Chimeric selenium point probiotics as well as preparation method and application thereof

Abstract

The invention discloses a chimeric selenium dot probiotic as well as a preparation method and application thereof. The chimeric selenium dot probiotic comprises a probiotic and elemental selenium growing on the surface of the probiotic in situ. The probiotics with the selenium spots growing on the surface in situ are prepared through in-situ reduction by taking the probiotics as a main body structure, the preparation method is simple, the process operation is simple and easy to implement, and meanwhile, the selenium spot embedded probiotics not only keep the biological activity of the probiotics, but also have the function of nano-selenium, so that the selenium spot embedded probiotics can be used for preparing the selenium spot embedded probiotics. The compound shows a more excellent treatment effect on inflammation-related diseases. In addition, the preparation method provided by the invention enriches the preparation of engineering probiotics, and further expands the application of the probiotics in the fields of nano medicine and animal feeding.

Description

Technical Field

[0001] The invention relates to the technical field of functional materials, and in particular to a chimeric selenium-dot probiotic and a preparation method and application thereof. Background Art

[0002] The intestinal microbiota is composed of trillions of bacteria, fungi, viruses and other microorganisms, and is involved in the host's immune regulation process. Studies have shown that the intestinal microbiota is one of the driving factors for inflammatory bowel disease and other related diseases, and the occurrence of inflammatory bowel disease is usually accompanied by an imbalance of the intestinal microbiota. In recent years, researchers have used probiotics to intervene and regulate the intestinal microbiota, increase the colonization of beneficial microbial communities in the host, and thus regulate the imbalanced intestinal microbial homeostasis under inflammatory conditions. However, the treatment of inflammatory bowel disease with probiotics alone has limited effect, because the inflammatory response will produce excessive reactive oxygen free radicals, causing oxidative stress damage to the body's tissues, exacerbating and intensifying the inflammatory response. Therefore, while using probiotics to treat inflammatory-related diseases, it is crucial to remove reactive oxygen free radicals in the lesion site.

[0003] Selenium is an indispensable trace element for the human body and exists in the antioxidant activity center of glutathione peroxidase in the human body. It plays a key role in regulating human immune function and reducing free radical oxidative damage. Therefore, moderate supplementation of selenium helps to treat inflammation-related diseases. In addition, due to its higher biosafety and antioxidant activity, nano-selenium is considered to be a new generation of selenium supplement health products.

[0004] Although nano-selenium has antioxidant function, it has poor adhesion in the intestine and is easily metabolized and excreted from the body. Therefore, improving the enrichment efficiency of nano-selenium in the intestine and combining probiotics with nano-selenium to synergistically treat inflammatory-related diseases are issues that need to be urgently addressed. Summary of the invention

[0005] In order to solve at least some of the technical problems in the prior art, the present invention provides a chimeric selenium dot probiotic and a preparation method and application thereof. The chimeric selenium dot probiotic not only retains the biological activity of the probiotic itself, but also has the function of nano-selenium, such as antioxidant activity, because of the in-situ grown selenium dots, and thus has a good therapeutic effect on inflammation-related diseases. Specifically, the present invention includes the following contents.

[0006] The first aspect of the present invention provides a chimeric selenium-dot probiotic, which comprises a probiotic and a selenium element growing in situ on the surface of the probiotic.

[0007] In certain preferred embodiments, according to the chimeric selenium-dot probiotics of the present invention, the probiotics include intestinal probiotics.

[0008] In certain preferred embodiments, according to the chimeric selenium-dot probiotics of the present invention, the intestinal probiotics include at least one of the genera Lactobacillus, Bifidobacterium, Streptococcus, Bacillus, Clostridium, Bacteroides, Lactococcus, Leuconostoc, Propionibacterium, Pediococcus and Saccharomyces.

[0009] In certain preferred embodiments, according to the chimeric selenium-dot probiotics of the present invention, the selenium element has a particle size of 1-100 nm.

[0010] In certain preferred embodiments, the chimeric selenium-dot probiotics according to the present invention are prepared by in situ adsorption of a selenium precursor on the probiotics, followed by reduction with a reducing agent, to obtain a probiotic having selenium dots grown in situ on the surface.

[0011] The second aspect of the present invention provides a method for preparing a chimeric selenium-dot probiotic, comprising:

[0012] (1) culturing probiotics in a culture medium, and then co-incubating the cultured probiotics in a solvent containing a selenium precursor to obtain probiotics adsorbed with the selenium precursor;

[0013] (2) subjecting the probiotics adsorbed with selenium precursor to a reduction reaction with a reducing agent to obtain the chimeric selenium-dot probiotics.

[0014] In certain embodiments, according to the method for preparing chimeric selenium-dot probiotics of the present invention, the ratio of the probiotics to the selenium precursor is 10:1. 7-10 CFU: (0.002-10)mmol.

[0015] In certain embodiments, according to the method for preparing the chimeric selenium-dot probiotics of the present invention, the selenium precursor comprises organic selenium and / or inorganic selenium;

[0016] Preferably, the selenium precursor is selected from any one or a combination of sodium selenite, potassium selenite, sodium selenate, potassium selenate, selenium dioxide, selenium oxide, selenomethionine, hydroxyselenomethionine, selenocysteine, selenocysteine, selenomethylselenocysteine, selenomethylselenomethionine or seaweed selenium polysaccharide, preferably sodium sulfite.

[0017] In certain embodiments, according to the method for preparing chimeric selenium-dot probiotics of the present invention, the reducing agent includes any one or a combination of ascorbic acid, glutathione, sodium borohydride, hydrazine hydrate and polyphenols (such as catechins, hydroquinone, gallic acid, quercetin, resveratrol, etc.).

[0018] In certain embodiments, according to the method for preparing chimeric selenium-dot probiotics of the present invention, the solvent is selected from at least one of physiological saline, phosphate buffer solution, M17 medium, MRS medium, LB medium, BH medium, Bifidobacterium BS medium and Roche broth medium.

[0019] The third aspect of the present invention provides a use of a chimeric selenium-dot probiotic in the preparation of a medicine or dietary supplement for preventing, improving or treating inflammatory diseases.

[0020] The fourth aspect of the present invention provides a pharmaceutical composition or dietary supplement containing the chimeric selenium-dot probiotics of the present invention.

