A probiotic product and its preparation method and medicine

Abstract

The present invention discloses a probiotic product and a preparation method and medicine thereof, belonging to the technical field of probiotic products. The preparation of the probiotic product includes: co-incubating and culturing the probiotic with a substance having an azide group, metabolizing the substance having an azide group by the endogenous metabolic pathway of the probiotic itself to obtain a probiotic expressing an azide group on the surface; after co-incubation, first adding a functional substance modified with a DBCO group to the resuspension of the probiotic expressing an azide group on the surface, then adding a polysaccharide modified with a DBCO group, and then reacting at 30°C to 38°C for 2h to 16h to obtain a probiotic covalently grafted with a functional substance and a polysaccharide. The method is simple and easy to operate, and can safely and efficiently prepare a probiotic product with a stable structure, enhance the colonization of probiotics in the intestine, and the product has a high survival rate of probiotics, and can be used to prepare drugs for treating intestinal diseases, especially drugs for treating colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic products, and in particular to a probiotic product and a preparation method and a medicine thereof. Background Art

[0002] Probiotics are a type of microorganism that can improve the balance of microorganisms in the host's intestines and produce positive benefits to the host. Due to the adverse effects of gastric acid, bile and various digestive enzymes in the human body, the survival rate of probiotics is greatly reduced when they reach the intestines. At the same time, only when probiotics are stably colonized in the host's intestines can they reproduce and exert their own probiotic effects. Therefore, whether a high survival rate can be maintained and the colonization of probiotics in the intestines can be enhanced is a key factor in the probiotic effect of probiotics.

[0003] The current common method for maintaining the activity of probiotics is to coat the surface of the probiotics with a protective film, which is a simple and effective method to improve the survival rate of probiotics in the digestive tract. However, the product structure obtained by the above coating method is relatively loose, and there are risks such as the protective film falling off and easy penetration of gastrointestinal digestive juices, and the corresponding product has poor effect.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a probiotic product and a preparation method and a medicine thereof, so as to solve or improve the above technical problems.

[0006] This application can be implemented as follows:

[0007] In a first aspect, the present application provides a method for preparing a probiotic product, which comprises the following steps: co-incubating a probiotic with a substance having an azide group, and metabolizing the substance having an azide group by an endogenous metabolic pathway of the probiotic to obtain a probiotic expressing an azide group on its surface;

[0008] After co-incubation, the functional substance modified with DBCO group is first added to the resuspended solution of the probiotics expressing azide groups on the surface, and then the polysaccharide modified with DBCO group is added, and then the reaction is carried out at 30°C to 38°C for 2h to 16h to obtain the probiotics covalently grafted with the functional substance and the polysaccharide;

[0009] The substance having an azide group is selected from at least one of D-alanine azide, azidomannosamine, N-azidoacetylgalactosamine-tetraacylated, N-azidoacetylgalactosamine, 3-deoxy-D-mannose-2-octulose acid azidosugar and L-azidohomoalanine hydrochloride;

[0010] The functional substance is selected from at least one of dopamine, a material having photodynamic properties, a material having photothermal properties, and a functional vesicle;

[0011] The co-incubation comprises: adding a bacterial solution containing probiotics to a culture medium, followed by culturing to obtain a resuspended solution of probiotics expressing azide groups on the surface;

[0012] Among them, the number of viable probiotics in the bacterial solution is 0.5×10 5 CFU / mL~2×10 6 CFU / mL, the culture medium contains a substance having an azide group, the concentration of the substance having an azide group in the culture medium is 0.02 mg / mL~1.2 mg / mL, and the volume ratio of the probiotic bacterial liquid to the culture medium containing the substance having an azide group is 1:10~1:50;

[0013] The culture temperature is 30℃~38℃, and the culture time is 2h~16h;

[0014] Add 0.05mg~0.2mg of DBCO group-modified functional substance per ml of resuspension;

[0015] 0.1 mg to 0.8 mg of DBCO group-modified polysaccharide is added to each milliliter of resuspension.

[0016] In an alternative embodiment, the probiotics include at least one of Lactococcus lactis, Escherichia coli Nissle 1917, Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacterium, Lactobacillus plantarum, Lactobacillus salivarius and Bacteroides fragilis.

[0017] In an optional embodiment, the polysaccharide includes at least one of β-glucan, fucoidan, silver thread vine polysaccharide, low-ester pectin, tremella polysaccharide, tuckahoe polysaccharide, chitosan, starch, lentinan, ganoderma lucidum polysaccharide and cellulose.

[0018] In a second aspect, the present application provides a probiotic product prepared by the preparation method of any one of the aforementioned embodiments.

[0019] In a third aspect, the present application provides a drug for treating intestinal diseases, the raw materials for preparing the drug include the probiotic product of the aforementioned embodiment.

[0020] The beneficial effects of this application include:

[0021] The preparation method provided in the present application can firmly put on coats with different functional properties for probiotics. It grafts functional substances and polysaccharides through safe and non-toxic bioorthogonal reactions. On the one hand, the functional substances and polysaccharides grafted by the covalent method are very stable and firm, not easy to fall off, and have a stable protective effect; on the other hand, the polysaccharides grafted on the outer layer of the probiotics have good stability in the upper digestive tract and have a shield-like effect, protecting the probiotics from damage by external environments such as gastric acid, bile salts, UV and freeze-drying, thereby effectively improving their survival rate; in addition, as the polysaccharides are degraded by enzymes secreted by intestinal bacteria in the intestine, the exposed functional substances can give full play to their functions. It should also be emphasized that in the present application, the probiotics expressing azide groups on the surface are formed by the probiotics metabolizing substances with azide groups using their own endogenous metabolic pathways, rather than combining by chemical reactions, thereby avoiding adverse effects on the probiotics, such as activity and stability.

