Probiotic single cell encapsulation method based on metal-polyphenol network and photo-clicked thiol-ene gelatin layer-by-layer self-assembly

Abstract

The present application provides a kind of based on metal-polyphenol network and light click thiol-alkene gelatin layer layer self-assembly probiotic single cell coating method, first polyphenol is self-assembled with metal ion on the surface of probiotic cell and forms metal-polyphenol network layer, then guide adsorption alkenyl functionalized gelatin on the surface of probiotic cell and form the second layer coating, finally based on the principle of thiol-alkene light click reaction forms crosslinked alkenyl functionalized gelatin protective coating.The probiotic coating method of the present application simplifies the operation steps of existing layer layer self-assembly technology, shortens the encapsulation time, and the number of encapsulation layers is reduced, but the dense protective coating can protect the cell from the damage of gastrointestinal tract adversity environment to maintain the activity of probiotic bacteria, and improve the colonization of probiotic bacteria in intestinal tract.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food, daily chemical, medicine and biotechnology, and particularly relates to a method for single-cell coating of probiotics based on layer-by-layer self-assembly of metal-polyphenol network and photo-clicked thiol-ene gelatin. BACKGROUND

[0002] With the development of biological treatment technology, probiotics have great development prospects in the treatment of intestinal diseases. However, probiotics will face a series of challenges such as strong acidic gastric juice, bile salts and degrading enzymes after oral administration, which significantly reduces the activity and bioavailability of probiotics. Recent studies have found that single-cell encapsulation technology of probiotics has the advantages of high bioavailability, less demand for encapsulation materials, and significantly improved intestinal colonization of probiotics, and is simpler, more efficient, faster and safer than traditional multi-cell encapsulation technologies such as microcapsules, hydrogels and liposomes.

[0003] Among them, layer-by-layer self-assembly is the most common single-cell encapsulation technology of probiotics, which usually alternately exposes probiotics to negatively charged and positively charged coating materials based on electrostatic adsorption. Although related molecular materials such as chitosan, alginate, carboxymethyl cellulose, sodium phytate, etc. have been widely used in layer-by-layer self-assembly encapsulation technology, however, at least 4 layers of multi-layer encapsulation are still needed to provide good protection effect for probiotics, which results in a cumbersome operation process and waste of coating materials. In addition, too thick coating will inhibit the growth and reproduction ability of probiotics in the intestine. Therefore, it is necessary to find coating materials that can simplify the number of coatings and at the same time provide superior protection effect to improve the application potential of layer-by-layer self-assembly encapsulation technology.

[0004] Metal-polyphenol network is a non-covalent coordination complex of metal ions and natural polyphenols, which can form a stable self-assembled coating on various interfaces, and therefore has potential application value in probiotic encapsulation. However, metal-polyphenol network is easily degraded in acidic environment, and has limited protection effect on orally administered probiotics, resulting in low bioavailability of probiotics. SUMMARY

[0005] To solve the problems in the prior art, the present application provides a method for single-cell coating of probiotics based on layer-by-layer self-assembly of metal-polyphenol network and photo-clicked thiol-ene gelatin, which is simple, and the coated probiotics prepared by the method enhance the survival ability of probiotics in gastrointestinal juice and improve the protection effect on probiotics.

[0006] The allyl-functionalized modified gelatin can be cross-linked to form a compact three-dimensional polymer network in a short time based on thiol-ene photo-click reaction, and the gelatin-based material has good biocompatibility and low immunogenicity. Based on this, the present application uses the adsorption of metal-polyphenol network to the hydrogen bond of protein (gelatin) to guide the allyl-functionalized modified gelatin to the surface of probiotic cells, and uses the thiol-ene photo-click reaction to form a compact protective coating outside the metal-polyphenol network, thereby realizing a simplified and efficient layer-by-layer self-assembly coating of probiotics.

[0007] A method for coating single cells of probiotics based on metal-polyphenol network and photo-click thiol-ene gelatin layer-by-layer self-assembly, comprising the following steps:

[0008] (1) adding a polyphenol solution to a probiotic bacterial solution and mixing, then adding a metal ion solution, stirring and reacting to obtain metal-polyphenol network coated probiotics;

[0009] (2) adding an allyl-functionalized gelatin solution to the metal-polyphenol network coated probiotics, stirring and reacting to obtain metal-polyphenol network and gelatin coated probiotics;

[0010] (3) adding a thiol solution and a photo initiator to the metal-polyphenol network and gelatin coated probiotics in sequence, mixing uniformly, and then performing photo-click reaction under light to obtain metal-polyphenol network and photo-click thiol-ene gelatin coated probiotics.

