A probiotic product and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a probiotic product and its preparation method and application. The method co-incubates a probiotic solution with a substance having an azide group, resuspends, and obtains a probiotic resuspension with an azide group modified on the surface; then reacts with a polysaccharide modified with a dibenzocyclooctyne group to obtain a resuspension of probiotics covalently encapsulated in a monolayer of polysaccharides; finally, reacts with a polysaccharide modified with an azide group to finally obtain a probiotic product covalently encapsulated in layers of polysaccharides. The probiotic product has better gastric acid and bile salt resistance, can be protected from gastrointestinal environmental attacks, and achieves intestinal targeted delivery; and the probiotic product can reduce the weight loss caused by colitis, relieve the splenomegaly caused by colitis, relieve the shortening of the colon length caused by colitis, actively improve the composition of intestinal flora, and can improve or improve the therapeutic effect of intestinal diseases to a certain extent, especially the therapeutic effect of colitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a probiotic product and a preparation method and application thereof. Background Art

[0002] Probiotics are a type of microorganism that can improve the intestinal microbial balance of the host and produce positive benefits to the host. However, oral probiotics are inevitably affected by the gastric acid, bile and various digestive enzymes in the human body, resulting in insufficient number of probiotics and failure to reach the intestinal part where they should work, thus greatly affecting the efficacy of probiotics, which leaves room for further improvement. Therefore, protecting probiotics from gastrointestinal attacks and thus delivering enough live probiotics to the intestine is a key step in consolidating the effectiveness of probiotic therapeutic drugs.

[0003] In order to protect probiotics from gastrointestinal attacks, the method currently commonly used to maintain the activity of probiotics is to wrap a protective layer on the surface of probiotics to prevent gastrointestinal attacks, which has been proven to be a direct and effective method. Some encapsulation methods of probiotics are disclosed in the prior art, and these prior arts have improved the survival rate of probiotics to varying degrees, but the products obtained by the above-mentioned encapsulation methods are all encapsulated in a non-covalent and non-single bacteria level manner. The resulting protective layer structure is relatively loose and unstable, and there are risks such as the protective layer falling off and the gastrointestinal digestive fluid being easily permeable, and the protective effect on the single bacteria is also different, so the effect of the corresponding product is also relatively poor. Secondly, in some of the disclosed encapsulation methods of probiotics, most of the materials selected for encapsulating probiotics do not have additional probiotic effects themselves, and even have certain toxic and side effects. Therefore, how to protect probiotics while coordinating probiotics to play a probiotic role, and then improve the efficacy of probiotic products is also a matter of future consideration. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a probiotic product and a preparation method and application thereof, specifically adopting the following technical solutions:

[0005] The first aspect of the present invention provides a method for preparing a probiotic product, comprising the following steps:

[0006] S1: co-incubating the probiotic bacterial solution with a substance having an azide group, and resuspending the solution to obtain a resuspended solution of probiotics with azide groups modified on the surface;

[0007] S2: mixing the resuspended probiotics with azide groups on the surface and the polysaccharide with probiotic effects modified with dibenzocyclooctyne groups, reacting, and resuspending to obtain a resuspended probiotics with a covalent monolayer of polysaccharide encapsulating the probiotics;

[0008] S3: placing the azide group-modified polysaccharide with probiotic effect in the resuspension of the polysaccharide monolayer-encapsulated probiotics for reaction, and finally obtaining a probiotic product covalently encapsulated by polysaccharides layer by layer.

[0009] The present invention co-incubates probiotics with substances having azide groups. During the incubation process, the bacteria use the substances modified by the azide groups as substrates in bacterial structures such as synthetic cell walls, outer membranes or capsule polysaccharides through an endogenous synthesis mechanism, thereby obtaining probiotics modified by the azide groups. Then, by using a click chemical reaction, the probiotics modified by the azide groups on the surface are mixed with the polysaccharide dibenzocyclooctyne modified by the dibenzocyclooctyne group and having a probiotic effect, so that the azide groups expressed on the surface of the probiotics and the dibenzocyclooctyne groups of the dibenzocyclooctyne modified polysaccharides undergo a click chemical reaction and form a stable covalent bond, thereby obtaining a covalently linked first protective layer. Subsequently, the polysaccharide modified by the azide groups and having a probiotic effect is added, so that the remaining dibenzocyclooctyne groups on the first protective layer (polysaccharides modified by the dibenzocyclooctyne groups) undergo a click chemical reaction with the azide groups of the azide modified polysaccharides, thereby obtaining a covalently linked second protective layer, and finally obtaining a probiotic product covalently encapsulated by polysaccharides.

[0010] As a further preferred embodiment, the number of viable bacteria in the probiotic solution in step S1 is 0.5×10 5 CFU / mL-1×10 7 CFU / mL; the concentration of the substance having an azide group is 0.02 mg / mL-1.46 mg / mL; the volume ratio of the probiotic solution to the substance having an azide group is: 1:1-1:100.

[0011] As a further preferred embodiment, in the above step S2, the ratio of the probiotic resuspension with azide groups modified on the surface to the polysaccharide modified with dibenzocyclooctyne groups is 1 mL: 0.6 mg-0.8 mg; the reaction temperature is 30°C-38°C; and the reaction time is 2 h-16 h.

[0012] As a further preferred embodiment, in the above step S3, the ratio of the resuspension of the polysaccharide-encapsulated probiotics to the azide-modified polysaccharide is 1 mL: 0.1 mg-0.6 mg; the reaction temperature is 30°C-38°C; and the reaction time is 2 h-16 h.

