Double-layer hydrogel embedded probiotic synergetic delivery polyphenol microcapsule and preparation method and application thereof

Abstract

The invention discloses a polyphenol microcapsule synergistically delivered by double-layer hydrogel embedded probiotics as well as a preparation method and application of the polyphenol microcapsule. The preparation method comprises the following steps: mixing suspension at least containing polyphenol substances with zinc salt and gallic acid for reaction to prepare zinc-gallic acid embedded polyphenol nanoparticles; mixing the zinc-gallic acid embedded polyphenol nanoparticles with probiotics to react, so as to prepare a first solid product; and mixing the first solid product with pachymaran, then adding a solution at least containing quaternized chitosan and sodium tungstate dihydrate, and then preparing the obtained mixed solution into the double-layer hydrogel embedded probiotic synergistic delivery polyphenol microcapsule by adopting an electrospray mode. According to the preparation method disclosed by the invention, the probiotics are embedded by adopting the double-layer hydrogel microcapsule with outer layer removal and inner layer colonization for the first time, so that the protection performance and functionality of the microcapsule are enhanced, efficient embedding and colonization of the probiotics are realized, the activity of the probiotics is effectively prevented from being damaged by high temperature and machinery, and industrial production is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic delivery and microcapsules, and particularly relates to a double-layer hydrogel-embedded probiotic synergistically delivered polyphenol microcapsule and a preparation method and application thereof. Background Art

[0002] Probiotics play an important role in intestinal health, including improving intestinal microbial balance and enhancing the immune system, so they are currently widely used in food. However, probiotics are easily inactivated during processing and storage, especially during gastrointestinal digestion. Their activity is affected by conditions such as temperature and pH, and by competition between probiotics and host flora. Probiotics need to compete with host flora for nutrients and adhesion sites. This competitive relationship affects the success rate of probiotic colonization. In order to overcome the above problems, developing effective probiotic encapsulation technology to improve its stability and survival rate is an important topic in the field of probiotic research.

[0003] Microencapsulation is an effective probiotic delivery system. Microencapsulation technology provides a physical barrier for probiotics, which helps to resist or delay the penetration of harmful substances such as gastric acid and bile salts, protects them from adverse environments such as food processing, storage and digestion in the gastrointestinal tract after consumption, improves the survival rate of probiotics, and ensures the activity of probiotics. However, different types of wall materials and encapsulation technologies have different effects on the activity and intestinal transport performance of probiotics. Therefore, when preparing microcapsules, the combination of wall materials and encapsulation technologies should be analyzed and selected on a case-by-case basis to maximize the effectiveness of probiotics. Traditional microencapsulation technology has shortcomings, such as low efficiency of extrusion method, high temperature damage to bacteria by spray drying method, high energy consumption of freeze drying method, and different particle size distribution of emulsification method.

[0004] Traditional probiotic microencapsulation focuses only on protecting probiotics from gastrointestinal damage under physiological conditions, ignoring the fact that pathological conditions of colitis can also severely hinder the colonization of probiotics. In the disease state, the large occupation of the ecological niche by Enterobacteriaceae severely limits the colonization of probiotics. Therefore, achieving efficient colonization by destroying the ecological niche of harmful bacteria is a daunting challenge. Summary of the invention

[0005] The main purpose of the present invention is to provide a double-layer hydrogel-embedded probiotic synergistically delivered polyphenol microcapsule and its preparation method and application. The present invention aims to provide a method for preparing probiotic microcapsules based on electrostatic spray technology, using Poria polysaccharide, zinc-gallic acid-embedded polyphenol nanoparticles, quaternized chitosan and sodium tungstate as raw materials, so as to improve the stability and survival rate of probiotics during processing and storage, while protecting the probiotics from damage by gastrointestinal digestive juices, improving their colonization and oral bioavailability in the colon, so as to overcome the shortcomings of the prior art.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0007] The embodiment of the present invention provides a method for preparing a double-layer hydrogel-embedded probiotic synergistically delivered polyphenol microcapsule, which comprises:

[0008] Provides Poria polysaccharide and probiotics;

[0009] Mixing a suspension containing at least polyphenols with zinc salt and gallic acid to produce zinc-gallic acid-embedded polyphenol nanoparticles;

[0010] The zinc-gallic acid-embedded polyphenol nanoparticles are mixed with probiotics to produce a first solid product;

[0011] And, the first solid product is mixed with Poria cocos polysaccharide, and then a solution containing at least quaternized chitosan and sodium tungstate dihydrate is added, and then the obtained mixed solution is electrosprayed to prepare double-layer hydrogel-embedded probiotics-co-delivered polyphenol microcapsules.

[0012] The embodiment of the present invention also provides a double-layer hydrogel-embedded probiotics synergistically delivered polyphenol microcapsule prepared by the aforementioned preparation method.

