Probiotic-embedded silk fibroin-chitosan microgel ball and preparation method thereof

Abstract

The preparation method comprises the following steps: uniformly mixing a probiotic bacterial suspension, a silk fibroin solution, a chitosan acetic acid aqueous solution, a cross-linking agent transglutaminase (TGase) and CaCl2 to obtain a water phase, dropwise adding the water phase into an oil phase under a stirring condition, continuously stirring and reacting for 2-4 hours, filtering, washing and drying to obtain the silk fibroin-chitosan microgel ball with the embedded probiotics. And centrifugally collecting and cleaning to obtain the product. According to the preparation method, TGase is utilized to catalyze silk fibroin and chitosan for crosslinking, meanwhile, Ca < 2 + > is introduced, the stable and compact silk fibroin-chitosan microgel ball with a three-dimensional network structure is prepared, and compared with single silk fibroin or chitosan, the silk fibroin-chitosan microgel ball has more excellent thermal stability. According to the present invention, with the probiotic embedding material as the carrier, the high embedding efficiency is provided, the probiotic can be effectively protected to resist the stress effects of processing, storage and gastrointestinal tract environment, the directional efficient release of the probiotic in the intestinal tract can be achieved, and the biological safety of the product can be ensured;

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic encapsulation, and relates to a silk fibroin-chitosan microgel sphere for encapsulating probiotics and a preparation method thereof. Background Art

[0002] Probiotics play an important role in improving intestinal health, enhancing immunity, preventing hyperglycemia, hyperlipidemia, cancer, etc., and are widely used in the fields of food, medicine, feed, etc. According to the definition of the Food and Agriculture Organization of the United Nations / World Health Organization (FAO / WHO) (2001): Probiotics are a group of live microorganisms that can exert beneficial effects on the host when ingested in sufficient quantities. It can be seen that sufficient quantity and viable state are the core characteristics of probiotics. However, during processing, storage, and gastrointestinal digestion, adverse factors such as temperature, oxygen, and gastrointestinal digestive juices (gastric acid, bile salts, digestive enzymes) will greatly affect the survival rate of probiotics, resulting in a very small number of probiotics that can reach the intestine alive, seriously affecting the function of probiotics, which to a certain extent limits the wide application of probiotics.

[0003] The encapsulation technology has become an effective solution to improve the stability and survival rate of probiotics. The encapsulation technology is to embed probiotics in a protective wall material to form a stable protective barrier, so that the probiotics are protected from the influence of environmental stress and can reach the target site smoothly for release and play a role. In the prior art, there are many problems with commonly used encapsulation materials such as chitosan, sodium alginate, gelatin, pectin, etc.: insufficient mechanical strength, easy to break under external pressure or shear force, resulting in the leakage of encapsulated probiotics; poor stability, easy to swell or dissolve under conditions of high temperature, high pH or high ionic strength, affecting the encapsulation effect; and so on. Therefore, it is of great significance to develop a new encapsulation material that can improve the stability and survival rate of probiotics.

[0004] Silk fibroin is a natural biomaterial that can be safely taken orally, with good biocompatibility, degradability, thermal stability, and easy availability. In addition, silk fibroin also has the ability to emulsify and form gels, which can improve the appearance, texture, and taste of foods such as yogurt, and at the same time endow them with rich nutritional value, and has received increasing attention in the field of functional food production. However, the disadvantages such as brittleness caused by the specific composition and structure of silk fibroin limit its application in various fields.

[0005] Chinese Patent Application CN119138589A discloses a preparation method and application of a silk fibroin-based hydrogel. This hydrogel can protect probiotics from the damage of gastric acid and digestive enzymes and is used for the treatment of enteritis. However, the chemical reagents used in the process of preparing the hydrogel have potential toxicity. For example, lithium bromide used in the silk fibroin degumming stage, as well as N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) used in the cross-linking stage, are all chemical reagents with potential toxicity to the human body. Secondly, it is not clear about the embedding efficiency of this hydrogel for probiotics and whether this hydrogel still has good protective effects on probiotics under high-temperature stress and long-term storage. In fact, before being ingested by the human body, probiotics often also face the stress brought about during the processing and storage processes. For example, the high-temperature treatment used in the pasteurization process and the temperature, humidity, and oxygen stress during storage will all greatly affect the survival rate of probiotics, thus seriously affecting the functions of probiotics. Summary of the Invention

[0006] In view of the above problems, the purpose of the present invention is to provide a novel probiotic embedding system and its preparation method. This system needs to meet the following requirements: it not only has a very high embedding efficiency and can effectively protect probiotics against the stress effects of processing, storage, and gastrointestinal environment, but also can ensure the biosafety of the materials themselves and various reagents used in the preparation process.

