Bacillus species strain expressing superoxide dismutase, bacillus species strain spore and / or superoxide dismutase, and use thereof for preventing or treating of fibrotic disease

Abstract

The present invention relates to a composition for the prevention, alleviation, or treatment of fibrotic diseases, containing a Bacillus species strain, a Bacillus species strain spore, each expressing superoxide dismutase, and / or superoxide dismutase. When orally administered, the composition allows the polypeptide with superoxide dismutase activity to remove reactive oxygen species in vivo, thus being helpful for the prevention, improvement, or treatment of fibrotic diseases such as pulmonary fibrosis or hepatic fibrosis.

Description

Bacillus species strain expressing superoxide dismutase, Bacillus species strain spore and / or superoxide dismutase and use thereof for preventing or treating fibrotic diseases

[0001] This application claims priority to Korean Patent Application No. 2022-0111060, filed September 1, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to research (No. 2020R1A2C1008431, 'Exploring a new treatment strategy for COPD, an intractable fatal disease, using microbial symbiosis') conducted with the support of the National Research Foundation of Korea's Individual Basic Research Program funded by the Ministry of Science and ICT of the Republic of Korea in 2022.

[0003] Sequence list

[0004] This application includes a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of said sequence listing, created on September 1, 2023, is named KC23094SEQ.xml and is 64.0 kilobytes in size.

[0005] The present invention relates to a Bacillus species strain expressing superoxide dismutase, a Bacillus species strain spore and / or a composition comprising superoxide dismutase (SOD), and its use for preventing, improving, or treating fibrotic diseases.

[0006] Fibrosis is a disease in which extracellular matrix (ECM), such as collagen, accumulates in tissues, causing structural and functional problems. It can be idiopathic with an unknown cause or caused by various factors. For example, pulmonary fibrosis can be caused by several conditions, including chronic inflammation (e.g., sarcoidosis, Wegener's granulomatosis, etc.), infection, environmental factors (e.g., asbestos, exposure to certain gases, etc.), exposure to ionizing radiation (e.g., radiation therapy for breast cancer), chronic diseases, and side effects of certain medications (e.g., bleomycin, amiodarone, busulfan, methotrexate, and nitrofurantoin, etc.). In addition, idiopathic pulmonary fibrosis with an unknown cause is a disease that typically causes fibrosis in the lungs, and oxidative stress is known to play a significant role in the development of idiopathic pulmonary fibrosis. Patients with idiopathic pulmonary fibrosis have been found to have higher concentrations of reactive oxygen species and reduced antioxidant activity than normal individuals, which can progress to irreversible lung damage and fibrosis. However, the etiology of idiopathic pulmonary fibrosis is not precisely known, and although drugs such as IFN-γ are under development, there is no effective drug yet. As another example, liver fibrosis can be caused by nonalcoholic fatty liver disease (NAFLD) such as nonalcoholic steatohepatitis (NASH), viral hepatitis (HBV, HBC, etc.), alcohol, liver toxic substances (acetaminophen overdose, chemicals such as arsenic, etc.), genetic factors, etc. In particular, NASH is a representative disease that causes liver fibrosis in which fat deposition is accompanied by liver cell damage and inflammation. If liver fibrosis worsens, it can progress to cirrhosis, and if it gets worse, it can develop into liver cancer.There is a need for drugs to treat nonalcoholic steatohepatitis and prevent the progression of the disease, but currently, there are no drugs that effectively improve fibrosis and steatohepatitis.

[0007] Therefore, there is a need to develop drugs that can treat fibrotic diseases, including pulmonary fibrosis, NASH, and liver fibrosis.

[0008] The purpose of the present invention is to solve all of the problems of the above-mentioned prior art.

[0009] The present invention aims to provide a Bacillus species strain, a Bacillus species strain spore and / or a superoxide dismutase (SOD) for the prevention or treatment of fibrotic diseases.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrotic diseases, comprising a Bacillus species strain, a Bacillus species strain spore and / or SOD.

[0011] Another object of the present invention is to provide a food composition for preventing or improving fibrotic diseases, comprising a Bacillus species strain, a Bacillus species strain spore, and / or SOD.

[0012] Another object of the present invention is to provide a method for preventing, improving or treating a fibrotic disease, comprising administering to a subject a Bacillus species strain, a Bacillus species strain spore and / or SOD.

[0013] Another object of the present invention is to provide a use of Bacillus species strains, Bacillus species strain spores and / or superoxide dismutase for preventing, improving or treating fibrotic diseases.

[0014] The purpose of the present invention is not limited to the purposes mentioned above. The purpose of the present invention will become clearer from the following description, and can be realized by the means and combinations thereof described in the claims.

[0015] A representative configuration of the present invention to achieve the above purpose is as follows.

[0016] According to one aspect of the present invention, a composition for treating, improving or preventing a fibrotic disease is provided, comprising as an active ingredient at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore and a polypeptide having superoxide dismutase (SOD) activity.

[0017] In one embodiment, the Bacillus species strain may be a strain that expresses SOD or a strain that has been mutated or recombined to overexpress SOD.

[0018] In another embodiment, the Bacillus species strain spores may comprise spores derived from a strain that expresses SOD or a strain that has been mutated or recombined to overexpress SOD.

[0019] In another embodiment, the Bacillus species strain may be a Bacillus velezensis species strain.

[0020] In another embodiment, the Bacillus species strain may be at least one strain selected from the Bacillus velezensis strain deposited with the accession number KCTC 13222 BP, the Bacillus velezensis strain deposited with the accession number KCTC 13227 BP, and the Bacillus velezensis strain deposited with the accession number KCTC 15552 BP.

[0021] In one embodiment, the polypeptide having SOD activity can be Mn-SOD.

[0022] In another embodiment, the polypeptide having SOD activity can be deamidated Mn-SOD.

[0023] In another embodiment, the polypeptide having SOD activity may be derived from a Bacillus species strain.

[0024] In another embodiment, the strain from which the polypeptide having SOD activity is derived may be a Bacillus velezensis strain.

[0025] In another embodiment, the Bacillus species strain from which the polypeptide having SOD activity is derived may be at least one strain selected from the Bacillus velezensis strain deposited under accession number KCTC 13222 BP, the Bacillus velezensis strain deposited under accession number KCTC 13227 BP, and the Bacillus velezensis strain deposited under accession number KCTC 15552 BP.

[0026] In another embodiment, the polypeptide having SOD activity can comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0027] In one embodiment, the polypeptide having SOD activity may be coated with a coating agent.

[0028] In another embodiment, the coating agent of the polypeptide having SOD activity may comprise shellac.

[0029] In one embodiment, the fibrotic disease may be a pulmonary fibrotic disease or a hepatic fibrotic disease.

[0030] In another embodiment, the pulmonary fibrotic disease may be selected from the group consisting of interstitial lung disease, pulmonary fibrosis, and idiopathic pulmonary fibrosis.

[0031] In another embodiment, the liver fibrosis disease may be selected from the group consisting of nonalcoholic steatohepatitis, hepatic fibrosis, idiopathic hepatic fibrosis, cirrhosis, alcoholic steatohepatitis, chronic liver disease, and viral hepatitis.

[0032] In one embodiment, the active ingredient of the composition can be administered orally.

[0033] In another embodiment, the composition comprises two or more selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and a polypeptide having the SOD activity, wherein the two or more components may be administered simultaneously, sequentially, or in reverse order.

[0034] In one embodiment, the composition may be a pharmaceutical composition.

[0035] In one embodiment, the composition may be a food composition.

[0036] In one embodiment, the composition may be a veterinary composition.

[0037] In one embodiment, the composition may be a feed composition.

[0038] According to another aspect of the present invention, a method for preventing, improving or treating a fibrotic disease is provided, comprising administering to a subject a composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore and a polypeptide having superoxide dismutase activity.

[0039] According to another aspect of the present invention, there is provided a use for preventing, improving or treating fibrotic diseases, comprising a composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore and a polypeptide having superoxide dismutase activity.

[0040] The present inventors have confirmed that a composition comprising at least one selected from the group consisting of a Bacillus species strain of the present invention, a Bacillus species strain spore, and superoxide dismutase has a preventive or therapeutic effect on fibrotic diseases. In one embodiment of the present invention, the Bacillus species strain spore of the present invention showed an effect of improving fibrosis both when administered before or after inducing pulmonary fibrosis, and it was confirmed that a Bacillus species strain spore with increased SOD expression showed a better preventive or therapeutic effect. In addition, the effect of SOD in removing reactive oxygen species in the body was superior when administered in the form of a spore of the strain rather than when free SOD was administered, which indicates that the spore form is more stable in the body. Meanwhile, oral SOD with improved stability in gastric acid was effective in improving pulmonary fibrosis even when administered alone, and showed an increased effect when administered in combination with normal SOD strain spores. In another embodiment of the present invention, the Bacillus species strain of the present invention exhibited an excellent therapeutic effect on liver fibrosis in an animal model of nonalcoholic steatohepatitis, and it was confirmed that this effect was greater when a strain variant overexpressing SOD was administered. In addition, when the composition was administered, not only was liver fibrosis improved, but also nonalcoholic steatohepatitis indicators (whole blood glucose level, plasma ALT level, plasma CK-18 level, liver triglyceride content, etc.) were improved and the NAFLD activity score (NAS) was significantly reduced. Thus, it was confirmed that the composition disclosed in the present invention is very effective in the prevention, improvement, or treatment of fibrotic diseases.

[0041] Figure 1 shows the overall procedure for producing a recombinant production strain (BSBA310) expressing SodA2.

[0042] Figure 2 shows an expression vector for overexpression of the sodA2 gene. Here, rrnBT1T2 represents a transcription terminator; rep(pBR322) represents a replicon from pBR322 that operates in E. coli; rep(pUB110) is a replicon from pUB110 that operates in B. subtilis; KanR represents a kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); and BJ27 promoter represents a strong promoter for B. subtilis.

[0043] Figure 3 is a schematic diagram showing the cloning process of the GF427 strain, which has increased SOD activity by replacing the promoter of the GF423 strain. Figure 3a shows the process of producing pUori-cm-amp-10sod using a PCR product obtained using GF423 genomic DNA as a template, and Figure 3b shows the process of producing pUori-cm-amp-P3-SOD using a PCR product obtained using pUori-cm-amp-10sod as a template.

[0044] Figure 4 illustrates the administration schedule of test substances and bleomycin to confirm the fibrosis prevention effect in a pulmonary fibrosis mouse model.

[0045] Figure 5 shows the results of measuring hydroxyproline content according to the concentration of GF424 spores administered.

[0046] Figure 6 illustrates the results of measuring the number of immune-related cells in lung lavage fluid according to the administration of GF424 spores at different concentrations. Figure 6a shows the number of total immune cells (total BAL cells), Figure 6b shows the number of neutrophils, Figure 6c shows the number of lymphocytes, and Figure 6d shows the number of macrophages.

[0047] Figure 7 shows the results of measuring TGF-β1 concentration according to the administration of GF424 spores at different concentrations.

[0048] Figure 8 shows the staining results (H&E and Masson's trichrome staining) of lung tissue sections according to the administration of GF424 spores at different concentrations. The scale bar represents 500 μm.

[0049] Figure 9 shows the results of confirming the expression level of lung fibrosis-related gene markers (Figure 9a, Col1a1; Figure 9b, TGF-β; Figure 9c, CTGF, and Figure 9d, IL-6) according to the administration of GF424 spores at different concentrations using real-time polymerase chain reaction.

[0050] Figure 10 shows the results of measuring reactive oxygen byproducts (Figure 10a, MDA; and Figure 10b, 4-HNE) according to the concentration-specific administration of GF424 spores.

[0051] Figure 11 shows the results of measuring hydroxyproline content according to administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression).

[0052] Figure 12 shows the results of measuring the number of immune-related cells in lung lavage fluid following administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression). Figure 12a shows the number of total immune cells (Total BAL cells), Figure 12b shows the number of neutrophils, Figure 12c shows the number of lymphocytes, and Figure 12d shows the number of macrophages.

[0053] Figure 13 shows the results of measuring TGF-β1 concentration according to administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression).

[0054] Figure 14 shows the staining (H&E and Masson's trichrome staining) results of lung tissue sections following administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression). The scale bar represents 500 μm.

[0055] Figure 15 shows the results of confirming the expression level of lung fibrosis-related gene markers (Figure 15a, Col1a1; Figure 15b, α-SMA; Figure 15c, TGF-β; Figure 15d, CTGF; Figure 15e, TNF-α, and Figure 15f, IL-6) by real-time polymerase chain reaction according to administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression).

[0056] Figure 16 shows the results of Western blots of lung fibrosis-related markers following administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression).

[0057] Figure 17 shows the results of estimating the survival rate of a lung fibrosis mouse model according to the administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression).

[0058] Figure 18 shows the results of measuring reactive oxygen byproducts (Figure 18a, MDA; and Figure 18b, 4-HNE) according to administration of strain spores showing differences in SOD expression levels (SOD knockout, normal expression, and overexpression).

[0059] Figure 19 shows the results of measuring hydroxyproline content according to administration of glass SOD and strain spores.

[0060] Figure 20 shows the results of measuring TGF-β1 concentration according to administration of glass SOD and strain spores.

[0061] Figure 21 shows the staining results (H&E and Masson's trichrome staining) of lung tissue sections following administration of free SOD and strain spores. The scale bar represents 500 μm.

[0062] Figure 22 shows the results of confirming the expression level of lung fibrosis-related gene markers (Figure 22a, Col1a1; Figure 22b, α-SMA; Figure 22c, TGF-β; Figure 22d, CTGF; Figure 22e, TNF-α, and Figure 22f, IL-6) according to administration of free SOD and strain spores using real-time polymerase chain reaction.

[0063] Figure 23 shows the results of Western blot of markers related to pulmonary fibrosis following administration of free SOD and strain spores.

[0064] Figure 24 shows the results of measuring reactive oxygen byproducts (Figure 24a, MDA; and Figure 24b, 4-HNE) according to the administration of glass SOD and strain spores.

[0065] Figure 25 shows the results of immunofluorescence staining in primary lung fibroblasts isolated from a mouse model of lung fibrosis. Each lung fibroblast was stained with antibodies against α-smooth muscle actin (α-SMA), Collagen 1 (Col1a1), and DAPI. The scale bar represents 50 μm.

[0066] Figure 26 shows the results of confirming the expression level of lung fibrosis-related gene markers (Figure 26a, TGF-β; Figure 26b, Col1a1; Figure 26c, TNF-α, and Figure 26d, IL-6) in primary lung fibroblasts isolated from a lung fibrosis mouse model using real-time polymerase chain reaction.

[0067] Figure 27 shows the results of measuring hydroxyproline content according to oral SOD (GF103), strain spores, and co-administration of oral SOD and strain spores in a pulmonary fibrosis mouse model.

[0068] Figure 28 shows the results of measuring TGF-β1 concentration according to oral SOD (GF103), strain spores, and co-administration of oral SOD and strain spores in a pulmonary fibrosis mouse model.

[0069] Figure 29 shows the staining results (H&E and Masson's trichrome staining) of lung tissue sections following oral SOD (GF103), strain spores, and combined oral SOD and strain spores in a mouse model of pulmonary fibrosis. The scale bar represents 500 μm.

[0070] Figure 30 shows the results of confirming the expression level of lung fibrosis-related gene markers (Figure 30a, Col1a1; Figure 30b, α-SMA; Figure 30c, TGF-β; Figure 30d, IL-6; Figure 30e, IL-1β, and Figure 30f, TNF-α) by real-time polymerase chain reaction according to oral SOD (GF103), strain spores, and co-administration of oral SOD and strain spores in a lung fibrosis mouse model.

[0071] Figure 31 shows the results of measuring reactive oxygen byproducts (Figure 31a, MDA; Figure 31b, 4-HNE; and Figure 31c, 8-OHdG) following oral SOD (GF103), strain spores, and co-administration of oral SOD and strain spores in a pulmonary fibrosis mouse model.

[0072] Figure 32 illustrates the administration schedule of test substances and bleomycin to confirm the anti-fibrotic effect in a pulmonary fibrosis mouse model.

[0073] Figure 33 shows the results of measuring hydroxyproline content following administration of spores of GF424, a SOD overexpressing strain, and GF427, a strain expressing more SOD than GF424, in a pulmonary fibrosis mouse model.

[0074] Figure 34 shows the results of measuring TGF-β1 concentration according to spore administration of SOD overexpressing strains (GF424 and GF427) in a pulmonary fibrosis mouse model.

[0075] Figure 35 shows the staining results (H&E and Masson's trichrome staining) of lung tissue sections following administration of spores of SOD-overexpressing strains (GF424 and GF427) in a pulmonary fibrosis mouse model. The scale bar represents 500 μm.

[0076] Figure 36 shows the results of confirming the expression level of lung fibrosis-related gene markers (Figure 36a, Col1a1; Figure 36b, TGF-β; Figure 36c, CTGF; Figure 36d, TNF-α; Figure 36e, IL-6, and Figure 36f, IL-1β) by real-time polymerase chain reaction following administration of spores of SOD overexpressing strains (GF424 and GF427) in a lung fibrosis mouse model.