[0013] As a further preferred embodiment, the probiotics in the above-mentioned probiotic liquid include at least one of Lactococcus lactis, Escherichia coli Nissle1917, Lactobacillus reuteri, Lactobacillus rhamnosus, Bifidobacterium, Lactobacillus plantarum, Lactobacillus johnsonii, Clostridium butyricum, Lactobacillus salivarius and Bacteroides fragilis.

[0014] As a further preferred embodiment, the above-mentioned substance having an azide group includes 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.

[0015] As a further preferred embodiment, the polysaccharide with prebiotic effects includes at least one of β-glucan, fucoidan, silver thread polysaccharide, golden thread polysaccharide, low-ester pectin, tremella polysaccharide, tuckahoe polysaccharide, chitosan, inulin, licorice polysaccharide, astragalus polysaccharide, lentinan, ganoderma polysaccharide and lemon polysaccharide. The above polysaccharide is a polysaccharide with prebiotic effects. As a macromolecular substance of natural origin, it can not only be used as a protective wall material with targeted intestinal delivery, but also have beneficial effects on the human body.

[0016] The second aspect of the present invention provides a probiotic product prepared by the above preparation method.

[0017] The third aspect of the present invention also provides the use of the above probiotic product in the preparation of a drug for preventing and / or treating intestinal diseases. More preferably, the above intestinal disease is colitis.

[0018] The beneficial effects of the present invention are:

[0019] (1) The present invention provides a layer-by-layer assembly method. The preparation method is simple and efficient. Through the layer-by-layer assembly, probiotics can be firmly "dressed with a coat with different probiotic functional properties". The probiotic polysaccharides are grafted layer by layer through safe and non-toxic bioorthogonal reactions. On the one hand, the polysaccharides grafted by the covalent layer-by-layer assembly 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 probiotic polysaccharides are degraded by enzymes secreted by intestinal bacteria in the intestine, the functional substances produced can also play their probiotic functions.

[0020] (2) The present invention also provides a probiotic product prepared by the above preparation method, and the experimental data of the present invention show that compared with probiotic products of the same category, the probiotic product has better resistance to gastric acid and bile salts, can be protected from gastrointestinal environment attack, and achieve intestinal targeted delivery; and the probiotic product can reduce the weight loss caused by colitis, relieve the splenomegaly caused by colitis, relieve the shortening of colon length caused by colitis, and actively improve the composition of intestinal flora; in addition, the probiotic product also has good anti-freeze-drying ability, anti-ultraviolet ability, and storage stability. By using the above probiotic product in medicine, the therapeutic effect of intestinal diseases, especially the therapeutic effect of colitis, can be improved to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is a scanning electron microscope image of the probiotic product prepared in Example 2;

[0022] Figure 2 Shown is a scanning electron micrograph of Lactobacillus plantarum;

[0023] Figure 3 Shown is a transmission electron micrograph of the probiotic product prepared in Example 2;

[0024] Figure 4 Shown is a transmission electron micrograph of Lactobacillus plantarum;

[0025] Figure 5 Shown is the laser confocal scanning of the probiotic product, Lactobacillus plantarum and β-glucan prepared in Example 2;

[0026] Figure 6 Shown is a scanning electron microscope image of the probiotic product prepared in Example 5;

[0027] Figure 7 Shown is a scanning electron micrograph of Lactobacillus reuteri;

[0028] Figure 8 The figure shows the resistance results of β-glucan covalently encapsulated Lactobacillus plantarum layer by layer and β-glucan covalently encapsulated Lactobacillus plantarum monolayer and Lactobacillus plantarum prepared in Example 2 in simulated gastric fluid;

[0029] Fig. 9 The figure shows the resistance results of β-glucan covalently encapsulated Lactobacillus plantarum layer by layer and β-glucan covalently encapsulated Lactobacillus plantarum monolayer and Lactobacillus plantarum prepared in Example 2 in simulated bile;

[0030] Fig.10 The figure shows the intestinal targeted release ability result of the covalent layer-by-layer encapsulation of Lactobacillus plantarum by β-glucan prepared in Example 2;

[0031] Fig.11 The figure shows the result of the covalent layer-by-layer encapsulation of Lactobacillus plantarum and the anti-freeze-drying performance of Lactobacillus plantarum by β-glucan prepared in Example 2;

[0032] Fig.12 The graph shows the storage stability of the covalently layered encapsulation of Lactobacillus plantarum and Lactobacillus plantarum by β-glucan prepared in Example 2;

[0033] Fig.13 The figure shows the result of the UV resistance of the β-glucan covalently encapsulated Lactobacillus plantarum prepared in Example 2;

[0034] Fig.14 The figure shows the resistance results of the Ganoderma lucidum polysaccharide covalently encapsulated Lactobacillus plantarum layer by layer and the Ganoderma lucidum polysaccharide covalently encapsulated Lactobacillus plantarum monolayer and Lactobacillus plantarum in simulated gastric fluid prepared in Example 4;

[0035] Fig.15 The figure shows the resistance results of fucoidan covalently encapsulated Lactobacillus reuteri prepared in Example 5 in simulated gastric fluid, and fucoidan covalently encapsulated Lactobacillus reuteri in simulated gastric fluid;

[0036] Fig.16 The figure shows the resistance results of the covalent layer-by-layer encapsulation of Lactobacillus salivarius by polysaccharide of Lepidium argyi prepared in Example 6 and the covalent monolayer encapsulation of Lactobacillus salivarius by polysaccharide of Lepidium argyi prepared in Example 6 and Lactobacillus salivarius in simulated gastric fluid;

[0037] Fig.17 The figure shows the weight change results of colitis mice during 9 days of oral gavage;

