Postbiotic based on selenium-rich bacillus amyloliquefaciens c-1 and its use

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a post-fermentation product based on selenium-rich Bacillus amyloliquefaciens C-1 and application thereof. The culture method of the selenium-rich Bacillus amyloliquefaciens C-1 is as follows: Bacillus amyloliquefaciens is inoculated into a culture medium with a final concentration of 60-100 mu g / mL of sodium selenite at an inoculation amount of 7-10% of bacterial liquid, and aerated culture is carried out at room temperature for 24-30 hours, and the culture medium is LB culture medium containing 1% glucose; the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens C-1 with a preservation number of CCTCC NO:M2012177. The Bacillus amyloliquefaciens C-1 has strong selenium-rich metabolic capacity, and converts extracellular selenite into intracellular biological nanometer selenium through an oxidation-reduction pathway. The selenium-rich post-fermentation product obtained by inactivating the fermentation liquid of the Bacillus amyloliquefaciens C-1 has properties such as anti-inflammatory, anti-oxidation, anti-tumor and intestinal protection, has development potential of composite nanometer selenium material, and can be applied to multiple fields such as special medical and special diet food, health food, healthy ecological food, intestinal micro-ecological preparation, cosmeceutics, biological medicine and health.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a metagenetic agent based on selenium-enriched Bacillus amyloliquefaciens C-1 and its application. Background Technology

[0002] Selenium is a typical semi-metallic element, one of the fourteen essential trace elements for living organisms, and a component of many biological enzymes in the body. It possesses various functions, including antioxidant properties, enhanced immunity, tumor suppression, increased oxygen-carrying capacity of red blood cells, free radical scavenging, and cardiovascular protection. The human body cannot obtain sufficient selenium from a normal diet. Currently, traditional selenium supplements are mainly inorganic selenium, but these have drawbacks such as a narrow toxicity range and low bioavailability. Therefore, finding a safe and low-concentration selenium supplement has become a research hotspot.

[0003] Selenium in nature is mostly inorganic, primarily existing as selenite, which is toxic to the body. Nano-selenium has attracted attention due to its low toxicity and high activity. Currently, there are three synthetic methods for nano-selenium: physical, chemical, and biotransformation. Industrial nano-selenium can be produced through chemical reagent redox reactions and electrochemical techniques, but this is limited by high temperature, high pressure, and catalysts. Furthermore, the produced nano-selenium exhibits poor stability, uneven size, and low activity. Compared to the other two methods, biosynthesis is a key focus due to its green, environmentally friendly, and safe nature. Biosynthesis involves adding selenite during the normal growth of microorganisms, which then reduce the selenite to a zero-valent state nano-selenium through metabolism. Bio-nano-selenium possesses various regular nanostructures and high activity, making it suitable as a biological template for preparing nanomaterials. By utilizing the unique template proliferation function of bacteria, template diversification and morphological control of the final synthesized material can be achieved, yielding nanomaterials different from those obtained through conventional chemical synthesis methods. The selenium nanoparticles synthesized through microbial transformation are uniform in size and have high electron density. Furthermore, the bacterial SefA protein (selenium factor A) regulates the polymerization of the selenium nanoparticles, encapsulating the protein on the surface, resulting in more stable and higher-activity biosynthetic selenium nanoparticles. This method of biosynthesizing selenium nanoparticles offers advantages such as low cost, high safety, simple operation, stable structure, uniform size, and high bioactivity.

[0004] Numerous studies have demonstrated that microorganisms such as lactic acid bacteria, Bacillus, and yeast can convert inorganic selenium into nano-selenium. Due to the rapid growth, fast reproduction, strong metabolic capacity, and high adaptability of bacteria, and the fact that the conversion of selenium using probiotics is unaffected by seasons, has a short production cycle, is pollution-free, and has high biosafety, the production of nano-selenium using probiotics will become a new research hotspot, and its applications will become increasingly widespread.

[0005] In the process of producing nano-selenium using probiotics, two aspects need to be considered. First, the main factors that generally affect the synthesis of nano-selenium by bacteria include carbon source, nitrogen source, oxygen, temperature, fermentation pH, etc., and different bacteria have different selenate reductase and structure, so it is necessary to screen for strains with high selenium conversion rate and strong stress resistance. Second, probiotics produce many active secondary metabolites during metabolism. If only the nano-selenium produced by the selenium-enriched metabolism of probiotics is used, other beneficial components produced during fermentation will be greatly wasted.

[0006] Current research on the established probiotic mechanisms of probiotics is based on a certain number of live bacteria. In recent years, increasing research has found that, in addition to live bacteria, probiotic metabolites, lysate extracts, cell wall components, and even culture supernatants exhibit significant probiotic effects, with different components exhibiting different probiotic mechanisms. These non-probiotic components are called postbiotics. Many types of postbiotics have been identified, including short-chain fatty acids, enzymes, peptides, endogenous or exogenous polysaccharides, cell surface proteins, and organic acids. Their probiotic functions include, but are not limited to, antibacterial, antioxidant, and regulation of intestinal barrier function and immune responses. Besides maintaining the host's gut microbiota, protecting the intestinal epithelial barrier function, immunomodulation, and antioxidant functions, postbiotics also have advantages that probiotics cannot match, including well-defined chemical structures, safe dosage parameters, and longer shelf life. Furthermore, postbiotics can achieve similar probiotic effects while avoiding the problems of low bioavailability, unstable effects, and easy transmission of drug resistance genes inherent in live bacteria, making them a new direction for future research in the field of probiotics.

[0007] Bacillus has been used as a probiotic for at least 60 years. Combining the performance of probiotics in reducing and preparing nano-selenium with their probiotic effects, a small amount of probiotic-nano-selenium complex can supplement the human body with sufficient selenium, while also promoting intestinal health and improving immunity. Bacillus amyloliquefaciens C-1 is a patented strain isolated in the applicant's laboratory that produces high levels of extracellular polysaccharides, lipopeptides, proteases, and amylases (accession number CCTCC NO: M2012177), and has been granted two patents (ZL201210268667.0; ZL201410260574.2). It exhibits excellent growth and stress resistance, tolerating acid, high concentrations of bile salts, and NaCl, and can adhere to the intestinal mucosa. This bacterium has significant antibacterial, antitumor, antiviral, and larvicidal functions; it is sensitive to 14 common antibiotics, does not exhibit hemolytic or gelatin hydrolytic virulence phenotypes, tests for related virulence genes are negative, and it is non-toxic to the large wax moth. Currently, there are no reports on the biotransformation of sodium selenite by Bacillus amyloliquefaciens C-1 and the related characteristics of the generation of selenium nanoparticles (SeNPs).

