Superoxide dismutase and uses thereof for preventing or treating kidney injury

Abstract

The present invention relates to a use of Bacillus-derived superoxide dismutase for preventing or treating kidney injury. More specifically, the present invention relates to a pharmaceutical composition or a therapeutic method for prevention or treatment of kidney injury. The composition and method according to the present invention exhibit at least one effect selected from the group consisting of a change in intestinal microbial composition, a reduction in intestinal epithelial cell death, a reduction in intestinal permeability, a reduction in oxidative stress in the kidney, an alleviation of tubular injury, and an improvement in kidney function, and thus can be effectively used for preventing or treating kidney injury.

Description

Superoxide dismutase and its use for preventing or treating kidney damage

[0001] This application claims priority to Korean Patent Application No. 10-2023-0147282, filed October 30, 2023, Korean Patent Application No. 10-2023-0147284, filed October 30, 2023, and Korean Patent Application No. 10-2024-0075277, filed June 10, 2024, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Sequence list

[0003] 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 the sequence listing, dated October 23, 2024, is named KC24164.xml and is 28.6 kilobytes in size.

[0004] The present invention relates to superoxide dismutase and its use for the prevention, improvement, or treatment of renal damage. More specifically, the present invention relates to the use, composition, or method of Bacillus-derived superoxide dismutase for the prevention, improvement, or treatment of renal damage.

[0005] Acute kidney injury (AKI), a representative example of kidney damage, is defined as the sudden loss of kidney excretory function. AKI can occur following abrupt events such as accidents, burns, or trauma. Furthermore, AKI is a common complication in critical care, and patients with underlying medical conditions are considered particularly high-risk. AKI can be life-threatening, often requiring kidney transplantation, and contributes to increased hospital stays, mortality, and medical costs. Because AKI can lead to chronic kidney disease and cardiovascular disease in the long term, preventing, detecting, and treating AKI early is essential. However, AKI has a wide range of causes and severity, making it difficult to approach it as a single disease. Furthermore, clinical trials have failed to demonstrate consistent treatment options. Currently, fluid therapy, which involves continuous monitoring of fluid status and administration of appropriate fluids and diuretics, is the primary treatment for AKI.

[0006] Despite the severity of acute kidney injury, there is no clear treatment, so there is a need to develop agents to prevent or treat acute kidney injury.

[0007] Meanwhile, the kidney is an organ responsible for maintaining homeostasis, and kidney damage affects nearly every system in the body in various ways. Recent basic and clinical research data have shown that acute kidney injury induces severe systemic inflammation, inducing damage to distant organs such as the heart, lungs, spleen, brain, liver, and intestines. These findings suggest the potential for kidney-gut crosstalk in kidney injury. However, unlike diabetes, obesity, and inflammatory bowel disease, only a few studies have demonstrated a kidney-gut connection in kidney injury, necessitating further research.

[0008] [Prior Art Literature]

[0009] [Non-patent literature]

[0010] (Non-patent Document 1) Kellum, J. A., Romagnani, P., Ashuntantang, G. et al. Acute kidney injury. Nat Rev Dis Primers 7, 52 (2021). https: / / doi.org / 10.1038 / s41572-021-00284-z

[0011] (Non-patent Document 2) Doi K, Rabb H. Impact of acute kidney injury on distant organ function: recent findings and potential therapeutic targets. Kidney Int 2016; 89: 555-564.

[0012] (Non-patent Document 3) Se won Oh, The Cause and Treatment of Acute Kidney Injury, Korean J Med. 2019; 94(4): 315-321.

[0013] The present invention aims to solve one or more of the problems of the above-mentioned prior art.

[0014] The present invention aims to provide a Bacillus species strain, a Bacillus species strain spore and / or a superoxide dismutase (SOD) for preventing or treating kidney damage.

[0015] Another object of the present invention is to provide a composition for preventing, improving or treating kidney damage comprising a Bacillus species strain, a Bacillus species strain spore and / or SOD.

[0016] Another object of the present invention is to provide a method for preventing, improving or treating kidney damage, comprising administering to a subject a Bacillus species strain, a Bacillus species strain spore and / or SOD.

[0017] Another object of the present invention is to provide a use of a Bacillus species strain, a Bacillus species strain spore and / or a superoxide dismutase for preventing, improving or treating renal damage.

[0018] 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.

[0019] A representative configuration of the present invention to achieve the above purpose is as follows.

[0020] According to one aspect of the present invention, a pharmaceutical composition for preventing or treating kidney damage is provided, comprising a Bacillus species strain, a Bacillus species strain spore and / or a polypeptide having superoxide dismutase (SOD) activity as an active ingredient.

[0021] In some embodiments, the renal injury may be renal failure, acute kidney injury, or chronic kidney injury. In some embodiments, the renal injury may be contrast-induced acute kidney injury or ischemic acute renal failure.

[0022] In some embodiments, the composition may exhibit one or more effects selected from the group consisting of altering the composition of the intestinal microbiota, reducing intestinal epithelial cell death, reducing intestinal permeability, reducing oxidative stress in the kidney, alleviating tubular damage, and improving renal function.

[0023] In some embodiments, the polypeptide having superoxide dismutase (SOD) activity or SOD may be in the form of a Bacillus strain producing or containing the same or a spore thereof or may be present in this form.

