Single-cell nano-encapsulated probiotic microcapsules and methods of making same

Abstract

This invention discloses a single-cell nanoencapsulated probiotic microcapsule and its preparation method. The probiotic microcapsule consists of a core material encapsulated by a wall material. The wall material comprises two layers: an outer layer of methacrylate copolymer and an inner layer of metal-polyphenol network loaded with small-molecule oligosaccharides. The core material is probiotics. This probiotic microcapsule utilizes a single-cell nanoencapsulation method, which is simple and easy to operate, and can form a nanoscale functional coating on the surface of a single probiotic. The single-cell nanoencapsulated probiotic microcapsule provided by this invention not only has a particle size almost similar to that of a single probiotic, but also, through the synergistic effect of the inner and outer wall materials, endows the probiotic with excellent resistance to gastrointestinal digestion and intestinal targeted adhesion and colonization capabilities. Furthermore, the addition of small-molecule oligosaccharides makes the network structure of the coating denser and enhances the stability of the probiotics during storage and transportation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a single-cell nanoencapsulated probiotic microcapsule and its preparation method. Background Technology

[0002] Probiotics are a class of microorganisms that benefit the host's health. They can regulate the balance of intestinal flora, inhibit the growth of harmful bacteria, enhance immunity, and prevent and treat some diseases. However, probiotics are affected by various adverse factors during processing, transportation, and delivery within the body, such as temperature, oxygen, gastric acid, and bile salts, leading to reduced activity and stability, thus affecting their colonization and function in the gut. Therefore, how to effectively protect and deliver probiotics and increase their survival rate and activity in the host is an important research topic in probiotic formulations.

[0003] To address this issue, a common method is to encapsulate probiotics in suitable carrier materials, creating probiotic microcapsules. These microcapsules provide a physical barrier for the probiotics, extending their shelf life and effectiveness, and enhancing their resistance to gastrointestinal digestion. Currently, most commercially available probiotics utilize mass-encapsulated hydrogel systems, such as polysaccharide hydrogels and protein hydrogels. While these hydrogel systems can protect probiotics to some extent from external environmental and digestive fluid contamination, their drawbacks are also significant. For example, probiotic microcapsules formed using hydrogel systems have relatively large particle sizes, ranging from tens to thousands of micrometers, making them unsuitable for addition to food as food ingredients. Furthermore, the large particle size also makes it difficult for some probiotics to break through the cell wall and be released into the intestines, resulting in their excretion in feces. In addition, hydrogel probiotic microcapsules generally suffer from poor adhesion, making them difficult to colonize in the intestines.

[0004] Therefore, under the premise of enhancing the basic resistance of probiotics, there is an urgent need to develop a new probiotic encapsulation system with small particle size, strong adhesion, and even diversified functions. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a probiotic microcapsule with small particle size, strong stress resistance, and good adhesion, encapsulated in single-cell nanoparticles.

[0006] The second objective of this invention is to provide a simple and easy-to-operate method for preparing probiotic microcapsules using single-cell nano-encapsulation.

[0007] One of the objectives of this invention is achieved through the following technical solution:

[0008] A single-cell nanoencapsulated probiotic microcapsule, wherein the probiotic microcapsule is composed of a wall material encapsulating a core material, the wall material comprising two layers: an outer wall material being a methacrylate copolymer and an inner wall material being a metal-polyphenol network loaded with small molecule oligosaccharides; the core material being probiotics.

[0009] Furthermore, the outer wall material is formed by the self-assembly of a divalent metal ion-induced methacrylate copolymer on the surface of the inner wall material.

[0010] Furthermore, the divalent metal ions include Ca... 2+ Mg 2+ Zn 2+ Fe 2+ One or more of the following in any proportion: the methacrylate copolymer includes one or more of Eudragit L100-55, Eudragit L100, and Eudragit S100 in any proportion.

[0011] Furthermore, the metal-polyphenol network of the inner wall material is formed by the complexation of transition metal element ions with polyphenols containing catechol and / or gallic acid functional groups.

[0012] Furthermore, the transition metal ions include Fe. 3+ Al 3+ Cu 2+ Mn 2+ Zr 4+ Ti 4+ The polyphenols include one or more of the following in any proportion: tea polyphenols, grape polyphenols, tannins, and apple polyphenols.

[0013] Furthermore, the small molecule oligosaccharides include one or more of galacto-oligosaccharides, fructo-oligosaccharides, xyloo-oligosaccharides, isomaltooligosaccharides, chitosan oligosaccharides, and human milk oligosaccharides in any proportion.

[0014] Furthermore, the probiotics include one or more of the following probiotics in any proportion: Lactobacillus, Bifidobacterium, Streptococcus, and Escherichia coli.

