Taming of mucor circinelloides and its application

Abstract

The application discloses a domesticated total Mucor and application thereof. The application provides the domesticated total Mucor, which is preserved in the China General Microbiological Culture Collection Center on September 19, 2022, and the preservation number is CGMCC NO. 40320. The application research finds that the domesticated total Mucor can grow in an anaerobic environment at 36-38 DEG C, can be used for preventing and / or treating radioactive diseases, expands the application field of the total Mucor, and provides theoretical guidance for clinical application. Experimental data show that the domesticated total Mucor can improve the inflammation level, integrity and function of the colon and small intestine of a mouse with intestinal type radiation injury.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a domesticated Mucor racemosus and application thereof. BACKGROUND

[0002] Mucor racemosus has a rich protease system and is important in the field of traditional fermented food. The optimal growth temperature of Mucor racemosus is 23℃ and it is aerobic, and it cannot grow in an anaerobic environment at 37℃.

[0003] Reference document 1 discloses a high-temperature-resistant Mucor racemosus isolated from Mucor bean douchi koji produced in Sichuan, named M-THF-02, and the accession number is CCTCC NO: M 2020552; the optimal growth temperature of the strain is 28-30℃.

[0004] At present, Mucor racemosus is mainly used in the production of Mucor bean douchi. For example, reference document 2 discloses a Mucor racemosus M-THF-03 obtained by mutagenesis and domestication of the Mucor racemosus M-THF-02 provided in reference document 1, and the optimal growth temperature of the Mucor racemosus M-THF-03 is 28-30℃. The Mucor racemosus M-THF-03 is applied to disc koji making, which can significantly reduce the moisture of the material and improve the enzyme activity of the koji.

[0005] Reference document 3 discloses another use of Mucor racemosus. It is found that the Mucor racemosus containing β-glucosidase obtained by fermentation and crushing can hydrolyze soybean isoflavones to produce free aglycone under suitable temperature and pH conditions.

[0006] Since the culture of Mucor racemosus in the prior art can only be carried out at a temperature below 30℃, the application environment is also concentrated under this condition, and the function is also limited to fermentation, which greatly limits the exploration and development of new functions of Mucor racemosus.

[0007] Reference document 1: CN 112226372 A

[0008] Reference document 2: CN 114276933 A

[0009] Reference document 3: CN 103923956 A SUMMARY

[0010] Problem to be solved by the invention

[0011] At present, the culture conditions of Mucor racemosus provided in the prior art are limited to aerobic environment below 30 DEG C, and the culture at higher temperature and / or anaerobic environment cannot be carried out, which limits the functional development of Mucor racemosus. In view of the above technical problems, the present application provides a domesticated Mucor racemosus which can be cultured and grown in vitro to simulate the intestinal environment in vivo, and further provides the application thereof in repairing radiation intestinal injury.

[0012] Solution for solving the problem

[0013] The first aspect of the present application provides a domesticated Mucor racemosus, wherein the domesticated Mucor racemosus is deposited with the China General Microbiological Culture Collection Center on September 19, 2022, and the deposit number is CGMCC NO. 40320.

[0014] Further, the growth conditions of the domesticated Mucor racemosus are 36-38 DEG C and anaerobic environment. This makes it possible to realize in vitro culture close to the intestinal environment in vivo, and provides help for expanding the new functions and uses of Mucor racemosus.

[0015] The second aspect of the present application provides the use of the domesticated Mucor racemosus in the first aspect in the preparation of a medicament for preventing and / or treating a radiation disease.

[0016] Further, the radiation disease is caused by ionizing radiation, and the ionizing radiation includes ionizing radiation generated by tumor radiotherapy.

[0017] Further, the radiation disease is caused by gamma rays.

[0018] Further, the radiation disease is intestinal radiation injury.

[0019] Preferably, the intestinal radiation injury has at least one of the following symptoms: shortening of colon length, sparseness of small intestinal villi, shortness of small intestinal villi, decreased expression level of small intestinal integrity marker, and increased level of intestinal inflammation.

[0020] More preferably, the small intestinal integrity marker includes Occludin.

[0021] More preferably, the increased level of inflammation includes increased expression of inflammatory immune factors.