[0021] The fifth aspect of the present invention provides a feed additive containing the chimeric selenium-dot probiotics of the present invention.

[0022] The technical solution provided by the present invention has the following advantages compared with the prior art:

[0023] (1) The present invention uses probiotics as the main structure, in situ adsorbs selenium precursors, and then uses a reducing agent to reduce them to obtain probiotics with selenium dots growing in situ on the surface. The preparation method is simple and the process operation is simple and easy;

[0024] (2) The chimeric selenium dot probiotics provided by the present invention not only retain the biological activity of the probiotics themselves but also have the function of nano-selenium. Compared with the aqueous-phase synthesized nano-selenium (100-400 nm) reported in traditional literature, the chimeric selenium dot probiotics provided by the present invention have more outstanding functionality. For example, in terms of antioxidant activity, the chimeric selenium dot probiotics provided by the present invention can show a better therapeutic effect on inflammatory-related diseases;

[0025] (3) The preparation method provided by the present invention not only enriches the preparation method of engineered probiotics, but also provides a theoretical basis and technical guidance for the application of probiotics in the fields of nanomedicine and animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Transmission electron micrographs of chimeric selenium-dot Lactobacillus casei are shown.

[0027] Figure 2 The elemental analysis spectrum of the chimeric selenium-dot Lactobacillus casei is shown.

[0028] Figure 3 Transmission electron micrographs of chimeric selenoid Streptococcus thermophilus and Pediococcus acidilactici are shown.

[0029] Figure 4 A transmission electron micrograph of the preparation of chimeric selenium-dot Lactobacillus casei using selenomethionine is shown.

[0030] Figure 5The growth curve of chimeric selenium-dot Lactobacillus casei is shown.

[0031] Figure 6 Shown is the survival quantification of chimeric selenium-dot Lactobacillus casei and Lactobacillus casei in simulated gastric fluid.

[0032] Figure 7 The fluorescence imaging images and quantitative curves of the chimeric selenium-dot Lactobacillus casei and Lactobacillus casei are shown.

[0033] Figure 8 The data of body weight, colon length and HE section score of ulcerative colitis mice after treatment are shown, wherein the columns from left to right are the healthy group, model group, Lactobacillus casei group, Lactobacillus casei + nano-selenium mixed group, and chimeric selenium-dot Lactobacillus casei group.

[0034] Fig. 9 Data showing gut microbiota diversity, genus-level composition, and relative abundance of beneficial and harmful bacterial genera in mice treated with ulcerative colitis.

[0035] Fig.10 Shown are data on the scavenging of reactive oxygen species in vivo after treatment of ulcerative colitis mice.

[0036] Fig.11 Shown are flow cytometry data of colonic immune cells after treatment of ulcerative colitis mice.

[0037] Fig.12 Shown are the quantitative data of cytokines in the colon of mice with ulcerative colitis after treatment.

[0038] Fig.13 Data on colon length and HE section scores after delayed treatment of ulcerative colitis mice with chimeric selenium-containing Lactobacillus casei are shown.

[0039] Fig.14 The data of colon length and HE section score after treatment of TNBS-induced ulcerative colitis mice with chimeric selenium-containing Lactobacillus casei are shown.

[0040] Fig.15 Data on colon length and HE section scores after treatment of chronic ulcerative colitis mice with chimeric selenium-containing Lactobacillus casei are shown.

[0041] Among them, Figure 13-15 In the figure, the columns from left to right are the healthy group, the model group, and the chimeric selenium-dot Lactobacillus casei group.

[0042] Fig.16 The evaluation of the biosafety of short-term oral administration of chimeric selenium-containing Lactobacillus casei in healthy mice is shown.

[0043] Fig.17The evaluation of the biosafety of long-term oral administration of chimeric selenium-containing Lactobacillus casei in healthy mice is shown.

[0044] Fig.18 Shown are the contents of reactive oxygen free radicals and the composition of the genus level of intestinal flora after the treatment of ulcerative colitis cynomolgus monkeys with chimeric selenium-dot Lactobacillus casei.

[0045] Fig.19 Colonoscopic results after treatment of ulcerative colitis in cynomolgus monkeys with chimeric selenium-containing Lactobacillus casei are shown.

[0046] Fig. 20 The figure shows the evaluation of the biosafety of chimeric selenium-dot Lactobacillus casei in cynomolgus monkeys (Note: ND means not detected). DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0050] Chimeric Selenium Probiotics

[0051] The first aspect of the present invention provides an engineered probiotic, which can be any microorganism that exists naturally in the intestine and can adhere to and colonize in the intestine. The present invention has found through research that combining probiotics with nano-selenium can not only solve the poor targeting problem of nano-selenium, but also enhance the antioxidant capacity of probiotics, and achieve the synergistic therapeutic effect of probiotics and nano-selenium on inflammatory-related diseases.

[0052] The chimeric selenium dot probiotics of the present invention are based on probiotics as the main structure, and utilize the surface of the probiotics to achieve in-situ adsorption of selenium precursors, which are then reduced using a reducing agent to obtain probiotics with selenium dots grown in-situ only on the surface.

[0053] The selenium single substance particle size (average particle size) on the surface of the chimeric selenium dot probiotics of the present invention is 1-100nm, preferably 2-80nm, also preferably 2-50nm, preferably 2-20nm, preferably 2-10nm such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50nm, etc. The inventors found that compared to large particle size (e.g., greater than 100, 200, 300 or more particle size), nano selenium within the particle size range of the present invention has more excellent functions, such as antioxidant effects, and the chimeric selenium dot probiotics containing the nano selenium can show a more excellent therapeutic effect for inflammation-related diseases. In the present invention, selenium single substance can also be referred to as selenium dot single substance.