[0022] The probiotic product prepared by the above preparation method has better resistance to gastric acid and bile salts, can be protected from gastrointestinal environment attacks, and achieve intestinal colonization and adhesion; moreover, the probiotic product can reduce the weight loss caused by colitis, alleviate the splenomegaly caused by colitis, alleviate the shortening of colon length caused by colitis, and effectively increase the retention of probiotics in the intestine; in addition, the probiotic product also has good anti-freeze-drying ability, anti-UV ability, and storage stability.

[0023] By using the above-mentioned probiotic products in medicines, the therapeutic effect of intestinal diseases, especially the therapeutic effect of colitis, can be enhanced or improved to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a scanning electron microscope image of the probiotic product in Experimental Example 1;

[0026] Figure 2 is a scanning electron microscope image of Lactococcus lactis in Experimental Example 1;

[0027] Figure 3 This is a transmission electron micrograph of the probiotic product in Experimental Example 1;

[0028] Figure 4 is a transmission electron microscope image of Lactococcus lactis in Experimental Example 1;

[0029] Figure 5 This is the confocal scanning result of the probiotic product in Experimental Example 1;

[0030] Figure 6 This is a scanning electron microscope image of the probiotic product in Experimental Example 2;

[0031] Figure 7 is a scanning electron microscope image of Lactobacillus plantarum in Experimental Example 2;

[0032] Figure 8 This is a graph showing the resistance results of dopamine-β-glucan-embedded Lactococcus lactis, Lactococcus lactis and β-glucan-embedded Lactococcus lactis in simulated gastric fluid in Experimental Example 3;

[0033] Fig. 9 This is a graph showing the resistance results of dopamine-β-glucan-embedded Lactococcus lactis, Lactococcus lactis and β-glucan-embedded Lactococcus lactis in simulated bile in Experimental Example 3;

[0034] Fig.10 This is a graph showing the intestinal retention ability results of dopamine-β-glucan-embedded Lactococcus lactis, Lactococcus lactis and β-glucan-embedded Lactococcus lactis in Experimental Example 4;

[0035] Fig.11 The result diagram of the anti-freeze-drying performance of Lactococcus lactis and dopamine-β-glucan-embedded Lactococcus lactis in Experimental Example 5;

[0036] Fig.12 The result diagram of the storage stability of Lactococcus lactis and dopamine-β-glucan-embedded Lactococcus lactis in Experimental Example 5;

[0037] Fig.13 The results of the UV resistance of Lactococcus lactis and dopamine-β-glucan-embedded Lactococcus lactis in Test Example 6 are shown in FIG.

[0038] Fig.14 This is a graph showing the resistance results of dopamine-Ganoderma lucidum polysaccharide-embedded Lactococcus lactis, Lactococcus lactis and Ganoderma lucidum polysaccharide-embedded Lactococcus lactis in simulated gastric fluid in Experimental Example 7;

[0039] Fig.15 This is a graph showing the resistance results of dopamine-fucoidan-embedded Lactobacillus salivarius, Lactobacillus salivarius and fucoidan-embedded Lactobacillus salivarius in simulated gastric fluid in Experimental Example 7;

[0040] Fig.16 This is a graph showing the resistance results of dopamine-silver thread vine polysaccharide-embedded Lactobacillus plantarum, Lactobacillus plantarum and silver thread vine polysaccharide-embedded Lactobacillus plantarum in simulated gastric fluid in Experimental Example 7;

[0041] Fig.17The graph is a result of the weight change of each group of colitis mice in Experimental Example 8 during the 7-day oral gavage period;

[0042] Fig.18 This is the spleen index result diagram of each group of colitis mice in Experimental Example 8 after 7 days of intragastric administration;

[0043] Fig.19 The colon length results of each group of colitis mice in Experimental Example 8 after 7 days of intragastric administration;

[0044] Fig. 20 The graph is a result of the absorbance value at a wavelength of 600 nm after overnight culture of the culture medium containing different concentrations of D-alanine azide and the bacterial solution of Lactococcus lactis in Experimental Example 9;

[0045] Fig.21 The potential result diagram of the resuspension containing the functional substance modified with DBCO group at different concentrations in Experimental Example 9;

[0046] Fig. 22 This is a graph showing the potential results of the resuspension containing different concentrations of DBCO group-modified polysaccharides in Experimental Example 9. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0048] The probiotic product and its preparation method and medicine provided in this application are described in detail below.

[0049] The present application proposes a method for preparing a probiotic product, which comprises the following steps: co-incubating probiotics with a substance having an azide group, metabolizing the substance having an azide group by the endogenous metabolic pathway of the probiotics themselves to obtain probiotics expressing an azide group on the surface;

[0050] Probiotics expressing azide groups on their surfaces are mixed with functional substances modified with dibenzocyclooctyne groups (hereinafter referred to as "DBCO") and polysaccharides modified with dibenzocyclooctyne groups, so that part of the azide groups expressed on the surface of the probiotics are covalently bonded to the dibenzocyclooctyne groups of the modified functional substances, and the remaining azide groups expressed on the surface of the probiotics are covalently bonded to the dibenzocyclooctyne groups of the modified polysaccharides, thereby obtaining probiotics covalently grafted with functional substances and polysaccharides.

