A probiotic encapsulation material, an encapsulated probiotic and a preparation method thereof

Abstract

The present invention belongs to the technical field of biological preparations, and specifically relates to a probiotic encapsulation material, an encapsulated probiotic and a preparation method thereof. The probiotic encapsulation material provided by the present invention includes a protective layer and a functional layer stacked from the inside to the outside; the protective layer is a proanthocyanidin metal complex; the functional layer is a functionally active molecule that is physically adsorbed or chemically bonded to the proanthocyanidin group in the protective layer, and the chemical bonding is a chemical bonding of the functionally active molecule to the phenolic hydroxyl group in the proanthocyanidin group, and the functionally active molecule includes one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and its derivatives. The probiotic encapsulation material provided by the present invention increases the number of probiotics reaching the colon, and at the same time significantly improves the adhesion, colonization and survival rate of probiotics in a complex gastrointestinal environment, thereby improving the oral availability and in vivo survival rate of probiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparations, and in particular relates to a probiotic encapsulation material, an encapsulated probiotic and a preparation method thereof. Background Art

[0002] Humans are "superorganisms" composed of human cells and symbiotic microbial communities. There is a complex interaction between the human body and the microbial community, so all physiological processes of the human body need to consider the influence of symbiotic microorganisms. The intestinal flora is an extremely complex symbiotic microbial ecosystem in the human body, with a population of up to 10 13 . There are more than 1,000 species of anaerobic microorganisms in the intestine, encoding about 3.3 million specific genes, which is more than 150 times the number of genes encoded by the human genome. The human intestinal flora has a variety of physiological functions due to its diversity. Under normal circumstances, the intestinal flora is in a relatively steady-state balance. However, in the process of co-evolution of the intestinal flora and the host, this balance will be broken under certain conditions, leading to the occurrence of diseases. The intestinal flora is closely related to physiological functions such as metabolism, nutrition, immunity and host health. Intestinal flora disorders can lead to the occurrence of many host diseases, such as obesity, diabetes, enteritis and even malignant tumors. Therefore, intestinal microbial homeostasis plays an extremely important role in the host's immune regulation and health maintenance.

[0003] The intestinal flora has good plasticity, and personalized and precise regulation of it can have a positive impact on the treatment of various diseases. A large number of basic research and clinical trial results have shown that probiotics play an important role in regulating the intestinal flora and maintaining flora homeostasis. Fecal microbiota transplantation is an effective means of preventing and treating diseases caused by dysbiosis, but this interventional therapy inevitably reduces patient compliance, and there may also be gastrointestinal irritation and potential complications.

[0004] Oral administration is a common and most acceptable way of administration for patients, and is an ideal way to supplement probiotics. Therefore, regulating intestinal microorganisms through oral intervention with specific probiotics may become a new method for treating a variety of diseases including metabolic-related diseases, cardiovascular diseases, and even tumors. Oral probiotics can significantly inhibit the colonization of pathogens, regulate intestinal flora, and enhance intestinal immunity. However, oral probiotics also face a series of problems and limitations. The complex gastrointestinal environment and continuous intestinal peristalsis will lead to low oral utilization and limited intestinal colonization rate. First, the low pH in gastric juice and a large amount of digestive enzymes and bile acids in the intestine can all lead to the inactivation of probiotics, thereby reducing their oral utilization and therapeutic effect; secondly, the rapid peristalsis of the gastrointestinal tract reduces the retention time of probiotics in the intestine, thereby reducing the adhesion and colonization of probiotics in the intestine. In addition, the integrity of the intestinal barrier function will also affect the colonization of oral probiotics in the intestine. These problems have greatly limited the clinical application and market transformation of oral probiotics.

[0005] Therefore, there is an urgent need to precisely control oral probiotics and regulate their biological behaviors, so as to improve the oral availability and in vivo survival rate of probiotics. Existing methods include genetic modification of probiotics using synthetic biology and other methods, which produces genetically engineered bacteria with stronger stress resistance, and also enables them to have some characteristics that the original probiotics do not have. However, this modification method is based on gene recombination, which is an irreversible process and may bring about some potential gene mutations. There are certain safety risks in its application on the human body. In recent years, researchers have tried to improve the in vivo survival rate of oral probiotics by means of enteric-coated capsules, hydrogels and dry powders, but so far no very ideal results have been achieved. In addition, the complex preparation process and the encapsulation materials used will also affect the activity of probiotics, further increasing the difficulty of their clinical transformation.

[0006] With the development of technology, surface modification of bacteria has become a simple and effective strategy. The surface of bacteria is extremely important for maintaining their biological behavior. There are many inherent antigens, adhesion factors, and "motor organs" such as flagella on the surface of bacteria, which play a very critical role in the physiological functions of bacteria, such as bacterial adhesion and colonization in the intestine. Therefore, on the basis of not affecting the vitality of living probiotics, developing a material chemical modification method for the surface of living organisms, while protecting probiotics from gastrointestinal damage and giving them additional drug activity, has far-reaching clinical significance and huge market value. Summary of the invention

[0007] The purpose of the present invention is to provide a probiotic encapsulation material, an encapsulated probiotic and a preparation method thereof. The probiotic encapsulation material provided by the present invention increases the number of probiotics reaching the colon, and at the same time significantly improves the adhesion, colonization and survival rate of probiotics in a complex gastrointestinal environment, thereby improving the oral availability and in vivo survival rate of probiotics.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The invention provides a probiotic encapsulation material, which comprises a protective layer and a functional layer stacked from the inside to the outside; the protective layer is a proanthocyanidin metal complex; the functional layer is a functionally active molecule that is physically adsorbed or chemically combined with a proanthocyanidin group in the protective layer, wherein the chemical combination is a chemical combination of a thiol group or an amino group in the functionally active molecule with a phenolic hydroxyl group in the proanthocyanidin group through Michael addition or Schiff base reaction, and the functionally active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof.