[0013] The embodiment of the present invention also provides the use of the aforementioned double-layer hydrogel-embedded probiotics co-delivery polyphenol microcapsules in the preparation of products with colon-targeted co-delivery of hydrophobic polyphenols and probiotics.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention provides a method for preparing probiotic microcapsules based on layer-by-layer self-assembly and electrospray technology, which utilizes electrospray technology combined with specific encapsulation wall materials to achieve efficient encapsulation of probiotics and improve the intestinal targeted delivery and controlled release capabilities of probiotics;

[0016] (2) The present invention is the first to use a double-layer hydrogel microcapsule of "outer layer removal + inner layer colonization" to encapsulate probiotics, thereby enhancing the protection and functionality of the microcapsule and achieving efficient encapsulation and colonization of probiotics;

[0017] (3) The double-layer hydrogel-embedded probiotics synergistically delivered polyphenol microcapsules provided by the present invention not only improve the survival rate and colonization effect of probiotics, but also provide a more favorable environment for the effective action of probiotics in the intestine, and have important application prospects and development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is a growth curve diagram of Lactobacillus rhamnosus LGG and Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 at 0-24 hours;

[0020] Figure 2 Surface potential diagrams of Lactobacillus rhamnosus LGG and microcapsules prepared in Example 1 and Comparative Examples 1-4 of the present invention;

[0021] Figure 3 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated gastric fluid in vitro for 2 hours;

[0022] Figure 4 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated small intestinal fluid in vitro for 2 hours;

[0023] Figure 5 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated colon fluid in vitro for 2 hours;

[0024] Figure 6 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated bile salt in vitro for 2 hours;

[0025] Figure 7 The graph is a graph showing the growth inhibition of different Enterobacteriaceae by Lactobacillus rhamnosus LGG and the embedded Lactobacillus rhamnosus LGG in Example 1 of the present invention and Comparative Examples 1-4. DETAILED DESCRIPTION

[0026] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The present invention uses zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide as the inner layer of probiotic microcapsules, and quaternized chitosan and sodium tungstate as the outer layer of microcapsules to prepare oral colon-targeted delivery microcapsules of probiotics through electrospray technology. This delivery system is applicable to a variety of probiotics, which not only improves the resistance of probiotics to harsh environments and enhances the ability to colonize the intestines, but also acts as a prebiotic to enhance the growth and reproduction of probiotics.

[0027] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Specifically, as one aspect of the technical solution of the present invention, a method for preparing a double-layer hydrogel-embedded probiotic synergistically delivered polyphenol microcapsule comprises:

[0029] Provides Poria polysaccharide and probiotics;

[0030] Mixing a suspension containing at least polyphenols with zinc salt and gallic acid to produce zinc-gallic acid-embedded polyphenol nanoparticles;

[0031] The zinc-gallic acid-embedded polyphenol nanoparticles are mixed with probiotics to produce a first solid product;

[0032] And, the first solid product is mixed with Poria cocos polysaccharide, and then a solution containing at least quaternized chitosan and sodium tungstate dihydrate is added, and then the obtained mixed solution is electrosprayed to prepare double-layer hydrogel-embedded probiotics-co-delivered polyphenol microcapsules.

[0033] In some preferred embodiments, the preparation method specifically comprises:

[0034] (1) crushing, defatting, drying, extracting, precipitating, dialysis, and freeze-drying the Poria cocos brick tea to obtain Poria cocos polysaccharide;

[0035] (2) Provide a bacterial concentration of 1 0 8 ~10 9 CFU / mL of probiotic concentrate;

[0036] (3) dissolving the polyphenols in ethanol to form a polyphenols ethanol solution, then adding the solution to a polyvinyl pyrrolidone solution to form a polyphenols suspension, and then dispersing the polyphenols suspension in an aqueous solution containing zinc salt and gallic acid, performing ultrasonication, centrifugation, washing, precipitation, and PBS dispersion treatment to obtain a zinc-gallic acid-encapsulated polyphenols nanoparticle solution;

[0037] (4) mixing the probiotic concentrate with the zinc-gallic acid-encapsulated polyphenol nanoparticle solution, stirring and centrifuging the mixture to obtain a first solid product;

[0038] (5) adding Pachymaran to a PBS solution and then adding the first solid product and mixing thoroughly to prepare a first suspension;

[0039] (6) mixing the quaternized chitosan, sodium tungstate dihydrate and PBS solution uniformly to obtain a solution containing quaternized chitosan and sodium tungstate dihydrate;

[0040] (7) vortex mixing the first suspension with a solution containing quaternized chitosan and sodium tungstate dihydrate to prepare a probiotic suspension;

[0041] (8) The probiotic suspension is injected into a sodium tripolyphosphate solution by electrospraying, allowed to stand and solidify, and then filtered to obtain a double-layer hydrogel-embedded probiotic-co-delivered polyphenol microcapsule.

[0042] Furthermore, the degreasing treatment in step (1) includes: mixing the crushed Poria cocos brick tea with ethanol and heating and stirring the mixture in a water bath at 70-90° C. for 2-6 hours, followed by filtering.

[0043] Furthermore, the mass volume ratio of the Poria brick tea to ethanol is 0.1-0.5 mg:1 mL.

[0044] Furthermore, the extraction and precipitation treatment in step (1) includes: dissolving the dried product in water at 80-100°C and heating it for 2-4 hours, then collecting the supernatant, concentrating it, and then mixing the concentrated solution with ethanol and precipitating it at 40-50°C for 8-16 hours.

[0045] Furthermore, the probiotics include any one or more combinations of Lactobacillus rhamnosus LGG strain, Bifidobacterium, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus acidophilus, but are not limited thereto.

[0046] Furthermore, in step (3), the volume ratio of the polyphenol ethanol solution to the polyvinyl pyrrolidone solution is 1:10-1:5; wherein the concentration of the polyvinyl pyrrolidone solution is 0.2-0.6 mg / mL.

[0047] Furthermore, the mass ratio of the zinc salt to gallic acid is 1:5-1:10.

[0048] Furthermore, the polyphenolic substances include any one or more combinations of magnolol, baicalin, luteolin, ginsenosides, puerarin, and berberine, but are not limited thereto.

[0049] Further, the zinc salt includes zinc chloride, but is not limited thereto.

