A liver-protecting product containing monascus ruber ceswgf001

Abstract

The application discloses a liver-protecting product containing Monascus purpureus CESWGF001, wherein the Monascus purpureus strain is named as Monascus purpureus CESWGF001, has been preserved in the China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M 20231512; and the efficacy experiment result shows that the strain has obvious effects of degrading alcohol and protecting liver, and can be used for preparing liver-protecting products such as liver-protecting drugs and liver-protecting probiotic preparations.

Description

[0001] The present application is a divisional application, the parent application of which has an application date of October 31, 2023, an application number of 202311431255.9, and an invention patent name of "Monascus purpureus CEWL18 and its application in preparing liver-protecting products". TECHNICAL FIELD

[0002] The present application belongs to the field of microorganisms, and more specifically relates to a liver-protecting product containing Monascus purpureus CESWGF001. BACKGROUND

[0003] Monascus is an important microorganism for producing pigments and has been widely used worldwide. It contains multiple species, among which, Monascus purpureus is included in the 8 important Monascus species officially collected and cataloged by the Institute of Microbiology, Chinese Academy of Sciences and the Food Institute of the Light Industry Department. Monascus purpureus is a saprophytic fungus that has attracted widespread attention due to its ability to produce large amounts of natural red pigment. It is commonly found in environments such as trees, soil, and accumulations, and has been listed as one of the top ten health food fungus species.

[0004] Among them, due to the application value of Monascus pigment being much higher than that of other pigments, most existing researches focus on the improvement of Monascus purpureus species to further improve the yield of Monascus pigment. For example, patent CN 108641968 A "Space Monascus purpureus FuH-23-4 screening and its application in producing Monascus pigment" uses space mutagenesis to screen a space Monascus purpureus strain with higher Monascus pigment production capacity. In recent years, it has been found that the secondary metabolites lovastatin and its derivatives produced by Monascus fermentation have the effects of reducing cholesterol and lowering blood pressure, and have attracted widespread attention. However, there is still a need to explore more functional strains of Monascus purpureus. SUMMARY

[0005] In view of the above defects or improvement needs of the prior art, the present application provides a Monascus purpureus CEWL18 and its application in preparing liver-protecting products, which aims to discover a new strain of Monascus purpureus (Monascus purpureus CEWL18). It has been preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 20231512. Experimental results show that the acetaldehyde dehydrogenase activity of the Monascus purpureus CEWL18 strain is higher than 4 U / mL and can significantly improve chemical liver injury, with good alcohol metabolism and liver protection effects. It can be used for preparing liver-protecting products, thereby solving the technical problems of the existing Monascus purpureus having single function and narrow application range.

[0006] To achieve the above object, according to one aspect of the present application, there is provided a purple Monascus strain CEWL18, which is named Monascus purpureus CESWGF001, has been preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO: M 20231512.

[0007] Preferably, the purple Monascus strain CEWL18 has an acetaldehyde dehydrogenase activity of more than 4 U / mL.

[0008] According to another aspect of the present application, there is also provided an application of the purple Monascus strain CEWL18 in the preparation of a liver-protecting product.

[0009] Preferably, the application of the purple Monascus strain CEWL18 in the preparation of a liver-protecting product is applied to the preparation of a medicine for preventing or improving liver damage.

[0010] Preferably, the application of the purple Monascus strain CEWL18 in the preparation of a liver-protecting product is applied to the preparation of a medicine for preventing alcoholic liver damage.

[0011] Preferably, the application of the purple Monascus strain CEWL18 in the preparation of a liver-protecting product is applied to the preparation of a medicine for improving chemical liver damage.

[0012] In addition, the present application also provides a medicine for preventing or improving liver damage, which comprises the purple Monascus strain CEWL18 as an effective component.

[0013] Preferably, the medicine for preventing or improving liver damage has a viable count of the purple Monascus strain CEWL18 of more than 3 x 10 7 CFU / mL.