[0010] Preferably, the metal ions in the proanthocyanidin metal complex include Fe 3+ 、Zn 2+ , Ca 2+ Mg 2+ 、Ti 4+ 、Ce 4 + , Cu 2+ and Mn 2+ One or more of .

[0011] The present invention provides a method for preparing encapsulated probiotics, comprising the following steps:

[0012] Proanthocyanidins, water-soluble metal salts and water are mixed, and the obtained mixed solution undergoes coordination chelation reaction to obtain a protective layer;

[0013] The protective layer and the probiotic solution are mixed for first encapsulation to obtain probiotics encapsulated by the protective layer;

[0014] The probiotics encapsulated in the protective layer are mixed with the functional active molecule solution to undergo physical adsorption or chemical combination for second encapsulation, and a functional layer is formed on the outer surface of the protective layer to obtain the encapsulated probiotics.

[0015] Preferably, in the mixed solution, the mass concentration of proanthocyanidins is 0.5-50 mg / mL, and the mass concentration of metal ions is 0.1-5 mg / mL.

[0016] Preferably, the pH value of the mixed solution ranges from 4 to 10.

[0017] Preferably, the concentration of probiotics in the probiotic solution is 1×10 5 ~1×10 9 CFU / mL.

[0018] Preferably, the probiotics include one or more of enterococci, streptococci, bifidobacteria, lactobacilli, propionibacteria, bacillus, yeast and Escherichia coli.

[0019] Preferably, the mass concentration of the functionally active molecule solution is 0.5-50 mg / mL.

[0020] Preferably, the temperature of the coordination chelation reaction is 25 to 37° C., and the time of the coordination chelation reaction is 1 to 2 hours;

[0021] The temperature of the first encapsulation is 25 to 37° C., and the insulation time is 1 to 2 hours;

[0022] The temperature of the second encapsulation is 1-4° C., and the insulation time is 1-2 hours.

[0023] The present invention provides encapsulated probiotics prepared by the preparation method described in the above technical solution, including probiotics and an encapsulation material encapsulating the probiotics, wherein the encapsulation material is the probiotic encapsulation material described in the above technical solution, and the protective layer in the encapsulation material is in contact with the probiotics.

[0024] The present invention provides a probiotic encapsulation material, which comprises a protective layer and a functional layer stacked from the inside to the outside; the protective layer is a proanthocyanidin metal complex; the functional layer is a functional active molecule that is physically adsorbed or chemically combined with a proanthocyanidin group in the protective layer, wherein the chemical combination is a chemical combination of a thiol group or an amino group in the functional active molecule with a phenolic hydroxyl group in the proanthocyanidin group through Michael addition or Schiff base reaction, and the functional active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof. The present invention uses a proanthocyanidin metal complex with a three-dimensional network structure formed by coordination chelation reaction between proanthocyanidins and metal ions as a protective layer of the encapsulation material of probiotics, which can significantly improve the survival rate of probiotics in complex gastrointestinal environments (for example: strong acid conditions, antibiotics, strong oxidation, etc.), and increase the number of probiotics reaching the colon; at the same time, the present invention uses one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and their derivatives as functional active molecules to react with the proanthocyanidin groups in the proanthocyanidin metal complex to form a functional layer. The formed functional layer has both anionic surface charge and mucosal adhesion, and can also interact with inflammatory receptors, which can not only significantly increase the adhesion and colonization of probiotics in the intestine, but also avoid non-targeted immunosuppression, can effectively adhere to cationic inflammatory surfaces, enhance the probiotic drug concentration in the local inflammatory area, and achieve site-specific delivery of probiotics in the intestinal inflammatory area. Thus, the probiotic encapsulation material provided by the present invention increases the number of probiotics reaching the colon part, and significantly improves the adhesion, colonization and survival rate of probiotics in a complex gastrointestinal environment, thereby improving the oral availability and in vivo survival rate of probiotics. The results of the embodiment show that, compared with healthy mice, in a colitis mouse model, the presence of the functional layer of the probiotic encapsulation material provided by the present invention can effectively adhere the encapsulated probiotics to the inflammatory area and enhance the probiotic drug concentration in the inflammatory area. However, after the functional molecules are physically mixed with the probiotics with only a protective layer, the adhesion of the probiotics in the inflammatory area cannot be significantly increased, and the probiotic encapsulation material provided by the present invention encapsulates the probiotics and significantly inhibits the secretion of colon inflammatory factors. The above embodiment results show that the probiotic encapsulation material provided by the present invention can effectively adhere to the cationic inflammatory surface, enhance the probiotic drug concentration in the local inflammatory area, and achieve site-specific delivery of probiotics in the intestinal inflammatory area.