[0007] Through research, the present invention provides the following technical solutions:

[0008] 1. A preparation method of silk fibroin-chitosan microgel beads embedding probiotics, comprising the following steps:

[0009] a. Mix a probiotic suspension, a silk fibroin solution, a chitosan acetic acid aqueous solution, a cross-linking agent transglutaminase, and CaCl2 evenly to form an aqueous phase; the concentration of the silk fibroin solution is 1%-2% (g / mL), the concentration of chitosan in the chitosan acetic acid aqueous solution is 1.5%-2.5% (g / mL), the mass ratio of silk fibroin to chitosan is 3:3.5-4.5, the dosage of transglutaminase is 60-70 U / g of silk fibroin, and the final concentration of CaCl2 is 40-50 mM;

[0010] b. Under stirring conditions, drop the aqueous phase obtained in step a into the oil phase, continuously stir and react for 2-4 hours, centrifuge, collect, and wash to obtain silk fibroin-chitosan microgel beads embedding probiotics.

[0011] Furthermore, the probiotic suspension in step a is prepared by culturing probiotics, centrifuging to collect the bacterial cells, and resuspending them in PBS.

[0012] Further, the probiotics described in step a are Lactobacillus paracasei and / or Pediococcus pentosaceus.

[0013] Further, the silk fibroin solution described in step a is prepared by the following method: The silkworm cocoons are broken into small pieces and placed in a 0.5% (g / mL) sodium carbonate aqueous solution, boiled for 25 - 35 min to remove sericin, and the degummed silk fibroin fibers are washed with deionized water and dried at 60°C; The dried silk fibroin fibers are added to a ternary solution composed of calcium chloride, ethanol, and water with a molar ratio of 1:2:8 at a ratio of 1 g:10 - 20 mL, stirred and dissolved in a water bath at 80°C for 20 - 40 min, then transferred to an 8 - 14KDa dialysis bag and dialyzed with water for 2 - 3 days, the solution is collected and centrifuged to obtain the silk fibroin solution, and the concentration of the silk fibroin solution is measured and adjusted.

[0014] Further, the chitosan acetic acid aqueous solution described in step a is prepared by dissolving chitosan in a 2% (mL / mL) acetic acid aqueous solution.

[0015] Further, the oil phase described in step b is edible vegetable oil. Including but not limited to: edible rapeseed oil, soybean oil, peanut oil, sunflower seed oil, corn oil, sesame oil, and edible vegetable blended oil, etc. Rapeseed oil is preferably used.

[0016] Further, the concentration of the silk fibroin solution described in step a is 1.5% (g / mL), the concentration of chitosan in the chitosan acetic acid aqueous solution is 2% (g / mL), the mass ratio of silk fibroin to chitosan is 3:4, the dosage of transglutaminase is 65 - 68 U / g silk fibroin, and the final concentration of CaCl2 is 45 mM.

[0017] In step a, the concentration and added volume of the probiotic suspension can be adjusted according to the use and requirements of the final probiotic product. As one of the implementation examples, the concentration of the probiotic suspension in step a is 10 11 CFU / mL, the concentration of the silk fibroin solution is 1.5% (g / mL), the concentration of chitosan in the chitosan acetic acid aqueous solution is 2% (g / mL), the volume ratio of the probiotic suspension, the silk fibroin solution, and the chitosan acetic acid aqueous solution is 1:10:10, the dosage of transglutaminase is 67 U / g silk fibroin, and the final concentration of CaCl2 is 45 mM.

[0018] Further, in step b, the aqueous phase obtained in step a is dropped into the oil phase at a stirring speed of 2000 revolutions per minute, continuously stirred and reacted for 2 - 4 hours, and collected and washed by centrifugation at 500 rpm / min to obtain the silk fibroin - chitosan microgel beads encapsulating probiotics.