[0014] Overall, compared with the prior art, the above technical solutions conceived by the present application can achieve the following beneficial effects due to the discovery that the purple Monascus strain CEWL18 has a significant liver-protecting effect:

[0015] The purple Monascus strain CEWL18 provided by the present application can reduce the activities of ALT and AST after being verified by a liver damage model, and has a better effect on reducing the activities of transaminases than the existing liver-protecting medicine bifendate; HE staining shows that the damaged liver cells are significantly reduced, and the improvement effect on liver damage caused by carbon tetrachloride is equivalent to that of the existing liver-protecting medicine bifendate, and the strain can be used for preparing a liver-protecting product. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is an ALT activity detection result;

[0017] Figure 2 AST activity detection results are as follows:

[0018] Figure 3 HE staining results are as follows. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely intended to explain the present application and not to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0020] The present application screens Monascus purpureus from wine starter, and unexpectedly discovers that a strain CEWL18 has a high acetaldehyde dehydrogenase activity of 4.18 U / mL, which has obvious effects of degrading alcohol and protecting liver. The strain is identified as Monascus purpureus CESWGF001, has been preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20231512.

[0021] The present application provides a Monascus purpureus strain CEWL18, which has been preserved in China Center for Type Culture Collection in Wuhan on August 21, 2023, and the preservation number is CCTCC NO: M 20231512, and is named Monascus purpureus CESWGF001.

[0022] The Monascus purpureus strain CEWL18 has an acetaldehyde dehydrogenase activity of more than 4 U / mL.

[0023] Liver damage is caused by damage to liver cells due to various reasons, including viral hepatitis, fatty liver disease, alcoholic liver disease, drug-induced liver damage, and autoimmune liver disease, which further affects a series of functions of the liver such as digestion, detoxification, metabolism, synthesis, etc., and among them, alcohol abuse easily leads to alcoholic liver damage.

[0024] Alcohol is catalyzed by ethanol dehydrogenase (ADH) on the gastric mucosa to oxidize ethanol to acetaldehyde, and acetaldehyde is converted to acetic acid by acetaldehyde dehydrogenase (ALDH). Therefore, accelerating the oxidative metabolism of ethanol in the stomach is beneficial to reducing the bioavailability of ethanol in the body, and can reduce the toxic effects of alcohol on the liver. The Monascus purpureus strain CEWL18 provided by the present application has a high acetaldehyde dehydrogenase activity of up to 4.18 U / mL, which can promote the oxidative metabolism of ethanol in the stomach, is beneficial to reducing the damage of alcohol to the liver, and can be used for preventing alcoholic liver damage.

[0025] In addition, the application also provides an application of the Monascus purpureus strain CEWL18 in preparation of a liver-protecting product.

[0026] The application is applied to preparation of a medicine for preventing or improving liver injury.

[0027] Preferably, the application is applied to preparation of a medicine for preventing alcoholic liver injury.

[0028] Preferably, the application is applied to preparation of a medicine for improving chemical liver injury.

[0029] A medicine for preventing or improving liver injury, wherein an effective component of the medicine comprises the Monascus purpureus strain CEWL18.

[0030] The medicine, wherein a viable count of the Monascus purpureus strain CEWL18 is greater than or equal to 3x10 7 CFU / mL.

[0031] In some embodiments, the content of the Monascus purpureus strain CEWL18 is 10 9 CFU / mL.

[0032] The following is an example:

[0033] Example 1: Isolation of the Monascus purpureus strain CEWL18

[0034] Sample: distiller's yeast, the distiller's yeast used in the brewing of a certain distillery is used to isolate the strain, which indicates that the safety and genetic stability of the strain in the distiller's yeast are verified in practice, and it is ensured that the screened strain can be directly used in other fields.

[0035] Potato medium: potato 200 g, glucose 20 g, pure water 1 L, and natural pH.