[0077] Figure 37 shows the results of Western blot of lung fibrosis-related markers following administration of spores of SOD overexpressing strains (GF424 and GF427) in a lung fibrosis mouse model.

[0078] Figure 38 shows the results of measuring reactive oxygen byproducts (Figure 38a, MDA; Figure 38b, 4-HNE; and Figure 38c, 8-OHdG) and thiols (Figure 38d), which are important markers of oxidative stress, following administration of spores of SOD overexpressing strains (GF424 and GF427) in a pulmonary fibrosis mouse model.

[0079] Figure 39 is a graph confirming the expression level of biomarkers that induce lung fibrosis following spore administration of SOD-overexpressing strains (GF424 and GF427) in a pulmonary fibrosis mouse model. The expression levels of epithelial damage markers (Figure 39a, SP-D), fibrosis markers (Figure 39b, MMP-7; Figure 39c, Tenascin-C, and Figure 39d, Periostin), inflammatory markers (Figure 39e, CXCL13), and thrombosis markers (Figure 39f, PAI-1) are shown.

[0080] Figure 40 shows the survival rate estimation results of a pulmonary fibrosis mouse model following administration of spores of SOD overexpressing strains (GF424 and GF427) in a pulmonary fibrosis mouse model.

[0081] Figure 41 is a drawing showing the part to be used in the experiment by isolating and dissecting the left outer lobe of a liver sample in an experiment to confirm the effect of Bacillus velezensis strain on the treatment of liver fibrosis in a non-alcoholic steatohepatitis STAM™ model.

[0082] Figure 42 is a graph showing the change in body weight of mice belonging to 10 groups over 21 days of inducing and treating non-alcoholic steatohepatitis (NASH, STAM™ model).

[0083] Figure 43 is a graph showing the changes in organ weights of mice belonging to 10 groups on the 21st day after inducing non-alcoholic steatohepatitis (NASH, STAM™ model) and when all treatments were completed. Figure 43a is a table summarizing the results of body weight, liver weight, and liver-to-body weight ratio, Figure 43b is a body weight graph, Figure 43c is a liver weight graph, and Figure 43d is a graph showing the results of liver-to-body weight ratio.

[0084] Figure 44 shows the results of biochemical analyses using plasma and liver samples from mouse models that were induced with non-alcoholic steatohepatitis (NASH, STAM™ model) and at the end of treatment. Figure 44a is a table summarizing the results of whole blood glucose levels, plasma ALT levels, plasma CK-18 levels, and liver triglyceride content. Figure 44b is a graph showing whole blood glucose levels, Figure 44c is a graph showing plasma ALT levels, Figure 44d is a graph showing plasma CK-18 levels, and Figure 44e is a graph showing liver triglyceride content.

[0085] Figure 45 shows the results of H&E staining and NAFLD activity score evaluation using liver samples of mouse models in which non-alcoholic steatohepatitis (NASH, STAM™ model) was induced and all treatments were completed. Figures 45a to 45c show the results of H&E staining of liver samples from mice belonging to 10 groups, and Figure 45d shows the results of H&E staining showing liver sections from the disease control group. Figure 45e is a table showing the numerical value of the NAFLD activity score (NAS) calculated according to Kleiner's criteria, and Figure 45f is a graph showing the NAS of each group. Figures 45g to 45i show the details of NAS, such as steatosis score (Figure 45g), inflammation score (Figure 45h), and ballooning score (Figure 45i).

[0086] Figure 46 shows the results of Sirius red staining using liver samples from mouse models that were induced with non-alcoholic steatohepatitis (NASH, STAM™ model) and completely treated. Figure 46a is a photomicrograph of a Sirius red-stained liver section, Figure 46b is a table quantifying the degree of fibrosis, and Figure 46c is a graph showing the degree of fibrosis (Sirius red positivity) in each group.

[0087] The detailed description of the present invention that follows will be described with reference to specific embodiments in which the present invention may be practiced, with reference to the drawings. However, the present invention is not limited thereto, but is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled, if properly described. It should be understood that the various embodiments / embodiments of the present invention, while different, are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be modified from one embodiment / embodiment to another, or may be combined among embodiments / embodiments, without departing from the spirit and scope of the present invention. Unless otherwise indicated, the terms presented in the description of the present invention should generally be construed in their ordinary meanings, and apply to the same terms used herein as well as to the aspects or embodiments of the invention in which the term is defined. For purposes of interpreting this specification, the following definitions will apply, and terms used in the singular will include the plural where appropriate, and vice versa.

[0088] definition

[0089] The term "subject" is used interchangeably with "subject" or "patient" and may be a mammal in need of prevention, improvement, or treatment of a fibrotic disease, such as a primate (e.g., human, monkey, chimpanzee, etc.), companion animal (e.g., dog, cat, etc.), livestock animal (e.g., cow, pig, horse, sheep, goat, etc.), and laboratory animal (e.g., rat, mouse, guinea pig, etc.). In one embodiment of the present invention, the subject is a human.

[0090] The term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. Such effect is therapeutic in that it partially or completely cures a disease and / or other unwanted or undesirable condition (e.g., a fibrotic disease such as pulmonary fibrosis or liver fibrosis). Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, improving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. Preferably, "treatment" can refer to medical intervention for an already present disease or disorder.

[0091] The term "prevention" means obtaining a desired preventive pharmacological and / or physiological effect with a view to partially or completely preventing a disease or its symptoms.

[0092] The term "administration" means providing an active ingredient to a subject to achieve a preventive or therapeutic purpose (e.g., preventing or treating a fibrotic disease such as pulmonary fibrosis or liver fibrosis).

[0093] Use of Bacillus species strains, Bacillus species strain spores and / or SOD for the prevention or treatment of fibrotic diseases

[0094] The present invention is based, at least in part, on the surprising discovery that administration of a composition comprising at least one selected from the group consisting of a Bacillus sp. strain, a Bacillus sp. strain spore, and superoxide dismutase (SOD) is effective in preventing, ameliorating, or treating a fibrotic disease, such as a pulmonary fibrotic disease (e.g., interstitial lung disease, pulmonary fibrosis, idiopathic pulmonary fibrosis, etc.) or a liver fibrotic disease (e.g., nonalcoholic steatohepatitis, liver fibrosis, idiopathic hepatic fibrosis, cirrhosis, alcoholic steatohepatitis, chronic liver disease, viral hepatitis, etc.). Accordingly, according to one aspect of the present invention, there is provided a use for preventing, ameliorating, or treating at least one fibrotic disease selected from the group consisting of a Bacillus sp. strain, a Bacillus sp. strain spore, and superoxide dismutase. In some embodiments, the use of a Bacillus sp. strain, a Bacillus sp. strain spore, or SOD alone for preventing, ameliorating, or treating a fibrotic disease is provided. In some embodiments, a use is provided for preventing, ameliorating or treating a fibrotic disease of a combination of two or more selected from a Bacillus species strain, a Bacillus species strain spore and SOD.

[0095] Fibrotic diseases, or fibrosis, are characterized by the abnormal accumulation of collagen matrix following damage or inflammation that alters the structure and function of various tissues. Fibrotic diseases, or fibrosis, can occur in tissues of various organs, including the kidney, liver, lungs, heart, bone marrow, and skin. Examples of such fibrotic diseases, or fibrosis, include, but are not limited to, liver fibrosis, cirrhosis, vocal cord scarring, vocal cord mucosal fibrosis, laryngeal fibrosis, pulmonary fibrosis, pancreatic fibrosis, bone marrow fibrosis, myocardial infarction, post-myocardial fibrosis, myocardial fibrosis, endomyocardial fibrosis, splenic fibrosis, mediastinal fibrosis, submucosal fibrosis, intestinal fibrosis (e.g., associated with inflammatory bowel disease), retroperitoneal fibrosis, uterine fibrosis, scleroderma, and mammary fibrosis.

[0096] In some embodiments, the fibrotic disease may be pulmonary fibrosis. Pulmonary fibrosis includes not only pulmonary fibrosis (PF) in the narrow sense, but also interstitial lung disease (ILD) (pulmonary fibrosis or lung damage caused by autoimmune conditions, viral infections, bacterial infections, medications such as anticancer drugs like bleomycin and antibiotics, toxic substances, etc.) or idiopathic pulmonary fibrosis (IPF).

[0097] In some embodiments, the fibrotic disease may be pulmonary fibrosis (PF) or idiopathic pulmonary fibrosis (IPF).

[0098] In another embodiment, the fibrotic disease may be interstitial lung disease (ILD).

[0099] In some embodiments, the fibrotic disease may be liver fibrosis. Liver fibrosis includes not only liver fibrosis in the narrow sense, but also non-alcoholic steatohepatitis, (progressive) hepatic fibrosis, idiopathic liver fibrosis, cirrhosis, alcoholic steatohepatitis, chronic liver disease, or viral hepatitis.

[0100] In some embodiments, the fibrotic disease may be nonalcoholic steatohepatitis (NASH).

[0101] In one embodiment of the present invention, when Bacillus velezensis strain spores were orally administered before and after induction of pulmonary fibrosis (bleomycin treatment), the degree of pulmonary fibrosis was significantly improved, and this effect was confirmed to be greater when spores of a SOD overexpressing strain mutant with a high degree of SOD expression were administered.

[0102] In another embodiment of the present invention, after administration of SOD-overexpressing strain spores, the 21-day survival rate of a bleomycin-induced lung fibrosis mouse model was confirmed to be excellent enough to be almost similar to that of normal mice not induced with lung fibrosis (preventive effect). In addition, when SOD-overexpressing strain spores were administered to a mouse model in which lung fibrosis was induced by bleomycin treatment, the survival rate was not as high as that of normal mice, but was significantly increased compared to the bleomycin-administered group (therapeutic effect).

[0103] In another embodiment of the present invention, oral SOD with increased stability in gastric acid was effective in improving pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis mouse model even when administered alone, and an inhibitory effect on pulmonary fibrosis was confirmed even when the oral SOD was administered in combination with SOD normal strain spores.

[0104] In another embodiment of the present invention, when a Bacillus velezensis (Bacillus amyloliquefaciens) strain was orally administered to an animal model of NASH induced through streptozotocin solution and a high-fat diet, the degree of liver fibrosis was significantly improved, and this effect was more effective when the strain had a significantly improved degree of SOD expression. In addition, improvements in non-alcoholic steatohepatitis indices (such as total blood glucose level, plasma ALT level, plasma CK-18 level, and hepatic triglyceride content) and a significant decrease in the NAFLD activity score (NAS) were also observed.

[0105] Composition comprising Bacillus species strain, Bacillus species strain spore and / or SOD

[0106] According to another aspect of the present invention, a composition for preventing, improving or treating a fibrotic disease is provided, comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore and SOD as an active ingredient. Specifically, the composition may comprise (i) a Bacillus species strain, (ii) a Bacillus species strain spore, (iii) SOD, (iv) a Bacillus species strain and a Bacillus species strain spore, (v) a Bacillus species strain and SOD, (vi) a Bacillus species strain spore and SOD or (vii) a Bacillus species strain, a Bacillus species strain spore and SOD.

[0107] The term "spore" as used herein may be used interchangeably with "spore", and the Bacillus species strain spores may be obtained by culturing the Bacillus species strain in a suitable medium, inducing sporulation, and then isolating the produced spores.

[0108] In some embodiments, the Bacillus species strain may be sourced from a generally recognized as safe (GRAS) bacterium that is generally regarded as safe for use in drugs or foods. Bacillus species strain spores are known to be resistant to proteases and low pH (see Cutting SM. Bacillus probiotics. Food Microbiol. 2011; 28:214-220. doi: 10.1016 / j.fm. 2010.03.007; and Wang Y, et al., In vitro assessment of probiotic properties of Bacillus isolated from naturally fermented congee from inner Mongolia of China. World J. Microb. Biot. 2010; 26:1369-1377. doi: 10.1007 / s11274-010-0309-7).

[0109] Additionally, Bacillus species strains are GRAS probiotics approved in several countries. Specifically, the Bacillus species strains may be, but are not limited to, B. velezensis, B. amyloliquesfaciens, B. methylotrophicus, B. siamensis, B. subtilis, B. tequilensis, B. atrophaeus, B. mojavensis, or B. vallismortis. The Bacillus species strain spores may be derived from, but are not limited to, the strains described above. Preferably, the Bacillus species strain may be a Bacillus velezensis (Bacillus amyloliquefaciens) strain (e.g., GF423 strain, GF424 strain, or GF427 strain). The Bacillus species strain spores may be derived from a Bacillus velezensis (Bacillus amyloliquefaciens) strain (e.g., GF423 strain, GF424 strain, or GF427 strain). The GF423 strain, the GF424 strain, and the GF427 strain were deposited with the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017, March 13, 2017, and August 14, 2023, respectively (Accession No. KCTC 13222 BP, Accession No. KCTC 13227 BP, and Accession No. KCTC 15552 BP). Additionally, the characteristics and cultivation method of the GF423 strain are described in Korean Patent No. 1762199, the entire disclosure of which is incorporated herein by reference.The above-deposited Bacillus strain was classified and described as Bacillus amyloliquefaciens in a previous application, but as a result of comparison using genome-based classification methods such as DDH and ANI, it was suggested that it should be classified as a microorganism belonging to Bacillus velezensis because it did not meet the species level distinction criteria, and it was also registered as "Bacillus velezensis" in LPSN (List of Prokaryotic names with Standing in Nomenclature; https: / / lpsn.dsmz.de / species / bacillus-velezensis) (see literature [Fan, Ben, et al. "Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an "operational group B. amyloliquefaciens" within the B. subtilis species complex." Frontiers in microbiology 8 (2017): 22.]). Bacillus Given that velezensis and Bacillus amyloliquefaciens are heterotypic synonyms, the two names can be used interchangeably.

[0110] In some embodiments, Bacillus amyloliquefaciens strains may be used interchangeably with Bacillus velezensis strains. Accordingly, Bacillus amyloliquefaciens strains GF423, GF424, and GF427 may be understood to be the same strains as Bacillus velezensis strains GF423, GF424, and GF427.

[0111] The process by which the deposited Bacillus species strains were classified as Bacillus velezensis was as follows. The deposited Bacillus species strains were isolated and compared with three highly homologous reference strains through DDH, ANI, and AAI analyses (gene-based classification method) for their 16S rRNA genes and whole genomes (see Table 1).

[0112] Bacillus amyloliquefaciens plantarum Bacillus amyloliquefaciens Amyloliquefaciens Bacillus subtilis sp. spp. Species differentiation criteria (threshold) (%) Standard strain FZB42DSM7NRRL B-23049-16S rRNA 99.67~99.73% 99.46~99.66% 99.18~99.39% 98.65% DDH 92.10% 78.60% 32.70% 70% ANI 98.65% 93.59% 80.04% 94% AAI 98.79% 95.09% 79.88% 95%

[0113] Referring to Table 1, the deposited Bacillus species strains through DDH and ANI results were found to be more similar to Bacillus amyloliquefaciens subspecies plantarum than to Bacillus amyloliquefaciens subspecies amyloliquefaciens (the DDH species discrimination criterion is 70% or higher, and the ANI species discrimination criterion is 94% or higher). Meanwhile, Bacillus amyloliquefaciens subsp. plantarum did not satisfy the species distinction criteria when compared with Bacillus amyloliquefaciens DSM7, the type strain of Bacillus amyloliquefaciens subsp. amyloliquefaciens, using the genome-based classification methods DDH and ANI, and was classified as a microorganism belonging to Bacillus velezensis rather than being the same species as Amyloliquefaciens subsp. amyloliquefaciens. In some embodiments, the Bacillus species strain may be a strain that expresses or produces SOD or a mutant strain that has been mutated or recombined to overexpress or overproduce SOD. In some embodiments, the Bacillus species strain spore may be a spore of a strain that expresses or produces SOD. Furthermore, the Bacillus species strain spore may be derived from a strain that has been mutated or recombined to overexpress or overproduce SOD. For example, the Bacillus species strain may be a naturally isolated strain (e.g., GF423 strain) or a mutant strain that has been mutated to overexpress or overproduce SOD (e.g., GF424 or GF427 strain).

[0114] In another embodiment, the Bacillus species strain spores may be derived from another Bacillus species strain that has been recombinantly engineered to express the SOD of the Bacillus velezensis strain. Such recombinant strains are further described below.

[0115] Sporulation of Bacillus species strains can be induced using conventional techniques known in the art. For example, spores can be obtained by inducing sporulation in media such as DSM (Difco Sporulation Medium), NB (Nutrient broth), SYP (Starch Yeast extract Peptone medium), and LB2 (Luria-Bertani 2) after pre-cultivating the strain and then performing the main culture. Spores can also be separated by culturing various sources including natural, mutant, or recombinant hosts, and then filtering, concentrating, or centrifuging after removing vegetative cells.