[0019] The beneficial effects of the present invention are as follows:

[0020] (1) Chitosan is a natural alkaline polysaccharide with excellent properties such as biocompatibility, degradability, and gelation, and has broad application prospects in the fields of food and biomedicine. In addition, chitosan also exhibits good acid resistance, bile resistance, and adhesiveness, making it an ideal material for drug delivery systems. However, chitosan has low mechanical strength and poor stability. The present invention uses transglutaminase to catalyze the cross-linking of silk fibroin and chitosan to form stable and dense silk fibroin-chitosan microgel spheres with a three-dimensional network structure; at the same time, Ca 2+ , Ca 2+ forms coordination bonds with silk fibroin and chitosan respectively to further enhance the network structure stability of the microgel spheres. The introduction of chitosan for cross-linking with silk fibroin in the present invention can promote the secondary structure of silk fibroin to transform from the silk I structure to the more stable silk II structure, thereby enhancing the stability of silk fibroin and improving the properties of silk fibroin. The test results confirm that the microgel spheres formed by the composite of silk fibroin-chitosan have better thermal stability than single silk fibroin or chitosan.

[0021] (2) Using the silk fibroin-chitosan microgel spheres as a probiotic embedding carrier not only has a high embedding efficiency for probiotics, but also can play a good protective role for probiotics under high-temperature treatment, long-term low-temperature storage, and adverse gastrointestinal environments, can significantly enhance the stability of probiotics during storage and their tolerance to high-temperature environments, can effectively improve the survival rate of probiotics in gastric acid and bile salts, and can achieve the targeted and efficient release of probiotics in the intestine.

[0022] (3) Using the silk fibroin-chitosan microgel spheres as a probiotic embedding carrier, since both silk fibroin and chitosan have antioxidant activity, they can scavenge the free radicals generated by probiotics during storage and reduce the damage of free radicals to probiotics, thereby improving the storage stability of the encapsulated probiotics; at the same time, after the silk fibroin-chitosan microgel spheres enter the intestine, silk fibroin is hydrolyzed into silk peptides under the action of trypsin, and silk peptides have excellent antioxidant activity, which can enhance the antioxidant function of the host and have a positive effect on human health.

[0023] (4) The preparation method of the present invention is simple, easy to operate, and suitable for industrial production. Silk fibroin is derived from silk, and chitosan is derived from natural chitin. Both are biodegradable natural polymer materials with good biocompatibility, no toxic and side effects, and are more friendly to the human body and the environment than some synthetic polymers. Other reagents and materials used in the preparation process are harmless to the human body, ensuring the biosafety of the product.

[0024] (5) The silk fibroin-chitosan microgel beads encapsulating probiotics of the present invention have good application prospects in the fields of food, health products, pharmaceuticals, feeds, etc., and are particularly suitable for products with high requirements for probiotic activity.

[0025] This invention is funded by the National Key Research and Development Program (No. 2023YFF1103800). Brief Description of the Drawings

[0026] Figure 1 It is a schematic diagram of the preparation process of silk fibroin-chitosan microgel beads encapsulating probiotics.

[0027] Figure 2 It is the microscopic structure of silk fibroin-chitosan microgel beads encapsulating probiotics observed under an optical microscope.

[0028] Figure 3 It is the Fourier transform infrared spectroscopy (FTIR) graph (A) and differential scanning calorimetry (DSC) graph (B) of silk fibroin (SF), chitosan (CS), and silk fibroin-chitosan microgel beads (Microgel).

[0029] Figure 4 It is the antioxidant activity of silk fibroin-chitosan microgel beads as a probiotic encapsulating wall material, where A is the DPPH radical scavenging rate and B is the ABTS radical scavenging rate.

[0030] Figure 5 It is the survival rate of probiotics encapsulated in silk fibroin-chitosan microgel beads under heat treatment at different temperatures.

[0031] Figure 6 It is the stability of probiotics encapsulated in silk fibroin-chitosan microgel beads during storage at 4°C.

[0032] Figure 7 It is the survival rate (A, B) of probiotics encapsulated in silk fibroin-chitosan microgel beads in simulated gastric juice and bile salt solution and the release rate (C) in intestinal juice.

[0033] Figure 8 It is the fluorescence graph of silk fibroin-chitosan microgel beads encapsulating stained probiotics after being treated in simulated gastric juice and intestinal juice for 2 h. Detailed Embodiments

[0034] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below.