[0024] In some embodiments, the polypeptide may be an isolated or purified protein.

[0025] In some embodiments, the polypeptide may be included as a strain lysate, strain culture, strain culture concentrate, strain culture extract, or a dried form thereof.

[0026] In some embodiments, the polypeptide may be Mn-SOD.

[0027] In some embodiments, the polypeptide may be deamidated Mn-SOD.

[0028] In some embodiments, the polypeptide may be derived from a Bacillus species strain.

[0029] In some embodiments, the Bacillus species strain may be a Bacillus velezensis species.

[0030] In some embodiments, the Bacillus species strain may be 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.

[0031] In some embodiments, the composition may comprise as an active ingredient a polypeptide having superoxide dismutase activity comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0032] In some embodiments, the polypeptide may be coated with a coating agent.

[0033] In some embodiments, the coating may include shellac.

[0034] In some embodiments, the composition may be administered orally.

[0035] In some embodiments, the composition may be administered to the subject before, after, or both before and after the onset of kidney damage.

[0036] In some embodiments, the composition may be co-administered to a subject prior to, concurrently with, and / or following administration of another drug that causes or is at risk of causing renal damage.

[0037] In some embodiments, an oral composition for preventing, ameliorating, or treating renal damage is provided, comprising a superoxide dismutase as disclosed herein as an active ingredient.

[0038] According to another aspect of the present invention, a food composition for preventing, improving or treating kidney damage is provided, comprising superoxide dismutase as disclosed herein as an active ingredient.

[0039] According to another aspect of the present invention, a feed composition for preventing, improving or treating kidney damage is provided, comprising superoxide dismutase as disclosed herein as an active ingredient.

[0040] According to another aspect of the present invention, a veterinary composition for preventing, improving or treating kidney damage is provided, comprising superoxide dismutase as disclosed herein as an active ingredient.

[0041] According to another aspect of the present invention, a method for preventing or treating kidney damage is provided, comprising administering to a subject a polypeptide as disclosed herein.

[0042] According to another aspect of the present invention, there is provided a use of a superoxide dismutase as disclosed herein for preventing or treating renal damage.

[0043] According to another aspect of the present invention, there is provided a use for the manufacture of a medicament for preventing or treating renal damage of a superoxide dismutase as disclosed herein.

[0044] A composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and superoxide dismutase (SOD) of the present invention exhibits a preventive or therapeutic effect on renal damage (e.g., renal failure, acute renal damage, or chronic renal damage). In one embodiment, when the composition of the present invention was orally administered to an experimental animal (mouse) and renal damage was induced, a protective effect on renal damage was observed, such as changes in the composition of intestinal microflora, reduction in intestinal epithelial cell death, reduction in intestinal permeability, reduction in oxidative stress in the kidney, alleviation of renal tubular damage, and improvement in renal function. Therefore, the superoxide dismutase according to the present invention can be effectively used to prevent, improve, or treat renal damage. In one embodiment, the SOD of the present invention exhibits an effect of preventing or treating acute kidney damage when administered together with a contrast agent that induces acute kidney damage, and thus can be used as a drug administered concomitantly in an imaging diagnostic examination using a contrast agent to protect against renal damage. In another embodiment, the SOD of the present invention is derived from a Bacillus species strain (e.g., a Bacillus velezensis strain) that is generally regarded as safe (GRAS) bacteria, thereby ensuring oral administration efficacy and safety, and also having the process advantage of being able to be recovered directly from the supernatant during culture.

[0045] Figure 1 shows the procedure for producing a recombinant production strain (BSBA310) expressing SodA2.

[0046] 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.

[0047] Figures 3a and 3b are schematic diagrams 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 from GF423 genomic DNA as a template, and Figure 3b shows the process of producing pUori-cm-amp-P3-SOD using a PCR product obtained from pUori-cm-amp-10sod as a template.

[0048] Figure 4a is a graph showing principal coordinates analysis performed by 16s RNA sequencing on stool samples from the unadministered Sham group (Sham), the SOD-BA-administered Sham group (SOD+Sham), the unadministered IRI group (IRI), and the SOD-BA-administered IRI group (SOD+IRI).

[0049] Figure 4b shows the composition ratio of families of the intestinal microbiota by analyzing the intestinal microbiome using 16s RNA sequencing for stool samples from the unadministered Sham group (Sham), the SOD-BA-administered Sham group (SOD+Sham), the unadministered IRI group (IRI), and the SOD-BA-administered IRI group (SOD+IRI).

[0050] Figure 5 is a graph showing changes in intestinal permeability according to administration of SOD.

[0051] Figures 6a and 6b are diagrams showing the degree of intestinal epithelial cell death according to the administration of SOD. Figure 6a is a graph showing the quantification of TUNEL-positive epithelial cells, and Figure 6b is a slide scanner photograph of the stained tissue (scale bar = 20 μm).

[0052] Figures 7a and 7b are diagrams showing changes in the distribution of intestinal immune cells according to the administration of SOD. Figure 7a is a graph showing the quantification of Ly6G-positive cells, and Figure 7b is a slide scanner photograph of stained tissue (scale bar = 20 μm).