[0011] In the above coating method, the reactions in steps (1) and (2) are both carried out under mild stirring conditions; after the reaction in step (1) is completed, a first protective layer, i.e. a metal-polyphenol network layer, is formed on the surface of the probiotic cells; after the reaction in step (2) is completed, a second protective layer, i.e. an allyl-functionalized gelatin layer, is formed outside the metal-polyphenol network layer; in step (3), the allyl-functionalized gelatin is cross-linked to form a compact cell protective layer by adding thiol and photo initiator to the probiotics prepared in step (2) and performing photo-click reaction, and finally the layer-by-layer self-assembly coated probiotics are obtained. The above single cell coating process of probiotics is carried out at 0-50°C.

[0012] As a preferred option, the probiotics are Escherichia coli Nissle 1917, Lactobacillus plantarum or Saccharomyces cerevisiae.

[0013] As a preferred option, the probiotic bacterial solution is prepared by suspending probiotic cells in a weakly alkaline buffer. The probiotic cells (enriched cells) are obtained by collecting the cells cultured overnight to the logarithmic phase with a sterile tube.

[0014] As a preferred option, the polyphenol is one or more of tannic acid, tea polyphenol and procyanidine. As a further preferred option, the polyphenol is one of tannic acid, tea polyphenol and procyanidine.

[0015] As a preference, the metal ion solution is a ferric chloride solution or a calcium chloride solution.

[0016] As a preference, the mass ratio of the polyphenol to the metal ion is 1:20-20:1. Further preferably, it is (1-10):1. More preferably, it is 3:1.

[0017] As a preference, in step (1), the stirring reaction time is 20-50 min. Further preferably, it is 30 min.

[0018] As a preference, the mass ratio of the polyphenol to the alkenyl-functionalized gelatin is 20:1-1:20. Further preferably, it is 1:(1-10). More preferably, it is 1:(2-5).

[0019] As a preference, in step (2), the stirring reaction time is 20-50 min. Further preferably, it is 30 min.

[0020] As a preference, the solvents of the polyphenol solution, the metal ion solution, and the alkenyl-functionalized gelatin solution are selected from weakly alkaline buffers, respectively. The weakly alkaline buffer as the solvent can stabilize the metal-polyphenol network and promote the complexation reaction.

[0021] As a preference, the thiol is dithiothreitol (DTT). The thiol is used to provide the required thiol group for the photoclick reaction.

[0022] As a preference, the solvent of the thiol solution is a weakly alkaline buffer.

[0023] As a preference, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP).

[0024] As a preference, the photoinitiator is added in the form of its weakly alkaline buffer solution.

[0025] As a preference, the mass ratio of the thiol to the alkenyl-functionalized gelatin is 1:(5-50). Further preferably, it is 1:(5-10). More preferably, it is 1:8.

[0026] As a preference, the mass ratio of the photoinitiator to the thiol is 1:(5-100). Further preferably, it is 1:(40-60). More preferably, it is 1:50.

[0027] As a preference, in step (3), the light irradiation time is 3-8 min. Further preferably, it is 5 min.

[0028] As a preference, the light source used for the photoclick reaction is visible light with a wavelength of 405 nm.

[0029] As preferred, the weak alkaline buffer solution described herein can be selected from Tris-HCl solution with pH value of 8.0.

[0030] As a specific preferred, a probiotic single cell coating method based on metal-polyphenol network and thiol-alkene photo-click gelatin layer-by-layer self-assembly, comprising the following steps:

[0031] (1) First, the probiotic bacteria are suspended in a weak alkaline buffer solution to obtain a probiotic bacteria solution; then a polyphenol solution prepared with a weak alkaline buffer solution as a solvent is added dropwise into the probiotic bacteria solution and mixed to obtain a mixed solution; a metal ion solution prepared with a weak alkaline buffer solution as a solvent is added dropwise into the mixed solution, and a mild stirring reaction is performed to obtain a bacteria slurry containing metal-polyphenol network coated probiotic bacteria;

[0032] (2) The bacteria slurry obtained in step (1) is added with an alkyl-functionalized gelatin solution prepared with a weak alkaline buffer solution as a solvent, and a mild stirring reaction is performed to obtain a bacteria slurry containing metal-polyphenol network and gelatin coated probiotic bacteria;

[0033] (3) The bacteria slurry obtained in step (2) is added with dithiothreitol solution (solvent is a weak alkaline buffer solution) and LAP solution (solvent is a weak alkaline buffer solution) in sequence, and after stirring, it is irradiated under 405 nm wavelength light to obtain metal-polyphenol network and thiol-alkene photo-click gelatin layer-by-layer self-assembly coated probiotic bacteria.