[0025] The present invention provides a method for preparing encapsulated probiotics, comprising the following steps: mixing proanthocyanidins, water-soluble metal salts and water, subjecting the obtained mixed solution to a coordination chelation reaction to obtain a protective layer; mixing the protective layer and the probiotic solution for a first encapsulation to obtain probiotics encapsulated by the protective layer; mixing the probiotics encapsulated by the protective layer, an aqueous solution of functional active molecules and a buffer solution for physical adsorption or chemical combination to perform a second encapsulation, forming a functional layer on the outer surface of the protective layer to obtain the encapsulated probiotics. The encapsulation method provided by the present invention is simple, convenient, rapid and safe, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention detects the growth of the probiotics, EcN@PC-Fe and EcN@PC-Fe / HA in Example 1 respectively exposed to simulated gastric fluid (SGF) containing pepsin and bile acid (pH = 2);

[0027] Figure 2 Live-dead staining of the probiotics, EcN@PC-Fe and EcN@PC-Fe / HA in Example 1 of the present invention exposed to simulated gastric fluid (SGF) containing pepsin and bile acid (pH=2) respectively;

[0028] Figure 3 Live-dead staining of LaC, LaC@PC-Ca and LaC@PC-Ca / Alg in Example 2 of the present invention exposed to simulated gastric fluid (SGF) containing pepsin and bile acid (pH=2) respectively;

[0029] Figure 4 The present invention detects the growth of the probiotics, EcN@PC-Fe and EcN@PC-Fe / HA in Example 1 respectively exposed to simulated intestinal fluid (SiF) containing trypsin (pH = 6.8);

[0030] Figure 5 The morphological characterization of the probiotics, EcN@PC-Fe and EcN@PC-Fe / HA in Example 1 of the present invention under TEM;

[0031] Figure 6 The in vitro growth curves of the probiotics, EcN@PC-Fe and EcN@PC-Fe / HA in Example 1 of the present invention are shown;

[0032] Figure 7 The in vivo distribution of EcN@PC-Fe, EcN@PC-Fe / HA and unencapsulated EcN prepared in Example 1 at different time points after oral administration to mice in the present invention;

[0033] Figure 8The distribution of the encapsulated EcN and the unencapsulated EcN prepared in Example 1 in the present invention in the intestines of mice 120 hours after oral administration;

[0034] Fig. 9 The survival rates of different parts of the intestine of mice 120 hours after intragastric administration of EcN@PC-Fe, EcN@PC-Fe / HA and unencapsulated EcN prepared in Example 1 of the present invention;

[0035] Fig.10 The changes in body weight and disease activity index of the colitis mouse model treated by intragastric administration of encapsulated EcN prepared in Example 1 and unencapsulated EcN in the present invention;

[0036] Fig.11 The results of hematoxylin and eosin staining of the intestine of mice with colitis treated by intragastric administration using EcN@PC-Fe, EcN@PC-Fe / HA and unencapsulated EcN prepared in Example 1 in the present invention were used to evaluate the pathological histological damage of the colon;

[0037] Fig.12 The distribution and fluorescence intensity statistics of the encapsulated EcN prepared in Example 1 in the colon of healthy mice and colitis mice after intragastric administration;

[0038] Fig.13 The changes of intestinal inflammatory factors in the colitis mouse model treated by intragastric administration of EcN@PC-Fe, EcN@PC-Fe / HA and unencapsulated EcN prepared in Example 1 of the present invention;

[0039] Fig.14 This is a TEM characterization image of EcN@PC-Fe / HA prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0040] The invention provides a probiotic encapsulation material, which comprises a protective layer and a functional layer stacked from the inside to the outside; the protective layer is a proanthocyanidin metal complex; the functional layer is a functionally active molecule that is physically adsorbed or chemically combined with a proanthocyanidin group in the protective layer, wherein the chemical combination is a chemical combination of a thiol group or an amino group in the functionally active molecule with a phenolic hydroxyl group in the proanthocyanidin group through Michael addition or Schiff base reaction, and the functionally active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof.

[0041] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0042] In the present invention, the metal ions in the proanthocyanidin metal complex preferably include Fe 3+ 、Zn2+ , Ca 2+ Mg 2 + 、Ti 4+ 、Ce 4+ , Cu 2+ and Mn 2+ One or more of, more preferably Fe 3+ , Ca 2+ or Mg 2+ .

[0043] In the present invention, the functionally active molecule includes one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof, preferably hyaluronic acid, sodium alginate, chondroitin sulfate or heparan sulfate.

[0044] In the present invention, the molecular weight of the sodium hyaluronate is preferably 30 to 500 kDa, more preferably 360 kDa.

[0045] In the present invention, the thickness ratio of the protective layer to the functional layer is preferably (1-2):3.

[0046] The present invention provides a method for preparing encapsulated probiotics, comprising the following steps:

[0047] Proanthocyanidins, water-soluble metal salts and water are mixed, and the obtained mixed solution undergoes coordination chelation reaction to obtain a protective layer;

[0048] The protective layer and the probiotic solution are mixed for first encapsulation to obtain probiotics encapsulated by the protective layer;

[0049] The probiotics encapsulated in the protective layer are mixed with the functional active molecule solution to undergo physical adsorption or chemical combination for second encapsulation, and a functional layer is formed on the outer surface of the protective layer to obtain the encapsulated probiotics.