[0008] In view of this, the present invention is hereby proposed. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a metabiotic based on selenium-enriched Bacillus amyloliquefaciens C-1 and its applications.

[0010] In a first aspect, the present invention provides a method for culturing selenium-enriched Bacillus amyloliquefaciens, which involves inoculating Bacillus amyloliquefaciens at a bacterial inoculum volume of 7-10% into a culture medium with a final sodium selenite concentration of 60-100 μg / mL, and culturing at room temperature with aeration for 24-30 h, wherein the culture medium is LB medium containing 1% glucose.

[0011] The Bacillus amyloliquefaciens mentioned is Bacillus amyloliquefaciens C-1, with accession number CCTCC NO:M2012177.

[0012] Secondly, the present invention provides a selenium-enriched Bacillus amyloliquefaciens obtained by the aforementioned method for culturing Bacillus amyloliquefaciens.

[0013] Thirdly, the present invention provides a selenium-enriched post-biotic, which is obtained by inactivation treatment of the fermentation broth of the selenium-enriched Bacillus amyloliquefaciens.

[0014] Fourthly, the present invention provides the application of the selenium-enriched post-biotic in the preparation of anti-tumor drugs.

[0015] Furthermore, the tumor includes lung cancer.

[0016] Fifthly, the present invention provides a drug for treating lung cancer, wherein the active ingredient is the selenium-enriched post-biotic.

[0017] Sixthly, the present invention provides the application of the selenium-enriched postbiotic in the preparation of intestinal protection drugs.

[0018] In a seventh aspect, the present invention provides an intestinal protection drug, the active ingredient of which is the aforementioned selenium-enriched postbiotic.

[0019] Eighthly, the present invention provides the use of the selenium-enriched post-biotic in the preparation of a drug with both anti-inflammatory and antioxidant effects.

[0020] In a ninth aspect, the present invention provides a drug with both anti-inflammatory and antioxidant effects, wherein the active ingredient is the aforementioned selenium-enriched post-biotic.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Current research on the established probiotic mechanisms of probiotics is based on a certain number of live bacteria. In recent years, an increasing number of studies have found that, in addition to live bacteria, the metabolites, lysate extracts, cell wall components, and even culture supernatants of probiotics exhibit significant probiotic effects, with different components exhibiting different probiotic mechanisms. These non-probiotic components are called postbiotics. Besides maintaining the host's gut microbiota, protecting the intestinal epithelial barrier function, immunomodulation, and antioxidant functions, postbiotics also have advantages that probiotics cannot match, including well-defined chemical structures, safe dosage parameters, and longer shelf life. Furthermore, postbiotics can achieve similar probiotic effects while avoiding the problems of low bioavailability, unstable effects, and easy transmission of drug resistance genes inherent in live bacteria, making them a new direction for future research in the field of probiotics.

[0023] This invention uses the patented strain Bacillus amyloliquefaciens C-1 as a probiotic strain to prepare and characterize bio-nano selenium through a biological template method. Simultaneously, starting from the nano-selenium producing strain C-1, it conducts post-biotic efficacy evaluation of the Bacillus selenium complex. Based on selenium-enriched probiotics, it provides sufficient and reliable data support for the development of novel nano-selenium composite post-biotic preparations. The product is expected to be used in the preparation of antimicrobial peptide-modified medical implants, medical materials, etc., and can also be directly applied in multiple fields such as special medical dietary foods, health foods, healthy ecological foods, intestinal microecological preparations, and biomedicine and health. Attached Figure Description

[0024] Figure 1 To determine the growth curves of Bacillus amyloliquefaciens C-1 under different selenium-enriched culture conditions.

[0025] Figure 2 The fermentation growth status of Bacillus amyloliquefaciens C-1 culture at different times (from left to right: growth status of bacterial solutions at 0h, 6h, 12h, 18h, 24h, and 30h).

[0026] Figure 3 To collect cells from Bacillus amyloliquefaciens C-1 culture at different time points after centrifugation, the state of the selenium-enriched cells is shown (from left to right, the aggregation state of cells after centrifugation of bacterial culture at 0h, 6h, 12h, 18h, 24h, and 30h are shown).

[0027] Figure 4 To investigate the growth curves and selenium conversion rate of Bacillus amyloliquefaciens C-1 culture at different time points.

[0028] Figure 5 The morphological observation results of Bacillus amyloliquefaciens C-1 under a non-selenium-enriched (a) and selenium-enriched (b) electron microscope (SEM) are shown (magnification of 10000×).

[0029] Figure 6Figure 1 shows the morphological observation results of Bacillus amyloliquefaciens without selenium (a) and with selenium (b) under a transmission electron microscope (magnification of 17500×). The arrow in Figure 2b points to the formed selenium nanoparticles.

[0030] Figure 7 The results of X-ray photoelectron spectroscopy (XPS) analysis of the elemental composition of Bacillus amyloliquefaciens C-1 cells after selenium-enriched metabolism.

[0031] Figure 8 This study aimed to detect the elemental content of Bacillus amyloliquefaciens C-1 cells after selenium-enriched metabolism using energy dispersive spectroscopy (EDS).

[0032] Figure 9 The scavenging effects of C-1 post-genetic agents (C-1) and selenium-enriched C-1 post-genetic agents (C1-SeNPs) on superoxide anions (a), hydroxyl radicals (b), and DPPH radicals (c) were compared between the selenium-enriched and non-selenium-enriched groups at the same concentration. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0033] Figure 10 The effects of C-1 postbiotic and selenium-enriched C-1 postbiotic antagonism against Salmonella on LDH activity in Caco-2 cells were investigated (Control: no bacterial treatment; S: treatment with Salmonella Typhimurium ATCC14028 alone for 2 h; C-1+S: C-1 postbiotic pretreatment for 2 h, followed by Salmonella Typhimurium ATCC14028 treatment for 2 h; C-1-Se+S: selenium-enriched C-1 postbiotic pretreatment for 2 h, followed by Salmonella Typhimurium ATCC14028 treatment for 2 h; S+C-1: Salmonella Typhimurium ATCC14028 pretreatment for 2 h, followed by C-1 postbiotic treatment for 2 h; S+C-1-Se: Salmonella Typhimurium ATCC14028 pretreatment for 2 h, followed by selenium-enriched C-1 postbiotic treatment for 2 h; compared with group S, *** indicates P<0.001).