[0053] Figures 8a to 8e are drawings showing the kidney improvement effect according to the administration of SOD. Figures 8a and 8b are a graph (Figure 8a) and a micrograph (Figure 8b), respectively, showing the distribution of SOD in the kidney according to the administration of SOD as a percentage of positive staining compared to the total tissue area. Figures 8c and 8d are a graph (Figure 8c) and a micrograph (Figure 8d), respectively, showing the distribution of catalase in the kidney according to the administration of SOD as a percentage of positive staining compared to the total tissue area. Figure 8e illustrates the oxidative stress alleviation process of SOD. (All scale bars in the drawings except for those marked with * (1000 μm) are 500 μm.)

[0054] Figures 9a to 9d are diagrams showing changes in immune cell distribution (changes in infiltration level) in renal tissue according to SOD administration. Figures 9a and 9b are graphs (Figure 9a) and micrographs (Figure 9b) showing the quantification of Ly6G-positive cells (neutrophils) (scale bar = 20 μm). Figures 9c and 9d are graphs (Figure 9c) and micrographs (Figure 9d) showing the quantification of F4 / 80-positive cells (lymphocytes) (scale bar = 20 μm).

[0055] Figure 10a is a graph showing changes in blood creatinine concentration according to administration of SOD (unpaired t-test; *, p <0.05; **, p <0.05).

[0056] Figure 10b is a graph showing changes in blood urea nitrogen (BUN) concentration according to administration of SOD (unpaired t-test; *, p <0.05; **, p <0.05; #, p <0.05).

[0057] Figures 11a and 11b are a graph (Figure 11a) and a microscopic photograph (Figure 11b) showing changes in the degree of tubular necrosis according to the administration of SOD (scale bar = 50 μm).

[0058] Figure 12 is a schematic diagram for creating a contrast-induced acute kidney injury (CI-AKI) model.

[0059] Figures 13a to 13c are diagrams showing the preventive or ameliorating effects of SOD administration on renal damage in a contrast agent-induced acute renal injury model. Figure 13a is a graph showing changes in blood creatinine concentration, Figure 13b is a graph showing changes in blood urea nitrogen (BUN) concentration, and Figure 13c is a graph showing NGAL distribution as a positive ratio relative to the total tissue area.

[0060] The detailed description of the present invention, which follows, will be described with reference to specific drawings (where drawings exist) regarding specific embodiments in which the present invention may be practiced; however, the present invention is not limited thereto, but is defined solely by the appended claims to the full scope equivalent to or equivalent to what the claims describe. It should be understood that the various embodiments / embodiments of the present invention, while different from each other, are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be changed from one embodiment / embodiment to another, or multiple embodiments / embodiments may be combined, without departing from the spirit and scope of the present invention. Technical and scientific terms used herein, unless otherwise defined, have the same meaning as commonly used in the art to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and terms expressed in the singular should be construed to also refer to the plural (i.e., at least one), unless the context makes it inappropriate.

[0061] definition

[0062] The term "subject" is used interchangeably with "subject" or "patient" and can be a mammal in need of prevention, amelioration, or treatment of kidney damage, 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 some embodiments, the subject is a human.

[0063] 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., kidney damage, such as renal failure, acute kidney injury, or chronic kidney injury). Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a 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" may refer to medical intervention for an already present disease or disorder.

[0064] 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.

[0065] The term "administration" means providing an active ingredient to a subject for a preventive or therapeutic purpose (e.g., renal impairment, such as renal failure, acute renal injury, or chronic renal injury).

[0066] For use in preventing or treating kidney damage

[0067] The present invention is based, at least in part, on the surprising discovery that administering a composition comprising as an active ingredient a polypeptide having superoxide dismutase (SOD) activity is effective in preventing, ameliorating, or treating renal damage, such as acute kidney injury. The polypeptide having SOD activity may be included in the composition in the form of a Bacillus strain expressing SOD, a spore of the Bacillus strain, or free SOD. Accordingly, according to one aspect of the present invention, there is provided a use for preventing or treating renal damage of at least one selected from the group consisting of a Bacillus strain expressing SOD, a spore of the Bacillus strain expressing SOD, and a superoxide dismutase.

[0068] In some embodiments, a use is provided for preventing, improving or treating kidney damage of a combination of two or more selected from a Bacillus species strain, a Bacillus species strain spore and SOD.

[0069] In some embodiments, uses of SOD for preventing or treating kidney damage are provided.

[0070] Kidney damage refers to a deterioration in kidney function, particularly characterized by a reduced ability to filter waste products from the blood. Kidney damage may be due to, but is not limited to, trauma, infection, drug toxicity, vascular abnormalities, tumors, aging, surgery, etc. In some embodiments, kidney damage may be (acute or chronic) renal failure, acute renal injury, or chronic renal injury, such as contrast-induced acute renal injury, ischemic acute renal failure, or chronic renal failure or chronic renal injury caused by these.

[0071] In some embodiments of the present invention, it was confirmed that when Bacillus-derived SOD was orally administered during, before, or after induction of renal damage (acute renal failure due to ischemia-reperfusion injury or contrast agent-induced acute kidney injury), the degree of renal damage was significantly improved (see Examples 1 to 5). For example, a mouse model of renal damage induced by ischemia-reperfusion injury after administration of Bacillus-derived SOD showed improved intestinal permeability and alleviated intestinal epithelial cell death (improved intestinal environment) compared to a mouse model of renal damage not administered SOD. For example, administration of Bacillus-derived SOD increased the distribution of SOD, an oxidative stress factor, in the kidney, and showed effects of protecting renal function and alleviating the degree of renal tubular damage (renal protective effect).