[0116] Meanwhile, superoxide dismutase (SOD) is an enzyme that decomposes superoxide (O2 ㆍ- ) As an enzyme that alternately catalyzes the dismutation of radicals into ordinary molecular oxygen (O2) and hydrogen peroxide (H2O2), SOD plays an important role in reducing oxidative stress by removing reactive oxygen species. SODs are widely distributed in prokaryotic and eukaryotic cells and are classified into four classes according to the different types of metal centers (copper / zinc, nickel, manganese, and iron). Manganese-containing SODs [Mn-SODs] are widely present in the chloroplasts, mitochondria, and cytoplasm of many bacteria or eukaryotic cells. The term "SOD" in the present invention may be used interchangeably with a (poly)peptide having superoxide dismutase activity. In addition, SOD may include a polypeptide having superoxide dismutase activity, a fragment thereof, or a fusion comprising the same.

[0117] In some embodiments, the SOD may be manganese-binding (Mn-SOD). Preferably, the SOD may be deamidated Mn-SOD. In addition, the SOD may comprise or consist of the amino acid sequence represented by SEQ ID NO: 2 (SodA). Preferably, the SOD may have amino acid residues 74 and 137 of SEQ ID NO: 2 substituted with Asp. More preferably, the SOD may comprise or consist of the amino acid sequence represented by SEQ ID NO: 4 (SodA2). In some embodiments, the SOD may be in a form in which the methionine (Met), which is the start codon during protein translation, is deleted. Preferably, the SOD may be a form in which the methionine (Met) of the deamidated Mn-SOD is deleted. The SOD may have amino acid residue 1 Met deleted based on SEQ ID NO: 2. Preferably, the SOD may comprise or consist of the amino acid sequence represented by SEQ ID NO: 5 (Met-deleted SodA). Additionally, SOD may be one in which amino acid residue Met, which is at position 1 based on sequence number 4, is deleted. More preferably, SOD may comprise or consist of an amino acid sequence represented by sequence number 6 (Met-deleted SodA2).

[0118] The SOD or polypeptide having SOD activity of the present invention is interpreted to also include an amino acid sequence that exhibits substantial identity to the amino acid sequence described above. The substantial identity refers to an amino acid sequence that exhibits a sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98%, when analyzing aligned sequences using an algorithm commonly used in the art.

[0119] In some embodiments, the SOD is a modified or engineered polypeptide having SOD enzymatic activity, which may or may not include one or more mutations, such as deletions, insertions, or substitutions of one or more amino acids, which may or may not affect various aspects (e.g., in vivo, in vitro, or ex vivo stability, uniformity, and / or conformational changes). In addition, the polypeptide may further include a heterologous material (e.g., a tag known in the art, including a HIS tag, an HA tag, a myc tag, a GFC, and / or an Fc domain of an antibody) for purification, detection, in vivo delivery, or increased stability.

[0120] In some embodiments, the SOD of the present invention may be derived from various sources, including natural, mutant, or recombinant microorganisms. For example, the SOD may be derived from bacteria. Preferably, the SOD may be derived from a bacterium generally regarded as safe (GRAS) for use in drugs or foods, such as a Bacillus sp. strain or a mutant or recombinant thereof. Specific examples of the Bacillus sp. strain are as described above. More preferably, the SOD may be obtained from a Bacillus velezensis strain (e.g., GF423 strain, GF424 strain, or GF427 strain) or a culture supernatant thereof. The Bacillus velezensis GF424 strain is a strain obtained by mutagenizing the Bacillus velezensis GF423 strain by UV irradiation to improve the expression of the sod gene. The above Bacillus velezensis GF427 strain is a strain obtained by replacing the promoter sequence of the Bacillus velezensis GF423 strain with a base sequence having a stronger promoter performance in order to improve the expression of the sod gene. The SOD enzyme (SodA) derived from the Bacillus velezensis GF423, GF424 or GF427 strain is Mn-SOD and may comprise or consist of the amino acid sequence of SEQ ID NO: 2 (the nucleotide sequence thereof is represented by SEQ ID NO: 1). In addition, the SOD may be a recombinant polypeptide. For example, it may be a deamidated SOD in which amino acid residues 74 and 137 of SEQ ID NO: 2 are substituted with Asp (SodA2), and this may comprise or consist of the amino acid sequence of SEQ ID NO: 4 (the nucleotide sequence thereof is represented by SEQ ID NO: 3). In addition, the SOD may be a polypeptide in which the start codon methionine (Met) is removed.For example, it may be an SOD with amino acid residue 1 deleted based on SEQ ID NO: 2 or SEQ ID NO: 4, and may include or consist of an amino acid sequence of SEQ ID NO: 5 or 6. The sequences of SEQ ID NOs: 1 to 6 are as shown in Table 2.

[0121] Sequence number Nucleotide sequence of SodA Sequence number 1 Amino acid sequence of SodA MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNNGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNNGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAKS Sequence number 2 Nucleotide sequence of SodA2서열ATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAAGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACGATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATCTCCGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACGACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCGTCCTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAATAA서열번호 3SodA2의 아미노산 서열MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNDGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNDGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAK서열번호 4Met-deleted SodA의 아미노산Sequence AYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNNGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNNGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAKS SEQ ID NO: 5 Amino acid of Met-deleted SodA2 SequenceAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNDGGGHANHSLFWTLLSPNGGGEPTGELAE EIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNDGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAKSEQ ID NO. 6

[0122] In addition, SOD can be derived from another recombinant strain (e.g., a Bacillus subtilis strain) containing the SOD expression gene of the Bacillus velezensis strain. The recombinant strain can be produced by recombinant technology using a conventional protein production strain known in the art. For example, the Bacillus subtilis strain (which is the parent strain of the recombinant strain) can be KCTC 3135, and the KCTC 3135 strain can be obtained from the Korea Research Institute of Bioscience and Biotechnology (KCTC). In addition, the recombinant strain can have one or more of the genes shown in Table 3 below deleted to facilitate post-processing.

[0123] Gene nameProtein nameFunctionAprESerine alkaline protease(subtilisin E)Extracellular proteaseNprEStercellular neutral metalloproteaseExtracellular proteaseBprBacillopeptidase FExtracellular proteaseEprExtracellular serine proteaseExtracellular proteaseNprBExtracellular neutral protease BExtracellular proteaseVprExtracellular serine proteaseExtracellular proteaseMprExtracellular metalloproteaseExtracellular proteaseIspAIntracellular serine proteaseIntracellular proteaseSrfACsurfactin synthaseSurfactin synthesisspoIIACRNA polymerase sporulation-specific sigma factor (sigma-F)SporulationEpsEPutative glycosyltransferaseExtracellular polysaccharideXpfRNA polymerase sigma factorPBSXTranscription of prophage genes

[0124] For example, the recombinant strain can be produced through the process illustrated in Fig. 1. In addition, the recombinant strain can include the expression vector illustrated in Fig. 2. In the drawing, sodA and sodA2 represent genes encoding SOD. Since the SOD derived from the strain is an enzyme secreted outside the cell, when producing SOD using the strain, efficient production is possible because SOD can be produced in large quantities with guaranteed safety for the organism without going through an expensive purification process (e.g., column purification).

[0125] In some embodiments, SOD can be obtained by culturing the natural, mutant, or recombinant microorganism in various culture media. For example, SOD can be isolated from the culture supernatant of Bacillus velezensis strain GF423, GF424, or GF427. Specifically, the Bacillus velezensis strain can be first cultured in various types of media to obtain a culture solution. For example, the strain is grown at about 25°C to about 42°C for about 1 to about 4 days using a complex medium (pH 6.0 to 7.0). Other suitable media for culturing the above Bacillus velezensis strain include Luria-Bertani (LB) medium, International Streptomyces Project (ISP) medium, nutrient agar (NA) medium, brain heart infusion agar (BHI) medium, sabouraud dextrose agar (SDA) medium, potato dextrose agar (PDA) medium, nutrient broth (NB) medium, etc. In a preferred embodiment, LB medium, ISP medium, BHI medium, SDA medium, or NB medium can be used. Additionally, SOD can be sourced from other natural, mutant, or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).

[0126] In some embodiments, the SOD may be isolated or purified from a culture of a natural, mutant, or recombinant strain, wherein the isolated or purified SOD or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it was derived. For example, the purified product may be purified from a culture of the strain by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or may be a culture concentrate obtained by ultrafiltration, concentration, etc. The phrase "substantially free of cellular material" includes preparations of the protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In some embodiments, the phrase "substantially free of cellular material" includes preparations of the protein in which undesired proteins are less than about 30% by dry weight, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5%.

[0127] SOD may be purified using, but is not limited to, the following purification methods. For example, the culture medium obtained by culturing Bacillus velezensis strains is centrifuged to collect the culture supernatant. The supernatant fraction is pretreated by solid phase extraction, and then isolated and purified by chromatography. SOD can be purified using various chromatographic methods. Hydrophobic interaction chromatography is preferably used.

[0128] In some embodiments, SOD may be included in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof. Here, "strain lysate" means something obtained by culturing a strain and mechanically or chemically disrupting the strain, and may include anything that has undergone additional processes such as extraction, dilution, concentration, and purification therefrom. "Strain culture" may mean the culture solution itself obtained by culturing a strain or its supernatant. "Strain culture concentrate" refers to something that is purified from a strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained through ultrafiltration, concentration, etc. "Strain culture extract" means something extracted from the culture solution or its concentrate, and may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a preparation or purification thereof, and a fraction obtained by fractionating the extract. The dried form may include a freeze-dried form.

[0129] In some embodiments, the SOD may comprise cellular material, such as extracellular vesicles, from the cell or tissue source from which it is derived. In this case, the cellular material comprising the SOD can be cultured from various sources, including natural, mutant, or recombinant host cells, using conventional techniques known in the art, as described above, and isolated from the culture solution by filtration, concentration, or other methods.

[0130] Methods for preventing or treating fibrotic diseases

[0131] According to another aspect of the present invention, a method for preventing, improving, or treating a fibrotic disease is provided, comprising administering to a subject a composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and SOD as disclosed herein. For example, the method may comprise administering to a subject who has developed or is likely to develop a fibrotic disease a composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and SOD as disclosed herein in a therapeutically or nutritionally effective amount. The fibrotic disease is as described herein.

[0132] In some embodiments, the method may comprise administering to the subject a Bacillus species strain, a Bacillus species strain spore, or a SOD as disclosed herein alone. In other embodiments, the method may comprise administering to the subject a combination of one or more selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and a SOD as disclosed herein. The administration may be oral or parenteral, and is preferably oral. In one embodiment of the present invention, it was confirmed that even when a Bacillus species strain, a Bacillus species strain spore, or a SOD is orally administered to the subject, fibrotic diseases can be effectively prevented or treated.

[0133] The effective amount or effective non-toxic amount of a composition according to the present invention can be determined by routine experimentation. For example, the therapeutically active amount of a composition comprising the Bacillus species strain, the Bacillus species strain spore, and / or the SOD of the present invention may vary depending on factors such as the stage of the disease, the severity of the disease, the age, sex, medical complications, and body weight of the subject, and the ability of the species strain spore to express SOD to induce the desired response in the subject. The dosage and administration regimen of the composition of the present invention can be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, weekly, every two weeks, every three weeks, every four weeks, etc., and / or the dosage may be proportionally reduced or increased depending on the exigencies of the therapeutic situation.

[0134] For example, a Bacillus species strain or a Bacillus species strain spore may be 10 4 10 inland 12 , 10 4 10 inland 10 , 10 4 10 inland 9 , 10 5 10 inland 12 , 10 5 10 inland 11 , 10 5 10 inland 10 or 10 5 10 inland 9 CFU can be included in the composition.

[0135] The composition according to the present invention may be administered together with one or more other agents that cause or are likely to cause a fibrotic disease for the purpose of preventing, improving, or treating a fibrotic disease. Such other agents include, but are not limited to, antibiotics, immunosuppressants, drugs for heart disease, and cancer chemotherapy drugs, which are administered for the purpose of improving or ameliorating other diseases, but which have the potential to inadvertently cause a fibrotic disease. When a composition comprising one or more selected from the group consisting of a Bacillus species strain of the present invention, a Bacillus species strain spore, and / or a SOD is administered together with another agent, they may be administered simultaneously, sequentially, or in reverse order, and may be administered before the other agent is administered. Each component may be administered to the subject at a different time than when the other components are administered. In certain embodiments, each administration may be given non-simultaneously (e.g., separately or sequentially) at multiple intervals over a given period of time. Furthermore, the individual components may be administered to the subject by the same or different routes. The routes by which the drug may be administered are detailed in the pharmaceutical composition.

[0136] In one embodiment, the Bacillus species strain, the Bacillus species strain spore, and / or the polypeptide having the SOD activity of the present invention can be administered simultaneously, sequentially, or in reverse order. That is, a composition comprising the following components (i) to (vii) can be administered simultaneously, sequentially, or in reverse order to exhibit a preventive or therapeutic effect on a fibrotic disease: (i) the Bacillus species strain and the Bacillus species strain spore, (ii) the Bacillus species strain and SOD, (iii) the Bacillus species strain spore and SOD, or (iv) the Bacillus species strain, the Bacillus species strain spore, and SOD. Preferably, the Bacillus species strain spore and SOD can be administered simultaneously, sequentially, or in reverse order to exhibit a preventive or therapeutic effect on a fibrotic disease.

[0137] Pharmaceutical composition

[0138] According to another aspect of the present invention, a pharmaceutical or veterinary composition comprising the composition described above is provided. In other words, a pharmaceutical or veterinary composition is provided comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and SOD as an active ingredient. The pharmaceutical composition of the present invention may be used interchangeably with the term "veterinary composition" when applied to animals other than humans.

[0139] In some embodiments, the pharmaceutical composition may be used for the prevention or treatment of fibrotic diseases. Fibrotic diseases are described above.

[0140] The pharmaceutical or veterinary composition of the present invention may further comprise one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients and diluents. The pharmaceutically acceptable carriers, excipients and / or diluents may be those commonly used in the art. Examples of the carriers, excipients or diluents include, but are not limited to, mineral oils such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and silicon dioxide.

[0141] When formulating, additives such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants can be used. The additives for the formulation can be selected from those commonly used in the pharmaceutical field.

[0142] In addition, the pharmaceutical or veterinary composition of the present invention may be formulated in a desirable form depending on the method of use, and may be formulated by adopting a method known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Specific examples of such formulations include tablets, pills, powders, granules, syrups, solutions, capsules, suspensions, emulsions, injectable solutions, plasters, lotions, liniments, limonades, aerosols, extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, patches, etc.

[0143] Furthermore, the pharmaceutical or veterinary composition of the present invention may be preferably formulated using any suitable method known in the art or using the method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

[0144] The pharmaceutical or veterinary composition of the present invention may be administered orally or parenterally, depending on the intended method. Parenteral administration may include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intrapulmonary, intraarterial, intramuscular, rectal, intravaginal, intraarticular, intraprostatic, intranasal, intraocular, intravesical, intrathecal, or intraventricular administration (e.g., intracerebroventricular administration).

[0145] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. into a complex composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid dosage forms for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be used.

[0146] For oral administration, SOD may be coated with shellac for protection from gastric acid, but the coating is not limited thereto. Examples of coatings suitable for use in the present invention include shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof. When SOD is coated, the SOD may be coated in solution. Specifically, a purified solution and a shellac-containing solution are mixed, followed by lyophilization. The lyophilized sample may be powdered and stored at about 4°C until use. In some embodiments, the SOD may be an oral SOD with enhanced stability in gastric acid through shellac coating. Preferably, the SOD may be a shellac-coated SOD in which amino acid residue 1, Met, is deleted based on SEQ ID NO: 4. More preferably, the SOD may be a shellac-coated SOD comprising or consisting of an amino acid sequence represented by SEQ ID NO: 6.

[0147] The pharmaceutical or veterinary composition of the present invention is administered in a pharmaceutically or veterinarily effective amount. The term "pharmaceutically effective amount" or "veterinarily effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical or veterinary treatment, and the effective dosage level may be determined based on factors including the patient's or animal's body weight, sex, age, health status, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0148] The pharmaceutical or veterinary composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, either sequentially or simultaneously. Furthermore, the pharmaceutical composition can be administered singly or in multiple doses, as needed. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with the minimum amount possible without causing side effects. Such an amount can be readily determined by those skilled in the art.

[0149] In some embodiments, the composition may have various forms suitable for human administration known in the art, including liquid form, solid form, gel form, powder form, paste form, and the like.

[0150] Food or feed composition

[0151] According to another aspect of the present invention, a food composition is provided comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and SOD as described above. This food composition includes a medical or nutraceutical food composition. Accordingly, the food composition is provided as a food composition for preventing or improving fibrotic diseases.

[0152] The term "medical food" or "nutraceutical food" refers to a food manufactured from raw materials or ingredients that have the potential to have a beneficial function for the human body, and that maintains or improves health by maintaining normal functions or activating physiological functions of the human body, and is regulated by the Ministry of Food and Drug Safety, but is not limited thereto, and does not exclude any conventional health food from its meaning.