[0051] It should be noted that the conventional microencapsulation method can protect probiotics from tolerating the gastrointestinal environment, but it is of little help to the colonization of probiotics. This application utilizes the properties of polysaccharides to protect probiotics from tolerating the gastrointestinal environment and delivers probiotics to the intestinal tract in a targeted manner, and uses different functional substances to exert different effects as needed. In addition, compared with conventional non-covalently embedded probiotics, this application uses metabolic labeling and click chemistry to covalently react functional substances and polysaccharides with azide groups to graft functional substances and polysaccharides onto the surface of probiotics, so that probiotic products exhibit more compact and firm characteristics, which can ensure that probiotics can exert their effects in the intestines to a greater extent.

[0052] For reference, the above co-incubation may include: adding a bacterial solution containing probiotics to a culture medium, followed by culturing, during which the probiotics metabolize substances with azide groups using their own endogenous metabolic pathways, and resuspending to obtain a resuspended solution of probiotics expressing azide groups on the surface.

[0053] In the present application, the probiotics expressing azide groups on their surfaces are formed by the probiotics utilizing their own endogenous metabolic pathways to metabolize substances with azide groups, rather than combining through chemical reactions, thereby avoiding adverse effects on the probiotics, such as activity and stability.

[0054] The number of viable probiotics in the bacterial solution can be 0.5×10 5 CFU / mL~2×10 6 CFU / mL, such as 0.5×10 5 CFU / mL, 1×10 5 CFU / mL, 1.5×10 5 CFU / mL or 2×10 5 CFU / mL, etc., can also be 0.5×10 5 CFU / mL~2×10 6 Any other value within the CFU / mL range.

[0055] The above-mentioned culture medium contains a substance having an azide group. Exemplarily, the concentration of the substance having an azide group in the culture medium can be 0.02 mg / mL to 1.2 mg / mL, such as 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 1.0 mg / mL or 1.2 mg / mL, or it can be any other value within the range of 0.02 mg / mL to 1.2 mg / mL.

[0056] The volume ratio of the probiotic bacterial solution to the culture medium containing the substance having an azide group can be 1:10-1:50, such as 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50, or any other value within the range of 1:10-1:50.

[0057] The culture temperature can be 30°C to 38°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 36.5°C, 37°C, 37.5°C or 38°C, etc. The culture time can be 2h to 16h, such as 2h, 4h, 6h, 10h, 12h, 14h or 16h, etc.

[0058] After co-incubation, the functional substance modified with the DBCO group is first added to the resuspended solution of the probiotic expressing the azide group on the surface, and then the polysaccharide modified with the DBCO group is added, followed by the reaction at 30°C to 38°C (such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 36.5°C, 37°C, 37.5°C or 38°C, etc.) for 2h to 16h (such as 2h, 4h, 6h, 10h, 12h, 14h or 16h, etc.).

[0059] Among them, 0.05 mg~0.2 mg (such as 0.05 mg, 0.1 mg, 0.15 mg or 0.2 mg, etc.) of DBCO group-modified functional substance can be added to each milliliter of resuspension.

[0060] 0.1 mg to 0.8 mg (such as 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg or 0.8 mg, etc.) of DBCO group-modified polysaccharide can be added to each milliliter of resuspension.

[0061] In the present application, the probiotics may include, by way of example but not limitation, at least one of Lactococcus lactis, Escherichia coli Nissle 1917, Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacterium, Lactobacillus plantarum, Lactobacillus salivarius and Bacteroides fragilis. In addition, other probiotics beneficial to the human body may also be used as needed.

[0062] Wherein, the culture medium used for Lactococcus lactis can be M17 culture medium, the culture medium used for Escherichia coli Nissle1917 can be LB broth culture medium, the culture medium used for Lactobacillus reuteri can be MRS culture medium, the culture medium used for Lactobacillus rhamnosus and Lactobacillus plantarum can be MRS culture medium, the culture medium used for Bifidobacterium can be MRS culture medium, the culture medium used for Lactobacillus salivarius can be MRS culture medium, and Bacteroides fragilis can be used for BHI culture medium. The above-mentioned culture mediums of various types can directly adopt commercially available related culture mediums, or can be configured by themselves according to the conventional formulas of various types of culture mediums.

[0063] The substance having an azide group is selected from at least one of D-alanine azide, azidomannosamine, N-azidoacetylgalactosamine-tetraacylated, N-azidoacetylgalactosamine, 3-deoxy-D-mannos-2-octulose acid azidosugar and L-azidohomoalanine hydrochloride.

[0064] The functional substance is selected from at least one of dopamine, materials with photodynamic properties, materials with photothermal properties, and functional vesicles. Among them, dopamine has strong adhesion and can act as a gripper to grasp the intestinal mucosal layer, which is beneficial to improving the colonization effect and intestinal retention time of probiotics. Materials with photodynamic properties can include, for example, gold cluster composite materials, which are beneficial to clear or reduce the active oxygen produced in the intestine and improve the antioxidant effect; photothermal materials can include, for example, black phosphorus composite materials, indocyanine green (ICG), etc., which are beneficial to killing tumors; functional vesicles can include, for example, AKK bacteria vesicles, which can improve enteritis.

