Highly active composite wall material probiotic microcapsule and preparation method thereof
By using a composite wall material system composed of konjac glucomannan, soy protein isolate, and nano-titanium dioxide, the stability problem of probiotic microcapsules in complex environments has been solved, improving the survival rate and antibacterial properties of probiotics and achieving higher environmental adaptability and storage stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MINSHENG ZHONGKE JIAYI (SHANDONG) BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-04-29
- Publication Date
- 2026-05-29
AI Technical Summary
The wall material of existing probiotic microcapsules has poor stability, especially in complex environments where its ability to protect against light and external microorganisms is limited, leading to a decrease in probiotic activity and affecting its application effect.
A composite wall material system composed of konjac glucomannan, soy protein isolate, and nano-titanium dioxide is used. Through hydrogen bonding and molecular chain entanglement, a dense three-dimensional network structure is formed. Nano-titanium dioxide provides photocatalytic and antibacterial properties, and enhances the mechanical strength and environmental stability of the microcapsules.
It significantly improves the survival rate and stability of probiotics in complex environments, enhances antioxidant, UV-resistant and antibacterial capabilities, and improves the survival rate and stability of probiotics during storage and digestion.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of probiotic microcapsule technology, specifically to a highly active composite wall material probiotic microcapsule and its preparation method. Background Technology
[0002] Probiotics are a class of live microorganisms that can produce beneficial effects on the host's health. They maintain the balance of the intestinal microecology by regulating the structure of the intestinal flora, inhibiting the growth of harmful microorganisms, and promoting the absorption of nutrients. They have wide applications in food, medicine, health products, and animal feed. However, probiotics are easily affected by factors such as temperature, oxygen, humidity, gastric acid, and bile salts during production, processing, storage, and passage through the gastrointestinal environment, leading to a decrease in bacterial activity or even inactivation, thus affecting their efficacy. Therefore, improving the stability and survival rate of probiotics during processing, storage, and digestion has always been a key technical issue in the industrial application of probiotics.
[0003] Currently, to improve the stability of probiotics, probiotic microencapsulation technology is commonly used to protect them. This technology uses materials such as polysaccharides or proteins as wall materials to encapsulate probiotics into micron- or submicron-sized capsule structures, thus forming a protective layer outside the bacteria to reduce the adverse effects of the external environment on the bacteria and improve the survival rate of probiotics in storage and digestion environments. For example, Chinese patent CN116763755B discloses a probiotic microcapsule and its preparation method. The probiotic microcapsule structure includes a probiotic core material, a freeze-drying protective layer formed by a freeze-drying protectant, and a wall material formed by a polysaccharide material, wherein the freeze-drying protective layer is disposed between the core material and the wall material. This technology improves the stability of probiotics to a certain extent during freeze-drying and storage by setting a protective layer between the probiotics and the wall material and using polysaccharide materials such as sodium alginate, pectin, and chitosan to form the outer shell of the probiotic microcapsule. However, this technology primarily relies on sodium alginate, pectin, and chitosan to form a polysaccharide composite wall material structure, whose stability remains limited under complex environmental conditions. Furthermore, this type of wall material system has limited protection against light and external microorganisms, and may still lead to a decrease in probiotic activity during long-term storage or under complex environmental conditions. In addition, this type of probiotic microcapsule structure mainly focuses on freeze-drying protection, and there is still room for further improvement in enhancing the structural stability, environmental adaptability, and overall protective ability of probiotic microcapsules.
[0004] Therefore, developing a probiotic microcapsule system with structural stability, excellent protective properties, and the ability to further improve the survival rate and environmental tolerance of probiotics, as well as its preparation method, is of great significance for improving the quality of probiotic products and expanding their application scope. Summary of the Invention
[0005] To address the technical problems of poor stability and limited protection against light and external microorganisms in existing probiotic microcapsule wall materials, this invention provides a highly active composite wall material probiotic microcapsule and its preparation method. The specific technical solution is as follows: In a first aspect, the present invention provides a highly active composite wall material probiotic microcapsule, comprising a composite wall material and a core material, wherein the raw materials of the composite wall material include konjac glucomannan, soy protein isolate and nano titanium dioxide.