[0050] Furthermore, in step (4), the volume ratio of the probiotic concentrate to the zinc-gallic acid-encapsulated polyphenol nanoparticle solution is 1:5-1:10.

[0051] Furthermore, step (5) specifically includes: mixing pachymaran with PBS solution to form a pachymaran PBS solution with a concentration of 5-10 mg / mL, and then adding the first solid product and mixing thoroughly to obtain a first suspension.

[0052] Furthermore, in step (6), the mass ratio of the quaternized chitosan to sodium tungstate dihydrate is 2:1-5:1.

[0053] Furthermore, in step (7), the volume ratio of the first suspension to the solution containing quaternized chitosan and sodium tungstate dihydrate is 1:5-1:10.

[0054] Furthermore, the mass concentration of the sodium tripolyphosphate solution in step (8) is 0.1-0.3%.

[0055] Furthermore, the process parameters adopted by the electrospraying method in step (8) include: an operating voltage of 12 to 16 kV, an injection flow rate of 0.025 to 0.100 ml / min, and a distance of 8 to 16 cm.

[0056] In some more specific embodiments, the preparation method of the double-layer hydrogel-embedded probiotics synergistically delivered polyphenol microcapsules comprises the following steps:

[0057] S1, preparing Poria cocos polysaccharide;

[0058] S2, preparing a probiotic concentrate;

[0059] S3, preparing zinc-gallic acid-embedded polyphenol nanoparticles;

[0060] S4, mixing the probiotic concentrate with zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide in sequence to serve as the inner layer of the probiotic microcapsule;

[0061] S5, preparing a mixed solution of quaternized chitosan and sodium tungstate;

[0062] S6, mixing the probiotic mixture containing zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide with quaternary ammonium chitosan and sodium tungstate to form the outer layer of the probiotic microcapsule;

[0063] S7, spraying the double-layer embedded probiotic mixed solution into the sodium tripolyphosphate solution by electrospraying and letting it stand to solidify;

[0064] S8. Filter and collect the solidified microcapsules.

[0065] As a further technical solution, the preparation of Poria cocos polysaccharide in step S1 comprises the following steps:

[0066] The water-soluble mixture of the Poria cocos brick tea is extracted after crushing, defatting and drying, and then the protein and other small molecules are removed by dialyzing after ethanol precipitation, and the Poria cocos polysaccharide is obtained by freeze drying and stored at room temperature for later use;

[0067] Preferably, the defatting of the tuckahoe brick tea comprises mixing the tuckahoe brick tea with anhydrous ethanol in a ratio of 1:10 (m / v), heating in a water bath with continuous stirring for 4 hours, and filtering to collect the tuckahoe brick tea residue.

[0068] Preferably, the drying comprises placing the tuckahoe brick tea in a vacuum drying oven, drying at 60° C. for 8 hours, and grinding to obtain a powder of the defatted tuckahoe brick tea residue.

[0069] Preferably, the dry powder is dissolved in 90°C water and heated for 1 hour, and the process is repeated three times. The supernatant is collected and concentrated by rotary evaporation.

[0070] Preferably, the collected concentrate is mixed with 80% ethanol and the polysaccharide is precipitated at 4°C for 12 hours.

[0071] Preferably, the dialysis process comprises using a 8KDa-14kDa dialysis bag, dialysis in ultrapure water for three days, and changing the ultrapure water every 4 hours.

[0072] As a further technical solution, the preparation method of the probiotic concentrate described in step S2 comprises the following steps: resuscitating and activating the frozen Lactobacillus rhamnosus LGG in solid LB medium, selecting a single colony and growing it in MRS medium to the logarithmic phase, and then freezing and centrifuging it, washing the precipitate three times with sterile PBS and resuspending it in sterile PBS again to make the concentration of Lactobacillus rhamnosus LGG 10 8 ~10 9 CFU / mL.

[0073] Preferably, the probiotics in step S2 are Lactobacillus rhamnosus LGG strain.

[0074] Preferably, the parameters of the refrigerated centrifugation are: temperature 4°C, rotation speed 6000 rpm, and centrifugation time 15 minutes.

[0075] As a further technical solution, the preparation of zinc-gallic acid-embedded polyphenol nanoparticles described in step S3 comprises the following steps:

[0076] The polyphenols were dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL, and then added to a PVP solution with a concentration of 0.5 mg / mL at a volume ratio of 1:10, and ultrasonically dispersed at 120W for 10 minutes. Zinc chloride and gallic acid were fully mixed at a mass ratio of 1:8, and then added to the polyphenol PVP solution to prepare a mixed solution with a concentration of 10 mg / mL. After sufficient stirring, the solution was centrifuged, and the precipitate was washed three times and freeze-dried to obtain nanoparticles.

[0077] As a further technical solution, the step of sequentially mixing the probiotic concentrate described in step S4 with the zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide comprises:

[0078] Zinc-gallic acid-embedded polyphenol nanoparticles and pachymaran were dissolved in PBS buffer, respectively, added to the probiotic concentrate in sequence, and stirred vigorously to obtain a mixed probiotic suspension.

[0079] Preferably, the concentration of the PBS solution of zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide is 10 mg / mL.

[0080] Preferably, the probiotic concentrate and zinc-gallic acid-embedded polyphenol nanoparticles are vortexed, centrifuged, and the precipitate is washed three times with PBS buffer, and then vortexed with the tuckahoe polysaccharide solution to obtain a probiotic mixture of zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide coating.