[0015] Furthermore, the probiotics are Lactobacillus rhamnosus LGG, Lactobacillus rhamnosus HN001, Lactobacillus fermentum CECT5716, Lactobacillus reuteri DSM17938, Lactobacillus acidophilus NCFM, Bifidobacterium breve M-16V, Bifidobacterium lactis HN019, Bifidobacterium lactis Bi-07, and Bifidobacterium animalis Bb-12. One or more of Bb-12 and Escherichia coli Nissle1917 in any proportion.

[0016] The second objective of this invention is achieved by the following technical solution:

[0017] A method for preparing single-cell nanoencapsulated probiotic microcapsules includes the following steps:

[0018] 1) Preparation of probiotic suspension:

[0019] The activated probiotics were cultured to the late logarithmic growth phase, centrifuged to collect the cells, washed, and resuspended in sterile physiological saline or neutral phosphate buffer containing small molecule oligosaccharides to obtain a bacterial suspension.

[0020] 2) Preparation of probiotic microcapsules:

[0021] (1) Take the bacterial suspension from step 1) above, add the polyphenol aqueous solution dropwise to the bacterial suspension under vortex conditions, and then let it stand for 2 to 5 minutes.

[0022] (2) Under vortex conditions, add metal salt aqueous solution dropwise to the bacterial suspension after step (1) has been settling. After the addition is complete, continue vortexing for 3 to 8 minutes to allow for a full reaction.

[0023] (3) Add weakly alkaline phosphate buffer to the bacterial suspension after vortexing in step (2), mix thoroughly, let stand for 2-5 minutes, centrifuge, remove the supernatant, and continue to centrifuge and wash 2-3 times with weakly alkaline phosphate buffer.

[0024] (4) The bacterial cells cleaned in step (3) are resuspended in a neutral phosphate buffer containing divalent metal salts, and an aqueous solution of methacrylate copolymer is added. After vortexing and mixing, the pH is adjusted to weak acidity and vortexed for 5 to 10 minutes.

[0025] (5) Add weakly acidic phosphate buffer to the bacterial suspension after vortexing in step (4), mix thoroughly, let stand for 2-5 minutes, centrifuge, remove the supernatant, and repeat this step with weakly acidic phosphate buffer to continue centrifuging and washing 2-3 times.

[0026] 3) Freeze-drying of probiotic microcapsules:

[0027] The probiotic microcapsules collected after centrifugation and washing were pre-frozen in a freezer at -60 to -100°C, and then transferred to a vacuum freeze dryer for freeze drying to obtain single-cell nano-encapsulated probiotic microcapsules.

[0028] Further, the culture conditions in step 1) are: temperature 30–40℃, pH 6–7, anaerobic environment; the washing involves washing 2–3 times with sterile physiological saline or neutral phosphate buffer to remove the culture medium; the concentration of the small molecule oligosaccharides in the sterile physiological saline or neutral phosphate buffer is 5–15 mg / ml; the concentration of the bacterial suspension is 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL.

[0029] More preferably, the culture temperature is 37°C.

[0030] Furthermore, the concentration of the polyphenol aqueous solution in step (1) is 10–30 mg / ml, and the volume ratio of the polyphenol aqueous solution to the bacterial suspension is 1:40–60.

[0031] Furthermore, the concentration of the metal salt aqueous solution in step (2) is 4 to 10 mg / ml, and the volume ratio of the metal salt aqueous solution to the bacterial suspension is 1:40 to 80.

[0032] Furthermore, the metal salt mentioned in step (2) is a transition metal salt.

[0033] Furthermore, the weakly alkaline phosphate buffer solution described in step (3) has a pH of 7.4 to 8.0 and is added in an amount that is 1 to 2 times the volume of the bacterial suspension.

[0034] Furthermore, in step (4), the concentration of the divalent metal salt in the neutral phosphate buffer containing the divalent metal salt is 1–6 mg / ml; the divalent metal salt is calcium chloride; and the concentration of the bacterial suspension after resuspending the bacteria in the neutral phosphate buffer containing the divalent metal salt is 1 × 10⁻⁶ mg / ml. 7 ~1×109 CFU / mL; the concentration of the aqueous solution of the methacrylate copolymer is 2-10 mg / mL, and the volume ratio of the aqueous solution of the methacrylate copolymer to the bacterial suspension is 1:20-30.

[0035] Furthermore, the centrifugal cleaning described in steps (3) and (5) uses a centrifugal speed of 3000-9000 rpm and a centrifugation time of 2-8 min.

[0036] Furthermore, the pH adjustment in step (4) is in the range of 4.0 to 5.5, which is weakly acidic; the pH range of the weakly acidic phosphate buffer in step (5) is 4.0 to 5.5, and the amount added is 1 to 2 times the volume of the bacterial suspension.