[0006] Konjac glucomannan possesses excellent film-forming and gelling properties, enabling it to form a continuous three-dimensional network structure, providing a preliminary physical barrier for probiotics. Soy protein isolate, rich in various amino acids, not only enhances the flexibility and stability of the cell wall material but also provides some nutritional support for probiotics. Nano-titanium dioxide exhibits excellent photocatalytic properties, antibacterial activity, and UV shielding capabilities, inhibiting the growth of harmful microorganisms in the environment surrounding the probiotic microcapsules while simultaneously blocking UV damage to the probiotics.
[0007] Konjac glucomannan and soy protein isolate form a composite network structure through hydrogen bonding and molecular chain entanglement, improving the density and mechanical properties of the wall material. Soy protein isolate can adsorb onto the surface of nano-titanium dioxide, forming a stable interface layer and improving the dispersibility of nano-titanium dioxide in the system. Nano-titanium dioxide is embedded in the polysaccharide-protein composite network, forming an inorganic-organic composite reinforcing structure. Konjac glucomannan serves as the skeletal support matrix of the microcapsules, nano-titanium dioxide acts as structural reinforcing particles, and soy protein isolate provides flexibility enhancement. The synergistic effect of these three components not only significantly improves the mechanical strength and environmental stability of the microcapsules but also endows them with multiple protective properties such as antioxidant, UV protection, and antibacterial activity, thereby effectively improving the survival ability of probiotics in complex environments.
[0008] Furthermore, the mass ratio of konjac glucomannan, soy protein isolate, and nano-titanium dioxide is (3-5):(2-4):(0.5-1.5).
[0009] Furthermore, the core material is a probiotic suspension with a concentration of 10. 8 ~10 10 CFU / mL. This concentration range ensures a high viable bacterial load while avoiding uneven encapsulation or decreased activity due to excessive bacterial density, thereby improving the efficacy and stability of the microcapsules.
[0010] Secondly, the present invention provides a method for preparing the above-mentioned highly active composite wall material probiotic microcapsules, comprising the following steps: (1) Preparation of composite wall material solution: Weigh konjac glucomannan and soy protein isolate, and stir to dissolve them to prepare uniform konjac glucomannan solution and soy protein isolate solution; disperse nano titanium dioxide in a small amount of water, and obtain uniform nano titanium dioxide dispersion by ultrasonic treatment; mix konjac glucomannan solution, soy protein isolate solution and nano titanium dioxide dispersion, and stir to obtain uniform composite wall material solution; (2) Preparation of probiotic suspension: After the probiotics are cultured to the logarithmic growth phase, the bacterial cells are collected, washed and mixed, and resuspended in sterile physiological saline to obtain probiotic suspension; (3) Add the probiotic suspension to the composite wall material solution and stir evenly to obtain a mixture; spread the mixture and then pre-freeze and vacuum freeze-dry it to obtain highly active composite wall material probiotic microcapsules.
[0011] Stepwise dissolution and dispersion processes prevent component aggregation or phase separation, improving system homogeneity and ensuring the uniformity and stability of the final microcapsule structure. Selecting bacteria in the logarithmic growth phase ensures maximum activity, while washing removes residual culture medium, enhancing the purity and stability of the microcapsule product. Low-temperature freeze-drying technology forms porous microcapsules while avoiding high-temperature inactivation, which helps maintain probiotic activity and improves rehydration and release performance.
[0012] Furthermore, in step (1), the dissolution temperature of konjac glucomannan is 50-60℃, the dissolution temperature of soy protein isolate is 40-50℃, and the pH value of the solution is controlled at 7-8. Under these temperature and pH conditions, soy protein isolate can be fully dissolved. These conditions are conducive to the unfolding of protein structure, improve its interaction with polysaccharides, and thus enhance the stability of the composite wall material.
