic, and less prone to breakage during subsequent curing, washing and freeze-drying processes.

[0019] The low-temperature ripening and settling step in step C, combined with intermittent gentle stirring, allows for further rearrangement of molecular chains and strengthening of hydrogen bonds in the gel network, resulting in a secondary enhancement of the microsphere's mechanical strength. Simultaneously, this temperature condition maintains slow bacterial metabolism, preventing nutrient consumption and the accumulation of metabolic waste. The freeze-drying protectant is a combination of trehalose, sucrose, and vitamin C, adjusted to a specific osmotic pressure range. The soaking process allows the protectant to fully penetrate the gel network. During the pre-freezing stage of vacuum freeze-drying, these glassy formations displace the water surrounding the bacteria, inhibiting ice crystal nucleation and growth that could damage the cell membrane. The temperature control and vacuum conditions during the primary and secondary drying processes are progressively optimized to ensure an appropriate rate of water sublimation while preventing excessively high shelf temperatures that could denature bacterial proteins. After freeze-drying, the bacteria within the microcapsule powder are in a dormant state, and the wall material forms a porous yet rigid glassy structure with extremely low oxygen and water permeability.

[0020] The microcapsule powder was mixed and homogenized with pharmaceutical-grade vegetable oil and natural antioxidants to obtain the final drops. Because the microcapsule particle size is controlled between tens and two hundred micrometers, and the surface has a certain degree of lipophilicity due to chitosan modification, it disperses evenly in the oil phase without the need for additional surfactants. The natural antioxidants can scavenge free radicals in the oil phase caused by light or trace metal ions, delaying the oxidation of the vegetable oil and thus protecting the microcapsule wall material from oxidative degradation, while also preventing oxidative byproducts from damaging the internal bacteria. When the drops are ingested, the microcapsules remain intact in the mouth and esophagus. Upon reaching the stomach, the alginate-chitosan composite gel shrinks in the acidic environment, further slowing calcium ion dissociation and delaying microcapsule rupture. After entering the intestines, the pH increases, the gel network swells and undergoes enzymatic hydrolysis, achieving targeted release of the bacteria. The released live bacteria retain a high metabolic recovery capacity due to the multi-level protection in the early stages. All parameters in the entire preparation process are mild physicochemical processes, without introducing toxic cross-linking agents or organic solvents, and the final product safety meets pharmaceutical-grade standards. This method can adapt to the encapsulation requirements of different probiotic strains. By adjusting the concentration of the bacterial suspension and microfluidic parameters, the product specifications can be flexibly changed. It has strong potential for continuous production and high process quality control. Attached Figure Description

[0021] Figure 1 This diagram illustrates the working steps of a method for preparing a probiotic drop according to the present invention. Detailed Implementation

[0022] This invention discloses a method for preparing probiotic drops. It employs microfluidic microcapsule encapsulation combined with vacuum freeze-drying technology to encapsulate probiotics in a double-layer gel and protect them at low temperatures. The drops are then formulated with pharmaceutical-grade plant oil and natural antioxidants, effectively solving the technical problems of low viable bacteria rate, poor storage stability, easy probiotic inactivation, and uneven particle size in traditional probiotic drops. The raw materials used in the following examples and comparative examples are all commercially available food-grade and pharmaceutical-grade raw materials. The equipment used is all conventional commercial equipment, including an anaerobic incubator, a high-speed refrigerated centrifuge, a microfluidic droplet generator, a vacuum freeze dryer, a high-pressure homogenizer, an osmometer, a laser particle size analyzer, and a colony counter. The probiotic used is *Lactobacillus rhamnosus*, and the strain activation and culture process are standardized. All preparation operations comply with food production safety regulations.

[0023] All experimental groups had the same initial probiotic inoculation amount and basic culture medium composition. The only difference was in the preparation according to the limited process parameters of each group. Finally, the core indicators such as probiotic viable count, encapsulation rate, droplet particle size, and storage survival rate were uniformly tested to ensure that the experimental data were comparable and authentic.