[0034] Figure 11 The effects of C-1 postbiotic and selenium-enriched C-1 postbiotic on the growth of Caco-2 cells under oxidative stress were investigated. In the subdivisions, a represents the effect of C-1 postbiotic on the growth of Caco-2 cells under oxidative stress, b represents the effect of selenium-enriched C-1 postbiotic on the growth of Caco-2 cells under oxidative stress, c represents the effect of C-1 postbiotic pretreatment on the growth of Caco-2 cells under oxidative stress, and d represents the effect of selenium-enriched C-1 postbiotic pretreatment on the growth of Caco-2 cells under oxidative stress (Control: RMPI 1640 medium without antibiotics; H2O2 group: treatment with 200 μM H2O2; compared with the H2O2 group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001).

[0035] Figure 12 The effects of C-1 post-biotic and selenium-enriched C-1 post-biotic on the expression levels of inflammatory factors in Caco-2 cells after oxidative stress were investigated. a represents the relative expression level of IL-8 mRNA, b represents the relative expression level of IL-1β mRNA, and c represents the relative expression level of TNF-α mRNA (Control: RMPI 1640 medium without antibiotics; H2O2 group: treated with 200 μM H2O2; * indicates P < 0.05, *** indicates P < 0.001 compared to the H2O2 group).

[0036] Figure 13 The effects of C-1 postbiotic and selenium-enriched C-1 postbiotic on cell membrane permeability under oxidative stress in Caco-2 cells were investigated. a represents Claudin-1 expression level, b represents ZO-1 expression level, and c represents Occludin expression level (Control: RMPI 1640 medium without antibiotics; H2O2 group: treated with 200 μM H2O2; compared with the H2O2 group, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001).

[0037] Figure 14 The effect of co-incubation of C-1 postbiotic and selenium-enriched C-1 postbiotic on the survival rate of human lung cancer cells A549 (Control: no bacterial treatment; compared with the control group, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001). Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0039] The following examples involve information on the experimental strains, cells, and primers used:

[0040] Bacillus amyloliquefaciens C-1 is a patented strain isolated and preserved in our laboratory; Salmonella typhimurium ATCC14028 is a strain preserved in our laboratory. Both strains are preserved in cryovials containing 30% glycerol at a ratio of 1:1, and the cryovials are placed in a freezer at -80°C.

[0041] The human colon cancer epithelial cells Caco-2 and human non-small cell lung cancer cells A549 were commercially available cells purchased and cryopreserved in liquid nitrogen.

[0042] The primers used were synthesized by Sangon Biotech (Shanghai) Testing Co., Ltd. The gene names and sequences are shown in Table 1.

[0043] Table 1. Gene names and sequences of primers used in the experiment.

[0044]

[0045] Example 1: Bacterial Culture

[0046] Remove the glycerol cryovials of *Salmonella Typhimurium* ATCC14028 stored at -80°C and thaw them slowly at 4°C. Transfer 60 μL of the bacterial culture to 3 mL of sterile LB broth and incubate at 37°C with shaking for 24 hours. Then, transfer the culture to a 37°C incubator. After 24 hours, use a disposable inoculation loop to streak the culture onto LB agar plates and incubate overnight at 37°C. Then, store the culture at 4°C. For subsequent use, single colonies can be picked from the plates.

[0047] Take out the solid plate of Bacillus amyloliquefaciens C-1LB+1% glucose (LB+1%G) from the 4℃ refrigerator, place it in the clean bench, pick a single colony with a disposable inoculation loop, inoculate it into 3mL of sterile LB+1%G liquid medium, and incubate overnight at 30℃ in a constant temperature shaking incubator.

[0048] Example 2: Optimization of Culture Conditions for Selenium-Enriched Bacillus Amyloliquefaciens

[0049] 1 Experimental Methods

[0050] (1) Determination of selenium concentration and bacterial culture transfer rate

[0051] Sodium selenite was dissolved in sterile water to a final concentration of 100 mg / mL, sterilized by filtration through a 0.22 μM disposable filter, aliquoted, and stored at -20°C for later use. Single colonies from *Bacillus amyloliquefaciens* C-1 solid plates were picked and inoculated into 3 mL of LB + 1% G liquid medium, and cultured overnight at 30°C using a constant temperature shaking incubator. Different final concentration gradients of sodium selenite (0, 15, 30, 60, 100, 150 μg / mL) and different bacterial transfer volumes (1%, 3%, 5%, 7%, 10%) were added to the strains. The absorbance at 600 nm for each group was measured using a 96-well plate microplate reader and recorded as OD. 600 Fresh LB + 1% G liquid medium was used as the control group, and the cultures were continuously incubated for 22 hours, with measurements taken every hour. Based on time and OD... 600The growth curves were plotted to determine the optimal selenium concentration and bacterial culture transfer rate.

[0052] (2) Determination of culture time

[0053] The bacterial strain was cultured in selenium-enriched medium according to the method in (1). At culture times of 0h, 6h, 12h, 18h, 24h, and 30h, the cells were collected by centrifugation at 4℃, 10000g, and 15min. The cells were then washed three times sequentially with PBS and Tris-HCl (0.1mM, pH=7), dried to constant weight in a 45℃ oven, and stored at -20℃ for analysis. The samples were analyzed using atomic fluorescence spectrometry, and bacterial growth (OD) was measured at different time points. 600 The optimal culture time for the strain was determined by the selenium conversion rate (measured by atomic fluorescence spectrometry and then converted to selenium conversion per OD).