[0037] Preferably, the pH range of the neutral phosphate buffer solution is 6.8 to 7.2.

[0038] The present invention has the following beneficial effects:

[0039] The single-cell nano-encapsulated probiotic microcapsules of the present invention not only have a particle size almost similar to that of a single probiotic, but also endow the probiotics with excellent resistance to gastrointestinal digestion and intestinal targeted adhesion and colonization ability through the synergistic effect of the inner and outer double-layer wall materials. The addition of small molecule oligosaccharides makes the network structure of the coating more compact and enhances the stability of the probiotics during storage and transportation.

[0040] The method for preparing single-cell nanoencapsulated probiotic microcapsules of the present invention can form a nanoscale functional coating on the surface of probiotics. This preparation method is simple, easy to operate and does not require special equipment, which is conducive to realizing large-scale industrial production. Attached Figure Description

[0041] Figure 1 The images are transmission electron microscopy (TEM) images of Lactobacillus rhamnosus LGG single-cell nano-encapsulation before (left) and after (right) in Example 4.

[0042] Figure 2 A bar chart showing the particle size change of Lactobacillus rhamnosus LGG before and after single-cell nano-encapsulation.

[0043] Figure 3 A bar chart showing the change in the number of *Lactobacillus rhamnosus* LGG freeze-dried before and after single-cell nano-encapsulation.

[0044] Figure 4 A bar chart showing the change in the number of Lactobacillus rhamnosus LGG cells after digestion with gastric juice before and after single-cell nano-encapsulation.

[0045] Figure 5 A bar chart showing the change in the number of Lactobacillus rhamnosus LGG cells after digestion with intestinal fluid before and after single-cell nano-encapsulation.

[0046] Figure 6 A dotted line graph showing the change in the number of Lactobacillus rhamnosus LGG before and after single-cell nano-encapsulation during storage. Detailed Implementation

[0047] The present invention will now be described in detail with reference to specific embodiments. It should be understood that the following embodiments are only for the purpose of helping to understand the present invention and should not be regarded as limiting the scope of the present invention. That is, the present invention may have other embodiments in addition to the following embodiments.

[0048] The pH of the neutral phosphate buffer in the following examples is 7.0.

[0049] Example 1

[0050] A single-cell nanoencapsulated Lactobacillus rhamnosus LGG microcapsule is prepared by the following steps:

[0051] 1. Preparation of probiotic suspension:

[0052] Activated Lactobacillus rhamnosus LGG was cultured to the late logarithmic growth phase. The cells were collected by centrifugation, washed three times with neutral phosphate buffer to remove the culture medium, and then resuspended in neutral phosphate buffer at a concentration of 2 × 10⁻⁶. 8 CFU / mL available;

[0053] 2. Preparation of probiotic microcapsules:

[0054] (1) Take 4.8 ml of the above bacterial suspension, and under vortex conditions, add 100 μL of epigallocatechin solution with a concentration of 20 mg / ml dropwise to the bacterial suspension, and then let it stand for 2 min.

[0055] (2) Under vortex conditions, add 100 μL of ferric chloride solution with a concentration of 5 mg / ml dropwise to the bacterial suspension after standing. After the addition is complete, continue vortexing for 3 min.

[0056] (3) Add 5 ml of phosphate buffer solution with pH 7.6, mix thoroughly, let stand for 5 min, and then add 5000 ml of phosphate buffer solution.

[0057] Centrifuge at rpm for 3 min, discard the supernatant, and repeat this step twice more with phosphate buffer at pH 7.6.

[0058] 3. Freeze-drying of probiotic microcapsules:

[0059] The probiotic microcapsules collected after washing and centrifugation were pre-frozen at -80℃ for 3 hours, and then transferred to a vacuum freeze dryer and freeze-dried at -60℃ and 30Pa for 24 hours. After drying, the microcapsules were transferred to a -80℃ refrigerator, sealed and stored at low temperature until use.

[0060] Example 2

[0061] A single-cell nanoencapsulated Lactobacillus rhamnosus LGG microcapsule is prepared by the following steps:

[0062] 1. Preparation of probiotic suspension:

[0063] Activated Lactobacillus rhamnosus LGG was cultured to the late logarithmic growth phase. The cells were collected by centrifugation, washed three times with neutral phosphate buffer to remove the culture medium, and then resuspended in neutral phosphate buffer at a concentration of 2 × 10⁻⁶. 8 CFU / mL available;

[0064] 2. Preparation of probiotic microcapsules:

[0065] (1) Take 4.8 ml of the above bacterial suspension, add 7.2 mg of anhydrous calcium chloride to the bacterial suspension under vortex conditions to dissolve it completely, and then let it stand for 2 min.