[0050] In the present invention, proanthocyanidin, water-soluble metal salt and water are mixed (hereinafter referred to as the first mixing), and the obtained mixed solution undergoes a coordination chelation reaction to obtain a protective layer.

[0051] In the present invention, the proanthocyanidins are preferably mixed in the first step in the form of a proanthocyanidin solution.

[0052] In the present invention, the solvent in the proanthocyanidin solution is preferably a Tris-HCl buffer. In the present invention, the pH value of the Tris-HCl buffer is preferably 8.5, and the molar concentration of the Tris-HCl buffer is preferably 10 mmol / L.

[0053] In the present invention, the water-soluble metal salt is preferably one or more of a water-soluble trivalent iron salt, a water-soluble zinc salt, a water-soluble calcium salt, a water-soluble magnesium salt, a water-soluble titanium salt, a water-soluble cerium salt, a water-soluble copper salt and a water-soluble manganese salt, more preferably a water-soluble trivalent iron salt, a water-soluble zinc salt, a water-soluble calcium salt or a water-soluble magnesium salt, and specifically preferably a water-soluble trivalent iron salt and a water-soluble calcium salt.

[0054] In a specific embodiment of the present invention, the water-soluble metal salt is preferably ferric chloride and calcium dichloride.

[0055] In the present invention, the first mixing preferably comprises the following steps: dissolving the water-soluble metal salt in water to obtain a metal salt solution; mixing the proanthocyanidin solution and the metal salt solution. In the present invention, the proanthocyanidin solution and the metal salt solution are preferably mixed in a vortex oscillator, and the mixing time in the vortex oscillator is preferably 20 seconds.

[0056] In the present invention, in the mixed solution, the mass concentration of proanthocyanidins is preferably 0.5-50 mg / mL, more preferably 1-45 mg / mL. In the present invention, in the mixed solution, the mass concentration of metal ions is 0.1-5 mg / mL, more preferably 0.2-4.5 mg / mL.

[0057] In the present invention, the pH range of the mixed solution is 4-10, preferably 5-8.

[0058] In the present invention, the temperature of the coordination chelation reaction is preferably 25-37°C, more preferably 37°C; the time of the coordination chelation reaction is preferably 1-2h, more preferably 1h; the coordination chelation reaction is preferably carried out under stirring, and the stirring speed is preferably 150rpm.

[0059] In the present invention, in the mixed solution, the proanthocyanidins and metal ions undergo coordination and chelation reactions to generate proanthocyanidin metal complexes with a three-dimensional network macromolecular structure.

[0060] In the present invention, a protective layer solution is obtained after the coordination chelation reaction. In the present invention, the protective layer solution is preferably directly encapsulated.

[0061] After obtaining the protective layer, the present invention mixes the protective layer with the probiotic solution (hereinafter referred to as the second mixing) for first encapsulation to obtain probiotics encapsulated by the protective layer.

[0062] In the present invention, the concentration of probiotics in the probiotic solution is preferably 1×10 5 ~1×10 9 CFU / mL, more preferably 2×10 5 ~0.5×109 CFU / mL.

[0063] In the present invention, the probiotic solution is preferably a PBS buffer solution of probiotics.

[0064] The present invention has no special requirements on the source or preparation method of the probiotic solution, and it can be obtained by a resin method known to those skilled in the art.

[0065] In the present invention, the probiotics preferably include one or more of enterococci, streptococci, bifidobacteria, lactobacilli, propionibacteria, bacillus, yeast and Escherichia coli, more preferably one or more of Escherichia coli, propionibacteria, bacillus and lactobacilli.

[0066] In the present invention, the second mixing is preferably performed in a vortex oscillator, and the second mixing time is preferably 30 s.

[0067] In the present invention, the temperature of the first encapsulation is preferably 25-37°C, more preferably 37°C; the reaction time of the encapsulation is preferably 1-2h, more preferably 1h. The first encapsulation is preferably carried out under stirring, and the stirring speed is preferably 150rpm.

[0068] In the present invention, during the first encapsulation, the three-dimensional mesh nanofilm formed by the protective layer encapsulates the probiotics to obtain probiotics encapsulated by the protective layer.

[0069] In the present invention, the first encapsulation reaction liquid is obtained after the first encapsulation. The present invention preferably performs post-treatment on the first encapsulation reaction liquid to obtain the probiotics encapsulated by the protective layer. In the present invention, the post-treatment preferably includes the following steps: performing solid-liquid separation on the first encapsulation reaction liquid to obtain a solid product; washing the solid product to obtain the probiotics encapsulated by the protective layer. In the present invention, the solid-liquid separation is preferably centrifugation, the centrifugal speed is preferably 1000-5000rpm, more preferably 1500-3500rpm, and the centrifugal time is preferably 5min. The washing medium is preferably a sodium chloride solution, and the molar concentration of the sodium chloride solution is preferably 0.15mol / L. The number of washings is preferably 3 times, and the present invention preferably removes the unreacted raw materials by washing.

[0070] After obtaining the probiotics encapsulated by the protective layer, the present invention mixes the probiotics encapsulated by the protective layer with the functional active solution to undergo physical adsorption or chemical combination for a second encapsulation, and forms a functional layer on the outer surface of the protective layer to obtain the encapsulated probiotics.

[0071] In the present invention, the solvent in the functional active solution is preferably a Tris-HCl buffer. In the present invention, the pH value of the Tris-HCl buffer is preferably 8.5, and the molar concentration of the Tris-HCl buffer is preferably 10 mmol / L.