[0054] 2 Experimental Results

[0055] (1) Optimization of selenium addition and bacterial culture transfer amount

[0056] Bacillus amyloliquefaciens C-1 was inoculated into 96-well plates under different treatment conditions and cultured continuously for 22 h, with OD being measured continuously. 600 Plot the growth curve, such as Figure 1 As shown. The results indicate that when the initial selenium concentration is constant, the bacterial density (OD) 600 The OD increased with increasing bacterial inoculum transfer volume. Under conditions of a final selenium concentration of 100 μg / mL and an inoculum volume of 10%, after culturing for 22 h, the OD... 600 It can reach 0.666, which is the optimal value for bacterial growth.

[0057] (2) Determination of the optimal time point for selenium-enriched culture

[0058] To determine the optimal growth time for selenium-enriched culture, C-1 strains were continuously cultured in selenium-enriched medium for 30 hours, and the growth status of the strains at different time points (0h, 6h, 12h, 18h, 24h, 30h) was observed. Figure 2 After shaking and mixing, take 1 mL of the suspension, centrifuge, and discard the supernatant. The precipitation result is as follows: Figure 3 As shown in the figure. The results indicate that the liquid color changed significantly with increasing culture time, gradually turning from the original color of the bacteria to red. An increase in red precipitate was observed after centrifugation.

[0059] The selenium content at each time point was determined by atomic fluorescence absorption spectrometry, and the selenium conversion rate per OD was calculated. The results are as follows: Figure 4 As shown, the maximum selenium conversion rate per OD of Bacillus amyloliquefaciens C-1 is 55.51%, and the optimal culture time is 30 h.

[0060] Example 3: Characterization of selenium nanoparticles

[0061] 1 Experimental Methods

[0062] A single colony of *Bacillus amyloliquefaciens* C-1 solid culture plate was picked and inoculated into a 3 mL LB + 1% G liquid culture tube. The culture was then incubated overnight at 30°C with shaking. The OD of the cultured bacterial solution was adjusted. 600 To 1.0, 10% of the bacterial culture was transferred to a culture flask containing 100 mL LB + 1% G medium with a final concentration of 100 μg / mL sodium selenite. After overnight incubation at 30°C with shaking for 30 h, the cells were collected by centrifugation at 10000g for 15 min at 4°C and washed three times with PBS. For morphological analysis, the collected cells were fixed with 2% glutaraldehyde fixative and sent to the Analytical and Testing Center of Xi'an Jiaotong University for scanning electron microscopy (SEM / EDS) and transmission electron microscopy (TEM). For the detection of elements in the selenium-enriched bacterial cells, the cells were collected by centrifugation, washed with PBS, dried at 45°C to constant weight, and detected by X-ray photoelectron spectroscopy (XPS).

[0063] 2 Experimental Results

[0064] (1) Results observed by scanning electron microscopy

[0065] SEM was performed on *Bacillus amyloliquefaciens* C-1 (cultured in LB + 1% G medium at 30℃ for 30 h) and *Bacillus amyloliquefaciens* C-1 (inoculated in LB + 1% G medium with a final selenium concentration of 100 μg / mL, 10% inoculum, cultured at 30℃ for 30 h) to determine the morphological changes in C-1 cells. The results are as follows: Figure 5 As shown, there is no significant morphological change between unenriched and selenium-enriched C-1, both exhibiting a rod-like shape.

[0066] (2) Results of transmission electron microscopy

[0067] TEM observations were performed on C-1 (incubated in LB+1%G medium at 30℃ for 30 h) and selenium-enriched C-1 (incubated in LB+1%G medium with a final selenium concentration of 100 μg / mL, inoculum size of 10%, and cultured at 30℃ for 30 h). The results are as follows: Figure 6 As shown in the figure. Transmission electron microscopy revealed that selenium-enriched Bacillus amyloliquefaciens C-1 cells exhibited clearly enriched selenium particles. The nano-selenium particles were mainly aggregated within the cells (indicated by the arrow) and were mostly spherical with a diameter of 43-143 nm. The morphology of the unenriched and selenium-enriched cells did not change significantly.

[0068] (3) X-ray photoelectron spectroscopy results

[0069] Energy dispersive spectroscopy (EDS) was performed on selenium nanoparticles synthesized by Bacillus amyloliquefaciens C-1 through biotransformation. The results are as follows: Figure 7As shown, the elements carbon (C), nitrogen (N), oxygen (O), and selenium (Se) were detected.

[0070] (4) Energy Dispersion Spectroscopy Results

[0071] Energy dispersive spectroscopy (EDS) was performed on selenium nanoparticles synthesized by Bacillus amyloliquefaciens C-1 through biotransformation. The results are as follows: Figure 8 As shown in the figure. Energy dispersive spectroscopy results showed that the selenium-enriched Bacillus amyloliquefaciens C-1 contained detectable amounts of carbon (C), oxygen (O), selenium (Se), osmium (Os), sulfur (S), and copper (Cu), with contents of 74.62%, 8.46%, 5.22%, 0.62%, 8.84%, and 2.23%, respectively.

[0072] Example 4: Study on the antioxidant properties of selenium-enriched post-biotics

[0073] 1 Experimental Methods

[0074] Selenium-enriched Bacillus amyloliquefaciens C-1 was obtained by culturing Bacillus amyloliquefaciens s C-1 according to the method in Example 2. The fermentation product was diluted and plated for counting; the calculated viable cell count per milliliter of fermentation broth was 1.2 × 10⁻⁶. 7 CFU. The diluted Bacillus amyloliquefaciens C-1 full fermentation broth and the selenium-enriched Bacillus amyloliquefaciens C-1 full fermentation broth were inactivated at 121℃ for 20 min to obtain postgenes, denoted as C1 postgene and selenium-enriched C-1 postgene.

[0075] (1) Experiment on scavenging superoxide anions

[0076] Using the inhibition and generation of superoxide anions (O2) - The free radical scavenging kit is operated according to the kit instructions. The specific operation is as follows: Dilute the selenium-enriched C-1 post-biotic to 2.5, 5, 10, 20, and 30 mg / mL according to the selenium concentration per OD selenium conversion rate. Dilute the C1 post-biotic to the same factor. Take 50 μL of sample into a 5 mL centrifuge tube, and add the three reagents sequentially according to the instructions. Mix thoroughly and incubate at 37°C for 40 min. Then add 2 mL of chromogenic reagent, mix well, and let stand at room temperature for 10 min. Take 200 μL of the supernatant into an ELISA plate and measure the OD. 550 200 μL of double-distilled water was used as a blank control. The calculation formula is as follows:

[0077]

[0078] Where: A0—average OD value of blank control; A1—average OD value of test sample.