[0066] (2) Under vortex conditions, add 200 μL of Eudragit S100 solution with a concentration of 3 mg / ml dropwise to the bacterial suspension after standing. After mixing thoroughly, adjust the pH of the solution to 5.0 and continue vortexing for 5 min.

[0067] (3) Add 5 ml of phosphate buffer solution with pH 5.0, mix thoroughly, let stand for 5 min, and then add 5000 ml of phosphate buffer solution.

[0068] Centrifuge at rpm for 3 min, discard the supernatant, and repeat this step twice more with phosphate buffer at pH 5.0.

[0069] 3. Freeze-drying of probiotic microcapsules:

[0070] The probiotic microcapsules collected after washing and centrifugation were pre-frozen at -80℃ for 3 hours, and then transferred to a vacuum freeze dryer and freeze-dried at -60℃ and 30Pa for 24 hours. After drying, the microcapsules were transferred to a -80℃ refrigerator, sealed and stored at low temperature until use.

[0071] Example 3

[0072] A single-cell nanoencapsulated Lactobacillus rhamnosus LGG microcapsule is prepared by the following steps:

[0073] 1. Preparation of probiotic suspension:

[0074] Activated Lactobacillus rhamnosus LGG was cultured to the late logarithmic growth phase. The cells were collected by centrifugation, washed three times with neutral phosphate buffer to remove the culture medium, and then resuspended in neutral phosphate buffer at a concentration of 2 × 10⁻⁶. 8 CFU / mL available;

[0075] 2. Preparation of probiotic microcapsules:

[0076] (1) Take 4.8 ml of the above bacterial suspension, and under vortex conditions, add 100 μL of epigallocatechin solution with a concentration of 20 mg / ml dropwise to the bacterial suspension, and then let it stand for 2 min.

[0077] (2) Under vortex conditions, add 100 μL of ferric chloride solution with a concentration of 5 mg / ml dropwise to the bacterial suspension after standing. After the addition is complete, continue vortexing for 3 min.

[0078] (3) Add 5 ml of phosphate buffer solution with pH 7.6, mix thoroughly, let stand for 5 min, and then add 5000 ml of phosphate buffer solution.

[0079] Centrifuge at rpm for 3 min, discard the supernatant, and repeat this step twice more with phosphate buffer at pH 7.6.

[0080] (4) The cleaned bacterial cells were resuspended in 4.8 ml of neutral phosphate buffer containing 1.5 mg / ml calcium chloride. 200 μL of Eudragit S100 solution with a concentration of 3 mg / ml was added under vortex conditions. After thorough mixing, the pH of the solution was adjusted to 5.0 and vortexed for 5 min.

[0081] (5) Add 5 ml of phosphate buffer solution with pH 5.0, mix thoroughly, let stand for 5 min, and then add 5000 ml of phosphate buffer solution.

[0082] Centrifuge at rpm for 3 min, discard the supernatant, and repeat this step twice more with phosphate buffer at pH 5.0.

[0083] 3. Freeze-drying of probiotic microcapsules:

[0084] The probiotic microcapsules collected after washing and centrifugation were pre-frozen at -80℃ for 3 hours, and then transferred to a vacuum freeze dryer and freeze-dried at -60℃ and 30Pa for 24 hours. After drying, the microcapsules were transferred to a -80℃ refrigerator, sealed and stored at low temperature until use.

[0085] Example 4

[0086] A single-cell nanoencapsulated Lactobacillus rhamnosus LGG microcapsule is prepared by the following steps:

[0087] 1. Preparation of probiotic suspension:

[0088] Activated Lactobacillus rhamnosus LGG was cultured to the late logarithmic growth phase. The cells were collected by centrifugation, washed three times with neutral phosphate buffer to remove the culture medium, and then resuspended in neutral phosphate buffer containing 5 mg / ml 2'-fucosylated lactose (2'-FL), adjusting the concentration to 2 × 10⁻⁶. 8 CFU / mL available;

[0089] 2. Preparation of probiotic microcapsules:

[0090] (1) Take 4.8 ml of the above bacterial suspension, and under vortex conditions, add 100 μL of epigallocatechin solution with a concentration of 20 mg / ml dropwise to the bacterial suspension, and then let it stand for 2 min.

[0091] (2) Under vortex conditions, add 100 μL of ferric chloride solution with a concentration of 5 mg / ml dropwise to the bacterial suspension after standing. After the addition is complete, continue vortexing for 3 min.

[0092] (3) Add 5 ml of phosphate buffer solution with pH 7.6, mix thoroughly, let stand for 5 min, and then add 5000 ml of phosphate buffer solution.

[0093] Centrifuge at rpm for 3 min, discard the supernatant, and repeat this step twice more with phosphate buffer at pH 7.6.