[0072] Bacillus species strain expressing SOD, spores of the Bacillus species strain expressing SOD and / or a composition comprising SOD

[0073] According to another aspect of the present invention, a composition for preventing, improving or treating kidney damage is provided, comprising as an active ingredient at least one selected from the group consisting of a Bacillus species strain expressing SOD, a Bacillus species strain spore expressing SOD, and SOD. 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.

[0074] The term "spore" as used herein may be used interchangeably with "spore", and Bacillus species strain spores may be obtained by culturing a Bacillus species strain in a suitable medium, inducing sporulation, and then isolating the produced spores.

[0075] 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).

[0076] Bacillus species strains are GRAS probiotics approved in several countries. Specifically, the Bacillus species strains may be, but are not limited to, Bacillus velezensis, Bacillus amyloliquesfaciens, B. methylotrophicus, Bacillus 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 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 listed 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.

[0077] In some embodiments, the Bacillus amyloliquefaciens strains may be used interchangeably with the Bacillus velezensis strains. Accordingly, the Bacillus amyloliquefaciens GF423 strain, the GF424 strain, and the GF427 strain may be understood to be the same strains as the Bacillus velezensis GF423 strain, the GF424 strain, and the GF427 strain, respectively.

[0078] 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).

[0079] Bacillus amyloliquefaciens plantarum Bacillus amyloliquefaciens amyloliquefaciens Bacillus subtilis spizigeni Species Differentiation criteria (threshold) (%) Existing 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%

[0080] 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 DDH and ANI, which are genome-based classification methods, and was therefore classified as a microorganism belonging to Bacillus velezensis, rather than the same species as Bacillus 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 may be a mutant strain that has been mutated to overexpress or overproduce SOD (e.g., GF424 or GF427 strain). In another embodiment, the Bacillus species strain spores may be derived from another Bacillus species strain that has been recombinantly modified to express the SOD of the Bacillus velezensis strain. Such recombinant strains are further described below. Sporulation of the Bacillus species strain may be induced using conventional techniques known in the art.For example, spores can be obtained by inducing spore formation 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 culturing the strain, and can be separated by culturing various sources including natural, mutant, or recombinant hosts, and then filtering, concentrating, or centrifuging the vegetative cells.

[0081] 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.

[0082] 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).

[0083] 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.

[0084] 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.

[0085] 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 bacteria 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 Bacillus sp. strains are as described above. More preferably, the SOD can be obtained from a Bacillus velezensis strain (e.g., a GF423 strain, a GF424 strain or a 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 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 to improve the expression of the sod gene. The SOD enzyme (SodA) derived from the Bacillus velezensis GF423, GF424 or GF427 strain is a Mn-SOD and may comprise or consist of an amino acid sequence of SEQ ID NO: 2. For example, its nucleotide sequence may be represented by SEQ ID NO: 1. Additionally, SOD may be a recombinant polypeptide. For example, it may be a deamidated SOD (SodA2) in which amino acid residues 74 and 137 of SEQ ID NO: 2 are substituted with Asp, and which may comprise or consist of the amino acid sequence of SEQ ID NO: 4. For example, its nucleotide sequence may be represented by SEQ ID NO: 3. Additionally, 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.

[0086] 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

[0087] In another example, SOD may be derived from another recombinant strain (e.g., a Bacillus subtilis strain) containing the SOD expression gene of a Bacillus velezensis strain. The recombinant strain may be produced by recombinant technology using conventional protein-producing strains known in the art. For example, the Bacillus subtilis strain (which is the parent strain of the recombinant strain) may be KCTC 3135, and the KCTC 3135 strain may be provided by the Korea Research Institute of Bioscience and Biotechnology (KCTC). For example, the recombinant strain may have one or more of the genes shown in Table 3 below deleted to facilitate post-processing.

[0088] 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

[0089] For example, a 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 SOD is produced using the strain, efficient production is possible because SOD can be produced in large quantities while ensuring safety for the organism without going through an expensive purification process (e.g., column purification). In some embodiments, SOD can be obtained by culturing a natural, mutant, or recombinant microorganism in various culture media. For example, SOD can be isolated from the culture supernatant of Bacillus velezensis GF423 strain, GF424 strain, or GF427 strain. Specifically, a culture solution can be obtained by first culturing a Bacillus velezensis strain in various types of media. 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 Bacillus velezensis strains 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, and the like. 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).In some embodiments, the SOD may be isolated or purified from a culture of a natural, mutant, or recombinant strain. 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 is 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%.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Methods for preventing, improving, or treating kidney damage

[0094] According to another aspect of the present invention, a method for preventing, improving, or treating renal impairment is provided, comprising administering to a subject a composition comprising SOD as disclosed herein. For example, this method may comprise administering to a subject who has or is likely to have renal impairment a therapeutically or nutritionally effective amount of a composition comprising a Bacillus species strain, a Bacillus species strain spore, and / or a SOD as an active ingredient as disclosed herein. The renal impairment is as described herein.

[0095] In some embodiments, the method may comprise administering to the subject a Bacillus species strain, a Bacillus species strain spore, or SOD alone, as disclosed herein. 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 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 SOD can effectively prevent or treat kidney damage even when administered orally to the subject.

