Mineralizing microorganisms and their preparation and application

Abstract

The present application discloses a mineralized microorganism and its preparation and application, belonging to the field of biomedical technology. The mineralized microorganism is a probiotic containing a photothermal effect material, which can be taken orally and adhere to the intestines. When irradiated with near-infrared light that can penetrate the skin, the photothermal effect material can quickly convert light energy into heat energy, perform local hyperthermia, promote visceral fat browning, increase fat uptake of glucose, improve impaired glucose tolerance and / or alleviate insulin resistance, enhance heat production and metabolism, and consume visceral fat to treat obesity and / or diabetes, etc.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a mineralized microorganism and its preparation and application, and more specifically to a probiotic containing a photothermal effect material and its application in the preparation of a drug for treating obesity and / or diabetes. Background Art

[0002] Obesity is a chronic and complex disease defined by excessive fat deposition that may cause health damage. Obesity can cause other metabolic diseases, such as type 2 diabetes, fatty liver and cardiovascular disease. At present, the means of preventing and treating obesity and diabetes are limited to physical and chemical means such as diet, exercise, surgery and drug intervention. These interventions are generally indirect or act on the central nervous system to suppress appetite, or act on the gastrointestinal tract to inhibit fat absorption. The effects are not ideal, compliance is poor, and there are even more serious side effects. For example, taking the drug β3-adrenaline can easily induce cardiovascular diseases.

[0003] The fat in the human body includes subcutaneous fat and visceral fat. When visceral fat accumulates excessively, it can damage organs such as the liver, causing fatty liver, disrupting metabolism, and causing type 2 diabetes and infertility. In addition, visceral fat can easily lead to an increase in subcutaneous fat. If visceral fat is not reduced, reducing subcutaneous fat alone is equivalent to treating the symptoms but not the root cause, which is also an unhealthy way to lose weight.

[0004] Currently, targeted fat reduction methods for improving obesity and diabetes are mostly focused on subcutaneous white adipose tissue, and are mainly delivered by insulin needles and microneedles. However, metabolic inflammation such as insulin resistance is often related to the accumulation of visceral fat, and there is an urgent need to develop new simple, easy, safe and non-invasive strategies to solve the problem of difficult visceral fat reduction. Therefore, developing simple, easy, safe and efficient treatments to reduce the deposition of visceral fat or increase the consumption of visceral fat is of great significance for controlling the occurrence of obesity and related diseases such as type 2 diabetes. Summary of the invention

[0005] 1. Purpose of the Invention

[0006] The invention purpose of the present application is to provide a mineralized microorganism and its preparation and application. The mineralized microorganism is a probiotic containing a photothermal effect material. The mineralized microorganism can enter the body through oral administration and adhere to the intestine. It is irradiated with near-infrared light (NIR) that can penetrate the skin. The photothermal effect material can quickly convert light energy into heat energy to perform local hyperthermia therapy (LHT), promote the browning of visceral fat, increase the uptake of glucose by brown fat, enhance heat production and metabolism, and consume visceral fat to treat obesity and / or diabetes, etc.

[0007] 2. Technical solution

[0008] In order to achieve the above purpose, the technical solution adopted in this application is as follows:

[0009] The present application provides a mineralized microorganism, which includes a microorganism containing a photothermal effect material in the cell and / or on the cell surface, and the microorganism includes a microorganism that can adhere to the intestine.

[0010] Furthermore, the above-mentioned microorganisms adhere to the intestine for 0 to 24 hours.

[0011] Furthermore, the above-mentioned microorganisms include one or more of Bifidobacterium, Lactobacillus, Clostridium butyricum, Streptococcus faecalis, Bacillus, Bacillus subtilis, Saccharomyces boulardii, and Saccharomyces cerevisiae. Most of these microorganisms are probiotics that adhere to the intestines and can not only be used for local hyperthermia therapy, but also can regulate intestinal microecological imbalance, maintain microecological balance, and improve the health level or health status of the host.

[0012] Furthermore, the microorganisms include Saccharomyces boulardii and / or Saccharomyces cerevisiae. S. boulardii )、Saccharomyces cerevisiae( Saccharomyces cerevisiae ) is a probiotic fungus that can improve the intestinal immune microenvironment through oral administration. Compared with probiotic bacteria, probiotic fungi are microorganisms with eukaryotic cell structures. They have complete cell structures, will not be transported with the blood and lymph, and are resistant to gastric acid corrosion. They can target visceral fat through modification and oral administration to exert the effect of local hyperthermia, and their safety is guaranteed.