[0081] As a further technical solution, the step of preparing the mixed solution of quaternized chitosan and sodium tungstate in step S5 includes: dissolving quaternized chitosan and sodium tungstate dihydrate in PBS solution at a mass ratio of 5:1 to prepare a mixed solution with a concentration of 10 mg / mL.

[0082] As a further technical solution, the probiotic mixture containing the adsorbed zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide as described in step S6 is mixed with quaternized chitosan and sodium tungstate to form the outer layer of the probiotic microcapsule.

[0083] Preferably, the probiotic mixed solution obtained in step S4 is uniformly mixed with the mixed solution of quaternized chitosan and sodium tungstate at a volume ratio of 1:10.

[0084] As a further technical solution, the electrospray parameters include an operating voltage of 12.0 to 16.0 kV, an injection flow rate of 0.025 to 0.100 mL / min, a distance of 8 to 16 cm, and an inner diameter of a syringe needle of 0.6 mm.

[0085] Preferably, the electrospray operating voltage is 14.0 kV, the injection flow rate is 0.050 mL / min, and the distance is 12 cm.

[0086] As a further technical solution, a sodium tripolyphosphate solution having a mass fraction of 0.1 to 0.3% is sprayed in and then allowed to stand and solidify for 30 minutes.

[0087] Another aspect of the embodiments of the present invention further provides double-layer hydrogel-embedded probiotics synergistically delivered polyphenol microcapsules prepared by the aforementioned preparation method.

[0088] In some preferred embodiments, the double-layer hydrogel-encapsulated probiotics and synergistically delivered polyphenol microcapsules include probiotics, an inner layer of the microcapsule encapsulating the probiotics, and an outer layer of the microcapsule; wherein the inner layer of the microcapsule is composed of polyphenol nanoparticles encapsulated by zinc-gallic acid and adsorbed with Poria cocos polysaccharide; the outer layer of the microcapsule is composed of sodium tungstate and quaternized chitosan; the inner layer of the microcapsule and the outer layer of the microcapsule are self-assembled layer by layer through electrostatic interaction to encapsulate the probiotics.

[0089] The preparation of zinc-gallic acid-embedded polyphenol nanoparticles in the present invention not only realizes the efficient embedding of polyphenols, but also realizes the co-delivery of hydrophobic polyphenols and probiotics; at the same time, the present invention combines the dual protection and probiotic effects of tuckahoe polysaccharide and zinc-gallic acid-embedded polyphenol nanoparticles, and synergistically realizes the colon-targeted delivery and efficient colonization of probiotics. The combined use of quaternized chitosan and sodium tungstate as the outer layer of probiotic microcapsules can effectively remove Enterobacter bacteria in the intestine, provide more intestinal sites for the colonization of probiotics, thereby improving the colonization rate and biological activity of probiotics in the intestine.

[0090] The sodium tungstate in the present invention, as a novel microcapsule outer material, has unique antibacterial properties and can effectively remove Enterobacter bacteria in the intestine. Enterobacter bacteria occupy a large number of ecological niches in the intestine, and their overgrowth will inhibit the colonization and growth of probiotics. Through the antibacterial effect of sodium tungstate, more intestinal sites can be provided for the colonization of probiotics, thereby improving the colonization rate and biological activity of probiotics in the intestine. At the same time, quaternized chitosan, as a commonly used microcapsule wall material, has good biocompatibility and biodegradability, and can effectively protect probiotics from being destroyed by digestive juices such as gastric acid and bile salts. In addition, the positively charged quaternized chitosan can bind to the negatively charged mucin in the intestinal mucus layer through electrostatic interaction, thereby prolonging the retention time of probiotics in the colon.

[0091] The present invention uses zinc-gallic acid-embedded polyphenol nanoparticles and tuckahoe polysaccharide as the inner layer of probiotic microcapsules, and uses quaternized chitosan and sodium tungstate as the outer layer of microcapsules to prepare oral colon-targeted delivery microcapsules of probiotics through electrospray technology. The delivery system is applicable to a variety of probiotics, which not only improves the resistance of probiotics to harsh environments and enhances the ability to colonize the intestines, but also acts as a prebiotic to enhance the growth and reproduction of probiotics.

[0092] Another aspect of the embodiments of the present invention further provides the use of the aforementioned double-layer hydrogel-embedded probiotics co-delivery polyphenol microcapsules in the preparation of products with colon-targeted co-delivery of hydrophobic polyphenols and probiotics.

[0093] The present invention innovatively provides a method for preparing a double-layer hydrogel probiotic microcapsule with colon-targeted delivery characteristics. The inner layer of the probiotic microcapsule is composed of polyphenol nanoparticles wrapped in zinc-gallic acid adsorbing tuckahoe polysaccharide, and the outer layer is composed of sodium tungstate and quaternized chitosan. The inner and outer layers are self-assembled and embedded with probiotics layer by layer through electrostatic interaction, and the probiotic microcapsule is prepared by electrospray technology. Tuckahoe polysaccharide can protect probiotics from damage to the gastrointestinal environment, and acts as a prebiotic to improve the vitality of probiotics during processing, storage and oral delivery. Zinc-gallic acid metal phenolic network embeds polyphenols to prepare carrier-free encapsulated hydrophobic polyphenol nanoparticles, which not only achieves efficient embedding of polyphenols but also improves their retention time and retention rate in the intestine. Sodium tungstate can selectively destroy the ecological niche occupied by abnormally expanded Enterobacteriaceae during colitis, thereby promoting the colonization of probiotics. Quaternized chitosan can increase the adhesion and cross-linking of probiotics in the intestine to ensure the colonization of probiotics in the colon. Therefore, the outer layer of the double-layer hydrogel in the present invention can preferentially remove the Enterobacter species in the intestine that hinder the colonization of probiotics, and then continuously release polyphenols and probiotics through the inner layer of hydrogel to synergistically maintain intestinal health. Compared with the prior art, the method provided by the present invention is energy-saving and environmentally friendly, and the raw material source for preparation is green, non-toxic and biodegradable. It not only improves the survival rate of probiotics during processing and storage, but also improves the bioavailability of probiotics in the intestine. In addition, the present invention uses the double-layer hydrogel microcapsules of "outer layer removal + inner layer colonization" for the first time to embed probiotics, enhance the protective performance and functionality of the microcapsules, achieve efficient embedding and colonization of probiotics, and effectively avoid high temperature and mechanical damage to the activity of probiotics. The microcapsules prepared by the method provided by the present invention have uniform particle size distribution and regular appearance, which is conducive to industrial large-scale production.