[0054] In the present invention, the type of probiotics is not particularly limited. In a preferred embodiment, the probiotics are intestinal probiotics, and examples thereof include, but are not limited to, at least one or a combination of the following: Lactobacillus, Bifidobacterium, Streptococcus, Bacillus, Clostridium, Bacteroides, Lactococcus, Leuconostoc, Propionibacterium, Pediococcus and Saccharomyces. Among them, the probiotics of the genus Lactobacillus are preferably selected from at least one of Lactobacillus casei, Lactobacillus bulgaricus and Lactobacillus lactis. The probiotics of the genus Bifidobacterium are preferably selected from at least one of Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium infantis. The probiotics of the genus Streptococcus are preferably Streptococcus thermophilus. The probiotics of the genus Bacillus are preferably at least one of Bacillus subtilis and Bacillus licheniformis. The probiotics of the genus Pediococcus are preferably Pediococcus acidilactici. The probiotics of the genus Clostridium are preferably Clostridium butyricum. The genus Bacteroides is preferably Bacteroides fragilis. The genus Lactococcus is preferably Lactococcus lactis. The genus Leuconostoc is preferably Leuconostoc mesenteroides. The genus Propionibacterium is preferably Propionibacterium freudenreichii.

[0055] In a preferred embodiment, the probiotic used in the present invention is Lactobacillus casei, and is also preferably Lactobacillus casei with a preservation number of CICC20280, Streptococcus thermophilus with a preservation number of CICC6222, and Pediococcus acidilactici with a preservation number of JCM8797.

[0056] In a preferred embodiment of the present invention, the selenium element located on the surface of the probiotics is obtained by an in-situ reduction method, which is described in detail below.

[0057] Preparation method

[0058] The preparation method of the chimeric selenium-dot probiotics of the present invention comprises: (1) co-incubating the cultured probiotics in a solvent containing a selenium precursor to obtain probiotics adsorbed with the selenium precursor; (2) subjecting the probiotics adsorbed with the selenium precursor to a reduction reaction with a reducing agent to obtain the chimeric selenium-dot probiotics.

[0059] Step (1)

[0060] In step (1) of the present invention, the probiotics are first cultured in a culture medium. The culture medium preferably contains factors that promote the production of polysaccharides, and then the probiotics are allowed to adsorb selenium precursors in situ under appropriate co-incubation conditions. Suitable culture medium and culture conditions can be selected according to the specific type of the probiotics to be cultured, and are not particularly limited thereto, as long as the desired number of probiotics can be achieved.

[0061] In a preferred embodiment, the culture medium used in the present invention is Msgg culture medium. Also preferably, the culture medium contains 0.2-1.5 g / L K2HPO4, 10-40 g / L MOPS, 1-10 g / L glycerol, 1-10 g / L amino acids (glutamic acid, phenylalanine and tryptophan), 0.1-1.5 mg / L B1, and 5-50 mg / L metal salts. Further preferably, the culture medium contains 0.5-1.0 g / L K2HPO4, 15-25 g / L MOPS, 2-8 g / L glycerol, 3-8 g / L amino acids, 0.4-0.6 mg / L B1, and 10-30 mg / L metal salts. Wherein the metal salt is selected from at least one or a combination of MgCl2, CaCl2, FeCl3·6H2O, MnCl2·4H2O, and ZnCl2. The amino acids are selected from at least one or a combination of glutamic acid, phenylalanine, and tryptophan.

[0062] In a preferred embodiment of the present invention, the ratio of probiotics to the selenium precursor is 10 7-10 CFU: (0.002-100) mmol, preferably 10 7-10 CFU: (0.002-10) mmol, wherein, preferably, the number of probiotics is preferably 10 8-10 CFU, preferably 10 9 CFU, the selenium precursor is preferably 0.002-10mmol, and further preferably 0.006-10mmol. Further, the ratio of probiotics to the selenium precursor is preferably 10 7-10 CFU: (0.006-1)mmol, more preferably 10 7-10 CFU: (0.006-0.2)mmol, for example, 0.006, 0.008, 0.010, 0.012, 0.02, 0.1, 0.15, 0.2mmol, etc.

[0063] In a preferred embodiment of the present invention, any high-valent selenium precursor compound that can obtain nano-selenium element through reduction reaction can be used in the present invention. The high-valent selenium precursor can be a divalent, tetravalent, or hexavalent selenium precursor compound, and can be organic selenium and / or inorganic selenium. Examples include, but are not limited to, sodium selenite, potassium selenite, sodium selenate, potassium selenate, selenium dioxide, selenium oxide, selenomethionine, hydroxyselenomethionine, selenocysteine, selenocysteine, selenomethylselenocysteine, selenomethylselenomethionine, or any one of seaweed selenium polysaccharides, or a combination of at least two of them.

[0064] In a preferred embodiment of the present invention, the type of solvent is not particularly limited, and examples thereof include, but are not limited to, any one of physiological saline, buffer (e.g., phosphate buffer solution), M17 medium, MRS medium, LB medium, BH medium, Bifidobacterium BS medium and Roche broth medium, or a combination of at least two thereof.

[0065] In a preferred embodiment of the present invention, the co-incubation temperature is 4-45°C, preferably 4-40°C, and also preferably 36-38°C, such as 36°C, 36.5°C, 37°C, 37.5°C, 38°C, etc. The co-incubation temperature should not be too high or too low. The inventors have found that co-incubation at the above temperature, especially 36-38°C, can make the selenium precursor adsorbed on the surface of the probiotics as quickly as possible. If the co-incubation temperature is high, it is not conducive to the survival of the probiotics. If the co-incubation temperature is low, the therapeutic effect (including antioxidant effect, etc.) of the chimeric selenium point probiotics is poor.

[0066] In the present invention, the co-incubation time is not particularly limited and can be adjusted as needed, but is preferably 1-12 h, preferably 1-8 h, for example 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, etc.

[0067] In the present invention, the pH of the co-incubation is not particularly limited and can be adjusted as needed, but is preferably 3-11, preferably 4-8, for example 4, 5, 6, 7, 8, etc.

[0068] In addition, the probiotics co-cultured with the selenium precursor are preferably probiotics that do not contain free polysaccharides. Therefore, the method of the present invention further includes pre-treating the cultured probiotics to remove free extracellular polysaccharides. The pre-treatment method is not particularly limited, for example, free extracellular polysaccharides can be removed by low-speed centrifugation, wherein low speed refers to a centrifugal force of no more than 6000g, preferably no more than 3000g.

[0069] In a preferred embodiment of the present invention, the order of adding the solvent, the probiotics and the selenium precursor is not particularly limited. Preferably, the probiotics can be mixed with the solvent first and then with the selenium precursor.