[0024] Example 1 See appendix Figure 1 This embodiment provides a method for preparing probiotic drops, the specific steps of which are as follows: Step A: Probiotic Activation Culture and Suspension Preparation: Lyophilized *Lactobacillus rhamnosus* powder was selected and inoculated into liquid activation medium for strain activation. After activation, the strain was transferred to fresh liquid medium and cultured anaerobicly at 37°C for 15 hours until the strain reached the logarithmic growth phase. The cultured bacterial suspension was centrifuged at 5000 rpm for 12 minutes using a high-speed refrigerated centrifuge. The supernatant was discarded, and the bottom bacterial precipitate was collected. The bacterial precipitate was washed with sterile physiological saline, repeated twice. After each wash, the bacterial cells were fully resuspended and centrifuged again for purification. A protective agent solution was prepared by mixing skim milk powder, glycerol, and monosodium glutamate in a mass ratio of 10:5:1 in sterile water, stirring thoroughly to dissolve and sterilize before use. The washed and purified bacterial suspension was resuspended in the protective agent solution, and the OD600 value of the suspension was adjusted to 2.5 to obtain a homogeneous and stable probiotic suspension.

[0025] Step B, Microfluidic Microsphere Preparation: Prepare an aqueous solution containing 2.0% sodium alginate and 0.8% gelatin by mass using sterile deionized water as the solvent. Place the prepared aqueous solution in a vacuum degassing device and degas for 15 minutes. After degassing, cool to 12°C for later use. Mix the bacterial suspension prepared in Step A with the cooled and degassed aqueous solution at a volume ratio of 1:2 and stir thoroughly to obtain a homogeneous mixture. Select liquid paraffin as the continuous phase and the mixture as the dispersed phase. Connect them to the channels of a microfluidic droplet generator, setting the flow rate of the dispersed phase to 1.2 mL / h and the flow rate of the continuous phase to 10 mL / h. Shear the microfluidic structure to generate uniformly sized droplets with a diameter controlled at 120 μm. Prepare a coagulation bath solution containing 0.2 mol / L calcium chloride and 0.5% chitosan by mass using sterile deionized water as the solvent. The coagulation bath temperature is kept constant at 30°C. The uniform droplets generated by microfluidics were slowly dropped into the coagulation bath, and the mixture was stirred at a low speed of 150 rpm throughout the process. The gelation reaction lasted for 20 minutes, and the microspheres were initially formed.

[0026] Step C: Microsphere maturation, soaking, and vacuum freeze-drying: The gelled microspheres, along with the coagulation bath, are transferred to a low-temperature environment and allowed to mature at 6°C for 4 hours. During maturation, the microspheres are stirred at 80 rpm for 1 minute every 30 minutes to ensure uniform maturation and prevent agglomeration. After maturation, the microspheres are collected by filtration through a sterile filter cloth and washed three times with sterile deionized water to remove residual coagulation bath components and unreacted colloids from the surface. A freeze-drying protective solution is prepared by dissolving trehalose, sucrose, and vitamin C in sterile water at a mass ratio of 3:2:1, and adjusting the osmotic pressure of the solution to 700 mOsm / kg. The washed microspheres are completely immersed in the freeze-drying protective solution at room temperature for 45 minutes to allow the protective solution to fully penetrate the microspheres. After soaking, the microspheres are removed, laid flat on a freeze-drying tray, and subjected to segmented vacuum freeze-drying. Pre-freezing stage: Pre-freezing temperature -45℃, pre-freezing time 3h, to ensure that the microspheres are completely frozen and there is no liquid residue; First drying stage: shelf temperature -5℃, vacuum degree 15Pa, drying time 10h, to remove free moisture inside the microspheres; Second drying stage: shelf temperature 25℃, vacuum degree 8Pa, drying time 5h, to remove bound moisture, finally obtaining loose and dry probiotic microcapsule powder.