[0072] In the present invention, the mass concentration of the functionally active molecule solution is preferably 0.5 to 50 mg / mL, more preferably 1 to 45 mg / mL.

[0073] In the present invention, the temperature of the second encapsulation is preferably 1-4°C, more preferably 4°C; the insulation time of the second encapsulation is preferably 1-2h, more preferably 1h, and the second encapsulation is preferably carried out under stirring.

[0074] In the present invention, during the second encapsulation, the functionally active molecules undergo physical adsorption or chemical binding reaction with the proanthocyanidin groups in the protective layer to form a functional layer on the surface of the protective layer.

[0075] In the present invention, after the second encapsulation, a second encapsulation reaction liquid is obtained. In the present invention, the second encapsulation reaction liquid is preferably post-processed to obtain the encapsulated probiotics. In the present invention, the post-processing preferably includes the following steps: the second encapsulation reaction liquid is subjected to solid-liquid separation to obtain a solid product; the solid product is washed to obtain the encapsulated probiotics. In the present invention, the solid-liquid separation is preferably centrifugation, the centrifugal speed is preferably 1000-5000rpm, more preferably 1500-3500rpm, and the centrifugal time is preferably 5min. The washing medium is preferably a sterile PBS solution, and the molar concentration of the PBS solution is preferably 0.01M. The number of washings is preferably 3 times. After the washing, the present invention preferably centrifuges to collect the solid product to obtain the encapsulated probiotics. The centrifugal speed is preferably 1000-5000rpm, more preferably 1500-3500rpm, and the centrifugal time is preferably 5min.

[0076] The present invention provides an encapsulated probiotic, comprising probiotics and an encapsulation material encapsulating the probiotics, wherein the encapsulation material is the probiotic encapsulation material described in the above technical solution, and a protective layer in the encapsulation material is in contact with the probiotics.

[0077] In the present invention, the encapsulated probiotics are preferably prepared according to the encapsulation method described in the above technical solution.

[0078] The encapsulated probiotic provided by the present invention includes a nano coating film for encapsulating probiotics; the nano coating includes two layers, the first layer is a protective layer, a three-dimensional mesh nano film formed by coordination and chelation of proanthocyanidins and metal ions, and the second layer is a functional layer, which is formed by physical adsorption or chemical binding reaction of functional molecules and proanthocyanidin groups. The preparation process is simple, convenient, fast and safe, and the formed nano coating can significantly improve the survival rate of probiotics in a complex gastrointestinal environment, and can significantly increase the adhesion and colonization of probiotics in the intestine.

[0079] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0080] Example 1

[0081] In this example, Escherichia coli Nissle 1917 (hereinafter referred to as EcN) was encapsulated and characterized. The specific encapsulation method includes the following steps:

[0082] Take 1 mL 1×10 8 The EcN with the number of CFU or the EcN carrying pBBR1MCS2-Tac-mCherry was centrifuged at 3000 rpm for 3 min, the supernatant was taken, and then the above-mentioned centrifugation and supernatant taking operations were repeated 3 times, and then 0.01 mol / L PBS was added to adjust the total volume to 200 μL, and the mixture was fully mixed to obtain the EcN bacterial solution for use.

[0083] 15 mL of 1 mg / mL proanthocyanidin solution was prepared with pH=8.5, 10 mM Tris-HCl buffer, and then 10 μL of 225 mg / mL ferric chloride aqueous solution was added, vortexed for 20 s to mix the two thoroughly, and reacted at 37°C, 150 rpm for 1 h to allow the proanthocyanidin to react with Fe 3+ Coordinate to form a three-dimensional network macromolecule. Then add 200μL EcN bacterial solution, vortex and shake for 30s, and react at 37℃ and 150rpm for 1h. After the reaction, centrifuge at 4000rpm for 5min, wash the solid product 3 times with 0.15M NaCl solution to remove the unreacted raw materials, and obtain the probiotics encapsulated by the protective layer, which is recorded as EcN@PC-Fe.

[0084] Subsequently, 10 mL of 10 mM Tris-HCl buffer (pH = 8.5) containing 1 mg / mL sodium hyaluronate with a molecular weight of 360 kDa was added for resuspending, and the reaction was continued at 4°C, 150 rpm for 1 h. The completely encapsulated bacteria were obtained by centrifugation at 4000 rpm for 5 min, and washed three times with sterile 0.01 M PBS, centrifuged at 4000 rpm for 5 min, and the precipitated product was collected to obtain encapsulated probiotics, which were recorded as EcN@PC-Fe / HA.

[0085] Example 2

[0086] In this example, Lactobacillus crispatus (hereinafter referred to as LaC) was encapsulated and characterized. The specific encapsulation method includes the following steps:

[0087] Take 1 mL 1×10 6 The LaC with the number of CFU was centrifuged at 3500 rpm for 5 min, the supernatant was taken, and then the above centrifugation and supernatant taking operations were repeated 3 times, and then 0.01 mol / L PBS was added to adjust the total volume to 200 μL, and the mixture was fully mixed to obtain the EcN bacterial solution for use.