[0094] (4) The cleaned bacterial cells were resuspended in 4.8 ml of neutral phosphate buffer containing 1.5 mg / ml calcium chloride. 200 μL of Eudragit S100 solution with a concentration of 3 mg / ml was added under vortex conditions. After thorough mixing, the pH of the solution was adjusted to 5.0 and vortexed for 5 min.

[0095] (5) Add 5 ml of phosphate buffer solution with pH 5.0, mix thoroughly, let stand for 5 min, and then add 5000 ml of phosphate buffer solution.

[0096] Centrifuge at rpm for 3 min, discard the supernatant, and repeat this step twice more with phosphate buffer at pH 5.0.

[0097] 3. Freeze-drying of probiotic microcapsules:

[0098] The probiotic microcapsules collected after washing and centrifugation were pre-frozen at -80℃ for 3 hours, and then transferred to a vacuum freeze dryer and freeze-dried at -60℃ and 30Pa for 24 hours. After drying, the microcapsules were transferred to a -80℃ refrigerator, sealed and stored at low temperature until use.

[0099] Effect evaluation

[0100] 1. Transmission electron microscopy imaging

[0101] The Lactobacillus rhamnosus LGG bacterial suspension before and after single-cell nano-encapsulation in Example 4 was appropriately diluted in sterile deionized water, and after being vortexed and dispersed evenly, a drop of bacterial suspension was dropped onto a copper grid covered with a carbon film layer. After it was completely dried, the sample was imaged by a transmission electron microscope.

[0102] Figure 1 The images are transmission electron microscopy images of Lactobacillus rhamnosus LGG single-cell nano-encapsulation before and after Example 4.

[0103] Depend on Figure 1 As can be seen from the transmission electron microscope, the original LGG cell surface is smooth, flat and very clean. However, after single-cell nano-encapsulation, a rough nano-coating is formed on the surface of a single Lactobacillus rhamnosus LGG, indicating the successful preparation of a single-cell nano-encapsulated probiotic microcapsule.

[0104] 2. Average particle size measurement

[0105] The Lactobacillus rhamnosus LGG suspension before and after single-cell nano-encapsulation was appropriately diluted in sterile deionized water, vortexed and dispersed evenly, and then about 1 ml of the diluted solution was added to the sample cell of the Malvern nanoparticle size analyzer for particle size detection.

[0106] Table 1. Particle size changes of Lactobacillus rhamnosus LGG single-cell nano-encapsulation before and after.

[0107]

[0108] Figure 2 A bar chart showing the particle size change of Lactobacillus rhamnosus LGG before and after single-cell nano-encapsulation.

[0109] From Table 1, Figure 2It can be seen that the average particle size of Lactobacillus rhamnosus LGG after single-cell nano-encapsulation only increased by tens to hundreds of nanometers. The particle size of Example 1, which was only encapsulated with a metal-polyphenol coating, increased by about 70 nm. The particle size of Example 2, which was only encapsulated with an acrylic resin material, increased by about 100 nm. The particle size of Example 3, which was encapsulated with methacrylate copolymer in addition to the metal-polyphenol coating, increased by about 90 nm on the basis of Example 1. The particle size of Example 4, which added 2'-FL on the basis of Example 3, was similar to that of Example 3. This shows that the addition of 2'-FL does not significantly affect the particle size of the microcapsules.

[0110] 3. Freeze-drying test

[0111] Before and after wet single-cell nano-encapsulation, Lactobacillus rhamnosus LGG was pre-frozen at -80℃ for 3 hours, then transferred to a vacuum freeze dryer and freeze-dried at -60℃ and 30 Pa for 24 hours. After drying, 0.5 g of microcapsules were decapsulated, resuspended in PBS, and the viable count was detected by plate count method. The survival rate was calculated according to the following formula.

[0112] Uncapsulation procedure: Soak probiotic microcapsules in phosphate buffer solution with pH 7.2 and osmotic pressure of 300 Pa for 10 min, then centrifuge at 5000 rpm for 5 min, remove the supernatant, add 10 ml of 18 mM EDTA-2Na aqueous solution, vortex mix at 2000 rpm for 3 min, add 60 μL of Tween 80, sonicate at 35 kHz for 30 s, and finally repeat the washing process three times under the condition of centrifugation at 5000 rpm for 5 min. After removing the supernatant, the uncapsulated probiotic sludge can be obtained.

[0113] Plate colony counting: Take 1 mL of bacterial suspension and dilute it 10-fold sequentially to the appropriate dilution. Select 3 appropriate dilutions, take 1 mL of each dilution into an empty sterile petri dish, pour 15 mL of dissolved and cooled culture medium to about 45℃ into each dish, and immediately place them on the table to mix well. Make three petri dishes for each dilution. After solidification, invert the dishes and incubate them at 37℃ under anaerobic conditions for 48 hours. Then take them out and record the colony count.