[0153] In addition, the above foods include, but are not limited to, various foods, food additives, beverages (e.g., functional beverages, natural fruit juices and vegetable beverages), gum, tea, vitamin complexes, health functional foods, and other functional foods.

[0154] The above food can be manufactured by conventional methods known in the art. For example, the medical food, nutraceutical food, or health functional food can be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations by further including one or more of a carrier, diluent, excipient, and additive in addition to the SOD for the purpose of preventing or improving fibrotic diseases. Specific examples of the carrier, excipient, diluent, and additive are well known in the art, and a person skilled in the art can manufacture the product by combining appropriate ingredients according to the formulation.

[0155] The content of the Bacillus species strain, strain spore and / or SOD according to the present invention as an effective ingredient in the above-described formulation can be appropriately adjusted depending on the form and purpose of use, the patient's condition, the type and severity of symptoms, etc., and may be 0.001 to 99.9 wt%, preferably 0.01 to 50 wt%, based on the solid weight, but is not limited thereto.

[0156] The dosage of the food of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist. For example, the daily dosage may be 10 to 1,000 mg / kg based on the content of the active ingredient. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences. If the daily dosage of the health functional food of the present invention is less than the above dosage, a significant effect may not be obtained, and if it exceeds it, it is not only uneconomical but also goes beyond the range of commercial dosage, so undesirable side effects may occur.

[0157] According to another aspect of the present invention, a feed composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and SOD as described above is provided. In addition, the feed composition of the present invention can be prepared in any formulation commonly used in the art. For example, the feed composition of the present invention may further comprise adjuvant ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes, and other microbial preparations in the form of live cells; grains such as ground or crushed wheat, oats, barley, corn, and rice; plant-based protein feeds such as those mainly composed of rapeseed, soybeans, and sunflower; animal-based protein feeds such as blood meal, meat meal, bone meal, and fish meal; dry ingredients composed of sugars and dairy products such as various types of milk powder and whey powder; lipids such as animal fats and vegetable fats optionally liquefied by heating; and additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventive agents.

[0158] The feed composition of the present invention may be in the form of a powder or liquid formulation, and may include an excipient for feed addition (calcium carbonate, malt powder, zeolite, corn powder, or rice bran, etc.).

[0159] Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are presented to aid understanding of the present invention and are not intended to limit its scope in any way, nor should they be construed as limiting it.

[0160] Example

[0161] Manufacturing Example 1. Manufacturing of SOD-expressing Bacillus velezensis strain spores

[0162] The spores of the Bacillus velezensis (Bacillus amyloliquefaciens) strain expressing SOD used in this example were prepared from the Bacillus velezensis strain deposited at the Korea Center for Biological Resources (KCTC) (Accession No.: KCTC 13227BP, Deposit Date: March 13, 2017) (hereinafter referred to as the "GF424 strain"), the Bacillus velezensis strain deposited at the Korea Center for Biological Resources (KCTC) (Accession No.: KCTC 13222BP, Deposit Date: March 6, 2017) (hereinafter referred to as the "GF423 strain"), and the Bacillus velezensis strain deposited at the Korea Center for Biological Resources (KCTC) (Accession No.: KCTC 15552BP, Deposit Date: August 14, 2023) (hereinafter referred to as the "GF427 strain") according to the following method.

[0163] A single colony of the Bacillus velezensis strain was inoculated into 1 mL of LB in a 14 mL tube and cultured at 37°C and 200 rpm for 12 hours. 1 mL of the culture was transferred to 50 mL of LB medium in a 500 mL flask and cultured at 37°C and 200 rpm for 12 hours. 20 mL of the culture medium was then transferred to 1 L of SYP or DSM in a 2.5 L baffled flask. The inoculated culture was cultured at 37°C and 200 rpm for 24 to 120 hours.

[0164] SYP medium contains 1.5% soy tone, 0.5% yeast extract, 0.5% K2HPO4, 0.1% MnSO4, 0.1% MgSO4, 10 mM FeSO4, 0.04% (NH4)2SO4, 0.04% (NH4)2PO4, 0.1% CaCl2 and 2% glucose, and DSM medium contains 8 g / L bacto nutrient broth, 1 g / L KCl, 0.25 g / L MgSO4, 0.16415 g / L Ca(NO3)2, 0.9521 mg / L MnCl2 and 0.152 mg FeSO4. MnSO4, MgSO4, FeSO4, (NH4)2SO4, (NH4)2PO4 and CaCl2 added to the above medium were dissolved in ddH2O before use.

[0165] After incubation, lysozyme (0.5 g / L) was added to the culture broth and incubated at 37°C and 200 rpm for 1 hour to remove remaining vegetative cells. The crude spores were collected by centrifugation at 6,000 rpm for 10 minutes. The collected crude spores were washed twice with water, washed with 0.02% SDS, washed twice with water again, and then suspended in PBS solution to purify them. The spore suspension was stored at -20°C. The number of spores was determined by counting colonies after spreading the diluted spore solution on LB agar plates.

[0166] The above spores were dissolved in phosphate-buffered saline (PBS) according to the experimental conditions and prepared in a volume of 100 μl.

[0167] Manufacturing Example 2. Manufacturing of GF427 strain

[0168] The Bacillus velezensis GF427 strain expressing SOD was prepared from the GF423 strain by replacing the promoter sequence of the GF423 sodA gene with a base sequence having a stronger promoter performance to increase SOD activity according to the following method.

[0169] The promoter sequences of the original GF423 strain and the mutant GF427 strain are as follows: GF423 promoter sequence (5'-TTGATTACCACGCTTTCTTTT―GTTACATT-3') (SEQ ID NO: 7) and GF427 promoter sequence (5'-TTGACTTT-ACGCTTTCTTATAGGTTATAAT-3') (SEQ ID NO: 8).

[0170] Substitution of base sequences in the genome was performed by double crossover recombination (Fig. 3). PCR was performed using GF423 genomic DNA as a template and the primers SOD up F and Psodmut R to obtain PCR products, and PCR was performed using GF423 genomic DNA as a template and the primers Psodmut F and SOD dw R to obtain PCR products. Next, the PCR products were cloned into pUori-cm-amp-tsrepA digested with BamHI using the LIC method, thereby constructing pUori-cm-amp-10sod (Fig. 3a).

[0171] Next, PCR was performed using pUori-cm-amp-10sod as a template and the SOD PF and SOD P3R primers to prepare the PCR products, and the PCR products were also prepared using the same template and the SOD P3F and SOD PR primers to prepare the PCR products. Using the LIC method, the PCR products were cloned into pUori-cm-amp-10sod digested with HindIII and ClaI, thereby constructing pUori-cm-amp-P3-SOD to be used for replacement (Fig. 3b). The vector was constructed using E. coli, E. coli C2984H, and the base sequences of the primers used, PCR conditions, and LIC reaction solution compositions are shown in Tables 4, 5, and 6, respectively.

[0172] Primer sequence (5'→3') SEQ ID NO: SOD up Ftccagatcctctacgggcgctgcgtatgctggaa9Psodmut Raacttttctaatctcattataacaaaagaaagcg10Psodmut Fcgctttcttttgttataatgagattagaaaagtt11SOD dw Rgagttttcgttcggatctgtcctccggcactgcg12SOD PFgtaatggaataagccgaaagcttccagagctg13SOD P3Rattataacctataagaaagcgtaaagtcaatgaaaaagctcacaatcc14SOD P3Fttgactttacgctttcttataggttataatgagattagaaaagttc15SOD PRcatcgtttccttatcgatatgaggttctaaag16Cm C Fccgctatctttacaggtacatca17SOD mid Rgaaccgaaacggcctgcag18424 conf Fggcaggcattatattaggcc19424 conf Rgcactgcgttcaaatcagca20SOD DC F2ggattcagcgcttcaaaatc21-10 SOD Rcgcgaacttttctaatctcatta22SOD P2-Rctaatctcattataacctat23cm conf Facctttctgatgtagagaaatataatggttcggg24cm conf Rcggcattatctcatattataaaagccagtcatt25

[0173] PCR Step Temperature Time Initial Denaturation 95℃ 30 sec 30 cycles 95℃ 50℃ 72℃ 30 sec 30 sec 1 min / kb Final Elongation 72℃ 10 min The DNA polymerase used in PCR is Taq DNA polymerase (TAKARA, JAPAN).

[0174] Mixture volume (ul) Linearized vector 2 PCR product 1 1 PCR product 2 1 10X T4 polymerase buffer 1 10X BSA 1 T4 polymerase 0.5 DW 3.5 (~to 10 ul) Total 10

[0175] pUori-cm-amp-10sod was transformed into the GF423 strain by electroporation. The GF423 strain was inoculated into LB-Sor medium (1x LB, 0.5 M sorbitol) and cultured at 37°C and 200 rpm. When the OD reached 0.8, glycine was added to a final concentration of 10 mg / ml and cultured for an additional 1.5 h. After culture, the cells were placed on ice, cooled for 15 min, and centrifuged at 4000 rpm, 4°C, and 10 min. The harvested cells were washed three times with electrotransfection buffer (containing 0.5 M sorbitol, 0.5 M mannitol, and 10% glycerol), and the cells were resuspended in 1 / 50 of the culture volume using the same buffer and used for electroporation. 0.5 ug of pUori-cm-amp-10sod DNA was mixed with the prepared cells and placed in a chilled 1 mm gap electroporation cuvette and incubated on ice for 3 min. Next, a single electric pulse was applied using a MicroPulser Electroporation system (purchased from Bio-rad) at a field strength of 2.5 V, 25 uF, and 200 Ω (time constant = 4.8-5.8). Immediately after the pulse, 1 mL of LB-Sor medium was added and the cells were recovered at 37°C and 200 rpm for 2 h. Afterwards, the cells were plated on antibiotic solid medium (1x LB, cm 2.5 ug / ml, 1.5% agar) and cultured in a 37°C incubator for 48 h. The resulting colonies were confirmed to be transformed by PCR using cm conf F and cm conf R primers. Induction and confirmation of double crossover recombination were performed as follows. The confirmed colonies were added to LB medium and cultured for 24 hours at 37°C and 200 rpm, and then 1 / 10 5After diluting the culture medium, 100 μl was spread on antibiotic solid medium (1x LB, agar 1.5%) and cultured in a 37°C incubator for 20 hours. Among the colonies obtained by culture, PCR was performed using primers Cm CF and SOD mid R to obtain colonies in which single crossover recombination occurred. The obtained colonies were inoculated again into LB broth and cultured for 20 hours at 28°C, and then 1 / 10 5It was diluted and spread on LB solid medium and cultured. The confirmed colonies were stamped on both antibiotic solid medium and general solid medium to confirm the colonies from which the plasmid had been removed. Among the colonies from which the plasmid had been removed, PCR conditions were set so that only colonies that successfully engineered using primers containing the mutation site were confirmed to show a band, and colonies that successfully mutated were selected. The primers used were SOD DC F2, -10 SOD R pair, SOD DC F2, SOD P2 R pair. The selected colonies were PCR-treated using 424 conf F and 424 conf R primers, and after gel purification of the PCR product, the sample was sequenced to confirm the correct substitution and named 'GF427'. This strain was deposited with the Korea Research Institute of Bioscience and Biotechnology on August 14, 2023 (KCTC 15552 BP). To confirm the increase in SOD expression level of the GF427 strain, it was compared with the SOD expression level of the GF424 strain, which has improved SOD activity. The SOD activity of the culture supernatant was measured as follows. Both GF424 and GF427 strains were inoculated with colonies in 1x SYPG (1.5% soybean, 1% yeast extract, 0.5% potassium phosphate dibasic, 1% glucose) containing 50 ug / mL of MnSO4 and cultured for 20 hours. The culture solution was centrifuged at 12,000 rpm for 10 minutes, and the culture supernatant was recovered and used to measure SOD activity. The SOD activity was measured as 22.2 U / mL for GF424 and 67.8 U / mL for GF427, which was about 3 times higher than that of GF424.

[0176] Additionally, GF427 strain spores were prepared using the same method as in Manufacturing Example 1.

[0177] Example 1. Confirmation of the fibrosis prevention effect of SOD-overexpressing strain spores in an animal model of pulmonary fibrosis.

[0178] Example 1.1. Establishment and Testing of an Animal Model of Pulmonary Fibrosis

[0179] In order to confirm whether the SOD overexpressing strain spore (GF424 spore) has a preventive effect in a pulmonary fibrosis animal model, a pulmonary fibrosis animal model was established and an experiment was conducted using the following method. After a one-week acclimatization period, 7-week-old C57BL / 6 male mice were observed for general symptoms to confirm their health status, and healthy animals were used for the test. The mice were divided into three groups: a control group (CTL), a bleomycin-treated group (Bleo), and a test group (GF424 spore-administered group; B+Spore SOD). The test group mice were again divided into three groups and administered GF424 spores prepared in Manufacturing Example 1 at three concentrations (1x10 6 CFU, 1x10 7 CFU and 1x10 8 Each test group was orally administered 100 CFU) once a day (total 5 times per week) for 3 weeks to each test group, and on the 7th day (D0) from the start of spore administration (-D7), bleomycin (Bleomycin sulfate, Millipore) was administered intratracheally once at a dose of 3 U / kg to induce lung fibrosis. The schedule of spore and bleomycin administration for a total of 21 days is shown in Fig. 4. Meanwhile, the control group (CTL) did not induce lung fibrosis and was not administered spores. The bleomycin-treated group (Bleo) was administered 3 U / kg of bleomycin intratracheally once on D0 without spore administration to induce lung fibrosis (see Fig. 4).

[0180] The experiment was terminated 21 days after the first spore administration, and the mice in each group were sacrificed.

[0181] Example 1.2. Measurement of collagen concentration by measuring hydroxyproline concentration.

[0182] Hydroxyproline is an amino acid found almost exclusively in collagen in mammals and is a representative indicator that can directly measure the amount of collagen. The amount of hydroxyproline was measured through the following process. The left lung of a mouse model of pulmonary fibrosis was extracted, 100 μl of sterile water was added per 10 mg of lung tissue, and the tissue was thoroughly ground. The same amount of hydrochloric acid (HCl, ~12 N) as the suspension of the finely ground tissue was added, and the tube was sealed tightly and boiled at 120°C for 3 hours. The cell suspension after the reaction was centrifuged (4°C, 10,000 × g, 3 min), and only the clear supernatant was transferred to a new tube, and the colorimetric assay kit (purchased from Biovison) and a multiplate spectrometer were used to measure the colorimetric response at 560 nm.

[0183] As a result, referring to Figure 5, in the bleomycin-treated group, where pulmonary fibrosis progressed due to bleomycin and the amount of collagen increased, hydroxyproline was measured to be higher than in the control group. On the other hand, in the GF424 spore-administered group, the amount of hydroxyproline (i.e., the amount of collagen) was confirmed to be decreased compared to the bleomycin-treated group at all concentrations. In particular, in the 1x10 7 The decrease was most significant in the CFU concentration administration group.

[0184] Example 1.3. Measurement of the number of immune cells in lung lavage fluid

[0185] The trachea of ​​the pulmonary fibrosis mouse model was partially incised, 1 ml of sterile PBS was added, and the process of collecting it was repeated twice. Approximately 2 ml of the collected bronchoalveolar lavage fluid (BALF) was rapidly centrifuged at 2,200 rpm for 10 minutes at 4°C. The centrifuged supernatant was transferred to a new 2 ml tube, and 0.5 ml of fresh PBS was added to the cell pellet to resuspend the cells. The total cell number of the cell suspension was measured using a hemocytometer, and 1 X 10 per sample was counted. 5 The cells were diluted in PBS at a concentration of 10 cells / 0.5 ml. The prepared cells were attached to microscope slides using a cytocentrifuge at 600 rpm for 5 minutes. The cells on the slides were stained using Diff-Quik reagent (purchased from Sysmex), and the number of macrophages, lymphocytes, and neutrophils among the total lung lavage fluid cells was measured.

[0186] As a result, referring to Figures 6a to 6d, in the bleomycin-treated group, the total cell number (Total BAL cells) and the number of each cell increased compared to the control group, and in the GF424 spore-administered group, the total cell number and the number of each cell decreased compared to the bleomycin-treated group. In particular, most of the immune cells found in lung fibrosis are macrophages, and 1x10 7 It was confirmed that the number of macrophages and lymphocytes was most significantly reduced in the CFU concentration administration group.

[0187] Example 1.4. Measurement of the concentration of transforming growth factor beta 1 (TGF-β1) in lung lavage fluid.

[0188] Transforming growth factor beta 1 (TGF-β1) is a factor that plays a very important role in the development of pulmonary fibrosis. When cells are stimulated by reactive oxygen species or various factors, TGF-β1 is activated, which progresses pulmonary fibrosis through a sub-mechanism. To measure TGF-β1, the lung lavage fluid from Example 1.3 was centrifuged, and the supernatant collected separately was concentrated fourfold. The concentrated lung lavage fluid was measured at 450 nm using a TGF-β1 ELISA kit (purchased from: R&D systems) and a multiplate spectrometer to confirm the concentration of TGF-β1 in the lung lavage fluid.