[0065] The polysaccharide may include, by way of example but not limitation, at least one of β-glucan, fucoidan, silver thread vine polysaccharide, low-ester pectin, tremella polysaccharide, tuckahoe polysaccharide, chitosan, starch, lentinan, ganoderma polysaccharide and cellulose. In addition, other polysaccharide substances may also be used as required.

[0066] As mentioned above, the preparation method provided in the present application can firmly dress the probiotics with outer coats of different functional properties. It grafts functional substances (taking dopamine as an example) and polysaccharides through safe and non-toxic bioorthogonal reactions. On the one hand, the dopamine and polysaccharides grafted by the covalent method are very stable and firm, not easy to fall off, and have a stable protective effect; on the other hand, the polysaccharides grafted on the outer layer of the probiotics have good stability in the upper digestive tract and have a shield-like effect, protecting the probiotics from damage by external environments such as gastric acid, bile salts, UV and freeze-drying, thereby effectively improving their survival rate; in addition, as the polysaccharides are degraded in the intestine by enzymes secreted by intestinal bacteria, the exposed dopamine has strong adhesion properties, so it can act as a gripper to grasp the intestinal mucosal layer, thereby improving the colonization effect of the probiotics, and thus maximizing the function of the probiotics.

[0067] Correspondingly, the present application also provides a probiotic product, which is prepared by the above preparation method.

[0068] This probiotic product has good resistance to gastric acid and bile salts, can be protected from gastrointestinal environment attacks, and achieve intestinal colonization and adhesion; moreover, this probiotic product can reduce the weight loss caused by colitis, alleviate the splenomegaly caused by colitis, alleviate the shortening of colon length caused by colitis, and effectively increase the retention of probiotics in the intestine; in addition, this probiotic product also has good anti-freeze-drying ability, anti-UV ability, and storage stability.

[0069] Furthermore, the present application also provides a medicine for treating intestinal diseases, the raw materials for preparing the medicine include the probiotic product of the aforementioned embodiment.

[0070] By using the above-mentioned probiotic products in medicines, the therapeutic effect of intestinal diseases, especially the therapeutic effect of colitis, can be enhanced or improved to a certain extent.

[0071] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0072] Example 1

[0073] This embodiment provides a probiotic product, and the preparation method thereof comprises:

[0074] Step (1): 0.016 mL of a bacterial solution containing Lactococcus lactis was added to 0.8 mL of fresh M17 medium, wherein the number of viable Lactococcus lactis in the bacterial solution was 0.5×10 5 CFU / mL, the above fresh culture medium contains 0.016 mg of D-alanine azide, and is cultured overnight (37°C, 220 rpm, 16 h), followed by centrifugation and washing, and the solid matter is resuspended in 0.4 mL of PBS (pH 6.5) to obtain a resuspension of Lactococcus lactis expressing azide groups on the surface (the number of viable Lactococcus lactis in the resuspension is > 1×10 9 CFU / mL);

[0075] Step (2): Add 0.02 mg of DBCO-modified dopamine to the above resuspension, vortex for 60 seconds, then add 0.04 mg of DBCO-modified β-glucan, vortex for 60 seconds, and react overnight (37°C, 220 rpm, 16 hours);

[0076] Step (3): Wash twice with PBS and centrifuge to remove excess DBCO-modified β-glucan and DBCO-modified dopamine. The centrifugation speed is 6000 rpm and the centrifugation time is 4 min to obtain Lactococcus lactis covalently grafted with β-glucan and dopamine (i.e., a probiotic product).

[0077] Example 2

[0078] This embodiment provides a probiotic product, and the preparation method thereof comprises:

[0079] Step (1): Add 0.032 mL of a bacterial solution containing Lactococcus lactis to 0.8 mL of fresh M17 medium, wherein the number of viable Lactococcus lactis in the bacterial solution is 1×10 6 CFU / mL, the above fresh culture medium contains 0.44 mg of D-alanine azide, and is cultured overnight (37°C, 220 rpm, 16 h), followed by centrifugation and washing, and the solid matter is resuspended in 0.4 mL of PBS (pH 6.5) to obtain a resuspension of Lactococcus lactis expressing azide groups on the surface (the number of viable Lactococcus lactis in the resuspension is > 1×10 9 CFU / mL);

[0080] Step (2): Add 0.04 mg of DBCO-modified dopamine to the above resuspension, vortex for 60 seconds, then add 0.24 mg of DBCO-modified β-glucan, vortex for 60 seconds, and react overnight (37°C, 220 rpm, 16 hours);

[0081] Step (3): Wash twice with PBS and centrifuge to remove excess DBCO-modified β-glucan and DBCO-modified dopamine. The centrifugation speed is 6000 rpm and the centrifugation time is 4 min to obtain Lactococcus lactis covalently grafted with β-glucan and dopamine (i.e., a probiotic product).