[0094] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0095] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0096] Example 1

[0097] A method for preparing a double-layer hydrogel-embedded probiotic synergistically delivered hydrophobic polyphenol microcapsule comprises the following steps:

[0098] S1. Preparation of Poria cocos polysaccharide: Crush the Poria cocos brick tea and mix it with anhydrous ethanol in a ratio of 1:10 (m / v), heat in a water bath and stir continuously for 4 hours, filter and collect the Poria cocos brick tea residue, place the residue in a vacuum drying oven, dry it at 60°C for 8 hours, and grind to obtain the defatted Poria cocos brick tea residue powder. Dissolve the dry powder in 90°C water to prepare a 10% (m / v) solution, stir it in a 90°C water bath for 1 hour, let it stand and cool, collect the supernatant and concentrate it by rotary evaporation. After collecting the concentrated solution, mix it with 80% ethanol, precipitate the polysaccharide at 4°C for 12 hours, place it in a 8k-14kDa dialysis bag and dialyze it in ultrapure water for three days, changing the ultrapure water every 4 hours.

[0099] S2. Preparation of probiotic condensate: The frozen Lactobacillus rhamnosus LGG was revived and activated in solid LB medium, and a single colony was selected and grown in liquid MRS medium for 16-18 hours until the logarithmic phase, and centrifuged at 6000 rpm for 15 minutes at 4°C. The precipitate was washed three times with sterile PBS and then resuspended in sterile PBS to make the concentration of Lactobacillus rhamnosus LGG 10 8 ~10 9 CFU / mL.

[0100] S3. Preparation of zinc-gallic acid-embedded magnolol nanoparticles: magnolol was dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL, and then added to a PVP solution with a concentration of 0.5 mg / mL at a volume ratio of 1:10, and ultrasonically dispersed at 120W for 10 minutes. Zinc chloride and gallic acid were fully mixed at a mass ratio of 1:8, and then added to the magnolol PVP solution to prepare a mixed solution with a concentration of 10 mg / mL. After sufficient stirring, the solution was centrifuged, and the precipitate was washed three times and freeze-dried to obtain nanoparticles.

[0101] S4. The probiotic concentrate was sequentially mixed with zinc-gallic acid-embedded magnolol nanoparticles and tuckahoe polysaccharide as the inner layer of the probiotic microcapsule: zinc-gallic acid-embedded magnolol nanoparticles and tuckahoe polysaccharide were dissolved in PBS buffer, respectively, with a concentration of 10 mg / mL. First, the zinc-gallic acid-embedded magnolol nanoparticles and the bacterial suspension prepared in S2 were thoroughly vortexed and then centrifuged and the precipitate was washed three times with PBS buffer, and then vortexed with the tuckahoe polysaccharide solution to obtain a probiotic mixture of zinc-gallic acid-embedded magnolol nanoparticles and tuckahoe polysaccharide coating.

[0102] S5. Prepare a mixed solution of quaternized chitosan and sodium tungstate: dissolve quaternized chitosan and sodium tungstate dihydrate in PBS solution at a mass ratio of 5:1 to prepare a mixed solution with a concentration of 10 mg / mL.

[0103] S6. Preparing a double-layer embedded probiotic mixed solution: uniformly mixing the probiotic mixed solution obtained in S4 with a mixed solution of quaternized chitosan and sodium tungstate at a volume ratio of 1:10.

[0104] S7, spraying the double-layer embedded probiotic mixed solution into the sodium tripolyphosphate solution by electrospraying and letting it stand to solidify, wherein the electrospraying working voltage is 14.0 kV, the injection flow rate is 0.050 mL / min, and the distance is 12 cm. After spraying the sodium tripolyphosphate solution with a mass fraction of 0.2%, let it stand to solidify for 30 minutes.

[0105] S8. Filter and collect the solidified microcapsules.

[0106] Comparative Example 1

[0107] A method for preparing zinc-gallic acid-embedded magnolol nanoparticles / pachymaran / quaternized chitosan-embedded probiotic microcapsules comprises the following steps:

[0108] S1. Preparation of Poria cocos polysaccharide: Crush the Poria cocos brick tea and mix it with anhydrous ethanol in a ratio of 1:10 (m / v), heat in a water bath and stir continuously for 4 hours, filter and collect the Poria cocos brick tea residue, place the residue in a vacuum drying oven, dry it at 60°C for 8 hours, and grind to obtain the defatted Poria cocos brick tea residue powder. Dissolve the dry powder in 90°C water to prepare a 10% (m / v) solution, stir it in a 90°C water bath for 1 hour, let it stand and cool, collect the supernatant and concentrate it by rotary evaporation. After collecting the concentrated solution, mix it with 80% ethanol, precipitate the polysaccharide at 4°C for 12 hours, place it in a 8k-14kDa dialysis bag and dialyze it in ultrapure water for three days, changing the ultrapure water every 4 hours.

