A silk fibroin-gelatin hydrogel material embedding bacterial strains and a preparation method and application thereof
By using genipin crosslinking technology of silk fibroin-gelatin hydrogel, an embedding system with both biocompatibility and mechanical strength was constructed, which solved the shortcomings of traditional materials, improved the survival rate and functional expression of microbial strains in the gastrointestinal tract, and is suitable for food, agriculture and environmental remediation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MBIOU
- Filing Date
- 2026-03-09
- Publication Date
- 2026-05-29
AI Technical Summary
Traditional embedding materials suffer from poor biocompatibility, insufficient mechanical strength, mismatched degradation rates, and potential to induce chronic inflammation during oral delivery of microbial strains, leading to decreased bacterial activity and limited functional expression.
Using silk fibroin-gelatin hydrogel as a composite embedding material, a dual network structure is formed through genipin crosslinking, providing biocompatibility, mechanical toughness and controllable degradation, to construct an embedding system that shields against gastric acid and allows for controlled release from the intestine.
It significantly improved the activity retention rate and stress resistance of the strain, enhanced functional expression, improved the survival rate and colonization efficiency of the bacteria in the gastrointestinal environment, and the degradation products of the material are non-toxic, making it suitable for food, agriculture and environmental remediation.
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Figure CN122103618A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a silk fibroin-gelatin hydrogel material with embedded bacterial strains, its preparation method, and its application. Background Technology
[0002] Microbial strains are widely used in agriculture, food, and environmental remediation, but in practical applications, they often face environmental stresses (such as temperature, pH, and salt concentration) leading to decreased activity, low survival rates, and limited functional expression. During oral delivery, microbial strains must continuously traverse the harsh "internal environment" of the gastrointestinal tract: a 30-second retention period in the oral cavity followed by a 2-hour retention period at a gastric acid pH of 1.5-2.0, emulsification by bile salts at 0.3-0.5%, pancreatic enzyme hydrolysis, and intestinal hypoxia and hyperosmolar fluctuations. The combined effect of these factors causes lipid peroxidation and protein denaturation in the free bacterial membrane, resulting in a decrease in viable bacterial count of 2-4 lg CFU / mL before reaching the target site. -1 The expression level of functional genes is reduced by more than 50%, and the ability to colonize and benefit plants is basically lost.
[0003] Encapsulation technology can construct a biocompatible barrier on the surface of bacteria, achieving gastric acid shielding, bile isolation, and controlled release, and is widely recognized as a core strategy for improving the intestinal survival rate of oral probiotics. Traditional encapsulation materials such as sodium alginate (SA) and polyvinyl alcohol (PVA) are widely used due to their ease of gelation and low cost, but their performance defects remain significant: SA gels easily swell and experience a sharp drop in mechanical strength after high salt levels or repeated fermentation in the intestine, even exhibiting a "melting bead" phenomenon, leading to sudden bacterial release; while PVA, although highly resilient, requires repeated freeze-thaw cycles or saturated boric acid treatment, and residual borate causes lipid peroxidation of the cell membrane, inhibiting bacterial activity. Furthermore, both are chemically inert, and their degradation rates are not synchronized with the needs of bacterial colonization, potentially leading to chronic inflammation with long-term retention. Therefore, constructing novel encapsulation carriers that combine excellent biocompatibility, controllable degradation, and high mechanical toughness has become a key breakthrough for improving the survival rate and colonization efficacy of oral probiotics. Summary of the Invention
[0004] In view of this, the purpose of this invention is to provide the application of silk fibroin-gelatin hydrogel in embedding bacterial strains, as well as a silk fibroin-gelatin hydrogel material for embedding bacterial strains, its preparation method, and its application. This invention uses a composite hydrogel embedding system with silk fibroin-gelatin as the matrix, which, after embedding bacterial strains, can significantly enhance the activity, stress resistance, and functional expression of the strains, solving the problems of poor biocompatibility and rapid decline in bacterial activity associated with traditional embedding materials.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of silk fibroin-gelatin hydrogel in embedding bacterial strains; The silk fibroin-gelatin hydrogel is obtained by cross-linking gelatin solution and silk fibroin solution as matrices through genipin.
[0006] Preferably, the strain includes one or more of lactic acid bacteria, yeast, and nitrogen-fixing bacteria.
[0007] Preferably, the preparation method of the silk fibroin-gelatin hydrogel includes the following steps: Gelatin solution, genipin solution and silk fibroin solution were mixed and cross-linked to obtain silk fibroin-gelatin hydrogel.
[0008] Preferably, the mass concentration of the gelatin solution is 2-8%, the mass concentration of the silk fibroin solution is 2-6%, and the mass concentration of the genipin solution is 0.01-1%. The mass ratio of the gelatin solution, genipin solution and silk fibroin solution is (2~8):(0.1~2):(8~2). The cross-linking reaction is carried out at a temperature of 35-38 °C for 4-6 h.