[0082] Example 3

[0083] This embodiment provides a probiotic product, and the preparation method thereof comprises:

[0084] Step (1): Add 0.064 mL of a bacterial solution containing Lactococcus lactis to 0.8 mL of fresh M17 medium, wherein the number of viable Lactococcus lactis in the bacterial solution is 2×10 6 CFU / mL, the above fresh culture medium contains 0.64 mg of D-alanine azide, and is cultured overnight (37°C, 220 rpm, 16 h), followed by centrifugation and washing, and the solid matter is resuspended in 0.4 mL of PBS (pH 6.5) to obtain a resuspension of Lactococcus lactis expressing azide groups on the surface (the number of viable Lactococcus lactis in the resuspension is > 1×10 9 CFU / mL);

[0085] Step (2): Add 0.08 mg of DBCO-modified dopamine to the above resuspension, vortex for 60 seconds, then add 0.32 mg of DBCO-modified β-glucan, vortex for 60 seconds, and react overnight (37°C, 220 rpm, 16 hours);

[0086] Step (3): Wash twice with PBS and centrifuge to remove excess DBCO-modified β-glucan and DBCO-modified dopamine. The centrifugation speed is 6000 rpm and the centrifugation time is 4 min to obtain Lactococcus lactis covalently grafted with β-glucan and dopamine (i.e., a probiotic product).

[0087] Example 4

[0088] The difference between this embodiment and embodiment 2 is that an equal amount of β-glucan is replaced with Ganoderma lucidum polysaccharide.

[0089] Example 5

[0090] The difference between this embodiment and embodiment 2 is that an equal amount of β-glucan is replaced by fucoidan, and an equal amount of Lactococcus lactis is replaced by Lactobacillus salivarius.

[0091] Example 6

[0092] The difference between this embodiment and embodiment 2 is that an equal amount of β-glucan is replaced by leucophylla polysaccharide, and an equal amount of Lactococcus lactis is replaced by Lactobacillus plantarum.

[0093] Example 7

[0094] The difference between this embodiment and embodiment 2 is that an equal amount of β-glucan is replaced by silver thread vine polysaccharide, an equal amount of Lactococcus lactis is replaced by Escherichia coli Nissle1917 (hereinafter referred to as "EcN"), and an equal amount of D-alanine azide is replaced by 3-deoxy-D-manno-2-octulose acid azide sugar.

[0095] Example 8

[0096] The difference between this embodiment and embodiment 2 is that an equal amount of β-glucan is replaced with low-ester pectin.

[0097] Example 9

[0098] The difference between this embodiment and embodiment 2 is that an equal amount of β-glucan is replaced by a mixture of Tremella fuciformis polysaccharide and Poria cocos polysaccharide (in the mixture, the mass ratio of Tremella fuciformis polysaccharide to Poria cocos polysaccharide is 1:1).

[0099] Example 10

[0100] The difference between this embodiment and embodiment 2 is that: the β-glucan is replaced by an equal amount of a mixture of chitosan, starch and cellulose (in the mixture, the mass ratio of chitosan, starch and cellulose is about 1:1:1).

[0101] Embodiment 11

[0102] The difference between this embodiment and embodiment 2 is that an equal amount of Lactococcus lactis is replaced by Lactobacillus reuteri.

[0103] Example 12

[0104] The difference between this embodiment and embodiment 2 is that an equal amount of Lactococcus lactis is replaced by Lactobacillus rhamnosus.

[0105] Embodiment 13

[0106] The difference between this embodiment and embodiment 2 is that an equal amount of Lactococcus lactis is replaced by a mixture of Bifidobacterium and Lactobacillus plantarum (in the mixture, the mass ratio of Bifidobacterium to Lactobacillus plantarum is 1:1).

[0107] Test Example 1

[0108] Taking Example 2 as an example, the obtained probiotic product was subjected to the following tests:

[0109] ①. Scanning electron microscopy was performed on the probiotic products obtained and the Lactococcus lactis used in their preparation (without any treatment). The results are as follows: Figure 1 and Figure 2 As shown. Among them, Figure 1 This is a scanning electron microscope image of a probiotic product. Figure 2 This is a scanning electron micrograph of Lactococcus lactis.

[0110] ②. The obtained probiotic products and the Lactococcus lactis used in their preparation (without any treatment) were observed under transmission electron microscopy. The results are as follows: Figure 3 and Figure 4 As shown. Among them, Figure 3 This is a transmission electron micrograph of a probiotic product. Figure 4 This is a transmission electron micrograph of Lactococcus lactis.

[0111] ③. Perform confocal scanning on the obtained probiotic product, and the results are as follows: Figure 5 Among them, a is a bright field image of the probiotic product, b is a laser confocal image of dopamine in the probiotic product (dopamine modified with DBCO group labeled with rhodamine), c is a laser confocal image of β-glucan in the probiotic product (β-glucan modified with DBCO group labeled with fluorescein), and d is a laser confocal image of the probiotic product.

[0112] Combination Figures 1 to 5It can be seen that dopamine and β-glucan successfully encapsulated Lactococcus lactis.

[0113] Test Example 2

[0114] Taking Example 6 as an example, the obtained probiotic product was subjected to the following tests:

[0115] ①. Scanning electron microscopy was performed on the probiotic product obtained and the Lactobacillus plantarum (without any treatment) used in its preparation process. The results are as follows: Figure 6 and Figure 7 As shown. Among them, Figure 6 This is a scanning electron microscope image of a probiotic product. Figure 7 This is a scanning electron micrograph of Lactobacillus plantarum.