[0109] S2. Preparation of probiotic condensate: The frozen Lactobacillus rhamnosus LGG was revived and activated in solid LB medium, and a single colony was selected and grown in liquid MRS medium for 16-18 hours until the logarithmic phase, and centrifuged at 6000 rpm for 15 minutes at 4°C. The precipitate was washed three times with sterile PBS and then resuspended in sterile PBS to make the concentration of Lactobacillus rhamnosus LGG 10 8 ~10 9 CFU / mL.

[0110] S3. Preparation of zinc-gallic acid-embedded magnolol nanoparticles: magnolol was dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL, and then added to a PVP solution with a concentration of 0.5 mg / mL at a volume ratio of 1:10, and ultrasonically dispersed at 120W for 10 minutes. Zinc chloride and gallic acid were fully mixed at a mass ratio of 1:8, and then added to the magnolol PVP solution to prepare a mixed solution with a concentration of 10 mg / mL. After sufficient stirring, the solution was centrifuged, and the precipitate was washed three times and freeze-dried to obtain nanoparticles.

[0111] S4. The probiotic concentrate was sequentially mixed with zinc-gallic acid-embedded magnolol nanoparticles and tuckahoe polysaccharide as the inner layer of the probiotic microcapsule: zinc-gallic acid-embedded magnolol nanoparticles and tuckahoe polysaccharide were dissolved in PBS buffer, respectively, with a concentration of 10 mg / mL. First, the zinc-gallic acid-embedded magnolol nanoparticles and the bacterial suspension prepared in S2 were thoroughly vortexed and then centrifuged and the precipitate was washed three times with PBS buffer, and then vortexed with the tuckahoe polysaccharide solution to obtain a probiotic mixture of zinc-gallic acid-embedded magnolol nanoparticles and tuckahoe polysaccharide coating.

[0112] S5. Prepare quaternary ammonium chitosan solution: dissolve quaternary ammonium chitosan in PBS solution to prepare a mixed solution with a concentration of 10 mg / mL.

[0113] S6. Preparing a double-layer embedded probiotic mixed solution: uniformly mixing the probiotic mixed solution obtained in S4 and the quaternized chitosan solution at a volume ratio of 1:10.

[0114] S7, spraying the double-layer embedded probiotic mixed solution into the sodium tripolyphosphate solution by electrospraying and letting it stand to solidify, wherein the electrospraying working voltage is 14.0 kV, the injection flow rate is 0.050 mL / min, and the distance is 12 cm. After spraying the sodium tripolyphosphate solution with a mass fraction of 0.2%, let it stand to solidify for 30 minutes.

[0115] S8. Filter and collect the solidified microcapsules.

[0116] Comparative Example 2

[0117] A method for preparing zinc-gallic acid-encapsulated magnolol nanoparticles / poochrysanthemum polysaccharide-encapsulated probiotic microcapsules comprises the following steps:

[0118] S1. Preparation of Poria cocos polysaccharide: Crush the Poria cocos brick tea and mix it with anhydrous ethanol in a ratio of 1:10 (m / v), heat in a water bath and stir continuously for 4 hours, filter and collect the Poria cocos brick tea residue, place the residue in a vacuum drying oven, dry it at 60°C for 8 hours, and grind to obtain the defatted Poria cocos brick tea residue powder. Dissolve the dry powder in 90°C water to prepare a 10% (m / v) solution, stir it in a 90°C water bath for 1 hour, let it stand and cool, collect the supernatant and concentrate it by rotary evaporation. After collecting the concentrated solution, mix it with 80% ethanol, precipitate the polysaccharide at 4°C for 12 hours, place it in a 8k-14kDa dialysis bag and dialyze it in ultrapure water for three days, changing the ultrapure water every 4 hours.

[0119] S2. Preparation of probiotic condensate: The frozen Lactobacillus rhamnosus LGG was revived and activated in solid LB medium, and a single colony was selected and grown in liquid MRS medium for 16-18 hours until the logarithmic phase, and centrifuged at 6000 rpm for 15 minutes at 4°C. The precipitate was washed three times with sterile PBS and then resuspended in sterile PBS to make the concentration of Lactobacillus rhamnosus LGG 10 8 ~10 9 CFU / mL.

[0120] S3. Preparation of zinc-gallic acid-embedded magnolol nanoparticles: magnolol was dissolved in anhydrous ethanol to prepare a solution with a concentration of 10 mg / mL, and then added to a PVP solution with a concentration of 0.5 mg / mL at a volume ratio of 1:10, and ultrasonically dispersed at 120W for 10 minutes. Zinc chloride and gallic acid were fully mixed at a mass ratio of 1:8, and then added to the magnolol PVP solution to prepare a mixed solution with a concentration of 10 mg / mL. After sufficient stirring, the solution was centrifuged, and the precipitate was washed three times and freeze-dried to obtain nanoparticles.

[0121] S4. Mix the probiotic concentrate with zinc-gallic acid-encapsulated magnolol nanoparticles and tuckahoe polysaccharide: dissolve zinc-gallic acid-encapsulated magnolol nanoparticles and tuckahoe polysaccharide in PBS buffer, respectively, with the concentration of both being 10 mg / mL. First, thoroughly vortex-mix the zinc-gallic acid-encapsulated magnolol nanoparticles and the bacterial suspension prepared in S2, then centrifuge and wash the precipitate three times with PBS buffer, and then vortex with the tuckahoe polysaccharide solution to obtain a probiotic mixture of zinc-gallic acid-encapsulated magnolol nanoparticles and tuckahoe polysaccharide coating.