[0038] Fig.18 Shown is the spleen index result of colitis mice after 9 days of oral gavage;

[0039] Fig.19 The figure shows the colon length results of colitis mice 9 days after oral administration;

[0040] Fig. 20 Shown is the result of pro-inflammatory factors in colitis mice after 9 days of oral gavage;

[0041] Fig.21 The figure shows the results of anti-inflammatory factors in colitis mice after oral gavage for 9 days;

[0042] Fig. 22 The figure shows the absorbance value result at 600 nm after overnight culture of culture medium containing different concentrations of D-alanine azide and bacterial solution of Lactobacillus plantarum;

[0043] Fig.23Shown are the potential results of the resuspension containing different concentrations of dibenzocyclooctyne group-modified polysaccharides;

[0044] Fig.24 Shown are potential results of resuspensions containing polysaccharides modified with azide groups at different concentrations;

[0045] Fig.25 Shown are the results of the anti-whole stomach and small intestine performance of β-glucan covalently encapsulated Lactobacillus plantarum, β-glucan non-covalently encapsulated Lactobacillus plantarum, and Lactobacillus plantarum. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments and drawings to clearly and completely describe the concept, specific structure and technical effects of the present invention, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other without conflict.

[0047] Example 1

[0048] A method for preparing a probiotic product, which specifically comprises the following steps:

[0049] Step 1: Add 0.004 mL of Lactobacillus plantarum culture solution to 0.4 mL of fresh MRS medium, where the number of viable Lactobacillus plantarum in the culture solution is 1×10 6 CFU / mL, the above fresh MRS medium contained 0.008 mg of D-alanine azide, cultured overnight (37°C, 220 rpm, 16 h), then centrifuged (centrifugal speed was 6000 rpm, centrifugal time was 4 min), washed twice with phosphate buffered saline, and the solid was resuspended in 0.4 mL of phosphate buffered saline (pH 7.0) to obtain a resuspension of Lactobacillus plantarum with azide groups modified on the surface (the number of viable Lactobacillus plantarum in the resuspension was >1×10 9 CFU / mL);

[0050] Step 2: Add 0.24 mg of β-glucan modified with dibenzocyclooctyne group to the above-obtained resuspension of Lactobacillus plantarum with azide group modified on the surface, vortex for 60 s, and react overnight (37°C, 220 rpm, 10 h); after the reaction, wash twice with phosphate buffered saline solution, centrifuge (centrifugal speed of 6000 rpm, centrifugal time of 4 min), remove excess β-glucan modified with dibenzocyclooctyne group, and resuspend the solid in 0.4 mL of phosphate buffered saline solution (pH 7.0) to obtain a β-glucan covalent monolayer embedded Lactobacillus plantarum resuspension;

[0051] Step 3: Add 0.04 mg of azide-modified β-glucan to the above-mentioned β-glucan covalently monolayer-embedded Lactobacillus plantarum resuspension, vortex for 60 s, and react overnight (37°C, 220 rpm, 10 h); then wash twice with phosphate buffered saline solution, centrifuge (centrifugation speed is 6000 rpm, centrifugation time is 4 min), remove excess azide-modified β-glucan, and finally obtain β-glucan covalently monolayer-embedded Lactobacillus plantarum (i.e., probiotic product).

[0052] The preparation process of the above-mentioned dibenzocyclooctyne group-modified β-glucan (250 kDa) is as follows:

[0053] 100 mg of carboxylated β-glucan was weighed and dissolved in 10 mL of dimethyl sulfoxide. Dibenzocyclooctyne-amine (3.0 eq.), benzotriazol-1-yl-oxytripyrrolidinophosphorus reagent (9.0 eq.) and N,N-diisopropylethylamine (15.0 eq.) were added and dissolved completely. The mixture was reacted at room temperature for 1 h. After ethanol was added for dispersion, a large amount of icy ether was poured into the mixture for precipitation. The product was collected by filtration and washed with ethanol for several times to obtain β-glucan modified with dibenzocyclooctyne group.

[0054] The preparation process of the above-mentioned azide-modified β-glucan (250 kDa) is as follows:

[0055] Weigh 100 mg of carboxylated β-glucan and dissolve it in 10 mL of dimethyl sulfoxide. Add azidopropylamine (3.0 eq.), benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate reagent (9.0 eq.) and N,N-diisopropylethylamine (15.0 eq.) to dissolve completely. React at room temperature for 1 h. Add ethanol to disperse it, pour a large amount of ice ether to precipitate, filter and collect the product, and wash it with ethanol several times to obtain azide-modified β-glucan.

[0056] Example 2

[0057] A method for preparing a probiotic product, which specifically comprises the following steps:

[0058] Step 1: Add 0.008 mL of Lactobacillus plantarum culture solution to 0.4 mL of fresh MRS medium, where the number of viable Lactobacillus plantarum in the culture solution is 2×10 6CFU / mL, the above fresh MRS medium contained 0.44 mg of D-alanine azide, cultured overnight (37°C, 220 rpm, 16 h), then centrifuged (centrifugal speed was 6000 rpm, centrifugal time was 4 min), washed twice with phosphate buffered saline, and the solid was resuspended in 0.4 mL of phosphate buffered saline (pH 7.0) to obtain a resuspension of Lactobacillus plantarum with azide groups modified on the surface (the number of viable Lactobacillus plantarum in the resuspension was >1×10 9 CFU / mL);