[0079] (2) Hydroxyl radical scavenging experiment

[0080] Using hydroxyl radicals (OH)- The assay kit is as follows: Dilute the selenium-enriched C-1 post-genetic agent to 2.5, 5, 10, 20, and 30 mg / mL according to the selenium concentration per OD selenium conversion rate. Dilute the C-1 post-genetic agent by the same factor. Take 200 μL of sample into a 5 mL centrifuge tube, follow the instructions, mix well, and react at 37℃ for 1 min. From the time the reagent 3 is added until the 1-minute mark, immediately add 2 mL of colorimetric reagent to stop the reaction; mix well, let stand at room temperature for 20 min, zero the chamber with double-distilled water, and measure the OD of each tube. 550 The calculation formula is as follows:

[0081] OH - Clearance rate (%) = [(A0-A1) / A0] × 100%

[0082] Where: A0—average OD value of blank control; A1—average OD value of test sample.

[0083] (3) DPPH free radical scavenging experiment

[0084] The DPPH radical scavenging kit was used with the following procedure: Selenium-enriched C-1 post-biotics were diluted to 2.5, 5, 10, 20, and 30 mg / mL according to their selenium concentration, based on the OD selenium conversion rate per unit volume. All C-1 post-biotics were diluted by the same factor. 400 μL of sample was placed in a 1.5 mL EP tube, and 600 μL of the working solution from the kit was added. The mixture was thoroughly mixed and incubated at 25°C in the dark for 30 min. The tubes were then centrifuged at 4000 rpm for 5 min. The tube was zeroed with anhydrous ethanol, and the OD values ​​of each tube were measured. 517 The calculation formula is as follows:

[0085] DPPH clearance rate (%) = [(A0-A1) / A0] × 100%

[0086] Where: A0—average OD value of blank control; A1—average OD value of test sample.

[0087] 2 Experimental Results

[0088] This embodiment investigated the scavenging abilities of selenium-enriched and selenium-enriched Bacillus amyloliquefaciens postbiotics against superoxide anions, hydroxyl radicals, and DPPH radicals. The results are shown in […]. Figure 9 .Depend on Figure 9 As shown in Figure a, both selenium enrichment and non-selenium enrichment demonstrated the ability to scavenge superoxide anions. Specifically, when the concentration exceeded 10 mg / mL, the scavenging ability of the non-selenium enrichment group was significantly higher than that of the selenium enrichment group at the same concentration (10 mg / mL: 75.95% vs 69.47%, P = 0.001; 20 mg / mL: 77.42% vs 67.93%, P < 0.001; 30 mg / mL: 78.24% vs 65.99%, P = 0.006). After selenium enrichment, there was no significant difference in scavenging ability among different concentrations, all remaining around 68%.

[0089] The scavenging abilities of C-1 post-biotics and selenium-enriched C-1 post-biotics against hydroxyl radicals are shown in the results. Figure 9 b. As shown in the figure, both selenium-enriched and unenriched groups exhibited excellent hydroxyl radical scavenging capabilities. After selenium enrichment, there was no significant difference in scavenging capacity across different concentrations, remaining around 92%. Furthermore, when the concentration was below 5 mg / mL, the scavenging capacity of the selenium-enriched group was significantly higher than that of the unenriched group (2.5 mg / mL: 92.90% vs 84.81%, P<0.001; 5 mg / mL: 92.10% vs 88.75%, P<0.001).

[0090] The scavenging abilities of C-1 postbiotics and selenium-enriched C-1 postbiotics against DPPH free radicals are shown in the results. Figure 9 c. As shown in the figure, both the selenium-enriched and unenriched groups exhibited the ability to scavenge DPPH free radicals. With increasing concentration, the DPPH scavenging ability of both groups gradually increased, showing a certain dose-dependent effect. When the concentration was greater than 10 mg / mL, the scavenging ability of the selenium-enriched group was significantly higher than that of the unenriched group (10 mg / mL: 71.98% vs 52.95%, P = 0.001; 20 mg / mL: 75.85% vs 52.31%, P = 0.019; 30 mg / mL: 80.16% vs 50.42%, P < 0.001).

[0091] Example 5: Effects of C-1 post-genetic agents and selenium-enriched C-1 post-genetic agents on Caco-2 cell viability

[0092] Caco-2 cells, with similar structure and function to small intestinal epithelial cells, are widely used to construct intestinal models. C-1 postbiotics and selenium-enriched C-1 postbiotics were regulated using RMPI 1640 medium (antibody-free, 20% fetal bovine serum, the same below) to achieve bacterial concentrations of 8 × 10⁻⁶. 6 1×10 6 8×10 5 1×10 5 8×10 4 1×10 4 8×10 3 CFU / mL.

[0093] After Caco-2 cells were seeded into a monolayer, the culture medium was gently aspirated and removed. 100 μL of C-1 and selenium-enriched C-1 bacterial suspensions (inactivated) at different concentration gradients were added to each well. Antibiotic-free RMPI 1640 medium was used as a control. After incubation for 20 h, the supernatant was removed, and 50 μL of 1×MTT was added to each well for another 4 h of incubation. The supernatant was then removed again, and 150 μL of DMSO was added to each well. OD was then measured. 490 Results are expressed as cell viability:

[0094] Survival rate (%) = (B1 / B2) × 100

[0095] In the formula: B1——C-1 / C-1-Se treatment group OD 490 B2 – Control group OD 490 .