[0035] Preparation Example 1. Preparation of Silk Fibroin-Chitosan Microgel Beads Encapsulating Probiotics (mass ratio of silk fibroin to chitosan is 3:4)

[0036] The schematic diagram of the preparation process of silk fibroin-chitosan microgel beads embedding probiotics is as Figure 1 shown. It includes the following steps:

[0037] (1) Preparation of probiotic suspension: Take out the probiotic strains preserved with glycerol from the -80°C refrigerator, streak them on the MRS solid medium, and after culturing at 37°C for 48 h, pick a single colony and culture it in 2 mL of MRS liquid medium for 16 h. Then inoculate the cultured bacterial liquid into 200 mL of MRS liquid medium for enlarged culture. After 16 h, centrifuge at 4500 rpm for 5 min to collect the bacterial cells, wash the bacterial cells twice with sterile PBS (pH 7.4), and then resuspend the bacterial cells with 2 mL of sterile PBS (pH 7.4) to prepare a bacterial suspension with a concentration of 10 11 CFU / mL;

[0038] (2) Preparation of silk fibroin solution: Cut the silkworm cocoons into small pieces, add them to 0.5% (g / mL) sodium carbonate solution according to the ratio of 1 g:50 mL, boil for 30 minutes to remove sericin, wash the degummed silk fibroin fibers 3 times with deionized water and then wring them dry, dry them in an oven at 60°C for later use; Add the dried silk fibroin fibers to a ternary solution composed of calcium chloride, ethanol, and water with a molar ratio of 1:2:8 according to the ratio of 1 g:10 mL, stir and dissolve in a water bath at 80°C for 30 min, then transfer it to a dialysis bag with a molecular weight cut-off of 8 - 14 KDa, dialyze with ultrapure water for 3 days, collect the solution, and centrifuge at 10000 rpm for 10 min to obtain a silk fibroin solution. Measure the concentration of silk fibroin and adjust the concentration to 1.5% (g / mL).

[0039] (3) Preparation of chitosan acetic acid aqueous solution: Under magnetic stirring, dissolve chitosan in 2% (mL / mL) acetic acid aqueous solution to prepare a chitosan acetic acid aqueous solution with a chitosan concentration of 2% (g / mL).

[0040] (4) Preparation of silk fibroin-chitosan microgel beads embedding probiotics: Mix 1 mL of the probiotic suspension prepared in step (1) with 10 mL of the silk fibroin solution prepared in step (2) evenly, and then sequentially add 10 mL of the chitosan acetic acid aqueous solution prepared in step (3), 0.2 g of transglutaminase at 50 U / g, and 1 mL of 1 M CaCl2 solution, mix evenly to obtain the aqueous phase; Use 100 mL of edible rapeseed oil as the oil phase, and under the conditions of magnetic stirring at room temperature and 2000 rpm / min, dropwise add the aqueous phase into the oil phase, continuously stir and react for 3 h. The generated microgel beads will precipitate from the emulsion system, centrifuge at 500 rpm / min for 1 min to collect and wash, and then the silk fibroin-chitosan microgel beads embedding probiotics can be obtained.

[0041] According to the above preparation method, two probiotics were selected: Lactobacillus paracasei and Pediococcus pentosaceus. Silk fibroin-chitosan microgel beads embedding Lactobacillus paracasei (Microgels / L.paracasei) and silk fibroin-chitosan microgel beads embedding Pediococcus pentosaceus (Microgels / P.pentosaceus) were respectively prepared.

[0042] The optical microscope images of the silk fibroin-chitosan microgel beads embedding probiotics are as Figure 2 shown, being spherical, complete and plump, with uniform morphology and an average particle size of about 800 μm.

[0043] Comparative Preparation Example 1: Without adding transglutaminase

[0044] The preparation was carried out according to the preparation method described in Preparation Example 1, except that: transglutaminase was not added in step (4).

[0045] It was found that the whole reaction system could not be fully gelated, and the amount of microgel beads formed was significantly reduced compared with Preparation Example 1.

[0046] Comparative Preparation Example 2: Without adding CaCl2

[0047] The preparation was carried out according to the preparation method described in Preparation Example 1, except that: CaCl2 was not added in step (4).

[0048] It was found that the structure of the formed microgel beads was unstable and easy to break, and they could not be used as a stable carrier for embedding probiotics.

[0049] Comparative Preparation Example 3: Without adding chitosan

[0050] The preparation was carried out according to the preparation method described in Preparation Example 1, except that: the chitosan acetic acid aqueous solution was not added in step (4).

[0051] It was found that during the continuous stirring reaction process, a large amount of irregular white flocculates precipitated from the emulsion system and no gel could be formed.

[0052] Comparative Preparation Example 4: The mass ratio of silk fibroin to chitosan was 3:2

[0053] The preparation was carried out according to the preparation method described in Preparation Example 1, except that: the volume of the chitosan acetic acid aqueous solution added in step (4) was different. It was changed to 5 mL, and the mass ratio of silk fibroin to chitosan was 3:2.