[0013] Furthermore, in step (1), the ultrasonic treatment time is 30-60 minutes, and the ultrasonic frequency is 40kHz. Ultrasonic treatment can effectively break up the nano-titanium dioxide agglomerates, improve their dispersion uniformity, and thus enhance the overall performance of the composite material.
[0014] Furthermore, in step (2), the probiotic is one or more of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, and Bifidobacterium bifidum.
[0015] Furthermore, in step (2), the probiotics are cultured at 36-38°C for 18-24 hours under anaerobic conditions. These conditions are close to the optimal growth environment for probiotics and are conducive to obtaining highly active bacteria.
[0016] Furthermore, in step (3), the pre-freezing conditions are -40 to -60°C for 2 to 4 hours. Rapid pre-freezing is beneficial for forming a fine and uniform ice crystal structure, thereby improving the pore structure and mechanical properties of the microcapsules.
[0017] Furthermore, in step (3), the vacuum freeze-drying conditions are a vacuum degree of 10~30 Pa, a temperature of -30~-20℃, and a drying time of 12~24 hours. These conditions allow for gentle sublimation of moisture, maximizing the preservation of probiotic activity and the integrity of the microcapsule structure.
[0018] Furthermore, in step (3), the spread thickness of the mixture is 5-10 mm. Controlling the spread thickness helps to improve heat and mass transfer efficiency, making the freeze-drying process uniform and stable, thereby ensuring consistent product quality.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a highly active composite wall material probiotic microcapsule and its preparation method. By constructing a composite wall material system composed of konjac glucomannan, soy protein isolate, and nano-titanium dioxide, the polysaccharide framework structure, the flexible protein network, and the functional enhancement effect of nanoparticles synergistically complement each other. Konjac glucomannan is rich in hydroxyl groups, and soy protein isolate is rich in amide groups. The two form a dense three-dimensional network structure through hydrogen bonding and molecular chain entanglement. Soy protein isolate acts as an interface stabilizer and dispersant for nano-titanium dioxide, while nano-titanium dioxide is embedded in the composite network to form an organic-inorganic synergistic enhancement structure, thereby significantly improving the mechanical properties, structural stability, and environmental tolerance of the microcapsules. Simultaneously, nano-titanium dioxide endows the microcapsules with excellent antibacterial properties and UV shielding capabilities, effectively inhibiting the influence of harmful microorganisms and reducing the damage of light to probiotic activity, thus significantly improving the survival rate of probiotics in storage and complex environments.
[0020] 2. This invention employs a low-temperature vacuum freeze-drying process, which avoids the damage to probiotic activity caused by high temperatures while enabling the microcapsules to form a uniform porous structure, thus improving their rehydration performance and probiotic release efficiency. The combination of composite wall materials and low-temperature vacuum freeze-drying technology effectively reduces the loss of probiotic activity during preparation and storage. After treatment in simulated gastric fluid for 2 hours, the probiotic survival rate reaches over 90%; after treatment in simulated intestinal fluid for 4 hours, the survival rate remains above 85%; after storage at 4℃ for 6 months, the survival rate is no less than 80%; and after storage in an accelerated aging environment at 40℃ and 75% relative humidity for 3 months, the survival rate is no less than 74%. This invention significantly improves the gastrointestinal tolerance and long-term storage stability of probiotic products, and the preparation process is simple, highly controllable, suitable for large-scale production, and has good application prospects. Detailed Implementation
[0021] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with preferred embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.
[0022] The probiotic used in the following examples is Lactobacillus rhamnosus JYLR-127, and the strain information has been disclosed in CN117586927B.
[0023] Example 1: Preparation of Probiotic Microcapsules for Composite Wall Materials (1) Preparation of wall material solution: Weigh 3g of konjac glucomannan, add it to 200mL of deionized water, and stir to dissolve at 50℃; weigh 2g of soy protein isolate, add it to 150mL of deionized water, stir to dissolve at 40℃, and adjust the pH value to 7; weigh 0.5g of nano titanium dioxide, disperse it in 50mL of deionized water, and ultrasonically disperse it for 30min; mix the above three solutions and stir for 30min to obtain a uniform composite wall material solution.