[0070] Step (2)

[0071] Step (2) of the present invention is a step of obtaining chimeric selenium dot probiotics by surface in situ reduction with a reducing agent. The type of reducing agent is not particularly limited, and examples thereof include but are not limited to any one or a combination of ascorbic acid, glutathione, sodium borohydride and polyphenols.

[0072] In a preferred embodiment of the present invention, in order to obtain selenium dots or nano-selenium with a fixed particle size, the molar ratio of the reducing agent to the selenium atoms in the selenium precursor is preferably (1-320):1, also preferably (1-100):1, further preferably (1-80):1, more preferably (1-60):1, for example 1:1, 4:1, 8:1, 10:1, 20:1, 40:1, 60:1, etc.

[0073] In a preferred embodiment of the present invention, the temperature of the reduction reaction is 4-40°C, preferably 5-35°C, for example 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, etc.

[0074] In a preferred embodiment of the present invention, the reduction reaction time is 0.5-24 h, preferably 1-10 h, further preferably 2-10 h, for example 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, etc.

[0075] In a preferred embodiment of the present invention, the pH of the reduction reaction is 3-11, preferably 4-8, further preferably 5-8, such as 5, 6, 7, 8, etc.

[0076] In addition, the present invention also uses infrared spectroscopy and nuclear magnetic resonance hydrogen spectrum to characterize the interaction between hydroxyl and selenium points in polysaccharides on the surface of Lactobacillus casei, confirming that hydroxyl is a ligand of nano-selenium and proving that selenium can disrupt the hydrogen bonding system of sugar.

[0077] use

[0078] One aspect of the present invention provides a pharmaceutical composition comprising the chimeric selenium-dot probiotics of the present invention.

[0079] In one aspect of the present invention, a dietary supplement is provided, which contains the chimeric selenium-dot probiotics of the present invention.

[0080] One aspect of the present invention provides a feed additive, which contains the chimeric selenium-dot probiotics of the present invention.

[0081] The present invention also provides the use of the chimeric selenium-dot probiotic in the preparation of the following medicines, dietary supplements or feed additives, wherein the medicines, dietary supplements or feed additives can be:

[0082] (1) Prevent, improve, alleviate or treat intestinal inflammatory diseases;

[0083] (2) Drugs for preventing, improving, alleviating or treating nasal inflammatory diseases;

[0084] (3) Increase or improve the efficiency of scavenging reactive oxygen free radicals;

[0085] (4) increasing the diversity of intestinal flora or improving the abundance of probiotics;

[0086] (5) regulating immune cells associated with intestinal inflammation, preferably restoring immune cells associated with intestinal inflammation to normal levels;

[0087] (6) Regulating intestinal inflammation-related cytokines, preferably promoting the expression or amount of anti-inflammatory cytokines, and / or inhibiting the expression or amount of pro-inflammatory cytokines.

[0088] Those skilled in the art will appreciate that the chimeric selenium dot probiotics of the present invention can be used for diagnostic or therapeutic purposes, or for non-diagnostic or therapeutic purposes. Based on this, the present invention also provides a method for increasing or improving the efficiency of scavenging reactive oxygen free radicals in vitro, or regulating inflammation-related cytokines in vitro, which includes the step of contacting the chimeric selenium dot probiotics of the present invention with cells.

[0089] As used herein, the terms "prevent, ameliorate, alleviate or treat" refer to improving a condition before or after the onset of a disease or disorder. Such alleviation or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique compared to an untreated control group under the same conditions. In the present invention, the term "treat" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as reducing the risk of inflammatory diseases. Beneficial or desired clinical results include, but are not limited to, the following, whether detectable or undetectable, including relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and reduction (whether partial or complete). Those in need of treatment include those who already have a disease or disorder, as well as those who are susceptible to a disease or disorder, or those who need to prevent the disease or disorder.

[0090] Example 1

[0091] The following is an exemplary description using Lactobacillus casei as a probiotic.

[0092] 1. Preparation of chimeric selenium-dot Lactobacillus casei

[0093] (1) Lactobacillus casei (CICC20280) cultured overnight in Msgg medium was centrifuged at low speed to remove polysaccharides in the solution, and the supernatant was discarded after centrifugation at 3000×g for 5 min. The cells were resuspended in physiological saline solution and the ratio of Lactobacillus casei to selenium precursor was 10:1. 9 CFU: 0.012mmol sodium selenite was added, and after incubation at 37°C for 3h, Lactobacillus casei adsorbed with selenium precursors was obtained;

[0094] (2) adding ascorbic acid at a molar ratio of the reducing agent to the selenium atom in the selenium precursor of 40:1, and performing a reduction reaction at 37° C. for 2 h to obtain a chimeric selenium-dot Lactobacillus casei.

[0095] 2. Performance test of chimeric selenium-dot Lactobacillus casei

[0096] The microscopic morphology of chimeric selenium-dot Lactobacillus casei was observed using transmission electron microscopy. Figure 1 This is a transmission electron microscope image of the chimeric selenium-dot Lactobacillus casei obtained in Example 1, wherein the embedded image is a selenium-dot lattice. Figure 2 is the elemental analysis spectrum of Lactobacillus casei with chimeric selenium dots (the arrow indicates the energy spectrum position of selenium), Figure 1 and Figure 2 It can be seen that the preparation method provided by the present invention can successfully grow selenium dots on the surface of Lactobacillus casei in situ, that is, obtain the product chimeric selenium dot Lactobacillus casei. At the same time, it can be seen from the figure that the average particle size of the selenium dots is about 5 nm.

[0097] Example 2

[0098] Preparation of chimeric selenium-dot Lactobacillus casei:

[0099] (1) Lactobacillus casei (CICC20280) cultured overnight in Msgg medium was centrifuged at low speed to remove polysaccharides in the solution, and the supernatant was discarded after centrifugation at 3000×g for 5 min. The cells were resuspended in physiological saline solution and the ratio of Lactobacillus casei to selenium precursor was 10:1. 9 CFU: 0.006mmol sodium selenite was added, and after incubation at 37°C for 3h, Lactobacillus casei adsorbed with selenium precursors was obtained;

[0100] (2) adding ascorbic acid at a molar ratio of the reducing agent to the selenium atom in the selenium precursor of 40:1, and performing a reduction reaction at 37° C. for 2 h to obtain a chimeric selenium-dot Lactobacillus casei.