[0036] Specific implementation steps:

[0037] a Diluted sample: 2 g of the crushed distiller's yeast is weighed and mixed in 10 ml of phosphate buffer solution, and then diluted to obtain a diluted solution, which is coated on a potato medium plate, and cultured anaerobically at 30°C for 5 days.

[0038] b Single colony purification: single colonies are selected according to colony morphology, color, and size, and are streaked on a potato medium plate, and the edge mycelium of the single colonies is selected for purification multiple times, and then the pure bacteria obtained are scraped for 16s identification and preservation, and the strain is identified as Monascus purpureus, and the strain is named Monascus purpureus CESWGF001.

[0039] On August 21, 2023, the above-mentioned new Monascus purpureus strain isolated was sent to the China Center for Type Culture Collection (Wuhan University Preservation Center) for preservation, and the preservation number was CCTCC NO: M 20231512.

[0040] Example 2 Effect of Monascus purpureus CEWL18 strain on degradation of alcohol

[0041] (1) Degradation of ethanol

[0042] The Monascus purpureus CEWL18 strain was taken out and inoculated in a liquid medium incubator at 37°C for 24 h at an inoculation amount of 10% to obtain a 1st generation bacterial suspension. The 1st generation bacterial suspension was inoculated in MRS broth at an inoculation amount of 10% and incubated at 37°C for 18 h to obtain a 2nd generation bacterial suspension. The activated strain was inoculated in 10%, 15%, and 24% ethanol culture liquid at an inoculation amount of 5% and mixed uniformly, and then incubated at 37°C for 24 h under anaerobic conditions. MRS ethanol culture liquid without the strain was used as a control. The residual amount of ethanol was determined by the potassium dichromate-sulfuric acid method to analyze the ethanol degradation capacity of the strain, and the results are shown below.

[0043] The degradation rates of the Monascus purpureus CEWL18 strain on 10% ethanol, 15% ethanol, and 24% ethanol were 32.44%, 14.63%, and 5.9%, respectively. It can be seen that the strain has good alcohol degradation effect and is beneficial to the prevention of alcoholic liver damage.

[0044] (2) ADH and ALDH activity determination

[0045] The Monascus purpureus CEWL18 strain was activated. The strain was taken out from a -80°C refrigerator and inoculated in a potato solid culture medium incubator at 30°C for 4-7 d. 5 mL of physiological saline was added to the potato culture medium plate, and the spores were washed and collected to prepare a 10 6 CFUm / L spore suspension, which was inoculated in potato liquid culture medium at 30°C at 200 rpm for 48 h.

[0046] The fungus was separated by centrifugation at 4100 rpm for 10 min, and detected by ethanol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) detection kits. The results showed that the ALDH activity in the Monascus purpureus CEWL18 strain was 4.18 U / mL.

[0047] First-pass metabolism (FPM) of ethanol refers to the phenomenon that part of ethanol is oxidatively metabolized in the stomach after drinking. The ethanol metabolism process in the body is as follows: ethanol is oxidized to acetaldehyde by ethanol dehydrogenase (ADH) on the gastric mucosa, and acetaldehyde is converted to acetic acid by acetaldehyde dehydrogenase (ALDH). Therefore, accelerating the oxidative metabolism of ethanol in the stomach is beneficial to reducing the bioavailability of ethanol in the body and can reduce the toxic effects of alcohol on the liver, brain, and other organs.

[0048] The purple red koji mold CEWL18 provided by the application can produce ALDH and has high ALDH activity, has good alcohol metabolism effect, can promote the oxidation metabolism of the body to ethanol, is beneficial to the protection of organs such as liver and brain, and can be used for preventing alcoholic liver injury.

[0049] Example 3: Hepatoprotective effect of the purple red koji mold CEWL18 strain

[0050] Experimental materials:

[0051] Mice: C57B male mice, 8 weeks old, weighing 20-25 g;

[0052] Strain: Monascus purpureus CEWL18.

