Method for assembling probiotics by polyphenol nanoparticles and application

Abstract

The invention provides a method for assembling probiotics by polyphenol nanoparticles and application, and belongs to the technical field of probiotic assembling. According to the invention, an EGCG (epigallocatechin gallate) aqueous solution is used as a suspending agent of a probiotic suspension, and the probiotic with polyphenol attached on the surface is obtained by utilizing the action characteristic of adhesion of the EGCG on a cell interface. The preparation method comprises the following steps: firstly, preparing probiotics, then carrying out Mannich condensation reaction on polyphenol on the surfaces of the probiotics and amino acid in the presence of formaldehyde (aldehyde group), and rapidly forming nano-particles in a short time, so that the EGCG-amino acid nano-particles which are insoluble and enteric in the stomach are assembled on the surfaces of the probiotics, the activity of the probiotics in gastric juice is protected, and the colonization rate of the probiotics in intestinal tracts is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic assembly, and in particular relates to a method and application of assembling probiotics with polyphenol nanoparticles. Background Art

[0002] Human intestinal microorganisms are closely related to diseases, such as obesity, allergies, inflammatory bowel disease, depression, cancer, etc. The balance of intestinal microbiota is easily affected by various endogenous factors and external factors, such as food and antibiotic treatment, leading to "intestinal disorders". At this time, oral probiotics can help restore the dynamic balance of intestinal flora. However, during in vitro production, processing and storage, probiotics are prone to low bacterial survival rates due to oxygen, temperature, etc., and in vivo, they are prone to poor biological activity due to low gastric juice pH, bile salts, lipase, etc., and only a sufficient number of live bacteria can be released into the human intestine to exert their probiotic effects. In order to ensure the survival rate of probiotics and their colonization and release in the human intestine, probiotics need to be encapsulated.

[0003] Nanoengineered assembly is a single bacteria encapsulation technology. Currently, the commonly used single bacteria encapsulation technology is mainly metal-polyphenol network (MPN) encapsulation, but MPN will disassemble under acidic conditions of pH = 2, exposing probiotics to gastric juice, reducing the gastric activity and intestinal colonization rate of probiotics during oral administration. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application of assembling probiotics with polyphenol nanoparticles. The method of the present invention can protect the activity of probiotics in gastric juice and improve the colonization rate of probiotics in the intestine.

[0005] The present invention provides a method for assembling probiotics with polyphenol nanoparticles, comprising the following steps:

[0006] Probiotics, an aqueous solution of epigallocatechin gallate, amino acids and formaldehyde are mixed and subjected to a Mannich condensation reaction to complete the assembly of polyphenol nanoparticles on the bacterial surface.

[0007] Preferably, the concentration of epigallocatechin gallate in the epigallocatechin gallate aqueous solution is 2.5-3 mg / mL.

[0008] Preferably, the reaction concentration of the amino acid is 0.5-0.75 mg / mL; the amino acid includes at least one of arginine, lysine, histidine, glycine and leucine.

[0009] Preferably, the reaction concentration of the probiotics is (1-2)×10 9 CFU / mL.

[0010] Preferably, the volume ratio of formaldehyde to probiotic suspension is (1-3):2000.

[0011] Preferably, the reaction temperature of the Mannich condensation reaction is 20-30° C.; the reaction time of the Mannich condensation reaction is 1 min-2 h.

[0012] Preferably, after the Mannich condensation reaction is carried out, the process further comprises centrifuging the reaction product of the Mannich condensation reaction to collect the precipitate.

[0013] Preferably, the probiotics include Escherichia coli Nissle 1917 and / or Lactobacillus plantarum.

[0014] The present invention also provides a probiotic preparation obtained by the method described in the above scheme, wherein polyphenol nanoparticles are assembled on the surface of the probiotic.

[0015] The present invention also provides the use of the probiotic preparation described in the above scheme in the preparation of a probiotic oral product.