[0027] Step D: Homogenization and Filling of Probiotic Drops: Pharmaceutical-grade medium-chain triglyceride oil was selected as the base oil, and rosemary extract was added as a natural antioxidant. The ingredients were prepared at a ratio of 4:1 (plant oil to microcapsule powder by mass), with the antioxidant added at 0.12% of the plant oil mass. The microcapsule powder, medium-chain triglyceride oil, and rosemary extract were sequentially added to the mixing tank and initially mixed evenly with low-speed stirring. Homogenization was performed using a high-pressure homogenizer at a pressure of 30 MPa, with three consecutive homogenization cycles. After homogenization, the droplet particle size was measured, and D90 was controlled to be ≤10 μm. After homogenization, the solution was aseptically filtered and aseptically filled to obtain the finished probiotic drops.

[0028] Example 2 This embodiment provides a method for preparing probiotic drops, the specific steps of which are as follows: Step A: Probiotic activation culture and bacterial suspension preparation: The strain activation method is the same as in Example 1. After activation, it is anaerobically cultured at 37℃ for 12 hours until the logarithmic growth phase. The bacterial suspension is centrifuged at 4000 rpm for 10 minutes and washed twice with sterile physiological saline. A protective agent solution is prepared using skim milk powder, glycerol, and sodium glutamate in a mass ratio of 10:5:1. After resuspending the bacterial suspension, the OD600 value of the suspension is adjusted to 2.0 to obtain the bacterial suspension.

[0029] Step B, Microfluidic Microsphere Preparation: Prepare an aqueous solution containing 1.5% sodium alginate and 0.5% gelatin by mass, degas under vacuum for 10 min, and cool to 10℃. Mix the bacterial suspension and aqueous solution at a volume ratio of 1:2. Using vegetable oil as the continuous phase, the flow rate of the microfluidic dispersed phase is 0.5 mL / h, and the flow rate of the continuous phase is 5 mL / h, controlling the droplet diameter to 50 μm. Prepare a coagulation bath containing 0.1 mol / L calcium chloride and 0.2% chitosan by mass, and gel at 25℃ with stirring at 100 rpm for 10 min to complete microsphere formation.

[0030] Step C: Microsphere ripening, soaking, and vacuum freeze-drying: The microspheres were allowed to ripen at 4℃ for 2 hours, with stirring at 50 rpm for 1 minute every 30 minutes during the ripening process. After filtration and washing, they were soaked in a trehalose-sucrose-vitamin C freeze-drying protective solution with an osmotic pressure of 600 mOsm / kg and a mass ratio of 3:2:1 for 30 minutes. Vacuum freeze-drying parameters: pre-freezing at -40℃ for 2 hours; drying at -10℃ and 10 Pa for 8 hours; and drying at 20℃ and 5 Pa for 4 hours to obtain microcapsule powder.

[0031] Step D: Homogenization and Filling of Probiotic Drops: The mass ratio of medium-chain triglyceride oil to microcapsule dry powder is 3:1, and the amount of tocopherol added is 0.05% of the mass of vegetable oil. The homogenization pressure is 20 MPa, and the homogenization cycle is 2 times. After homogenization, the product is aseptically filled to obtain the finished drops.

[0032] Example 3 This embodiment provides a method for preparing probiotic drops, the specific steps of which are as follows: Step A: Probiotic activation culture and bacterial suspension preparation: After activation, the strain was anaerobically cultured at 37℃ for 18 hours until the logarithmic growth phase. The bacterial suspension was centrifuged at 6000 rpm for 15 minutes and washed three times with sterile physiological saline. The bacterial cells were resuspended in a fixed-ratio cryoprotectant solution, and the OD600 value of the bacterial suspension was adjusted to 3.0 to prepare a high-concentration bacterial suspension.

[0033] Step B, Microfluidic Microsphere Preparation: Prepare an aqueous solution containing 2.5% sodium alginate and 1.0% gelatin by mass, degas under vacuum for 20 min, and cool to 15℃. Mix the bacterial suspension and aqueous solution at a volume ratio of 1:2. Using liquid paraffin as the continuous phase, the dispersed phase flow rate is 2.0 mL / h, and the continuous phase flow rate is 15 mL / h, controlling the droplet diameter to 200 μm. Prepare a coagulation bath containing 0.3 mol / L calcium chloride and 0.8% chitosan by mass, and gel at 35℃ with stirring at 200 rpm for 30 min to complete microsphere formation.