[0013] Furthermore, the above-mentioned microorganisms include Saccharomyces boulardii, which is a probiotic fungus with the "Generally Recognized as Safe" (GRAS) attribute of the U.S. Food and Drug Administration (FDA). It is safe and reliable, can adhere to the intestines through oral administration, and improve the intestinal immune environment. At the same time, Saccharomyces boulardii has excellent stress tolerance, antioxidant and high temperature resistance (tolerance temperature is about 50°C) characteristics.

[0014] Furthermore, the above-mentioned microorganisms include Saccharomyces cerevisiae.

[0015] Furthermore, the above-mentioned photothermal effect materials include nanogold. As a nanomaterial, nanogold has good photostability and photothermal conversion capabilities. The use of near-infrared laser irradiation can enable mineralized microorganisms to convert light energy into thermal energy, achieve local hyperthermia through photothermal effect, promote visceral fat browning, enhance heat production and metabolism, consume visceral fat, and improve metabolic diseases such as obesity and diabetes.

[0016] The present application also provides a method for preparing the above-mentioned mineralizing microorganism, wherein the microorganism is co-incubated with gold ions, the microorganism absorbs and / or adsorbs the gold ions, and the bioactive molecules are self-assembled into gold nanoparticles in a biosynthetic manner, and the gold nanoparticles are adsorbed on the surface of the microbial cells and / or contained in the cells.

[0017] Furthermore, the above-mentioned method for preparing a mineralizing microorganism specifically comprises the following steps:

[0018] The microorganisms and the acid or salt containing gold are mixed in a solution, the mixed solution is shaken and incubated, centrifuged after incubation, and the precipitate is collected to obtain the mineralized microorganisms.

[0019] Furthermore, the above-mentioned microorganisms include Saccharomyces boulardii and / or Saccharomyces cerevisiae.

[0020] Furthermore, the gold-containing acid or salt includes one or more of chloroauric acid, sodium gold thiosulfate and gold chloride.

[0021] Furthermore, the gold-containing acid or salt includes chloroauric acid.

[0022] Furthermore, in the above blended solution, the concentration of chloroauric acid is 0.1-1 mg / mL. Furthermore, in the above blended solution, the concentration of chloroauric acid is 0.5 mg / mL.

[0023] Furthermore, in the above blended solution, the concentration of microorganisms is 1×10 7 ~1×10 8 Furthermore, in the above blended solution, the concentration of microorganisms is 5×10 7 Pieces / mL.

[0024] Furthermore, the above-mentioned solution for blending includes PBS.

[0025] Furthermore, the shaking incubation conditions include 37±1°C and 200±50 rpm.

[0026] Furthermore, the above incubation time is 18 to 48 hours.

[0027] Furthermore, the incubation time is 18 to 24 hours. Furthermore, the incubation time is 24 hours.

[0028] Furthermore, the centrifugation after incubation includes centrifugation at 5000±50 g for 10±2 min.

[0029] The present application also provides the use of the above-mentioned mineralized microorganism in the preparation of a drug for treating obesity and / or diabetes.

[0030] Furthermore, the above applications include promoting glucose uptake and enhancing fat thermogenesis.

[0031] Furthermore, the above application includes improving impaired glucose tolerance and / or alleviating insulin resistance.

[0032] Furthermore, the above-mentioned medicine is an oral preparation.

[0033] Furthermore, the therapeutically effective amount of the above-mentioned drug is not less than 2.5×10 7 indivual.

[0034] Furthermore, the therapeutically effective amount of the above-mentioned drug includes 2.5×10 7 ~1×10 8 indivual.

[0035] The present application also provides a composition, which comprises the above-mentioned mineralizing microorganism.

[0036] The present application also provides an oral preparation for treating obesity and / or diabetes, wherein the oral preparation comprises the above-mentioned mineralized microorganism.

[0037] The present application also provides an oral preparation for improving impaired glucose tolerance and / or alleviating insulin resistance, wherein the oral preparation comprises the above-mentioned mineralized microorganism.

[0038] 3. Beneficial effects

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] (1) The mineralized microorganisms provided in the present application and their preparation and application, the mineralized microorganisms include microorganisms containing photothermal effect materials in the cells and / or on the cell surface that can adhere to the intestine, especially probiotics such as Saccharomyces boulardii and Saccharomyces cerevisiae containing nanogold in the cytoplasm and / or on the cell surface, which can be taken orally and fixed in the intestine; the nanogold has a high photothermal conversion rate, good biocompatibility, stability, low cost and easy preparation, no toxicity of other metal ions, and high safety; it adheres to the intestine and can not only be activated by near-infrared light (NIR) that can penetrate the skin, quickly converting light energy into high heat to promote the effect of local hyperthermia, but also can regulate the imbalance of intestinal microecology, maintain the balance of microecology, and improve the health level or health status of the host.