[0189] As a result, referring to Figure 7, in the bleomycin-treated group, the amount of TGF-β1 due to lung fibrosis significantly increased compared to the control group. On the other hand, in the GF424 spore-administered group, the amount of TGF-β1 decreased compared to the bleomycin-treated group at all concentrations, and in particular, in the 1x10 7 It was significantly reduced in the CFU concentration administration group.

[0190] Example 1.5. Confirmation of pulmonary fibrosis through pathological tissue staining.

[0191] Lung tissues excised from mice sacrificed in Example 1.1 were pretreated by soaking in a 4% paraformaldehyde (PFA) solution for more than a day. Next, the lung tissues were made into paraffin blocks, sectioned into 5-μm-thick sections, and mounted on slides. Tissue sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome stain to compare histopathological changes and observed under a microscope.

[0192] As a result of the observation, referring to Figure 8, it was confirmed that the increased lung fibrosis by bleomycin was effectively reduced at all concentrations of the GF424 spore administration group, and in particular, at 1x10 7 The effect was most effective in the group administered with a concentration of CFU.

[0193] Example 1.6. Measurement of expression levels of pulmonary fibrosis gene markers.

[0194] The expression levels of representative gene markers known to be activated as pulmonary fibrosis progresses, Col1a1 (a factor that increases collagen synthesis), TGF-β1 (a factor that activates myofibroblasts and promotes fibrosis), CTGF (connective tissue growth factor), and IL-6 (inflammatory cytokine), were confirmed using real-time polymerase chain reaction (qRT-PCR).

[0195] In Example 1.1, a portion of the right lung lobe extracted from the sacrificed mouse was finely ground using TRIzol reagent (Purchased from Thermo Fisher Scientific), and total RNA was extracted. The extracted RNA was synthesized into cDNA using M-MLV RT reagent (Purchased from Promega), and real-time polymerase chain reaction (qRT-PCR) was performed using the synthesized cDNA according to the Go Taq qPCR master mix (Purchased from Promega) protocol. The relative mRNA expression level was corrected using 18S rRNA as an internal control, and the value was calculated using the 2-ΔΔCq method. The primer information used in this experiment and the following qRT-PCR experiments is summarized in Table 7 below.

[0196] Gene name Primer sequence (5'→3') Sequence number Col1a1 (Type 1 collage) F: GCTCCTCTTAGGGGCCACT 26 R: CCACGTCTCACCATTGGGG 27 α-SMA (α-Smooth Muscle Actin) F: GCTCCTCTTAGGGGCCACT 28 R: CCCAGACATCAGGGAGTAATG 29 TGF-β1 (Transforming growth factor beta 1) F: AGACCACATCAGCATTGAGTG 30 R: GGTGGCAACGAATGTAGCTGT 31 CTGF (Connective tissue growth factor) F: GGGCCTCTTCTGCGATTTC 32 R: ATCCAGGCAAGTGCATTGGTA 33 IL-6 (InterLeukin-6) F: CAAAGCCAGAGTCCTTCAGAG 34 R: GTCCTTAGCCACTCCTTCTG 35 TNF-α (tumor necrosis factor-α) F: CTTCTGTCTACTGAACTTCGGG36R: CAGGCTTGTCACTCGAATTTTG37IL-1βF: ACGGACCCCAAAAGATGAAG38R: TTCTCCACAGCCACAATGAG3918S rRNAF: CAGCGTGGTCAGGATAGAAC40R: CTTGATTTGAATGCAGCGGAC41

[0197] As a result, referring to Figures 9a to 9d, the expression of all four genes tended to decrease in the GF424 spore administration group compared to the bleomycin treatment group.

[0198] Example 1.7. Measurement of the concentration of reactive oxygen byproducts

[0199] Lipid peroxidation is a representative mechanism of cell damage in both animals and plants. The lung tissue concentrations of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), the most common byproducts of lipid peroxidase, were measured as follows.

[0200] A portion of the lung tissue extracted from the mouse sacrificed in Example 1.1 was finely ground in 1X RIPA buffer (including protease inhibitor cocktail, purchased from Abcam). The tissue suspension was centrifuged at 4℃, 12,000 rpm, and 10 min, and only the clear supernatant was separated and transferred to a new 1.5 ml tube. Protein quantification was measured at 562 nm using a BCA assay kit (purchased from Thermo Fisher Scientific) on a multiplate spectrometer. Byproduct quantification was performed using the same amount of sample as the MDA ELISA kit (purchased from Cell Biolabs, cat#. STA-832) and HNE ELISA kit (purchased from Cell biolabs, cat#. STA-838) according to the manufacturers' manuals, and the concentration was measured at 450 nm using a multiplate spectrometer.

[0201] As a result, referring to Figures 10a and 10b, it was confirmed that the amount of reactive oxygen byproducts (MDA and 4-HNE) present in lung tissue was statistically significantly reduced in the GF424 spore administration group compared to the bleomycin treatment group. Overall, 1x10 7 It was more effectively reduced in CFU concentration.

[0202] Example 2. Confirmation of the preventive effect of strains with different SOD expression levels in an animal model of pulmonary fibrosis.

[0203] Example 2.1. Preparation and testing of spores of strains with different SOD expression levels.

[0204] To determine whether the level of superoxide dismutase (SOD) expression directly affects the preventive effect of pulmonary fibrosis, spores of three strains of Bacillus velezensis with different levels of SOD expression were prepared. The three strains are a strain with the SOD gene deleted (knock-out, indicated by '-', GF423Δ), a wild strain with SOD expression isolated from nature (wild, indicated by '+', GF423), and an overexpression strain genetically engineered to overexpress SOD (overexpression, indicated by '++', GF424).

[0205] The knockout strain Bacillus velezensisΔsodA lacking sodA function was constructed by double crossover recombination as follows. First, a pair of DNA fragments flanking the sodA gene for homologous recombination were amplified by PCR from its chromosome using primers for the upstream fragment (5'aaacagctgggatgaacacaagtgagag 3' (SEQ ID NO: 42) and 5' cacactcttaagtttgcttccaattctggaagtttgtaag 3' (SEQ ID NO: 43)) and primers for the downstream fragment (5' ctactgacagcttccaaggatacctgaactaccaaaaccg 3' (SEQ ID NO: 44) and 5' aaacagctgaagctcatgaccacagcaag 3' (SEQ ID NO: 45)). The erythromycin resistance (EmR) gene was amplified by PCR from pDG1664 (Guerout-Fleury, AM ey et al., Gene 180:57-61 (1996)) using primers (5' gaagcaaacttaagagtgtg 3' (SEQ ID NO: 46) and 5' tccttggaagctgtcagtag 3' (SEQ ID NO: 47)). The upstream fragment, the EmR gene, and the downstream fragment were sequentially assembled and cloned into the PvuII site of the pUori-ts plasmid, which contains a temperature-sensitive replication origin operating in Bacillus subtilis (J Bacteriol 176, no. 6; 1761-1763). The resulting plasmid was transformed into Bacillus velezensis GF423 by electroporation (see literature [Zhang GQ et al., Anal Biochem. 409:130-137 (2011)]). The sodA knockout mutant was selected from the transformants resistant to erythromycin (5 μg / ml) at 42°C, followed by plasmid curing at 30°C. The genomic status around the sodA region was confirmed by PCR and DNA sequencing of the PCR products.

[0206] The normal strain is Bacillus velezensis GF423 strain (Accession No.: KCTC 13222BP, Accession Date: 20170306), and the overexpressing strain is Bacillus velezensis GF424 strain (Accession No.: KCTC 13227BP, Accession Date: 20170313). The Bacillus velezensis GF424 strain is a strain obtained by inducing a mutation in the Bacillus velezensis GF423 strain by irradiating it with UV to improve the expression of the sodA gene.

[0207] Spores prepared by the method described in Manufacturing Example 1 from the above strain were administered to mice with pulmonary fibrosis induced by bleomycin at a dose of 1x10 7 Oral administration was performed at a concentration of 10 CFU. Spore administration and bleomycin treatment were performed in the same manner as described in Example 1.1, and the control and bleomycin-treated groups were prepared in the same manner. The experiment was terminated 21 days after the first spore administration, and mice in each group were sacrificed (see Figure 4). For information on Bacillus species strains and species strain spores, see Table 8.

[0208] Spore substance name description strain name SOD activity (relative value) GF423 spore Wild-type GF4231 GF424 spore SOD overexpression strain GF424 ~ 3.5 times GF427 spore SOD overexpression strain GF427 ~ 11 times GF423Δ spore SOD knockout strain GF423Δ 0.04 times

[0209] Example 2.2. Measurement of collagen concentration by measuring hydroxyproline concentration.

[0210] The measurement of hydroxyproline, a representative indicator that can directly measure the amount of collagen, was conducted in the same manner as in Example 1.2.

[0211] As a result of the measurement, referring to Figure 11, the group administered with spores of the SOD knockout strain (B+Spore SOD(-)) had a higher amount of hydroxyproline than the bleomycin-treated group. On the other hand, the groups administered with spores of the SOD normal strain (B+Spore SOD(+)) and spores of the SOD overexpressing strain (B+Spore SOD(++)) showed a decrease in hydroxyproline. In particular, the group administered with spores of the SOD overexpressing strain showed the greatest decrease.

[0212] Example 2.3. Measurement of the number of immune cells in lung lavage fluid

[0213] The method for measuring the number of macrophages, lymphocytes, and neutrophils among total lung lavage fluid cells was the same as that of Example 1.3.

[0214] As a result of the measurement, referring to Figures 12a to 12d, a decrease in the number of immune cells in the lung lavage fluid was observed in all groups administered SOD knockout strain spores, SOD normal strain spores, and SOD overexpression strain spores compared to the bleomycin treatment group. The decrease in the number of immune cells was greater in the group administered SOD normal strain spores than in the group administered SOD knockout strain spores, and the most significant decrease was observed in the group administered SOD overexpression strain spores.

[0215] Example 2.4. Measurement of the concentration of growth factor beta 1 in lung lavage fluid

[0216] Measurement of the concentration of TGF-β1 using lung lavage fluid was performed in the same manner as in Example 1.4.

[0217] As a result of the measurement, referring to Figure 13, the amount of TGF-β1, which had increased due to lung fibrosis in the bleomycin treatment group, decreased more significantly in the order of the SOD knockout strain spore administration group, the SOD normal strain spore administration group, and the SOD overexpression strain spore administration group.

[0218] Example 2.5. Confirmation of pulmonary fibrosis through pathological tissue staining

[0219] The pathological tissue staining method using the extracted lung tissue was performed in the same manner as in Example 1.5.

[0220] Referring to Figure 14, which shows the tissue staining results of the control group, bleomycin-treated group, and groups administered spores of strains with different SOD expression levels (spores of knockout, normal, and overexpressing strains), it was confirmed that bleomycin-induced lung fibrosis was improved by spore SOD. In particular, the improvement in lung fibrosis was most remarkable in the group administered spores of the SOD-overexpressing strain.

[0221] Example 2.6. Measurement of expression levels of pulmonary fibrosis gene markers

[0222] Representative gene markers known to be activated as pulmonary fibrosis progresses, Col1a1, α-SMA, TGF-β1, CTGF, TNF-α, and IL-6, were identified for gene expression by real-time polymerase chain reaction using the primers in Table 7 (same method as Example 1.6).

[0223] As a result of the measurement, referring to Figures 15a to 15f, it was confirmed that the expression of gene markers that had been increased by bleomycin significantly decreased depending on the SOD expression level of the administered spores. In the SOD knockout strain spore administration group, the expression levels of Col1a1 (Figure 15a), α-SMA (Figure 15b), and TGF-β1 (Figure 15c) showed values ​​similar to or higher than those of the bleomycin treatment group. On the other hand, in the SOD normal strain spore administration group and the SOD overexpression strain spore administration group, the expression of the six markers was significantly decreased compared to the bleomycin treatment group and the SOD knockout strain spore administration group. In particular, in the SOD overexpression strain spore administration group, the expression levels of the TGF-β1 (Figure 15c), CTGF (Figure 15a), and IL-6 (Figure 15f) genes were statistically significantly decreased.

[0224] Example 2.7. Measurement of protein expression levels of pulmonary fibrosis markers

[0225] A portion of the right lung lobe of the pulmonary fibrosis model mouse sacrificed in Example 2.1 was finely ground in 1X RIPA buffer (containing protease and phosphatase inhibitor cocktail, purchased from Abcam). The tissue suspension was centrifuged (4°C, 12,000 rpm, 10 min), and only the clear supernatant was separated and transferred to a new 1.5 ml tube. For Western blotting, protein quantification was performed using a BCA assay kit (purchased from Thermo Fisher Scientific) and measured at 562 nm with a multiplate spectrometer. An equal amount of sample obtained through protein quantification was dispensed onto each line of the gel, followed by electrophoresis. The gel was transferred to a PVDF membrane, and the primary antibody (TGF-β (1:1000, purchased from: abcam), phospho-Smad2 / 3 (1:1000, purchased from: abcam), Smad2 / 3 (1:1000, purchased from: abcam), α-SMA (1:1000, purchased from: CST), Col1a1 (1:1000, purchased from: CST), and GAPDH (1:2000, purchased from: CST)) was attached and reacted overnight at 4°C. The next day, the membrane was thoroughly washed with 1X PBST buffer and then reacted with the secondary antibody (anti-rabbit) for 1 hour at room temperature. The band pattern was measured using an imaging device (ChemiDoc imaging system, purchased from: Bio-rad).

[0226] As a result of the measurement, referring to Figure 16, in the case of the bleomycin treatment group, it was confirmed that the TGF-β1-Smad2 / 3 signaling, which is a signaling pathway related to lung fibrosis, was activated, and the expression of TGF-β1 and phospho-Smad2 / 3 proteins increased. In addition, the production of α-SMA and Col1a1, which are the results of signaling, also increased (Lane 2). Meanwhile, in the SOD knockout strain spore administration group and the SOD normal strain spore administration group, the expression of phospho-Smad2 / 3 protein was slightly decreased compared to the bleomycin treatment group, and the expression levels of α-SMA and Col1a1 were also slightly decreased (Lane 3 and Lane 4). In the group administered with SOD overexpressing strain spores, it was confirmed that the expression of the upper TGF-β1 was suppressed, the phosphorylation of the lower Smad2 / 3 was reduced, and the production of α-SMA and Col1a1, which are the results of signal transduction, was significantly reduced (Lane 5).

[0227] Example 2.8. Survival rate measurement

[0228] To determine the survival rate of a lung fibrosis mouse model induced after administering spores of strains with different expression levels of SOD, the survival rate was estimated using the Kaplan-Meier method, and the log-rank test was used to calculate statistical differences.

[0229] As a result of the estimation, referring to Figure 17, the survival rate of the control group (CTL, N=13 / 13) was 100%, while the survival rate of the bleomycin treatment group (Bleo, N=10 / 19) decreased to 52.6%. On the other hand, the survival rate of the SOD knockout strain spore administration group (B+Spore SOD(-), N=5 / 7) was 71.4%, and the survival rate of the SOD normal strain spore administration group (B+Spore SOD(+), N=5 / 7) also showed 71.4%, which was no different from the SOD knockout strain spore administration group. On the other hand, the survival rate of the SOD overexpressing strain spore administration group (B+Spore SOD(++), N=19 / 20) significantly increased to 95%. The survival rate results of the experimental groups are shown in Table 9 below.

[0230] Model name Survival rate (%), 21-day standard P-value CTL vs. group Bleo vs. group Control group (CTL) 100-** Bleomycin treatment group (Bleo) 52.6** SOD knockout strain spore (Spore SOD (-)) administration group 71.4* nsSOD normal strain spore (Spore SOD (+)) administration group 71.4* nsSOD overexpressing strain spore (Spore SOD (++)) administration group 95ns**

[0231] Through this, it was confirmed that SOD has a preventive effect on pulmonary fibrosis and is also effective in increasing the survival rate.

[0232] Example 2.9. Measurement of the concentration of reactive oxygen byproducts

[0233] The method for measuring the concentration of reactive oxygen byproducts (MDA and 4-HNE) in the extracted lung tissue was the same as that in Example 1.7.

[0234] As a result, it was confirmed that the concentration of by-products increased by bleomycin significantly decreased depending on the SOD expression level of the administered spores. The concentrations of MDA and 4-HNE significantly decreased in the order of the SOD knockout strain spore administration group, the SOD normal strain spore administration group, and the SOD overexpressing strain spore administration group (Fig. 18a and Fig. 18b).

[0235] Example 3. Comparison of the preventive effects of SOD-overexpressing strain spores and SOD in an animal model of pulmonary fibrosis.

[0236] Example 3.1. Preparation and testing of samples

[0237] SOD is difficult to maintain protein activity and stability when administered orally alone, so it can be mostly decomposed by gastric acid before it becomes effective in the body. On the other hand, spores produced by bacteria are resistant to harsh environments (e.g., extreme temperatures, acids or alkalis, drying, and toxic chemicals), so they are expected to be effective even when administered orally. To compare the effects of the two substances, SOD was administered in a dose-dependent manner with free SOD (B+Free SOD, 2 units / mouse) and GF424 spores (B+Spore SOD, 1x10 7 The preventive effect on lung fibrosis was compared in a lung fibrosis model mouse by oral administration in the form of (CFU / mouse).