[0096] 2 Experimental Results

[0097] The effects of different concentrations of C-1 postbiotics and selenium-enriched C-1 postbiotics on the growth of Caco-2 cells were detected using the MTT assay. Cell viability results are shown in Table 4. The results showed that, compared with the blank control group, the concentration of bacterial culture added to the culture medium was 8 × 10⁻⁶. 3 1×10 4 8×10 4 1×10 5 CFU / mL C-1 postbiotics and selenium-enriched C-1 postbiotics had no significant effect on Caco-2 cell growth (P>0.05); with increasing bacterial concentration, the selenium-enriched group had no significant effect on cell viability, reaching 8×10⁻⁶. 5 After reaching CFU / mL, a decreasing trend emerged. Subsequent experiments used 8×10⁻⁶ CFU / mL. 3 1×10 4 8×10 4 1×10 5 The bacterial concentration of CFU / mL was used as the concentration of the treated cells.

[0098] Table 4. Effects of C-1 post-biotics and selenium-enriched C-1 post-biotics on Caco-2 cell viability.

[0099]

[0100] Example 6: Effects of C-1 post-genetic agents and selenium-enriched C-1 post-genetic agents on the integrity of Caco-2 cells

[0101] 1 Experimental Methods

[0102] Once the cells have grown to form a complete monolayer, 1 mL of 0.25% trypsin is added to digest them. The cells are then counted and divided into groups of 2 × 10⁻⁶ cells per well. 5 1 mL of standard cells was added to each clean 24-well cell culture plate. After the cells formed a monolayer, the concentrations of Salmonella Typhimurium ATCC14028, C-1 postbiotic, and selenium-enriched C-1 postbiotic were adjusted to 1 × 10⁻⁶ using RMPI 1640 medium. 7 CFU / mL. Four treatment groups were set up: C-1+S group, C-1-Se+S group, S+C-1 group, and S+C-1-Se group. The sample treatment process is as follows:

[0103] C-1+S group: 1 mL of diluted C-1 post-gene (inactivated) was added to each well of the treatment group and cultured in a 37℃, 5% CO2 cell culture incubator for 2 h. The supernatant was collected. The cells were then gently washed 3 times with sterile PBS, and 1 mL of diluted Salmonella ATCC14028 bacterial culture was added. The cells were cultured in a 37℃, 5% CO2 cell culture incubator for another 2 h. The supernatant was collected.

[0104] C-1-Se+S group: 1 mL of diluted selenium-enriched C-1 postbiotic (inactivated) was added to each well of the treatment group and cultured in a 37℃, 5% CO2 cell culture incubator for 2 h. The supernatant was collected. The cells were then gently washed 3 times with sterile PBS, and 1 mL of diluted Salmonella ATCC14028 bacterial culture was added. The cells were cultured in a 37℃, 5% CO2 cell culture incubator for another 2 h. The supernatant was collected.

[0105] S+C-1 group: 1 mL of diluted Salmonella ATCC14028 bacterial suspension was added to each well of the treatment group and cultured in a 37℃, 5% CO2 cell culture incubator for 2 h. The supernatant was collected. The cells were then gently washed 3 times with sterile PBS, and 1 mL of diluted C-1 post-biotic (inactivated) was added. The cells were cultured in a 37℃, 5% CO2 cell culture incubator for another 2 h. The supernatant was collected.

[0106] S+C-1-Se group: 1 mL of diluted Salmonella ATCC14028 bacterial suspension was added to each well of the treatment group and cultured in a 37℃, 5% CO2 cell culture incubator for 2 h. The supernatant was collected. The cells were then gently washed 3 times with sterile PBS, and 1 mL of diluted selenium-enriched C-1 post-biotic (inactivated) was added. The cells were cultured in a 37℃, 5% CO2 cell culture incubator for another 2 h and the supernatant was collected.

[0107] 2 Experimental Results

[0108] Lactate dehydrogenase (LDH) is a cytoplasmic enzyme. When the cell membrane ruptures, intracellular LDH leaks into the culture medium. Therefore, a significant increase in LDH activity in the culture medium can serve as a direct indicator of cell membrane damage. This study evaluated the effect of selenium-enriched C-1 metabiotic on the cell membrane integrity of Caco-2 cells using LDH activity in cell supernatant. The results are as follows: Figure 10As shown in the figure. The results showed that, compared with the control group, the model group showed cell membrane rupture and significantly increased LDH activity after treatment with Salmonella typhimurium ATCC14028 (292.99 vs 219.94, P<0.001). Compared with the model group, Caco-2 cells pretreated with C-1 postbiotic and selenium-enriched C-1 postbiotic before Salmonella were added, both of which had a protective effect on the cells, and the protective effect of selenium-enriched C-1 postbiotic was higher than that of C-1 postbiotic pretreatment. However, when Salmonella treatment was performed first to model the cells, and then C-1 postbiotic and selenium-enriched C-1 postbiotic were added for intervention, the LDH activity of the cells was significantly lower than that of the Salmonella group (C-1: 144.58 vs 219.94, P<0.001; C-1-Se: 211.35 vs 219.94, P<0.001).

[0109] Example 7: Effects of C-1 post-genetic agents and selenium-enriched C-1 post-genetic agents on a Caco-2 cell oxidative stress model 1. Experimental methods

[0110] In the experiment, Caco-2 cells were treated with 200 μM H2O2 for 4 h to construct an oxidative stress model.

[0111] (1) Effects of C-1 postbiotic and selenium-enriched C-1 postbiotic pretreatment on the growth of Caco-2 cells under oxidative stress

[0112] After Caco-2 cells were seeded into a monolayer, the culture medium was gently aspirated and removed. Then, 100 μL of a concentration gradient prepared with RMPI 1640 medium was added to each cell to achieve a bacterial concentration of 1 × 10⁻⁶. 5 8×10 4 1×10 4 8×10 3 CFU / mL of C-1 post-biotic or selenium-enriched C-1 post-biotic (inactivated) was placed in a cell culture incubator and cultured at 37°C for 20 h. A blank control group was also set up, containing RMPI 1640 medium without C-1 post-biotic or selenium-enriched C-1 post-biotic. The medium was then carefully aspirated, and 100 μL of H2O2 prepared with RMPI 1640 medium was added. The blank control group was incubated with 100 μL of RMPI 1640 medium, and cultured at 37°C for 4 h. After incubation, the supernatant was removed, and 50 μL of 1×MTT was added to each well for another 4 h. The supernatant was then removed, and 150 μL of DMSO was added to each well to measure OD. 490 Results are expressed as cell viability:

[0113] Survival rate (%) = (B1 / B2) × 100

[0114] In the formula: B1——C-1 / C-1-Se treatment group OD 490 B2 – Control group OD490 .