[0041] (2) The mineralized microorganisms and their preparation and application provided in this application propose for the first time a new strategy for local hyperthermia treatment of visceral fat for the treatment of obesity and / or diabetes. Under the action of mineralized microorganisms and local mild temperature hyperthermia, the browning of adipocytes in visceral fat can be enhanced without affecting the safety and metabolism of other organs, thereby increasing the uptake of glucose by body fat, thereby improving glucose tolerance and insulin sensitivity. The application also specifically provides a mineralized probiotic fungus that uses probiotic fungi to synthesize gold nanoparticles with photothermal effects through biomineralization, which is used for local hyperthermia treatment of visceral fat to improve obesity and / or diabetes. The application is simple and easy to operate, has strong clinical transformation capabilities, and is expected to be widely promoted and applied.

[0042] (3) The mineralized microorganisms provided in the present application and their preparation and application, specifically, microorganisms containing photothermal effect materials that can adhere to the cells in the intestine and / or on the cell surface, especially probiotics such as Saccharomyces boulardii and Saccharomyces cerevisiae that contain nanogold in the cytoplasm and / or on the cell surface, can be safely and stably adhered to the intestine by oral administration, and can be activated by near-infrared light (NIR) for local thermal therapy of visceral fat to improve obesity and / or diabetes, thereby solving practical and urgent problems in the prior art of treating metabolic diseases, such as difficulty in local weight loss and painful administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the specific biomineralization of Saccharomyces boulardii.

[0044] Figure 2 It is the color change of the mixed solution of Saccharomyces boulardii cells and HAuCl4 after oscillation incubation for 0 to 48 h.

[0045] Figure 3 This is a transmission electron microscope image of the mixed solution after oscillation incubation for 4 h. The red arrow points to the gold nanoparticles.

[0046] Figure 4 It is the temperature change curve of different samples under near-infrared laser irradiation.

[0047] Figure 5 This is an infrared thermal image of a mineralized Saccharomyces boulardii solution.

[0048] Figure 6 It is the result of the temperature of the mineralized Saccharomyces boulardii solution rising to a steady state under near-infrared laser irradiation and then naturally cooling to room temperature.

[0049] Figure 7 It is the result of four cycles of mineralized Saccharomyces boulardii solution under near-infrared laser irradiation.

[0050] Figure 8These are the results of characterization of the photothermal properties of mineralized Saccharomyces boulardii solutions with different concentrations.

[0051] Fig. 9 The results are the effects of different laser power densities on the photothermal heating of mineralized Saccharomyces boulardii solution.

[0052] Fig.10 The temperature change of the mineralized Saccharomyces boulardii solution obtained by mineralization of different metal ions under continuous irradiation of near-infrared laser.

[0053] Fig.11 These are the small animal imaging results of the mouse's abdominal cavity and intestines.

[0054] Fig.12 This is the thermal image of mice after Funsome+NIR treatment.

[0055] Fig.13 This is the PET-CT detection result of Funsome+NIR treated mice.

[0056] Fig.14 The results of glucose tolerance test (GTT) and insulin tolerance test (ITT) in Funsome+NIR treated mice.

[0057] Fig.15 These are the test results of ALT, AST and BUN in each group of mice. DETAILED DESCRIPTION

[0058] The present application is further described below in conjunction with specific embodiments.

[0059] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0061] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0062] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0063] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.

[0064] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is used only for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly stated as the range limit, but also including all individual numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly stated.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly stated, but also including individual numbers (such as 2,3,4) and sub-ranges (such as 1 to 3, 2 to 4, etc.).The same principle is applicable to the range of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this interpretation should be applied.

[0065] Hyperthermia Therapy (HT) refers to a method of using different physical factors (radio frequency, microwave, ultrasound and laser, etc.) to increase the temperature and utilize the high temperature effect for treatment. Hyperthermia therapy is a comprehensive treatment method following surgery, radiotherapy, chemotherapy and immunotherapy.

[0066] Local hyperthermia therapy (LHT), a form of hyperthermia therapy, induces a local temperature increase of ±0.5°C to 41°C without causing significant stress to the skin or adjacent tissues and does not alter norepinephrine levels, norepinephrine turnover, or cortisol levels in mice and human subjects.

