Single cell technology encapsulated probiotics and preparation method thereof
By encapsulating probiotics using single-cell technology and utilizing covalent binding and functional polysaccharide grafting, the limitations of existing probiotic encapsulation technologies have been overcome. This has enabled tolerance to harsh environments and functional release, thereby improving the intestinal colonization effect of probiotics.
Patent Information
- Application Number
- CN202510985896.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-17
- Publication Date
- 2025-10-28
AI Technical Summary
Current probiotic encapsulation technologies are easily limited by packaging materials and preparation processes, resulting in unstable release effects, difficulty in regulating intercellular interactions, and intolerance to environments such as gastric acid, bile salts, oxygen, and storage temperature.
Probiotics are encapsulated using single-cell technology, and functional polysaccharides are grafted onto the surface of the probiotics through a covalent binding strategy. Azobenzene molecules are used to endow the system with hypoxia and enzyme-responsive release mechanisms, thereby improving its resistance to harsh environments and functional properties.
It significantly enhances the probiotics' resistance to environments such as gastric acid, bile salts, ethanol, and high temperature, improves antioxidant activity, and achieves stimulus-responsive release and targeted colonization, thereby improving intestinal adhesion and colonization capabilities.
Smart Images
Figure CN120836748A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of probiotic microcapsule technology, and in particular to a single-cell technology for encapsulating probiotics and its preparation method. Background Technology
[0002] Probiotics are beneficial microorganisms that regulate gut microbiota, enhance immunity, and lower cholesterol, and are widely used in food, biology, and other fields. To ensure that probiotics can improve the host's gut health, it is essential to ensure that a sufficient number of live probiotics colonize the gut. However, probiotics are highly sensitive to pH, bile salts, oxygen, and storage temperature. To overcome the adverse effects of harsh in vitro and in vivo environments on probiotic survival, researchers have designed and constructed various encapsulation systems based on food-derived materials to protect and deliver probiotics.
[0003] Currently, most research focuses on microencapsulating probiotics using encapsulation technology, which involves packaging billions of probiotic individuals as a whole and releasing them at the same site. This traditional multi-cell encapsulation technology can improve the viability of probiotics in food and the human gut. However, this encapsulation technology is limited by packaging materials and manufacturing processes, and suffers from drawbacks such as unstable release effects and difficulty in controlling intercellular interactions. Summary of the Invention
[0004] The purpose of this invention is to provide a single-cell technology for encapsulating probiotics and its preparation method, thereby addressing the problems existing in the prior art. This invention innovatively improves upon existing probiotic delivery technologies, aiming to construct a novel delivery system with multiple functional advantages.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] One of the technical solutions of this invention is a method for preparing probiotics encapsulated using single-cell technology, comprising the following steps:
[0007] (1) Add 3-(4,5-dimethylthiazol-2-yl)propionaldehyde dropwise to an aqueous solution of 6-aminohexanoic acid and N-methylmorpholine, and stir overnight at room temperature to obtain an azobenzene compound;
[0008] (2) Dissolve the azobenzene compound with N,N'-dicyclohexylcarboimide and 4-dimethylaminopyridine in dimethyl sulfoxide and react at 25-45°C for 3-4 hours.
[0009] (3) Triethylamine was added to the β-glucan solution and then dropped into the reaction system of step (2). The mixture was stirred overnight at room temperature to obtain trans-Dex-AZO. Cis-Dex-AZO was purified by irradiation under 350 nm ultraviolet light for 30 min.
[0010] (4) Add EDC and NHS to the cis-Dex-AZO solution and react at room temperature for 1-2 h; then add Tris-HCl and continue the reaction for 30 min. After the reaction is complete, dialyze at 3500 Da to remove excess reagents; add probiotic suspension and stir at 37 °C for 2 h; centrifuge to remove unbound Dex-AZO, and freeze dry to obtain the single-cell encapsulated probiotics.
[0011] The second technical solution of the present invention is that the single-cell encapsulated probiotics prepared by the preparation method are obtained.
[0012] Based on the above technical solution, the present invention has the following technical effects:
[0013] 1. Enhanced protective efficiency. Employing a covalent binding strategy, it significantly improves resistance to harsh environments such as stomach acid, bile salts, ethanol, and high temperatures.