[0016] The present invention provides a method for assembling probiotics with polyphenol nanoparticles, comprising the following steps: mixing probiotics, an aqueous solution of epigallocatechin gallate, amino acids and formaldehyde, performing a Mannich condensation reaction, and completing the assembly of polyphenol nanoparticles on the bacterial surface. The present invention uses an EGCG aqueous solution as a suspending agent for a probiotic suspension, and utilizes the action characteristics of plant polyphenols (EGCG) in cell interface adhesion to obtain probiotics with polyphenols attached to the surface. Then, in the presence of formaldehyde (aldehyde group), the polyphenols on the surface of the probiotics undergo a Mannich condensation reaction with amino acids, and nanoparticles are rapidly formed in a short time, so that gastric-insoluble and enteric-coated EGCG-amino acid nanoparticles are assembled on the probiotic surface, protecting the activity of the probiotics in gastric juice and improving the colonization rate of the probiotics in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 is the Fourier infrared spectrum;

[0019] Figure 2 is a scanning electron microscope image;

[0020] Figure 3It is a transmission electron microscope image;

[0021] Figure 4 The CFU counts and growth curves of naked bacteria and assembled bacteria after 1 and 2 hours of treatment with simulated gastric fluid;

[0022] Figure 5 The CFU counts of naked bacteria and assembled bacteria after 2 hours of simulated intestinal fluid treatment; the left picture is the colony spot plate picture, and the right picture is the colony number statistics result;

[0023] Figure 6 This is a curve chart showing the effects of nanoparticles, naked bacteria, and assembled bacteria on the body weight of mice with ulcerative colitis;

[0024] Figure 7 This is a scatter plot of the effects of nanoparticles, naked bacteria, and assembled bacteria on the DAI score of mice with ulcerative colitis on day 14;

[0025] Figure 8 The results of the effects of nanoparticles, naked bacteria, and assembled bacteria on the colon length of mice with ulcerative colitis. The upper picture is a colon photo, and the lower picture is a colon length statistic.

[0026] Fig. 9 This is the HE staining of colon tissue of ulcerative colitis mice by nanoparticles, naked bacteria, and assembled bacteria;

[0027] Fig.10 This is a bar graph showing the effects of nanoparticles, naked bacteria, and assembled bacteria on serum LPS, LBP, and inflammatory factors in mice with ulcerative colitis;

[0028] Fig.11 This is an analysis chart of the effects of nanoparticles, naked bacteria, and assembled bacteria on the diversity of intestinal flora in mice with ulcerative colitis. DETAILED DESCRIPTION

[0029] The invention provides a method for assembling probiotics with polyphenol nanoparticles, comprising the following steps: mixing probiotics, an aqueous solution of epigallocatechin gallate, amino acids and formaldehyde, performing a Mannich condensation reaction, and completing the assembly of polyphenol nanoparticles on the surface of the bacteria.

[0030] The present invention utilizes the adhesion characteristics of plant polyphenols (EGCG) at the cell interface to obtain probiotics with polyphenols attached to the surface. Then, in the presence of aldehyde groups, the polyphenols on the surface of the probiotics undergo a Mannich condensation reaction with amino acids to rapidly form nanoparticles in a short time, so that gastric-insoluble and enteric-coated EGCG-amino acid nanoparticles are assembled on the surface of the probiotics, thereby protecting the activity of the probiotics in gastric juice and improving the colonization rate of the probiotics in the intestine.

[0031] In the specific implementation process of the present invention, the probiotics include Escherichia coli Nissle1917 and / or Lactobacillus plantarum, which are purchased from China Industrial Microbiological Culture Collection Center CICC; the present invention first resuspends the probiotic mud in an epigallocatechin gallate aqueous solution to obtain a probiotic suspension, and then mixes it with amino acids and formaldehyde; the concentration of epigallocatechin gallate in the epigallocatechin gallate aqueous solution is 2.5-3 mg / mL; the concentration of probiotics in the probiotic suspension is (1-2)×10 9 CFU / mL; the probiotic sludge is prepared by the following method: inoculating probiotic strains into a culture medium and culturing them to obtain a probiotic liquid; centrifuging the probiotic liquid, collecting the bacterial precipitate, and washing the bacterial precipitate to obtain a sludge; the probiotic strains are obtained by thawing the frozen liquid at -80°C; the inoculation amount of the probiotic strain accounts for 1% of the volume fraction of the culture medium; the culturing time is 16 to 24 hours; the culturing temperature is a temperature suitable for the growth of probiotics; the culture medium is a liquid culture medium suitable for the growth of probiotics; the centrifugal temperature is 4°C; the centrifugal speed is 4000 to 6000 rpm; the centrifugal time is 5 to 8 min; the reagent used for washing includes deionized water; the number of washings is 3 times; the washing is used to remove the culture medium residue; the resuspension method includes vortexing.

