Bacillus amyloliquefaciens and its application

Abstract

The present invention relates to the field of microorganisms, and in particular to a strain of Bacillus amyloliquefaciens and its application. The Bacillus amyloliquefaciens is named Bacillus amyloliquefaciens ( Bacillus amylolyticus )Wang, and was deposited in the China Center for Type Culture Collection on May 20, 2024, with the deposit number CCTCC NO:M 20241004. The Bacillus amyloliquefaciens described in the present invention can degrade copper ions in the environment, and also has a biological elimination effect on copper ions. It can reduce the accumulation of copper in the body by promoting the expression of ATP7A gene and ATP7B gene, reduce copper absorption by inhibiting the expression of copper transporter gene SLC31A1, and reduce oxidative stress by upregulating the expression of antioxidant genes SOD1 and SOD3. In addition, the Bacillus amyloliquefaciens described in the present invention has a protective effect on visceral organs, which is manifested in reducing the activity of enzymes (alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase) in visceral organs, especially liver organs, and reducing visceral organ stress response and damage.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Bacillus amyloliquefaciens and application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Copper is one of the essential trace elements for animal bodies. It plays an irreplaceable role in the body's hematopoiesis, metabolism, growth and reproduction, maintaining production performance, and enhancing the body's resistance. Therefore, it is widely used in livestock and poultry farming as an efficient and inexpensive growth-promoting additive. Copper, as a growth promoter, is added to pig feed, which has the ability to inhibit bacteria and significantly promote growth, and can accelerate the growth rate of pigs and improve feed efficiency. Excessive use of copper in animal feed can lead to animal poisoning and copper accumulation in animal products. This excessive use not only harms the health of livestock, but also affects public health. When excessive copper sulfate enters the host, it causes poisoning. Soluble copper sulfate is a protein coagulant with irritating and corrosive effects. When the host ingests copper, a complex - ceruloplasmin (CER) is formed, which is stored in tissues such as the liver, brain, and kidney. When the absorbed copper exceeds the liver's storage capacity, the copper in the liver is released in large quantities, resulting in a variety of different copper poisoning symptoms. Copper enters red blood cells, increasing their fragility, causing hemolysis, jaundice, and hemoglobinuria; copper ions oxidize ferrous hemoglobin to methemoglobin, reducing the blood's ability to transport oxygen, resulting in dyspnea and wheezing; when copper enters the digestive tract in large quantities, it can cause necrotizing gastroenteritis, leading to diarrhea and death. Therefore, strengthening the prevention of copper poisoning is one of the tasks that cannot be ignored in the global pig farming industry. Summary of the invention

[0004] The object of the present invention is to provide a strain of Bacillus amyloliquefaciens, which can not only degrade copper ions in the environment, but also has a biological elimination effect on copper ions in animals, and the biological elimination effect is embodied in that it can not only reduce the accumulation and absorption of copper in the body and increase the excretion of copper by promoting the expression of copper transporter genes, but also can reduce oxidative stress by up-regulating the expression of antioxidant genes. In addition, the strain also shows a protective effect on internal organs, which is manifested in that it can reduce the activity of enzymes in the internal organs and reduce the stress response and damage of the internal organs.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect of the present invention, a strain of Bacillus amyloliquefaciens is provided, which is named Bacillus amyloliquefaciens ( Bacillus amylolyticus) Wang, was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on May 20, 2024, with the deposit number CCTCC NO: M 20241004.

[0007] The Bacillus amyloliquefaciens of the present invention has good growth performance, can enter the logarithmic growth phase after 4 hours of cultivation, and can well tolerate artificial intestinal juice (survival rate of 92%) and artificial gastric juice (survival rate of 85%).

[0008] In a second aspect of the present invention, a fermentation product of Bacillus amyloliquefaciens described in the first aspect is provided.

[0009] In the third aspect of the present invention, there is provided a use of the Bacillus amyloliquefaciens strain described in the first aspect or the fermentation product of the Bacillus amyloliquefaciens described in the second aspect in preparing a product for environmental remediation, wherein the product is a bioremediation agent for degrading copper ions in the environment.

[0010] In some embodiments of the present invention, an in vitro copper ion adsorption test was conducted to test and compare the adsorption capacity of Bacillus amyloliquefaciens of the present invention and other strains (such as Lactobacillus salivarius, Lactobacillus rumenicus, Lactobacillus plantarum WSCFI, Lactobacillus rhamnosus, Bacillus licheniformis, Lactobacillus reuteri LAC, and Clostridium bifermentans para) for copper ions. The results show that the Bacillus amyloliquefaciens of the present invention has the strongest adsorption capacity for copper ions. This shows that the application of Bacillus amyloliquefaciens of the present invention in environmental remediation, especially in heavy metal treatment, has obvious advantages, and this efficient adsorption capacity makes it a preferred choice for reducing copper accumulation in environmental remediation, wastewater treatment and feed additives.