[0034] The probiotic single cell coating method of the present application utilizes the metal chelating ability of polyphenol to coordinate and self-assemble polyphenol and iron ions or calcium ions on the surface of probiotic bacteria cells to form a metal-polyphenol network layer, and at the same time, an alkyl-functionalized gelatin layer is self-assembled on the basis of the interaction between polyphenol and protein, and finally a cross-linked alkyl-functionalized gelatin dense protective layer is formed based on the principle of thiol-alkene photo-click reaction; the dense protective layer can protect the probiotic bacteria cells from the damage of the gastrointestinal stress environment to maintain the activity of the probiotic bacteria, and improve the colonization of the probiotic bacteria in the intestinal tract. The probiotic single cell coating material of the present application has good safety, and is a simple, efficient, rapid and safe probiotic bacteria packaging method.

[0035] Compared with the prior art, the present application has the following advantages:

[0036] (1) The probiotic bacteria coating method of the present application is a single cell packaging technology, the material preparation is simple, and the preparation process has less pollution;

[0037] (2) The probiotic bacteria coating material of the present application has good safety;

[0038] (3) The probiotic bacteria coating material of the present application can enhance the survival ability of probiotic bacteria in gastrointestinal juice, improve the bioavailability of probiotic bacteria in the intestinal tract, etc., and can be used as an enhanced therapy for prevention and treatment of intestinal diseases.

[0039] The single-cell coating method of the probiotic bacteria of the present application first coordinates the polyphenol with the metal ion to form a metal-polyphenol network layer on the surface of the probiotic bacteria cells through self-assembly, then guides the adsorption of the alkenyl functionalized gelatin on the surface of the probiotic bacteria cells to form a second layer of coating, and finally forms a cross-linked alkenyl functionalized gelatin protective coating based on the principle of thiol-alkene photo-click reaction. The probiotic bacteria coating method of the present application simplifies the operation steps of the existing layer-by-layer self-assembly technology, shortens the encapsulation time, and reduces the number of encapsulation layers while forming a dense protective coating, which can protect the cells from the damage of the gastrointestinal adverse environment to maintain the activity of the probiotic bacteria and improve the colonization of the probiotic bacteria in the intestinal tract. The coating process of the present application is simple, biodegradable polyphenol and gelatin are used as coating materials, which can enhance the survival ability of the probiotic bacteria in the gastrointestinal fluid, improve the bioavailability of the probiotic bacteria in the intestinal tract, and can be used as a biological therapy for the prevention and treatment of intestinal diseases. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Transmission electron micrographs of probiotic bacteria and different substances prepared in Example 1 coated probiotic bacteria. DETAILED DESCRIPTION

[0041] The present application will be further described in conjunction with the examples below, but the embodiments of the present application are not limited thereto.

[0042] In the following examples:

[0043] The buffer used is Tris-HCl buffer with a pH value of 8.0.

[0044] The alkenyl functionalized gelatin (GelAGE) used is prepared according to the reference method (Adv. Mater. 2017, 29, 1703404.).

[0045] Preparation of tannic acid coated probiotic bacteria in Example 1

[0046] The Escherichia coli Nissle 1917 (EcN) (5 x 10 8 CFUs) was suspended in 1 mL of buffer, and 3 mL of tannic acid (TA) solution (1 mg / mL) prepared with the buffer as the solvent was added dropwise into the probiotic bacteria solution and mixed to obtain a mixed solution;

[0047] Then, 1 mL of ferric chloride or calcium chloride solution (1 mg / mL) prepared with the buffer as the solvent was added dropwise into the above mixed solution, and the reaction was gently stirred (25°C, 30 min) to obtain the iron / calcium-tannic acid coated EcN bacteria paste, and the iron / calcium-tannic acid coated EcN was denoted as EcN@TA-Fe 3+ / Ca 2+ .

[0048] To the EcN slurry coated with iron / calcium-tannic acid, 4 mL of alkenyl-functionalized gelatin solution (2 mg / mL) prepared in buffer was added, and the reaction was gently stirred (25 °C, 30 min) to obtain the EcN slurry coated with iron / calcium-tannic acid and gelatin, denoted as EcN@TA-Fe 3+ / Ca 2+ @Gelatin.