[0101] Example 3

[0102] Preparation of chimeric selenium-dot Lactobacillus casei:

[0103] (1) Lactobacillus casei (CICC20280) cultured overnight in Msgg medium was centrifuged at low speed to remove polysaccharides in the solution, and the supernatant was discarded after centrifugation at 3000×g for 5 min. The cells were resuspended in physiological saline solution and the ratio of Lactobacillus casei to selenium precursor was 10:1. 9 CFU: 0.02mmol sodium selenite was added, and after incubation at 37°C for 3h, Lactobacillus casei adsorbed with selenium precursors was obtained;

[0104] (2) adding ascorbic acid at a molar ratio of the reducing agent to the selenium atom in the selenium precursor of 40:1, and performing a reduction reaction at 37° C. for 2 h to obtain a chimeric selenium-dot Lactobacillus casei.

[0105] Example 4

[0106] Preparation of chimeric selenium-dot Lactobacillus casei:

[0107] (1) Lactobacillus casei (CICC20280) cultured overnight in Msgg medium was centrifuged at low speed to remove polysaccharides in the solution, and the supernatant was discarded after centrifugation at 3000×g for 5 min. The cells were resuspended in physiological saline solution and the ratio of Lactobacillus casei to selenium precursor was 10:1. 9 CFU: 0.012mmol sodium selenite was added, and after incubation at 40°C for 3h, Lactobacillus casei adsorbed with selenium precursors was obtained;

[0108] (2) adding ascorbic acid at a molar ratio of the reducing agent to the selenium atom in the selenium precursor of 40:1, and performing a reduction reaction at 37° C. for 2 h to obtain a chimeric selenium-dot Lactobacillus casei.

[0109] Example 5

[0110] Preparation of chimeric selenium-dot Streptococcus thermophilus and chimeric selenium-dot Pediococcus acidilactici:

[0111] (1) The thermophilic Streptococcus (CICC6222) or the lactic Pediococcus (JCM8797) cultured overnight in Msgg medium was centrifuged at low speed to remove the polysaccharide in the solution. After centrifugation at 3000×g for 5 min, the supernatant was discarded and the cells were resuspended in physiological saline solution. The ratio of thermophilic Streptococcus or lactic Pediococcus to selenium precursor was 10:1. 9 CFU: 0.012mmol sodium selenite was added, and after incubation at 37°C for 3h, thermophilic Streptococcus or lactic acid bacteria adsorbed with selenium precursors were obtained;

[0112] (2) Ascorbic acid was added at a molar ratio of 40:1 between the reducing agent and the selenium atom in the selenium precursor, and the reduction reaction was carried out at 37°C for 2 hours to obtain chimeric selenium-dot thermophilic Streptococcus and chimeric selenium-dot lactic Pediococcus. The microscopic morphology of the chimeric selenium-dot Lactobacillus casei was observed using a transmission electron microscope. Figure 3The transmission electron micrographs of the chimeric selenium-dot thermophilic Streptococcus and the chimeric selenium-dot lactic Pediococcus obtained in Example 5 show that the preparation method provided by the present invention can successfully grow selenium dots in situ on the bacterial surface, i.e., obtain the chimeric selenium-dot thermophilic Streptococcus or the chimeric selenium-dot lactic Pediococcus as products.

[0113] Example 6

[0114] Preparation of chimeric selenium-containing Lactobacillus casei using selenomethionine as a selenium precursor:

[0115] (1) Lactobacillus casei (CICC20280) cultured overnight in Msgg medium was centrifuged at low speed to remove polysaccharides in the solution, and the supernatant was discarded after centrifugation at 3000×g for 5 min. The cells were resuspended in physiological saline solution and the ratio of Lactobacillus casei to selenium precursor was 10:1. 9 CFU: 0.1mmol selenomethionine was added, and then the pH of the solution was adjusted to 7.0. After incubation at 37°C for 3h, Lactobacillus casei adsorbed with selenium precursors was obtained;

[0116] (2) Ascorbic acid was added according to the molar ratio of the reducing agent to the selenium atom in the selenium precursor of 60:1, and then the pH of the solution was adjusted to 6.0, and the reduction reaction was carried out at 37° C. for 9 hours to obtain the chimeric selenium-dot Lactobacillus casei. The microscopic morphology of the chimeric selenium-dot Lactobacillus casei was observed using a transmission electron microscope. Figure 4 This is a transmission electron micrograph of the chimeric selenium-dot Lactobacillus casei obtained in Example 6. The preparation method provided by the present invention can utilize selenomethionine as a precursor to successfully grow selenium dots in situ on the bacterial surface, thereby obtaining the chimeric selenium-dot Lactobacillus casei product.

[0117] Comparative Example 1

[0118] Preparation of Nano-Selenium:

[0119] (1) Sodium selenite (same mass as in Example 1) was added to a phosphate buffer solution, and after being placed at 37° C. for 3 h, ascorbic acid was added in a molar ratio of the reducing agent to the selenium atoms in the selenium precursor of 40:1, and a reduction reaction was carried out at room temperature for 12 h to obtain nano-selenium.

[0120] Comparative Example 2

[0121] Preparation of the mixed solution of nano-selenium and Lactobacillus casei:

[0122] The Lactobacillus casei provided in Example 1 was mixed with the nano-selenium provided in Comparative Example 1 to obtain a mixed solution of nano-selenium and Lactobacillus casei provided in this comparative example (the amount of the solvent used was the same as that in Example 1).

[0123] Comparative Example 3

[0124] This comparative example provides a kind of Lactobacillus casei exopolysaccharide secreted by separation, and is used for synthesizing nano-selenium. The specific extraction method of the Lactobacillus casei exopolysaccharide secreted by the described Lactobacillus casei is as follows: the culture solution of Lactobacillus casei (CICC20280) is inactivated in a boiling water bath (30min), cooled to room temperature, and centrifuged (12000×g, 30min) to remove impurities such as bacterial cells and enzymolysis proteins. The supernatant is then taken, and 80.0% trichloroacetic acid solution is added while stirring to a final content of 4.0%, and the remaining denatured protein is removed by centrifugation (12000×g, 30min) after standing overnight at 4°C. The supernatant is taken while vigorously stirring and adding 3 times the volume of 95% ice ethanol, standing at 4°C for 24h, and centrifuged again (12000×g, 30min) to take the precipitate, which is redissolved with appropriate deionized water, and the solution is dialyzed for 48h (dialysis bag with a molecular weight cutoff of 8-14kDa) and water is changed once every 8h. The dialyzate is the following polysaccharide solution used. Sodium selenite (same mass as in Example 1) was added to the polysaccharide solution, and after being placed at 37° C. for 2 h, ascorbic acid was added according to a molar ratio of the reducing agent to the selenium atom in the selenium precursor of 40:1, and a reduction reaction was carried out at room temperature for 2 h to obtain a connection product of extracellular polysaccharide and nano-selenium.