[0059] Step 2: Add 0.24 mg of β-glucan modified with dibenzocyclooctyne group to the resuspension of Lactobacillus plantarum with azide group modification on the surface, vortex for 60 seconds, react overnight (37°C, 220 rpm, 10 hours), wash twice with phosphate buffered saline after the reaction, centrifuge (centrifugal speed of 6000 rpm, centrifugal time of 4 minutes), remove excess β-glucan modified with dibenzocyclooctyne group, and resuspend the solid in 0.4 mL of phosphate buffered saline (pH 7.0) to obtain a β-glucan covalent monolayer embedded Lactobacillus plantarum resuspension;

[0060] Step 3: Add 0.24 mg of azide-modified β-glucan to the above-obtained β-glucan covalently monolayer-embedded Lactobacillus plantarum resuspension, vortex for 60 s, and react overnight (37°C, 220 rpm, 10 h); then wash twice with phosphate buffered saline solution, centrifuge (centrifugation speed is 6000 rpm, centrifugation time is 4 min), remove excess azide-modified β-glucan, and finally obtain β-glucan covalently monolayer-embedded Lactobacillus plantarum (i.e., probiotic product).

[0061] The preparation process of the above-mentioned dibenzocyclooctyne group-modified β-glucan (250 kDa) is as follows:

[0062] 100 mg of carboxylated β-glucan was weighed and dissolved in 10 mL of dimethyl sulfoxide. Dibenzocyclooctyne-amine (3.0 eq.), benzotriazol-1-yl-oxytripyrrolidinophosphorus reagent (9.0 eq.) and N,N-diisopropylethylamine (15.0 eq.) were added and dissolved completely. The mixture was reacted at room temperature for 1 h. After ethanol was added for dispersion, a large amount of icy ether was poured into the mixture for precipitation. The product was collected by filtration and washed with ethanol for several times to obtain β-glucan modified with dibenzocyclooctyne group.

[0063] The preparation process of the above-mentioned azide-modified β-glucan (250 kDa) is as follows:

[0064] Weigh 100 mg of carboxylated β-glucan and dissolve it in 10 mL of dimethyl sulfoxide. Add azidopropylamine (3.0 eq.), benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate reagent (9.0 eq.) and N,N-diisopropylethylamine (15.0 eq.) to dissolve completely. React at room temperature for 1 h. Add ethanol to disperse it, pour a large amount of ice ether to precipitate, filter and collect the product, and wash it with ethanol several times to obtain azide-modified β-glucan.

[0065] Example 3

[0066] A method for preparing a probiotic product, which specifically comprises the following steps:

[0067] Step 1: Add 0.016 mL of Lactobacillus plantarum culture solution to 0.4 mL of fresh MRS medium, where the number of viable Lactobacillus plantarum in the culture solution is 4×10 5 CFU / mL, the above fresh culture medium contained 0.56 mg of D-alanine azide, cultured overnight (37°C, 220 rpm, 16 h), then centrifuged (centrifugal speed was 6000 rpm, centrifugal time was 4 min), washed twice with phosphate buffered saline, and the solid was resuspended in 0.4 mL of phosphate buffered saline (pH 7.0) to obtain a resuspension of Lactobacillus plantarum with azide groups modified on the surface (the number of viable Lactobacillus plantarum in the resuspension was >1×10 9 CFU / mL);

[0068] Step 2: Add 0.32 mg of β-glucan modified with dibenzocyclooctyne group to the above-obtained resuspension of Lactobacillus plantarum with azide group on the surface, vortex for 60 s, and react overnight (37°C, 220 rpm, 10 h); after the reaction, wash twice with phosphate buffered saline solution, centrifuge (centrifugal speed of 6000 rpm, centrifugal time of 4 min), remove excess β-glucan modified with dibenzocyclooctyne group, and resuspend the solid in 0.4 mL of phosphate buffered saline solution (pH 7.0) to obtain a β-glucan covalent monolayer embedded Lactobacillus plantarum resuspension;

[0069] Step 3: Add 0.24 mg of azide-modified β-glucan to the above-obtained β-glucan covalently monolayer-embedded Lactobacillus plantarum resuspension, vortex for 60 s, and react overnight (37°C, 220 rpm, 10 h); then wash twice with phosphate buffered saline solution, centrifuge (centrifugation speed is 6000 rpm, centrifugation time is 4 min), remove excess azide-modified β-glucan, and finally obtain β-glucan covalently monolayer-embedded Lactobacillus plantarum (i.e., probiotic product).

[0070] The preparation process of the above-mentioned dibenzocyclooctyne group-modified β-glucan (250 kDa) is as follows:

[0071] 100 mg of carboxylated β-glucan was weighed and dissolved in 10 mL of dimethyl sulfoxide. Dibenzocyclooctyne-amine (3.0 eq.), benzotriazol-1-yl-oxytripyrrolidinophosphorus reagent (9.0 eq.) and N,N-diisopropylethylamine (15.0 eq.) were added and dissolved completely. The mixture was reacted at room temperature for 1 h. After ethanol was added for dispersion, a large amount of icy ether was poured into the mixture for precipitation. The product was collected by filtration and washed with ethanol for several times to obtain β-glucan modified with dibenzocyclooctyne group.

[0072] The preparation process of the above-mentioned azide-modified β-glucan (250 kDa) is as follows:

[0073] Weigh 100 mg of carboxylated β-glucan and dissolve it in 10 mL of dimethyl sulfoxide. Add azidopropylamine (3.0 eq.), benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate reagent (9.0 eq.) and N,N-diisopropylethylamine (15.0 eq.) to dissolve completely. React at room temperature for 1 h. Add ethanol to disperse it, pour a large amount of ice ether to precipitate, filter and collect the product, and wash it with ethanol several times to obtain azide-modified β-glucan.

[0074] Example 4

[0075] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of β-glucan is replaced with Ganoderma lucidum polysaccharide.