[0032] In the present invention, the EGCG can have a strong interaction with intestinal mucin, promote the colonization of probiotics and enhance the intestinal mucus barrier. Moreover, EGCG can promote the production of short-chain fatty acids such as butyrate by affecting the changes in intestinal flora, thereby triggering significant antioxidant and anti-inflammatory effects, thereby synergizing with probiotics to reduce intestinal inflammation and damage.

[0033] In the specific implementation process of the present invention, the reaction concentration of the amino acid is 0.5-0.75 mg / mL; the amino acid includes at least one of arginine, lysine, histidine, glycine and leucine, and further is glycine or leucine.

[0034] The present invention can adjust the shape and size of nanoparticles by changing the type of amino group, and can be used for the assembly of different strains and different needs. When the amino acid is arginine, the average diameter of the nanoparticles is 470nm; When the amino acid is Lysine When the average diameter of the nanoparticles is 280nm; When the amino acid is Histidine When the average diameter of the nanoparticles is 604nm; When the amino acid is GlycineWhen the average diameter of the nanoparticles is 437nm; When the amino acid is Leucine When the average diameter of the nanoparticles is 679nm.

[0035] In the specific implementation process of the present invention, the volume ratio of formaldehyde to probiotic suspension is (1-3):2000.

[0036] In the specific implementation process of the present invention, the reaction temperature of the Mannich condensation reaction is 20-30°C; the reaction time of the Mannich condensation reaction is 1min-2h; the Mannich condensation reaction is carried out under static conditions. After the Mannich condensation reaction, the present invention also includes centrifuging the product after the Mannich condensation reaction and collecting the precipitate; the speed of the centrifugation is 4000-6000rpm; the time of the centrifugation is 5-8min; the role of the centrifugation is to remove free EGCG, amino acids and formaldehyde in the supernatant; after collecting the precipitate, the present invention also includes redissolving the precipitate in water.

[0037] The method of the present invention is simple and quick to assemble, and only requires mixing EGCG, amino acids, formaldehyde and probiotics to form nanoparticles through a Mannich condensation reaction.

[0038] The present invention also provides a probiotic preparation obtained by the method described in the above scheme, wherein polyphenol nanoparticles are assembled on the surface of the probiotic.

[0039] The probiotic preparation of the present invention has pH responsiveness, can stably exist in gastric juice with low pH, and protect the activity of probiotics, and will disassemble in intestinal juice with pH=7, thereby releasing probiotics.

[0040] The present invention also provides the use of the probiotic preparation described in the above scheme in the preparation of a probiotic oral product.

[0041] In a specific implementation of the present invention, the product includes a drug.

[0042] The method of the present invention is applicable to the development and utilization of various probiotic oral drugs, and provides a possible new development strategy for probiotic delivery systems in the future.

[0043] In order to further illustrate the present invention, the method and application of assembling probiotics with polyphenol nanoparticles provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] 1) After thawing the frozen Gram-negative bacteria Escherichia coli 1917 (purchased from China Industrial Microbiological Culture Collection Center) at -80°C, inoculate it into LB liquid medium at a volume ratio of 1% and culture for 16 hours to obtain a concentration of about 1×10 9 CFU / mL of bacterial solution.

[0046] 2) Centrifuge the bacterial solution at 4000 rpm for 5 min, remove the supernatant, wash the bacterial precipitate with deionized water, repeat three times, and remove the culture medium residue. Then add an EGCG aqueous solution with an equal volume to the initial bacterial solution, wherein the concentration of EGCG is 2.5 mg / mL, and vortex to suspend the bacterial solution in the EGCG aqueous solution.

[0047] 3) Add glycine to the solution in 2) to a final concentration of 0.75 mg / mL, stir evenly, and then add formaldehyde. The volume ratio of formaldehyde to solution is 2:2000.

