CN116115652A

CN116115652A - Polysaccharide embedded probiotics and preparation method and medicine thereof

Info

Publication number: CN116115652A
Application number: CN202310409447.3A
Authority: CN
Inventors: 万昊; 谢安琪; 万益群
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2023-04-18
Filing date: 2023-04-18
Publication date: 2023-05-16

Abstract

The invention discloses polysaccharide-embedded probiotics, a preparation method thereof and a medicament thereof, and belongs to the technical field of biological medicines. The polysaccharide embedded probiotics comprise probiotics positioned on the inner layer, a metal-polyphenol network structure layer coated on the surface of the probiotics on the inner layer, and a polysaccharide layer coated on the surface of the metal-polyphenol network structure layer. The polysaccharide embedded probiotics have good stability in intestinal tracts, can effectively improve the storage stability of the probiotics under the condition of not damaging the inherent biological characteristics of the probiotics, and can safely and effectively enhance the survival rate of the probiotics in the intestinal tracts and enhance the colonisation of the probiotics in the intestinal tracts. The preparation method of the polysaccharide-embedded probiotics is simple to operate, mild in reaction conditions and easy to industrialize, and the prepared polysaccharide-embedded probiotics can be used for preparing medicines for treating gastrointestinal diseases, in particular medicines for treating colonitis.

Description

Polysaccharide embedded probiotics and preparation method and medicine thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to polysaccharide embedded probiotics and a preparation method and a medicine thereof.

Background

Probiotics are a class of microorganisms that improve the intestinal microbial balance of a host and produce positive benefits to the host. Probiotics themselves digest food, produce useful products to destroy harmful microorganisms, complement the functions of those deficient digestive enzymes, and maintain the pH of the digestive system. The survival rate of probiotics is greatly reduced when they reach the intestinal tract due to the adverse effects of gastric acid, bile and various digestive enzymes in the human body. Meanwhile, probiotics can reproduce flora only by stably colonising in the intestinal canal of a host, so that the probiotics effect of the probiotics can be exerted. Therefore, whether to maintain a high survival rate and enhance the colonization of the intestinal tract by the probiotics is a key factor for the probiotics to exert their probiotic effect.

A common method for maintaining the activity of probiotics is a microcapsule embedding method. The microcapsule embedding method is to embed probiotics in a wall material solution, enhance the resistance of the probiotics to the external adverse environment, and control the release time and release position of the probiotics, thereby improving the survival rate of the probiotics. However, the microcapsule method has defects such as waste of raw materials of strains and microbial contamination caused by easy adhesion of bacterial powder or bacterial liquid to the outer wall of the embedding layer during the embedding process and the freeze-drying process. In addition, few methods have been reported to enhance colonization of the gut by probiotics.

In view of this, the present invention has been made.

Disclosure of Invention

One of the purposes of the invention is to provide a polysaccharide-embedded probiotic which has good stability in the intestinal tract, can effectively improve the storage stability of the probiotic without damaging the inherent biological characteristics of the probiotic, and can safely and effectively enhance the survival rate of the probiotic in the intestinal tract and enhance the colonisation of the probiotic in the intestinal tract.

The second purpose of the invention is to provide a preparation method of the polysaccharide-embedded probiotics.

The third object of the present invention is to provide a medicament containing the polysaccharide-embedded probiotic.

The application can be realized as follows:

in a first aspect, the present application provides a polysaccharide-embedded probiotic comprising a probiotic located in an inner layer, a metal-polyphenol network structure layer coated on the surface of the inner layer probiotic, and a polysaccharide layer coated on the surface of the metal-polyphenol network structure layer;

the preparation of the polysaccharide-embedded probiotics comprises the following steps: mixing the suspension of the probiotics with a metal solution to "anchor" the metal ions on the surface of the probiotics by electrostatic adsorption to obtain a probiotic-metal mixture;

mixing a probiotic-metal mixture with a polyphenol raw material to form a metal-polyphenol network structure layer on the surface of the probiotic to obtain a probiotic-metal-polyphenol compound;

mixing the probiotic-metal-polyphenol compound with a polysaccharide raw material to form a polysaccharide layer on the surface of the probiotic-metal-polyphenol compound, thereby obtaining polysaccharide-embedded probiotic;

the number of live probiotics in the suspension of probiotics is 0.8X10 ⁹ CFU/mL-1.2×10 ⁹ CFU/mL；

The concentration of the metal solution is 0.075-0.75 mg/mL;

the volume ratio of the suspension of probiotics to the metal solution is 100-200:800-900.

In alternative embodiments, the gastrointestinal disorder comprises colitis.

In an alternative embodiment, the polysaccharide layer corresponds to a polysaccharide material comprising at least one of beta-glucan, fucoidan, clematis polysaccharide, low ester pectin, tremella polysaccharide, pachyman, chitosan, starch, and cellulose.

In an alternative embodiment, the probiotic bacteria include at least one of escherichia coli Nissle1917 (hereinafter "EcN"), lactobacillus reuteri, lactobacillus rhamnosus, bifidobacterium, lactobacillus plantarum and lactobacillus salivarius.