[0053] Experimental procedure:

[0054] (1) Establishing a mouse liver injury model and grouping

[0055] Thirty-two healthy C57B male mice were selected, and the mice were adaptively fed for one week before the experiment, so that the mice were familiar with the environment and recovered the original rhythm of the mice. According to the initial body weight, the mice were randomly divided into four groups, namely a normal control group, a liver injury group (CCl4 group), a liver injury and bifendate administration group (CCl4+bifendate), and a liver injury and probiotic administration group (CCl4+CEWL18). The specific treatment of each group of mice is as follows:

[0056] ① Normal control group: free drinking water, the mice were administered with normal saline for 1-12 days, 3 times a week, and the mice were injected intraperitoneally with olive oil for 12 days.

[0057] ② Liver injury model group: free drinking water, the mice were administered with normal saline for 1-12 days, 3 times a week, and the mice were injected intraperitoneally with 10% carbon tetrachloride (CCl4) for 12 days.

[0058] ③ Positive control group (CCl4+bifendate): free drinking water, the mice were administered with bifendate (2.9 mg / kg) for 1-12 days, 3 times a week, and the mice were injected intraperitoneally with 10% carbon tetrachloride (CCl4) for 12 days.

[0059] ④ Strain experimental group (CCl4+CEWL18): free drinking water, the mice were administered with strain spore suspension (3×10 7 CFU / kg) for 1-12 days, 3 times a week, and the mice were injected intraperitoneally with 10% carbon tetrachloride (CCl4) for 12 days. According to the dose ratio of mice to adults being 10:1, in this example, it is equivalent to an adult taking 3×10 6 CFU / kg of CEWL18.

[0060] Carbon tetrachloride and olive oil were mixed in a ratio of 1:9 to prepare a 10% carbon tetrachloride solution, which was administered to the mice by intraperitoneal injection at a dose of 5 mL / kg. The normal control group was injected with olive oil at the same dose.

[0061] (2) Sampling detection

[0062] The mice were fasted for 24 hours after gavage, and the serum of the mice was collected, and the mice were dissected, and the liver was taken for detection, as follows:

[0063] (2-1) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity detection

[0064] Collect mouse serum samples: after the administration time ends, fast the mice for 24 hours, and then take the eyeball blood of each group of mice, respectively, into a clean EP tube with a label, and place it at 4°C for detection of serum aminotransferase (ALT and AST);

[0065] Sample detection: take out the above-mentioned mouse eyeball blood; centrifuge at 4°C, 3000 rpm for 30 min in a low-temperature high-speed centrifuge; after centrifugation, take the supernatant into a new 1.5 ml EP, and use the aspartate aminotransferase (glutamic oxalate transaminase / AST / GOT) test box and alanine aminotransferase (glutathione transaminase / ALT / GPT) test box purchased from Nanjing Jiancheng Biological Engineering Institute for detection, and the results are shown in Figure 1 and Figure 2 , wherein Figure 1 is the ALT activity detection result, Figure 2 is the AST activity detection result.

[0066] Alanine aminotransferase mainly exists in hepatocytes, and aspartate aminotransferase mainly exists in cardiac cells, and the content of alanine aminotransferase in hepatocytes is 100 times that in blood. Under normal circumstances, the content of ALT and AST in serum is very low, and the normal value is in the range of 0-40 U / L. Generally, the simultaneous elevation of these two enzymes is seen in liver damage, and when 1% of hepatocytes are damaged, alanine aminotransferase can be doubled. At present, alanine aminotransferase is the most sensitive indicator of liver damage. In clinical practice, alanine aminotransferase and aspartate aminotransferase are collectively referred to as aminotransferase, which is used to evaluate liver damage and function.