[0048] 4) After the mixture is stirred evenly, it is allowed to stand at room temperature for 30 minutes to complete the assembly of nanoparticles on the bacterial surface. It is then centrifuged at 5000 rpm for 8 minutes to remove free EGCG, amino acids, and formaldehyde in the supernatant. Deionized water is added to the precipitate to obtain a probiotic solution with nanoparticles assembled on the surface, which is named assembled bacteria Ec-NPs.

[0049] After assembly and centrifugation, liquid phase detection of formaldehyde residue showed a concentration of 0.005 mg / L, which was lower than the detection value of 1 mg / L.

[0050] Example 2

[0051] 1) Thaw the frozen Lactobacillus plantarum (purchased from China Industrial Microbiological Culture Collection Center) culture liquid at -80℃ and inoculate it into the corresponding culture medium at a volume ratio of 1% and culture for 24 hours to obtain a concentration of about 10 9 CFU / mL of bacterial solution.

[0052] 2) Centrifuge the bacterial solution at 6000 rpm for 5 min, remove the supernatant, wash the bacterial precipitate with deionized water, repeat three times, and remove the culture medium residue. Then add an EGCG aqueous solution with an equal volume to the initial bacterial solution at a concentration of 3 mg / mL, and vortex to suspend the bacterial solution in the EGCG aqueous solution.

[0053] 3) Add glycine to the solution in 2) to a final concentration of 0.5 mg / mL, stir evenly, and then add formaldehyde. The volume ratio of formaldehyde to solution is 1:2000.

[0054] 4) After the mixture is stirred evenly, it is allowed to stand at room temperature for 30 minutes to complete the assembly of nanoparticles on the bacterial surface. Then it is centrifuged at 6000 rpm for 7 minutes to remove free EGCG, amino acids, and formaldehyde in the supernatant. Deionized water is added to the precipitate to obtain a probiotic solution with nanoparticles assembled on the surface, which is named assembled bacteria Lp-NPs.

[0055] Example 3

[0056] 1) Thaw the Gram-negative bacteria Escherichia coli 1917 culture solution frozen at -80℃ and inoculate it into the corresponding culture medium at a volume ratio of 1% and culture for 16 hours to obtain a concentration of about 1*10 9 CFU / mL of bacterial solution.

[0057] 2) Centrifuge the bacterial solution at 5000 rpm for 6 min, remove the supernatant, wash the bacterial precipitate with deionized water, repeat three times, and remove the culture medium residue. Then add an EGCG aqueous solution of the same volume as the initial bacterial solution at a concentration of 2.5 mg / mL, and vortex to suspend the bacterial solution in the EGCG aqueous solution.

[0058] 3) Add leucine to the solution in 2) to a final concentration of 0.6 mg / mL, stir evenly, and then add formaldehyde. The volume ratio of formaldehyde to solution is 1.5:2000.

[0059] 4) After the mixture is stirred evenly, it is allowed to stand at room temperature for 1 hour to complete the assembly of nanoparticles on the surface of the bacteria. Then it is centrifuged at 6000 rpm for 5 minutes to remove free EGCG, amino acids and formaldehyde in the supernatant. Deionized water is added to the precipitate to obtain a probiotic solution with nanoparticles assembled on the surface.

[0060] Comparative Example 1

[0061] CN118160929A discloses a nanocapsule for embedding lactic acid bacteria and its application, wherein the lactic acid bacteria are resuspended in a mixed solution prepared by a hyaluronic acid solution, a quinoa protein solution and a nano zinc oxide solution, and then freeze-dried at 4°C overnight to obtain a nanocapsule for embedding lactic acid bacteria. Although single-cell embedding of bacteria can be achieved, the preparation process of the raw material quinoa protein solution is complex and time-consuming, and freeze-drying is required after embedding overnight.