[0009] The present invention provides a silk fibroin-gelatin hydrogel material for embedding bacterial strains, comprising silk fibroin-gelatin hydrogel and bacterial strains embedded in the silk fibroin-gelatin composite hydrogel. The silk fibroin-gelatin hydrogel is obtained by cross-linking gelatin solution and silk fibroin solution as matrices through genipin.
[0010] Preferably, the strain includes one or more of lactic acid bacteria, yeast, and nitrogen-fixing bacteria; The encapsulation amount of the bacterial strain in the silk fibroin-gelatin hydrogel is ≥5×10⁻⁶. 8 CFU g -1 ; The silk fibroin-gelatin hydrogel material used to embed the bacterial strain is in the form of granules, microspheres, or lyophilized agents.
[0011] This invention provides a method for preparing the silk fibroin-gelatin hydrogel material with the above-mentioned embedded strain, comprising the following steps: (1) Mix gelatin solution, genipin solution and silk fibroin solution to carry out cross-linking reaction to obtain silk fibroin-gelatin hydrogel; (2) The bacterial suspension is mixed with the silk fibroin-gelatin hydrogel to obtain the silk fibroin-gelatin hydrogel material with the encapsulated strain.
[0012] Preferably, the crosslinking reaction is carried out at a temperature of 35-38 °C for 4-6 h.
[0013] Preferably, when the silk fibroin-gelatin hydrogel material for embedding the bacterial strain is a microsphere, it further includes: Under shear conditions, the silk fibroin-gelatin hydrogel material of the embedded strain was dropped into an oil phase containing a surfactant to obtain an emulsion. The emulsion was physically cross-linked under ice-water bath conditions to obtain an emulsion containing microspheres. After washing and freeze-drying, silk fibroin-gelatin hydrogel microspheres were obtained.
[0014] This invention provides the application of the silk fibroin-gelatin hydrogel material containing the above-mentioned embedded strains in the fields of food, agriculture, environmental remediation, or pharmaceutical preparation.
[0015] This invention provides the application of silk fibroin-gelatin hydrogel in embedding bacterial strains; the silk fibroin-gelatin hydrogel is obtained by cross-linking gelatin solution and silk fibroin solution as matrices through genipin. This invention uses silk fibroin (SF) and gelatin as embedding raw materials. Silk fibroin is derived from degummed silkworm silk and is a highly crystalline block copolymer composed of heavy chains (≈390 kDa) and light chains (≈26 kDa) linked by disulfide bonds. Its sequence is rich in Gly-Ala-Gly-Ala repeating units, which can self-assemble under mild conditions to form two reversible conformations: β-sheet crystals (Silk II) and random coils (Silk I). By adjusting temperature, pH, or shear force, the proportion of secondary structures can be precisely controlled, thereby endowing the material with an adjustable elastic modulus of 50~200 MPa, a controllable degradation rate (weeks to months), and extremely low immunogenicity. It has been approved by the FDA for in vivo implantation. Gelatin is a triple-helical fragment of collagen after partial hydrolysis, containing >90% amino acid residues. The Gly-Pro-Hyp repeat sequence provides Arg-Gly-Asp (RGD) cell adhesion sites, and it undergoes a reversible sol-gel phase transition around 37 °C, with degradation products being amino acids that can be directly utilized by cells. When gelatin is cross-linked with silk fibroin, it forms a "double-network" hydrogel. The β-sheet domain of silk fibroin provides a rigid framework, while the gelatin helical chains provide elastic and cell-binding sites. Both possess high mechanical toughness and excellent cell affinity, making them ideal carriers for encapsulating probiotics or therapeutic strains. This invention crosslinks gelatin and silk fibroin with genipin. The β-sheet network of silk fibroin provides a dense barrier, significantly reducing gastric acid permeation. After reacting with the primary amino group of gelatin or silk fibroin, genipin generates a new intermediate with a "nitrogen-carbon double bond" similar to a Schiff base, which then cyclizes into a stable pyridine crosslinking point. This Schiff base crosslinking structure can be broken at pH 7.4 in the small intestine, achieving an intelligent "acid protection-intestinal release" response.
[0016] This invention provides a silk fibroin-gelatin hydrogel material for embedding bacterial strains, comprising a silk fibroin-gelatin hydrogel and bacterial strains embedded in the silk fibroin-gelatin composite hydrogel. The results of the embodiments show that the silk fibroin-gelatin hydrogel material for embedding bacterial strains of this invention has the following advantages: ① Improved activity retention rate: After storage at 4 ℃ for 30 days, the activity retention rate of the encapsulated strain was ≥85%, which was significantly higher than that of the unencapsulated group (<50%).