In an alternative embodiment, the metal ion corresponding to the metal-polyphenol network layer comprises Fe ³⁺ 、Al ³⁺ 、Ti ⁴⁺ And Cr (V) ³⁺ At least one of them.

In an alternative embodiment, the polyphenol material corresponding to the metal-polyphenol network layer comprises at least one of flavonoids, tannins, and phenolics.

In an alternative embodiment, the polyphenol material corresponding to the metal-polyphenol network layer is tannic acid.

In a second aspect, the present application provides a method of preparing a polysaccharide-embedded probiotic according to any one of the preceding embodiments, comprising the steps of: and sequentially coating a metal-polyphenol network structure layer and a polysaccharide layer on the surface of the inner layer probiotics according to a preset position.

In an alternative embodiment, the mixing is performed in a vortex fashion.

In an alternative embodiment, the vortex time is 40-80 s.

In an alternative embodiment, the concentration of polyphenol feedstock in the probiotic-metal-polyphenol complex is from 0.25 to 0.4 mg/mL.

In an alternative embodiment, the mixing of the probiotic-metal mixture with the polyphenol feedstock is performed in a vortex manner.

In an alternative embodiment, the vortex time is 40-80 s.

In an alternative embodiment, 0.01-0.1 mg of polysaccharide starting material per milliliter of probiotic-metal-polyphenol complex is used.

In an alternative embodiment, the mixing of the probiotic-metal-polyphenol complex with the polysaccharide material is performed in a vortexing manner.

In an alternative embodiment, the vortex time is 40-80 s.

In an alternative embodiment, after obtaining the polysaccharide-embedded probiotic, further comprising: excess polysaccharide-metal-polyphenol complex is removed.

In an alternative embodiment, the removal of excess polysaccharide-metal-polyphenol complex is achieved by centrifugation.

In an alternative embodiment, the centrifugation speed is 3000-7000 rpm and the centrifugation time is 5-10 min.

In a third aspect, the present application provides a medicament comprising the polysaccharide-embedded probiotic of any one of the preceding embodiments.

In an alternative embodiment, the drug is a drug for treating gastrointestinal disorders.

In an alternative embodiment, the drug is a drug for treating colitis.

The beneficial effects of this application include:

the method comprises the steps of firstly mixing metal cations with probiotics with negatively charged surfaces, anchoring the cations on the surfaces of the probiotics, then adding polyphenol, and forming a metal-polyphenol network structure on the surfaces of the probiotics through chelation between the polyphenol and the metal ions. The network structure has a general adhesion effect and can be settled or adhered to the surfaces of various substances (such as cells or bacteria). Since polyphenol has more hydroxyl groups and has higher adhesion, polysaccharide can be adsorbed on the surface of the polyphenol through hydrogen bonding to form a polysaccharide-metal-polyphenol composite structure, and probiotics are wrapped. Since the polysaccharide located at the outermost layer is not decomposed in the upper digestive tract but is decomposed by intestinal flora in the large intestine. Therefore, by arranging the polysaccharide embedded probiotics with the structure, the probiotics can be protected to remain intact in the stomach and the small intestine and reach the large intestine, the outermost polysaccharide is decomposed by intestinal flora in the large intestine to expose the metal-polyphenol network structure and adhere to the inner wall of the large intestine, so that the probiotics can continuously play a role in the large intestine. That is, the method protects the probiotic from damage in the upper digestive tract, reaches the intended site of action, and enhances its colonization at the intended site of action. In addition, the polysaccharide can be used as a physical barrier to protect probiotics from being damaged by digestive tracts, and meanwhile, the polysaccharide is also a prebiotic, plays a role in probiotics, can cooperate with the probiotics to play a role in biological activity, and integrally improves the treatment effect of the system.

The corresponding polysaccharide embedded probiotics have good stability in intestinal tracts, can effectively improve the storage stability of the probiotics under the condition of not damaging the inherent biological characteristics of the probiotics, and can safely and effectively enhance the survival rate of the probiotics in the intestinal tracts and enhance the colonisation of the probiotics in the intestinal tracts, and have the effects of gastric acid resistance and/or bile salt resistance, so that the polysaccharide embedded probiotics can be used for preparing medicines for treating gastrointestinal diseases, in particular medicines for treating colonitis.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIGS. 1 and 2 are scanning electron microscope images of EcN and beta-glucan-embedded EcN, respectively, of test example 1;

FIGS. 3 and 4 are transmission electron microscope images of EcN and beta-glucan-embedded EcN, respectively, of test example 1;

FIG. 5 is a laser confocal plot of test example 1 for beta-glucan, ecN, and beta-glucan embedded EcN;

FIG. 6 is an inverted fluorescence microscope image of EcN and Nelumbo nucifera Gaertn polysaccharide entrapped EcN of test example 2;

FIG. 7 is a graph showing the protective effect of EcN, metal-phenolic structural layer entrapping EcN and beta-glucan entrapping EcN in simulated gastric fluid in test example 3;

FIG. 8 is a graph showing the protective effect of EcN, metal-phenolic structural layer embedding EcN and beta-glucan embedding EcN in simulated bile in test example 3;