[0054] It was found that due to the large proportion of silk fibroin in the reaction system, irregular white flocs precipitated from the emulsion system during the continuous stirring reaction, and the generated microgel balls adhered to each other, resulting in the inability to obtain many microgel balls with complete and uniform shapes.

[0055] Comparative Preparation Example 5, the mass ratio of silk fibroin to chitosan is 3:8

[0056] Prepared according to the preparation method described in Preparation Example 1, the difference is that: the volume of the chitosan acetic acid aqueous solution added in step (4) is different, changed to 20 mL, then the mass ratio of silk fibroin to chitosan is 3:8.

[0057] It was found that due to the large proportion of chitosan in the reaction system, although microgel balls could be formed, most of the balls would break during centrifugal washing and could not effectively entrap probiotics.

[0058] Test Example 1, Detection of the Structural Stability and Thermal Stability of Silk Fibroin-Chitosan Microgel Balls

[0059] The chemical structures of silk fibroin, chitosan and silk fibroin-chitosan microgel balls (prepared according to the preparation method described in Preparation Example 1 but without adding probiotic suspension in step (4)) were analyzed by Fourier transform infrared spectroscopy, and the results are as Figure 3 shown in A. The main characteristic peaks of silk fibroin: 3423 cm-1 (-OH and -NH stretching vibrations), 1650 cm-1 (amide I band), 1536 cm-1 (amide II band) and 1240 cm-1 (amide III band), indicating that silk fibroin is mainly in a random coil structure. The main characteristic peaks of chitosan: 3424 cm-1 (-OH and -NH stretching vibrations), 1632 cm-1 (amide I band, C=O stretching vibration), 1594 cm-1 (amide II band, C–N stretching vibration and N–H bending vibration). Compared with the characteristic peaks of silk fibroin and chitosan, the silk fibroin-chitosan microgel balls showed new characteristic peaks at 1629 cm-1, 1554 cm-1 and 1526 cm-1 in the amide I band and amide II band, indicating the formation of new amide bonds, and the secondary structure of silk fibroin changed from silk I (random coil) to a more stable silk II (β-sheet) structure; in addition, the -OH and -NH stretching vibration peaks of the silk fibroin-chitosan microgel balls became narrower and shifted to 3421 cm-1, indicating the formation of hydrogen bonds between silk fibroin and chitosan. Based on the above experimental results, due to the combined action of the new amide bonds (isopeptide bonds) formed by silk fibroin and chitosan under the catalysis of transglutaminase and the hydrogen bonds formed by the close proximity of molecules in space, silk fibroin and chitosan were successfully crosslinked together to form stable microgel balls. At the same time, Ca 2+They respectively form coordination bonds with silk fibroin and chitosan, further enhancing the network structure stability of the microgel spheres.

[0060] Silk fibroin, chitosan and silk fibroin-chitosan microgel spheres (prepared according to the preparation method described in Preparation Example 1 but without adding the probiotic suspension in step (4)) were analyzed by differential scanning calorimetry, and the results are as Figure 3 shown in B. The Tm (129.71 °C) of the silk fibroin-chitosan microgel spheres exceeded that of silk fibroin (118.96 °C) and chitosan (121.81 °C), indicating that the microgel spheres formed by the combination of silk fibroin and chitosan have more excellent thermal stability compared with single silk fibroin or chitosan, which is particularly important in food processing (such as pasteurization).

[0061] The combination of the above Fourier transform infrared spectroscopy analysis results and differential scanning calorimetry analysis results confirmed the interaction between silk fibroin and chitosan. This interaction helps to form a stable and dense gel network structure, ultimately resulting in enhanced thermal stability of the silk fibroin-chitosan microgel spheres.

[0062] Test Example 2: Detection of the entrapment efficiency of silk fibroin-chitosan microgel spheres for probiotics

[0063] Take 1 g of the silk fibroin-chitosan microgel spheres (Microgels / L.paracasei, Microgels / P.pentosaceus) entrapping probiotics prepared in Preparation Example 1, add 10 mL of PBS (0.2 mol / L, pH 7.4), treat with a high-speed disperser at 4000 rpm for 10 min, then perform 10-fold serial dilutions, coat on MRS solid medium, and count after static culture at 37 °C for 48 h to calculate the number of probiotics entrapped in all the microgel spheres prepared in step (4) of Preparation Example 1 (n); at the same time, perform 10-fold serial dilutions on the probiotic suspension used in Preparation Example 1 and plate coat and count to calculate the number of probiotics contained in 1 mL of the probiotic suspension (N), and calculate the entrapment rate (ER) according to the following formula:

[0064]

[0065] The results are shown in Table 1. The entrapment rate is the mean ± standard error (n = 3). The same lowercase letters indicate no significant difference between the two groups (p > 0.05). It can be seen that the silk fibroin-chitosan microgel spheres have a high entrapment efficiency (>80%) for both Lactobacillus paracasei and Pediococcus pentosaceus, and the difference between them is not obvious, indicating that the silk fibroin-chitosan microgel spheres have relatively stable entrapment efficiency for different probiotics.