[0125] Test Case

[0126] The following shows the performance test of the chimeric selenium-dot Lactobacillus casei prepared in Examples 1-4.

[0127] (1) Antioxidant activity test of chimeric selenium-dot Lactobacillus casei

[0128] The ability to scavenge the classic free radical DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) was used as a representative test: 50 μg / mL of the chimeric selenium-dot Lactobacillus casei was reacted with 0.5 mmol / L of the purple free radical DPPH solution, and the change in absorbance at 526 nm after the entire reaction system reacted for 30 minutes was tested. The scavenging rate of the DPPH free radical was calculated. The results are shown in Table 1:

[0129] Table 1

[0130] sample Clearance rate / % sample Clearance rate / % Example 1 58.2 Comparative Example 1 28.5 Example 2 42.6 Comparative Example 2 36.4 Example 3 40.2 Comparative Example 3 29.6 Example 4 41.1 Control group 1 0.4

[0131] Note: Control group 1 is the untreated control group (containing only Lactobacillus casei).

[0132] As shown in Table 1, the chimeric selenium-dot Lactobacillus casei provided by the present invention has excellent antioxidant activity. As shown in the comparison between Examples 1-3 and Comparative Example 1, the ratio of Lactobacillus casei to selenium precursor in the present invention has a better effect within a certain range; as shown in the comparison between Examples 1 and 4, the present invention is co-incubated at 36-40°C, and the chimeric selenium dots on the surface of Lactobacillus casei have a good antioxidant effect. As shown in the comparison between Example 1 and Comparative Examples 1 and 2, the preparation method provided by the present invention can obtain a chimeric selenium-dot Lactobacillus casei with excellent antioxidant effect.

[0133] (2) In vitro growth ability of the chimeric selenium-dot Lactobacillus casei prepared in Example 1

[0134] 1×10 9 CFU of chimeric selenium-containing Lactobacillus casei with 1 × 10 9 CFU Lactobacillus casei was added to a culture tube containing MRS liquid culture medium, and the absorbance value of the bacterial solution at 600nm after different culture times was tested, and a growth curve was drawn based on this. Figure 5 It can be seen that the growth ability of Lactobacillus casei was not affected after the chimeric selenium dots were added.

[0135] (3) Gastric acid environment tolerance of the chimeric selenium-dot Lactobacillus casei prepared in Example 1

[0136] A certain amount of chimeric selenium-dot Lactobacillus casei and Lactobacillus casei were added to the gastric acid simulation solution for co-incubation. An appropriate amount of bacterial suspension was taken for dilution at different time points, and then spread on a pre-prepared plate (MRS solid culture medium), inverted and placed in a 37°C incubator for 24 hours for counting. Figure 6 It can be seen that after 4 hours of co-incubation, 1% of the chimeric selenium-dot Lactobacillus casei bacteria still survived, while the Lactobacillus casei itself was inactivated.

[0137] (4) Intestinal adhesion ability of the chimeric selenium-dot Lactobacillus casei prepared in Example 1

[0138] Female BALB / c mice aged 5-6 weeks were used. The abdominal hair of the mice was removed with a depilatory cream and 1×10 8 CFU chimeric selenium-dot Lactobacillus casei, fluorescence imaging at different time points. Figure 7 It can be seen that within 4-6 hours, the abdominal fluorescence intensity of mice in the chimeric selenium-dot Lactobacillus casei group was significantly higher than that in the Lactobacillus casei group, proving that the chimeric selenium-dot Lactobacillus casei can adhere to and remain in the intestine better, which is beneficial to relieve inflammation in the intestine.

[0139] (5) Therapeutic effect of chimeric selenium-dot Lactobacillus casei in ulcerative colitis model mice

[0140] Female BALB / c mice aged 5-6 weeks were adaptively raised for 7 days, and their drinking water was replaced with a 3% aqueous solution of dextran sulfate sodium (DSS) (w / v); on the 1st to 7th day of modeling, the mice were given different treatments (PBS, Lactobacillus casei, a mixture of nano-selenium and Lactobacillus casei prepared in the above comparative example 2, and a chimeric selenium-dot Lactobacillus casei prepared in the above example 1); the body weight of the mice was recorded every day during the 9-day modeling period. On the 9th day of modeling, the mice were killed, and the colon tissues of the mice were removed and the lengths were measured for analysis. The results are shown in FIG. Figure 8 As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, which is beneficial to relieve inflammation in mice with ulcerative colitis and reduce intestinal damage, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has a good therapeutic effect in, for example, inflammatory disease models.

[0141] (6) Effect of chimeric selenium-dot Lactobacillus casei on the recovery of intestinal flora in ulcerative colitis model mice

[0142] Female BALB / c mice aged 5-6 weeks were adaptively fed for 7 days, and their drinking water was replaced with a 3% DSS aqueous solution (w / v); on the 1st to 7th day of modeling, the mice were given different treatments (PBS, Lactobacillus casei, a mixture of nano-selenium and Lactobacillus casei prepared in the above comparative example 2, and a chimeric selenium-dot Lactobacillus casei prepared in the above example 1); on the 9th day of modeling, the mouse feces were collected for 16SrRNA sequencing analysis. The results are shown in Fig. 9 As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, which is beneficial to alleviate the imbalanced intestinal flora in mice with ulcerative colitis, increase the diversity of intestinal flora, and improve the abundance of probiotics, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has a good recovery effect on the imbalanced intestinal flora.