[0014] 2. Functional properties. Surface modification with polysaccharides possessing functional properties significantly enhances the antioxidant activity of the microcapsules.
[0015] 3. Stimulus-responsive release mechanism. By grafting azobenzene molecules onto the carrier backbone, the system acquires dual response characteristics: hypoxia targeting and enzyme responsiveness. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] Figure 1 This is the hydrogen NMR spectrum of DEX.
[0018] Figure 2 This is the hydrogen NMR spectrum of DEX-AZO.
[0019] Figure 3 The hypoxia response of DEX-AZO.
[0020] Figure 4 This refers to the enzyme responsiveness of DEX-AZO.
[0021] Figure 5 This is the growth curve.
[0022] Figure 6 This is a SEM image of the microcapsules.
[0023] Figure 7The image shows flow cytometry data of the microcapsules. In the diagram, a represents the unlabeled group, and b represents the dual-labeled group.
[0024] Figure 8 Microcapsules mimic gastrointestinal digestion.
[0025] Figure 9 This refers to the ethanol resistance of the microcapsules.
[0026] Figure 10 This refers to the heat resistance of the microcapsules.
[0027] Figure 11 The DPPH free radical scavenging rate of the microcapsules.
[0028] Figure 12 The free radical scavenging rate of ABTS microcapsules.
[0029] Figure 13 This represents the hypoxia-responsive release of the microcapsules. In the diagram, a is the UV spectrum, and b is the solution color change graph.
[0030] Figure 14 This represents the enzyme-responsive release from the microcapsules. In the diagram, a is the UV spectrum, and b is the solution color change graph. Detailed Implementation
[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.
[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0036] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0037] This invention provides a method for preparing probiotics encapsulated using single-cell technology, comprising the following steps:
[0038] (1) Add 3-(4,5-dimethylthiazol-2-yl)propionaldehyde dropwise to an aqueous solution of 6-aminohexanoic acid and N-methylmorpholine, and stir overnight at room temperature to obtain an azobenzene compound;
[0039] (2) Dissolve the azobenzene compound with N,N'-dicyclohexylcarboimide and 4-dimethylaminopyridine in dimethyl sulfoxide and react at 25-45°C for 3-4 hours.
[0040] (3) Triethylamine was added to the β-glucan solution and then dropped into the reaction system of step (2). The mixture was stirred overnight at room temperature to obtain trans-Dex-AZO. Cis-Dex-AZO was purified by irradiation under 350 nm ultraviolet light for 30 min.
[0041] (4) Add EDC and NHS to the cis-Dex-AZO solution and react at room temperature for 1-2 h; then add Tris-HCl and continue the reaction for 30 min. After the reaction is complete, dialyze at 3500 Da to remove excess reagents; add probiotic suspension and stir at 37 °C for 2 h; centrifuge to remove unbound Dex-AZO, and freeze dry to obtain the single-cell encapsulated probiotics.
[0042] In some specific embodiments, the ratio of 6-aminohexanoic acid, N-methylmorpholine and 3-(4,5-dimethylthiazol-2-yl)propionaldehyde is (0.4-0.6)g:(0.4-0.6)mL:(0.2-0.4)g.
[0043] In some specific embodiments, the mass ratio of the azobenzene compound, N,N'-dicyclohexylcarboimide, and 4-dimethylaminopyridine is 12:9:1.
[0044] In some specific implementations, the concentration of the cis-Dex-AZO solution is 0.1 mol / L; the mass ratio of the cis-EDC, NHS and Dex-AZO is 1:1:0.1.
[0045] In some specific implementations, the probiotics include Escherichia coli ECN·1917.
[0046] This invention also provides single-cell encapsulated probiotics prepared by the aforementioned method.
[0047] This invention provides a method based on single-cell encapsulation technology, which uses covalent bonding to directly graft functional polysaccharides onto the surface of probiotics, thereby improving the protective effect while enabling the probiotics to possess functional characteristics such as antioxidant activity, in vitro colonization adhesion, and responsive release.