[0096] The effective amount or effective non-toxic amount of the 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.

[0097] For example, Bacillus species strains or Bacillus species strain spores are administered at doses of 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.

[0098] The composition of the present invention may be administered simultaneously with, or before or after, one or more other agents that cause or are likely to cause renal damage to prevent, improve, or treat renal damage when administered. When administering one or more other agents that cause or are likely to cause renal damage, it may be preferable to administer the composition of the present invention before administration of one or more other agents for effective prevention of renal damage. Such other agents include, but are not limited to, any agent that is administered for the purpose of improving or ameliorating other diseases, such as contrast agents, antibiotics, immunosuppressants, drugs for heart disease, and cancer chemotherapy drugs, and that has the potential to induce unintended renal damage. When a composition comprising one or more selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and / or a SOD disclosed herein 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 a subject at a different time than when the other components are administered. In certain embodiments, each dose may be administered 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 via the same or different routes. The routes of administration are detailed in the pharmaceutical composition.

[0099] In some embodiments, the Bacillus species strain, the Bacillus species strain spore, and / or the polypeptide having SOD activity of the present invention can be administered simultaneously, sequentially, or in reverse order. For example, a composition comprising the following components (i) to (iv) can be administered simultaneously, sequentially, or in reverse order to exhibit a preventive or therapeutic effect on renal damage: (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 renal damage.

[0100] Pharmaceutical or veterinary compositions

[0101] According to another aspect of the present invention, a pharmaceutical or veterinary composition 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 SOD. The pharmaceutical composition of the present invention may be used interchangeably with the term "veterinary composition" when applied to animals other than humans.

[0102] In some embodiments, the pharmaceutical composition may be used for the prevention or treatment of kidney damage as described herein.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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, intrarectal, intravaginal, intraarticular, intraprostatic, intranasal, intraocular, intravesical, intrathecal, intraventricular, or intracerebroventricular administration.

[0108] 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.

[0109] For oral administration, SOD may be coated with shellac for protection from gastric acid, but the coating agent is not limited thereto. Examples of coating agents 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] Food or feed composition

[0114] 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 can be used for the prevention or improvement of kidney damage as described herein.

[0115] 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.

[0116] Additionally, food includes, but is not limited to, various food products, food additives, beverages (e.g., functional beverages, natural fruit juices and vegetable beverages), gum, tea, vitamin complexes, health functional foods, and other functional foods.

[0117] Foods can be manufactured by conventional methods known in the art. For example, medical foods, nutraceutical foods, or health functional foods can be formulated into tablets, pills, powders, granules, powders, capsules, and liquid formulations by further including one or more carriers, diluents, excipients, and additives in addition to the SOD for the purpose of preventing or improving kidney damage. Specific examples of the carriers, excipients, diluents, and additives are well known in the art, and those skilled in the art can prepare them by combining appropriate ingredients according to the formulation.

[0118] 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.

[0119] 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.

[0120] 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 disclosed herein is provided. In addition, the feed composition of the present invention may 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, antimicrobials, 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 powdered milk 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.

[0121] 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.).

[0122] 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.

[0123] Example

[0124] Manufacturing Example 1. SOD-expressing Bacillus velezensis strain

[0125] The Bacillus velezensis (Bacillus amyloliquefaciens) strain expressing SOD used in this example was 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"), 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"), or 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").

[0126] Spores of the strain were prepared according to the following method. A single colony of 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. 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 of bacto nutrient broth, 1 g / L of KCl, 0.25 g / L of MgSO4, 0.16415 g / L of Ca(NO3)2, 0.9521 mg / L of MnCl2, and 0.152 mg of FeSO4. MnSO4, MgSO4, FeSO4, (NH4)2SO4, (NH4)2PO4, and CaCl2 added to the above medium were dissolved in ddH2O before use. 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. 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 phosphate buffered saline (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. The spores were dissolved in phosphate-buffered saline (PBS) according to the experimental conditions and prepared in a volume of 100 μl.

[0127] Manufacturing Example 2. Manufacturing of GF427 strain

[0128] 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.

[0129] 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).

[0130] Substitution of base sequences in the genome was performed by double crossover recombination (Fig. 3a and Fig. 3b). 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).

[0131] 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.

[0132] 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

[0133] PCR Step Temperature Time Initial Denaturation 95℃ 30 sec 30 cycles 95℃ 30 sec 50℃ 30 sec 72℃ 1 min / kb Final Elongation 72℃ 10 min The DNA polymerase used in PCR is Taq DNA polymerase (TAKARA, JAPAN).

[0134] Mixture volume (μl) 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 μl) Total 10

[0135] 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 μg of pUori-cm-amp-10sod DNA was mixed with the prepared cells and placed in a chilled 1 mm gap electroporation cuvette, followed by incubation 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 μF, 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 μg / ml, 1.5% agar) and cultured in a 37°C incubator for 48 h. The transformed colonies were confirmed by PCR using cm conf F and cm conf R primers. Induction and confirmation of double crossover recombination were performed as follows. Confirmed colonies were added to LB medium and cultured at 37°C and 200 rpm for 24 hours, 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, the PCR conditions were set so that only the colonies that successfully engineered using primers containing the mutation site were confirmed to show a band, and the 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 the 424 conf F and 424 conf R primers, and after gel purification of the PCR product and sequencing the sample to confirm the correct substitution, it was named 'GF427'. This strain was deposited at the Korea Research Institute of Bioscience and Biotechnology on August 14, 2023 (KCTC 15552 BP). To confirm the increase in the 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 the GF424 and GF427 strains were inoculated with colonies in 1x SYPG (1.5% soytone, 1% yeast extract, 0.5% potassium phosphate dibasic, 1% glucose) containing 50 μg / 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 activity of SOD was measured at 22.2 U / mL for GF424 and 67.8 U / mL for GF427, which was approximately three times higher than that of GF424. In addition, GF427 strain spores were prepared using the same method as in Manufacturing Example 1.