[0062] Test Example 1

[0063] 1. The assembled bacteria Ec-NPs obtained in Example 1 and the assembled bacteria Lp-NPs obtained in Example 2, as well as naked Gram-negative bacteria Escherichia coli 1917 (Ec) and Lactobacillus plantarum (Lp) were dried and then sampled for infrared spectroscopy detection. The results are shown in Figure 1 ,Depend on Figure 1 It can be seen that the Mannich reaction forms two characteristic peaks of glycine nanoparticles (Gly-NPs): 1204C-NC and 1118C-OC. The naked bacteria group (Escherichia coli Ec and Lactobacillus plantarum Lp) does not show characteristic peaks, while the assembled bacteria group (Ec-NPs and Lp-NPs) shows characteristic peaks, which preliminarily indicates that the bacteria in the assembled bacteria group are assembled with nanoparticles.

[0064] 2. The assembled bacteria Ec-NPs obtained in Example 1 and the assembled bacteria Lp-NPs obtained in Example 2, as well as naked Gram-negative bacteria Escherichia coli 1917 (Ec) and Lactobacillus plantarum (Lp) were prepared and sprayed with gold and observed under a scanning electron microscope. The results are shown in Figure 2 .Depend on Figure 2 It can be seen that compared with naked bacteria (Ec, Lp), spherical nanoparticles are attached to the surface of assembled bacteria (Ec-NPs, Lp-NPs) and the morphology is more complete. While verifying the successful assembly, it also shows that the nanoparticle assembly has a protective effect during the vacuum process.

[0065] 3. The assembled bacteria Ec-NPs obtained in Example 1 and the assembled bacteria Lp-NPs obtained in Example 2, as well as naked Gram-negative bacteria Escherichia coli 1917 (Ec) and Lactobacillus plantarum (Lp) were observed by transmission electron microscopy. The results are as follows: Figure 3 The results show that for naked bacteria (Ec, Lp) and assembled bacteria (Ec-NPs, Lp-NPs), due to the presence of Gly-NPs on the surface of assembled bacteria, the electron scattering angle is large, fewer electrons pass through, the image brightness is darker, and the surroundings are hemispherical.

[0066] 4. Simulated gastric fluid, wherein the formula of simulated gastric fluid is prepared by adding 3.2 g pepsin, 2 g sodium chloride, 7 mL hydrochloric acid and 993 mL deionized water to prepare 1 L of stock SGF solution to dissolve to 1000 mL, and then adjusting the pH to 2. After treating the assembled bacteria Ec-NPs obtained in Example 1 and the assembled bacteria Lp-NPs obtained in Example 2, as well as naked Gram-negative bacteria Escherichia coli1917 (Ec) and Lactobacillus plantarum (Lp) for 1 and 2 hours, the naked bacteria and assembled bacteria were counted and the growth curve was drawn. The CFU counts and growth curves of the naked bacteria and assembled bacteria are shown in Figure 4 The results showed that assembly can improve the activity of bacteria under acidic conditions.

[0067] 5. Simulated intestinal fluid, wherein 1L of simulated intestinal fluid should contain: 4.0857g of ox bile salt, 266mg of CaCl, and 400mg of pancreatic enzyme. After treating the assembled bacteria Ec-NPs obtained in Example 1 and the assembled bacteria Lp-NPs obtained in Example 2, as well as naked Gram-negative bacteria Escherichia coli 1917 (Ec) and Lactobacillus plantarum (Lp) for 2 hours, the naked bacteria and assembled bacteria were counted and growth curves were drawn. The CFU counts and growth curves of the naked bacteria and assembled bacteria are shown in Figure 5 The results showed that there was a statistical difference in the activity of naked / assembled bacteria after treatment, and the surface nanoparticles could also play a certain protective role against bile salts and pancreatic enzymes during the slow disassembly process of SIF.

[0068] Test Example 2

[0069] After the adaptation period, 50 C57BL / 6 mice were divided into 5 groups, namely the control group (Water), model group (Water+DSS), nanoparticle group (NPs+DSS), naked bacteria group (EcN+DSS), and assembled bacteria group (EcN / NPs+DSS). In the first and second weeks, the control group and the model group were gavaged with 200 μL of deionized water, the nanoparticle group was gavaged with 200 μL of 0.2 mg of nanoparticles, and the naked bacteria group was gavaged with 200 uL of 5×10 8 CFU of bacteria, the assembled bacterial group was gavaged with 200 μL of 5 × 10 8 Starting from the second week, the drinking water of the model group, nanoparticle group, naked bacteria group, and assembled bacteria group was changed to 1.5% DSS solution.