[0143] (7) Effect of chimeric selenium-dot Lactobacillus casei on scavenging reactive oxygen free radicals in ulcerative colitis model mice

[0144] Female BALB / c mice aged 5-6 weeks were adaptively fed for 7 days, and their drinking water was replaced with a 3% DSS aqueous solution (w / v); on the 1st to 7th day of modeling, the mice were given different treatments (PBS, Lactobacillus casei, a mixture of nano-selenium and Lactobacillus casei prepared in the above comparative example 2, and a chimeric selenium-dot Lactobacillus casei prepared in the above example 1); on the 9th day of modeling, the mouse intestines were stained with dihydroethidium dye for 30 minutes, and then the mice were dissected to remove the colon and observed and photographed under a two-photon microscope. The results are shown in Fig.10As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is beneficial to the removal of excessive reactive oxygen free radicals produced in the body of mice with ulcerative colitis, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has an excellent effect of removing reactive oxygen free radicals in vivo.

[0145] (8) Effect of chimeric selenium-containing Lactobacillus casei on immune cell regulation in ulcerative colitis model mice

[0146] Female BALB / c mice aged 5-6 weeks were adaptively fed for 7 days, and their drinking water was replaced with a 3% DSS aqueous solution (w / v); on the 1st to 7th day of modeling, the mice were given different treatments (PBS, Lactobacillus casei, a mixture of nano-selenium and Lactobacillus casei prepared in the above comparative example 2, and a chimeric selenium-dot Lactobacillus casei prepared in the above example 1); on the 9th day of modeling, the mice were dissected to remove the colon and separate the lamina propria cells for flow cytometry staining. The results are shown in Fig.11 As shown, when the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, the proportion of anti-inflammatory Treg cells is increased, and the proportion of pro-inflammatory Th17 and Macrophage cells is reduced, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has a regulatory effect on intestinal inflammation-related immune cells.

[0147] (9) Effect of chimeric selenium-containing Lactobacillus casei on cytokine regulation in ulcerative colitis model mice

[0148] Female BALB / c mice aged 5-6 weeks were adaptively fed for 7 days, and their drinking water was replaced with a 3% DSS aqueous solution (w / v); on the 1st to 7th day of modeling, the mice were given different treatments (PBS, Lactobacillus casei, a mixture of nano-selenium and Lactobacillus casei prepared in the above comparative example 2, and a chimeric selenium-dot Lactobacillus casei prepared in the above example 1); on the 9th day of modeling, the mice were dissected to remove the colon and homogenized to extract protein, and cytokine ELISA was performed. The results are shown in Fig.12 As shown, when the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, the anti-inflammatory cytokines IL-10 and TGF-β are increased, and the proportions of pro-inflammatory TNF-α, IL-6, and IL-1β are reduced, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has a regulatory effect on intestinal inflammation-related cytokines.

[0149] (10) Effect of chimeric selenium-containing Lactobacillus casei in delayed treatment of ulcerative colitis model

[0150] Female BALB / c mice aged 5-6 weeks were adaptively fed for 7 days. On the 1st to 7th day of modeling, the drinking water was replaced with a 3% DSS aqueous solution (w / v). On the 8th to 11th day, the mice were given different treatments (PBS, chimeric selenium-dot Lactobacillus casei prepared in Example 1 above); on the 12th day, the mice were killed, and the colon tissues of the mice were removed and the lengths were measured for analysis. The results are shown in Figure 2. Fig.13 As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, which is beneficial to relieve inflammation in mice with ulcerative colitis and reduce intestinal damage, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has a good therapeutic effect in, for example, inflammatory disease models.

[0151] (11) Therapeutic effect of chimeric selenium-containing Lactobacillus casei in the TNBS-induced ulcerative colitis model

[0152] Female c57BL / 6 mice aged 5-6 weeks were adaptively raised for 7 days. On the 0th day of modeling, the hair on the head of the mice was removed after anesthesia, and TNBS solution was absorbed percutaneously. On the 7th day, TNBS solution was administered rectally. On the 8th to 11th day, the mice were given different treatments (PBS and the chimeric selenium-dot Lactobacillus casei prepared in Example 1 above); on the 12th day, the mice were killed, and the colon tissues of the mice were removed and the length was measured for analysis. The results are shown in Figure 2. Fig.14 As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, which is beneficial to relieve inflammation in mice with ulcerative colitis and reduce intestinal damage, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has a good therapeutic effect in, for example, inflammatory disease models.

[0153] (12) Therapeutic effect of chimeric selenium-containing Lactobacillus casei in a chronic ulcerative colitis model

[0154] Female BALB / c mice aged 5-6 weeks were adaptively fed for 7 days. In the first, third and fifth weeks of modeling, drinking water was replaced with a 3% DSS aqueous solution (w / v). In the second, fourth and sixth weeks, mice were given drinking water and different treatments (PBS and chimeric selenium-dot Lactobacillus casei prepared in Example 1 above). On the 43rd day, the mice were killed, and the colon tissues of the mice were removed and the lengths were measured for analysis. The results are shown in Figure 2. Fig.15 As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, which is beneficial to relieve inflammation in mice with ulcerative colitis and reduce intestinal damage, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has a good therapeutic effect in, for example, inflammatory disease models.

[0155] (13) Evaluation of the biosafety of chimeric selenium-containing Lactobacillus casei in mice after short-term / long-term oral administration

[0156] Female BALB / c mice aged 5-6 weeks were adaptively raised for 7 days, and were given drinking water and the chimeric selenium-dot Lactobacillus casei prepared in Example 1 for one week; on the 8th day, the mice were killed, and the whole blood, heart, liver, spleen, lung, and kidney of the mice were collected for physiological and pathological tests. Female BALB / c mice aged 5-6 weeks were adaptively raised for 7 days, and were given drinking water and the chimeric selenium-dot Lactobacillus casei prepared in Example 1 for four weeks; on the 29th day, the mice were killed, and the whole blood, heart, liver, spleen, lung, and kidney of the mice were collected for physiological and pathological tests. The results are as follows: Fig.16 and Fig.17 As shown, when the chimeric selenium-dot Lactobacillus casei provided by the present invention is orally administered, all indicators of mice are within the normal range.