[0034] Step C: Microsphere ripening, soaking, and vacuum freeze-drying: The microspheres were allowed to ripen at 8℃ for 6 hours, with stirring at 100 rpm for 1 minute every 30 minutes during the ripening process. After filtration and washing, they were soaked in a freeze-drying protective solution with an osmotic pressure of 800 mOsm / kg for 60 minutes. Vacuum freeze-drying parameters: pre-freezing at -50℃ for 4 hours; drying at 0℃ and 20 Pa for 12 hours; and drying at 30℃ and 10 Pa for 6 hours to obtain microcapsule powder.

[0035] Step D: Homogenization and Filling of Probiotic Drops: The mass ratio of medium-chain triglyceride oil to microcapsule dry powder is 5:1, and the amount of rosemary extract added is 0.2% of the mass of the vegetable oil. The homogenization pressure is 40 MPa, and the homogenization cycle is 4 times. After homogenization, the product is aseptically filled to obtain the finished drops.

[0036] Example 4 This embodiment provides a method for preparing probiotic drops, the specific steps of which are as follows: Step A: Probiotic activation culture and bacterial suspension preparation: After activation, the strain was anaerobically cultured at 37℃ for 14h, centrifuged at 5500rpm for 13min, washed twice with sterile physiological saline, and the bacterial cells were resuspended using a fixed ratio of protectant. The OD600 value of the bacterial suspension was adjusted to 2.8 to obtain the bacterial suspension.

[0037] Step B, Microfluidic Microsphere Preparation: Prepare an aqueous solution containing 2.2% sodium alginate and 0.7% gelatin by mass, degas under vacuum for 18 min, and cool to 13℃. Mix the bacterial suspension and aqueous solution at a volume ratio of 1:2, with a dispersed phase flow rate of 1.5 mL / h and a continuous phase flow rate of 12 mL / h, resulting in droplet diameters of 150 μm. Use a coagulation bath containing 0.25 mol / L calcium chloride and 0.6% chitosan, at 32℃, and stir at 180 rpm for 25 min to gel.

[0038] Step C: Microsphere ripening, soaking, and vacuum freeze-drying: The microspheres were ripened at 5℃ for 5 hours, with stirring at 90 rpm for 1 minute every 30 minutes. They were then soaked in a 750 mOsm / kg freeze-drying protective solution for 50 minutes. Freeze-drying parameters: pre-freezing at -48℃ for 3.5 hours; primary drying at -8℃ and 18 Pa for 11 hours; secondary drying at 28℃ and 9 Pa for 5.5 hours, yielding the microcapsule powder.

[0039] Step D: Homogenization and filling of probiotic drops: The mass ratio of vegetable oil to microcapsule dry powder is 4.5:1, the amount of rosemary extract added is 0.15%, the homogenization pressure is 35MPa, the homogenization is repeated 3 times, and the finished drops are aseptically filled.

[0040] Comparative Example 1 This comparative example provides a method for preparing probiotic drops, differing from Example 1 only in that the microfluidic droplet generation process is omitted, and the traditional stirred droplet method is used to prepare microspheres. All other raw material ratios, culture parameters, freeze-drying parameters, and filling parameters are completely consistent with Example 1. The specific steps are as follows: All operating parameters, raw material ratios, and process conditions in steps A, C, and D are exactly the same as in Example 1.

[0041] Step B: Preparation of microspheres using traditional methods: Prepare a mixture of aqueous solution and bacterial suspension according to the ratio in Example 1. Manually drip the mixture into the coagulation bath using a regular syringe instead of a microfluidic droplet generator. Without precise flow rate control, the mixture naturally drips to form gel microspheres. The coagulation bath conditions, gelation time, and stirring parameters are the same as in Example 1, thus completing the microsphere preparation.

[0042] Comparative Example 2 This comparative example provides a method for preparing probiotic drops, which differs from Example 1 only in that the lyophilization protective solution soaking step C is omitted, and the microspheres are directly subjected to vacuum freeze-drying after washing. All other process parameters, raw material ratios, and operating steps are completely consistent with Example 1.