[0115] (2) Effects of C-1 postbiotics and selenium-enriched C-1 postbiotics on the growth of Caco-2 cells under oxidative stress

[0116] After Caco-2 cells formed a monolayer, the culture medium was gently aspirated and removed. 100 μL of H2O2 prepared with RMPI 1640 medium was added to each cell. 100 μL of RMPI 1640 medium was added to the blank control group. Cells were incubated at 37°C for 4 hours. The culture medium was then gently aspirated and removed. 100 μL of a mixture prepared with RMPI 1640 medium was added to each cell, resulting in bacterial concentrations of 1 × 10⁻⁶ cells. 5 8×10 4 1×10 4 8×10 3 CFU / mL of C-1 post-biotic or selenium-enriched C-1 post-biotic (inactivated) was placed in a cell culture incubator and cultured at 37°C for 20 h. A blank control group was also provided, containing RMPI 1640 medium without selenium-enriched C-1 or C-1. After incubation, the supernatant was removed, and 50 μL of 1×MTT was added to each well for another 4 h of incubation. The supernatant was then removed again, and 150 μL of DMSO was added to each well. OD was measured using a microplate reader. 490 Results are expressed as cell viability:

[0117] Survival rate (%) = (B1 / B2) × 100

[0118] In the formula: B1——C-1 / C-1-Se treatment group OD 490 B2 – Control group OD 490 .

[0119] (3) qRT-PCR method was used to detect the expression of inflammatory factors and genes related to tight junction proteins in cells.

[0120] Total RNA was extracted from cells, reverse transcribed, and cDNA was synthesized. After preparation, the cDNA was placed in a PCR instrument at 42°C for 5 min. After the reaction, the cDNA was placed on ice. The reaction product was then used to prepare a system for reverse transcription. The system was mixed and placed in a PCR instrument at 50°C for 15 min. The reaction product can be used directly for qRT-PCR or stored at -80°C.

[0121] Follow the instructions for the qRT-PCR kit. The reaction mixture is shown in Table 2.

[0122] Table 2 qRT-PCR system

[0123]

[0124] After the system was prepared, the reaction was carried out using a real-time PCR instrument. The program settings are shown in Table 3.

[0125] Table 3 qRT-PCR reaction conditions

[0126]

[0127] After the real-time quantitative PCR reaction, the validity of the results was analyzed based on the melting curves and amplification curves obtained from the PCR. The relative expression difference of the target gene between each group and the control group was calculated using the ΔΔCt method based on the Ct values ​​of the target gene and internal reference gene in each group. ΔΔCt = (Ct...) 目的基因 -Ct 内参基因 Processing group - (Ct) 目的基因 -Ct 内参基因 The control group, with gene expression results as 2 -ΔΔCt express.

[0128] 2 Experimental Results

[0129] (1) Effects of C-1 post-biotics and selenium-enriched C-1 post-biotics on the growth of Caco-2 cells under H2O2-induced oxidative stress

[0130] The effects of C-1 post-genetic agents and selenium-enriched C-1 post-genetic agents on H2O2-induced oxidative stress cell growth were investigated using the MTT assay. The results are as follows: Figure 11 As shown.

[0131] Depend on Figure 11 From a and b, we can see that when cells are subjected to oxidative stress, the addition of selenium-enriched C-1 post-biotics and C-1 post-biotics, compared with the H2O2 group, resulted in a bacterial concentration of 8 × 10⁻⁶. 3 1×10 4 8×10 4 CFU / mL of C-1 postbiotics can promote cell growth (82.75% vs 68.85%, P = 0.009; 81.63% vs 68.85%, P = 0.014; 73.35% vs 68.85%, P = 0.007); when the bacterial concentration is 1×10⁻⁶, the effect is more pronounced. 5 CFU / mL of selenium-enriched C-1 postgenetic agent can promote cell growth (77.21% vs 68.85%, P = 0.024).

[0132] Depend on Figure 11 From c and d, it can be seen that compared with the H2O2 group, pretreatment with C-1 post-biotic and selenium-enriched C-1 post-biotic in Caco-2 cells can alleviate H2O2-induced cell growth inhibition. Specifically, when the bacterial concentration was 8 × 10⁻⁶... 3 1×10 4The effect was most significant at CFU / mL (C-1: 108.51% vs 93.06%, P<0.001; 114.5% vs 93.06%, P<0.001; selenium-enriched C-1: 111.53% vs 93.06%, P<0.001; 115.88% vs 93.06%, P<0.001). In conclusion, Caco-2 cells showed higher survival rates after treatment with C-1 postbiotics and selenium-enriched C-1 postbiotics.

[0133] Therefore, 8 × 10⁸ g of pre-added Caco-2 cells will be added. 3 1×10 4 CFU / mL of selenium-enriched C-1 postbiotic and C-1 postbiotic were used for subsequent studies.

[0134] (2) Effects of C-1 post-biotics and selenium-enriched C-1 post-biotics on the expression of inflammatory factors in Caco-2 cells under oxidative stress

[0135] The relative mRNA expression levels of cytokines IL-8, IL-1β, and TNF-α in a H2O2-induced Caco-2 cell inflammation model were detected by qRT-PCR. The results are as follows: Figure 12 As shown.