[0067] from Figure 1 and Figure 2It can be seen that the ALT activity and AST activity of the normal control group mice are lower than 20 U / L, while the ALT activity and AST activity of the liver injury model group mice are more than 40 U / L, compared with the normal control group mice, the ALT activity and AST activity of the liver injury model mice are significantly increased, and are more than 40 U / L, which indicates that the liver injury model is successfully established; while the mice are given bifendate or CEWL18 strain by gavage, the ALT activity and AST activity of the liver injury model mice decrease significantly, and the ALT activity and AST activity of the mice given CEWL18 strain by gavage decrease to a lesser extent, which indicates that compared with the existing liver-protecting drug bifendate, the liver-protecting effect of CEWL18 strain is better.

[0068] (2-2) Detection of liver tissue damage by HE staining

[0069] Collection of liver tissue samples: after the end of the administration time, the mice were sacrificed by cervical dislocation, the abdominal cavity was quickly and carefully opened, the removed liver tissue was carefully washed clean with normal saline, and a small part of the liver tissue about 2.0 cm x 2.0 cm x 1 cm was cut into a liver tissue block and immersed in formalin solution for HE staining. After the operation, the mouse carcasses were uniformly recovered and disposed of.

[0070] Sample detection: HE staining was used to detect liver tissue damage, and the experimental method was briefly as follows:

[0071] (1) The aforementioned liver tissue samples were prepared into paraffin sections (the material should be small and thin, so that the fixative can quickly penetrate the interior. The thickness is not more than 2 mm, and the size is not more than 5 x 5 mm2);

[0072] (2) The paraffin sections were immersed in xylene for 5-10 min for deparaffinization;

[0073] (3) The paraffin sections were immersed in new xylene again for 5-10 min for deparaffinization;

[0074] (4) The sections were sequentially immersed in anhydrous ethanol for 5 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and finally distilled water for 2 min;

[0075] (5) The sections were stained in hematoxylin staining solution for 5-10 min (the staining time is determined according to the staining results and requirements);

[0076] (6) The sections were immersed in tap water to remove excess staining solution, for about 10 min;

[0077] (7) The sections were immersed in new distilled water again and washed again (for a few seconds); the sections were stained in eosin staining solution for 30 seconds-2 minutes (the time is determined according to the staining results and requirements);

[0078] (8) Wash the slices twice in 70% ethanol;

[0079] (9) Then, place the slices in 70% ethanol for 10 seconds, 80% ethanol for 10 seconds, 90% ethanol for 10 seconds, and anhydrous ethanol for 10 seconds, and finally clear them with xylene for 5 minutes (the concentration of anhydrous ethanol is increased from low to high in order to avoid strong tissue shrinkage caused by violent diffusion).

[0080] (10) Put the slice back into fresh xylene and let it clear for 5 minutes.

[0081] (11) Place the slides in a ventilated area and mount them with neutral resin;

[0082] (12) Observe under an inverted microscope. The cell nucleus appears blue, while the cytoplasm appears pink or red. Take pictures, save, and analyze the data; the results are as follows: Figure 3 As shown.

[0083] Depend on Figure 3 It can be seen that, compared with the normal control group, the liver tissue of mice in the liver injury model group was significantly damaged. However, when the existing hepatoprotective drug biphenyl diester and the CEWL18 strain were administered by gavage, the liver tissue damage of the mice in the liver injury model group was significantly restored. This indicates that the CEWL18 strain has a significant hepatoprotective effect, and its effect is comparable to or even better than that of the existing hepatoprotective drug biphenyl diester. It can be used to prepare hepatoprotective drugs or probiotic preparations.

[0084] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A medicament for preventing or ameliorating liver injury, characterized by comprising a sirtuin 3-activating substance. The effective component includes Monascus purpureus CESWGF001, which has been preserved in China Typical Culture Collection Center, and the preservation number is CCTCC NO: M 20231512; the acetaldehyde dehydrogenase activity of the Monascus purpureus CESWGF001 strain is higher than 4 U / mL; the medicine is a medicine for preventing or improving alcoholic liver injury and chemical liver injury.

2. The medicament for preventing or improving liver injury according to Claim 1, wherein The Monascus purpureus CESWGF001 viable cell number is above 3 x 10 7 CFU / mL.

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