[0017] ② Enhanced stress resistance: Under stress conditions such as pH 2.0, 0.3% bile salts, and 50 ℃ heat shock, the survival rate of the embedded strains is over 80%.
[0018] ③ Enhanced functional expression: Encapsulated lactic acid bacteria have a 20-40% higher acid production capacity, and encapsulated yeast has a 15-30% higher fermentation rate. Simulating gastric juice and enteritis environments allows for the dual advantages of gastric survival, rapid intestinal colonization, and acid production.
[0019] ④ Good reusability: The encapsulated bacterial cells can be reused 3-5 times with an activity decrease of <20%.
[0020] ⑤ High biocompatibility: The hydrogel degradation products are non-toxic and suitable for use in food, agriculture, environmental remediation, or medicine.
[0021] This invention provides a method for preparing silk fibroin-gelatin hydrogel material with the above-mentioned embedded strain. This method is simple to operate, low in cost, and easy to realize for continuous industrial production. Attached Figure Description
[0022] Figure 1 Survival rate of SF-Gel-encapsulated Lactococcus lactis microspheres in simulated gastric juice and simulated enteritis environments; Figure 2 The activity retention rate of SF-Gel-encapsulated Lactococcus lactis microspheres and free Lactococcus lactis at different time points under 4℃ conditions; Figure 3 Survival rate of SF-Gel-encapsulated Saccharomyces cerevisiae microspheres in simulated gastric juice and simulated enteritis environments. Detailed Implementation
[0023] This invention provides the application of silk fibroin-gelatin hydrogel in embedding bacterial strains; The silk fibroin-gelatin hydrogel is obtained by cross-linking gelatin solution and silk fibroin solution as matrices through genipin.
[0024] In this invention, the strain includes one or more of lactic acid bacteria, yeast, and nitrogen-fixing bacteria. Preferably, the lactic acid bacteria are *Lactococcus lactis*, and the yeast is preferably *Saccharomyces cerevisiae*. Preferably, the strain is commercially available.
[0025] In this invention, the preparation method of the silk fibroin-gelatin hydrogel includes the following steps: Gelatin solution, genipin solution and silk fibroin solution were mixed and cross-linked to obtain silk fibroin-gelatin hydrogel.
[0026] In this invention, the method for preparing the silk fibroin preferably includes the following steps: After the silkworm cocoons are cut into pieces, they are mixed with sodium carbonate solution and boiled. The mixture is then washed and dried to obtain silk fibroin fibers. The silk fibroin fibers were dissolved in a ternary solution, and the solution was obtained by centrifugation, filtration and dialysis.
[0027] In this invention, the mass concentration of the sodium carbonate solution is preferably 0.5%; the number of boiling times is preferably 2; the washing is preferably done with deionized water; and the drying temperature is preferably 45°C.
[0028] In this invention, the ternary solution is preferably a mixture of CaCl2, anhydrous ethanol, and deionized water, and the molar ratio of CaCl2, anhydrous ethanol, and deionized water is preferably 1:2:8. In this invention, the dissolution method is preferably heating and stirring, the centrifugation rate is preferably 4000 r / min, and the time is preferably 10 min. In this invention, the molecular weight cutoff for dialysis is preferably M. W =8000 kDa, and the dialysis time is preferably 48 h. After dialysis, a silk fibroin solution with a mass concentration of 2% is preferably obtained. Preferably, the 2% silk fibroin solution is concentrated, and the concentration method is preferably: transferring the 2% silk fibroin solution to M... W Reverse osmosis is performed in a dialysis bag with a molecular weight of 8000 kDa in a PEG solution. In this invention, the molecular weight of the PEG in the PEG solution is preferably 20000, the mass concentration of the PEG solution is preferably 20%, and the reverse osmosis time is preferably 24 h.
[0029] In this invention, the mass concentration of the gelatin solution is preferably 2-8%, more preferably 5%; the mass concentration of the silk fibroin solution is preferably 2-6%, more preferably 5%; and the mass concentration of the genipin solution is preferably 1%. In this invention, the mass ratio of the gelatin solution, genipin solution, and silk fibroin solution is preferably (2-8):(0.1-2):(8-2), more preferably 4-6:(0.5-1.5):6-4. In this invention, the mixing method is preferably: first, the gelatin solution and genipin solution are vortexed, and then the silk fibroin solution is added for a second vortex mixing. In this invention, the first vortex mixing rate is preferably 1500-2000 rpm, and the time is preferably 1-3 min; the second vortex mixing rate is preferably 1000-1500 rpm, and the time is preferably 2-4 min.