[0066] Table 1 Entrapment efficiency of silk fibroin-chitosan microgel spheres for different probiotics

[0067]

[0068] Test Example 3: Detection of the antioxidant activity of silk fibroin-chitosan microgel beads as an embedding wall material for probiotics

[0069] Dilute the silk fibroin solution and chitosan acetic acid aqueous solution described in Preparation Example 1 respectively, so that the concentrations of silk fibroin and chitosan are 6.0, 3.0, and 1.5 mg / mL respectively; subject the silk fibroin to trypsin hydrolysis, with the addition amount of trypsin being 6% of the substrate mass, adjust the pH value of the reaction system to 8.0, stir and hydrolyze for 6 h, centrifuge to collect the supernatant to obtain a silk fibroin peptide (SFP) solution, and adjust the concentrations to 6.0, 3.0, and 1.5 mg / mL respectively; take the DPPH and ABTS free radical scavenging activities as indicators to measure the antioxidant activity of the probiotic embedding wall material, and refer to the methods described in the instructions of the corresponding kits to detect the antioxidant activities of silk fibroin, chitosan, and silk fibroin peptide respectively.

[0070] The results are as Figure 4 shown. Silk fibroin has strong DPPH and ABTS free radical scavenging activities, chitosan has strong DPPH free radical scavenging activity, and silk fibroin peptide has stronger DPPH and ABTS free radical scavenging activities. These results indicate that using silk fibroin-chitosan microgel beads as an embedding wall material for probiotics, since both silk fibroin and chitosan have antioxidant activities, they can scavenge the free radicals generated by probiotics during storage, reduce the damage of free radicals to probiotics, and thus can improve the storage stability of the encapsulated probiotics; after the silk fibroin-chitosan microgel beads enter the intestine, the silk fibroin is hydrolyzed into silk fibroin peptides under the action of trypsin, and the silk fibroin peptides have excellent antioxidant activities, which can enhance the antioxidant function of the host and have a positive effect on human health. Therefore, compared with other probiotic embedding wall materials, silk fibroin-chitosan microgel beads have superior biological activities and are natural antioxidants for stabilizing probiotics.

[0071] Test Example 4: Detection of the thermal stability of probiotics embedded in silk fibroin-chitosan microgel beads

[0072] Take 1 g of the silk fibroin-chitosan microgel beads (Microgels / L.paracasei) embedding probiotics prepared in Preparation Example 1, add 9 mL of PBS (0.2 mol / L, pH 7.4), and carry out the following four heat treatments respectively: heat at 65 °C for 30 min, heat at 75 °C for 10 min, heat at 85 °C for 3 min, and heat at 95 °C for 3 min. Take another probiotic suspension (unembedded probiotics) prepared in Preparation Example 1 for the same heat treatment. After the heat treatment is completed, immediately carry out the cooling operation, and then plate count to calculate the probiotic survival rate under different temperature treatments.

[0073] The results are as follows Figure 5 shown. Under the 4 temperature treatments, no viable (Nd) free L.paracasei was detected, while the Microgels / L.paracasei showed relatively high survival rates. Among them, the survival rate was over 80% under the simulated pasteurization conditions (heating at 65 °C for 30 min), and even reached over 70% under higher temperature treatments, indicating that the Microgels / L.paracasei showed stronger thermal stability.

[0074] Test Example 5: Detection of the storage stability of Microgels / L.paracasei

[0075] Take the Microgels / L.paracasei and the probiotic suspension (free L.paracasei) prepared in Preparation Example 1, store them in the dark at 4 °C, sample every 1 week for the detection of the survival rate of probiotics, and detect for a total of 6 weeks.

[0076] The results are as follows Figure 6 shown. Compared with the free L.paracasei, the survival rate of the Microgels / L.paracasei was significantly increased. At the 6th week, the survival rate of the free L.paracasei was 0, while the survival rate of the Microgels / L.paracasei was over 70%, indicating that the Microgels / L.paracasei had a significant effect on improving the storage stability of probiotics. The results of Test Example 3 have shown that both silk fibroin and chitosan have the ability to scavenge free radicals, which can scavenge the free radicals harmful to probiotics generated during storage, thereby improving the storage stability of the encapsulated probiotics. Therefore, the Microgels / L.paracasei can significantly extend the shelf life and efficacy of probiotic products.