[0024] (2) Preparation of probiotic suspension: Lactobacillus rhamnosus JYLR-127 was inoculated into MRS liquid medium and cultured at 37℃ for 24 h to obtain the above-mentioned probiotics in the logarithmic growth phase. The cells were collected by centrifugation, washed twice, mixed, and resuspended in sterile physiological saline. The concentration of the bacterial suspension was adjusted to 1×10⁻⁶. 9 CFU / mL was used to obtain a probiotic suspension.
[0025] (3) Preparation of probiotic microcapsules: The probiotic suspension was added to the composite wall material solution and stirred evenly. The solution was then poured into a shallow dish and spread into a liquid layer of about 5-10 mm thickness. The solution was pre-frozen at -40℃ for 2 hours and then freeze-dried at a vacuum of 10 Pa and a temperature of -30℃ for 12 hours to obtain probiotic microcapsules.
[0026] Example 2: Preparation of Probiotic Microcapsules for Composite Wall Materials (1) Preparation of wall material solution: Weigh 4g of konjac glucomannan, add it to 250mL of deionized water, and stir to dissolve at 55℃; weigh 3g of soy protein isolate, add it to 200mL of deionized water, stir to dissolve at 45℃, and adjust the pH value to 7.5; weigh 1g of nano titanium dioxide, disperse it in 80mL of deionized water, and ultrasonically disperse it for 45min; mix the above three solutions and stir for 45min to obtain a uniform composite wall material solution.
[0027] (2) Preparation of probiotic suspension: Lactobacillus rhamnosus JYLR-127 was inoculated into MRS liquid medium and cultured at 37℃ for 24 h to obtain the above-mentioned probiotics in the logarithmic growth phase. The cells were collected by centrifugation, washed 3 times, mixed, and resuspended in sterile physiological saline. The concentration of the bacterial suspension was adjusted to 1×10⁻⁶. 10 CFU / mL was used to obtain a probiotic suspension.
[0028] (3) Preparation of probiotic microcapsules: The probiotic suspension was added to the composite wall material solution and stirred evenly. The solution was then poured into a shallow dish and spread into a liquid layer of about 5-10 mm thickness. The solution was pre-frozen at -50℃ for 3 hours and then freeze-dried at a vacuum of 20 Pa and a temperature of -25℃ for 18 hours to obtain probiotic microcapsules.
[0029] Example 3: Preparation of Probiotic Microcapsules for Composite Wall Materials (1) Preparation of wall material solution: Weigh 5g of konjac glucomannan, add it to 300mL of deionized water, and stir to dissolve at 55℃; weigh 4g of soy protein isolate, add it to 250mL of deionized water, stir to dissolve at 45℃, and adjust the pH value to 8; weigh 1.5g of nano titanium dioxide, disperse it in 100mL of deionized water, and ultrasonically disperse it for 60min; mix the above three solutions and stir for 60min to obtain a uniform composite wall material solution.
[0030] (2) Preparation of probiotic suspension: Lactobacillus rhamnosus JYLR-127 was inoculated into MRS liquid medium and cultured at 37℃ for 24 h to obtain the above-mentioned probiotics in the logarithmic growth phase. After centrifugation to collect the bacterial cells, they were washed 3 times and then mixed together. The mixture was then resuspended in sterile physiological saline and the concentration of the bacterial suspension was adjusted to 1×10⁻⁶. 9 CFU / mL was used to obtain a probiotic suspension.
[0031] (3) Preparation of probiotic microcapsules: The probiotic suspension was added to the composite wall material solution and stirred evenly. The solution was then poured into a shallow dish and spread into a liquid layer of about 5-10 mm thickness. The solution was pre-frozen at -60℃ for 4 hours and then freeze-dried at a vacuum of 30 Pa and a temperature of -20℃ for 24 hours to obtain probiotic microcapsules.