FIG. 9 is a graph showing the results of retention of fluorescence intensity of EcN and beta-glucan embedded EcN in intestinal tract in test example 4;

FIG. 10 is a graph showing the storage stability of EcN and beta-glucan-embedded EcN of test example 5;

FIG. 11 is a graph showing the effect of the beta-glucan inclusion EcN and the carboxymethyl-modified beta-glucan inclusion EcN on gastric juice simulation in test example 6;

FIG. 12 is a graph showing the effect of Lactobacillus salivarius and fucoidan-embedded Lactobacillus salivarius in simulated gastric fluid in test example 6;

FIG. 13 is a graph showing the effect of Lactobacillus plantarum and Lactobacillus plantarum embedded with Leuconostoc polysaccharide in simulated gastric fluid in test example 6;

FIG. 14 is a graph showing the results of particle size of metal-polyphenol networks formed in correspondence with tannins at different concentrations in test example 7;

FIG. 15 is a graph showing the change in body weight of the colitis mice of test example 8 during 7 days of gastric lavage;

FIG. 16 is a graph of spleen index 7 days after gastric lavage of the colitis mice in test example 8;

FIG. 17 is a graph of colon length 7 days after gastric lavage of the colitis mice in test example 8.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The polysaccharide-embedded probiotics, the preparation method thereof and the medicament are specifically described below.

The application provides a polysaccharide-embedded probiotic, which comprises a probiotic positioned on an inner layer, a metal-polyphenol network structure layer coated on the surface of the inner layer probiotic, and a polysaccharide layer coated on the surface of the metal-polyphenol network structure layer. The polysaccharide embedded probiotics are used for preparing medicines for treating gastrointestinal diseases.

For reference, the polysaccharide raw material corresponding to the polysaccharide layer may include, for example, at least one of β -glucan, fucoidan, silverweed polysaccharide, low-ester pectin, tremella polysaccharide, pachyman, chitosan, starch, and cellulose.

In some preferred embodiments, the β -glucan is carboxymethylated β -glucan.

Illustratively, the preparation of carboxymethylated β -glucan can be referred to: dissolving beta-glucan in 50 mL isopropanol, and stirring for 30 min at room temperature; 10 mL of NaOH solution is added and stirred at room temperature for 1 h; then a certain amount of chloroacetic acid is slowly added while stirring, and the water bath is reacted with 4 h. And after the reaction is finished, regulating the pH value of the solution to 7, dialyzing, and freeze-drying to obtain the carboxymethylated beta-glucan.

In addition, the preparation of carboxymethylated β -glucan can be referred to in other prior art, and is not excessively limited herein.

It should be noted that carboxymethylated beta-glucan has higher water solubility and bioactivity than beta-glucan.

On the other hand, the outermost layer of the preparation method takes polysaccharide as a wall material, so that probiotics can be prevented from being degraded in the upper digestive tract, and the probiotics can enter the action part in a complete form. The polysaccharide gives the wall material the whole system good gastric acid resistance and cholate resistance. It is emphasized that in the absence of polysaccharides, protection of probiotics by metal-phenolic network structures alone is not resistant to gastric acid damage. The polysaccharide can be used as a physical barrier to protect probiotics from being damaged by digestive tracts, and meanwhile, the polysaccharide is also a prebiotic, plays a role in probiotics, can cooperate with the probiotics to play a role in biological activity, and integrally improves the treatment effect of the system. It is worth noting that the polysaccharide of the present application has the above effects that are not achieved by other types of wall materials.

For reference, the probiotics may include, for example, at least one of EcN, lactobacillus reuteri, lactobacillus rhamnosus, bifidobacterium, lactobacillus plantarum and lactobacillus salivarius.

Metal-polyphenol network structureThe metal ions corresponding to the structural layers can comprise Fe ³⁺ 、Al ³⁺ 、Ti ⁴⁺ And Cr (V) ³⁺ At least one of them.

It should be noted that Fe is not used ²⁺ Instead of Fe ³⁺ Or replacing Ti with titanium ions of other valence ⁴⁺ The reason for this is that: other valences are not sufficiently stable as complexes with polyphenols.

In the present application, the polyphenol raw material corresponding to the metal-polyphenol network structure layer may include at least one of flavonoids, tannins, and phenolic acids. Preferably, the polyphenol material corresponding to the metal-polyphenol network structure layer is tannic acid.

After oral intake, most probiotics are affected by the pH of gastric acid (probiotics are relatively sensitive to gastric acid), and under low pH conditions, the probiotics lose activity and cannot reach the large intestine or a specific action site by a certain order of magnitude.