[0077] Test Example 6: Detection of the survival rate of Microgels / L.paracasei in simulated gastric juice and bile salt solution and the release rate in intestinal juice

[0078] 1. Detection of the survival rate of probiotics in simulated gastric juice and bile salt solution

[0079] (1) Preparation of simulated gastric juice (SGJ): Adjust the pH value of PBS to 2.0, dissolve pepsin with it to make the final concentration of pepsin 3 g / L, and filter and sterilize it with a filter membrane with a pore size of 0.22 μm to obtain simulated gastric juice.

[0080] (2) Preparation of simulated bile salt solution (SBJ): The pH value of PBS was adjusted to 5.0, and bile salts were dissolved therein to make the final concentrations of bile salts 1 g / L and 3 g / L, respectively. The solution was sterilized by filtration using a filter membrane with a pore size of 0.22 μm to prepare two simulated bile salt solutions of different concentrations.

[0081] (3) Survival rate detection: Take 1 g of the silk fibroin-chitosan microgel beads (Microgels / L. paracasei, Microgels / P. pentosaceus) and 1 mL of the probiotic suspension (unencapsulated probiotics) prepared in Preparation Example 1, respectively, and place them in 9 mL of simulated gastric fluid and two different concentrations of simulated bile salt solutions, vortex and oscillate thoroughly, culture at 37°C and 100 rpm for 2 h, treat with a high-speed disperser at 4000 rpm for 10 min, apply gradient dilutions on plates, and count. Calculate the survival rate (SR) of probiotics in simulated gastric fluid and bile salt solution according to the following formula:

[0082]

[0083] Where m refers to the number of viable probiotics after simulation, and M refers to the number of viable probiotics before simulation.

[0084] The results are as follows Figure 7 A and 7B. Compared with the unencapsulated probiotics (free L. paracasei, free P. pentosaceus), the survival rates of the probiotics (Microgels / L. paracasei, Microgels / P. pentosaceus) encapsulated by silk fibroin-chitosan microgels were significantly improved in simulated gastric fluid and two different concentrations of simulated bile salt solutions, indicating that the encapsulation of silk fibroin-chitosan microgels played an effective protective role on the probiotics.

[0085] 2. Release rate detection of probiotics in simulated intestinal fluid

[0086] (1) Preparation of simulated intestinal fluid (SIJ): The pH value of PBS was adjusted to 8.0, and trypsin was dissolved therein to a final concentration of 1 g / L. The SIJ was sterilized by filtration using a filter membrane with a pore size of 0.22 μm to prepare simulated intestinal fluid.

[0087] (2) Release rate detection: Take 1 g of the silk fibroin-chitosan microgel beads (Microgels / L. paracasei, Microgels / P. pentosaceus) encapsulating probiotics prepared in Preparation Example 1, place in 9 mL of simulated intestinal fluid, vortex thoroughly, culture at 37°C, 100 rpm for 2 h, spread on a plate in gradient dilutions, and count. Calculate the release rate (RR) of probiotics in simulated intestinal fluid according to the following formula:

[0088]

[0089] Wherein, a refers to the viable count of probiotics after simulation, and A refers to the viable count of probiotics before simulation.

[0090] The results are as Figure 7 shown in C. The probiotics embedded in the silk fibroin-chitosan microgel beads were released in simulated intestinal fluid, and the release rate reached more than 95%, indicating that the probiotics embedded in the silk fibroin-chitosan microgel beads could be directionally and efficiently released in the intestine.

[0091] 3. Fluorescence microscopy observation of silk fibroin-chitosan microgel beads embedded with probiotics after being treated with simulated gastric juice and intestinal fluid

[0092] The probiotic suspension was added with 10 μg / mL acridine orange dye and stained for 20 min, centrifuged at 6000 rpm for 5 min to remove the excess dye, and the stained probiotics were washed with PBS and resuspended to obtain a stained probiotic suspension; the stained probiotics were embedded according to the method described in Preparation Example 1 to prepare silk fibroin-chitosan microgel beads embedded with stained probiotics. The microgel beads were respectively placed in simulated gastric juice and simulated intestinal fluid, vortexed thoroughly, cultured at 37 °C and 100 rpm for 2 h, and then observed and photographed under a fluorescence microscope, with the stained probiotic suspension as a control.