[0122] S5. Spray the mixed solution of zinc-gallic acid-encapsulated magnolol nanoparticles / Poria cocos polysaccharide-encapsulated probiotics into the sodium tripolyphosphate solution by electrospraying and let it stand to solidify, wherein the electrospraying voltage is 14.0 kV, the injection flow rate is 0.050 mL / min, and the distance is 12 cm. After spraying the sodium tripolyphosphate solution with a mass fraction of 0.2%, let it stand to solidify for 30 minutes.

[0123] S6. Filter and collect the solidified microcapsules.

[0124] Comparative Example 3

[0125] The method is the same as that of Example 1, except that chitosan is used instead of quaternized chitosan.

[0126] Comparative Example 4

[0127] The method is the same as Example 1, except that: no pachymaran is added;

[0128] Performance Guarantee: Figure 1 The graph is a growth curve of Lactobacillus rhamnosus LGG and the embedded Lactobacillus rhamnosus LGG in Example 1 and Comparative Examples 1-4 of the present invention at 0-24 hours; compared with free probiotics of LGG rhamnosus, Example 1 and Comparative Example 1 can significantly reduce the increase of probiotics and reduce the bacterial concentration in the first 15 hours. This is because the physical barrier provided by the nanoparticles and polysaccharide coating for the probiotics puts the probiotics into a dormant state. After 24 hours of bacterial activation, the coating is lysed and provides nutrients for the probiotics to make them proliferate rapidly. The similar growth curve trends of Example 1 and Comparative Example 1 indicate that the addition of sodium tungstate does not affect the proliferation of bacteria. The similarity between the growth curve trend of Comparative Example 2 and LGG is due to the fact that the probiotics lacking the outer layer of quaternized chitosan and sodium tungstate are more likely to exchange with external substances to promote bacterial proliferation; Comparative Example 3 is the growth curve of probiotics coated with chitosan and sodium tungstate. Since the solubility of chitosan is poorer than that of quaternized chitosan, the adsorption performance is weaker, so a complete probiotic outer coating cannot be formed, resulting in its failure to effectively restrict the proliferation of probiotics. Comparative Example 4 is the growth curve of probiotics without the addition of Poria cocos polysaccharide. This is because the preparation of the double-layer hydrogel-embedded probiotic delivery synergistic polyphenol microcapsules is based on electrostatic interaction. The lack of Poria cocos polysaccharide coating leads to electrostatic repulsion between quaternized chitosan and zinc-gallic acid-embedded magnolol, making it difficult to adsorb on the outer layer of the probiotics. Figure 2 Surface potential diagrams of Lactobacillus rhamnosus LGG and microcapsules prepared in Example 1 and Comparative Examples 1-4 of the present invention; ordinary rhamnosus LGG has a negative surface charge due to the polysaccharides and proteins on its surface, the outermost coating of the probiotic in Example 1 is positively charged quaternary ammonium chitosan and sodium tungstate, and therefore has a strong positive charge, the effect of the outer coating without adding sodium tungstate on the charge in Comparative Example 1 is negligible, and the surface of the probiotic exhibits the negative charge of Poria cocos polysaccharide in Comparative Example 2 because the positively charged quaternary ammonium chitosan is not coated, and the positive potential of Comparative Examples 3 and 4 is reduced compared with that of Example 1, which is due to the poor solubility of chitosan and electrostatic repulsion, which leads to a reduction in the amount of positive charge carried on the surface of the probiotic. Figure 3 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated gastric fluid in vitro for 2 hours; Figure 4 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated small intestinal fluid in vitro for 2 hours; Figure 5 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated colon fluid in vitro for 2 hours; Figure 6 It is a graph of the standardized bacterial survival rate of Lactobacillus rhamnosus LGG and the Lactobacillus rhamnosus LGG embedded in Example 1 of the present invention and Comparative Examples 1-4 after reacting in simulated bile salt in vitro for 2 hours; Figure 3-6 The standardized bacterial survival rate of free LGG is less than 0.4. The probiotics in Example 1 and Comparative Example 1 that are embedded in a double layer of hydrogel can effectively resist the damage of simulated gastric juice, intestinal juice and bile salts, and effectively improve the bacterial survival rate. In Comparative Examples 2-4, since the quaternary ammonium chitosan is not effectively coated on the outer layer of the probiotics to reduce its ability to resist the simulated digestive fluid, the bacterial survival rate decreases. Figure 7 The figure shows the growth inhibition of different Enterobacteriaceae by Lactobacillus rhamnosus LGG and the embedded Lactobacillus rhamnosus LGG in Example 1 of the present invention and Comparative Examples 1-4. In Example 1, sodium tungstate is added to the quaternized chitosan to make the outer layer of the probiotics have the ability to remove Escherichia coli, and its antibacterial effect on Escherichia coli is more significant than that of LGG. Comparative Examples 1-2 have poor removal ability for Escherichia coli because sodium tungstate and quaternized chitosan coating are not added. In Comparative Examples 3-4, although sodium tungstate is added, due to the poor solubility of chitosan and the electrostatic repulsion effect, sodium tungstate fails to be effectively wrapped on the outside of the LGG probiotics in large quantities, which weakens its ability to remove pathogenic bacteria Escherichia coli.