[0049] To the EcN slurry coated with iron / calcium-tannic acid and gelatin, 500 μL of dithiothreitol (DTT) solution (2 mg / mL) prepared in buffer and 500 μL of LAP solution (0.04 mg / mL) prepared in buffer were added successively, and the mixture was stirred and irradiated with light at a wavelength of 405 nm for 5 min to obtain the EcN coated with iron / calcium-tannic acid and gelatin and subjected to photo-click cross-linking reaction, denoted as EcN@TA-Fe 3+ / Ca 2+ @GelAGE.

[0050] In the same way, Lactobacillus plantarum and Saccharomyces cerevisiae cells coated with the above materials can be prepared.

[0051] Example 2 Preparation of tea polyphenol-coated probiotics

[0052] Tea polyphenol was dissolved in 3 mL of buffer (1 mg / mL), and the reaction was shaken at 60 °C for 3 h to obtain a tea polyphenol solution;

[0053] The tea polyphenol solution (3 mL) was added dropwise to 1 mL of E. coli Nissle 1917 (EcN) bacterial solution (solvent: weakly alkaline buffer, 5 x 10 8 CFUs) and mixed to obtain a mixed solution;

[0054] Subsequently, 1 mL of calcium chloride solution (1 mg / mL) prepared in buffer was added dropwise to the mixed solution, and the reaction was gently stirred (25 °C, 30 min) to obtain the EcN slurry coated with calcium-tea polyphenol.

[0055] To the EcN slurry coated with calcium-tea polyphenol, 4 mL of alkenyl-functionalized gelatin solution (2 mg / mL) prepared in buffer was added, and the reaction was gently stirred (25 °C, 30 min) to obtain the EcN slurry coated with calcium-tea polyphenol and gelatin.

[0056] To the calcium- tea polyphenol and gelatin coated EcN slurry, 500 μL of dithiothreitol (DTT) solution (solvent: buffer, 2 mg / mL) and 500 μL of LAP solution (solvent: buffer, 0.04 mg / mL) were added successively, and then the mixture was stirred uniformly and irradiated under light of 405 nm wavelength for 5 min to obtain the calcium- tea polyphenol and gelatin coated EcN which had undergone photo-click crosslinking reaction.

[0057] Preparation of probiotic bacteria coated with procyanidins

[0058] Escherichia coli Nissle 1917 (EcN) (5 x 10 8 CFUs) was suspended in 1 mL of buffer to obtain a probiotic bacteria solution; 3 mL of procyanidin solution (1 mg / mL) prepared with buffer as solvent was added dropwise into the probiotic bacteria solution and stirred uniformly to obtain a mixed solution;

[0059] Then, 1 mL of calcium chloride solution (1 mg / mL) prepared with buffer as solvent was added dropwise into the mixed solution, and the mixture was gently stirred and reacted (25°C, 30 min) to obtain the calcium- procyanidin coated EcN slurry.

[0060] To the calcium- procyanidin coated EcN slurry, 4 mL of alkenyl functionalized gelatin solution (2 mg / mL) prepared with buffer as solvent was added, and the mixture was gently stirred and reacted (25°C, 30 min) to obtain the calcium- procyanidin and gelatin coated EcN slurry.

[0061] To the calcium- procyanidin and gelatin coated EcN slurry, 500 μL of dithiothreitol (DTT) solution (solvent: buffer, 2 mg / mL) and 500 μL of LAP solution (solvent: buffer, 0.04 mg / mL) were added successively, and then the mixture was irradiated under light of 405 nm wavelength for 5 min to obtain the calcium- procyanidin and gelatin coated EcN which had undergone photo-click crosslinking reaction.

[0062] Characterization and tolerance test of the coated probiotic bacteria:

[0063] 1. Product characterization

[0064] The morphology and structure of EcN cells and the probiotic bacteria (EcN) coated with different substances prepared in Example 1 were characterized by transmission electron microscopy, and the results are shown in Figure 1 The transmission electron micrographs Figure 1 showed that the surfaces of EcN cells coated with different substances all had obvious and complete coating shells, reflecting the existence and integrity of the coating protective layer.

[0065] 2. Tolerance test

[0066] 1 x 10 8The uncoated and coated EcN cells of different materials prepared in Example 1 were placed in simulated gastric fluid (SGF) containing pepsin, bile salt solution, free radical generator AAPH solution and ampicillin solution for a period of time to evaluate the in vitro tolerance of EcN cells, and the number of surviving cells was detected using plate counting method.

[0067] After 2h treatment with SGF solution, EcN, EcN@TA-Ca 2+ and EcN@TA-Ca 2+ @Gelatin groups had no surviving cells; while EcN@TA-Ca 2+ @GelAGE still had 2.40 Log 10 CFUs of viable cells survived, indicating that the GelAGE layer formed by photoclick crosslinking could serve as a "armor" to prevent H + from entering the cell membrane, thereby improving the gastric acid tolerance of EcN cells.