[0238] Glass SOD was prepared by preparing a culture solution of Bacillus velezensis GF424 strain (KCTC 13227BP, deposit date: March 13, 2017) and extracting and purifying it using the following method.

[0239] First, a single colony formed on LB agar medium (LB (Luria-Bertani) agar; tryptophan 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L) was inoculated into 30 mL of LB medium and cultured at 37°C for 12 h. This seed culture was then inoculated into 3 L of LB medium containing 1 mM manganese sulfate (MnSO4) and cultured at 37°C for 20 h.

[0240] Next, the cell culture medium was centrifuged at 4℃, 3,578 xg for 20 minutes, and the supernatant was concentrated 10-fold using ultrafiltration (UF, MWCO 10,000). The concentrate was filtered through a sterilizing filter and then freeze-dried. SOD activity was analyzed using an SOD assay kit (purchased from: Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits superoxide radicals by 50%. The dried SOD enzyme was dissolved in PBS so that it exhibited an activity of 2 units and prepared in a volume of 100 μl.

[0241] Administration of GF424 spores or free SOD and bleomycin treatment of the two test groups were performed in the same manner as described in Example 1.1, and the control and bleomycin treatment groups were prepared in the same manner. The experiment was terminated 21 days after the first spore administration, and the mice in each group were sacrificed (see Fig. 4).

[0242] Example 3.2. Measurement of collagen concentration by measuring hydroxyproline concentration.

[0243] The measurement of hydroxyproline, which can confirm the level of collagen, was performed in the same manner as in Example 1.2.

[0244] As a result of the measurement, referring to Figure 19, the hydroxyproline concentrations of the free SOD administration group and the GF424 spore administration group were measured. Unlike the hydroxyproline concentrations of the free SOD administration group, which were almost the same as those of the bleomycin treatment group, the hydroxyproline value of the GF424 spore administration group was statistically significantly reduced. Therefore, it was confirmed that the administration of GF424 spores was effective in preventing pulmonary fibrosis.

[0245] Example 3.3. Measurement of the concentration of growth factor beta 1 in lung lavage fluid

[0246] Measurement of the concentration of TGF-β1 using lung lavage fluid was performed in the same manner as in Example 1.4.

[0247] As a result of the measurement, referring to Figure 20, the amount of TGF-β1 that had increased due to lung fibrosis in the bleomycin-treated group was very effectively reduced in the GF424 spore-administered group, and the effect of free SOD was minimal.

[0248] Example 3.4. Confirmation of pulmonary fibrosis through pathological tissue staining

[0249] The pathological tissue staining method using the extracted lung tissue was performed in the same manner as in Example 1.5.

[0250] Referring to Figure 21, which shows the tissue staining results of the control group, bleomycin-treated group, free SOD-administered group, and GF424 spore-administered group, bleomycin-induced lung fibrosis showed a slight improvement effect even with free SOD, but the GF424 spore-administered group showed a greater effect.

[0251] Example 3.5. Measurement of expression levels of pulmonary fibrosis gene markers

[0252] Representative gene markers known to be activated as pulmonary fibrosis progresses, Col1a1, α-SMA, TGF-β1, CTGF, TNF-α, and IL-6, were identified for gene expression by real-time polymerase chain reaction using the primers in Table 7 (same method as Example 1.6).

[0253] As a result of the measurement, referring to FIGS. 22a to 22f, the profibrotic markers (Col1a1, α-SMA, TGF-β1, and CTGF) and proinflammatory cytokines (TNF-α and IL-6) that were increased by bleomycin showed almost no change in the free SOD administration group, but were significantly reduced in the GF424 spore administration group.

[0254] Example 3.6. Measurement of protein expression levels of pulmonary fibrosis markers

[0255] Western blot to measure the protein expression level of lung fibrosis markers in lung fibrosis model mice was performed in the same manner as in Example 2.7.

[0256] As a result of the measurement, referring to Figure 23, it was confirmed that the expression of TGF-β1 and phospho-Smad2 / 3 proteins was reduced by inhibition of TGF-β1-Smad2 / 3 signaling, which is a major mechanism of pulmonary fibrosis, in both the free SOD administration group and the GF424 spore administration group. Meanwhile, the fibrosis markers α-SMA and Col1a1 showed a tendency to decrease in both administration groups, but in the case of Col1a1, it was reduced more effectively in the GF424 spore administration group (Lane 4).

[0257] Example 3.7. Measurement of the concentration of reactive oxygen byproducts

[0258] The method for measuring the concentration of reactive oxygen byproducts (MDA and 4-HNE) in the extracted lung tissue was the same as that in Example 1.7.

[0259] As a result, the concentrations of MDA and 4-HNE, which are reactive oxygen byproducts that were increased by bleomycin, showed little change in the free SOD administration group, but significantly decreased in the GF424 spore administration group (Fig. 24a and Fig. 24b).

[0260] Example 4. Confirmation of the preventive effect of SOD-overexpressing strain spores in primary lung fibroblasts isolated from an animal model of lung fibrosis.

[0261] Example 4.1. Isolation and culture of mouse primary lung fibroblasts

[0262] The GF424 spore administration group (B+Spore SOD) was administered 1x10 spores prepared by the method described in Manufacturing Example 1. 7 After oral administration of spores at a concentration of 10 CFU, pulmonary fibrosis was induced with bleomycin. Spore administration and bleomycin treatment were performed in the same manner as described in Example 1.1, and the control and bleomycin-treated groups were prepared identically. The experiment was terminated 21 days after the first spore administration, and mice in each group were sacrificed (see Figure 4).

[0263] After laparotomy of the pulmonary fibrosis model mouse, the lungs were sufficiently perfused with PBS (3% Penicillin / Streptomycin). The cleaned whole lungs were removed and washed with fresh PBS containing 3% antibiotics (Penicillin / Streptomycin). The washed lung tissues were transferred to a 6-well plate and dissected with sterile scissors. The dissected tissues were transferred to a new 15 ml tube and added to 5 ml of RPMI-1640 (2% Penicillin / Streptomycin, glutamine-free) medium containing 600 units / ml DNase I (purchased from Sigma, product number: DN25) and 0.14 units / ml liberase (purchased from Roche, product number: 05401119001), and incubated at 37°C for 1 hour. After the reaction was completed, the tube containing the tissue was filtered sequentially through a 40 μm filter, a 70 μm filter, and a 100 μm filter, and the final collected tube was centrifuged at 520 xg for 10 minutes. Finally, the supernatant was removed, and the remaining cells were suspended in an appropriate amount of DMEM (15% FBS, 2% Penicillin / Streptomycin) medium to prepare a cell suspension.

[0264] Example 4.2. Confirmation of pulmonary fibrosis using immunofluorescence staining.

[0265] When lung fibroblasts are exposed to external stimuli and the TGF-β pathway is activated, morphological changes (elongated shape) and functional changes (increased production of ECM products such as α-SMA and collagen) occur, leading to the progression of lung fibrosis. To confirm these changes, primary lung fibroblasts isolated from the lungs of mice with induced lung fibrosis were stained for α-SMA and Col1a1, representative markers of lung fibrosis, using the following immunofluorescence staining method.

[0266] After measuring the cell number using the mouse cell suspension of Example 4.1, 2 X 10 were seeded in a 60 mm culture dish. 6 Cells were added and cultured for 1 day. The next day, the medium was replaced with fresh DMEM (15% FBS, 2% Penicillin / Streptomycin), and the medium was replaced with fresh medium once every 2 to 3 days until the cells filled 85% to 90% of the surface area of ​​a 60 mm culture dish. After that, the cells in the dish were removed, 40,000 cells per well were added to an 8-channel slide, and when the cells were more than 80% filled, the medium was removed, washed with fresh PBS (repeated 3 times), and then removed. After fixing with 4% PFA for 1 hour at room temperature, the cells were washed with PBS, and blocking buffer (purchased from: Invitrogen, Product No.: 00-4952-54) was added and reacted for 1 hour at room temperature. After PBS washing (repeated 3 times), the primary antibodies α-SMA (1:200, Mouse mAb, purchased from: CST, Product No.: 48938) and Col1a1 (1:200, Rabbit mAb, purchased from: CST, Product No.: 72026) were diluted in blocking buffer and added to each well and reacted overnight at 4℃. The next day, after PBS washing (repeated 3 times), the secondary antibodies anti-Rabbit-Alexa 555 and anti-Mouse-Alexa 488 were diluted 1:500 in PBS and added to each well, and reacted for 1 hour at room temperature under light blocking. Next, after PBS washing (repeated 3 times), the mounting solution containing DAPI was dropped on each stained cell from which PBS had been completely removed, and a cover glass was placed and observed under a confocal microscope (purchased from: Zeiss).

[0267] As a result of the measurement, referring to Figure 25, in the bleomycin-treated group, the cell shape changed to an elongated shape and the expression of α-SMA and Col1a1 increased. On the other hand, in the GF424 spore-administered group, morphological changes (shortened cell shape) and functional changes (decreased expression of α-SMA and Col1a1) similar to the control group were observed, confirming a preventive effect on pulmonary fibrosis.

[0268] Example 4.3. Measurement of expression levels of pulmonary fibrosis gene markers

[0269] To verify whether there were changes in the expression levels of lung fibrosis genes in primary lung fibroblasts as in lung tissues of mice induced with lung fibrosis, real-time polymerase chain reaction was performed using the primers in Table 7 (same method as Example 1.6).

[0270] As representative gene markers known to be activated as pulmonary fibrosis progresses, such as TGF-β1 (a factor that activates myofibroblasts and promotes fibrosis), Col1a1 (a factor that increases collagen synthesis), TNF-α, and IL-6 (inflammatory cytokines), were measured for their expression levels, and as shown in Figures 26a to 26d, the pro-fibrotic markers (TGF-β and Col1a1) and pro-inflammatory cytokines (TNF-α and IL-6), which were increased by bleomycin, were significantly decreased in the GF424 spore-administered group.

[0271] Example 5. Confirmation of the effect of combined oral SOD and strain spores in an animal model of pulmonary fibrosis.

[0272] Example 5.1. Preparation and testing of samples

[0273] An experiment was prepared to confirm the preventive effect of pulmonary fibrosis by combined administration of oral SOD and strain spores. First, GF103, an oral SOD with enhanced stability in gastric acid, was prepared at three concentrations (1, 10, 20 units / mouse), and 1X10 wild-type strain GF423 spores (B+Spore SOD(+)) prepared in Manufacturing Example 1 were added. 7 It was prepared at a concentration of CFU / mouse. In addition, as a comparison group for the parallel treatment effect, overexpressing strain GF424 spores (B+Spore SOD(++), 1x10 7 CFU / mouse) was prepared.

[0274] GF103 (SEQ ID NO: 6; Met-deleted SodA2) was prepared by coating methionine-deleted SodA2 with shellac as follows. Shellac (EXCELACS co., LTD., Bangkok) was dissolved in 100% ethanol to a concentration of 3% and filtered using a 0.2 μm sterile filter. The shellac solution dissolved in ethanol was diluted 1 / 20 with sterile 1x PBS to prepare a shellac solution. Lyophilized purified SodA2 was dissolved to 20 mg / ml and sterilized using a 0.2 μm pore filter. The prepared shellac solution and the dissolved SOD were mixed in a 1:1 ratio with stirring. Stirring was continued for 10 minutes. The well-stirred material was lyophilized. This resulted in the final lyophilized shellac-coated SodA2. Dextrin was added to freeze-dried shellac-coated SodA2 at a ratio of 1:9 to 12 (shellac-coated SodA2: dextrin), and the activity was measured to obtain a final activity of 90 to 110 U / mg.

[0275] Freeze-dried purified SodA2 without shellac coating was used as the glass SOD.

[0276] Spore administration and bleomycin treatment were performed in the same manner as described in Example 1.1, and the control and bleomycin treatment groups were prepared in the same manner. The experiment was terminated 21 days after the first spore administration, and the mice in each group were sacrificed (see Fig. 4).

[0277] Example 5.2. Measurement of collagen concentration by measuring hydroxyproline concentration.

[0278] The measurement of hydroxyproline, which can confirm the level of collagen, was performed in the same manner as in Example 1.2.

[0279] As a result of measuring the hydroxyproline concentration, the hydroxyproline concentration was decreased in all experimental groups compared to the bleomycin treatment group, i.e., it was confirmed that the hydroxyproline concentration decreased in a concentration-dependent manner even in the group administered oral SOD (GF103) alone. Meanwhile, in the group administered oral SOD and GF423 spores together, it was confirmed that there was a significant effect at the concentrations of 1 unit / mouse and 10 units / mouse of oral SOD. In particular, the group administered oral SOD and GF423 spores together at a concentration of 10 units / mouse showed the best effect, and the hydroxyproline concentration was measured to be lower than that of the group administered GF424 spores, which are spores of an overexpressing SOD strain (Fig. 27).

[0280] Example 5.3. Measurement of the concentration of growth factor beta 1 in lung lavage fluid

[0281] Measurement of the concentration of TGF-β1 using lung lavage fluid was performed in the same manner as in Example 1.4.

[0282] As a result of the measurement, referring to Figure 28, it was confirmed that the amount of TGF-β1, which had increased due to pulmonary fibrosis in the bleomycin-treated group, showed almost no change when oral SOD (GF103) was administered alone, but was effectively reduced in the group administered oral SOD and GF423 spores in combination. In particular, the group administered oral SOD and GF423 spores in combination at a concentration of 20 units / mouse showed the best effect, and the TGF-β1 concentration was measured to be lower than that of the group administered overexpressing SOD strain GF424 spores. In contrast, there was almost no change in the group administered oral SOD alone.

[0283] Example 5.4. Confirmation of pulmonary fibrosis through pathological tissue staining

[0284] The pathological tissue staining method using the extracted lung tissue was performed in the same manner as in Example 1.5.

[0285] Referring to Figure 29, which is the tissue staining results of the control group, the bleomycin treatment group, the GF103 administration group at different concentrations, the GF423 spore administration group, the GF103 and GF423 spore combination administration group at different concentrations, and the GF424 spore administration group, it was confirmed that lung fibrosis induced by bleomycin was improved in a concentration-dependent manner even in the oral SOD mono-administration group, and that it was also improved in a concentration-dependent manner in the group administered oral SOD and GF423 spore combination, confirming the synergistic effect of combination administration.

[0286] Example 5.5. Measurement of expression levels of pulmonary fibrosis gene markers

[0287] Representative gene markers known to be activated as pulmonary fibrosis progresses, Col1a1, α-SMA, TGF-β1, IL-6, TNF-α, and IL-1β, were identified for gene expression by real-time polymerase chain reaction using the primers in Table 7 (same method as Example 1.6).

[0288] As a result of the measurement, referring to FIGS. 30a to 30f, pro-fibrotic markers (Col1a1, α-SMA, and TGF-β) and pro-inflammatory cytokine markers (IL-6, TNF-α, and IL-1β) that were increased by bleomycin were effectively reduced by oral SOD, but there was no synergistic effect by combined administration of oral SOD and GF423 spores.

[0289] Example 5.6. Measurement of the concentration of reactive oxygen byproducts

[0290] In addition to MDA and 4-HNE, which are byproducts of lipid peroxides produced by reactive oxygen species, 8-hydroxydeoxyguanosine (8-OHdG), which is produced when oxidative DNA damage is induced by reactive oxygen species, is also an important marker of oxidative stress. Therefore, the concentrations of reactive oxygen species byproducts (MDA and 4-HNE) in the excised lung tissue were measured using the same method as in Example 1.7, and the concentration of 8-OHdG was measured using the following method, and the values ​​in each experimental group were compared.

[0291] 8-OHdG was extracted from lung tissues cut into small pieces using the DNeasy Blood & Tissue Kit (Qiagen, cat#. 69504), and then the same amount of DNA was used for Oxidative DNA damage ELISA kit (Cell Biolabs, cat#. STA-320) according to the manufacturer's manual. The concentration was measured at 450 nm using a multiplate spectrometer.

[0292] As a result, MDA, 4-HNE, and 8-OHdG present in lung tissue all showed a concentration-dependent tendency to decrease when treated with oral SOD, and were generally further decreased by combined administration of oral SOD and GF423 spores (Figs. 31a to 31c).

[0293] Example 6. Confirmation of the anti-fibrotic effect of SOD-overexpressing strain spores in an animal model of pulmonary fibrosis.

[0294] Example 6.1. Establishment and Testing of an Animal Model of Pulmonary Fibrosis

[0295] After administering GF424 spores to an animal model of pulmonary fibrosis and inducing pulmonary fibrosis, it was confirmed that spore administration had a preventive effect against pulmonary fibrosis (see Example 1, etc.). Accordingly, pulmonary fibrosis was first induced, and then spores of the GF424 strain and spores of the GF427 strain, which were developed to produce approximately four times more SOD than GF424, were administered, and related indicators were confirmed. For the production of the GF427 strain, see Manufacturing Example 2.