The method comprises the steps of firstly mixing metal cations with probiotics with negatively charged surfaces, anchoring the cations on the surfaces of the probiotics, then adding polyphenol, and forming a metal-polyphenol network structure on the surfaces of the probiotics through chelation between the polyphenol and the metal ions. The network structure has a general adhesion effect and can be settled or adhered to the surfaces of various substances (such as cells or bacteria). Since polyphenol has more hydroxyl groups and has higher adhesion, polysaccharide can be adsorbed on the surface of the polyphenol through hydrogen bonding to form a polysaccharide-metal-polyphenol composite structure, and probiotics are wrapped. Since the polysaccharide located in the outermost layer is not decomposed in the upper digestive tract, it needs to be decomposed by intestinal flora in the large intestine. Therefore, by arranging the polysaccharide embedded probiotics with the structure, the probiotics can be protected to remain intact in the stomach and the small intestine and reach the large intestine, the outermost polysaccharide is decomposed by intestinal flora in the large intestine to expose the metal-polyphenol network structure and adhere to the inner wall of the large intestine, so that the probiotics can continuously play a role in the large intestine. That is, the method protects the probiotic from damage in the upper digestive tract, reaches the intended site of action, and enhances its colonization at the intended site of action. That is, after the whole system enters the colon part, the polysaccharide layer is decomposed by intestinal flora, and then the metal-polyphenol network layer is exposed, so that the retention of probiotics in the intestinal tract can be increased, the retention time of the probiotics in the intestinal tract can be prolonged, and the probiotics can play a role and an effect permanently.

On the contrary, the polysaccharide embedded probiotics provided by the application have good stability in intestinal tracts, and can effectively improve the storage stability of the probiotics under the condition of not damaging the inherent biological characteristics of the probiotics, and simultaneously safely and effectively enhance the survival rate of the probiotics in the intestinal tracts and enhance the colonisation of the probiotics in the intestinal tracts.

Correspondingly, the application also provides a preparation method of the polysaccharide-embedded probiotics, which comprises the following steps: and sequentially coating a metal-polyphenol network structure layer and a polysaccharide layer on the surface of the probiotics which are arranged in the inner layer according to a preset position.

It is also understood that the metal-polyphenol network structure and the polysaccharide are self-assembled in the surface layer of the probiotic bacteria in an order from the inside to the outside.

Incidentally, the production method may include:

s1: the suspension of the probiotic bacteria is mixed with a metal solution to "anchor" the metal ions to the surface of the probiotic bacteria by electrostatic adsorption, resulting in a probiotic-metal mixture.

The number of live probiotics in the suspension of the probiotics is about 0.8X10 ⁹ CFU/mL-1.2×10 ⁹ CFU/mL, e.g. 0.8X10 ⁹ CFU/mL、0.9×10 ⁹ CFU/mL、1.0×10 ⁹ CFU/mL、1.1×10 ⁹ CFU/mL or 1.2X10 ⁹ CFU/mL, etc., may be 0.8X10 ⁹ CFU/mL-1.2×10 ⁹ Any other value within the CFU/mL range. Preferably 1X 10 ⁹ CFU/mL。

In some embodiments, the concentration of the metal solution may be 0.075-0.75 mg/mL, such as 0.075-mg/mL, 0.1 mg/mL, 0.15 mg/mL, 0.2 mg/mL, 0.25-mg/mL, 0.3-mg/mL, 0.35-mg/mL, 0.4-mg/mL, 0.45-mg/mL, 0.5-mg/mL, 0.55-mg/mL, 0.6-mg/mL, 0.65-mg/mL, 0.7-mg/mL, or 0.75-mg/mL, and the like, as well as other arbitrary values within the range of 0.075-0.75-mg/mL, preferably 0.075-0.2-mg/mL.

The volume ratio of the suspension of probiotics to the metal solution may be 100-200:800-900, such as 100:800, 100:850, 100:900, 150:800, 150:850, 150:900, 200:800, 200:850 or 200:900, etc., but may also be any other value in the range of 100-200:800-900.

It should be noted that, if the amount of the metal raw material is too small, the metal network structure is easy to be loose, and a good protection effect cannot be achieved; if the metal raw material is used too much, the metal network structure is easy to shrink, and the internal probiotics are extruded.

The above mixing may be performed, for example, by vortexing, for a period of time ranging from 40 to 80 s, such as 40 s, 50 s, 60 s, 70 s, or 80 s, or any other value within the range of 40 to 80 s.

S2: and mixing the probiotic bacteria-metal mixture with a polyphenol raw material to form a metal-polyphenol network structure layer on the surface of the probiotic bacteria, so as to obtain the probiotic bacteria-metal-polyphenol compound.

For reference, the concentration of the polyphenol feedstock in the probiotic-metal-polyphenol complex is 0.25-0.4 mg/mL, such as 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, or 0.4 mg/mL, etc., but may also be any other value in the range of 0.25-0.4 mg/mL.

If the concentration of the polyphenol raw material in the probiotic-metal-polyphenol compound is lower than 0.25 mg/mL, the metal-polyphenol network structure is easy to loosen, and the probiotic cannot be completely embedded; if the concentration of the polyphenol starting material in the probiotic-metal-polyphenol complex is higher than 0.4 mg/mL, deposition of the polyphenol starting material is liable to result.

For example, the probiotic-metal mixture may be mixed with the polyphenol feedstock in the form of a vortex, which may take the form of a vortex for a period of time ranging from 40 to 80 s (e.g., 40 s, 50 s, 60 s, 70 s, 80 s, etc.).

S3: and mixing the probiotic-metal-polyphenol compound with a polysaccharide raw material to form a polysaccharide layer on the surface of the probiotic-metal-polyphenol compound, thereby obtaining the polysaccharide-embedded probiotic.