[0068] After 2-6h treatment with bile salt solution, the survival rate of EcN@TA-Ca 2+ @GelAGE group was significantly higher than that of the other three groups. Especially after 6h treatment, compared with EcN, EcN@TA-Ca 2+ and EcN@TA-Ca 2+ @Gelatin groups (4.22, 5.59, 5.80 Log 10 CFUs, respectively), the survival amount of EcN@TA-Ca 2+ @GelAGE group (5.99 Log 10 CFUs) was increased by 1.77, 0.40 and 0.18 Log 10 CFUs (P<0.05), respectively. Thus, EcN@TA-Ca 2+ @GelAGE had better resistance to strong acid and bile salt erosion.

[0069] After 8h treatment with ampicillin solution, the survival rate of EcN@TA-Ca 2+ @GelAGE (4.19 Log 10 CFUs) was significantly increased by 1.38 and 0.82 Log 2+ CFUs (P<0.05) compared with the survival rates of EcN@TA-Ca 2+ and EcN@TA-Ca 10 @Gelatin groups (2.81, 3.37 Log 10 CFUs, respectively), indicating that the GelAGE layer after photoclick crosslinking provided better protection for EcN cells.

[0070] Finally, all uncoated EcN died after 2 h treatment with AAPH solution, while EcN@TA-Ca 2+ , EcN@TA-Ca 2+ @Gelatin and EcN@TA-Ca 2+ In the EcN@Gel AGE group, 2.13, 2.16 and 2.29 Log 10 CFUs (P<0.05) were still observed, respectively, indicating that the coated EcN showed good tolerance to ROS-induced cell death, with EcN@TA-Ca 2+ @Gel AGE showing the best tolerance after photoclick crosslinking.

Claims

1. A method for coating probiotic single cells based on layer-by-layer self-assembly of metal-polyphenol network and photo-clicked thiol-ene gelatin layer, characterized in that, The method comprises the following steps: (1) adding a polyphenol solution to a probiotic bacterial solution, mixing, adding a metal ion solution, stirring and reacting to obtain metal-polyphenol network coated probiotics; (2) adding an alkenyl-functionalized gelatin solution to the metal-polyphenol network coated probiotics, stirring and reacting to obtain metal-polyphenol network and gelatin coated probiotics; (3) adding a thiol solution and a photoinitiator to the metal-polyphenol network and gelatin coated probiotics, mixing, and then performing a photo-click reaction under light to obtain metal-polyphenol network and photo-click thiol-alkenyl gelatin coated probiotics; The probiotic bacterial solution is prepared by suspending probiotic cells in a weakly alkaline buffer solution; The solvents of the polyphenol solution, the metal ion solution, the alkenyl-functionalized gelatin solution and the thiol solution are selected from weakly alkaline buffer solutions; The photoinitiator is added in the form of a weakly alkaline buffer solution; The mass ratio of polyphenol to metal ion is 1:20-20:1; The mass ratio of polyphenol to alkenyl-functionalized gelatin is 20:1-1:20; The mass ratio of thiol to alkenyl-functionalized gelatin is 1:(5-50); and the mass ratio of photoinitiator to thiol is 1:(5-100).

2. The method of coating probiotic single cells based on layer-by-layer self-assembly of metal-polyphenol network with photo-click thiol-ene gelatin according to claim 1, characterized in that, The polyphenol is one or more of tannic acid, tea polyphenol and procyanidin.

3. The method of coating probiotic single cells based on layer-by-layer self-assembly of metal-polyphenol network with photo-click thiol-ene gelatin according to claim 1, characterized in that, The metal ion solution is a ferric chloride solution or a calcium chloride solution.

4. The probiotic single cell coating method based on layer-by-layer self-assembly of metal-polyphenol network with photo-click thiol-ene gelatin layer according to claim 1, characterized in that, The thiol is dithiothreitol.

5. The probiotic single cell coating method based on layer-by-layer self-assembly of metal-polyphenol network with photo-click thiol-ene gelatin layer according to claim 1, characterized in that, The photoinitiator is a lithium salt of phenyl (2,4,6-trimethylbenzoyl) phosphate.

6. The method of coating probiotic single cells based on layer-by-layer self-assembly of metal-polyphenol network with photo-click thiol-ene gelatin according to claim 1, characterized in that, The probiotics are Escherichia coli Nissle 1917, Lactobacillus plantarum or Saccharomyces cerevisiae.

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