[0088] 15 mL of 2 mg / mL proanthocyanidin solution was prepared with pH=8.5, 10 mM Tris-HCl buffer, and then 10 μL of 300 mg / mL calcium chloride aqueous solution was added, vortexed for 20 s to mix the two thoroughly, and reacted at 37°C, 150 rpm for 1 h to allow proanthocyanidin to react with Ca 2+ Coordinate to form a three-dimensional network macromolecule. Then add 200μL LaC bacterial solution, vortex and shake for 30s, and react at 37℃ and 150rpm for 1h. After the reaction, centrifuge at 4000rpm for 5min, wash the solid product 3 times with 0.15M NaCl solution to remove the unreacted raw materials, and obtain the probiotics encapsulated by the protective layer, which is recorded as LaC@PC-Ca.

[0089] Subsequently, 10 mL of 10 mM Tris-HCl buffer (pH = 8.5) containing 5 mg / mL sodium alginate was added for resuspending, and the reaction was continued at 4°C, 150 rpm for 1 h. The completely encapsulated bacteria were obtained by centrifugation at 4000 rpm for 5 min, and washed three times with sterile 0.01 M PBS, centrifuged at 4000 rpm for 5 min, and the precipitated products were collected to obtain encapsulated probiotics, which were recorded as LaC@PC-Ca / Alg.

[0090] Test Example 1

[0091] In vitro gastric juice resistance test

[0092] The same amount of probiotics in Example 1 (1×10 8 CFU) EcN, EcN@PC-Fe and EcN@PC-Fe / HA were resuspended in 1 mL artificial simulated gastric fluid SGF (pH = 2.0, each 100 mL SGF contains 20 mg NaCl, 3 mg bile acid, 32 mg pepsin and 70 μL hydrochloric acid), placed in a 37 ° C incubator at 120 rpm for 2 h, 50 μL of each sample was taken out, washed 3 times with PBS, and finally the sample was diluted with PBS and evenly spread on LB agar plates. After culturing at 37 ° C for 24 hours, the colonies were counted (such as Figure 1 Then, using the bacterial live-dead fluorescent probe (Molecular Probes' BacLight TM Bacterial Viability Kit was used to fluorescently stain the bacteria, and CLSM was used to observe and count the bacterial mortality rates in different treatment groups (such as Figure 2 The results showed that compared with the unencapsulated EcN, the tolerance of EcN@PC-Fe and EcN@PC-Fe / HA to SGF was significantly increased, among which EcN@PC-Fe / HA showed the best protection effect.

[0093] An equal amount of (1×10 6 CFU) of LaC, LaC@PC-Ca and LaC@PC-Ca / Alg were resuspended in 1 mL artificial simulated gastric fluid SGF (pH = 2.0, each 100 mL SGF contained 20 mg NaCl, 3 mg bile acid, 32 mg pepsin and 70 μL hydrochloric acid), incubated at 120 rpm in a 37 ° C incubator for 2 h, and 50 μL of each sample was taken out and washed three times with PBS. After the samples were diluted, the bacterial live-dead fluorescent probe (Molecular Probes' BacLight TM Bacterial Viability Kit was used to fluorescently stain the bacteria, and CLSM was used to observe and count the bacterial mortality rates in different treatment groups (such as Figure 3 The test results showed that compared with the unencapsulated LaC, the tolerance of encapsulated LaC to SGF was significantly increased, among which LaC@PC-Ca / Alg showed the best protection effect.

[0094] Test Example 2

[0095] In vitro intestinal fluid stress resistance test

[0096] An equal amount of (1×10 8EcN, EcN@PC-Fe and EcN@PC-Fe / HA with CFU (100 mL SIF) were resuspended in 1 mL artificial simulated intestinal fluid (SIF) (pH = 6.8, 0.68 g KH2PO4, 15 mL 0.2 M NaOH and 1 g trypsin per 100 mL SIF), incubated at 120 rpm in a 37 ° C incubator for 2 h, 50 μL of each sample was taken out, washed 3 times with PBS, and finally the sample was diluted with PBS and evenly spread on LB agar plates. Colony counts (such as Figure 4 The results showed that the tolerance of encapsulated EcN to SIF was significantly increased compared with that of unencapsulated EcN, among which EcN@PC-Fe / HA showed the best protection effect.

[0097] Test Example 3

[0098] Characterization and analysis of coated probiotics

[0099] The bacterial morphology of EcN, EcN@PC-Fe and EcN@PC-Fe / HA in Example 1 was observed using a transmission electron microscope (TEM). The specific operation of TEM is as follows: take 10 μL of the diluted sample and drop it onto a 200-mesh carbon film copper grid, let it stand for 5 minutes, and use absorbent paper to absorb the remaining liquid. Then wash it three times with PBS, and place the copper grid in a 37°C constant temperature incubator to dry for 12 hours. Use a transmission electron microscope (Hitachi H7500) for observation, and the detection voltage is set to 80 kV. The transmission electron microscope image shows that the surface of EcN@PC-Fe has a translucent film coating with a thickness of about 50 nm. The modification of the functional molecule HA increases the thickness of the bacterial surface coating to about 200 nm (such as Figure 5 a in the equation is EcN without coating. Figure 5 b in the equation is EcN@PC-Fe, Figure 5 c and Fig.14 (EcN@PC-Fe / HA). Figure 5 It can be seen that the EcN@PC-Fe / HA encapsulation layer prepared in Example 1 of the present invention is significantly thicker than that of EcN@PC-Fe, indicating that the encapsulation layer of EcN@PC-Fe / HA prepared in Example 1 of the present invention is two layers, the first layer is a protective layer, which is a three-dimensional network nanofilm formed by coordination chelation between proanthocyanidins and metal ions, and the second layer is a functional layer, which is formed by physical adsorption or chemical binding reaction between functional molecules and proanthocyanidin groups.