[0030] In this invention, the temperature of the crosslinking reaction is preferably 35~38 °C, more preferably 37 °C; the time is preferably 4~6 h, more preferably 5 h. By controlling the temperature and time of the crosslinking reaction, this invention can regulate the β-sheet crystallinity and the Schiff base crosslinking density.
[0031] The present invention provides a silk fibroin-gelatin hydrogel material for embedding bacterial strains, comprising silk fibroin-gelatin hydrogel and bacterial strains embedded in the silk fibroin-gelatin composite hydrogel. The silk fibroin-gelatin hydrogel is obtained by cross-linking gelatin solution and silk fibroin solution as matrices through genipin.
[0032] In this invention, the bacterial strains include one or more of lactic acid bacteria, yeast, and nitrogen-fixing bacteria; the encapsulation amount of the bacterial strains in the silk fibroin-gelatin hydrogel is preferably ≥5×10⁻⁶. 8 CFU g -1 .
[0033] In this invention, the silk fibroin-gelatin hydrogel material for embedding the bacterial strain is preferably in the form of granules, microspheres, or lyophilized agents. When microspheres are used, the particle size of the silk fibroin-gelatin hydrogel for embedding the bacterial strain is preferably 50-150 μm, more preferably 80-120 μm, and even more preferably 100 μm.
[0034] This invention provides a method for preparing the silk fibroin-gelatin hydrogel material with the above-mentioned embedded strain, comprising the following steps: (1) Mix gelatin solution, genipin solution and silk fibroin solution to carry out cross-linking reaction to obtain silk fibroin-gelatin hydrogel; (2) The bacterial suspension is mixed with the silk fibroin-gelatin hydrogel to obtain the silk fibroin-gelatin hydrogel material with the encapsulated strain.
[0035] This invention involves mixing a gelatin solution, a genipin solution, and a silk fibroin solution to perform a cross-linking reaction, thereby obtaining a silk fibroin-gelatin hydrogel. In this invention, the mass concentration of the gelatin solution is preferably 2-8%, more preferably 5%; the mass concentration of the silk fibroin solution is preferably 2-6%, more preferably 5%; and the mass concentration of the genipin solution is preferably 1%. In this invention, the mass ratio of the gelatin solution, genipin solution, and silk fibroin solution is preferably (2-8):(0.1-2):(8-2), more preferably 4-6:(0.5-1.5):6-4. In this invention, the mixing method is preferably as follows: first, the gelatin solution and the genipin solution are vortexed, and then the silk fibroin solution is added for a second vortex mixing. In this invention, the first vortex mixing rate is preferably 1500-2000 rpm, and the time is preferably 1-3 min; the second vortex mixing rate is preferably 1000-1500 rpm, and the time is preferably 2-4 min.
[0036] In this invention, the temperature of the crosslinking reaction is preferably 35~38 °C, more preferably 37 °C; the time is preferably 4~6 h, more preferably 5 h. By controlling the temperature and time of the crosslinking reaction, this invention can regulate the β-sheet crystallinity and the Schiff base crosslinking density.
[0037] After obtaining the silk fibroin-gelatin hydrogel, the present invention mixes the bacterial suspension with the silk fibroin-gelatin hydrogel to obtain a silk fibroin-gelatin hydrogel material with embedded bacterial strains. In the present invention, the concentration of the bacterial suspension is preferably 1×10⁻⁶. 9 ~1×10 11 CFU mL -1 The method for preparing the bacterial suspension preferably includes the following steps: The target strain was cultured to the logarithmic growth phase, and the bacterial cells were collected by centrifugation. The bacterial cells were then resuspended in sterile PBS solution or a protective agent solution to obtain a bacterial suspension.
[0038] In this invention, the volume ratio of the bacterial suspension to the hydrogel is preferably 1:5 to 15, more preferably 1:5 to 10.
[0039] In this invention, when the silk fibroin-gelatin hydrogel material for embedding the bacterial strain is a microsphere, it further includes: Under shear conditions, the silk fibroin-gelatin hydrogel material of the embedded strain was dropped into an oil phase containing a surfactant to obtain an emulsion. The emulsion was physically cross-linked under ice-water bath conditions to obtain an emulsion containing microspheres; after washing and freeze-drying, silk fibroin-gelatin hydrogel microspheres were obtained.
[0040] This invention involves adding the bacterial-hydrogel phase to an oil phase containing a surfactant under shear conditions to obtain an emulsion. In this invention, the surfactant includes one or more of Span-80, Span-60, Span-20, Span-85, lecithin, sucrose ester SE-5, and ethyl cellulose; the oil solvent of the oil phase preferably includes one or more of soybean oil, corn oil, liquid paraffin, and ethyl oleate; the mass concentration of the surfactant in the oil phase is preferably 0.2-2%, more preferably 0.5-1.5%. In this invention, the shear rate is preferably 8000-15000 rpm, more preferably 12000 rpm; the temperature is preferably 45-65℃, more preferably 55℃; after adding the oil phase, the shearing is preferably continued for 3-8 minutes, more preferably 5 minutes.