For reference, 0.01-0.1 mg (e.g., 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, or 0.1 mg/mL, etc., and any other value in the range of 0.01-0.1 mg/mL) of polysaccharide starting material may be used per milliliter of the probiotic-metal-polyphenol complex.

If the concentration of the polysaccharide solution is lower than 0.01 mg/mL, incomplete embedding of probiotics is easily caused; if the concentration of the polysaccharide solution is higher than 0.1 mg/mL, excessive deposition on the surface of the probiotics is easily caused, the growth of the probiotics is limited, and the waste of raw materials is caused.

The mixing of the probiotic-metal-polyphenol complex and the polysaccharide raw material can be performed in a vortex manner, and the vortex time can be 40-80 s.

Further, after obtaining the polysaccharide-embedded probiotics, the method further comprises the following steps: excess polysaccharide-metal-polyphenol complex is removed, such as by centrifugation.

The centrifugation speed may be, for example, 3000-7000 rpm, such as 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm or 7000 rpm, etc., or any other value in the range of 3000-7000 rpm.

The centrifugation time may be 5-10 min, such as 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, or any other value within 5-10 min.

On the premise, the preparation method is simple to operate, mild in reaction condition and easy for industrial production. The method can effectively improve the capability of probiotics to resist adverse environmental effects, and can reach and colonize specific sites (such as colon) with higher viable count so as to play a role of probiotics.

In addition, the application also provides a medicine, and the preparation raw material of the medicine contains the polysaccharide embedded probiotics.

For reference, the above drugs are drugs for treating gastrointestinal diseases.

In some preferred embodiments, the medicament is a medicament for treating colitis.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The embodiment provides a polysaccharide-embedded probiotic, which is prepared by the following method:

step (1): 0.1 mg FeCl ₃ Adding the mixture into a PBS buffer solution of 4 mL to obtain a metal solution; subsequently, 100. Mu.L of the EcN suspension (EcN, viable count: 1X 10) ⁹ CFU/mL) and 900 mu L of the metal solution are mixed and vortexed 60 and s, so that the fixation of metal ions on the surface of EcN is realized (namely, a probiotic-metal mixture is obtained);

step (2): adding 0.4. 0.4 mg tannic acid into the probiotic-metal mixture, and swirling 60. 60 s to form a metal-polyphenol network structure layer on the surface of the probiotic to obtain a probiotic-metal-polyphenol compound;

step (3): adding 0.08 mg beta-glucan into the probiotic-metal-polyphenol compound, and swirling 60 s to form a polysaccharide layer on the surface of the probiotic-metal-polyphenol compound so as to obtain polysaccharide embedded probiotic;

step (4): washing with PBS for 2 times, centrifuging to remove excessive polysaccharide-metal-polyphenol complex, centrifuging at 7000 rpm for 5 min to obtain EcN embedded by beta-glucan.

Example 2

step (1): 0.3 mg FeCl ₃ Adding the mixture into a PBS buffer solution of 4 mL to obtain a metal solution; subsequently 150. Mu.L of the EcN suspension (EcN with a viable count of 0.8X10) ⁹ CFU/mL) and 850 mu L of the metal solution are mixed and vortexed 60 and s, so that the fixation of metal ions on the EcN surface is realized (namely the probiotic bacteria-metal mixture is obtained);

step (2): adding 0.3. 0.3 mg tannic acid into the probiotic-metal mixture, and swirling 60. 60 s to form a metal-polyphenol network structure on the surface of the probiotic to obtain a probiotic-metal-polyphenol compound;

step (3): adding 0.02 mg beta-glucan into the probiotic-metal-polyphenol compound, and swirling 60 s to form a polysaccharide layer on the surface of the probiotic-metal-polyphenol compound so as to obtain polysaccharide embedded probiotic;

step (4): washing with PBS for 2 times, centrifuging to remove excessive polysaccharide-metal-polyphenol complex, centrifuging at 3000 rpm for 10 min to obtain EcN embedded by beta-glucan.

Example 3

step (1): 3 mg FeCl ₃ Adding the mixture into a PBS buffer solution of 4 mL to obtain a metal solution; subsequently 200. Mu.L of the EcN suspension (EcN with a viable count of 1.2X10) ⁹ CFU/mL) and 800 mu L of the metal solution are mixed and vortexed 60 and s, so that the fixation of metal ions on the surface of EcN is realized (namely, a probiotic-metal mixture is obtained);

step (2): adding 0.25 mg tannic acid into the probiotic-metal mixture, and swirling 60 s to form a metal-polyphenol network structure layer on the surface of the probiotic to obtain a probiotic-metal-polyphenol compound;

step (3): adding 0.04 mg beta-glucan into the probiotic-metal-polyphenol compound, and swirling 60 s to form a polysaccharide layer on the surface of the probiotic-metal-polyphenol compound so as to obtain polysaccharide embedded probiotic;

step (4): washing with PBS for 2 times, centrifuging to remove excessive polysaccharide-metal-polyphenol complex, centrifuging at 4000 rpm for 7.5 min to obtain EcN embedded by beta-glucan.

Example 4

This embodiment differs from embodiment 1 in that: the polysaccharide is carboxymethylated beta-glucan.