[0076] Example 5

[0077] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of β-glucan is replaced by fucoidan, and an equal amount of Lactobacillus plantarum is replaced by Lactobacillus reuteri.

[0078] Example 6

[0079] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of β-glucan is replaced with silver thread vine polysaccharide, and an equal amount of Lactobacillus plantarum is replaced with Lactobacillus salivarius.

[0080] Example 7

[0081] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of β-glucan is replaced by Ganoderma lucidum polysaccharide, an equal amount of Lactobacillus plantarum 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.

[0082] Example 8

[0083] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of β-glucan is replaced with low-ester pectin.

[0084] Example 9

[0085] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that β-glucan is replaced with tremella polysaccharide and tuckahoe polysaccharide (tremella polysaccharide modified with dibenzocyclooctyne group and tuckahoe polysaccharide modified with azide group) in equal amounts.

[0086] Example 10

[0087] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that β-glucan is replaced by chitosan and roxburghii polysaccharide (chitosan modified with dibenzocyclooctyne groups and roxburghii polysaccharide modified with azide groups) in equal amounts.

[0088] Embodiment 11

[0089] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of Lactobacillus plantarum is replaced by Lactobacillus rhamnosus.

[0090] Example 12

[0091] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of Lactobacillus plantarum is replaced by Lactococcus lactis.

[0092] Example 13

[0093] A method for preparing a probiotic product, wherein the preparation process is the same as that of Example 2, except that an equal amount of Lactobacillus plantarum is replaced by a mixture of Bifidobacterium and Lactobacillus reuteri (in the mixture, the mass ratio of Bifidobacterium to Lactobacillus reuteri is 1:1).

[0094] Embodiment 14

[0095] The probiotic product prepared in Example 2 was subjected to the following tests:

[0096] (1) The probiotic product prepared in Example 2 and the Lactobacillus plantarum used in the present invention (without any treatment) were observed by scanning electron microscopy. 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 Lactobacillus plantarum.

[0097] (2) The probiotic product prepared in Example 2 and the Lactobacillus plantarum used in the present invention (without any treatment) were observed by 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 Lactobacillus plantarum.

[0098] (3) The probiotic product prepared in Example 2 was subjected to confocal scanning, and the results are as follows: Figure 5 As shown. Among them, Figure 5 a in the figure is the bright field image of the probiotic product. Figure 5 b is the laser confocal image of the first layer of β-glucan in the probiotic product (fluorescein-labeled β-glucan modified with dibenzocyclooctyne groups). Figure 5 c in the figure is the laser confocal image of the second layer of β-glucan in the probiotic product (β-glucan modified with rhodamine-labeled azide group). Figure 5 Figure d is a laser confocal image of the probiotic product.

[0099] By combining Figures 1 to 5 It can be seen that the covalent layer-by-layer assembly of β-glucan successfully encapsulated Lactobacillus plantarum.

[0100] Embodiment 15

[0101] The probiotic product prepared in Example 5 was subjected to the following tests:

[0102] The probiotic product prepared in Example 5 and the Lactobacillus reuteri used in the present invention (without any treatment) were observed by scanning electron microscopy, and 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 reuteri. Figure 6 and Figure 7 It can be seen that fucoidan successfully encapsulated Lactobacillus reuteri.

[0103] Example 16

[0104] The probiotic product prepared in Example 2 was subjected to an in vitro resistance study, and the specific research method is as follows:

[0105] Step (1): The probiotic product prepared in Example 2 (referred to as "β-glucan covalently encapsulated Lactobacillus plantarum", 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 Lactobacillus plantarum without any treatment and Lactobacillus plantarum with only a single layer of β-glucan covalently encapsulated (referred to as "β-glucan covalently encapsulated Lactobacillus plantarum", the same below) were used as a control group.

[0106] The method for simulating gastric fluid is as follows: 5 g of pepsin is dissolved in 1000 mL of phosphate buffer, and the pH is adjusted to 2.5, and filtered through a 0.22 μm sterilizing filter membrane before use.

[0107] The method for simulating bile is as follows: prepare a 4% bile salt solution and filter it through a 0.22 μm sterilizing filter membrane before use.

[0108] Step (2): At different time points (0 h, 0.5 h, 1 h and 2 h), 100 μL of simulated gastric fluid and simulated bile from the experimental group and each control group were taken for dilution, coating and counting.

[0109] The results are as follows Figure 8 and Fig. 9 As shown, 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. 9 It can be seen that: β-glucan covalently encapsulated Lactobacillus plantarum has a strong ability to resist gastric acid and bile salts, and Lactobacillus plantarum with only β-glucan covalently encapsulated in a single layer also has a certain ability to resist gastric acid and bile salts. This result shows that β-glucan covalently encapsulated in layers is more compact and thick, which is conducive to better improving the survival rate of Lactobacillus plantarum in gastric juice and bile, so that it can play an effective role in the intestine.

[0110] Embodiment 17

[0111] The probiotic product prepared in Example 2 was subjected to a study on its intestinal targeted release ability, and the specific method was as follows:

[0112] Step (1): Lactobacillus plantarum and the probiotic product prepared in Example 2 were then resuspended in gastric digestive fluid and digested for 2 h (37° C., 180 rpm). After the gastric digestion was completed, a portion of the sample was taken for scanning electron microscopy (SEM) observation;

[0113] Step (2): The gastric digestion sample was centrifuged, washed and resuspended in the small intestinal digestion fluid for 2 h (37°C, 180 rpm). After the small intestinal digestion was completed, a portion of the sample was taken for scanning electron microscopy (SEM) observation;

[0114] Step (3): The above-mentioned small intestinal digestion samples were centrifuged, washed and resuspended in a phosphate buffered saline solution containing β-glucanase (100 U / mL) and enzymatically hydrolyzed for 3 h (37°C, 180 rpm). Part of the samples were taken for scanning electron microscopy (SEM) observation at 0.5 h, 1.5 h and 3 h of enzymatic hydrolysis.