[0067] As used in this application, PET-CT detection is a combined imaging technology of positron emission tomography (Positron Emission Tomography) and computed tomography (Computed Tomography), and its working principle is:

[0068] PET: uses radioactive isotope-labeled metabolites (tracers) such as glucose to detect metabolic activity in the body. When these tracers are metabolized in the body, they emit positrons, which annihilate upon encountering electrons in the body, producing a pair of high-energy photons in opposite directions. The PET scanner detects these photons and generates images through computer reconstruction.

[0069] CT: X-rays are taken around the body to create a series of cross-sectional images that are then processed by a computer to produce detailed images of the body's internal structures.

[0070] Example 1

[0071] This embodiment provides mineralizing microorganisms and their preparation.

[0072] In this embodiment, the mineralizing microorganisms are microorganisms containing photothermal effect materials inside the cells and on the cell surface.

[0073] In this embodiment, the photothermal effect material is nano-gold.

[0074] In this embodiment, the microorganism is Saccharomyces boulardii ( Saccharomyces boulardii , S. boulardii ), purchased from Shanghai Guyan Industrial Co., Ltd. Saccharomyces boulardii is a probiotic fungus with the “Generally Recognized as Safe” (GRAS) attribute of the U.S. Food and Drug Administration (FDA). It is safe and reliable, can adhere to the intestine through oral administration, improve the intestinal immune environment; and has excellent stress tolerance, antioxidant and high temperature resistance (tolerance temperature is about 50°C).

[0075] This embodiment also provides the preparation of Saccharomyces boulardii containing nano-gold in cells and on the cell surface (referred to as "mineralized Saccharomyces boulardii"), which is prepared by culturing Saccharomyces boulardii with chloroauric acid, wherein the Saccharomyces boulardii absorbs gold ions in the chloroauric acid and converts the gold ions into nano-gold (solid particles) in a biosynthetic manner, which remain in the cytoplasm and are adsorbed on the cell surface ( Figure 1 ), specifically including the following steps:

[0076] S1, Saccharomyces boulardii activation

[0077] 1.5 μL of frozen Saccharomyces boulardii was inoculated into 25 mL of YPD medium and cultured at 37°C with shaking at 200 rpm for 24 h (until the logarithmic growth phase). The number of Saccharomyces boulardii was counted in an electronic counter. A certain volume of culture medium was aspirated and centrifuged at 4000 g for 10 min to obtain a precipitate. 5 mL of PBS was added and vortexed to resuspend the precipitate into a single cell suspension (concentration of 2×10 8 / mL) to obtain the Saccharomyces boulardii cell suspension.

[0078] S2, mineralization of Saccharomyces boulardii

[0079] Add 5 mL of Saccharomyces boulardii cell suspension (1×10 91 mL of 1% HAuCl4 was added to the mixed solution of Saccharomyces boulardii cells and HAuCl4, and the volume of the mixed solution was adjusted to 20 mL with PBS; the mixed solution was placed in an incubator and incubated at 37°C and 200 rpm for 24 h or 48 h. After incubation, it was centrifuged at 5000 g for 10 min, and the precipitate was collected to obtain mineralized Saccharomyces boulardii.

[0080] Result analysis:

[0081] (1) During the oscillation incubation of the mixed solution for 0 to 48 h, the color of the mixed solution changes as follows: Figure 2 As shown, the color of the mixed solution gradually changed from yellow to wine red (purple), indicating that chloroauric acid was converted into nanogold by Saccharomyces boulardii in a biosynthetic manner.

[0082] (2) Transmission electron microscopy images of the mixed solution after oscillation incubation for 4 h are shown in Figure 2. Figure 3 As shown, the red arrow points to the gold nanoparticles, indicating that chloroauric acid is being converted into gold nanoparticles in a biosynthetic manner by Saccharomyces boulardii. The gold nanoparticles are successfully biosynthesized in Saccharomyces boulardii and remain in the cytoplasm and adsorbed on the cell surface.

[0083] Example 2

[0084] This embodiment provides mineralizing microorganisms and their preparation.

[0085] In this embodiment, the mineralizing microorganisms are microorganisms containing photothermal effect materials inside the cells and on the cell surface.

[0086] In this embodiment, the photothermal effect material is nano-gold.

[0087] In this embodiment, the microorganism is Saccharomyces cerevisiae ( Saccharomyces cerevisiae , S. cerevisiae ), purchased from Shanghai Guyan Industrial Co., Ltd., brewer’s yeast is a probiotic fungus that can adhere to the intestine through oral administration and improve the intestinal immune environment.