Example 5

This embodiment differs from embodiment 1 in that: the polysaccharide is fucoidin, and the probiotics is lactobacillus salivarius.

Example 6

This embodiment differs from embodiment 1 in that: the polysaccharide is flos Lonicerae polysaccharide, and the probiotic is Lactobacillus plantarum.

Example 7

This embodiment differs from embodiment 1 in that: the polysaccharide is flos Lonicerae polysaccharide, and the probiotic is EcN.

Example 8

This embodiment differs from embodiment 1 in that: the polysaccharide is low-ester pectin.

Example 9

This embodiment differs from embodiment 1 in that: the polysaccharide is a mixture of tremella polysaccharide and pachyman (mass ratio is 1:1).

Example 10

This embodiment differs from embodiment 1 in that: the polysaccharide is a mixture of chitosan, starch and cellulose (mass ratio is about 1:1:1).

Example 11

This embodiment differs from embodiment 1 in that: the probiotics are lactobacillus reuteri.

Example 12

This embodiment differs from embodiment 1 in that: the probiotics are lactobacillus rhamnosus.

Example 13

This embodiment differs from embodiment 1 in that: the probiotics are a mixture of bifidobacteria and lactobacillus plantarum (the mass ratio is 1:1).

Example 14

This embodiment differs from embodiment 1 in that: the metal ion is Ti ⁴⁺ 。

Test example 1

Taking example 1 as an example, the following tests were performed on beta-glucan entrapped EcN:

(1) the results of scanning electron microscope observation are shown in fig. 1 and 2.

Wherein, FIG. 1 is a scanning electron microscope image of EcN, and FIG. 2 is a scanning electron microscope image of beta-glucan embedding EcN.

(2) The result of transmission electron microscopy was shown in fig. 3 and 4.

Wherein, fig. 3 is a transmission electron microscope image of EcN, and fig. 4 is a transmission electron microscope image of beta-glucan embedding EcN.

(3) The result of confocal scanning is shown in fig. 5.

Wherein, (a) is a laser confocal map of beta-glucan, (b) is a laser confocal map of EcN, and (c) is a laser confocal map of beta-glucan embedded EcN.

As can be seen in connection with fig. 1 to 5: beta-glucan successfully entraps EcN.

Test example 2

Inverted fluorescence microscopy was performed on the polysaccharide-embedded EcN of the clematis obtained in example 7. The fluorescence grafting process is as follows: before the probiotic (EcN) -metal-polyphenol complex is added, the acidic polysaccharide of the clematis is subjected to fluorescence grafting in the following manner: dissolving acid polysaccharide of herba Lobeliae chinensis in MES buffer solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and fluorescamine, and stirring in dark to react 12 h. Dialyzing, and freeze-drying to obtain the fluorescent grafted polysaccharide of the clematis. The results are shown in FIG. 6.

Wherein a is a EcN inverted fluorescence microscopy image, and b is an inverted fluorescence microscopy image of the polysaccharide embedded EcN of the clematis.

As can be seen from fig. 6: the polysaccharide of the silverfish is successfully embedded in EcN.

Test example 3

The beta-glucan prepared in example 1 was embedded in EcN for in vitro resistance studies, and the specific study method is as follows:

step (1): the beta-glucan prepared in example 1 was embedded EcN in simulated gastric fluid and simulated bile environment and incubated at 37 ℃ for 2 h, respectively, with unencapsulated EcN and EcN embedded with only the metal-phenolic structural layer (i.e., without the outermost beta-glucan) as controls.

The preparation method of the simulated gastric fluid comprises the following steps: 5g pepsin, 8.5g sodium chloride are dissolved in 1000ml sterile water and the pH is adjusted to 2.5, and the solution is passed through a 0.22 μm sterilizing filter before use; the method for simulating bile is as follows: 4% bile salt solution is prepared and used after passing through a 0.22 μm sterilizing filter membrane.

Step (2): 50. Mu.L were taken at various time points (0 h, 0.5 h, 1 h, 2 h) and counted after dilution coating.

The results are shown in fig. 7 and 8. Wherein, fig. 7 corresponds to the protection effect in simulated gastric fluid, fig. 8 corresponds to the protection effect in simulated bile, and the beta-glucan embedding EcN in fig. 7 and fig. 8 is the polysaccharide embedded probiotic bacteria, and the same is true below.

As can be seen from fig. 7 and 8: ecN embedded with beta-glucan has stronger gastric acid resistance and cholate resistance. And no polysaccharide is embedded in the escherichia coli only by the metal-phenolic structure layer, so that the survival rate of the original escherichia coli in gastric juice simulation and bile simulation cannot be improved, the escherichia coli is endowed with good gastric acid resistance and bile resistance only after the polysaccharide is embedded in the outermost layer, namely, the effect of effectively resisting gastric acid and bile and improving the survival rate of the escherichia coli in gastric juice and bile can be achieved only by taking the whole of the probiotics, the metal-polyphenol network structure layer and the polysaccharide layer as a complete system, so that the effect of treating colonitis can be effectively exerted in intestinal tracts.