[0048] (1) Grafting azobenzene-4,4-dicarboxylic acid onto β-glucan: 3-(4,5-dimethylthiazol-2-yl)propionaldehyde (ITPADA, 0.3 g) dissolved in N,N-dimethylformamide (DMF, 1 mL) was added dropwise to an aqueous solution of 6-aminohexanoic acid (0.51 g) and N-methylmorpholine (0.5 mL). The mixture was stirred overnight at room temperature. After the reaction was complete, DMF was removed under vacuum, and the residue was dissolved in ethyl acetate (150 mL). The residue was washed three times with double-distilled water and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under vacuum to obtain the azobenzene compound. The synthesized compound underwent a structural transformation from a trans to a cis configuration upon irradiation with ultraviolet light for 20 min.
[0049] (2) Synthesis of dextran-azobenzene compound: ① The Dex-AZO compound (12.776 mg) synthesized in (1) was dissolved in dimethyl sulfoxide (DMSO, 5.0 mL) with N,N'-dicyclohexylcarboimide (DCC, 9.55 mg) and 4-dimethylaminopyridine (DMAP, 1.885 mg). After sonication for 10 min, the solution was transferred to a reaction flask and reacted at 30 °C for 3 h. ② β-glucan (120 mg, 40 kDa) was dissolved in DMSO (5.0 mL), and then triethylamine (40 μL) was added. This solution was then added dropwise to the reaction flask in step ① and stirred overnight at room temperature to obtain the Dex-AZO(trans) compound. The compound was then irradiated under 350 nm ultraviolet light for 30 min to obtain the Dex-AZO(cis) compound. ③ After the reaction was completed, the reaction mixture was centrifuged, the supernatant was collected, and acetone (100 mL) was added to precipitate the product. The collected solid residue was vacuum dried, dissolved in water, and then dialyzed.
[0050] (3) Covalent cross-linking of carrier material with probiotics: The prepared AZO-DN compound was dissolved in PBS buffer (2 mL, 0.1 mol / L, pH = 7.4), and then EDC (26 mg) and NHS (23 mg) were added. The mixture was shaken and reacted at room temperature for 1-2 h to activate the carboxyl groups of the dextran side chain. Tris-HCl (1 mL, 0.1 mol / L, pH = 8.0) was then added and the reaction continued for 30 min. After the reaction was completed, excess reagents were removed using a dialysis bag (3500 Da). Next, a 10% concentration of ECN·1917 suspension was reacted with the activated Dex-AZO in PBS to achieve bioconjugation. The mixture was stirred at 37 °C for 2 h to allow amide coupling. Dex-AZO-ECN was separated from unbound Dex-AZO by centrifugation at 3500 rpm, and then washed three times with PBS. The sample was resuspended in PBS at pH 7.4. It was then lyophilized in a freeze dryer.
[0051] This invention proposes a single-cell encapsulation technology for probiotics. This method not only enhances the resilience of probiotics but also endows them with more functions, such as the ability to release substances in response to stimuli, target inflammatory tumor sites, and improve intestinal adhesion and colonization. "Single-cell encapsulation technology" involves modifying the inherent antigens and adhesion factors on the bacterial surface using various physicochemical or biotechnological techniques to perfectly combine functional materials with probiotics, thereby encapsulating a single probiotic cell. This single-cell encapsulation technology can provide more functions through the surface of a single probiotic than multi-cell encapsulation systems.
[0052] Example 1
[0053] 1. A method for preparing azobenzene-dextran (Dex-AZO) nanostructures, comprising the following steps:
[0054] Step 1: Synthesis of azobenzene compounds
[0055] To an aqueous solution of 6-aminohexanoic acid (0.51 g) and N-methylmorpholine (0.5 mL), 3-(4,5-dimethylthiazol-2-yl)propionaldehyde (ITPADA, 0.3 g) dissolved in N,N-dimethylformamide (DMF, 1 mL) was added dropwise. The resulting mixture was stirred overnight at room temperature. After the reaction was complete, DMF was removed under vacuum, and the residue was dissolved in ethyl acetate (150 mL). The organic phase was washed three times with double-distilled water and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under vacuum to give the azobenzene compound. The synthesized compound was irradiated with ultraviolet light for 20 min to induce a structural transformation from a trans to a cis configuration.