[0136] Compared with the negative control group, the mRNA expression levels of IL-8, IL-1β, and TNF-α in the H2O2-induced Caco-2 cell inflammation model were significantly increased (3.4875 vs 1, P < 0.001; 5.000 vs 1, P < 0.001; 9.331 vs 1, P < 0.001); compared with the H2O2-induced model group, 1×10 4 Pretreatment with CFU / mL selenium-enriched C-1 postbiotics reduced IL-8 mRNA expression levels (1.687 vs 3.487, P = 0.01). There were no significant differences in IL-8 mRNA expression levels between the low-concentration selenium-enriched C-1 postbiotic group and the high-concentration C-1 postbiotic group. Compared with the H2O2-induced model group, all four treatment groups significantly reduced IL-1β mRNA expression levels (high-concentration C-1 postbiotic: 2.432 vs 5.000, P < 0.001; low-concentration C-1 postbiotic: 1.258 vs 5.000, P < 0.001; high-concentration selenium-enriched C-1 postbiotic: 1.031 vs 5.000, P < 0.001; low-concentration selenium-enriched C-1 postbiotic: 2.371 vs 5.000, P < 0.001). 5.000, P<0.001), where 1×10 4The lowest IL-1β mRNA expression level was observed after treatment with selenium-enriched C-1 postbiotics at CFU / mL. Compared with the H2O2-induced model group, all four treatment groups significantly reduced TNF-α mRNA expression levels (high concentration C-1 postbiotic: 7.837 vs 9.331, P < 0.001; low concentration C-1 postbiotic: 4.687 vs 9.331, P < 0.001; high concentration selenium-enriched C-1 postbiotic: 2.677 vs 9.331, P < 0.001; low concentration selenium-enriched C-1 postbiotic: 5.974 vs 9.331, P < 0.001), with 1×10 4 The mRNA expression level of TNF-α was lowest after treatment with selenium-enriched C-1 postbiotic at CFU / mL.

[0137] (3) Effects of C-1 postbiotics and selenium-enriched C-1 postbiotics on the permeability of Caco-2 cell membranes under oxidative stress

[0138] The gene expression levels of three proteins (Claudin-1, ZO-1, and Occludin) in the tight junction structure of Caco-2 cells were quantitatively analyzed using qRT-PCR. The results are as follows: Figure 13 As shown.

[0139] Compared with the control group, after H2O2-induced cell damage, the expression levels of Claudin-1, ZO-1, and Occludin were significantly downregulated (0.611 vs 1, P < 0.001; 0.472 vs 1, P < 0.001; 0.307 vs 1, P < 0.001); compared with the H2O2 group, 8 × 10 3 CFU / mL of selenium-enriched C-1 postbiotics significantly increased Claudin-1 mRNA expression levels (0.971 vs 0.611, P < 0.001); compared with the H2O2 group, there was no significant difference in ZO-1 mRNA expression levels among the four treatment groups (P > 0.05); compared with the H2O2 group, 1×10 4 The highest mRNA expression level of Occludin was observed after treatment with CFU / mL C-1 postbiotic (0.750 vs 0.307, P < 0.001).

[0140] Example 8: Antitumor effects of C-1 post-biotics and selenium-enriched C-1 post-biotics

[0141] 1 Experimental Methods

[0142] The following bacteria were prepared using RMPI 1640 medium: Bacillus amyloliquefaciens C-1 complete fermentation product (C-1 complete fermentation product, not inactivated), selenium-enriched Bacillus amyloliquefaciens C-1 complete fermentation product (selenium-enriched C-1 complete fermentation product, not inactivated), C-1 postbiotic, and selenium-enriched C-1 postbiotic, with bacterial concentrations of 8 × 10⁻⁶. 6 8×10 5 8×10 4 CFU / mL. After A549 cells have formed a monolayer, the culture medium is gently aspirated and removed. 100 μL of different concentration gradients of *Bacillus amyloliquefaciens* C-1, selenium-enriched *Bacillus amyloliquefaciens* C-1, C-1 post-generant, and selenium-enriched C-1 post-generant are added to each well. RMPI 1640 medium is used as a blank control. Incubation is performed for 20 h. After incubation, the supernatant is removed, and 50 μL of 1×MTT is added to each well. Incubation continues for 4 h, then the supernatant is removed again, and 150 μL of LDMSO is added to each well. OD is measured using a microplate reader. 490 Results are expressed as cell viability:

[0143] Survival rate (%) = (B1 / B2) × 100

[0144] In the formula: B1——C-1 / C-1-Se treatment group OD 490 B2 – Control group OD 490 .

[0145] 2 Experimental Results

[0146] The effects of C-1 post-genetic agents and selenium-enriched C-1 post-genetic agents on the survival rate of human non-small cell lung cancer A549 cells were detected by the MTT assay. The results are as follows: Figure 14 As shown.

[0147] Compared with the blank control group, selenium-enriched C-1 post-biotic significantly reduced the survival rate of A549 cells (8 × 10⁻⁶). 4 CFU / mL: 76.21% vs 100%, P<0.001; 8×10 5 CFU / mL: 57.63% vs100%, P<0.001; 8×10 6 CFU / mL (54.88% vs 100%, P<0.001) induced apoptosis in cells. Furthermore, cell viability gradually decreased with increasing concentration.

[0148] Compared with C-1 post-genetic agents, selenium-enriched C-1 whole fermentation product can increase its ability to promote apoptosis in A549 cells (8×10). 4 CFU / mL: 76.21% vs96.58%, P<0.001; 8×10 5CFU / mL: 57.63% vs86.39%, P<0.001; 8×10 6 CFU / mL: 54.88% vs 84.98%, P<0.001).

[0149] Meanwhile, the ability of selenium-enriched C-1 post-biotic to promote apoptosis in A549 cells was significantly higher than that of selenium-enriched C-1 whole fermentation product (8×10⁻⁶). 4 CFU / mL: 76.21% vs 90.37%, P<0.001; 8×10 5 CFU / mL: 57.63% vs 85.42%, P<0.001; 8×10 6 CFU / mL (54.88% vs 71.74%, P<0.001) induced cell apoptosis. Furthermore, cell viability gradually decreased with increasing concentration.

[0150] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0151] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a selenium-enriched metagenerant in the preparation of anti-lung cancer drugs, characterized in that, The selenium-enriched post-biotic was obtained by inactivating the diluted selenium-enriched Bacillus amyloliquefaciens fermentation broth at 121°C for 20 minutes. The diluted selenium-enriched Bacillus amyloliquefaciens fermentation broth was prepared by the following steps: Bacillus amyloliquefaciens was inoculated at a bacterial inoculum volume of 10% into a culture medium with a final sodium selenite concentration of 100 μg / mL, and cultured at room temperature with aeration for 30 h. The resulting fermentation broth was then diluted to obtain the diluted selenium-enriched Bacillus amyloliquefaciens fermentation broth. The culture medium was LB medium containing 1% glucose. The Bacillus amyloliquefaciens mentioned is the Bacillus amyloliquefaciens with accession number CCTCC NO:M2012177. Bacillusammyloliquefaciens C-1.

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