[0088] This embodiment also provides the preparation of Saccharomyces cerevisiae containing nano-gold in the cell and on the cell surface (referred to as "mineralized Saccharomyces cerevisiae"), which is prepared by culturing Saccharomyces cerevisiae with chloroauric acid, wherein the Saccharomyces cerevisiae absorbs gold ions in the chloroauric acid and converts the gold ions into nano-gold (solid particles) in a biosynthetic manner, which remain in the cytoplasm and are adsorbed on the cell surface, and specifically comprises the following steps:

[0089] S1, Saccharomyces cerevisiae activation

[0090] 1.5 μL frozen Saccharomyces cerevisiae was inoculated into 25 mL YPD medium and cultured at 37°C with shaking at 200 rpm for 24 h (until the logarithmic growth phase). The number of Saccharomyces cerevisiae was counted in an electronic counter. A certain volume of culture medium was aspirated and centrifuged at 4000 g for 10 min to obtain a precipitate. 5 mL PBS was added and vortexed to resuspend the precipitate into a single cell suspension (concentration of 2×10 8 / mL) to obtain the Saccharomyces cerevisiae cell suspension.

[0091] S2, mineralization of Saccharomyces cerevisiae

[0092] The cell suspension of Saccharomyces cerevisiae obtained in S1 (1×10 9 1 mL of 1% HAuCl4 was added to 1 mL of 1% HAuCl4 to obtain a mixed solution of Saccharomyces cerevisiae cells and HAuCl4, and the volume of the mixed solution was adjusted to 20 mL with PBS; the mixed solution was placed in an incubator and shaken at 37°C and 200 rpm for 24 h. After incubation, it was centrifuged at 5000 g for 10 min, and the precipitate was collected to obtain mineralized Saccharomyces cerevisiae.

[0093] Example 3

[0094] This example provides a study on the photothermal conversion performance of the mineralizing microorganisms of the present application.

[0095] The mineralized microorganisms in this example are the mineralized Saccharomyces boulardii and the mineralized Saccharomyces cerevisiae prepared in Examples 1 and 2.

[0096] (a) Take 1 mL of 1×10 8 Mineralized Saccharomyces boulardii and Saccharomyces cerevisiae cells were prepared by 1 mL of 1×10 8 Saccharomyces boulardii cell solution, 1 mL 1×10 8 1 mL of 0.5 mg / mL chloroauric acid solution, 1 mL of PBS solution, and 1 mL of 1.5 W / cm 2 The 808 nm near-infrared laser was continuously irradiated, and the temperature at 300 seconds was recorded in real time using an infrared thermal imaging camera, and a temperature change graph was drawn. 8 The mineralized Saccharomyces boulardii cell solution was prepared at a concentration of 100 μg / mL and the temperature change of the sample solution was monitored and recorded.

[0097] (b) Using 1.5 W / cm 2 , 808 nm near-infrared laser continuous irradiation 1×10 8The temperature of the sample solution was monitored and recorded using a FLIR E5 infrared thermal imager until the temperature reached a steady state. The laser was turned off and the solution was allowed to cool naturally to room temperature.

[0098] (c) Photothermal cycle experiment: using 1.5 W / cm 2 , 808 nm near-infrared laser continuous irradiation 1×10 8 / mL mineralized Saccharomyces boulardii solution, use FLIR E5 infrared thermal imager to monitor and record the temperature change of the sample solution until the temperature rises to a steady state, turn off the laser, wait for it to cool naturally to room temperature, and then turn on the laser for irradiation again. This cycle is repeated for 4 times. During the whole process, the FLIRE5 infrared thermal imager is used to monitor and record the temperature change of the sample solution.

[0099] (d) Photothermal performance characterization of different concentrations of mineralized Saccharomyces boulardii solutions: 2.5×10 7 , 5×10 7 , 7.5×10 7 , 1×10 8 1 mL of mineralized Saccharomyces boulardii solution was taken from each group of samples and 1.5 W / cm 2 , 808 nm near-infrared laser was continuously irradiated for 10 min. During the irradiation process, the temperature changes of the sample solution were monitored and recorded using a FLIRE5 infrared thermal imager.

[0100] (e) Characterization of photothermal properties of the solution irradiated with different laser powers:

[0101] Use 0.75 W / cm 2 , 1 W / cm 2 , 1.25 W / cm 2 , 1.5 W / cm 2 808 nm near-infrared laser continuous irradiation 1×10 8 / mL mineralized Saccharomyces boulardii solution, and the temperature changes of the sample solution were monitored and recorded using a FLIR E5 infrared thermal imager.