[0041] After obtaining the emulsion, the present invention performs physical cross-linking of the emulsion under ice-water bath conditions to obtain an emulsion containing microspheres. In the present invention, the physical cross-linking is preferably carried out under stirring conditions, the stirring rate is preferably 200~800 rpm, more preferably 500 rpm; the physical cross-linking time is preferably 20~60 min, more preferably 30~40 min. The present invention uses the ice-water bath conditions to cool and physically cross-link the inner phase droplets into spheres, which are then washed and freeze-dried to obtain silk fibroin-gelatin hydrogel microspheres.
[0042] In this invention, the washing reagent used is preferably a 0.9% NaCl solution, the washing temperature is preferably 4 ℃, and the washing is preferably performed 3 times. Through the washing process, this invention thoroughly removes the oil phase and free genipin to obtain clean microspheres.
[0043] In this invention, the freeze-drying method is preferably as follows: the washed microspheres are mixed with a 10% mannitol solution and subjected to gradient freeze-drying; the mass ratio of the microspheres to the 10% mannitol solution is preferably 1:2.
[0044] In this invention, the pressure of the gradient freeze-drying is preferably 0.1 mbar, and the drying process of the gradient freeze-drying preferably includes: Keep at -40 ℃ for 24 hours; The temperature is raised from -40 ℃ to -20 ℃ at a rate of 5 ℃ / h. Keep at -20 ℃ for 6 hours; The temperature is increased from -20 ℃ to 20 ℃ at a rate of 5 ℃ / h. Keep at 20 ℃ for 4 h.
[0045] This invention provides applications of the above-mentioned silk fibroin-gelatin hydrogel material in the fields of food, agriculture, environmental remediation, or medicine.
[0046] In this invention, when used in the pharmaceutical field, the silk fibroin-gelatin hydrogel material is preferably used to prepare oral probiotic agents.
[0047] The following detailed description, in conjunction with embodiments, illustrates the silk fibroin-gelatin hydrogel material for embedding strains provided by the present invention, its preparation method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.
[0048] In the following examples, *Lactococcus lactis* (accession number: CCTCC CB 20081699) was obtained from the China Center for Type Culture Collection; *Saccharomyces cerevisiae* (accession number: CCTCC KY 2008613) was obtained from the China Center for Type Culture Collection.
[0049] Example 1: Lactococcus lactis-silk fibroin-gelatin hydrogel embedding and its evaluation in a simulated gastric juice-enteritis cascade environment. The preparation of silk fibroin-gelatin hydrogels involves the following steps: (1) The silkworm cocoons were cut into pieces and boiled twice in a 0.5 wt% sodium carbonate solution. After washing with deionized water, they were dried in a 45 ℃ constant temperature drying oven. Subsequently, CaCl2, anhydrous ethanol, and deionized water were mixed in a molar ratio of 1:2:8, stirred, and filtered to obtain a ternary solution. The silk fibroin fibers were placed in the ternary solution, heated and stirred until the silk fibroin fibers were completely dissolved, centrifuged at 4000 r / min for 10 min, and filtered. This solution was then transferred to a dialysis bag (M W Dialyze the solution in a dialysis bag (8000 kDa) for 48 h to remove impurities, obtaining a 2 wt% silk fibroin solution. Transfer the 2 wt% silk fibroin solution to a dialysis bag (M... W =8000kDa) was subjected to reverse osmosis for 24 h in a 20wt% PEG 20000 environment to finally obtain a 5wt% silk fibroin solution.
[0050] (2) Weigh a certain amount of gelatin particles and place them in deionized water. Heat and stir until the gelatin particles are completely dissolved to obtain a 5 wt% gelatin solution. At the same time, use anhydrous ethanol to sonicate and dissolve genipin to obtain a 1 wt% genipin solution. Add genipin solution dropwise to the gelatin solution and vortex mix for 2 min. Then add the silk fibroin solution along with the gelatin solution and vortex mix for 3 min. The mass ratio of gelatin solution, genipin solution and silk fibroin solution is 5:0.5:5. Seal the mixed solution and incubate it in a 37 ℃ water bath for 4 h to obtain a silk fibroin-gelatin hydrogel, denoted as SG-Gel.
[0051] (3) Lactococcus lactis was cultured statically at 37 ℃ for 16 h on MRS medium, and the bacterial cells were collected by centrifugation. The cells were then centrifuged at 4 ℃ and 5000×g for 10 min. After discarding the supernatant, the cells were resuspended in a cryoprotectant containing 10% skim milk, 5% trehalose, and 0.5% L-cysteine, and the bacterial concentration was adjusted to 1×10⁻⁶. 11 CFU mL -1 .