[0115] The results are as follows Fig.10 As shown by Fig.10 It can be seen that the β-glucan coating in which β-glucan covalently encapsulates Lactobacillus plantarum remains intact in the stomach and small intestine, and can then deliver Lactobacillus plantarum to the large intestine in a targeted manner. As the β-glucanase secreted by intestinal microorganisms hydrolyzes the coating, the coating gradually degrades and releases Lactobacillus plantarum in a timely manner in the large intestine.

[0116] Embodiment 18

[0117] The probiotic product prepared in Example 2 was freeze-dried and the storage stability was studied, and the specific method was as follows:

[0118] Freeze drying: Take a portion of the plant lactobacillus 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.

[0119] Storage stability: Store at different temperatures (37°C, 25°C) for 7 days, resuspend the bacterial powder after 7 days, take an appropriate volume for dilution, and spread it on the culture medium, and count it after 48 hours. Compare the colony counts before and after storage to compare the protective effect of β-glucan on Lactobacillus plantarum.

[0120] The results are as follows Fig.11 and Fig.12 As shown, Fig.11 The results of Lactobacillus plantarum and β-glucan covalently encapsulated Lactobacillus plantarum before and after freeze-drying; Fig.12 The results of storage stability of Lactobacillus plantarum and β-glucan covalently encapsulated Lactobacillus plantarum are shown in Figure 2. Fig.11 and Fig.12 It can be seen that β-glucan covalently encapsulated Lactobacillus plantarum has better anti-freeze-drying ability and storage stability than Lactobacillus plantarum.

[0121] Embodiment 19

[0122] The probiotic product prepared in Example 2 was subjected to a study on its resistance to ultraviolet light, and the specific method is as follows:

[0123] Lactobacillus plantarum and the probiotic product prepared in Example 2 were exposed to ultraviolet light (900 μW / cm 2 ) and irradiated for 5 min and 10 min, samples were taken at the corresponding time points and diluted and spread, and the number of colonies was calculated after 48 h.

[0124] The results are as follows Fig.13 As shown, Fig.13 The results of the anti-ultraviolet effect of Lactobacillus plantarum and probiotic products are shown in the figure. Fig.13 It can be seen that β-glucan covalently encapsulated Lactobacillus plantarum has better UV resistance than Lactobacillus plantarum.

[0125] Embodiment 20

[0126] The probiotic products prepared in Examples 4 to 6 were subjected to in vitro resistance studies (protective effects in simulated gastric fluid), and the specific methods were as described in Example 16.

[0127] The results are as follows Figures 14 to 16 As shown by Figures 14 to 16 It can be seen that Ganoderma lucidum polysaccharide covalently encapsulated Lactobacillus plantarum, fucoidan covalently encapsulated Lactobacillus reuteri, and Lepidium salivae covalently encapsulated Lactobacillus salivarius polysaccharide all have strong gastric acid resistance.

[0128] Embodiment 21

[0129] The following test was carried out using the probiotic product prepared in Example 2 as an example:

[0130] Experimental animal grouping and treatment: C57BL / 6J mice (20 ± 0.2 g, 6-8 weeks old) were divided into normal group, model group, Lactobacillus plantarum group (referred to as "Lp group"), polysaccharide group, β-glucan covalently embedded Lactobacillus plantarum group (referred to as "polysaccharide layer-embedded Lp group") and β-glucan and Lactobacillus plantarum physical mixture group (referred to as "polysaccharide + Lp 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 drinking water throughout the experiment, while the other 5 groups of mice had free drinking water for the first 7 days, and drank 3% sodium dextran sulfate for the next 7 days to establish a colitis model. The modeling was stopped for the next 2 days and the mice were allowed to drink water freely. The mice were weighed every day during and after the modeling. During the whole experiment, the normal group and the model group were gavaged with 0.2 mL of phosphate buffered saline every 2 days, while the Lp group and the polysaccharide-embedded Lp group were gavaged with the corresponding bacterial suspension (the probiotic dose was 1×10 9CFU, among which, the bacterial suspension of the polysaccharide-layer-embedded Lp group contained polysaccharides in addition to probiotics, and the concentration of dibenzocyclooctyne-modified polysaccharides was 6 mg·kg -1 BW, the concentration of azide-modified polysaccharide was 6 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 + Lp group were intragastrically administered with the corresponding probiotics and polysaccharide mixture (the probiotic dose was 1×10 9 CFU, polysaccharide concentration was 40 mg kg -1 BW). After 2 days of removal of 3% dextran sulfate sodium, mice were killed by cervical dislocation. Colon tissues of mice were isolated, colon tissue length was measured, spleen weight was weighed, and spleen index = (spleen weight / mouse body weight) × 100%. All dissected tissues were frozen at -80°C for further analysis.

[0131] Body weight changes of colitis mice during 9 days of oral gavage Fig.17 As shown, Fig.17 It can be seen that intragastric administration of polysaccharide-encapsulated Lp can effectively reduce the weight loss caused by colitis in mice.

[0132] The spleen index of colitis mice after oral gavage for 9 days Fig.18 As shown, Fig.18 It can be seen that intragastric administration of polysaccharide-encapsulated Lp can effectively alleviate the phenomenon of spleen enlargement caused by colitis in mice.