[0116] Combination Figure 6 and Figure 7 It can be seen that the silver thread vine polysaccharide and dopamine successfully encapsulated Lactobacillus plantarum.

[0117] Test Example 3

[0118] The probiotic product prepared in Example 2 was subjected to an in vitro resistance study, and the specific research method is as follows:

[0119] Step (1): The probiotic product prepared in Example 2 (referred to as "dopamine-β-glucan-encapsulated Lactococcus lactis", the same below) was placed in an environment of simulated gastric fluid and simulated bile, and incubated at 37°C for 2 h respectively as an experimental group, and Lactococcus lactis without any treatment and Lactococcus lactis grafted with β-glucan only (i.e., without dopamine grafted, referred to as "β-glucan-encapsulated Lactococcus lactis", the same below) were used as a control group.

[0120] The method for simulating gastric fluid is as follows: 5 g of pepsin and 8.5 g of sodium chloride are dissolved in 1000 ml of sterile water, and the pH is adjusted to 2.5, and filtered through a 0.22 μm sterilization filter membrane before use.

[0121] The method for simulating bile is as follows: prepare 0.5% bile salt solution and filter it through a 0.22μm sterilizing filter membrane before use.

[0122] Step (2): At different time points (0h, 0.5h, 1h and 2h), 50 μL of simulated gastric fluid and simulated bile from the experimental group and each control group were taken for dilution and coating and then counted.

[0123] The results are as follows Figure 8 and Fig. 9 As shown. Among them, Figure 8 Corresponding to the protective effect in simulated gastric fluid, Fig. 9 Corresponding to the protective effect in simulated bile. Figure 8 and Fig. 9It can be seen that dopamine-β-glucan-embedded Lactococcus lactis has a strong ability to resist gastric acid and bile salts, and Lactococcus lactis with and only polysaccharide embedding also has a certain ability to resist gastric acid and bile salts. This result shows that the presence of dopamine may make the polysaccharide grafting more compact, which is conducive to better improving the survival rate of Lactococcus lactis in gastric juice and bile, so that it can play an effective role in the intestine.

[0124] Test Example 4

[0125] The intestinal retention capacity of the probiotic product prepared in Example 2 was studied, and the specific method was as follows:

[0126] Step (1): After fasting for 24 hours, the mice were randomly divided into three groups, with 6 mice in each group, and were gavaged with 200 μL of fluorescently labeled Lactococcus lactis, fluorescently labeled β-glucan-embedded Lactococcus lactis, and fluorescently labeled dopamine-β-glucan-embedded Lactococcus lactis respectively;

[0127] Step (2): Perform in vivo imaging of mice at different time points (0h and 10h) and perform fluorescence imaging analysis. The relative fluorescence intensity is the difference between the fluorescence intensity at 10h and the fluorescence intensity at 0h.

[0128] The results are as follows Fig.10 As shown by Fig.10 It can be seen that the fluorescence intensity of dopamine-β-glucan-embedded Lactococcus lactis retained in the intestine after 10 hours was significantly stronger than that of β-glucan-embedded Lactococcus lactis and Lactococcus lactis, indicating that the retention time of dopamine-β-glucan-embedded Lactococcus lactis in the intestine after 10 hours was significantly longer than that of β-glucan-embedded Lactococcus lactis and Lactococcus lactis.

[0129] Test Example 5

[0130] The probiotic product prepared in Example 2 was freeze-dried and the storage stability was studied, and the specific method was as follows:

[0131] Freeze drying: Take a portion of Lactococcus lactis and the probiotic product prepared in Example 2 for dilution and counting. Then, freeze-dry the remaining sample in one-tenth volume of the protective solution (citrate buffer). After freeze drying, take a portion of the bacterial powder for dilution and coating, and count the number of colonies after 48 hours.

[0132] Storage stability: Store at different temperatures (-20℃, 4℃) for 7 days. Resuspend the bacterial powder after 7 days, take an appropriate volume for dilution, and spread it on the culture medium. Count it after 24 hours. Compare the colony counts before and after storage to compare the protective effect of dopamine-β-glucan on Lactococcus lactis.

[0133] The results are as follows Fig.11 and Fig.12As shown, Fig.11 The results of Lactococcus lactis and dopamine-β-glucan-encapsulated Lactococcus lactis before and after freeze-drying are shown; Fig.12 This is a graph showing the storage stability results of Lactococcus lactis and dopamine-β-glucan-encapsulated Lactococcus lactis.

[0134] Depend on Fig.11 and Fig.12 It can be seen that dopamine-β-glucan-embedded Lactococcus lactis has better anti-freeze-drying ability and storage stability than Lactococcus lactis.

[0135] Test Example 6

[0136] The probiotic product prepared in Example 2 was subjected to a study on UV resistance, and the specific method is as follows:

[0137] Lactococcus lactis and the probiotic product prepared in Example 2 were exposed to UV (900 μW / cm 2 ) and irradiated for 5 min, 10 min and 15 min. Samples were taken at the corresponding time points and diluted and spread. The number of colonies was calculated after 48 h.

[0138] The results are as follows Fig.13 As shown, Fig.13 The results of the anti-UV of Lactococcus lactis and probiotic products are shown in the figure. Fig.13 It can be seen that dopamine-β-glucan-embedded Lactococcus lactis has better UV resistance than Lactococcus lactis.