[0052] (4) The bacterial suspension and the above SF-Gel were quickly mixed at a volume ratio of 1:5 and immediately transferred to a W / O emulsification device. 0.5wt% Span-80 was added to soybean oil in advance and the temperature was raised to 55 °C. Under high-speed shearing conditions of 12000 rpm, the bacterial-hydrogel phase was slowly dripped into the oil phase using a peristaltic pump. After the dripping was completed, shearing was continued for 5 min to form a uniform emulsion. Then the whole thing was transferred to an ice-water bath and stirred at low speed of 500 rpm for 30 min to cool the inner phase droplets and physically cross-link them into spheres to obtain microspheres with a particle size of 50~150 μm.
[0053] (5) The microspheres were washed three times by centrifugation with 0.9% NaCl at 4 ℃ to completely remove the oil phase and free genipin, resulting in clean microspheres. The microspheres were mixed with 10wt% mannitol solution at a mass ratio of 1:2, dispensed into 3 mL vials, pre-frozen at -40 ℃ for 12 h, and then placed in a vacuum freeze dryer and dried at 0.1 mbar at -40 ℃ for 24 h; the temperature was programmed to increase from -40 ℃ to -20 ℃ (5 ℃ h). -1 → -20 ℃ for 6 h → 20 ℃ (10 ℃ h) -1 Maintain for 4 hours, until the final residual water content is ≤3%, then seal with a pressure cap to obtain a concentration of 1.67 × 10⁻⁶. 10 CFU g -1 SF-Gel-encapsulated Lactococcus lactis microspheres are instant-dissolving microsphere powders.
[0054] Test Example 1 ① Simulated gastric juice (SGF) environment: 0.1 g of SF-Gel-encapsulated Lactococcus lactis microspheres were placed in 50 mL of pH 2.0 solution containing 3.2 g L... -1 In a USP-simulated gastric fluid, pepsin was shaken at 37 °C and 100 rpm for 2 h; the reaction was immediately terminated by an ice bath, and MRS pour counts were performed.
[0055] ② Simulated enteritis environment (neutral pH + MMP): The microspheres treated with SGF were directly transferred into an environment containing pH 7.4, 5% bile salts, 100 μM H2O2, and 10 μg / mL [unclear - possibly a specific chemical environment]. -1 IL-1β and 20 ng mL -1A DMEM enteritis model of TNF-α was established and anaerobically cultured at 37 ℃ and 5% CO2 for 6 h; samples were taken, counted, and MMP-9 activity was detected.
[0056] ③ Acid production capacity test: Take 0.1 g of SF-Gel-encapsulated Lactococcus lactis microspheres, rewarm at 37℃ for 5 min, add 10 mL of pre-warmed skim milk, vortex resuspend to obtain the test solution; incubate anaerobically at 42℃, and aseptically take 1 mL samples at 0, 2, 4, 6, and 8 h for pH measurement. Use a pH meter to measure, ΔpH = pH0 - pHt, and then calculate the acid production efficiency.
[0057] ④ Bile salt-heat shock tolerance test: Dissolve 0.1 g microspheres in 10 mL PBS (pH 7.0) and vortex to disperse, preparing a bacterial suspension; first add 0.3% (w / v) porcine bile salts, incubate at 37 ℃ for 30 min, then transfer to a 50 ℃ water bath for heat shock for 15 min, and immediately cool with ice water. Serially dilute the treated solution and pour into MRS, anaerobically incubate at 37 ℃ for 48 h, and count the microbes according to (N... t The survival rate is calculated as ( / N0) × 100%.
[0058] ⑤ Take 10g each of SF-Gel-encapsulated Lactococcus lactis microspheres and free Lactococcus lactis suspension. 10 CFU was stored in PBS at 4 ℃, and samples were taken and counted at weeks 0, 1, 2, 3, 4, and 5.
[0059] Evaluation results of silk fibroin-gelatin hydrogel encapsulation properties: The average particle size of the SF-Gel-encapsulated Lactococcus lactis microspheres was 82 μm, and the compressive modulus was 1.1 MPa. The survival rate of the SF-Gel-encapsulated Lactococcus lactis microspheres under simulated gastric juice and simulated enteritis environments was as follows: Figure 1 As shown, after 2 hours of treatment with simulated gastric juice (SGF) (pH 2.0, pepsin 0.3%), the survival rate was (80±3)%; after 6 hours of treatment with simulated enteritis environment (neutral pH + MMP), the survival rate was (63±3)%. Acid production capacity testing showed that the acid production capacity of encapsulated *Lactococcus lactis* increased by 28%, and the survival rate of encapsulated *Lactococcus lactis* under bile salt-heat shock combined stress was ≥82%, significantly higher than the ≤20% of free bacteria. The activity retention rates of SF-Gel-encapsulated *Lactococcus lactis* microspheres and free *Lactococcus lactis* at different times under 4℃ conditions are shown in the figure. Figure 2 As shown, under 4℃ conditions, the survival rate of live bacteria in the embedded group increased from 100% at week 0 to ≥60% at week 5, while the survival rate of live bacteria in the free group decreased to less than 13% during the same period. The embedded bacteria can be reused 3-5 times with a decrease in activity of <20%. They can be directly filled into hard capsules or tablets for oral probiotic preparations.