[0032] Comparative Example 1: Preparation of probiotic microcapsules with konjac glucomannan-soy protein isolate wall material The difference from Example 1 is that the wall material consists only of konjac glucomannan and soy protein isolate, and does not contain nano-titanium dioxide. All other treatments are the same.
[0033] Comparative Example 2: Preparation of Probiotic Microcapsules with Konjac Glucomannan Wall Material The difference from Example 1 is that the wall material contains only konjac glucomannan and does not contain soy protein isolate or nano-titanium dioxide. All other treatments are the same.
[0034] Comparative Example 3 The difference from Example 1 is that, in preparing the wall material solution, the weighed amounts of konjac glucomannan, soy protein isolate, and nano-titanium dioxide were 3g, 5g, and 3g, respectively. All other treatments were the same.
[0035] Comparative Example 4 The difference from Example 1 is that, in preparing the wall material solution, the masses of konjac glucomannan, soy protein isolate, and nano-titanium dioxide were 3g, 1g, and 0.1g, respectively. All other treatments were the same.
[0036] Test case 1. Gastric acid resistance test Simulated gastric fluid with a pH of 3 was prepared, and probiotic microcapsules prepared in Examples 1-3 and Comparative Examples 1-2 were added respectively. The mixtures were then shaken at 37°C for 2 hours. The viable count of probiotics in the microcapsules was determined using the plate count method, and the survival rate was calculated. The results showed that the survival rates of probiotics in Examples 1-3 were 91.2%, 93.8%, and 91.0%, respectively, while the survival rate of Comparative Example 1 was 75.6%, and the survival rate of Comparative Example 2 was only 60.7%.
[0037] 2. Bile salt tolerance test Simulated intestinal fluid with a bile salt concentration of 0.3% was prepared, and probiotic microcapsules prepared in Examples 1-3 and Comparative Examples 1-2 were added respectively. The mixtures were then shaken at 37°C for 4 hours. The viable bacteria count was determined using the plate count method, and the survival rate was calculated. The survival rates of the probiotics in Examples 1-3 were 88.2%, 87.7%, and 86.4%, respectively; the survival rate in Comparative Example 1 was 65.2%; and the survival rate in Comparative Example 2 was 51.0%.
[0038] 3. Simulated gastrointestinal transit test To simulate the human gastrointestinal environment, probiotic microcapsules were sequentially placed in simulated gastric juice (pH=3) for 2 hours, and then transferred to simulated intestinal juice (bile salt concentration 0.3%, pH=6.8) for 4 hours. The viable bacterial count was determined using a plate count method at different stages, and the survival rate after transit through the entire simulated gastrointestinal tract was calculated. The survival rates of Examples 1-3 reached 80.0%, 82.1%, and 81.5%, respectively, while the survival rates of Comparative Examples 1 and 2 were 55.3% and 45.8%, respectively, indicating that the probiotic microcapsules of this invention can better protect the activity of probiotics during simulated actual digestion.