[0133] The length of the colon of colitis mice after oral gavage for 9 days Fig.19 As shown, Fig.19 It can be seen that intragastric administration of polysaccharide-encapsulated Lp can effectively alleviate the phenomenon of shortening of colon length caused by colitis.

[0134] The expression level of pro-inflammatory factor (IL-6) in the colon tissue of colitis mice after oral administration for 9 days Fig. 20 As shown by Fig. 20 It can be seen that intragastric administration of polysaccharide-layer-embedded Lp can effectively reduce the expression of pro-inflammatory factors in the colon tissue of colitis mice.

[0135] The expression level of anti-inflammatory factor (IL-10) in the colon tissue of colitis mice after oral administration on the 9th day Fig.21 As shown by Fig.21 It can be seen that intragastric administration of polysaccharide-encapsulated Lp can effectively increase the expression of anti-inflammatory factors in the colon tissue of colitis mice.

[0136] The above results can prove that the polysaccharide layer-by-layer encapsulation of Lp provided in the present application can effectively alleviate intestinal inflammation caused by colitis in mice.

[0137] In addition, through Figures 17 to 21 It can be seen that by using polysaccharide to embed Lp layer by layer, compared with the direct and simple physical mixing of β-glucan and Lp, it can effectively reduce the weight loss caused by colitis in mice, effectively alleviate the phenomenon of spleen enlargement caused by colitis mice, effectively alleviate the phenomenon of shortened colon length caused by colitis, and effectively regulate the phenomenon of intestinal immune homeostasis disorder in colitis mice. It shows that the embedding form used in this application can make the polysaccharide with prebiotic effect cooperate with probiotics to better exert the therapeutic effect.

[0138] Embodiment 22

[0139] Referring to Example 2, the absorbance at 600 nm of the medium containing each concentration of D-alanine azide and the bacterial solution of Lactobacillus plantarum after overnight culture (37°C, 220 rpm, 20 h) was studied when the concentrations of D-alanine azide in the MRS medium were 0, 0.18 mg / mL, 0.37 mg / mL, 0.73 mg / mL, 1.10 mg / mL, 1.46 mg / mL, 1.83 mg / mL and 2.20 mg / mL, respectively. The results are as follows: Fig. 22 shown.

[0140] Depend on Fig. 22 It can be seen that when the concentration of D-alanine azide in the culture medium is above 1.83 mg / mL, it may have an adverse effect on probiotics. Therefore, the concentration of D-alanine azide in the culture medium is controlled at 1.46 mg / mL or below, for example, it can be controlled to 0.02 mg / mL-1.46 mg / mL.

[0141] Furthermore, taking the concentration of D-alanine azide in MRS medium as 1.1 mg / mL as an example, the appropriate amount of dibenzocyclooctyne group-modified polysaccharide added to each milliliter of resuspension was studied. Specifically, the potential results of the resuspension containing dibenzocyclooctyne group-modified polysaccharide after adding 0, 0.10 mg, 0.20 mg, 0.40 mg, 0.60 mg and 0.80 mg of dibenzocyclooctyne group-modified polysaccharide to each milliliter of resuspension were studied. The results are as follows: Fig.23 shown.

[0142] Depend on Fig.23It can be seen that when the amount of polysaccharide modified with dibenzocyclooctyne groups in each milliliter of resuspension exceeds 0.6 mg, the potential of the resuspension containing polysaccharide modified with dibenzocyclooctyne groups does not change significantly, indicating that when the amount of polysaccharide modified with dibenzocyclooctyne groups in each milliliter of resuspension is ≥ 0.6 mg, the amount of grafting on the surface of the probiotics reaches saturation. Therefore, in order to ensure that the grafting amount of the first protective layer reaches saturation, the amount of polysaccharide modified with dibenzocyclooctyne groups added in each milliliter of resuspension is controlled to be ≥ 0.6 mg, for example, it can be controlled to be 0.6 mg-0.8 mg.

[0143] Furthermore, taking the addition of 0.6 mg of dibenzocyclooctyne group-modified polysaccharide per milliliter of resuspension as an example, the appropriate amount of polysaccharide modified with azide group in each milliliter of resuspension was studied. Specifically, 0.10 mg, 0.20 mg, 0.40 mg, 0.60 mg and 0.80 mg of polysaccharide modified with azide group were added to each milliliter of resuspension, and the corresponding potential of the resuspension was measured. The results are shown in FIG. Fig.24 shown.

[0144] Depend on Fig.24 It can be seen that when the amount of azide-modified polysaccharide added to each milliliter of resuspension exceeds 0.6 mg, the potential of the resuspension will decrease instead. Therefore, the amount of azide-modified polysaccharide added to each milliliter of resuspension is controlled to not exceed 0.6 mg, for example, it can be controlled to 0.1 mg-0.6 mg.

[0145] Embodiment 23

[0146] The probiotic product prepared in Example 2 was compared with a similar technical product (application number 202310409447.3) in an in vitro resistance study. The specific research method is as follows:

[0147] Step (1): Prepare the same product (β-glucan non-covalently embedded Lactobacillus plantarum), add 0.1 mg FeCl3 into 4 mL PBS buffer solution to obtain a metal solution; then add 100 μL of Lactobacillus plantarum suspension (the number of viable cells of Lactobacillus plantarum suspension is 1×10 9CFU / mL) was mixed with 900 μL of the above metal solution and vortexed for 60 s to achieve the fixation of metal ions on the surface of the Lactobacillus plantarum suspension (that is, to obtain a probiotic-metal mixture); then 0.4 mg of tannic acid was added to the above probiotic-metal mixture and vortexed for 60 s to form a metal-polyphenol network structure layer on the surface of the probiotics to obtain a probiotic-metal-polyphenol complex; then 0.08 mg of β-glucan was added to the above probiotic-metal-polyphenol complex and vortexed for 60 s to form a polysaccharide layer on the surface of the probiotic-metal-polyphenol complex to obtain polysaccharide non-covalently embedded probiotics; finally, the mixture was washed twice with PBS and centrifuged to remove excess polysaccharide-metal-polyphenol complex at a centrifugal speed of 7000 rpm for 5 min to obtain Lactobacillus plantarum non-covalently embedded by β-glucan.