[0060] Example 2: Encapsulation of *Saccharomyces cerevisiae*-silk fibroin-gelatin in a hydrogel and its evaluation in a simulated gastric juice-enteritis cascade environment. (1) Prepare silk fibroin-gelatin hydrogel according to steps (1) and (2) of Example 1.
[0061] (2) Yeast activation and pre-culture: Lyophilized Saccharomyces cerevisiae was streaked onto YPD agar (2% glucose, 2% peptone, 1% yeast extract, pH 6.0) and incubated at 30 ℃ for 24 h; a single colony was picked and inoculated into 50 mL of YPD liquid, and shaken at 30 ℃ and 200 rpm for 16 h until the mid-log phase (OD) was reached. 600 ≈1.2). Collect bacterial cells by centrifugation at 4 ℃ and 3000×g for 5 min, wash twice with sterile PBS (pH 7.0), resuspend in cryoprotectant containing 5% trehalose + 2% glycerol, and adjust the concentration to 5×10. 9 CFU mL -1 Prepare for ice water bath.
[0062] (3) The bacterial suspension and the above SF-Gel were quickly mixed at a volume ratio of 1:5 and immediately transferred to a W / O emulsification device: 0.5wt% Span-80 was added to soybean oil in advance and the temperature was raised to 55 ℃. Under high-speed shearing conditions of 12000 rpm, the bacterial-hydrogel phase was slowly dripped into the oil phase using a peristaltic pump. After the dripping was completed, shearing was continued for 5 min to form a uniform emulsion. Then the whole thing was transferred to an ice water bath and stirred at low speed of 500 rpm for 30 min to cool the inner phase droplets and physically cross-link them into spheres to obtain microspheres with a particle size of 50~150 μm.
[0063] (4) The microspheres were washed three times by centrifugation with 0.9% NaCl at 4 ℃ to completely remove the oil phase and free genipin, resulting in clean microspheres. The microspheres were mixed with 10wt% mannitol solution at a mass ratio of 1:2, dispensed into 3 mL vials, pre-frozen at -40 ℃ for 12 h, and then placed in a vacuum freeze dryer and dried at 0.1 mbar at -40 ℃ for 24 h; the temperature program was: -40 ℃ → -20 ℃ (5 ℃ h). -1 → -20 ℃ for 6 h → 20 ℃ (10 ℃ h) -1 Keep for 4 hours, with a final residual water content ≤3%, then seal with a pressure cap to obtain a viable bacterial count of 8.33 × 10⁻⁶. 8 CFU g -1 SF-Gel-encapsulated Saccharomyces cerevisiae microspheres are produced as fast-dissolving microsphere powders.
[0064] Test Example 2 ① Simulate gastric juice environment: NaCl 2 g L -1 3.2 g L of pepsin -1The pH was adjusted to 2.0 with 1 M HCl, filtered through a 0.22 μm filter, and preheated to 37 ℃. 0.25 g of microspheres were placed in 50 mL of SGF and shaken at 100 rpm and 37 ℃. Samples of 200 μL were taken at 0, 30, 60, 90, and 120 min, and immediately terminated by ice bath. The microspheres were diluted with PBS and poured onto YPD agar, incubated at 30 ℃ for 48 h for counting. A control without encapsulated lyophilized yeast was also included.
[0065] ② Simulated enteritis environment (pH 7.4 + MMP-9 + oxidative stress): Enteritis culture medium (SCM) composition: DMEM high glucose supplemented with 10% fetal bovine serum, 5% porcine bile salts, 100 μM H2O2, 10 μg mL -1 IL-1β, 20 ng / mL -1 TNF-α, 50 mM HEPES, pH 7.4, sterilized by 0.22 μm filtration. Microspheres that had completed the SGF 2 h challenge were aseptically transferred to a microsphere culture medium (1:20, volume ratio) to form a "gastric → enteritis" cascade; anaerobic culture was performed at 37 ℃, 5% CO2, and 100 rpm for 6 h. 200 μL samples were collected at 0, 2, 4, and 6 h, and the culture was terminated by ice bath for YPD counting; the supernatant was stored at -80 ℃ for the determination of inflammatory factors (IL-8) and yeast beneficial activity (short-chain fatty acids).