[0039] 4. Antibacterial performance test Prepare Escherichia coli and Staphylococcus aureus bacterial suspensions (concentration 10) separately. 6(CFU / mL), for later use. Take 0.5g of each of the 5 groups of microcapsules and add 5mL of sterile physiological saline to prepare test solutions. Mix 1mL of test solution with 1mL of bacterial suspension and incubate at 37℃ for 24h. A blank control group (1mL sterile physiological saline + 1mL bacterial suspension) was also set up. After incubation, a serial dilution method (10⁻¹⁰ CFU / mL) was used. -1 -10 -8 Dilute the bacterial suspension, and spread 0.1 mL of the diluted solution onto LB agar plates. Incubate at 37°C for 24 hours. Count the colonies using a colony counter and calculate the antibacterial rate: Antibacterial rate = (Number of colonies in the blank control group - Number of colonies in the experimental group) / Number of colonies in the blank control group × 100%. The results are as follows: Table 1 Results of antibacterial test
[0040] Table 1 shows that Example 1, using a composite wall material of konjac glucomannan, soy protein isolate, and nano-titanium dioxide, achieved an antibacterial rate of 72.74% ± 2.33% against *Escherichia coli* and 73.33% ± 2.33% against *Staphylococcus aureus*, demonstrating a good level of antibacterial effect. Example 2, after optimization of the formulation, further improved its antibacterial performance, achieving an antibacterial rate of 90.95% ± 1.33% against *Escherichia coli* and 91.32% ± 1.28% against *Staphylococcus aureus*, with an average antibacterial rate exceeding 91%, the best among all groups. Example 3 exhibited slightly lower antibacterial performance than Example 2, but still maintained an excellent level of antibacterial activity, with an antibacterial rate exceeding 85% against both bacteria.
[0041] Comparative Example 1, without the addition of nano-titanium dioxide, used only konjac glucomannan and soy protein isolate as wall materials. The antibacterial rates against *Escherichia coli* and *Staphylococcus aureus* decreased to 66.47% ± 2.73% and 66.29% ± 2.74%, respectively, significantly lower than in Example 1. This indicates that nano-titanium dioxide is a key component for improving antibacterial performance. Comparative Example 2, using only konjac glucomannan as the wall material, showed antibacterial rates of only 17.05% ± 3.74% and 16.73% ± 3.75%, indicating poor antibacterial effect. This confirms a significant synergistic effect between soy protein isolate and konjac glucomannan, and that their combination can significantly enhance antibacterial capabilities. Comparative Example 3, by increasing the amount of soy protein isolate and nano-titanium dioxide, showed a significant decrease in antibacterial rate, which was only 47.89%±3.10% and 45.79%±3.20%, respectively. Comparative Example 4, by reducing the amount of both, showed antibacterial rates of 55.15%±2.95% and 54.21%±3.05%, which were also lower than those in Example 1. This indicates that there is an optimal range for the amount of wall material components added, and that both excessively high and low amounts will weaken the antibacterial effect.
[0042] Overall, all experimental groups showed a high degree of consistency in their antibacterial trends against *Escherichia coli* and *Staphylococcus aureus*, indicating that the composite wall material microcapsules have broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with a slightly better antibacterial effect against *Staphylococcus aureus* than against *Escherichia coli*. Comprehensive comparison shows that the optimal combination of konjac glucomannan, soy protein isolate, and nano-titanium dioxide is key to obtaining microcapsules with high antibacterial performance. Example 2 showed the best ratio, with an antibacterial rate consistently exceeding 90%.
[0043] 5. Ultraviolet shielding capability test Five groups of microcapsules, 1g each, were evenly spread in sterile petri dishes (0.5mm thickness) and placed 30cm below a UV lamp for 2 hours (simulating natural UV irradiation intensity). After irradiation, 5mL of sterile physiological saline was added to the microcapsules, and they were sonicated to release the probiotics inside. The bacterial solution was diluted using a serial dilution method, and 0.1mL of the diluted solution was spread on MRS agar plates and anaerobically cultured at 37℃ for 48 hours. The colony count was then recorded. A control group (the same group of microcapsules, without UV irradiation) was also set up. The survival rate of the probiotics was calculated as: survival rate = (number of colonies after UV irradiation / number of colonies without irradiation) × 100%. The UV absorption spectrum (200~400nm) of each group of microcapsules was measured using a UV spectrophotometer, and the absorbance at 365nm was recorded (higher absorbance indicates stronger UV shielding ability). The results are as follows: Table 2. Experimental results of ultraviolet shielding capability test
[0044] As shown in Table 2, the microcapsules of Example 2 exhibited the best UV shielding capability, with an absorbance of 0.82±0.05 at 365nm, significantly higher than other groups. The survival rate of its probiotics after UV irradiation was 82.49%±3.78%, indicating that this microcapsule formulation effectively blocks UV radiation from damaging the internal probiotics. The UV shielding effect and probiotic survival rate of Examples 1 and 3 were slightly inferior to Example 2, but still maintained a high level, both demonstrating good UV protection performance.