[0148] Step (2): The probiotic product prepared in Example 2 (referred to as "β-glucan covalently embedded Lactobacillus plantarum", the same below) and the β-glucan non-covalently embedded Lactobacillus plantarum prepared in step (1) were placed in simulated gastric fluid at 37°C for incubation for 2 h, centrifuged (centrifugation speed was 7000 rpm, centrifugation time was 5 min), and then resuspended in simulated small intestinal fluid and incubated at 37°C for 1 h. The β-glucan non-covalently embedded Lactobacillus plantarum and the β-glucan covalently embedded Lactobacillus plantarum were used as the experimental group, and the Lactobacillus plantarum without any treatment was used as the control group.

[0149] The method for simulating gastric fluid is as follows: 5 g of pepsin is dissolved in 1000 mL of phosphate buffer, and the pH is adjusted to 2.5, and filtered through a 0.22 μm sterilizing filter membrane before use.

[0150] The method for simulating small intestinal fluid is as follows: dissolve 1 g of pancreatic enzyme and 5 g of bile salt in 1000 mL of phosphate buffer, adjust the pH to 7.0, and use it after passing through a 0.22 μm sterilizing filter membrane.

[0151] Step (3): At different time points (0 h, 0.5 h and 1 h), 100 μL of the simulated small intestinal fluid from the experimental group and each control group was diluted, spread and counted.

[0152] The results are as follows Fig.25 As shown by Fig.25 It can be seen that β-glucan covalently encapsulated Lactobacillus plantarum has a strong ability to resist gastric acid and bile salts, and performs better than β-glucan non-covalently encapsulated Lactobacillus plantarum of similar technology products. The results show that β-glucan covalently encapsulated layer by layer is more compact and thick, which is conducive to better improving the survival rate of Lactobacillus plantarum in gastric juice and intestinal juice, so that it can play an effective role in the intestine.

[0153] 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-ultraviolet ability and storage stability of the probiotic without damaging the inherent biological characteristics of the probiotic, and at the same time safely and effectively enhance the survival rate of the probiotic in the intestine, thereby enhancing 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.

[0154] Although the description of the present invention has been quite detailed and specifically describes several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively covering the intended scope of the present invention. In addition, the above description of the present invention is based on the embodiments foreseeable by the inventor, and its purpose is to provide a useful description, and those non-substantial changes to the present invention that have not yet been foreseen may still represent equivalent changes to the present invention.

Claims

1. A method for preparing a probiotic product, characterized in that: The following steps are involved: S1: co-incubating the probiotic bacterial solution with a substance having an azide group, and resuspending the solution to obtain a resuspended solution of probiotics with azide groups modified on the surface; S2: mixing the resuspended probiotics with azide groups on the surface and the polysaccharide with probiotic effects modified with dibenzocyclooctyne groups, reacting, and resuspending to obtain a resuspended probiotics with a covalent monolayer of polysaccharide encapsulating the probiotics; S3: placing the azide group-modified polysaccharide with probiotic effect in the resuspension of the polysaccharide covalently monolayer-embedded probiotics to react, and finally obtaining a polysaccharide covalently layer-embedded probiotic product; The probiotics in the probiotic liquid include at least one of Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, Escherichia coli Nissle1917, Lactobacillus rhamnosus, Lactococcus lactis, and Bifidobacterium; The substance having an azide group includes at least one of D-alanine azide and 3-deoxy-D-manno-2-octulose acid azide sugar.

2. The preparation method according to claim 1, characterized in that: The number of viable bacteria in the probiotic solution in step S1 is 0.5×10 5 CFU / mL-1×10 7 CFU / mL; the concentration of the substance having an azide group is 0.02 mg / mL-1.46 mg / mL; the volume ratio of the probiotic liquid to the substance having an azide group is: 1:1-1:

100.

3. The preparation method according to claim 1, characterized in that: In step S2, the ratio of the resuspended probiotic solution with azide groups on the surface to the polysaccharide modified with dibenzocyclooctyne groups is 1 mL: 0.6 mg-0.8 mg; the reaction temperature is 30°C-38°C; and the reaction time is 2 h-16 h.

4. The preparation method according to claim 1, characterized in that: In step S3, the ratio of the resuspension of the polysaccharide monolayer-encapsulated probiotics to the polysaccharide modified with the azide group is 1 mL: 0.1 mg-0.6 mg; the reaction temperature is 30°C-38°C; and the reaction time is 2 h-16 h.

5. The preparation method according to claim 1, characterized in that: The polysaccharide with probiotic effects includes at least one of β-glucan, fucoidan, silver thread polysaccharide, roxburghii polysaccharide, low-ester pectin, tremella polysaccharide, tuckahoe polysaccharide, chitosan, inulin, licorice polysaccharide, astragalus polysaccharide, lentinan, ganoderma polysaccharide and lemon polysaccharide.

6. A probiotic product, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the probiotic product according to claim 6 in the preparation of a medicament for preventing and / or treating intestinal diseases, characterized in that: The intestinal disease is colitis.

Images