[0114]

[0115] Table 2 Survival rates of Lactobacillus rhamnosus LGG lyophilized before and after single-cell nanoencapsulation

[0116] Group LGG Example 1 Example 2 Example 3 Example 4 Survival rate (%) 50.7±0.21 77.6±0.23 79.5±0.11 84.7±0.08 87.3±0.15

[0117] Figure 3 A bar chart showing the change in the number of *Lactobacillus rhamnosus* LGG freeze-dried before and after single-cell nano-encapsulation.

[0118] From Table 2, Figure 3 It can be seen that the stability of probiotics encapsulated in single cells during the freeze-drying process is significantly improved. The survival rate of Example 1, which only encapsulates a layer of metal-polyphenol coating, is about 27% higher than that of the original LGG after freeze-drying. The survival rate of Example 2, which only encapsulates a layer of acrylic resin material, is similar to that of Example 1 after freeze-drying. The survival rate of Example 3, which encapsulates methacrylate copolymer in addition to metal-polyphenol coating, is 34% higher than that of the original LGG after freeze-drying. The survival rate of Example 4, which adds 2'-FL to the basis of Example 3, is about 3% higher than that of Example 3 after freeze-drying. This shows that the addition of 2'-FL can improve the storage stability of LGG to a certain extent.

[0119] 4. Gastrointestinal digestion test

[0120] Simulated gastric digestion: Take 1 mL of Lactobacillus rhamnosus LGG suspension before and after single-cell nano-encapsulation (2×10⁻⁶). 8 The CFU / mL solution was fully dispersed in 9 mL of simulated gastric fluid (0.32% pepsin, pH 2.5), and incubated at 37°C and 150 rpm for 60 min in a shaker. 1 mL was then removed for decapsulation, resuspended in PBS, and the viable count was determined using the plate count method.

[0121] Simulated intestinal digestion: Take 1 mL of Lactobacillus rhamnosus LGG suspension before and after single-cell nano-encapsulation (2×10⁻⁶). 8 The CFU / mL solution was fully dispersed in 9 mL of simulated intestinal fluid (1% trypsin, 0.3% bile salts, pH 6.8), and incubated at 37°C and 150 rpm for 60 min in a shaker. 1 mL was then transferred to the solution for decapsulation, resuspended in PBS, and the viable count was determined using the plate count method.

[0122] Table 3. Changes in the number of *Lactobacillus rhamnosus* LGG after gastric digestion before and after single-cell nanoencapsulation.

[0123]

[0124] Figure 4 A bar chart showing the change in the number of Lactobacillus rhamnosus LGG cells after digestion with gastric juice before and after single-cell nano-encapsulation.

[0125] From Table 3, Figure 4It can be seen that the survival rate of probiotics after single-cell nano-encapsulation was significantly improved after simulated gastric digestion. Example 1, which was only encapsulated with a single layer of metal-polyphenol coating, had a survival rate 0.75 logs higher than the original LGG after gastric digestion. Example 2, which was only encapsulated with a single layer of acrylic resin material, showed a more impressive performance in gastric juice than Example 1. Example 3, which was encapsulated with methacrylate copolymer in addition to the metal-polyphenol coating, had a survival rate 0.32 logs higher than that of Example 2. Example 4, which added 2'-FL to Example 3, also showed a slight increase in survival rate compared to Example 3.

[0126] Table 4. Changes in the number of *Lactobacillus rhamnosus* LGG after intestinal digestion before and after single-cell nanoencapsulation.

[0127]

[0128] Figure 5 A bar chart showing the change in the number of Lactobacillus rhamnosus LGG cells after digestion with intestinal fluid before and after single-cell nano-encapsulation.

[0129] From Table 4, Figure 5 It can be seen that the survival rate of probiotics after single-cell nano-encapsulation was significantly improved after simulated intestinal digestion. Example 1, which only encapsulated a single layer of metal-polyphenol coating, had a survival rate 1.41 logs higher than the original LGG after intestinal digestion. Example 2, which only encapsulated a single layer of acrylic resin material, performed slightly worse than Comparative Example 1 in intestinal digestion. Example 3, which encapsulated a methacrylate copolymer in addition to the metal-polyphenol coating, had a survival rate 0.69 logs higher than that of Example 1. However, Example 4, which added 2'-FL to Example 3, did not show a significant change in survival rate compared to Example 3.