[0066] ③ Fermentation rate test: Take 0.10 g of lyophilized microspheres, add 10 mL of sterile YPD for rehydration for 15 min, and activate at 37℃ for 30 min to obtain an embedded bacterial suspension; take the same amount of free lyophilized yeast as a control. Add 15 mL of 12°P malt extract (30 ℃) to a 50 mL centrifuge tube, and ferment at 2×10⁻⁶. 6 cells mL -1 Inoculate; incubate statically at 30 ℃. Weigh CO2 loss every 2 h until 24 h.
[0067] Evaluation of the encapsulation properties of silk fibroin-gelatin hydrogel: The survival rate of SF-Gel-encapsulated Saccharomyces cerevisiae microspheres in simulated gastric juice and simulated enteritis environments is as follows: Figure 3 As shown, in a simulated gastric fluid environment, the survival rate of the embedded group was (85±2)% after 120 min, while that of the free group was only (7±1)%. In a simulated enteritis environment, the survival rate of yeast in the embedded group was still (65±3)% after 6 h, while that of the free group was below the detection limit (<5%); the IL-8 level was 28% lower than that of the positive control, suggesting that the embedded yeast maintained its anti-inflammatory potential. The fermentation rate test results showed that within 8 h, the embedded group lost 1.25 g of weight, while the free group lost 0.96 g, indicating a 30% increase in fermentation rate and a 28% decrease in sugar content.
[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. Application of silk fibroin-gelatin hydrogel in embedding bacterial strains; The silk fibroin-gelatin hydrogel is obtained by cross-linking gelatin solution and silk fibroin solution as matrices through genipin.
2. The application according to claim 1, characterized in that, The strains include one or more of lactic acid bacteria, yeasts, and nitrogen-fixing bacteria.
3. The application according to claim 1, characterized in that, The preparation method of the silk fibroin-gelatin hydrogel includes the following steps: Gelatin solution, genipin solution and silk fibroin solution were mixed and cross-linked to obtain silk fibroin-gelatin hydrogel.
4. The application according to claim 3, characterized in that, The mass concentration of the gelatin solution is 2-8%, the mass concentration of the silk fibroin solution is 2-6%, and the mass concentration of the genipin solution is 0.01-1%. The mass ratio of the gelatin solution, genipin solution and silk fibroin solution is (2~8):(0.1~2):(8~2). The cross-linking reaction is carried out at a temperature of 35-38 °C for 4-6 h.
5. A silk fibroin-gelatin hydrogel material with an embedded bacterial strain, characterized in that, Including silk fibroin-gelatin hydrogel, and bacterial strains embedded in the silk fibroin-gelatin composite hydrogel; The silk fibroin-gelatin hydrogel is obtained by cross-linking gelatin solution and silk fibroin solution as matrices through genipin.
6. The silk fibroin-gelatin hydrogel material with embedded strains according to claim 5, characterized in that, The strains include one or more of lactic acid bacteria, yeasts, and nitrogen-fixing bacteria; The encapsulation amount of the bacterial strain in the silk fibroin-gelatin hydrogel is ≥5×10⁻⁶. 8 CFU g -1 ; The silk fibroin-gelatin hydrogel material used to embed the bacterial strain is in the form of granules, microspheres, or lyophilized agents.
7. The method for preparing the silk fibroin-gelatin hydrogel material with embedded strains as described in claim 5 or 6, characterized in that, Includes the following steps: (1) Mix gelatin solution, genipin solution and silk fibroin solution to carry out cross-linking reaction to obtain silk fibroin-gelatin hydrogel; (2) The bacterial suspension is mixed with the silk fibroin-gelatin hydrogel to obtain the silk fibroin-gelatin hydrogel material with the encapsulated strain.
8. The preparation method according to claim 7, characterized in that, The cross-linking reaction is carried out at a temperature of 35-38 °C for 4-6 h.
9. The preparation method according to claim 7, characterized in that, When the silk fibroin-gelatin hydrogel material for embedding the bacterial strain is a microsphere formulation, it further includes: Under shear conditions, the silk fibroin-gelatin hydrogel material of the embedded strain was dropped into an oil phase containing a surfactant to obtain an emulsion. The emulsion was physically cross-linked under ice-water bath conditions to obtain an emulsion containing microspheres. After washing and freeze-drying, silk fibroin-gelatin hydrogel microspheres were obtained.
10. The application of the silk fibroin-gelatin hydrogel material with embedded strains as described in claim 5 or 6, or the silk fibroin-gelatin hydrogel material with embedded strains prepared by the preparation method described in any one of claims 7 to 9, in the fields of food, agriculture, environmental remediation, or pharmaceutical preparation.