[0296] Seven-week-old C57BL / 6 male mice were acclimated for one week, and then observed for general symptoms to determine their health. Healthy animals were used in the experiment. The mice were divided into four groups: a control group (CTL), a bleomycin-treated group (Bleo), and a test group (i.e., a GF424 spore-administered group or a GF427 spore-administered group).

[0297] First, pulmonary fibrosis was induced by intratracheal administration of bleomycin (Bleomycin sulfate, Millipore) at a dose of 3 U / kg (D0). Five days later, the test group mice were administered 1x10 GF424 spores prepared in Preparation Example 1. 7 CFU concentration and three concentrations of spores (1x10) of the GF427 strain prepared in Manufacturing Example 2 5 CFU concentration, 1x10 6 CFU concentration and 1x10 7(CFU concentration) was administered orally to each test group of mice once a day for 12 days (total of 5 times per week). The experiment was terminated on the 16th day from the first bleomycin administration, and the mice in each group were sacrificed. The control group (CTL) did not induce pulmonary fibrosis and was not administered spores. The schedule of spore and bleomycin administration for a total of 16 days is shown in Figure 32.

[0298] Example 6.2. Measurement of collagen concentration by measuring hydroxyproline concentration.

[0299] The measurement of hydroxyproline, which can confirm the level of collagen, was performed in the same manner as in Example 1.2.

[0300] Hydroxyproline concentration measurements showed that hydroxyproline concentrations were reduced in all experimental groups compared to the bleomycin-administered group, and GF427 spores reduced collagen production even at low doses. In particular, 1x10, the same dose as GF424 spores, was administered. 7 In the group administered with CFU concentration, the GF427 spore administration group showed a significant decrease compared to the GF424 spore administration group (Fig. 33).

[0301] Example 6.3. Measurement of the concentration of growth factor beta 1 in lung lavage fluid

[0302] Measurement of the concentration of TGF-β1 using lung lavage fluid was performed in the same manner as in Example 1.4.

[0303] As a result of the measurement, referring to Figure 34, the amount of TGF-β1 that increased due to lung fibrosis in the bleomycin-treated group was lower in the group administered a small amount of GF427 spores (1x10 5 It was confirmed that the concentration of CFU decreased in the GF427 spore-administered group (1x10 6 CFU concentration and 1x10 7 The group administered with CFU concentration showed a more significant decrease than when administered with GF424 spores.

[0304] Example 6.4. Confirmation of pulmonary fibrosis through pathological tissue staining

[0305] The pathological tissue staining method using the extracted lung tissue was performed in the same manner as in Example 1.5.

[0306] Referring to Figure 35, which shows the tissue staining results of the control group, bleomycin-treated group, GF424 spore-administered group, and GF427 spore-administered group at different concentrations, it was confirmed that bleomycin-induced lung fibrosis was effectively reduced at all concentrations of GF427 spores, and in particular, at 1x10 7 The most significant improvement was observed in CFU concentration.

[0307] Example 6.5. Measurement of expression levels of pulmonary fibrosis gene markers

[0308] Representative gene markers known to be activated as pulmonary fibrosis progresses, Col1a1, TGF-β1, CTGF, IL-6, TNF-α, and IL-1β, were identified for gene expression by real-time polymerase chain reaction using the primers in Table 7 (same method as Example 1.6).

[0309] As a result of the measurement, referring to FIGS. 36a to 36f, the pro-fibrotic markers (Col1a1, TGF-β, and CTGF) and pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) that were increased by bleomycin were reduced by the administration of spores of all SOD overexpressing strains (GF424 and GF427), and in particular, the GF427 spore administration group showed a remarkable reduction effect.

[0310] Example 6.6. Measurement of protein expression levels of pulmonary fibrosis markers

[0311] Western blot to measure the protein expression level of lung fibrosis markers in lung fibrosis model mice was performed in the same manner as in Example 2.7.

[0312] As a result of the measurement, referring to Figure 37, in both groups administered with SOD overexpressing strains (GF424 and GF427), the expression of TGF-β1 decreased due to the inhibition of TGF-β1-Smad2 / 3 signaling, which is the main mechanism of lung fibrosis, and the phosphorylation of phospho-Smad2 / 3 decreased. In addition, fibrosis markers α-SMA and Col1a1 also decreased in both groups administered with SOD overexpressing strains (GF424 and GF427). In particular, the protein expression level was 1x10 of GF427 spores. 7 There was a significant decrease in CFU concentration.

[0313] Example 6.7. Measurement of the concentration of reactive oxygen byproducts

[0314] The concentrations of MDA and 4-HNE, byproducts generated from lipid peroxides by reactive oxygen species, were measured using the same method as in Example 1.7, and the concentration of 8-hydroxydeoxyguanosine (8-OHdG), which is generated when oxidative DNA damage is induced by reactive oxygen species, was measured using the same method as in Example 5.6. In addition, the degree of thiol loss in the human body due to ROS was measured to compare the degree of oxidative stress.

[0315] MDA, 4-HNE, and 8-OHdG present in lung tissue all showed a concentration-dependent decrease in the spores of the SOD-overexpressing strains (GF424 and GF427). Meanwhile, it was confirmed that free thiols lost by ROS were restored by the spores of the SOD-overexpressing strain. The restoration of the lost thiols was more effective as the concentration of the spores of the SOD-overexpressing strain increased (Figs. 38a to 38d).

[0316] 6.8. Biomarker Analysis in Implementation

[0317] A piece of lung tissue extracted from the mouse sacrificed in Example 6.1 was finely ground in 1X RIPA buffer (containing protease inhibitor cocktail, purchased from Abcam). The tissue suspension was centrifuged at 4°C, 12,000 rpm, and 10 min, and only the clear supernatant was separated and transferred to a new 1.5 ml tube. Protein quantification was measured at 562 nm using a BCA assay kit (purchased from Thermo Fisher Scientific) on a multiplate spectrometer. Real-time polymerase chain reaction was performed using RNA obtained from a portion of the right lung lobe to analyze SP-D, MMP-7, tenascin-C, periostin, CXCL13, and PAI-1. The relative mRNA expression level was corrected using 18S rRNA as an internal control, and the value was calculated using the 2-ΔΔCq method. Information on the primers used in this experiment is summarized in Table 10 below.

[0318] Gene name Primer sequence (5' to 3' direction) Sequence number SP-DF: AGGTCCAGTTGGACCCAAAGGA 48 R: CTGGTTTGCCTTGAGGTCCTATG 49 MMP-7F: AGGTGTGGAGTGCCAGATGTTG 50 R: CCACTACGATCCGAGGTAAGTC 51 tenascin-CF: GAGACCTGACACGGAGTATGAG 52 R: CTCCAAGGTGATGCTGTTGTCTG 53 periostinF: CAGCAAACCACTTTCACCGACC 54 R: AGAAGGCGTTGGTCCATGCTCA 55 CXCK 13 F: CATAGATCGGATTCAAGTTACGCC 56 R: GTAACCATTTGGCACGAGGATTC 57 PAI-1F: CCTCTTCCACAAGTCTGATGGC 58 R: GCAGTTCCACAACGTCATACTCG 59 18 S rRNAF: CAGCGTGGTCAGGATAGAAC40R: CTTGATTTGAATGCAGCGGAC41

[0319] As a result of comparing the gene expression levels of six major markers that can determine pulmonary fibrosis, as shown in Figures 39a to 39f, the epithelial damage marker (SP-D), fibrosis markers (MMP-7, Tenascin-C, and Periostin), inflammation marker (CXCL13), and thrombosis marker (PAI-1) were all reduced by the spores of the SOD overexpressing strains (GF424 and GF427), and the reduction was more effective as the concentration of the spores of the SOD overexpressing strain increased.

[0320] Example 6.9. Survival rate measurement

[0321] The effects of spore administration of SOD-overexpressing strains (GF424 and GF427) on the survival of a mouse model of pulmonary fibrosis were investigated in a therapeutic model of pulmonary fibrosis. Survival rates were estimated using the Kaplan-Meier method in the same manner as in Example 2.8, and the log-rank test was used to calculate statistical differences.

[0322] As a result of the estimation, referring to Figure 40, the survival rate of the control group (CTL, N=6 / 6) was 100%, while the survival rate of the bleomycin treatment group (Bleo, N=7 / 12) decreased to 58.3%. Meanwhile, the GF424 strain spore administration group (B+GF424 spore 1x10 7 CFU concentration, N=7 / 12) was 58.3%, which was no different from the bleomycin treatment group, and GF427 10 5 Spore administration group (B+GF427 spore 1x10 5 CFU concentration, N=9 / 12) showed a survival rate of 75%, GF427 10 6 Spore administration group (B+GF427 spore 1x10 6 CFU concentration, N=6 / 12) had a survival rate of 50%, GF427 10 7 Spore administration group (B+GF427 spore 1x10 7CFU concentration, N=9 / 12) showed a survival rate of 75%. GF427 10 7 In the spore-administered group, deaths occurred the latest, and while the number of survivors was the highest, the difference was not statistically significant. These results suggest that administering SOD to a model with pulmonary fibrosis may have a therapeutic effect on pulmonary fibrosis, thereby improving survival rates.

[0323] Statistical analysis

[0324] All numerical data were statistically analyzed using GraphPad Prism statistical software (Ver 9.0). All data were calculated as the mean ± SEM within the group, and data were analyzed by t-test (two-way analysis of variance). * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0325] conclusion

[0326] This example verified that oral administration of Bacillus velezensis strain spores and oral SOD is effective in preventing or treating pulmonary fibrosis. Specifically, it was confirmed that the progression of pulmonary fibrosis was inhibited and improved in both a mouse model in which pulmonary fibrosis was induced after oral administration of GF424 spores for a total of 3 weeks (preventive effect confirmation model) and a mouse model in which pulmonary fibrosis was induced and then oral administration of GF424 spores for a total of 12 days (therapeutic effect confirmation model).

[0327] The induction of idiopathic pulmonary fibrosis by bleomycin has been shown to be closely related to reactive oxygen species (ROS). Therefore, when measuring the concentrations of representative byproducts generated by ROS in lung tissue, it was confirmed that SOD reduces these byproducts, demonstrating that SOD effectively reduces ROS in the body.

[0328] It was confirmed that the SOD-overexpressing strain spore administration group or the SOD normal strain spore administration group was more effective in preventing and treating pulmonary fibrosis than the SOD knockout strain spore administration group, and the SOD-overexpressing strain spore administration group showed a particularly great effect. Through this, the preventive and therapeutic effect of SOD on pulmonary fibrosis through the removal of reactive oxygen species in the body was confirmed, and the increased survival rate of mice due to the preventive and therapeutic effect was also confirmed. Meanwhile, SOD in spore form showed a remarkable therapeutic effect by overcoming the disadvantage that can occur when SOD is orally administered in free form (decomposition before absorption in the body), further increasing the absorption rate in the body. In addition, oral SOD (GF103) with enhanced stability in the body's gastric acid was effective in improving pulmonary fibrosis even when administered alone, and the combined effect with the SOD normal strain spore administration group (Spore SOD(+)) was slightly increased, although not significant. Through this, we were able to confirm that oral SOD (GF103) has the potential to be administered alone or in combination as a treatment for pulmonary fibrosis. Additionally, spores of the GF427 strain, developed to produce approximately four times more SOD than GF424, showed an effect of improving pulmonary fibrosis even with a lower dose than GF424 strain spores.

[0329] In conclusion, it was confirmed that single and concurrent administration of Bacillus velezensis SOD-overexpressing strain spores (GF424 spores and GF427 spores), SOD-normal strain spores, and oral SOD (GF103) can be useful materials for the inhibition and treatment of pulmonary fibrosis.

[0330] Example 7. Confirmation of the therapeutic effect of Bacillus velezensis strain spores on liver fibrosis in the nonalcoholic steatohepatitis STAM™ model.

[0331] Example 7.1. Establishment of a nonalcoholic steatohepatitis model

[0332] To determine whether spores of GF427, an overexpressing SOD strain, exhibit a therapeutic effect on liver fibrosis in a nonalcoholic steatohepatitis (NASH) STAM™ animal model, a nonalcoholic steatohepatitis animal model was established and the results were compared and analyzed with spores of GF423, a normal SOD strain, and oral SOD (GF103).

[0333] C57BL / 6J mice (14-day-old pregnant females and 6-week-old males) (purchased from SLC, Japan) were maintained in a SPF facility with controlled temperature (23 ± 3 °C), humidity (50 ± 20%), lighting (12-h artificial light / dark cycle; light cycle from 08:00 to 20:00), and air exchange.

[0334] NASH was induced in all groups except the normal group of 8 mice as follows: Male mice born on the second day after birth were injected subcutaneously once with 200 μg of streptozotocin (STZ, purchased from Sigma-Aldrich) solution, and after 4 weeks of age, NASH was induced by feeding solid high-fat diet (HFD) (57 kcal% fat, purchased from CLEA Japan, Inc., Cat# HFD32) ad libitum through a metal lid on the top of the cage.

[0335] Ten-week-old mice with NASH were randomly assigned to nine groups of eight mice each based on body weight the day before the start of treatment. Randomization was performed using Excel software using weight-stratified random sampling. NASH model mice were stratified by body weight to calculate standard deviations (SD) and minimize the difference in mean body weight between groups. Mice were identified by ear punch, and each cage was also labeled with a specific identification code for identification.

[0336] The animal model for confirming the therapeutic effect on liver fibrosis was divided into 10 groups as shown in Table 11 and 8 animals were assigned to each group through the random assignment described above.

[0337] Group Experimental group Mice (number) Test substance Concentration (units / mouse) Dose (ml / kg) Dosage regimen 1 Normal group Normal (8)----2 Disease control group (Disease control) STAM (8)----3 GF427 lowest dose administration group STAM (8) GF427 0.05 x 10 7 CFU100 μLPO, QD, 10 - 13 weeks 4GF427 low dose administration group STAM(8)GF4270.1 x 10 7 CFU100 μLPO, QD, 10 - 13 weeks 5GF427 intermediate dose group STAM(8)GF4270.5 x 10 7 CFU100 μLPO, QD, 10 - 13 weeks 6GF427 high-dose administration group STAM(8)GF4271 x 10 7 CFU100 μLPO, QD, 10 - 13 weeks 7GF427 Highest dose group STAM(8)GF4275 x 10 7 CFU100 μLPO, QD, 10 - 13 weeks 8GF423 administration group STAM(8)GF4231 x 10 7 CFU 100 μLPO, QD, 10 - 13 weeks 9 GF103 administration group STAM(8) GF103 10 U 100 μLPO, QD, 10 - 13 weeks 10 Telmisartan administration group STAM(8) Telmisartan 10 mg / kg 10 mL / kgPO, QD, 10 - 13 weeks

[0338] Here, PO means oral administration and QD means once daily.

[0339] The GF427 administration group was 0.05 x 10 per mouse. 7 (lowest capacity), 0.1 x 10 7 (low dose), 0.5 x 10 7 (medium capacity), 1 x 10 7 (high capacity) and 5 x 10 7 Five dose levels of CFU (highest dose) were administered once daily. The GF423 group was administered 1 x 10 per mouse. 7CFU dose was administered once daily. GF103 was administered once daily at a dose of 10 U per mouse. Telmisartan was administered once daily at a dose of 10 mg / kg. GF427, GF423, and GF103 groups were administered orally in the form of vehicle in a volume of 100 μL, and telmisartan was administered orally in the form of supplemented RO water (Reverse Osmosis Water) in a volume of 10 mL / kg. The normal and disease control groups did not receive any treatment until sacrifice, and were sacrificed at 13 weeks of age by direct cardiac puncture under isoflurane (Pfizer Inc.) anesthesia. Animals were monitored daily for viability, clinical signs (lethargy, convulsions, respiratory distress), and behavior to observe for significant clinical signs of toxicity, coma, and death before and after test substance administration. If an animal lost less than 25% of its body weight within a week or showed signs of lethargy, such as a prone posture, it was euthanized before the end of the study, and no samples were collected. All animal care and experiments were conducted in compliance with the Animal Welfare and Management Act.

[0340] Example 7.2. Sample Preparation

[0341] The GF427 spores and GF423 spores used in Example 7.1 were manufactured in the same manner as in Manufacturing Example 1, and GF103 was manufactured in the same manner as in Example 5.1. Telmisartan (Micardis®) was purchased from Boehringer Ingelheim GmbH (Germany). The Bacillus velezensis GF427 strain expressing SOD used in this example was manufactured from the Bacillus velezensis strain deposited at the Korea Center for Biological Resources (KCTC) (Accession No.: KCTC 13222BP, Deposit Date: 20170306) (“GF423 strain”) by replacing the promoter sequence of the GF423 sodA gene with a base sequence having a stronger promoter performance to increase SOD activity (see Manufacturing Example 2). The GF427 and GF423 test materials were vortexed for at least 30 seconds before use. When performing aliquots, an appropriate amount of CFU / mL was used for aliquots to obtain the desired stock volume using a well-stirred solution. The GF103 test substance was diluted with an appropriate amount of distilled water from 10 mg of GF103 powder before daily administration. The GF427, GF423, and GF103 test substances were stored and maintained at 4°C before administration. The telmisartan test substance was freshly prepared before administration. One tablet of telmisartan was transferred to a mortar and pestle, and then slowly added with RO water and ground with a mortar to obtain a homogeneous suspension of 1 mg / mL.