[0043] Comparative Example 3 This comparative example provides a method for preparing probiotic drops, which differs from Example 1 only in that: step D does not add natural antioxidants, but only uses medium-chain triglyceride oil and microcapsule dry powder for homogenization and filling. All other process parameters, raw material ratios, and operation steps are completely consistent with Example 1.

[0044] The probiotic drops prepared in each embodiment and comparative example were tested for core performance indicators. The core test indicators included microsphere particle size uniformity, probiotic encapsulation rate, initial viable count after freeze-drying, viable count after 30 days of storage at room temperature, and drop D90 particle size. All tests were conducted using a unified testing standard. Each group of samples was tested in parallel three times, and the average value was taken as the final experimental data.

[0045] Table 1: Results of microsphere size and probiotic encapsulation efficiency in each experimental group The coefficient of variation (COP) is a core indicator for evaluating the uniformity of microsphere size. A lower COP indicates better microsphere size consistency. The encapsulation rate directly reflects the encapsulation effect of probiotics and is fundamental to ensuring probiotic activity. As shown in Table 1, the microspheres prepared using microfluidic technology in the four embodiments of this invention all have a COP below 4.0%, exhibiting extremely high particle size uniformity. Furthermore, the microsphere size can be precisely controlled within the 50–200 μm range by adjusting the microfluidic flow rate, adapting to different droplet application requirements. Simultaneously, the probiotic encapsulation rate in each embodiment is consistently above 92%, demonstrating excellent encapsulation effect and maximizing the encapsulation of probiotics within the microspheres, isolating them from adverse external environments.

[0046] All three comparative examples showed varying degrees of performance degradation. Comparative Example 1, which omitted the microfluidic process and used the traditional manual droplet method without precise flow rate and shear control, experienced a sharp increase in the microsphere particle size variation coefficient to 18.62%, resulting in inconsistent particle sizes. Furthermore, some microspheres exhibited voids, breakage, and adhesion, leading to probiotic leakage and an encapsulation rate of only 81.36%, a decrease of 12.92 percentage points compared to Example 1. This clearly demonstrates that the microfluidic droplet generation process is the core key to ensuring microsphere uniformity and high encapsulation rate. Comparative Example 2, which omitted the freeze-drying protective solution soaking step, showed that the microsphere structure was prone to micropore collapse and bacterial cell damage during freeze-drying, resulting in an encapsulation rate of 85.72%. This indicates that the freeze-drying protective solution effectively stabilizes the microsphere structure and reduces probiotic detachment damage. Comparative Example 3, lacking only the antioxidant, had no impact on the microsphere forming and encapsulation process. Therefore, the particle size index and encapsulation rate were basically consistent with Example 1, proving that the antioxidant does not participate in the early microsphere preparation process but only acts in the later storage stage.

[0047] Table 2: Results of probiotic viable counts and storage stability tests in each experimental group The initial viable count of probiotics directly reflects the protective effect of the preparation process on the activity of the strains. The viable cell survival rate after 30 days of storage at room temperature is the core indicator for evaluating the storage stability of the drops; a higher value indicates better shelf-life stability. As shown in Table 2, the initial viable count of the drops prepared in the four examples of this invention is higher than 2.5 × 10 CFU / g, and the viable cell survival rate after 30 days is higher than 89%. Example 3 has the optimal parameters, achieving a viable cell survival rate of 93.05%. This demonstrates that the synergistic process of microfluidic encapsulation, gradient freeze-drying, and protective solution immersion of this invention can maximize the protection of probiotic activity and reduce bacterial damage during the preparation process.

[0048] The viable cell performance of each comparative example was significantly inferior to that of the example. Comparative Example 1 suffered from poor microsphere uniformity and a high breakage rate, resulting in the inactivation of a large number of probiotics during freeze-drying and homogenization, leading to a significant reduction in the initial viable cell count. Furthermore, the bacteria were easily exposed to oxygen and oils during storage, resulting in oxidative inactivation, with a 30-day survival rate of only 74.18%. Comparative Example 2 lacked freeze-drying protection, and ice crystal puncture and osmotic pressure imbalance during freeze-drying caused damage to a large number of bacteria, resulting in a decrease in the initial viable cell count, poor storage stability, and a survival rate of only 80.43%. Comparative Example 3 lacked natural antioxidants, and the oil-based substrate underwent oxidative deterioration during long-term storage. The resulting peroxides damaged the probiotics. Although the initial viable cell count was similar to that of Example 1, the viable cell count decreased significantly after long-term storage, with a survival rate of only 74.65%. This verified the crucial role of natural antioxidants in improving the storage stability of the drops, inhibiting oil oxidation, and protecting probiotics.