[0129] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0130] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a double-layer hydrogel-embedded probiotic synergistically delivered polyphenol microcapsule, characterized in that: include: Provides Poria polysaccharide and probiotics; Mixing a suspension containing at least polyphenols with zinc salt and gallic acid to produce zinc-gallic acid-embedded polyphenol nanoparticles; The zinc-gallic acid-embedded polyphenol nanoparticles are mixed with probiotics to produce a first solid product; And, the first solid product is mixed with Poria cocos polysaccharide, and then a solution containing at least quaternized chitosan and sodium tungstate dihydrate is added, and then the obtained mixed solution is electrosprayed to prepare double-layer hydrogel-embedded probiotics-co-delivered polyphenol microcapsules.

2. The preparation method according to claim 1, characterized in that: Specifically include: (1) crushing, defatting, drying, extracting, precipitating, dialysis, and freeze-drying the Poria cocos brick tea to obtain Poria cocos polysaccharide; (2) Provide a bacterial concentration of 10 8 ~10 9 CFU / mL of probiotic concentrate; (3) dissolving the polyphenols in ethanol to form a polyphenols ethanol solution, then adding the solution to a polyvinyl pyrrolidone solution to form a polyphenols suspension, and then dispersing the polyphenols suspension in an aqueous solution containing zinc salt and gallic acid, performing ultrasonication, centrifugation, washing, precipitation, and PBS dispersion treatment to obtain a zinc-gallic acid-encapsulated polyphenols nanoparticle solution; (4) mixing the probiotic concentrate with the zinc-gallic acid-encapsulated polyphenol nanoparticle solution, stirring and centrifuging the mixture to obtain a first solid product; (5) adding Pachymaran to a PBS solution and then adding the first solid product and mixing thoroughly to prepare a first suspension; (6) mixing the quaternized chitosan, sodium tungstate dihydrate and PBS solution uniformly to obtain a solution containing quaternized chitosan and sodium tungstate dihydrate; (7) vortex mixing the first suspension with a solution containing quaternized chitosan and sodium tungstate dihydrate to prepare a probiotic suspension; (8) The probiotic suspension is injected into a sodium tripolyphosphate solution by electrospraying, allowed to stand and solidify, and then filtered to obtain a double-layer hydrogel-embedded probiotic-co-delivered polyphenol microcapsule.

3. The preparation method according to claim 2, characterized in that: The degreasing treatment in step (1) comprises: mixing the crushed Poria brick tea with ethanol and stirring at 70-90° C. for 2-6 hours, and then filtering; preferably, the mass volume ratio of the Poria brick tea to the ethanol is 0.1-0.5 mg: 1 mL; And / or, the extraction and precipitation treatment in step (1) includes: dissolving the dried product in water at 80-100°C and heating it for 2-4 hours, then collecting the supernatant, concentrating it, and then mixing the concentrated solution with ethanol and precipitating it at 40-50°C for 8-16 hours.

4. The preparation method according to claim 2, characterized in that: The probiotics include any one or more combinations of Lactobacillus rhamnosus LGG strain, Bifidobacterium, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus acidophilus.

5. The preparation method according to claim 2, characterized in that: The volume ratio of the polyphenol ethanol solution to the polyvinyl pyrrolidone solution in step (3) is 1:5-1:10; wherein the concentration of the polyvinyl pyrrolidone solution is 0.2-0.6 mg / mL; And / or, the mass ratio of the zinc salt to gallic acid is 1:5-1:10; And / or, the polyphenolic substances include any one or more combinations of magnolol, baicalin, luteolin, ginsenoside, puerarin, and berberine; And / or, the zinc salt comprises zinc chloride.

6. The preparation method according to claim 2, characterized in that: The volume ratio of the probiotic concentrate to the zinc-gallic acid-encapsulated polyphenol nanoparticle solution in step (4) is 1:5-1:

10.

7. The preparation method according to claim 2, characterized in that: Step (5) specifically comprises: mixing pachymaran with a PBS solution to form a pachymaran PBS solution with a concentration of 5-10 mg / mL, and then adding the first solid product and mixing thoroughly to obtain a first suspension; And / or, in step (6), the mass ratio of the quaternized chitosan to sodium tungstate dihydrate is 2:1-5:1; And / or, in step (7), the volume ratio of the first suspension to the solution containing quaternized chitosan and sodium tungstate dihydrate is 1:5-1:10; And / or, the mass concentration of the sodium tripolyphosphate solution in step (8) is 0.1-0.3%; And / or, the process parameters adopted by the electrospraying method in step (8) include: working voltage of 12-16 kV, injection flow rate of 0.025-0.100 ml / min, and distance of 8-16 cm.

8. Double-layer hydrogel-embedded probiotics synergistically delivered polyphenol microcapsules prepared by the preparation method according to any one of claims 1 to 7.

9. The double-layer hydrogel-embedded probiotics synergistically delivered polyphenol microcapsules according to claim 8, characterized in that: The invention comprises probiotics, a microcapsule inner layer encapsulating the probiotics and a microcapsule outer layer; wherein the microcapsule inner layer is composed of polyphenol nanoparticles encapsulated by zinc-gallic acid and adsorbed with tuckahoe polysaccharide; the microcapsule outer layer is composed of sodium tungstate and quaternized chitosan; and the probiotics are embedded in the microcapsule inner layer and the microcapsule outer layer by self-assembly layer by layer through electrostatic interaction.

10. Use of the double-layer hydrogel-embedded probiotics co-delivery polyphenol microcapsules according to claim 8 or 9 in preparing products with colon-targeted co-delivery of hydrophobic polyphenols and probiotics.

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