[0093] The results are as Figure 8 shown. In the stained probiotic suspension (L. paracasei), probiotics with green fluorescence could be seen; the silk fibroin-chitosan microgel beads embedded with stained probiotics (Microgels / L. paracasei) presented green and complete spherical shapes, and still maintained the integrity of the structure after being treated with simulated gastric juice for 2 h (SGJ-2 h), while the microgel beads ruptured after being treated with simulated intestinal fluid for 2 h (SIJ-2 h), releasing probiotics with green fluorescence.

[0094] All of the above results indicate that the probiotics embedded in the silk fibroin-chitosan microgel beads can resist the damage of gastric juice and bile salt solution and be released efficiently at a specific site in the intestine.

[0095] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing silk fibroin-chitosan microgel spheres encapsulating probiotics, characterized in that: The following steps are involved: a. The probiotic suspension, the silk fibroin solution, the chitosan acetate aqueous solution, the crosslinking agent transglutaminase and CaCl2 are mixed uniformly to form an aqueous phase; the concentration of the silk fibroin solution is 1%-2% (g / mL), the concentration of chitosan in the chitosan acetate aqueous solution is 1.5%-2.5% (g / mL), the mass ratio of silk fibroin to chitosan is 3:3.5-4.5, the amount of transglutaminase is 60-70U / g silk fibroin, and the final concentration of CaCl2 is 40-50mM; b. Under stirring conditions, the aqueous phase obtained in step a is dripped into the oil phase, and the reaction is continued by stirring for 2-4 hours. The mixture is collected by centrifugation and washed to obtain silk fibroin-chitosan microgel spheres encapsulating probiotics.

2. The method for preparing a probiotic-embedded silk fibroin-chitosan microgel sphere according to claim 1, characterized in that: The probiotic suspension in step a is prepared by culturing the probiotics, collecting the bacteria by centrifugation, and resuspending them in PBS.

3. The method for preparing a silk fibroin-chitosan microgel sphere encapsulating probiotics according to claim 1, characterized in that: The probiotics described in step a are Lactobacillus paracasei and / or Pediococcus pentosaceus.

4. The method for preparing a probiotic-embedded silk fibroin-chitosan microgel sphere according to claim 1, characterized in that: The silk fibroin solution in step a is prepared by the following method: crushing silk cocoons into small pieces, placing them in a 0.5% (g / mL) sodium carbonate aqueous solution, boiling them for 25-35 minutes to remove sericin, washing the degummed silk fibroin fibers with deionized water, and drying them at 60° C.; adding the dried silk fibroin fibers to a ternary solution consisting of calcium chloride, ethanol and water in a molar ratio of 1:2:8 at a ratio of 1 g:10-20 mL, stirring and dissolving them in a water bath at 80° C. for 20-40 minutes, then transferring them to a dialysis bag of 8-14 KDa, dialyzing them with water for 2-3 days, collecting the solution and centrifuging it to obtain a silk fibroin solution, and measuring and adjusting the concentration of the silk fibroin solution.

5. The method for preparing the silk fibroin-chitosan microgel spheres encapsulating probiotics according to claim 1, characterized in that: The chitosan acetic acid aqueous solution in step a is prepared by dissolving chitosan in a 2% (mL / mL) acetic acid aqueous solution.

6. The method for preparing the silk fibroin-chitosan microgel spheres encapsulating probiotics according to claim 1, characterized in that: The oil phase in step b is edible vegetable oil.

7. The method for preparing the silk fibroin-chitosan microgel spheres encapsulating probiotics according to claim 1, characterized in that: The concentration of the silk fibroin solution in step a is 1.5% (g / mL), the concentration of chitosan in the chitosan acetate aqueous solution is 2% (g / mL), the mass ratio of silk fibroin to chitosan is 3:4, the amount of transglutaminase used is 65-68U / g silk fibroin, and the final concentration of CaCl2 is 45mM.

8. The method for preparing the silk fibroin-chitosan microgel spheres encapsulating probiotics according to claim 1, characterized in that: Step b is to drop the water phase obtained in step a into the oil phase at a stirring speed of 2000 rpm, continue stirring and reacting for 2-4 hours, collect and wash by centrifugation at 500 rpm / min, and obtain silk fibroin-chitosan microgel spheres encapsulating probiotics.

9. Silk fibroin-chitosan microgel spheres encapsulating probiotics prepared by the preparation method according to any one of claims 1 to 8.

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