[0022] Further preferably, the inflammatory immune factors include IL-6.

[0023] The domesticated M. vinacea provided by the present application can repair intestinal radiation damage, specifically, can increase the length of the colon, improve the condition of sparse and short small intestinal villi, increase the expression level of the small intestinal integrity marker (Occludin), and reduce the expression level of the inflammatory immune factor IL-6.

[0024] The third aspect of the present application provides a domestication method of M. vinacea, the method comprising the step of culturing a strain to be domesticated in a culture medium containing human or animal feces at 36-38 DEG C in an anaerobic environment.

[0025] Preferably, the strain to be domesticated comprises M. vinacea with the accession number CGMCC 3.4941.

[0026] More preferably, the strain to be domesticated is M. vinacea with the accession number CGMCC 3.4941. The M. vinacea with the accession number CGMCC 3.4941 selected in the present study is a recognized model strain by the ATCC preservation center, and has complete DNA sequencing data. The model strain is usually the first to be studied in the species, and the microbiological characteristics are the most comprehensive. The model strain has been analyzed and studied in the prior art.

[0027] Preferably, the culture medium is a mixture of potato dextrose agar medium and brain heart infusion broth medium.

[0028] More preferably, the culture medium is a mixture of potato dextrose agar medium and brain heart infusion broth medium with a mass ratio of 1:1.

[0029] Preferably, the culture time is greater than or equal to 5 days.

[0030] Preferably, the human or animal feces is the feces of 6-8 week old C57BL / 6J mice.

[0031] Preferably, during the culture, the culture medium containing human or animal feces needs to be replaced more than once.

[0032] The present application repeats the domestication experiment several times, and can obtain M. vinacea which can significantly prevent and / or treat radiation diseases. And the experimental data shows that the domestication method provided by the present application can significantly improve the curative effect of M. vinacea in treating intestinal radiation damage.

[0033] Effects of the invention

[0034] Compared with the prior art, the present application provides a domesticated M. vinacea which can grow in an anaerobic environment at 36-38 DEG C, breaking through the culture conditions of M. vinacea in the prior art, so that it can be cultured in a simulated in-vitro intestinal environment in-vivo, expanding the functional exploration conditions and application range of M. vinacea.

[0035] Meanwhile, the domesticated M. circinelloides provided by the application can be used for preventing and / or treating radiation diseases, which expands the application field of M. circinelloides and provides theoretical guidance for clinical application. In some embodiments, the domesticated M. circinelloides provided by the application can efficiently and safely repair intestinal damage caused by ionizing radiation (including but not limited to tumor radiotherapy), and experimental data shows that the domesticated M. circinelloides can improve the inflammation level, integrity and function of the colon and small intestine of a mouse with intestinal radiation damage. Moreover, compared with the existing M. circinelloides, the domesticated M. circinelloides provided by the application has more outstanding efficacy in improving intestinal radiation damage.

[0036] The application provides a domestication method of M. circinelloides, which is repeatable and can significantly improve the efficacy of M. circinelloides in treating intestinal radiation damage. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The results of the colon length statistics of the mice in each group of Example 2 after being administered for 14 days.

[0038] Figure 2 The results of the small intestine tissue structure detection of the mice in each group of Example 2 after being administered for 14 days.

[0039] Figure 3 The results of the relative expression amount detection of the small intestine integrity marker (Occludin) of the mice in each group of Example 2 after being administered for 14 days.

[0040] Figure 4 The results of the IL-6 content detection in the small intestine of the mice in each group of Example 2 after being administered for 14 days.

[0041] Figure 5 The colony morphology of M. circinelloides CGMCC 3.4941 and domesticated M. circinelloides CGMCC NO.40320.

[0042] Figure 6 The results of the colon length statistics of the mice in each group of Example 3 after being administered for 14 days.

[0043] Figure 7 The results of the small intestine tissue structure detection of the mice in each group of Example 3 after being administered for 14 days.

[0044] Figure 8 The results of the relative expression amount detection of the small intestine integrity marker (Occludin) of the mice in each group of Example 3 after being administered for 14 days.

[0045] Figure 9 The results of the IL-6 content detection in the small intestine of the mice in each group of Example 3 after being administered for 14 days.