[0056] Step 2: Synthesis of dextran-azobenzene compounds
[0057] (1) The AZO compound synthesized in step 1 (12.776 mg) was dissolved with N,N'-dicyclohexylcarboimide (DCC, 9.55 mg) and 4-dimethylaminopyridine (DMAP, 1.885 mg) in dimethyl sulfoxide (DMSO, 5.0 mL). After sonication for 10 min, the solution was transferred to a reaction flask and reacted at 30 °C for 3 h.
[0058] (2) Dissolve β-glucan (120 mg, 40 kDa) in DMSO (5.0 mL), then add triethylamine (40 μL). This solution is then added dropwise to the reaction flask from step (1), and stirred overnight at room temperature to obtain the Dex-AZO (trans) compound. Irradiate the compound under 350 nm ultraviolet light for 30 min to obtain the Dex-AZO (cis) compound.
[0059] (3) After the reaction is complete, centrifuge the reaction mixture, collect the supernatant and add acetone (100 mL) to precipitate the product. Dry the collected solid residue under vacuum, dissolve it in water and dialyze it.
[0060] Step 3: Dialysis and lyophilization of dextran-azobenzene compounds
[0061] Cut the dialysis bag into 10cm segments. Boil the dialysis bag in 500mL of 2% (w / v) sodium bicarbonate and 1mmol / L EDTA (pH 8.0) for 10 minutes, then thoroughly rinse the dialysis bag with distilled water. Seal both ends of the dialysis bag with dialysis clamps, add 1L of deionized water, and place it on a thermostatic magnetic stirrer for dialysis for 2–3 days, changing the water intermittently during this period.
[0062] After dialysis, the product was rotary evaporated to remove excess water and organic solvent. The concentrated solution was transferred to an evaporating dish, pre-frozen at -80°C for 4 hours, and then freeze-dried to obtain an orange viscous substance (Dex-AZO).
[0063] Step 4: Incubation of probiotics
[0064] Escherichia coli ECN·1917 was inoculated into LB medium and cultured at 37°C with shaking at 200 rpm for 12 h until the optical density (λ = 600 nm) reached 0.4-0.5, indicating the growth had reached the logarithmic growth phase. Then, sterile physiological saline was added, and the culture medium was washed away by centrifugation twice at 4000 rpm for 10 min. Finally, ECN·1917 was resuspended in PBS buffer for subsequent experiments. At this point, the bacterial suspension was approximately 10... 9 CFU / mL.
[0065] Step 5: Covalent cross-linking of carrier material with probiotics
[0066] The prepared Dex-AZO compound was dissolved in PBS buffer (2 mL, 0.1 mol / L, pH 7.4), and then EDC (26 mg) and NHS (23 mg) were added. The mixture was shaken and reacted at room temperature for 1–2 h to activate the carboxyl groups of the dextran side chains. Tris-HCl (1 mL, 0.1 mol / L, pH 8.0) was then added, and the reaction continued for 30 min. After the reaction was complete, excess reagents were removed using a dialysis bag (3500 Da). Next, a 10% ECN·1917 suspension was reacted with the activated Dex-AZO in PBS to achieve bioconjugation. The mixture was stirred at 37 °C for 2 h to allow amide coupling. Dex-AZO-ECN was separated from unbound Dex-AZO by centrifugation at 3500 rpm, and the sample was washed three times with PBS. The sample was resuspended in PBS at pH 7.4 and then lyophilized.
[0067] Testing of 2 packets of sealed probiotics
[0068] The surface morphology of Dex-AZO-ECN was observed using cold field scanning electron microscopy (SEM). Before encapsulation, the ECN surface was smooth; after encapsulation, a sugar coating was observed on the surface of the microcapsules, indicating that DEX-AZO was successfully bound to the ECN surface. DEX-AZO was also labeled with the fluorescent marker FITC, and ECN was labeled with Rhodamine B, and the results were analyzed using flow cytometry. The results showed that cells in the dual-labeled group were mainly distributed in the Q6 region (Rhodamine B+ / FITC+), and the fluorescence intensity of the red and green superimposed cells was significantly higher than that of the control group without any fluorescent labeling. This further confirmed that the modified polysaccharide was successfully modified on the ECN surface.