[0157] (14) Effect of chimeric selenium-containing Lactobacillus casei on scavenging reactive oxygen free radicals in cynomolgus monkeys with ulcerative colitis

[0158] Male cynomolgus monkeys aged 4 to 4.5 years and weighing 4.5±0.5 kg were individually caged with free access to food and water. On days 1 to 10 of modeling, each cynomolgus monkey was gavaged with 50 mL of 2.5% DSS aqueous solution (w / v) per day. On days 2 to 10 of modeling, the cynomolgus monkeys were given different treatments (PBS and the chimeric selenium-dot Lactobacillus casei prepared in Example 1 above). On the 11th day after the end of the treatment, the feces of the cynomolgus monkeys were collected for detection of the content of reactive oxygen free radicals and 16S rRNA sequencing analysis. The results are shown in Figure 2. Fig.18 As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is beneficial for clearing the reactive oxygen free radicals overproduced in ulcerative colitis crab-eating monkeys, and is beneficial for alleviating the imbalanced intestinal flora in ulcerative colitis mice, increasing the diversity of intestinal flora, and improving the abundance of probiotics, indicating that the chimeric selenium-dot Lactobacillus casei provided by the present invention has an excellent scavenging effect on reactive oxygen free radicals in vivo, and a good recovery effect on the imbalanced intestinal flora.

[0159] (15) Effect of chimeric selenium-dot Lactobacillus casei on improving intestinal ulcers in cynomolgus monkeys with ulcerative colitis

[0160] Male cynomolgus monkeys aged 4 to 4.5 years and weighing 4.5±0.5 kg were individually caged with free access to food and water. On days 1 to 10 of modeling, each cynomolgus monkey was gavaged with 50 mL of 2.5% DSS aqueous solution (w / v) per day. On days 2 to 10 of modeling, the cynomolgus monkeys were given different treatments (PBS and the chimeric selenium-dot Lactobacillus casei prepared in Example 1 above). On day 11 after the end of treatment, the monkeys were subjected to colonoscopy. The results are shown in Table 1. Fig.19As shown, the chimeric selenium-dot Lactobacillus casei provided by the present invention is used for treatment, which is beneficial to improving the symptoms of bleeding, ulcers and the like in the intestinal cavity of cynomolgus monkeys.

[0161] (16) Safety evaluation of chimeric selenium-containing Lactobacillus casei in the treatment of ulcerative colitis in cynomolgus monkeys

[0162] Male cynomolgus monkeys aged 4 to 4.5 years and weighing 4.5±0.5 kg were individually caged with free access to food and water. On days 1 to 10 of modeling, each cynomolgus monkey was gavaged with 50 mL of 2.5% DSS aqueous solution (w / v) per day. On days 2 to 10 of modeling, the cynomolgus monkeys were given different treatments (PBS and the chimeric selenium-dot Lactobacillus casei prepared in Example 1 above). The conditions of the cynomolgus monkeys after treatment were observed every day. On the 11th day after the end of treatment, blood samples were taken from the cynomolgus monkeys for physiological testing. The results are shown in Figure 2. Fig. 20 As shown, the chimeric selenium-containing Lactobacillus casei provided by the present invention was orally administered to cynomolgus monkeys without adverse oral reactions, and all blood indexes detected were within normal ranges.

[0163] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A chimeric selenium-dot probiotic, characterized in that: The invention comprises probiotics and selenium element which grows in situ on the surface of the probiotics.

2. The chimeric selenium-dot probiotic according to claim 1, characterized in that The probiotics include intestinal probiotics.

3. The chimeric selenium-dot probiotic according to claim 2, characterized in that The intestinal probiotics include at least one of Lactobacillus, Bifidobacterium, Streptococcus, Bacillus, Clostridium, Bacteroides, Lactococcus, Leuconostoc, Propionibacterium, Pediococcus and Saccharomyces; Preferably, the intestinal probiotics are Lactobacillus casei, Streptococcus thermophilus or Pediococcus acidilactici.

4. The chimeric selenium-dot probiotic according to claim 3, characterized in that: The selenium element has a particle size of 1-100 nm.

5. A method for preparing a chimeric selenium-dot probiotic, characterized in that: include: (1) culturing probiotics in a culture medium, and then co-incubating the cultured probiotics in a solvent containing a selenium precursor to obtain probiotics adsorbed with the selenium precursor; (2) subjecting the probiotics adsorbed with selenium precursor to a reduction reaction with a reducing agent to obtain the chimeric selenium-dot probiotics.

6. The method for preparing the chimeric selenium-dot probiotics according to claim 5, characterized in that: The incubation temperature is 4-45°C.

7. The method for preparing the chimeric selenium-dot probiotics according to claim 6, characterized in that: The ratio of the probiotics to the selenium precursor is 10 7-10 CFU: (0.002-100) mmol, preferably 10 7-10 CFU: (0.002-10)mmol.

8. The method for preparing the chimeric selenium-dot probiotics according to claim 7, characterized in that: The selenium precursor includes organic selenium and / or inorganic selenium; Preferably, the selenium precursor is selected from any one or a combination of sodium selenite, potassium selenite, sodium selenate, potassium selenate, selenium dioxide, selenium oxide, selenomethionine, hydroxyselenomethionine, selenocysteine, selenocysteine, selenomethylselenocysteine, selenomethylselenomethionine or seaweed selenium polysaccharide; Preferably, the solvent is selected from at least one of physiological saline, phosphate buffer solution, M17 medium, MRS medium, LB medium, BH medium, Bifidobacterium BS medium and Roche broth medium.

9. The method for preparing the chimeric selenium-dot probiotics according to claim 8, characterized in that: The reducing agent includes any one or a combination of ascorbic acid, glutathione, sodium borohydride, hydrazine hydrate, catechin, hydroquinone, gallic acid, quercetin and resveratrol; Preferably, the temperature of the reduction reaction is 4-40°C; Preferably, the reduction reaction time is 0.5-24h; Preferably, the pH of the reduction reaction is 3-11.

10. Use of the chimeric selenium-dot probiotic according to any one of claims 1 to 9 in the preparation of a medicine, dietary supplement or feed additive for preventing, improving or treating inflammatory diseases.

11. A pharmaceutical composition or dietary supplement comprising the chimeric selenium-dot probiotic according to any one of claims 1 to 10.

12. A feed additive comprising the chimeric selenium-dot probiotic according to any one of claims 1 to 10.

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