[0046] Figure 10 Differential gene Venn diagram of the domesticated M. furfur and M. furfur with the accession number CGMCC NO. 40320 and the accession number CGMCC 3.4941. DETAILED DESCRIPTION

[0047] Hereinafter, the contents of the present application will be described in detail. The description of the technical features described below is based on representative embodiments, specific examples of the present application, but the present application is not limited to these embodiments, specific examples.

[0048] In the present application, the term "a" or "an" or "the" can mean "one", "one or more", "at least one", and "one or more than one".

[0049] In the present specification, the numerical range represented by "numerical value A ~ numerical value B" means a range including the end point values A, B.

[0050] In the present specification, the numerical range represented by "above" or "below" means a numerical range including the present number.

[0051] In the present specification, the meaning represented by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0052] In the present specification, the "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", and the like mentioned refer to the specific elements (for example, features, structures, properties, and / or characteristics) described in relation to the embodiments are included in at least one embodiment described herein, and can be present in other embodiments or can not be present in other embodiments. In addition, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0053] In the present application, the term "anaerobic" means that an organism or a cell can grow under conditions of lack or absence of molecular oxygen.

[0054] In the present application, the term "radiation disease" is commonly used with "radiation injury", which is a general term for various types and degrees of injury (or disease) of the human body caused by various ionizing radiations.

[0055] In the present application, the term "intestinal type radiation injury" refers to intestinal injury (or disease) caused by the body receiving a large dose (>1 Gy) of ionizing radiation in a short period of time.

[0056] In the present application, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0057] In the present application, "treatment" means that after suffering from a disease, the subject is contacted (e.g., administered) with the domesticated Mucor racemosus CGMCC NO.40320 described in the present application, so as to alleviate the symptoms of the disease compared with not being contacted, and does not mean that it is necessary to completely inhibit the symptoms of the disease. Suffering from a disease means that the body has symptoms of the disease.

[0058] In the present application, "prevention" means that before suffering from a disease, the subject is contacted with the domesticated Mucor racemosus CGMCC NO.40320 described in the present application, so as to reduce the probability of suffering from the disease and / or alleviate the symptoms after suffering from the disease compared with not being contacted, and does not mean that it is necessary to completely inhibit the disease.

[0059] Unless otherwise defined, other technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0060] Biological material preservation information

[0061] Accession No.: CGMCC NO.40320;

[0062] Classification name: Mucor racemosus;

[0063] Date of deposit: September 19, 2022;

[0064] Depository: China General Microbiological Culture Collection Center;

[0065] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No.1, Yitianli, Beijing, China.

[0066] Examples

[0067] The present application is further illustrated by the following examples, but any example or combination thereof should not be understood as limiting the scope or implementation of the present application. The scope of the present application is defined by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in combination with the present specification and general common knowledge in the art. Any modification or change to the technical solutions of the present application made by those skilled in the art without departing from the spirit and scope of the present application is also included in the scope of the present application.

[0068] Unless otherwise indicated, the following conditions were used in the examples: routine conditions or manufacturer's recommendations. Unless otherwise indicated, all reagents or instruments were commercially available and routine products. In order to better illustrate the present application, numerous specific details are set forth in the following detailed description. One skilled in the art will understand, however, that the application can be practiced without certain of the specific details herein. In other instances, well-known methods, apparatus, instruments and steps have not been described in detail in order to avoid obscuring the present application.

[0069] Example 1: Obtaining domesticated Mucor racemosus

[0070] 1. Sterile glass paper was laid on the surface of potato dextrose agar medium PDA (BD, model 213213400, formula: potato powder, glucose and agar);

[0071] 2. Mucor racemosus (preserved number CGMCC 3.4941) was inoculated on the sterile PDA medium with glass paper under sterile conditions, and incubated at 30°C under aerobic conditions for 48-96h, until the medium was covered with Mucor racemosus;

[0072] 3. Fresh feces of healthy 6-8 week old C57BL / 6J mice were taken, and sterile normal saline (0.1g / 1mL) was added in proportion and mixed thoroughly;