[0011] In the third aspect of the present invention, there is provided a use of the Bacillus amyloliquefaciens strain described in the first aspect or the fermentation product of the Bacillus amyloliquefaciens described in the second aspect in the preparation of a feed additive for enhancing antioxidant defense, reducing copper ion absorption and promoting copper excretion.

[0012] The feed additive reduces oxidative stress by upregulating the expression of antioxidant genes SOD1 and SOD3.

[0013] The feed additive reduces the accumulation of copper in the body by promoting the expression of ATPase copper transporter alpha (ATP7A) gene and ATPase copper transporter beta (ATP7B) gene.

[0014] And, the feed additive reduces copper absorption by inhibiting the expression of the copper transporter gene SLC31A1.

[0015] In some embodiments of the present invention, the present invention conducts an in vivo copper ion bioelimination experiment, in which the effect of the Bacillus amyloliquefaciens of the present invention on the body weight of test animals, the copper content in serum and feces, a relative quantitative analysis of copper ion transporters and antioxidant proteins, and the copper content in intestinal tissues, especially ileum and colon trunk tissues, is investigated.

[0016] The results of the investigation on body weight showed that the animal group using the Bacillus amyloliquefaciens of the present invention had better weight gain, which indicates that the strain has a promoting effect on animal growth. The results of the investigation on the copper content in serum and feces showed that a high-copper diet can lead to an increase in the copper ion content in animal serum, and the use of the Bacillus amyloliquefaciens of the present invention can significantly reduce the concentration of copper ions in animal serum, which suggests that the Bacillus amyloliquefaciens of the present invention may be able to effectively promote the removal or absorption of copper ions. At the same time, the results of the copper ion content in feces showed that in the animal group using the Bacillus amyloliquefaciens of the present invention, the copper ion content of feces was significantly higher, which suggests that under the action of the Bacillus amyloliquefaciens of the present invention, copper ions can be excreted from the body faster through feces. Furthermore, the relative quantitative results of copper ion transporters and antioxidant proteins showed that a high-copper diet can significantly reduce the expression levels of antioxidant protein genes SOD1 and SOD3, and copper transporter genes ATP7A and ATP7B that help copper ions be excreted from the body, and increase the expression level of the SLC31A1 gene responsible for copper ion intake and absorption. In the animal group using the Bacillus amyloliquefaciens of the present invention, the expression levels of ATP7A, ATP7B, SOD1 and SOD3 can be significantly increased, and the expression level of the SLC31A1 gene can be significantly reduced. This shows that the use of the Bacillus amyloliquefaciens of the present invention can antagonize the decrease in the expression levels of SOD1, SOD3, ATP7A and ATP7B genes caused by a high-copper diet, and antagonize the increase in the expression level of the SLC31A1 gene caused by a high-copper diet, indicating that the Bacillus amyloliquefaciens of the present invention can enhance the antioxidant defense ability and copper excretion of piglets, and reduce the absorption of copper, thereby avoiding the accumulation of copper in animals. In addition, in the results of copper content in colon and ileum trunk tissues, high-copper feed led to obvious copper accumulation, with the highest copper content in the intestinal tissue. The animal group using the Bacillus amyloliquefaciens of the present invention significantly reduced the copper content in the intestinal tissue, which further confirmed that the Bacillus amyloliquefaciens of the present invention can reduce the accumulation of copper in animals.

[0017] In the fourth aspect of the present invention, there is provided the use of the Bacillus amyloliquefaciens strain described in the first aspect or the fermentation product of the Bacillus amyloliquefaciens described in the second aspect in the preparation of medicines for protecting internal organs, wherein the protection of internal organs mainly refers to reducing the stress response and damage of internal organs by reducing the activity of internal organ enzymes.

[0018] The internal organs are mainly the liver, and the enzymes are alanine aminotransferase (ALT), γ-glutamyl transferase (γ-GT) and alkaline phosphatase (ALP).