[0070] The weight of the five groups of mice was monitored for 14 consecutive days. Figure 6The results showed that at the end of the experiment on the 14th day, compared with the control group, the body weight of the mice in the model group decreased by 11%, and the body weight of the assembled bacteria group decreased by 4%. This result shows that assembled bacteria can alleviate the weight loss of mice caused by DSS.

[0071] On day 14, a comprehensive evaluation was performed based on the weight loss score, stool viscosity score, and stool blood score of the mice. The results are shown in Figure 7 The results showed that compared with the nanoparticle group and the naked bacteria group, the assembled bacteria were more effective in alleviating the symptoms of sticky stool and bloody stool.

[0072] On the 15th day, the mice were sacrificed and samples were taken to record the colon length of the ulcerative colitis mice. The results are shown in Figure 8 The results showed that compared with the naked bacteria group, the assembled bacteria had a more significant alleviating effect on the shortening of the mouse colon.

[0073] On the 15th day, the mice were sacrificed and samples were collected. After HE staining, the colon sections were observed and photographed at 4x and 40x magnification using an inverted microscope. The colon tissue of each mouse was scored according to the histopathological scoring criteria. The histological damage score of each mouse was the sum of crypt destruction, goblet cell damage and inflammation. Fig. 9 The results showed that nanoparticles, naked bacteria, and assembled bacteria could improve the ulcerative symptoms of mouse colon tissue, among which the assembled bacteria group had the most significant effect.

[0074] On the 15th day, the mice were killed and samples were collected. Blood was collected from the eyeballs of the five groups of mice and centrifuged to obtain serum. The inflammatory factors in the serum were then detected using an ELISA kit. The results are shown in Fig.10 The results showed that the colon barrier integrity of mice in the assembled bacteria group was the highest, and it could significantly inhibit the release of pro-inflammatory factors and promote anti-inflammatory factors.

[0075] On the 15th day, the mice were sacrificed and samples were collected. The colon contents of the mice were sequenced and analyzed by 16sRNA. The results are shown in Fig.11 The results showed that the assembled bacterial group significantly increased the community richness and species diversity of mice after DSS treatment.

[0076] In summary, compared with naked bacteria, assembled bacteria can more effectively alleviate the symptoms of ulcerative colitis caused by DSS in terms of mouse weight, DAI index, colon morphology, and intestinal flora structure.

[0077] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for assembling probiotics with polyphenol nanoparticles, characterized in that: The following steps are involved: Probiotics, an aqueous solution of epigallocatechin gallate, amino acids and formaldehyde are mixed and subjected to a Mannich condensation reaction to complete the assembly of polyphenol nanoparticles on the bacterial surface.

2. The method according to claim 1, characterized in that The concentration of epigallocatechin gallate in the epigallocatechin gallate aqueous solution is 2.5-3 mg / mL.

3. The method according to claim 1, characterized in that The reaction concentration of the amino acid is 0.5-0.75 mg / mL; the amino acid includes at least one of arginine, lysine, histidine, glycine and leucine.

4. The method according to claim 1 or 3, characterized in that: The reaction concentration of the probiotics is (1-2)×10 9 CFU / mL.

5. The method according to claim 1, characterized in that The volume ratio of the formaldehyde to the probiotic suspension is (1-3):2000.

6. The method according to claim 1, characterized in that The reaction temperature of the Mannich condensation reaction is 20-30° C.; the reaction time of the Mannich condensation reaction is 1 min-2 h.

7. The method according to claim 1, characterized in that After the Mannich condensation reaction is performed, the process further includes centrifuging the reaction product of the Mannich condensation reaction to collect the precipitate.

8. The method according to claim 1, characterized in that The probiotics include Escherichia coli Nissle 1917 and / or Lactobacillus plantarum.

9. The probiotic preparation obtained by the method according to any one of claims 1 to 8, characterized in that The surface of the probiotics is assembled with polyphenol nanoparticles.

10. Use of the probiotic preparation according to claim 9 in the preparation of probiotic oral products.

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