[0102] Result analysis:

[0103] (a) Temperature changes of each sample under near-infrared laser irradiation. Figure 4 As shown in the figure, the mineralized Saccharomyces boulardii and Saccharomyces cerevisiae solutions can be heated up to nearly 30°C after 5 minutes, while the temperature rise of other solutions is less than 10°C, indicating that the mineralized Saccharomyces boulardii and Saccharomyces cerevisiae have excellent photothermal conversion capabilities. Figure 5As shown, after 5 minutes of near-infrared laser irradiation, the temperature of the mineralized Saccharomyces boulardii solution was able to rise to 59°C, indicating that the mineralized Saccharomyces boulardii has excellent photothermal conversion ability.

[0104] (b) The temperature of the mineralized Saccharomyces boulardii solution rose to a steady state under near-infrared laser irradiation and then naturally cooled to room temperature. Figure 6 As shown, mineralized Saccharomyces boulardii at 808 nm, 1.5 W / cm 2 Under the irradiation of near-infrared laser with high power density, the temperature reached a stable state after heating for 10 min and returned to room temperature after cooling for 10 min, indicating that the photothermal conversion efficiency of the mineralized Saccharomyces boulardii solution was high and controllable.

[0105] (c) The results of four cycles of mineralized Saccharomyces boulardii solution under near-infrared laser irradiation. Figure 7 As shown, the temperature curves during the four cycles were almost consistent, indicating that the mineralized Saccharomyces boulardii solution had good thermal stability.

[0106] (d) Photothermal performance characterization results of mineralized Saccharomyces boulardii solutions with different concentrations. Figure 8 As shown, the higher the concentration of the mineralized Saccharomyces boulardii solution, the faster the temperature rises under near-infrared laser irradiation, indicating that the mineralized Saccharomyces boulardii solution has concentration dependence.

[0107] (e) Effect of different laser power densities on photothermal heating of mineralized Saccharomyces boulardii solution. Fig. 9 As shown, the temperature of the mineralized Saccharomyces boulardii solution rises more under laser irradiation with higher power density, indicating that the heat generation capacity of the mineralized Saccharomyces boulardii solution is dependent on the laser power density.

[0108] The above results show that the mineralizing microorganisms of the present application have stable and controllable photothermal conversion capabilities.

[0109] Comparative Example 1

[0110] This comparative example provides a study on the photothermal conversion performance of mineralized Saccharomyces boulardii obtained after co-incubation of Saccharomyces boulardii with different metal ion solutions.

[0111] The preparation of mineralized Saccharomyces boulardii in this comparative example refers to Example 1, except that the Saccharomyces boulardii cell suspension is mixed with 0.5 mg / mL manganese chloride (manganese ion) solution and 0.5 mg / mL potassium aluminum sulfate (aluminum ion) solution, respectively, and the volume of the mixed solution is adjusted to 20 mL with PBS. After co-culturing for 24 h, mineralized Saccharomyces boulardii is obtained.

[0112] Take 1 mL of 1×10 8 / mL of mineralized Saccharomyces boulardii cell solution and 1 mL of PBS solution as control. 2 The sample solution was continuously irradiated with 808 nm near-infrared laser, and the temperature change of the sample solution was monitored and recorded with a FLIRE5 infrared thermal imager.

[0113] Result analysis:

[0114] Temperature changes of the mineralized Saccharomyces boulardii solution obtained by mineralization with different metal ions under continuous irradiation of near-infrared laser Fig.10 As shown, only the mineralized Saccharomyces boulardii obtained by co-incubation with gold ions had a photothermal conversion effect under near-infrared light irradiation.

[0115] Example 4

[0116] This example provides a study on the adhesion performance of the mineralized microorganisms of the present application in the mouse intestine.

[0117] The mineralized microorganism in this example is the mineralized Saccharomyces boulardii prepared in Example 1.

[0118] 1 mL of mineralized Saccharomyces boulardii (concentration 1×10 9 Add 60 μL of cy5.5 acetone solution to a PBS solution containing 100 μL of cy5.5 / mL, place in a light-proof Eppendorf tube at room temperature in a metal bath for 4 h, and wash twice with PBS;

[0119] C57BL / 6 male mice of similar physiological status and body weight were selected for 6 to 8 weeks. Each mouse was gavaged with 0.1 mL of the stained mineralized Saccharomyces boulardii solution (the effective concentration of mineralized Saccharomyces boulardii was 1×10 8 One mouse was killed by cervical dislocation at 0 h, 6 h, 12 h, and 24 h, and the entire intestinal segment was obtained for small animal imaging analysis.

[0120] Result analysis:

[0121] Small animal imaging results of the mouse abdominal cavity and intestines are as follows Fig.11 As shown in the figure, after oral administration, the mineralized Saccharomyces boulardii could detect fluorescence in the intestines at 6, 12 and 24 hours, indicating that the mineralized Saccharomyces boulardii could adhere to and stay in the intestines of mice for about 24 hours. Only some residual fluorescence could be detected at 24 hours, which also showed that after 24 hours, the mineralized Saccharomyces boulardii was basically eliminated from the body and did not colonize in the body for a long time, and had good safety.