[0045] Comparative Example 1, without the addition of nano-titanium dioxide, used only konjac glucomannan-soybean protein isolate as the composite wall material. The absorbance at 365 nm was 0.88 ± 0.06, but the probiotic survival rate was only 64.43% ± 3.32%, significantly lower than Example 1. This indicates that nano-titanium dioxide is crucial for enhancing the UV absorption capacity of microcapsules; its nanoscale structure can enhance the scattering and absorption of UV light, forming an effective protective barrier. Comparative Example 2, using only konjac glucomannan as the wall material, had an absorbance of only 0.25 ± 0.02 at 365 nm, and a probiotic survival rate of only 21.01% ± 1.71%, exhibiting the worst UV shielding performance. This confirms that the combination of soybean protein isolate and konjac glucomannan can form a dense network structure, significantly improving the UV blocking performance of the wall material.
[0046] Comparative Example 3, by increasing the amounts of soy protein isolate and nano-titanium dioxide (3g konjac glucomannan, 5g soy protein isolate, 3g nano-titanium dioxide), showed an absorbance of 0.42±0.03 at 365nm, and a probiotic survival rate of only 41.76%±2.55%. It is speculated that excessive soy protein isolate may lead to a decrease in the density of the wall material structure, thereby increasing the probability of ultraviolet light penetration. Comparative Example 4, by decreasing the amounts of soy protein isolate and nano-titanium dioxide, showed an absorbance of 0.49±0.03 at 365nm, and a probiotic survival rate of 49.34%±2.87%, indicating that excessively low amounts significantly weaken the ultraviolet shielding performance.
[0047] 6. Antioxidant performance test The probiotic microcapsules prepared in Examples 1-3 and Comparative Examples 1-2 were placed in a solution containing hydrogen peroxide to simulate an oxidative stress environment. The antioxidant capacity was assessed by detecting changes in the activity of probiotics within the microcapsules after 24 hours of treatment. The results showed that after 30 minutes of treatment under the same oxidative environment, the survival rates of the probiotics in Examples 1-3 were 85%, 86%, and 84%, respectively, while the survival rates in Comparative Examples 1 and 2 were only 60% and 50%, respectively. This indicates that konjac glucomannan, soy protein isolate, and nano-titanium dioxide construct a dense barrier structure through intermolecular synergy, effectively hindering hydrogen peroxide penetration, reducing oxidative stress damage to the internal probiotics, and significantly enhancing the antioxidant protection capacity of the microcapsules. Conversely, the binary composite wall material lacking nano-titanium dioxide (Comparative Example 1) and the single konjac glucomannan wall material (Comparative Example 2) had a loose structure and weak barrier properties, making them unable to withstand oxidative stress, resulting in a significantly reduced probiotic survival rate.
[0048] 7. Storage stability test The probiotic microcapsules prepared in Examples 1-3 and Comparative Examples 1-4 were stored at 4°C for 6 months. The viable count of probiotics in the microcapsules was determined monthly using the plate count method, and the survival rate was calculated. The results are shown in Table 3. After 6 months, the survival rates of probiotics in Examples 1-3 were 89%, 93%, and 90%, respectively, while the survival rates in Comparative Examples 1-4 were 70%, 60%, 73%, and 68%, respectively. This indicates that the rationally formulated composite wall material can effectively block external moisture, oxygen, and environmental stress, reducing the inactivation of probiotics during long-term storage and significantly improving the storage stability of the microcapsules.