[0100] Test Example 4

[0101] Growth curve monitoring of coated probiotics

[0102] In order to detect whether the encapsulation coating would affect bacterial growth, we diluted the EcN in Example 1 and the two encapsulated probiotics EcN@PC-Fe and EcN@PC-Fe / HA with fresh LB medium to a value of 0.2 for optical density (OD600), and then placed them in a 37°C incubator at a constant temperature of 200 rpm. The readings at OD600nm were recorded every 30 minutes using a microplate spectrophotometer to record the growth curve of the encapsulated probiotics. Figure 6 As shown, the logarithmic growth phase of EcN@PC-Fe and EcN@PC-Fe / HA lagged behind that of EcN by 1-2 h, indicating that it takes a certain amount of time for probiotics to break through the coating barrier, resulting in a relatively delayed logarithmic growth phase. The lag time is related to the coating thickness, but the presence of the coating does not affect the activity of the encapsulated probiotics.

[0103] Test Example 5

[0104] Evaluation of the retention and survival rate of encapsulated probiotics in mice

[0105] Six-week-old C57BL / 6J mice were selected as experimental animals. All mice were adaptively raised in a SPF-grade animal room with constant temperature (22±1°C) and constant humidity (50%-60%) for one week, with a 12-h light-dark cycle and free access to food and water. 8 The mice were given CFU coated probiotics and non-coated probiotics with pBBR1MCS2-Tac-mCherry plasmid by gavage. The mice were anesthetized with 2% isoflurane and oxygen mixed gas anesthesia at 0.5h, 4h, 12h, 24h, 48h and 72h, and then placed in a small animal in vivo imager (PerkinElmer, Waltham, MA, USA) for imaging. Figure 7 As shown in Figure 2, after 72 hours, the fluorescence intensity of the gastrointestinal tract of EcN@PC-Fe and EcN@PC-Fe / HA increased significantly compared with EcN. After 120 hours, the mice were euthanized, and the digestive tract of the mice was removed for in vitro fluorescence detection and the fluorescence signal intensity was recorded. The results are shown in Figure 2. Figure 7 As shown, the fluorescence in the intestine of the EcN group almost disappeared, while the fluorescence in the intestine of the EcN@PC-Fe / HA group was the strongest compared with the EcN@PC-Fe group.

[0106] The contents of the mouse colon and cecum were then scraped, resuspended and diluted with PBS, and evenly spread on LB agar plates containing 50 μg / mL kanamycin. After being cultured at 37°C for 24 hours, the colonies were counted to evaluate the retention and survival rate of the encapsulated probiotics in the mice. Fig. 9As shown, after 120 hours, the probiotics were mainly distributed in the cecum and colon. Compared with EcN, the number of encapsulated probiotics in the cecum and colon of mice increased significantly, among which the EcN@PC-Fe / HA group had the largest number of live bacteria. In the colon, the number of live bacteria was 150 times and 1.5 times that of the EcN group and the EcN@PC-Fe group, respectively. The above results are consistent with the in vitro stress resistance results. The coating-encapsulated probiotics showed significantly increased survival and colonization rates in the gastrointestinal tract, especially in the cecum and colon.

[0107] Test Example 6

[0108] Evaluation of the therapeutic effect of encapsulated probiotics on ulcerative colitis in mice

[0109] The most classic method of mice freely drinking dextran sulfate sodium DSS aqueous solution for 7 days was used to establish the model. The specific operation was as follows: DSS was dissolved in distilled water to prepare a 2.5% (w / w) solution, which was replaced every 2 days. Mice freely drank 2.5% DSS water for 7 days, and the mice's food intake, water intake, body weight, and fecal occult blood were recorded every day. The disease activity index (DAI) was calculated based on the weight loss, fecal consistency, and occult blood (Table 1).

[0110] Table 1 Disease activity index of mice

[0111]

[0112] DSS-induced colitis mice were given 1×10 8 The encapsulated probiotics of CFU were administered for 5 days to characterize the weight changes and disease activity index of mice, which was used to evaluate the improvement effect of encapsulated probiotics on ulcerative colitis in mice. Fig.10 As shown, compared with mice given PBS and EcN, EcN@PC-Fe and EcN@PC-Fe / HA were able to significantly improve the weight loss of colitis mice and reduce the disease activity index, among which EcN@PC-Fe / HA showed the best effect. The mice were euthanized and dissected, and about 1 cm of distal colon tissue was quickly removed, rinsed three times with pre-cooled saline, and then placed in 4% paraformaldehyde fixative to fully fix the tissue. The remaining colon tissue was placed in a sterile EP tube and stored in a -80°C refrigerator. The fixed distal colon tissue was dehydrated with gradient ethanol, embedded in paraffin, and the slice thickness was 5 μm. The pathological histological damage of the colon was evaluated by hematoxylin and eosin staining. Fig.11The results showed that the colon structure of healthy mice was intact, without inflammation and edema, and the glandular cells were neatly arranged; the colon epithelial layer of DSS model mice was incomplete, with the disappearance of crypts, glands and goblet cells, and a large number of inflammatory infiltrations in the colon, and severe tissue damage. After probiotic intervention, the degree of colon damage was significantly improved, and the inflammatory infiltration was alleviated. Among them, EcN@PC-Fe / HA showed the best improvement effect.