[0122] Example 5

[0123] This example provides research on the application of the mineralized microorganisms of the present application in the treatment of obesity and / or type 2 diabetes.

[0124] The mineralized microorganism in this example is the mineralized Saccharomyces boulardii prepared in Example 1.

[0125] Research on the application of mineralized microorganisms in the treatment of obesity and / or type 2 diabetes, including:

[0126] (1) Group treatment

[0127] Mouse husbandry: 6-8 week old healthy male C57BL / 6 mice (from Nanjing Cavens Biotechnology Co., Ltd.) were housed in conventional and pathogen-free facilities at the School of Medicine of Nanjing University. All mice were SPF grade and housed at a temperature of 23±2°C and a humidity of 55±5°C. A 12-hour light / dark cycle was adopted to ensure that the mice had normal drinking water. They were fed with a high-fat diet with 60% fat function (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.), and mice with similar physiological state and body weight (HFD Mice) at 6 weeks of age were selected and divided into groups for the following treatments for two weeks. The groups and treatments were as follows:

[0128] Con: control group, intragastrically administered with the same volume of PBS;

[0129] Boularii: 1 × 10 8 Saccharomyces boulardii (PBS) for two weeks;

[0130] Funsome: 1×10 were given to mice orally every day 8 mineralized Saccharomyces boulardii for two weeks;

[0131] NIR: 808 nm 1.5 W / cm2 for daily use 2 The laser was evenly irradiated on the abdomen of the mouse (the mouse abdomen was to be hairless) for 5 minutes for two weeks;

[0132] Boularii+NIR: mice were gavaged with 1×10 8 Saccharomyces boulardii. Within 0 to 4 hours after oral gavage, mice were anesthetized with isoflurane and illuminated with 808 nm 1.5 W / cm 2 The laser was evenly irradiated on the abdomen of the mouse (the mouse abdomen was to be hairless) for 5 minutes for two weeks;

[0133] Funsome+NIR: mice were gavaged with 1×10 8 Mineralized Saccharomyces boulardii. Mice were anesthetized with isoflurane within 0-4 hours after oral gavage and illuminated with 808 nm 1.5 W / cm 2 The laser was evenly irradiated on the abdomen of the mouse (the mouse abdomen needed to be hairless) for 5 minutes for two weeks. After two weeks of treatment, the temperature of the back and abdomen of the mouse was observed with an infrared thermal imager.

[0134] (2) PET-CT

[0135] Mice were taken for PET-CT examination after two weeks of treatment. Before PET-CT scanning, mice fasted for 8 h, and then injected 18F-FDG 3.7MBq (100 μCi) through the tail vein. During the absorption of 18F-FDG (fluorodeoxyglucose), they were placed in an anesthesia induction box filled with 2% isoflurane. Micro-PET / CT scanning was performed about 60 min later: CT scanning for 5 min and PET scanning for 10 min. The scanning process was always in an anesthesia state. By observing the distribution of labeled glucose, the changes in the ability of white fat and brown fat to absorb glucose and the changes in metabolic levels were observed.

[0136] (3) Glucose tolerance test (GTT)

[0137] After two weeks of treatment, mice were subjected to glucose tolerance test (GTT). Mice were fasted for 12 h before the test, and their body weight was measured after fasting. Blood was collected from the tail vein to measure fasting blood glucose. 10% glucose solution (1 g / kg) was injected intraperitoneally, and blood was collected from the tail vein at 30 minutes, 60 minutes, 90 minutes, and 120 minutes for blood glucose measurement.

[0138] (4) Insulin tolerance test (ITT)

[0139] Two weeks after treatment, mice were subjected to insulin tolerance test (ITT). After the weight of mice was measured, blood was collected from the tail vein to measure blood glucose, and 0.5 U / kg insulin solution was injected intraperitoneally. Blood was collected from the tail vein at 30 minutes, 60 minutes, 90 minutes and 120 minutes for blood glucose measurement.

[0140] (5) ALT (alanine aminotransferase), AST (aspartate aminotransferase), and BUN (blood urea nitrogen) were measured in each group of mice to detect toxic side effects.

[0141] Result analysis:

[0142] (1) After Funsome+NIR treatment, the thermal imaging of mice is shown below Fig.12 As shown, the temperatures in the back and abdominal areas of mice treated with Funsome+NIR were significantly higher than those of the control group (Con) that was gavaged with the same volume of PBS after treatment, indicating that photothermal therapy with mineralized Saccharomyces boulardii can enhance the efficiency of photothermal conversion.