[0136] Manufacturing Example 3. Preparation of glass SOD

[0137] Glass SOD was prepared by extracting and purifying the Bacillus belligerentis strain culture solution using the following method.

[0138] 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 hours. 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 hours. The cell culture obtained in this way was centrifuged at 3,578 xg at 4°C for 20 minutes, and the supernatant was collected and concentrated 10-fold using ultrafiltration (UF, MWCO 10,000). 390 g of ammonium sulfate was added per 1 L of the concentrated cell culture, stirred for 20 minutes, centrifuged, and the supernatant was collected. The collected supernatant was purified using a phenylsepharose HP column. The purification process was performed by equilibrating the column with 50 mM potassium phosphate, pH 7.0, containing 2 M ammonium sulfate, and then passing the obtained supernatant through the column. The SodA2 attached to the column was recovered using 50 mM potassium phosphate, pH 7.0, containing 1.6 M ammonium sulfate. The purified solution purified through the column was collected and concentrated while removing the high-concentration salts formed during the purification process through ultrafiltration. The concentrated solution was filtered through a sterilizing filter and then lyophilized. The activity of SodA2 was analyzed using a SOD assay kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits 50% of superoxide radicals.

[0139] Manufacturing Example 4. Manufacturing of Oral Administration Material

[0140] GF103 (Met-deleted SodA2 of SEQ ID NO: 6) was coated with shellac and prepared as an oral SOD as follows. Shellac (EXCELACS co., LTD., Bangkok) was dissolved in 100% ethanol to a concentration of 3% and prepared 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 size 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 resultant was freeze-dried. Through this, lyophilized shellac-coated SodA2 was finally prepared. 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.

[0141] Example 1. Preparation of an animal model of ischemia-reperfusion acute renal failure and administration of SOD.

[0142] Animal model production

[0143] To determine whether Bacillus-derived SOD exhibits a preventive effect in an animal model of renal injury, a renal injury animal model was established and experiments were conducted using the following methods. Six-week-old male C57BL / 6 mice were purchased from Orient Bio and housed in a specific pathogen-free 1 facility with free access to water and food. The mice were randomly assigned to the "untreated Sham group (Sham)", "untreated IRI group (IRI)", "SOD-BA-administered Sham group (SOD+Sham)", and "SOD-BA-administered IRI group (SOD+IRI)". The IRI group was a group in which ischemia-reperfusion injury was induced to induce acute renal failure, the "untreated IRI group" was a group in which ischemia-reperfusion injury was induced in mice that had not been administered SOD-BA, and the "SOD-BA-administered IRI group" was a group in which ischemia-reperfusion injury was induced 24 hours after SOD-BA administration. The Sham group was not induced with ischemia-reperfusion injury. Like the IRI group, they underwent dorsal incision after intraperitoneal anesthesia, but renal blood vessel ligation was not performed. Depending on whether SOD-BA was administered before surgery, they were referred to as the "non-administered Sham group" and the "SOD-BA-administered Sham group."

[0144] Induction of bilateral ischemic reperfusion injury (IRI)

[0145] Ischemia-reperfusion injury was induced by ligating both renal vessels through a dorsal incision under intraperitoneal anesthesia, blocking blood flow for 24 to 25 minutes, followed by reperfusion. The mice were monitored on a heating pad until they regained consciousness. This study was approved by the Institutional Review Board of the Animal Research Center, Korea University College of Medicine (IRB No. KOREA-2020-0061).

[0146] SOD-BA administration

[0147] The preparation of the administered substance (SOD-BA) in Manufacturing Example 4 was orally administered once per mouse at a dose of 20 U one day before vascular ligation.

[0148] Example 2. Measurement of changes in the intestinal environment

[0149] Example 2.1. Analysis of changes in intestinal microbiota

[0150] At least two fecal pellets per mouse were collected, stored at -70°C, and analyzed en bloc. 16S rRNA pyrosequencing was used for quantitative microbiome analysis.

[0151] The untreated Sham group (Sham), the SOD-BA-administered Sham group (SOD+Sham), the unadministered IRI group (IRI), and the SOD-BA-administered IRI group (SOD+IRI) showed different microbiome-gut microbiota compositions in the principal coordinate analysis (PCoA) (Fig. 4a). In the SOD-BA-administered IRI group (SOD+IRI), the gut microbiota diversity index remained similar to that of the unadministered IRI group (IRI), but the composition ratio of Bacteroidaceae increased. The composition ratio of Bacteroidaceae showed a greater difference than that between the unadministered Sham group (Sham) and the SOD-BA-administered Sham group (SOD+sham) (Fig. 4b).