[0049] Table 3: Results of Particle Size and Dispersibility Testing for Each Experimental Component Droplets The D90 particle size and dispersion coefficient of drops are core indicators for evaluating the physicochemical quality of drops. A D90 ≤ 10 μm and a lower dispersion coefficient indicate more uniform particle size, better dispersion stability, and less stratification / precipitation, meeting the quality standards for oral drops. As shown in Table 3, the finished drops prepared in the four examples of this invention all have a D90 particle size of less than 10 μm and a dispersion coefficient of less than 0.2, exhibiting uniform particle size and excellent dispersibility. After standing at room temperature for 7 days, no stratification or precipitation was observed, demonstrating excellent physicochemical stability and fully meeting the quality standards for pharmaceutical probiotic drops. Furthermore, the particle size of the finished drops can be flexibly adjusted by regulating the microsphere particle size and homogenization parameters to suit different application scenarios.

[0050] All comparative examples exhibited significant defects in their physicochemical properties. Comparative Example 1, due to uneven microsphere size and the presence of large impurities, could not be thoroughly refined after homogenization, resulting in a D90 particle size of 15.38 μm, exceeding the standard limit. This significantly increased the dispersion coefficient, leading to uneven system dispersion and stratification after standing, resulting in substandard product quality. Comparative Example 2, due to poor microsphere structural stability, contained a small amount of broken microsphere debris after freeze-drying, causing slight precipitation and a slight decrease in dispersibility. Comparative Example 3 lacked antioxidants, and slight oxidation of the oils led to decreased system compatibility, resulting in slight stratification and turbidity after standing, affecting product appearance and storage stability. These data fully demonstrate that this invention, through a synergistic process of microfluidic precision pelleting, freeze-drying protection, antioxidant compounding, and high-pressure homogenization, can prepare probiotic drops with uniform particle size, strong stability, and excellent quality. All core process parameters are indispensable.

[0051] Based on the above multi-dimensional experimental data, it can be seen that this invention has formed a complete and standardized probiotic drop preparation process by optimizing probiotic culture and purification parameters, employing microfluidic precision droplet forming technology, combining double-layer gel solidification and gradient ripening processes, adding a specific freeze-drying protective solution immersion step, segmented vacuum freeze-drying, and an antioxidant oil homogenization system. The probiotic drops prepared by this invention have advantages such as high probiotic encapsulation rate, sufficient viable bacteria count, excellent storage stability, uniform particle size, and no stratification or sedimentation, solving the technical pain points of traditional preparation processes such as easy probiotic inactivation, uneven microspheres, poor droplet stability, and short shelf life. Three sets of comparative experiments clearly demonstrate the inventiveness and necessity of the core technical features of this invention: the microfluidic droplet generation process is the core foundation for ensuring microsphere uniformity and improving encapsulation rate; the freeze-drying protective solution immersion step effectively protects the activity of probiotics during freeze-drying and stabilizes the microsphere structure; and natural antioxidants inhibit oil oxidation, significantly improving the long-term storage stability of the drops. The process parameters of this invention have strong adaptability and high fault tolerance, and the preparation process is stable, controllable, and reproducible, making it suitable for industrial-scale production. The prepared probiotic drops are of excellent quality and stable performance, and have extremely high market application value and promotion prospects.