[0143] (2) After Funsome+NIR treatment, the PET-CT results of mice were as follows Fig.13As shown in the figure, the parts indicated by the arrows represent iWAT (Inguinal White Adipose Tissue, subcutaneous fat), eWAT (Epididymal White Adipose Tissue, visceral fat), and BAT (Brown Adipose Tissue, brown fat), respectively, showing the ability of fat to absorb glucose for fat browning and heat production. The higher the fluorescence intensity, the more glucose is absorbed and the stronger the ability of browning and heat production. The fluorescence intensity of the three fat parts of mice after Funsome+NIR treatment was stronger than that of control mice. This shows that the heat production capacity of BAT, iWAT, and eWAT of mice treated with Funsome+NIR is significantly enhanced, the glucose uptake is increased, and the metabolic capacity is enhanced.

[0144] (3) The results of glucose tolerance test (GTT) and insulin tolerance test (ITT) of Funsome+NIR treated mice are as follows Fig.14 As shown, mice gavaged with mineralized Saccharomyces boulardii showed improved impaired glucose tolerance and alleviated insulin resistance after hyperthermia.

[0145] (4) The ALT, AST and BUN test results of each group of mice are as follows Fig.15 As shown, there was no significant change in ALT (alanine aminotransferase), AST (aspartate aminotransferase) and BUN (blood urea nitrogen) of mice gavaged with mineralized Saccharomyces boulardii, indicating that gavage with mineralized Saccharomyces boulardii would not cause hepatotoxicity and nephrotoxicity.

Claims

1. Use of a mineralized microorganism in the preparation of a drug for treating obesity and / or type 2 diabetes, characterized in that: The mineralized microorganism is a microorganism containing photothermal effect materials in its cells and / or on its cell surface, the microorganism is a microorganism that can adhere to the intestine, and the microorganism is Saccharomyces boulardii; the mineralized microorganism adheres to the intestine through oral administration and is used for local thermal therapy of visceral fat to improve obesity and / or type 2 diabetes; the photothermal effect material is nanogold; the preparation method of the mineralized microorganism includes incubating the microorganism with gold ions, the microorganism absorbs and / or adsorbs the gold ions, and uses bioactive molecules to self-assemble into nanogold particles in a biosynthetic manner, and the nanogold particles are adsorbed on the surface of microbial cells and / or contained in the cells.

2. Use of a mineralized microorganism in the preparation of a drug for treating obesity and / or type 2 diabetes, characterized in that: The mineralized microorganism is a microorganism containing photothermal effect materials in its cells and / or on its cell surface, the microorganism is a microorganism that can adhere to the intestine, and the microorganism is brewer's yeast; the mineralized microorganism adheres to the intestine through oral administration and is used for local thermal therapy of visceral fat to improve obesity and / or type 2 diabetes; the photothermal effect material is nanogold; the preparation method of the mineralized microorganism includes incubating the microorganism with gold ions, the microorganism absorbs and / or adsorbs the gold ions, and uses bioactive molecules to self-assemble into nanogold particles in a biosynthetic manner, and the nanogold particles are adsorbed on the surface of microbial cells and / or contained in the cells.

3. The use according to claim 1 or 2, characterized in that: The microorganisms adhere to the intestine for 24 hours.

4. The use according to claim 3, characterized in that: The preparation method comprises: The microorganisms and the acid or salt containing gold are mixed in a solution, the mixed solution is shaken and incubated, and after incubation, the solution is centrifuged to collect and obtain the mineralized microorganisms.

5. The use according to claim 4, characterized in that: The gold-containing acid or salt includes one or more of chloroauric acid, sodium gold thiosulfate and gold chloride.

6. The use according to claim 5, characterized in that: In the blended solution, the concentration of chloroauric acid is 0.1-1 mg / mL; and / or In the mixed solution, the concentration of microorganisms is 1×10 7 ~1×10 8 / mL; and / or The shaking incubation conditions include 37±1°C, 200±50 rpm; and / or The incubation time is 18 to 48 hours; and / or The post-incubation centrifugation includes centrifugation at 5000±50 g for 10±2 min.

7. The use according to claim 6, characterized in that: In the blended solution, the concentration of chloroauric acid is 0.5 mg / mL; In the mixed solution, the concentration of microorganisms is 5×10 7 Pieces / mL; The incubation time is 24 h.

8. The use according to claim 1 or 2, characterized in that: The dosage form of the drug is an oral preparation.

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