[0139] Test Example 7

[0140] The probiotic products obtained in Examples 4-6 were used to conduct in vitro resistance studies (protective effects in simulated gastric fluid), and the specific methods were as described in Test Example 3.

[0141] The results are as follows Figures 14 to 16 As shown by Figures 14 to 16 It can be seen that dopamine-Ganoderma lucidum polysaccharide-embedded Lactococcus lactis, dopamine-fucoidan-embedded Lactobacillus salivarius and dopamine-Cebuia argyi polysaccharide-embedded Lactobacillus plantarum all have strong gastric acid resistance.

[0142] Test Example 8

[0143] The following test was carried out using the probiotic product prepared in Example 12 as an example:

[0144] Experimental animal grouping and treatment: C57BL / 6J mice (20±0.2 g, 6-8 weeks old) were divided into normal group, model group, Lactobacillus rhamnosus group (referred to as "LGG group"), polysaccharide group, β-glucan-embedded Lactobacillus rhamnosus group (referred to as "polysaccharide-embedded LGG group"), dopamine-β-glucan-embedded Lactobacillus rhamnosus group (referred to as "dopamine-polysaccharide-embedded LGG group") and β-glucan and Lactobacillus rhamnosus physical mixture group (referred to as "polysaccharide + LGG group") according to their weight, with 4 mice in each group. The mice first underwent a 7-day adaptation period. After the adaptation period, the normal group had free access to water throughout the experiment, while the other 6 groups of mice drank 2.5% dextran sulfate sodium for 7 consecutive days. Salt, DSS) was used to construct the colitis model, and the mice were weighed every day during the modeling period. During the 7-day modeling period, the normal group and the model group were gavaged with 0.2 mL of PBS every day, while the LGG group, the polysaccharide-embedded LGG group, and the dopamine-polysaccharide-embedded LGG group were gavaged with the corresponding bacterial suspension (the probiotic dose was 1×10 9 CFU, among which, the bacterial suspension of the polysaccharide-embedded LGG group contained polysaccharides in addition to probiotics, and the bacterial suspension of the dopamine-polysaccharide-embedded LGG group contained polysaccharides and dopamine in addition to probiotics, with a polysaccharide concentration of 6 mg kg -1 BW, dopamine concentration was 1 mg kg - 1 The polysaccharide group was intragastrically administered at a concentration of 40 mg kg-1 according to the body weight of each mouse. -1 The polysaccharide solution of BW and the polysaccharide + LGG group were gavaged with the corresponding probiotics and polysaccharide mixture (the probiotic dose was 1×10 9 CFU, polysaccharide concentration is 40 mg kg -1 BW). After removing DSS, mice were killed by cervical dislocation. The colon tissue of mice was isolated, the length of colon tissue was measured, and the spleen weight was weighed. The spleen index = (spleen weight / mouse body weight) × 100%. All dissected tissues were frozen at -80 °C for further analysis.

[0145] Body weight changes of colitis mice during 7 days of oral gavage Fig.17 As shown, Fig.17 It can be seen that oral administration of dopamine-β-glucan-embedded Lactobacillus rhamnosus can effectively reduce the weight loss caused by colitis in mice.

[0146] The spleen index of colitis mice after 7 days of oral gavage Fig.18 As shown, Fig.18 It can be seen that intragastric administration of dopamine-β-glucan-embedded Lactobacillus rhamnosus can effectively alleviate the phenomenon of spleen enlargement caused by colitis in mice.

[0147] The length of the colon of colitis mice after 7 days of oral gavage Fig.19 As shown, Fig.19 It can be seen that intragastric administration of dopamine-β-glucan-embedded Lactobacillus rhamnosus can effectively alleviate the phenomenon of shortening of colon length caused by colitis.

[0148] The above results prove that the dopamine-β-glucan-embedded Lactobacillus rhamnosus provided in the present application can effectively alleviate intestinal inflammation caused by colitis in mice.

[0149] In addition, through Figures 17 to 19 It can be seen that by combining dopamine, β-glucan and LGG and β-glucan and LGG in a covalent encapsulation form, the weight loss caused by colitis in mice can be effectively reduced, the spleen enlargement caused by colitis in mice can be effectively alleviated, and the shortening of the colon length caused by colitis can be effectively alleviated. Among them, the dopamine-β-glucan-embedded Lactobacillus rhamnosus group is better than the β-glucan-embedded Lactobacillus rhamnosus group, indicating that containing dopamine can effectively increase the retention of LGG in the intestine, thereby making LGG play a better role. It shows that the encapsulation form used in this application can make dopamine and polysaccharides cooperate with probiotics to exert biological activity, and improve the therapeutic effect of the system as a whole.

[0150] Test Example 9

[0151] Referring to Example 1, the absorbance at 600 nm of the culture medium containing D-alanine azide at various concentrations of 0 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.6 mg / mL, 0.9 mg / mL, 1.2 mg / mL, 1.5 mg / mL and 1.8 mg / mL was studied. The results are as follows: Fig. 20 shown.

[0152] Depend on Fig. 20 It can be seen that when the concentration of D-alanine azide in the culture medium is above 1.5 mg / mL, it may have an adverse effect on probiotics. Therefore, the concentration of D-alanine azide in the culture medium is controlled at 1.2 mg / mL or below, for example, it can be controlled to 0.02 mg / mL~1.2 mg / mL.