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing probiotic drops, characterized in that, Includes the following steps: Step A: After activating the probiotic freeze-dried powder, inoculate it into a liquid culture medium, culture it under anaerobic conditions until the logarithmic growth phase, collect the bacterial cells by centrifugation, wash them with sterile physiological saline, and resuspend them in a protective agent solution to obtain a bacterial suspension. Step B: The bacterial suspension is mixed with an aqueous solution containing sodium alginate and gelatin at a volume ratio of 1:

2. The mixture is then used to generate uniformly sized droplets using a microfluidic droplet generator. The droplets fall into a coagulation bath containing calcium chloride and chitosan, where they gel to form microspheres. Step C: The microspheres are allowed to stand and mature at 4°C, then filtered and washed with deionized water, then soaked in a freeze-drying protective solution, and finally freeze-dried under vacuum to obtain probiotic microcapsule powder. Step D: The microcapsule powder is mixed with pharmaceutical-grade plant oil and natural antioxidants, homogenized, and then filled to obtain probiotic drops.

2. The method for preparing a probiotic drop as described in claim 1, characterized in that, The protective agent solution in step A comprises skim milk powder, glycerin and monosodium glutamate in a mass ratio of 10:5:1; Furthermore, the OD600 value of the bacterial suspension is adjusted to 2.0-3.

0.

3. The method for preparing a probiotic drop as described in claim 1, characterized in that, The anaerobic culture temperature in step A is 37±1℃, and the culture time is 12-18 hours; The centrifugation speed is 4000-6000 rpm, and the centrifugation time is 10-15 minutes; Wash 2-3 times, and resuspend in sterile saline after each wash.

4. The method for preparing a probiotic drop as described in claim 1, characterized in that, In step B, the aqueous solution contains 1.5%-2.5% sodium alginate and 0.5%-1.0% gelatin by mass. Furthermore, the aqueous solution is degassed under vacuum for 10-20 minutes and cooled to 10-15°C before mixing.

5. The method for preparing a probiotic drop as described in claim 1, characterized in that, In step B, the dispersed phase flow rate of the microfluidic droplet generator is 0.5-2.0 mL / h, and the continuous phase flow rate is 5-15 mL / h. The droplet diameter is controlled between 50-200 μm; The continuous phase is liquid paraffin or vegetable oil.

6. The method for preparing a probiotic drop as described in claim 1, characterized in that, In step B, the concentration of calcium chloride in the coagulation bath is 0.1-0.3 mol / L, and the mass fraction of chitosan is 0.2%-0.8%. The temperature of the coagulation bath is 25-35℃, and the gelation time is 10-30 minutes; During the gelation process, keep stirring slowly at a speed of 100-200 rpm.

7. The method for preparing a probiotic drop as described in claim 1, characterized in that, The temperature for the ripening step described in step C is 4-8℃, and the standing time is 2-6 hours; During the maturation process, stir for 1 minute at a speed of 50-100 rpm every 30 minutes.

8. The method for preparing a probiotic drop as described in claim 1, characterized in that, The freeze-drying protective solution described in step C contains trehalose, sucrose, and vitamin C in a mass ratio of 3:2:

1. Furthermore, the osmotic pressure of the freeze-drying protective solution was adjusted to 600-800 mOsm / kg, and the soaking time was 30-60 minutes.

9. The method for preparing a probiotic drop as described in claim 1, characterized in that, The vacuum freeze drying described in step C includes a pre-freezing stage, a primary drying stage, and a secondary drying stage; The pre-freezing temperature is -40℃ to -50℃, and the pre-freezing time is 2-4 hours; The shelf temperature for one-time drying is -10℃ to 0℃, the vacuum degree is 10-20 Pa, and the drying time is 8-12 hours; The shelf temperature for secondary drying is 20-30℃, the vacuum degree is 5-10 Pa, and the drying time is 4-6 hours.

10. The method for preparing a probiotic drop as described in claim 1, characterized in that, The pharmaceutical-grade plant oil mentioned in step D is a medium-chain triglyceride oil, and the natural antioxidant is rosemary extract or tocopherol; The mass ratio of vegetable oil to microcapsule powder is 3:1 to 5:1, and the amount of antioxidant added is 0.05%-0.2% of the mass of vegetable oil. The homogenization is performed using a high-pressure homogenizer with a homogenization pressure of 20-40 MPa and a homogenization cycle of 2-4 times. After homogenization, the particle size D90 of the drops is no greater than 10 μm.

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