[0049] Table 3 Survival rate of probiotic microcapsules stored at 4°C
[0050] 8. Long-term storage stability test The probiotic microcapsules prepared in Examples 1-3 and Comparative Examples 1-4 were placed in an accelerated aging environment of 40°C and 75% relative humidity for 3 months to simulate the harsh conditions of long-term storage. The viable bacteria count was determined monthly using the plate count method, and the survival rate was calculated. The results are shown in Table 4. After 3 months, the survival rates of the probiotics in Examples 1-3 were 75.79%, 78.47%, and 74.78%, respectively, while the survival rates in Comparative Examples 1-4 were 50.11%, 40.64%, 60.81%, and 60.28%, respectively. Adding an appropriate amount of nano-titanium dioxide combined with konjac glucomannan-soybean protein isolate composite wall material can significantly improve the high-temperature and high-humidity storage stability of the probiotic microcapsules. The microcapsule cell protection ability of binary wall materials without added nano-titanium dioxide or single konjac glucomannan wall materials is poor, and the survival rate of probiotics drops significantly after humid heat aging. When the proportions of the wall material components are too high or too low, a dense and stable encapsulation structure cannot be formed, making it difficult to block the damage of humid heat and oxygen to probiotics, and the storage survival rate is significantly lower than that of the optimal formulation example. The composite wall material of this invention has excellent synergistic effect, and the coating after freeze-drying has strong barrier properties, which can effectively delay the inactivation of probiotics and significantly extend the long-term shelf life of the product.
[0051] Table 4 Survival rate of probiotic microcapsules stored under accelerated aging conditions
[0052] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.
Claims
1. A highly active composite wall material probiotic microcapsule, characterized in that, It includes a composite wall material and a core material. The raw materials of the composite wall material include konjac glucomannan, soy protein isolate and nano titanium dioxide. The mass ratio of konjac glucomannan, soy protein isolate and nano titanium dioxide is (3-5):(2-4):(0.5-1.5).
2. The highly active composite wall material probiotic microcapsules as described in claim 1, characterized in that, The core material is a probiotic suspension with a concentration of 10. 8 ~10 10 CFU / mL.
3. A method for preparing probiotic microcapsules of a highly active composite wall material as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of composite wall material solution: Weigh konjac glucomannan and soy protein isolate, and stir to dissolve them to prepare uniform konjac glucomannan solution and soy protein isolate solution; disperse nano titanium dioxide in a small amount of water, and obtain uniform nano titanium dioxide dispersion by ultrasonic treatment; mix konjac glucomannan solution, soy protein isolate solution and nano titanium dioxide dispersion, and stir to obtain uniform composite wall material solution; (2) Preparation of probiotic suspension: After the probiotics are cultured to the logarithmic growth phase, the bacterial cells are collected, washed and mixed, and resuspended in sterile physiological saline to obtain probiotic suspension; (3) Add the probiotic suspension to the composite wall material solution and stir evenly to obtain a mixture; spread the mixture and then pre-freeze and vacuum freeze-dry it to obtain highly active composite wall material probiotic microcapsules.
4. The preparation method according to claim 3, characterized in that, In step (1), the dissolution temperature of the konjac glucomannan is 50~60℃; the dissolution temperature of the soy protein isolate is 40~50℃, and the pH value of the solution is controlled at 7~8.
5. The preparation method according to claim 3, characterized in that, In step (1), the ultrasonic treatment time is 30 to 60 minutes and the ultrasonic frequency is 40 kHz.
6. The preparation method according to claim 3, characterized in that, In step (2), the probiotic is one or more of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, and Bifidobacterium bifidum.
7. The preparation method according to claim 3, characterized in that, In step (2), the probiotics are cultured at 36-38°C for 18-24 hours under anaerobic conditions.
8. The preparation method according to claim 3, characterized in that, In step (3), the pre-freezing conditions are -40~-60℃ for 2~4 hours.
9. The preparation method according to claim 3, characterized in that, In step (3), the vacuum freeze-drying conditions are a vacuum degree of 10~30Pa, a temperature of -30~-20℃, and a drying time of 12~24 hours.
10. The preparation method according to claim 3, characterized in that, In step (3), the thickness of the mixture is 5~10mm.