[0069] A strong absorption peak appeared at 448 nm in DEX-AZO(cis)@ECN, which is the characteristic absorption peak of the azophenyl group. However, this characteristic absorption peak disappeared in the presence of 60 μm sodium dithionite (Na2S2O4), indicating that the azo group was cleaved. The color change of the DEX-AZO(cis)@ECN nanostructure in the oxygen deficiency response is shown in the figure. Figure 12 As shown in Figure b, the DEX-AZO(cis)@ECN solution is pale yellow, but the color of the solution becomes significantly lighter after mixing with Na2S2O4. These results indicate that DEX-AZO(cis)@ECN has reduction-mediated cleavage properties. In hypoxic sites such as inflammation or tumors, the azo bonds on its surface break, releasing probiotics. This allows for precise release of probiotics at the target site, thereby improving its bioavailability and efficacy.
[0070] To test the enzymatic responsiveness of DEX-AZO(cis)@ECN, it was co-incubated with a solution containing 1 mM NADH and 5 μg / mL azoreductase. The UV absorption spectrum is shown below. Figure 13 As shown in Figure a, the sharp drop in UV absorption peak at 458 nm indicates that the azo bond was successfully cleaved. Meanwhile, the strong absorption peak between 350 and 400 nm is mainly due to the presence of NADH. In the azo reductase-catalyzed reaction, NADH is required as an electron donor to provide hydrogen ions (H+). + ) and electrons (e - This causes the -N=N- group to break, thereby reducing the azo compound to an amine. Figure 13 The results further validated this conclusion. The DEX-AZO(cis)@ECN solution was initially orange-yellow, but gradually turned pale yellow in the presence of azoreductase and NADH. These results indicate that DEX-AZO(cis)@ECN has good enzymatic responsiveness and the potential to precisely control the release of probiotics from DEX-AZO(cis)@ECN at in vivo inflammatory tumor sites.
[0071] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for preparing probiotics encapsulated using single-cell technology, characterized in that, Includes the following steps: (1) Add 3-(4,5-dimethylthiazo-2-yl)propionaldehyde dropwise to an aqueous solution of 6-aminohexanoic acid and N-methylmorpholine, and stir overnight at room temperature to obtain an azobenzene compound; (2) Dissolve the azobenzene compound with N,N'-dicyclohexylcarboimide and 4-dimethylaminopyridine in dimethyl sulfoxide and react at 25-45°C for 3-4 hours. (3) Triethylamine was added to the β-glucan solution and then dropped into the reaction system of step (2). The mixture was stirred overnight at room temperature to obtain trans-Dex-AZO. Cis-Dex-AZO was purified by irradiation under 350 nm ultraviolet light for 30 min. (4) Add EDC and NHS to the cis-Dex-AZO solution and react at room temperature for 1-2 h; then add Tris-HCl and continue the reaction for 30 min. After the reaction is complete, dialyze at 3500 Da to remove excess reagents; add probiotic suspension and stir at 37 °C for 2 h. Unbound Dex-AZO is removed by centrifugation, and the resulting lyophilized product is the probiotic encapsulated using the single-cell technology.
2. The preparation method according to claim 1, characterized in that, The ratio of 6-aminohexanoic acid, N-methylmorpholine and 3-(4,5-dimethylthiazol-2-yl)propionaldehyde is (0.4-0.6)g:(0.4-0.6)mL:(0.2-0.4)g.
3. The preparation method according to claim 1, characterized in that, The mass ratio of the azobenzene compound, N,N'-dicyclohexylcarboimide, and 4-dimethylaminopyridine is 12:9:
1.
4. The preparation method according to claim 1, characterized in that, The concentration of the cis-Dex-AZO solution is 0.1 mol / L; the mass ratio of the cis-EDC, NHS and Dex-AZO is 1:1:0.
1.
5. The preparation method according to claim 1, characterized in that, The probiotics include Escherichia coli ECN·1917.
6. The single-cell encapsulated probiotics prepared by the preparation method according to any one of claims 1-5.