[0130] 5. Cell adhesion test

[0131] Take 500 μL 2×10 8 The Lactobacillus rhamnosus LGG suspension before and after single-cell nano-encapsulation was added to a Caco-2 cell culture plate and incubated at 37°C and 5% CO2 for 90 min. The cells were washed three times with PBS to remove unadhered probiotics. 150 μL of trypsin cell digestion solution was added. After the cells were completely detached, 350 μL of complete cell culture medium was added to stop the digestion. After vortexing and dispersing evenly, 1 mL was transferred out for decapsulation and resuspended in PBS. The viable cell count was detected by plate count method, and the adhesion rate was calculated according to the following formula.

[0132]

[0133] Table 5. Cell adhesion rate of Lactobacillus rhamnosus LGG before and after single-cell nanoencapsulation.

[0134] Group Cell adhesion rate (%) LGG 8.53±0.08 Example 1 29.21±0.15 Example 2 9.87±0.12 Example 3 12.46±0.24 Example 4 12.54±0.21

[0135] Table 5 shows that the cell adhesion rate of probiotics after single-cell nano-encapsulation increased. Since the catechol / gallic acid functional groups were directly exposed, Example 1, which only encapsulated a layer of metal-polyphenol coating, showed the largest increase in adhesion rate, about 21% higher than the original LGG. The adhesion rate of Example 2, which only encapsulated a layer of acrylic resin material, was much lower than that of Example 1, only about 1% higher. Although Example 3, which encapsulated a methacrylate copolymer in addition to the metal-polyphenol coating, showed an increase in adhesion rate compared to the original LGG, it was still much lower than that of Example 1, only about 4% higher. The adhesion rate of Example 4, which added 2'-FL to Example 3, did not change significantly compared to Example 3. However, during actual use, the methacrylate copolymer layer in Examples 3 and 4 will slowly decompose after entering the intestines from the stomach, exposing the catechol / gallic acid functional groups on the surface, thereby achieving an adhesion rate similar to that in Example 1. At the same time, it avoids excessive adhesion of probiotics in the upper digestive tract, preventing them from entering the intestines.

[0136] 6. Shelf life testing

[0137] Lactobacillus rhamnosus LGG bacteria before and after single-cell nano-encapsulation were stored in a sealed container at -20℃ in the dark for 56 days. Every 7 days, 0.5g of samples were taken for decapsulation, resuspended in PBS, and then the viable bacteria count in the samples was detected by plate count method.

[0138] Figure 6 A dotted line graph showing the change in the number of Lactobacillus rhamnosus LGG before and after single-cell nano-encapsulation during storage.

[0139] Depend on Figure 6 It can be seen that the rate of decline in the number of viable bacteria in probiotics after single-cell nano-encapsulation slowed significantly during storage. The number of viable bacteria in unencapsulated LGG began to decline rapidly in week 4, dropping to one-tenth of the initial number by week 8, while the number of surviving probiotics in Examples 1 to 4 showed an increasing trend. In particular, the number of surviving probiotics in Example 4, which added 2'-FL to Example 3, showed a significant increase compared to Example 3.

[0140] Based on all the charts and data, it is clear that the single-cell nano-encapsulated probiotic microcapsules mentioned in this invention, while imparting good stress resistance to probiotics, also possess extremely small particle size, making them easy to add to various foods without affecting their taste. Furthermore, the probiotic microcapsules of this invention can improve the cell adhesion rate of probiotics in the intestines, enabling them to colonize better. The addition of small-molecule oligosaccharides also enhances the stability of probiotics during processing and transportation.

[0141] The above description is only a partial embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A single-cell nanoencapsulated probiotic microcapsule, characterized in that, The probiotic microcapsules are composed of a wall material encapsulating a core material. The wall material comprises two layers: an outer wall material is a methacrylate copolymer, and an inner wall material is a metal-polyphenol network loaded with small-molecule oligosaccharides. The outer wall material is formed by the self-assembly of the methacrylate copolymer on the surface of the inner wall material induced by divalent metal ions. The core material is probiotics. The metal-polyphenol network of the inner wall material is formed by the complexation of transition metal ions with polyphenols containing catechol and / or gallic acid functional groups. The transition metal ion is Fe. 3+ The polyphenols include one or more of tea polyphenols, grape polyphenols, tannins, and apple polyphenols; the small molecule oligosaccharide is 2'-fucosylated lactose.

2. The single-cell nanoencapsulated probiotic microcapsule according to claim 1, characterized in that, The divalent metal ions include Ca. 2+ Mg 2+ Zn 2+ Fe 2+ One or more of the following: the methacrylate copolymer includes one or more of Eudragit L100-55, Eudragit L100, and Eudragit S100.

3. The single-cell nanoencapsulated probiotic microcapsule according to claim 1, characterized in that, The probiotics include one or more of the following: Lactobacillus probiotics, Bifidobacterium probiotics, Streptococcus probiotics, and Escherichia coli probiotics.