[0152] Example 2.2. Intestinal permeability analysis

[0153] To analyze intestinal permeability, an FITC-dextran assay was performed. For this, fluorescein isothiocyanate-conjugated dextran (FITC-dextran; catalog number FD4; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS) (100 mg / mL) and administered orally to mice overnight with restricted drinking water. Fluorescence was extracted from the blood 4 hours later.

[0154] The results showed that intestinal permeability increased significantly in the untreated IRI group, whereas the increase was smaller in the SOD-BA-treated IRI group (Fig. 5). This suggests that SOD-BA administration alleviates (reduces) the increase in intestinal permeability caused by acute renal failure.

[0155] Example 2.3. Assessment of intestinal epithelial cell damage

[0156] Apoptosis of colonic epithelial cells was quantified by counting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive epithelial cells in 5–8 high-power fields (×200, high power field, HPF). Images were digitized and analyzed using a slide scanner, an automatic image capture system (Axio Scan Z1, Zeiss Korea, Seoul).

[0157] The analysis results showed that colonic epithelial cell death (TUNEL) was less in the SOD-BA-administered IRI group than in the non-administered IRI group (Figures 6a and 6b).

[0158] Example 2.4. Evaluation of intestinal inflammatory cell infiltration

[0159] Colonic tissues were stained with F4 / 80 monoclonal antibody (1:100; mch-497-GA; Bio-Rad Laboratories, Hercules, CA, USA) and Ly6G monoclonal antibody (1:200; 14-59-85; eBioscience) to assess macrophage / neutrophil infiltration levels.

[0160] As a result of analyzing the changes in the distribution of intestinal immune cells accompanying acute renal failure in the non-administered IRI group and the SOD-BA-administered IRI group, the neutrophil marker (Ly6G) was significantly increased in the non-administered IRI group and decreased in the SOD-BA-administered IRI group compared to the non-administered IRI group (Figures 7a and 7b).

[0161] Example 3. Measurement of changes in the kidney environment

[0162] Example 3.1. Measurement of SOD and catalase distribution in the kidney

[0163] SOD and catalase are oxidative stress factors that play a crucial role in alleviating oxidative stress, leading to the final metabolic product, water. The renal distribution of SOD and catalase was measured to assess whether they improve renal oxidative stress in acute renal failure.

[0164] SOD and catalase were stained with SOD monoclonal antibody (1:500; MA1-105, ThermoFisher) and catalase monoclonal antibody (1:500, PA5-29183, ThermoFisher), respectively, and the distribution was evaluated comparatively as the percentage of positive staining compared to the total tissue area at low magnification.

[0165] As a result of SOD observation, the distribution of SOD was decreased in the non-administered IRI group compared to the non-administered Sham group, and the distribution of SOD was increased in the SOD-BA-administered IRI group compared to the non-administered IRI group (Figures 8a and 8b).

[0166] In the case of catalase, the distribution of catalase significantly increased in the non-administered IRI group, while it remained at normal levels in the SOD-BA-administered IRI group (Figures 8c and 8d). Maintaining normal levels of catalase distribution suggests that oxidative stress was improved.

[0167] The reason why no significant change in catalase was observed in the IRI group administered SOD-BA is thought to be that the need for catalase was relatively less because the preceding SOD was increased in the process of alleviating oxidative stress (Fig. 8e).

[0168] Example 3.2. Evaluation of inflammatory cell infiltration in the kidney

[0169] To assess macrophage / neutrophil infiltration levels, kidney tissues were stained with F4 / 80 monoclonal antibody (1:100; mch-497-GA; Bio-Rad Laboratories, Hercules, CA, USA) and Ly6G monoclonal antibody (1:200; 14-59-85; eBioscience).

[0170] The renal protective effect of SOD-BA appeared to be unrelated to the improvement of renal immune cell deposition (Ly6G = neutrophil marker, F4 / 80 = lymphocyte marker) (Figs. 9a to 9d).

[0171] Example 4. Assessment of the severity of kidney damage

[0172] We confirmed that the increased levels of blood urea nitrogen (BUN) and plasma creatinine, which are elevated in renal damage, were alleviated, and the renal tubular damage score (ATN score) was improved.

[0173] Example 4.1. Blood urea nitrogen and creatinine tests

[0174] Creatinine and urea nitrogen are normally filtered and excreted by the kidneys, so they exist in small amounts in the blood. However, when there is a problem with kidney function, they are not filtered normally, so their concentration in the blood increases and they are used as factors for evaluating kidney function.

[0175] Plasma creatinine and urea nitrogen were measured using a Beckman AU® 5821 Beckman (Beckman Coulter, USA). After intraperitoneal anesthesia, tissues were removed, and liver and colon tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Kidney tissues were evaluated using a standard light microscope.

[0176] As a result of the test, it was confirmed that the levels of plasma creatinine and blood urea nitrogen, which were increased in acute renal failure, were reduced (alleviated) in the IRI group administered SOD-BA, and from this, it can be seen that SOD-BA administration has a renal protective effect (Figures 10a and 10b).

[0177] Example 4.2. Assessment of tubular damage

[0178] The extent of tubular damage was assessed in a dark-blind manner in five random fields (×100) throughout the renal tissue using periodic acid-Schiff staining (PAS). The extent of damage was assessed semiquantitatively and classified into grade 0, 0–25% (grade 1), 25–50% (grade 2), 50–75% (grade 3), and 75–100% (grade 4) according to the level of visible necrosis.