[0073] 4. Brain heart infusion broth BHI (Qingdao Haibo, model HB8297-1, formula: proteose peptone, dehydrated calf brain infusion powder, dehydrated beef heart infusion powder, sodium chloride, glucose and disodium hydrogen phosphate) was mixed with PDA medium powder 1:1 to prepare PDA+BHI medium;

[0074] 5. 300μL of the fecal liquid prepared in step 3 was evenly smeared on the PDA+BHI medium;

[0075] 6. The glass paper covered with Mucor racemosus was transferred to the PDA+BHI medium containing fecal liquid, and incubated at 37°C under strict anaerobic conditions for 5 days for domestication, wherein the PDA+BHI medium with fresh fecal liquid was replaced on the third day of domestication;

[0076] 7. The domesticated Mucor racemosus was scraped off from the glass paper, added with sterile normal saline and grinding beads for grinding, and quantified for subsequent functional experiments. The domesticated Mucor racemosus is the domesticated Mucor racemosus;

[0077] The applicant domesticated Mucor racemosus (Mucor racemosus) obtained in this example on September 19, 2022, and preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC NO.40320.

[0078] Through multiple repeated domestication experiments, target strains can be obtained, among which the domesticated Mucor racemosus with the preservation number of CGMCC NO.40320 and three strains of Mucor racemosus with the preservation number of CGMCC 3.4941 are selected for transcriptome sequencing, and the difference gene Wayne diagram is as shown in Figure 10 .

[0079] Example 2: Functional application of domesticated Mucor racemosus in treating intestinal radiation damage

[0080] This example studies the function of the domesticated Mucor racemosus strain obtained from Example 1.

[0081] 1. Experimental animals:

[0082] 6-8 week old male and female C57BL / 6J mice, 18 in each gender, are randomly divided into three groups: blank group, simple irradiation group and domesticated Mucor racemosus group.

[0083] 2. Model establishment:

[0084] Use Exactor to perform γ-ray local irradiation on the whole abdomen of the mouse (40Exactor comes with a local irradiation mold, and the whole abdomen is fully exposed), and the absorbed dose is 13 Gy and the dose rate is 0.8 Gy / min.

[0085] 3. Administration method:

[0086] Domesticated Mucor racemosus group: administration starts on the irradiation day, administration method: domesticated Mucor racemosus with the preservation number of CGMCC NO.40320 is administered intragastrically, 1 time per day at 2 pm, and continuous administration for 14 days; domesticated Mucor racemosus amount: 100 μL per mouse per time; domesticated Mucor racemosus liquid concentration: 100 μL of bacterial liquid OD 600 = 1.

[0087] Simple irradiation group: administration starts on the irradiation day, administration method: normal saline is administered intragastrically, 1 time per day at 2 pm, and continuous administration for 14 days; administration dose: 100 μL per mouse per time.

[0088] Blank group: no irradiation, administration method: normal saline is administered intragastrically, 1 time per day at 2 pm, and continuous administration for 14 days; administration dose: 100 μL per mouse per time.

[0089] 4. Observation index:

[0090] After 14 days of intragastric administration, the mice are sacrificed, and the colon and small intestine tissues of the mice in each group are taken to detect the colon length and the structure, integrity and inflammation level of the small intestine of the mice in each group.

[0091] ​5、Results and Conclusion

[0092] Results as shown in Figures 1-4 compared with the blank group of mice, after the abdominal local irradiation of gamma rays, the colon length of the simple irradiation group of mice was shortened, the small intestinal tissue structure was severely damaged, the villi were sparse and short, the expression of the small intestinal integrity marker (tight junction protein, Occludin) was significantly decreased, and the inflammation level (such as the expression level of IL-6) was significantly increased.

[0093] However, compared with the simple irradiation group of mice, the liquid of the domesticated Mucor mucedo with the accession number CGMCC NO.40320 continuously gavaged for 14 days can significantly improve the inflammation level, integrity and function of the colon and small intestinal tissue of mice, the colon length of the domesticated Mucor mucedo group of mice is lengthened, the small intestinal villi are improved, the expression level of the small intestinal integrity marker (Occludin) is significantly increased, even more than that of the blank group, and the content of the inflammatory immune factor IL-6 in the small intestine is also reduced. It can be seen that the domesticated Mucor mucedo with the accession number CGMCC NO.40320 can repair the intestinal damage caused by ionizing radiation.