[0153] Furthermore, taking the concentration of D-alanine azide in M17 medium as 1.2 mg / mL as an example, the appropriate amount of DBCO group-modified functional substance added per milliliter of resuspension was studied. Specifically, the potential results of the resuspension containing DBCO group-modified functional substance after adding 0 mg, 0.05 mg, 0.10 mg, 0.20 mg and 0.30 mg of DBCO group-modified functional substance per milliliter of resuspension were studied. The results are as follows: Fig.21 shown.

[0154] Depend on Fig.21 It can be seen that when the amount of DBCO group-modified functional substance in each milliliter of resuspension exceeds 0.2 mg, the potential of the resuspension containing the DBCO group-modified functional substance will stop decreasing, that is, the amount of DBCO group-modified functional substance usage reaches saturation. Therefore, the amount of DBCO group-modified functional substance added in each milliliter of resuspension is controlled to not exceed 0.2 mg, for example, it can be controlled to 0.05 mg~0.2 mg.

[0155] Furthermore, taking the addition of 0.05 mg DBCO group-modified functional substance per milliliter of resuspension as an example, the appropriate amount of DBCO group-modified polysaccharide added per milliliter of resuspension was studied. Specifically, 0.6 mg, 0.8 mg and 1.0 mg DBCO group-modified polysaccharide were added per milliliter of resuspension, and the corresponding potential of the resuspension was measured. The results are as follows: Fig. 22 shown.

[0156] Depend on Fig. 22 It can be seen that when the amount of DBCO group-modified polysaccharide added to each milliliter of resuspension exceeds 0.8 mg, the potential of the resuspension will stop decreasing, that is, the amount of DBCO group-modified polysaccharide used has reached saturation. Therefore, the amount of DBCO group-modified polysaccharide added to each milliliter of resuspension is controlled to not exceed 0.8 mg, for example, it can be controlled to 0.1 mg~0.8 mg.

[0157] In summary, the probiotic product provided by the present application has good stability in the intestine, and can effectively improve the anti-freeze-drying ability, anti-UV ability and storage stability of the probiotics without damaging the inherent biological characteristics of the probiotics, and at the same time safely and effectively enhance the survival rate of the probiotics in the intestine and enhance its colonization ability in the intestine. The preparation method of the probiotic product is simple to operate, and the reaction conditions are mild. The probiotic product can have a multivariate combination, and probiotics and polysaccharides can also have different choices. Different probiotics and polysaccharide materials can be selected for different diseases to be combined, thereby expanding the use. The corresponding probiotic product can be used to further prepare drugs for the treatment of gastrointestinal and metabolic diseases, especially drugs for the treatment of colitis.

[0158] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a probiotic product in the preparation of a drug for treating colitis, characterized in that: The drug is a drug for alleviating at least one of weight loss, spleen enlargement and shortened colon length caused by colitis; The probiotic product has freeze-drying resistance, UV resistance and storage stability; The preparation of the probiotic product comprises the following steps: co-incubating the probiotics with a substance having an azide group, metabolizing the substance having an azide group by the endogenous metabolic pathway of the probiotics themselves to obtain probiotics expressing an azide group on the surface; After co-incubation, first add the functional substance modified with DBCO group to the resuspension of the probiotics expressing azide groups on the surface, then add the polysaccharide modified with DBCO group, and then react at 30°C to 38°C for 2h to 16h, so that part of the azide groups expressed on the surface of the probiotics are covalently bonded to the dibenzocyclooctyne groups of the modified functional substance, and the remaining azide groups expressed on the surface of the probiotics are covalently bonded to the dibenzocyclooctyne groups of the modified polysaccharide, to obtain the probiotics covalently grafted with the functional substance and the polysaccharide; The substance having an azide group is selected from at least one of D-alanine azide, azidomannosamine, N-azidoacetylgalactosamine-tetraacylated, N-azidoacetylgalactosamine, 3-deoxy-D-mannose-2-octulose acid azidosugar and L-azidohomoalanine hydrochloride; The functional substance is dopamine; The co-incubation comprises: adding a bacterial solution containing the probiotics into a culture medium, followed by culturing and resuspending to obtain a resuspended solution of the probiotics expressing azide groups on the surface; The number of viable probiotics in the bacterial solution is 0.5×10 5 CFU / mL~2×10 6 CFU / mL, the culture medium contains a substance having an azide group, the concentration of the substance having an azide group in the culture medium is 0.02 mg / mL to 1.2 mg / mL, and the volume ratio of the probiotic solution to the culture medium containing the substance having an azide group is 1:10 to 1:50; The culture temperature is 30℃~38℃, and the culture time is 2h~16h; 0.05 mg to 0.2 mg of the DBCO group-modified functional substance is added to each milliliter of the resuspension; 0.1 mg to 0.8 mg of the DBCO group-modified polysaccharide is added to each milliliter of the resuspension.

2. The use according to claim 1, characterized in that: The probiotics include at least one of Lactococcus lactis, Escherichia coli Nissle 1917, Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacterium, Lactobacillus plantarum, Lactobacillus salivarius and Bacteroides fragilis.

3. The use according to claim 1, characterized in that: The polysaccharide comprises at least one of β-glucan, fucoidan, silver thread vine polysaccharide, low-ester pectin, tremella polysaccharide, tuckahoe polysaccharide, chitosan, starch, lentinan, ganoderma lucidum polysaccharide and cellulose.

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