[0342] Example 7.3. Sample Preparation

[0343] After sacrificing 13-week-old mice, frozen plasma samples, frozen liver samples, OCT-embedded liver blocks, and paraffin-embedded liver blocks were collected and stored.

[0344] Plasma sample preparation

[0345] At the end of the study, non-fasting blood was collected via direct cardiac puncture using prechilled syringes. The collected blood was transferred to prechilled polypropylene tubes with an anticoagulant (novoheparin, purchased from Mochida Pharmaceutical Co. Ltd.) and stored on ice until centrifugation. Blood samples were centrifuged at 1,000 × g for 15 minutes at 4°C. After centrifugation, the supernatant was collected and stored at -80°C for biochemical experiments and transportation.

[0346] Liver sample preparation

[0347] At sacrifice, the entire livers of the mice were collected and washed in cold saline. Individual whole livers (parietal side and visceral side) were photographed. Next, the liver weight was measured, and the liver-to-body weight ratio was calculated. The left lateral lobe of the liver was isolated and dissected and stored as shown in Figure 41. The liver specimen, indicated as a in Figure 41, was embedded in optimal cutting temperature (OCT, Sakura Finetek Japan) compound and stored at -80°C for further analysis or transport. The liver specimen, indicated as b, was fixed in Bouin's solution (Sigma-Aldrich Japan) for 24 hours. After fixation, these specimens were paraffin-embedded for H&E and Sirius Red staining. The liver specimen, indicated as c, was flash-frozen in liquid nitrogen and stored at -80°C for gene expression analysis.

[0348] Additionally, the left and right medial lobes were flash frozen in liquid nitrogen for transport and stored at -80°C. The right lobe was flash frozen in liquid nitrogen for biochemical experiments and stored at -80°C. The caudate lobe was flash frozen in liquid nitrogen for transport and stored at -80°C.

[0349] Example 7.4. Checking the mouse status during the experiment

[0350] The body weight changes of mice belonging to 10 groups were observed over a 21-day period during which NASH was induced and treated. As a result, referring to Figure 42, the average body weight of the disease control group was significantly lower than that of the normal group on days 13, 15, 16, and 17. The average body weight of the telmisartan-administered group was significantly lower than that of the disease control group from days 17 to 21. Meanwhile, there was no significant difference in the average body weight changes between the disease control group and the other treatment groups (Groups 3 to 9) on any day during the treatment period.

[0351] During the treatment period, the following mice were found dead before day 21: In the disease control group, 1 out of 8 mice was euthanized. In the mid- and high-dose GF427 groups, 1 out of 8 mice was found dead. In the GF423 group, 2 out of 8 mice were found dead. In the telmisartan group, 2 out of 8 mice were euthanized.

[0352] Example 7.5. Confirmation of long-term weight changes

[0353] On the 21st day, when all treatments were completed, the average daily body weight, liver weight, and liver-to-body weight ratio of mice belonging to the 10 groups were measured (Fig. 43a).

[0354] Referring to Figures 43a and 43b, the average body weight on the day of sacrifice in the disease control group tended to decrease compared to the normal group. The telmisartan group showed a significant decrease in average body weight on the day of sacrifice compared to the disease control group. In all treatment groups, the average body weight on the day of sacrifice tended to decrease compared to the disease control group.

[0355] Referring to Figures 43a and 43c, the disease control group showed a significant increase in average liver weight compared to the normal group. Meanwhile, the GF427 high-dose and telmisartan-administered groups showed a significant decrease in average liver weight compared to the disease control group. The average liver weights of the GF427 lowest-dose, low-dose, and GF423-administered groups tended to decrease compared to the disease control group. There was no significant difference in average liver weight between the remaining groups and the disease control group.

[0356] Referring to Figures 43a and 43d, the disease control group showed a significantly increased mean liver-to-body weight ratio compared to the normal group. Meanwhile, the mean liver-to-body weight ratio in the high-dose GF427 and telmisartan groups tended to decrease compared to the disease control group. There was no significant difference in the mean liver-to-body weight ratio between the disease control group and the remaining groups.

[0357] Example 7.6. Biochemical Analysis of Plasma and Liver Tissue

[0358] After collecting plasma or liver samples from each mouse according to the plasma sample preparation method and liver sample preparation method of Example 7.3, the whole blood glucose level, plasma ALT level, plasma CK-18 level, and liver triglyceride content were measured and compared (Fig. 44a).

[0359] Whole blood sugar level measurement

[0360] Non-fasting blood glucose levels in whole blood were measured using a Stat Strip blood glucose meter (purchased from NIPRO CORPORATION). As shown in Figure 44b, the disease control group showed significantly higher blood glucose levels compared to the normal group. The blood glucose levels of the lowest and highest doses of GF427 and the GF423 administration groups tended to decrease compared to the disease control group. There was no significant difference in blood glucose levels between the disease control group and the other groups.

[0361] Measurement of plasma ALT levels

[0362] Plasma ALT levels were measured using the FUJI DRI-CHEM 7000 (purchased from Fujifilm Corporation). As shown in Figure 44c, the disease control group showed a significant increase in plasma ALT levels compared to the normal group. The GF427 high-dose group showed a significant decrease in plasma ALT levels compared to the disease control group. Plasma ALT levels in the lowest and highest GF427 dose and telmisartan groups tended to decrease compared to the disease control group. There was no significant difference in plasma ALT levels between the disease control group and the remaining groups.

[0363] Measurement of plasma CK-18 levels

[0364] Plasma CK-18 levels were quantified using the Mouse Cytokeratin 18-M30 ELISA kit (purchased from Cusabio Biotech Co., Ltd). As shown in Figure 44d, the disease control group showed a significant increase in plasma CK-18 levels compared to the normal group. All groups showed a significant decrease in plasma CK-18 levels compared to the disease control group.

[0365] Check liver triglyceride content

[0366] Total liver lipid extracts were obtained according to the method of Folch (see [Folch J. et al., J. Biol. Chem. 1957;226: 497]). Liver samples were homogenized in chloroform-methanol (2:1, v / v) and incubated overnight at room temperature. After washing with chloroform-methanol-water (8:4:3, v / v / v), the extracts were evaporated to dryness and dissolved in isopropanol. The liver triglyceride content was measured using the Triglyceride E-Test (purchased from FUJIFILM Wako Pure Chemical Corporation).

[0367] As shown in Figure 44e, the liver triglyceride content in the disease control group significantly increased compared to the normal group. Meanwhile, all groups, except the lowest-dose GF427 group, showed a significant decrease in liver triglyceride content compared to the disease control group. The liver triglyceride content in the lowest-dose GF427 group tended to decrease compared to the disease control group.

[0368] Example 7.7. Confirmation of liver fibrosis through H&E staining of liver tissue and assessment of NAFLD activity score.

[0369] Sections were cut from the paraffin blocks of liver tissue prepared in Example 7.3 using a rotary microtome (Leica Microsystems). After sectioning, each slide was coded for blind evaluation. Each number was generated using the RAND function in Excel software and assigned to the slides in ascending order. The tissue slides were stained with H&E and evaluated by an experimenter.

[0370] Sections were cut from paraffin blocks of liver tissues prefixed in Bouin's solution for H&E staining and stained with Lilly-Meyer hematoxylin (purchased from Muto Pure Chemicals Co., Ltd.) and eosin solution (purchased from FUJIFILM Wako Pure Chemical Corporation). NAFLD activity score (NAS) was calculated according to Kleiner's criteria as shown in Table 12 (reference [Kleiner DE. Et al., Hepatology, 2005;41:1313]). For NAS scoring, bright-field images of H&E-stained sections were captured at 50x and 200x magnification using a digital camera (purchased from Leica, product number: DFC295). The steatosis score in one section per mouse (one representative field of view at 50× magnification), the inflammation score in one section per mouse (one representative field of view around the central vein at 200× magnification), and the ballooning score in one section per mouse (one representative field of view around the central vein at 200× magnification) were evaluated.

[0371] Item Range Score Steatosis Steatosis at 50x magnification □ <5% □ 5-33% □ >33-66% □ >66% 0123 Lobular Inflammation Assessment of inflammatory foci □ No foci □ <2 foci / 200x □ 2-4 foci / 200x □ >4 foci / 200x 0123 Ballooning Assessment of number of ballooning cells □ None □ Few ballooning cells □ Many cells / marked ballooning 012

[0372] Representative microscopic photographs of H&E-stained liver sections were taken (Figs. 45a to 45c), and the scores and NAS values ​​for each item were presented in a table (Fig. 45e) and graph (Figs. 45f to 45i) to confirm the degree of steatosis, inflammatory cells, and hepatocyte ballooning in each group. A detailed examination of the liver sections from the disease control group showed microvesicular and macrovesicular fat deposition, hepatocyte ballooning, and inflammatory cell infiltration compared to the normal group (Fig. 45d). Meanwhile, compared to the disease control group, the degrees of steatosis, inflammatory cells, and hepatocyte ballooning tended to decrease in the remaining treatment groups. Referring to Figs. 45e to 45i, the disease control group showed a significant increase in NAS compared to the normal group. Meanwhile, the GF427 low-dose, mid-dose, high-dose, and highest-dose, GF423, and telmisartan treatment groups showed a significant decrease in NAS compared to the disease control group. NAS in the GF103 group tended to decrease compared to the disease control group. There was no significant difference in NAS between the disease control group and the lowest dose GF427 group.

[0373] Example 7.8. Confirmation of liver fibrosis through Sirius Red staining of liver tissue.

[0374] To visualize collagen deposition, Bouin-fixed liver sections were stained with picro-sirius red solution (purchased from FUJIFILM Wako Pure Chemical Corporation). First, the sections were deparaffinized and hydrophilized sequentially with xylene, a 100–70% alcohol series, and reverse osmotic pressure water. They were then treated with a 0.03% picro-sirius red solution (Cat No. 194-16202) for 60 minutes. After washing with a 0.5% acetic acid solution and reverse osmotic pressure water, the stained sections were dehydrated, washed with a 70–100% alcohol series and xylene, and then sealed with Entellan® new (purchased from Merck) for observation.

[0375] For quantitative analysis of fibrotic area, brightfield images of Sirius Red-stained sections were captured around the central vein at 200x magnification using a digital camera (Leica, Part No. DFC295), and the positive area within five fields of view per section was measured using ImageJ software (National Institutes of Health, USA). Representative photomicrographs of Sirius Red-stained sections for assessment of fibrotic area are shown in Figure 46a.

[0376] Referring to Figures 46a to 46c, liver sections from the disease control group showed increased collagen deposition (Sirius red positivity) in the central and peripheral areas of the liver lobules compared to the normal group. Furthermore, the disease control group showed a significant increase in the area of ​​fibrosis (Sirius red-positive area) compared to the normal group. Meanwhile, the area of ​​fibrosis in the highest dose of GF427, GF423, GF103, and telmisartan groups tended to decrease compared to the disease control group. There was no significant difference in the area of ​​fibrosis between the disease control group and the remaining groups.

[0377] Statistical analysis

[0378] Statistical analysis was performed using Prism software 6 (GraphPad Software, USA) and Bonferroni Multiple Comparison Test.

[0379] A P value <0.05 was considered statistically significant. Results are expressed as mean ± SD.

[0380] A trend or tendency was assumed when a one-sided t-test yielded a P value less than 0.1. All results were compared between the following groups:

[0381] 1) Group 2 (disease control group) vs. Group 1 (normal group)

[0382] 2) Group 2 (disease control group) vs. Group 3 (GF427 lowest dose group)

[0383] 3) Group 2 (disease control group) vs. Group 4 (GF427 low-dose group)

[0384] 4) Group 2 (disease control group) vs. Group 5 (GF427 intermediate dose group)

[0385] 5) Group 2 (disease control group) vs. Group 6 (GF427 high-dose group)

[0386] 6) Group 2 (disease control group) vs. Group 7 (GF427 highest dose group)

[0387] 7) Group 2 (disease control group) vs. Group 8 (GF423 administration group)

[0388] 8) Group 2 (disease control group) vs. Group 9 (GF103 administration group)

[0389] 9) Group 2 (disease control group) vs. Group 10 (telmisartan administration group)

[0390] conclusion

[0391] This example confirmed that administration of spores of Bacillus velezensis GF427, GF423, and oral SOD (GF103) of the above strains is effective in treating liver fibrosis in an animal model of non-alcoholic steatohepatitis. Specifically, in a mouse model in which NASH was induced through streptozotocin solution and a high-fat diet, GF427 strain spores, GF423 strain spores, and GF103 were orally administered as a vehicle at a dose of 100 uL once a day for 21 days, and it was confirmed that the progression of liver fibrosis was inhibited and improved.

[0392] The GF427-administered group showed a tendency for a decrease in whole blood glucose levels and fibrosis area compared to the disease control group, while the plasma ALT level, plasma CK-18 level, liver triglyceride content, and NAS showed a significant decrease compared to the disease control group. The GF423-administered group showed a significant decrease in plasma CK-18 level, liver triglyceride content, and NAS compared to the disease control group. In addition, the whole blood glucose level and fibrosis area tended to decrease compared to the disease control group. The GF103-administered group showed a significant decrease in plasma CK-18 level and liver triglyceride content compared to the disease control group. In addition, the NAS and fibrosis area tended to decrease compared to the disease control group.

[0393] In conclusion, it was confirmed that the spores of the GF427 strain engineered to overexpress SOD, the spores of the GF423 strain expressing wild-type SOD, and the oral SOD GF103 could all be useful materials for the inhibition and treatment of liver fibrosis when administered alone.

[0394] [Correction pursuant to Rule 91, October 29, 2024]

Claims

1. A pharmaceutical composition for treating or preventing fibrotic disease, comprising as an active ingredient at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and a polypeptide having superoxide dismutase (SOD) activity.

2. In paragraph 1, The above Bacillus species strain is a strain expressing SOD or a strain mutated or recombined to overexpress SOD. Pharmaceutical composition.

3. In paragraph 1, The above Bacillus species strain spores include spores derived from a strain expressing SOD or a strain mutated or recombinant to overexpress SOD. Pharmaceutical composition.

4. In paragraph 1, The above Bacillus species strain is a Bacillus velezensis species strain. Pharmaceutical composition.

5. In paragraph 1, The above Bacillus species strain is at least one selected from the Bacillus velezensis strain deposited with the accession number KCTC 13222 BP, the Bacillus velezensis strain deposited with the accession number KCTC 13227 BP, and the Bacillus velezensis strain deposited with the accession number KCTC 15552 BP. Pharmaceutical composition.

6. In paragraph 1, The above polypeptide is Mn-SOD. Pharmaceutical composition.

7. In paragraph 1, The above polypeptide is deamidated Mn-SOD. Pharmaceutical composition.

8. In paragraph 1, The above polypeptide is derived from a Bacillus species strain. Pharmaceutical composition.

9. In paragraph 8, The above Bacillus species strain is a Bacillus velezensis species strain. Pharmaceutical composition.

10. In paragraph 8, The above Bacillus species strain is at least one selected from the Bacillus velezensis strain deposited with the accession number KCTC 13222 BP, the Bacillus velezensis strain deposited with the accession number KCTC 13227 BP, and the Bacillus velezensis strain deposited with the accession number KCTC 15552 BP. Pharmaceutical composition.

11. In paragraph 1, The above polypeptide comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6. Pharmaceutical composition.

12. In paragraph 1, The above polypeptide is coated with a coating agent. Pharmaceutical composition.

13. In paragraph 12, The above coating agent contains shellac. Pharmaceutical composition.

14. In paragraph 1, The above fibrotic disease is pulmonary fibrotic disease or liver fibrotic disease. Pharmaceutical composition.

15. In paragraph 14, The above pulmonary fibrotic disease is selected from the group consisting of interstitial lung disease, pulmonary fibrosis and idiopathic pulmonary fibrosis. Pharmaceutical composition.

16. In paragraph 14, The above liver fibrosis disease is selected from the group consisting of nonalcoholic steatohepatitis, liver fibrosis, idiopathic liver fibrosis, cirrhosis, alcoholic steatohepatitis, chronic liver disease, and viral hepatitis. Pharmaceutical composition.

17. In paragraph 1, The above active ingredient is administered orally. Pharmaceutical composition.

18. In paragraph 1, A composition comprising two or more selected from the group consisting of the above Bacillus species strain, the Bacillus species strain spores and the polypeptide having the above SOD activity, wherein the two or more components are administered simultaneously, sequentially or in reverse order. Pharmaceutical composition.

19. A method for preventing, improving or treating a fibrotic disease, comprising administering to a subject a composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore and a polypeptide having superoxide dismutase activity.

20. A food composition for preventing or improving fibrotic disease, comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and a polypeptide having superoxide dismutase activity.

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