[0094] Example 3: Comparative study on the functional application of domesticated Mucor mucedo in rescuing intestinal radiation damage

[0095] This example studies the functions of the domesticated Mucor mucedo strain obtained from Example 1 and the existing Mucor mucedo strain (with the accession number CGMCC 3.4941). Figure 5 The colony morphology of the domesticated Mucor mucedo with the accession number CGMCC NO.40320 and the Mucor mucedo (with the accession number CGMCC 3.4941) is shown.

[0096] 1、Experimental animals:

[0097] 6-8 week old male and female C57BL / 6J mice, 18 in each, were randomly divided into three groups: simple irradiation group, Mucor mucedo group and domesticated Mucor mucedo group.

[0098] 2、Model establishment:

[0099] The whole abdomen of the mice was irradiated with gamma rays using Exactor (the whole abdomen was exposed), the absorbed dose was 13 Gy, and the dose rate was 0.8 Gy / min. Exactor comes with a local irradiation mold, the whole abdomen is exposed), the absorbed dose was 13 Gy, and the dose rate was 0.8 Gy / min.

[0100] 3、Administration method:

[0101] Acclimatization of M. vinacea group: start dosing on the day of irradiation, dosing method: M. vinacea with preservation number CGMCC NO.40320 was administered by gavage, 100 μL / once / time of M. vinacea with preservation number CGMCC NO.40320 was administered by gavage at 2 pm every day for 14 consecutive days; amount of M. vinacea: 100 μL / once / time; concentration of M. vinacea liquid: 100 μL of liquid OD 600 = 1.

[0102] M. vinacea group: start dosing on the day of irradiation, dosing method: M. vinacea with preservation number CGMCC 3.4941 was administered by gavage, 100 μL / once / time of M. vinacea with preservation number CGMCC 3.4941 was administered by gavage at 2 pm every day for 14 consecutive days; amount of M. vinacea: 100 μL / once / time; concentration of M. vinacea liquid: 100 μL of liquid OD 600 = 1.

[0103] Irradiation alone group: start dosing on the day of irradiation, dosing method: normal saline was administered by gavage, 100 μL / once / time of normal saline was administered by gavage at 2 pm every day for 14 consecutive days.

[0104] 4. Observation index:

[0105] After 14 days of gavage, the mice were sacrificed, and the colon and small intestine tissues of the mice in each group were taken to detect the colon length and the structure, integrity, and inflammation level of the small intestine of the mice in each group.

[0106] 5. Results and conclusion

[0107] The results are shown in Table 1. Figures 6-9 Compared with the irradiation alone group, both types of M. vinacea liquid can improve the inflammation level, integrity, and function of the colon and small intestine tissues of the mice to a certain extent after 14 days of continuous gavage.

[0108] At the same time, compared with the M. vinacea group, the increase in colon length, the improvement in small intestine villi, the expression level of the small intestine integrity marker (Occludin), and the decrease in the content of the inflammation immune factor IL-6 in the small intestine of the mice in the acclimatization of M. vinacea group were more significant. It can be seen that, because the growth environment of the acclimatization of M. vinacea (preservation number CGMCC NO.40320) is close to the natural environment of the intestine, as an intestinal resident fungus, it can more effectively repair the intestinal damage caused by ionizing radiation.

Claims

1. A domesticated Mucor racemosus ( Mucor racemosus ), characterized in that, The domesticated Mucor racemosus was deposited with the China General Microbiological Culture Collection Center on September 19, 2022, and the deposit number is CGMCC NO.40320.

2. Use of the domesticated Mucor racemosus according to claim 1 in the preparation of a medicament for preventing and / or treating a radiation disease; the radiation disease is intestinal type radiation injury caused by gamma rays.

3. Use according to claim 2, characterized in that, The intestinal type radiation injury has at least one of the following symptoms: shortening of the colon length, sparseness of small intestinal villi, shortness of small intestinal villi, decreased expression level of small intestinal integrity marker, and increased level of intestinal inflammation. The small intestinal integrity marker is a tight junction protein. The increased level of inflammation is an increase in the expression of inflammatory immune factors, and the inflammatory immune factors are IL-6.

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