[0019] In some embodiments of the present invention, the effect of the Bacillus amyloliquefaciens of the present invention on animal viscera was detected, and the effect on liver function was particularly detected. In these embodiments, the activities of ALT, γ-GT and ALP in the liver of animals were mainly detected. The results show that a high-copper diet can significantly increase the activities of ALT, γ-GT and ALP in the liver of animals, leading to obvious stress reactions and damage, while the activities of ALT, γ-GT and ALP in the liver tissue of the animal group using the Bacillus amyloliquefaciens of the present invention are significantly reduced, which suggests that the protective effect of the Bacillus amyloliquefaciens of the present invention on animal liver tissue can reduce the stress reactions and damage of internal organs.

[0020] In a fifth aspect of the present invention, a product is provided, comprising the Bacillus amyloliquefaciens strain described in the first aspect or the fermentation product of the Bacillus amyloliquefaciens described in the second aspect, wherein the product is a feed additive, a medicine for protecting internal organs, or a product for environmental remediation. The product for environmental remediation may be a bioremediation agent or a biosorbent.

[0021] In some embodiments of the present invention, the feed additive is used to enhance antioxidant defense, reduce copper ion absorption and promote copper excretion; the drug for protecting visceral organs is used to protect the liver, reducing the stress response and damage of the liver by reducing the activity of alanine aminotransferase, γ-glutamyl transferase and alkaline phosphatase in the liver; the product for environmental remediation is used to degrade copper ions in the environment.

[0022] Compared with the prior art, the advantages of the present invention include:

[0023] The Bacillus amyloliquefaciens provided by the present invention has the ability to degrade copper ions in vitro and biologically remove copper ions in vivo, and exhibits the ability to protect internal organs, and has the following advantages in different fields:

[0024] Highly efficient copper ion degradation and removal capability: The Bacillus amyloliquefaciens of the present invention not only has a strong copper ion adsorption and degradation capability in vitro, but also can reduce the accumulation of copper ions in vivo by enhancing the expression of copper transport and excretion genes (such as ATP7A and ATP7B). This gives it a significant advantage in heavy metal pollution treatment and copper poisoning prevention and treatment.

[0025] Antioxidant defense: The Bacillus amyloliquefaciens of the present invention can enhance the expression of antioxidant genes (such as SOD1 and SOD3), thereby reducing oxidative stress. This property helps protect cells from free radical damage and is essential for maintaining cell health.

[0026] Inhibition of copper absorption: By inhibiting the expression of copper uptake genes (such as SLC31A1), the Bacillus amyloliquefaciens of the present invention can effectively reduce the absorption of copper in the body. This property is very important for preventing toxic reactions caused by excessive copper.

[0027] Visceral protection: The Bacillus amyloliquefaciens of the present invention can reduce the activity of key enzymes in visceral organs (such as alanine aminotransferase, γ-glutamine transferase, alkaline phosphatase), reduce stress response and damage to visceral organs, especially liver protection. This is widely used in animal feed additives and helps improve the health of animals.

[0028] Comprehensive health benefits: The multiple mechanisms of the Bacillus amyloliquefaciens of the present invention, including heavy metal removal, anti-oxidation, reduction of heavy metal absorption and visceral protection, comprehensively improve the health status of animals (such as piglets) and counteract the negative effects caused by high-copper feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

[0030] Figure 1 Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Wang’s Gram staining pattern.

[0031] Figure 2 Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Wang’s 16S rRNA PCR results.

[0032] Figure 3 Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Wang’s growth curve.

[0033] Figure 4 Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Wang's tolerance results in simulated artificial intestinal fluid and artificial gastric fluid.

[0034] Figure 5 Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Copper ion content in the supernatant of the culture medium of S. amylovora (S. amylovora) and other strains after the copper ion adsorption experiment.

[0035] Figure 6 The copper ion concentration in the serum of the starch degrading group, the positive control group and the negative control group at 8 days (left graph) and 30 days (right graph) of the experiment is shown.

[0036] Figure 7 The graphs show the comparison of the copper ion content in the serum of the treatment group on the 17th and 28th days of the bacterial feeding treatment, respectively, with the negative control group and the positive control group.

[0037] Figure 8 The copper ion content in the feces of piglets in the starch-degrading group, positive control group and negative control group is shown.

[0038] Fig. 9 The quantitative results of ATP7A, ATP7B, SLC31A1, SOD1 and SOD3 in the ileum, colon and liver tissues of piglets in the starch-depleted group, negative control group and positive control group are shown.

[0039] Fig.10 The copper content in the ileum stem tissue and colon stem tissue of piglets in the starch-depleted group, negative control group and positive control group is shown.