Test example 4

The beta-glucan prepared in example 1 was embedded in EcN for the study of intestinal retention capacity, and the specific method is as follows:

step (1): mice were randomized into 2 groups of 6 after 24-h fasted, each filled with 200 μl of fluorescence labeled unencapsulated EcN and fluorescence labeled β -glucan-embedded EcN prepared in example 1, respectively;

step (2): mice were sacrificed at different time points (4 h, 48 h) by cervical dislocation and their colon was taken for fluoroscopic analysis.

The results are shown in FIG. 9, and FIG. 9 is a graph showing the results of fluorescence intensity of beta-glucan inclusion EcN retained in the intestinal tract.

As can be seen from fig. 9: the fluorescence intensity of beta-glucan-embedded EcN was significantly stronger in the intestine after 48 h than in unencapsulated EcN, indicating that beta-glucan-embedded EcN was significantly longer in the intestine after 48 h than in unencapsulated EcN.

Test example 5

The storage stability of the beta-glucan prepared in example 1 was investigated by embedding EcN as follows:

step (1): the beta-glucan-embedded EcN prepared in example 1 was lyophilized with non-embedded EcN in one tenth of the volume of the protective solution. After lyophilization, a portion of the bacterial powder was diluted and spread, and the colony count was calculated after 24 h.

Step (2): the bacteria were stored for 7 days at different temperatures (-20 ℃,4 ℃) and after 7 days the bacteria dry powder was resuspended, diluted with the appropriate volume and spread on the medium, 24 h and counted. The number of colonies before and after storage was compared, and the protective effect of β -glucan on EcN was compared.

The results are shown in FIG. 10, and FIG. 10 is a graph showing the storage stability of the beta-glucan inclusion EcN.

As can be seen from fig. 10: beta-glucan-embedded EcN had better storage stability than non-embedded EcN.

Test example 6

In vitro resistance studies were performed with the polysaccharide-embedded probiotics obtained in examples 4-6, and specific methods are referred to test example 3.

As a result, as shown in fig. 11 to 13, fig. 11 to 13 each correspond to the protective effect in simulated gastric fluid.

As can be seen from fig. 11: ecN embedded with carboxymethyl modified beta-glucan has better gastric acid resistance than EcN embedded with beta-glucan.

As can be seen from fig. 12 and 13: the lactobacillus salivarius after being embedded by fucoidan and the lactobacillus plantarum after being embedded by the silverline polysaccharide have stronger gastric acid resistance.

Test example 7

Taking the preparation method of beta-glucan embedding EcN in example 1 as an example, particle sizes of metal-polyphenol networks formed corresponding to polyphenols (tannins, TAs) at different concentrations were studied.

The results are shown in FIG. 14.

As can be seen from fig. 14: TA can achieve a suitable network size at a concentration of 0.25-0.4 mg/mL in the probiotic-metal-polyphenol complex.

If the concentration of TA in the probiotic-metal-polyphenol compound is lower than 0.25 mg/mL, a larger metal network structure can be formed, so that the probiotic can not be well protected; higher than 0.4 mg/mL may result in waste of raw materials.

In addition, the smaller the size of the metal-polyphenol network is, the more easily and compactly embedded on the surface of the probiotics, the more convenient for industrial application, the larger the size of the metal-polyphenol network is, the more easily causes the surface roughness of the probiotics, and the probiotics cannot be well protected and the raw materials are wasted.

Test example 8

The following experiments were performed using the beta-glucan entrapped EcN prepared in example 1 as an example:

grouping and treating experimental animals: the C57BL/6J experimental animals are equally divided into a normal group, a model group, an escherichia coli (EcN) group, a polysaccharide group, a beta-glucan embedded escherichia coli group (called a polysaccharide embedded EcN group for short) and a beta-glucan and escherichia coli physical mixed group (called a polysaccharide+ EcN group for short) according to body weight, and 4 mice are in each group. Mice first passed a 7 day habituation period. After the adaptation period, the normal group was free to drink water throughout the experiment, while the other 5 groups were free to drink water continuously for the first 7 days, and 2.5% dextran sodium sulfate (Dextran Sulfate Sodium Salt, DSS) was drunk for the last 7 days to construct a colitis model, and the weight of the mice was weighed daily during the modeling period. The normal and model groups were filled with 0.2 mL of PBS, the EcN group and the polysaccharide-embedded EcN group each day after 7 days, and the corresponding bacterial suspensions (bacterial dose: 1×10) were filled ⁹ CFU), polysaccharide group was 40 mg.kg of gastric lavage concentration per mouse weight ^-1 Polysaccharide solution of BW, polysaccharide+ EcN group of gastric lavage corresponding bacteria and polysaccharide mixed solution (bacterial dose: 1×10) ⁹ CFU, polysaccharide concentration: 40 mg.kg ^-1 BW), gastric lavage once every 2 days. After removal of DSS, mice were sacrificed using cervical dislocation. Colon tissue of the mice was isolated, the length of the colon tissue was measured, and the spleen weight was weighed, spleen index= (spleen weight/mouse weight) ×100%. All anatomical tissues were frozen at-80 ℃ for further analysis.