[0113] Test Example 7

[0114] Site-specific adhesion of encapsulated probiotics to inflamed areas of the intestine

[0115] DSS-induced colitis mice and healthy mice were fasted for 12 h, and then 1×10 8 CFU of EcN@PC-Fe / HA carrying pBBR1MCS2-Tac-mCherry plasmid was administered by oral gavage to colitis mice and healthy mice. The mice were euthanized 8 h later, and the colons of the mice were placed in a small animal in vivo imager (PerkinElmer, Waltham, MA, USA) for imaging. Fig.12 The results showed that compared with healthy mice, the adhesion of EcN@PC-Fe / HA in the colon tissue of enteritis mice was significantly increased, resulting in a significant increase in the concentration of probiotics in the local inflammatory area, achieving site-specific delivery of probiotics in the intestinal inflammatory area.

[0116] Test Example 8

[0117] Effects of encapsulated probiotics on the expression of inflammatory factors in colon tissue of mice

[0118] Take 50 mg of colon tissue sample, add 1 mL of pre-cooled TRIzon reagent, use a tissue homogenizer to fully lyse it, then let it stand on ice for 10 minutes, add 200 μL of chloroform, shake for 15 seconds, and then let it stand at room temperature for 3 minutes; centrifuge at 12000 rpm for 15 minutes at 4°C, take 500 μL of colorless aqueous phase (upper layer) and transfer it to a new RNase-free EP tube; add 500 μL of isopropanol, mix and let it stand for 10 minutes; centrifuge at 12000 rpm for 10 minutes at 4°C, and discard the supernatant; add 1 mL of 75% ethanol to wash the precipitate (prepared with 4°C DEPC water); centrifuge at 2000 rpm for 3 minutes at 4°C, and discard the supernatant; let it stand at room temperature for 5-8 minutes, add 30 μL of DEPC water to fully dissolve it; the extracted RNA was reverse transcribed to obtain cDNA, and the expression levels of inflammatory factors TNF-α, IL-6 and IL-1β in mouse colon tissue were determined by real-time fluorescence quantitative PCR. Fig.13The results showed that compared with normal mice, the expression of TNF-α, IL-6 and IL-1β in the colon tissue of the model group mice was significantly increased, indicating that DSS caused severe intestinal inflammation. The unencapsulated probiotic EcN was not effective in improving intestinal inflammation, but after the intervention of the coated probiotics, the expression of inflammatory factors was significantly inhibited, among which EcN@PC-Fe / HA showed the best inflammation inhibition effect.

[0119] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing encapsulated probiotics, characterized in that: The steps include: Mixing proanthocyanidin, water-soluble metal salt and water, and obtaining a mixed solution to undergo coordination chelation reaction to obtain a protective layer; the protective layer is a proanthocyanidin metal complex; the coordination chelation reaction temperature is 25-37° C., and the coordination chelation reaction time is 1-2 hours; The protective layer and the probiotic solution are mixed for first encapsulation to obtain probiotics encapsulated by the protective layer; The probiotics encapsulated in the protective layer and the functionally active molecule solution are mixed and secondly encapsulated through physical adsorption or chemical bonding, and a functional layer is formed on the outer surface of the protective layer to obtain the encapsulated probiotics; the functional layer is a functionally active molecule that is physically adsorbed or chemically bonded to the proanthocyanidin group in the protective layer, and the chemical bonding is that the sulfhydryl group or amino group in the functionally active molecule is chemically bonded to the phenolic hydroxyl group in the proanthocyanidin group through Michael addition or Schiff base reaction, and the functionally active molecule includes one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and their derivatives.

2. The preparation method according to claim 1, characterized in that: The metal ions in the proanthocyanidin metal complex include Fe 3+ 、Zn 2+ , Ca 2+ Mg 2+ 、Ti 4+ 、Ce 4+ , Cu 2+ and Mn 2+ One or more of .

3. The preparation method according to claim 1, characterized in that: In the mixed solution, the mass concentration of proanthocyanidins is 0.5-50 mg / mL, and the mass concentration of metal ions is 0.1-5 mg / mL.

4. The preparation method according to claim 1, characterized in that: The pH value of the mixed solution ranges from 4 to 10.

5. The preparation method according to claim 1, characterized in that: The concentration of probiotics in the probiotic solution is 1×10 5 ~1×10 9 CFU / mL.

6. The preparation method according to claim 5, characterized in that: The probiotics include one or more of enterococci, streptococci, bifidobacteria, lactobacilli, propionibacteria, bacillus, yeast and Escherichia coli.

7. The preparation method according to claim 1, characterized in that: The mass concentration of the functional active molecule solution is 0.5-50 mg / mL.

8. The preparation method according to claim 1, characterized in that: The temperature of the first encapsulation is 25-37°C, and the insulation time is 1-2h; The temperature of the second encapsulation is 1-4° C., and the insulation time is 1-2 hours.

9. The encapsulated probiotics prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The invention comprises probiotics and an encapsulating material for encapsulating the probiotics, wherein the protective layer in the encapsulating material is in contact with the probiotics.

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