[0179] Since the acute tubular necrosis (ATN score) value in the IRI group administered SOD-BA was significantly lower than that in the non-administered IRI group, it was confirmed that the renal protective effect was caused by inhibition of tubular necrosis (Figures 11a and 11b).

[0180] Example 5. Alleviation of renal damage in an animal model of contrast-induced acute kidney injury.

[0181] Example 5.1. Construction of a contrast agent-induced acute renal injury model

[0182] To create a contrast-induced acute kidney injury (CI-AKI) model, 5-week-old C57BL / 6 male mice were purchased from Orient Bio and acclimatized for 7 days in a specific pathogen-free environment with free access to food and water. After anesthesia, the mice underwent unilateral nephrectomy (Unx), and rested for 4 weeks before single kidney mode observation. After a 4-week rest, Lasix (active ingredient: furosemide), a contrast agent that can cause renal dysfunction, was intravenously injected into the tail at a concentration of 10 mg / kg. A schematic diagram for constructing the experimental model is shown in Figure 12. Mice were randomly assigned to the "Vehicle-Administered CI-AKI Group (PBS+CI-AKI)" and the "SOD-BA-Administered CI-AKI Group (SOD+CI-AKI)", with 5 to 7 mice per group.

[0183] Example 5.2. SOD-BA administration

[0184] The preparation of the administered substance (SOD-BA) in Manufacturing Example 4 was orally administered once or twice per mouse at a dose of 20 U before contrast injection, and the mice were maintained in a dehydrated state for 48 hours. The CI-AKI group administered vehicle was orally administered PBS in the same amount as SOD.

[0185] Example 5.3. Effect of alleviating kidney damage

[0186] As a result of measuring creatinine, blood urea nitrogen (BUN), and renal tissue NGAL using the same method as in Example 4.1, administration of SOD-BA did not show renal toxicity, and in the CI-AKI group administered SOD-BA, the levels of creatinine, blood urea nitrogen (BUN), and renal NGAL were all decreased compared to the CI-AKI group administered vehicle (Figures 13a to 13c).

[0187] Just as SOD showed an effect of improving renal function in acute renal failure caused by ischemia-reperfusion injury described above, administration of the SOD of the present invention also showed an effect of preventing or improving renal damage in contrast agent-induced acute renal injury.

[0188] Statistical analysis

[0189] Data are expressed as the mean ± standard error of the mean (SEM). Unpaired t-tests and one-way ANOVA (with Bonferroni post hoc test) were used to compare two or more groups and three or more groups, respectively. A P value <0.05 was considered statistically significant. Data were analyzed using GraphPad Prism version 9.3.1 (GraphPad Software Inc., La Jolla, CA).

[0190] [Accession number]

[0191] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0192] Accession number: KCTC13222BP

[0193] Date of acceptance: March 6, 2017

[0194] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0195] Accession number: KCTC13227BP

[0196] Date of acceptance: 20170313

[0197] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0198] Accession number: KCTC15552BP

[0199] Date of acceptance: 20230814

Claims

1. A pharmaceutical composition for preventing or treating kidney damage, comprising a polypeptide having superoxide dismutase (SOD) activity as an active ingredient.

2. In paragraph 1, The above renal damage is renal failure, acute renal injury, chronic renal injury, contrast-induced acute renal injury, or ischemic acute renal failure. Pharmaceutical composition.

3. In paragraph 1, The composition exhibits at least one effect selected from the group consisting of changes in the composition of intestinal microflora, reduction of intestinal epithelial cell death, reduction of intestinal permeability, reduction of oxidative stress in the kidney, alleviation of tubular damage, and improvement of renal function. Pharmaceutical composition.

4. In paragraph 1, The above polypeptide is an isolated or purified protein, Pharmaceutical composition.

5. In paragraph 1, The above polypeptide is present in a Bacillus species strain or a spore thereof, Pharmaceutical composition.

6. In paragraph 1, The above polypeptide is included in the form of a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract or a dried form thereof. Pharmaceutical composition.

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

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

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

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

11. In paragraph 9, The above Bacillus species strain is 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.

12. 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.

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

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

15. In paragraph 1, The above composition is administered orally. Pharmaceutical composition.

16. In paragraph 1, The above composition is administered to a subject in need thereof before, after, or both before and after the occurrence of kidney damage. Pharmaceutical composition.

17. In paragraph 1, The above composition is administered to a subject requiring administration of another drug before, simultaneously with, or after administration of another drug that causes or is at risk of causing renal damage. Pharmaceutical composition.

18. In paragraph 17, The other drugs mentioned above are contrast agents, antibiotics, immunosuppressants, drugs for heart disease, or cancer chemotherapy drugs. Pharmaceutical composition.

19. Containing a polypeptide having superoxide dismutase (SOD) activity as an active ingredient A veterinary composition for preventing or improving kidney damage.

20. Containing a polypeptide having superoxide dismutase (SOD) activity as an active ingredient A food composition for preventing or improving kidney damage.

21. Containing a polypeptide having superoxide dismutase (SOD) activity as an active ingredient Feed composition for preventing or improving kidney damage.

22. A method comprising administering to a subject in need thereof a pharmaceutical composition according to any one of claims 1 to 21 or a polypeptide according to any one of claims 1 to 21. A method for preventing or treating kidney damage.

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