The change in body weight of the colitis mice during 7 days of gavage is shown in fig. 15, and fig. 15 shows that: the gastric lavage beta-glucan embedded escherichia coli can effectively reduce the weight loss phenomenon of mice caused by colonitis.

Spleen index of colonitis mice 7 days after lavage is shown in fig. 16, and fig. 16 can be seen: the gastric lavage beta-glucan embedded escherichia coli can effectively relieve the phenomenon of splenomegaly of mice caused by colonitis.

The colon length 7 days after lavage of the colitis mice is shown in fig. 17, and fig. 17 shows that: the lavage beta-glucan embedding EcN can effectively relieve the phenomenon of colon length shortening caused by colonitis.

The above results prove that the beta-glucan embedding EcN provided by the application can effectively relieve intestinal inflammation of mice caused by colonitis.

In addition, as can be seen from fig. 15 to 17, by combining β -glucan with EcN in an embedding form, the physical mixing of β -glucan with EcN is simpler than that of β -glucan, which can effectively reduce the weight loss phenomenon caused by colitis on mice, effectively alleviate the splenomegaly phenomenon caused by colitis on mice, and effectively alleviate the colon length shortening phenomenon caused by colitis. The embedded form used in the application can enable the polysaccharide to play biological activity in cooperation with probiotics, and the therapeutic effect of the system is integrally improved.

In conclusion, the polysaccharide-embedded probiotics provided by the application have good stability in intestinal tracts, the storage stability of the probiotics can be effectively improved under the condition that the inherent biological characteristics of the probiotics are not damaged, and meanwhile, the survival rate of the probiotics in the intestinal tracts and the colonisation capacity of the probiotics in the intestinal tracts are safely and effectively enhanced. The preparation method is simple to operate, mild in reaction condition and easy to industrialize, the method for embedding the probiotics by the polysaccharide can be used for multiple combinations, the probiotics, the polysaccharide wall material on the outermost layer and the like can be selected differently, and different probiotics and polysaccharide materials can be selected for combination according to different diseases, so that the application is expanded. The prepared polysaccharide embedded probiotics can be used for medicines for treating gastrointestinal diseases, in particular for medicines for treating colonitis.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The polysaccharide-embedded probiotics are characterized by comprising probiotics in an inner layer, a metal-polyphenol network structure layer coated on the surface of the probiotics and a polysaccharide layer coated on the surface of the metal-polyphenol network structure layer;

the polysaccharide embedded probiotics are used for preparing medicines for treating gastrointestinal diseases;

the preparation of the polysaccharide-embedded probiotics comprises the following steps: mixing a suspension of probiotics with a metal solution to "anchor" metal ions on the surface of the probiotics by electrostatic adsorption to obtain a probiotic-metal mixture;

mixing the probiotic bacteria-metal mixture with a polyphenol raw material to coat the surface of the probiotic bacteria to form the metal-polyphenol network structure layer, so as to obtain a probiotic bacteria-metal-polyphenol compound;

mixing the probiotic-metal-polyphenol compound with a polysaccharide raw material to coat the surface of the probiotic-metal-polyphenol compound to form a polysaccharide layer, so as to obtain a polysaccharide embedded probiotic;

the number of live probiotics in the suspension of the probiotics is 0.8x10 ⁹ CFU/mL-1.2×10 ⁹ CFU/mL；

The concentration of the metal solution is 0.075-0.75 mg/mL;

the volume ratio of the suspension of the probiotics to the metal solution is 100-200:800-900.

2. The polysaccharide-embedded probiotic of claim 1, wherein the gastrointestinal disorder comprises colitis.

3. The polysaccharide-embedded probiotic of claim 1 or 2, wherein the polysaccharide layer corresponds to a polysaccharide material comprising at least one of beta-glucan, fucan, silverfish polysaccharide, low ester pectin, tremella polysaccharide, pachyman, chitosan, starch, and cellulose.

4. The polysaccharide-embedded probiotic of claim 1 or 2, wherein the probiotic comprises at least one of escherichia coli Nissle1917, lactobacillus reuteri, lactobacillus rhamnosus, bifidobacterium, lactobacillus plantarum and lactobacillus salivarius.

5. The polysaccharide-embedded probiotic of claim 1 or 2, wherein the metal ion corresponding to the metal-polyphenol network layer comprises Fe ³⁺ 、Al ³⁺ 、Ti ⁴⁺ And Cr (V) ³⁺ At least one of (a) and (b);

and/or the polyphenol raw materials corresponding to the metal-polyphenol network structure layer comprise at least one of flavonoids, tannins and phenolic acids.

6. A method of preparing a polysaccharide-embedded probiotic according to any one of claims 1 to 5, comprising the steps of: and coating the metal-polyphenol network structure layer and the polysaccharide layer on the surface of the probiotics in the inner layer according to a preset position.

7. The method of claim 6, wherein the concentration of the polyphenol feedstock in the probiotic-metal-polyphenol complex is from 0.25 to 0.4 mg/mL.

8. The method of claim 6, wherein 0.01-0.1/mg of the polysaccharide material is used per ml of the probiotic-metal-polyphenol complex.

9. A medicament comprising the polysaccharide-embedded probiotic of any one of claims 1-5.