4. The single-cell nanoencapsulated probiotic microcapsule according to claim 3, characterized in that, The probiotics are one or more of the following: Lactobacillus rhamnosus LGG, Lactobacillus rhamnosus HN001, Lactobacillus fermentum CECT5716, Lactobacillus reuteri DSM17938, Lactobacillus acidophilus NCFM, Bifidobacterium breve M-16V, Bifidobacterium lactis HN019, Bifidobacterium lactis Bi-07, Bifidobacterium animalis Bb-12, and Escherichia coli Nissle1917.

5. A method for preparing single-cell nanoencapsulated probiotic microcapsules as described in any one of claims 1 to 4, characterized in that, Includes the following steps: 1) Preparation of probiotic suspension: The activated probiotics were cultured to the late logarithmic growth phase, centrifuged to collect the cells, washed, and resuspended in sterile physiological saline or neutral phosphate buffer containing small molecule oligosaccharides to obtain a bacterial suspension. 2) Preparation of probiotic microcapsules: (1) Take the bacterial suspension from step 1) above, add the polyphenol aqueous solution dropwise to the bacterial suspension under vortex conditions, and then let it stand for 2~5 min; (2) Under vortex conditions, add metal salt aqueous solution dropwise to the bacterial suspension after step (1) has been settling. After the addition is complete, continue vortexing for 3 to 8 minutes to allow for full reaction. (3) Add weakly alkaline phosphate buffer to the bacterial suspension after vortexing in step (2), mix thoroughly, let stand for 2-5 minutes, centrifuge, remove the supernatant, and continue to centrifuge and wash 2-3 times with weakly alkaline phosphate buffer. (4) The bacterial cells cleaned in step (3) are resuspended in a neutral phosphate buffer containing divalent metal salts, and an aqueous solution of methacrylate copolymer is added. After vortexing and mixing, the pH is adjusted to weak acidity and vortexed for 5-10 min. (5) Add weakly acidic phosphate buffer to the bacterial suspension after vortexing in step (4), mix thoroughly, let stand for 2-5 minutes, centrifuge, remove the supernatant, and repeat this step with weakly acidic phosphate buffer to continue centrifuging and washing 2-3 times. 3) Freeze-drying of probiotic microcapsules: The probiotic microcapsules collected after centrifugation and washing were pre-frozen in a freezer at -60~-100℃, and then transferred to a vacuum freeze dryer for freeze drying to obtain single-cell nano-encapsulated probiotic microcapsules.

6. The method for preparing a single-cell nanoencapsulated probiotic microcapsule according to claim 5, characterized in that, Step 1) The culture conditions are: temperature 30-40℃, pH 6-7, anaerobic environment; the washing involves washing 2-3 times with sterile physiological saline or neutral phosphate buffer to remove the culture medium; the concentration of the small molecule oligosaccharides in the sterile physiological saline or neutral phosphate buffer is 5-15 mg / ml; the concentration of the bacterial suspension is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

7. The method for preparing a single-cell nanoencapsulated probiotic microcapsule according to claim 5, characterized in that, The concentration of the polyphenol aqueous solution in step (1) is 10~30 mg / ml, and the volume ratio of the polyphenol aqueous solution to the bacterial suspension is 1:40~60; The concentration of the metal salt aqueous solution in step (2) is 4~10 mg / ml, and the volume ratio of the metal salt aqueous solution to the bacterial suspension is 1:40~80; The weakly alkaline phosphate buffer solution mentioned in step (3) has a pH of 7.4 to 8.0 and is added in an amount that is 1 to 2 times the volume of the bacterial suspension.

8. A method for preparing a single-cell nanoencapsulated probiotic microcapsule according to claim 5, characterized in that, In step (4), the concentration of the divalent metal salt in the neutral phosphate buffer containing the divalent metal salt is 1-6 mg / ml; the divalent metal salt is calcium chloride; and the concentration of the bacterial suspension after resuspending the bacteria in the neutral phosphate buffer containing the divalent metal salt is 1×10⁻⁶ mg / ml. 7 ~1×10 9 CFU / mL; the concentration of the aqueous solution of the methacrylate copolymer is 2~10 mg / mL, and the volume ratio of the aqueous solution of the methacrylate copolymer to the bacterial suspension is 1:20~30.

9. The method for preparing a single-cell nanoencapsulated probiotic microcapsule according to claim 5, characterized in that, The centrifugal cleaning described in steps (3) and (5) uses a centrifugation speed of 3000~9000 rpm and a centrifugation time of 2~8 min; The pH adjustment in step (4) is a weakly acidic pH range of 4.0 to 5.5; the weakly acidic phosphate buffer in step (5) has a pH range of 4.0 to 5.5, and the amount added is 1 to 2 times the volume of the bacterial suspension.

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