[0040] Fig.11 The liver function of piglets in the starch-degrading group, negative control group and positive control group, namely the activities of alanine aminotransferase, γ-glutamyl transferase and alkaline phosphatase, are shown.

[0041] In the attached figure, * P <0.05, ** P <0.01,*** P <0.001, **** P <0.0001. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0043] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0044] Example 1 Bacillus amyloliquefaciens ( Bacillus amylolyticus )

[0045] 1 Materials

[0046] MRS medium: glucose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.2g / L, ammonium citrate 2g / L, sodium acetate 5g / L, Tween-80 1ml / L, pH 6.0±0.05, sterilized at 121℃ for 20min. Solid medium needs to add 1.2% agar.

[0047] 2.1 Bacillus amyloliquefaciens ( Bacillus amylolyticus )

[0048] Take fresh feces of Northeast Min pigs and use gradient dilution method to dilute the feces to 1, 10, -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , respectively, spread on MRS culture medium and placed in a 37°C shaker for 18 hours. After a single colony grew on the culture medium, pick a single colony and place it in liquid MRS. After culture, perform Gram staining respectively. The experiments were repeated three times.

[0049] 2.2 Identification of bacteria

[0050] 2.2.1 Gram staining method

[0051] Add a drop of saline to the slide, then pick a single colony from the plate and spread it in saline, fix it with a flame, and dye it after the liquid on the slide evaporates. First, add ammonium oxalate crystal violet to cover it, rinse with running water, then add Ruger's iodine solution to cover it, rinse with running water, add 95% alcohol to decolorize it, rinse with water, and finally add carbolic acid fuchsin to cover it, rinse with absorbent paper, and observe it under a microscope.

[0052] 2.2.2 Extraction of genomic DNA from Gram-positive bacteria

[0053] (1) Take 1-5 ml (10 6 -10 8 cells, not more than 2×10 9Place 100 cells in a centrifuge tube (prepare by yourself) and centrifuge at 12,000 rpm (13,400 × g) for 1 minute. Aspirate the supernatant as much as possible.

[0054] (2) Add 180 μL Enzymatic Lysis Buffer to resuspend the cells.

[0055] (3) Incubate at 37°C for 30 min.

[0056] (4) Add 20 μL Proteinase K and vortex to mix thoroughly. Add 200 μL Buffer GL and vortex to mix thoroughly. Note: Do not add Proteinase K directly to Buffer GL.

[0057] (5) Incubate at 56°C for 30 min.

[0058] (6) Add 200 μL of anhydrous ethanol and vortex to mix thoroughly.

[0059] (7) Add all the solution obtained in step 6 (including the precipitate formed) to the adsorption column in the collection tube. If the solution cannot be added in one go, add it in several batches. Centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0060] (8) Add 500 μL of Buffer GW1 to the adsorption column and centrifuge at 12,000 rpm for 1 min. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.

[0061] (9) Add 500 μL of Buffer GW2 to the adsorption column and centrifuge at 12,000 rpm for 1 min. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.

[0062] (10) Centrifuge at 12,000 rpm for 2 minutes and discard the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly. The purpose of this step is to remove the residual ethanol in the adsorption column, which will affect subsequent enzymatic reactions (enzyme digestion, PCR, etc.).

[0063] (11) Place the adsorption column in a new centrifuge tube, add 50-200 μL of Buffer GE to the middle of the adsorption column, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20°C.

[0064] PCR identification: The genomic DNA of multiple Gram-positive bacteria isolated was extracted and amplified by PCR using the 16S rDNA method. The primer sequences were as follows: 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 1), 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 2). The reaction system of 50 μL was as follows:

[0065] The reaction conditions were as follows: 98°C, 20s, 98°C, 10s, 59°C, 15s, 72°C, 30s, 72°C, 7min for a total of 35 cycles. After the PCR was completed, the PCR product was subjected to agarose gel electrophoresis to observe the experimental results, and then the PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were compared with the NCBI gene library.

[0066] 2.3 Plotting the growth curve

[0067] The isolated and identified strains were divided into 1×10 7 The amount of bacteria was added to 50 ml of MRS liquid culture medium, and the OD value was detected using a UV spectrophotometer, which was counted as 0 hour. The OD values ​​corresponding to 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, and 36 hours were then detected respectively, and the results were plotted into a growth curve.

[0068] 2.4 Comparison of tolerance to artificial intestinal fluid and artificial gastric fluid

[0069] The cultured strains were divided into 1×10 7 The amount of bacteria was added to simulated artificial intestinal fluid and artificial gastric fluid (the preparation of artificial intestinal fluid and gastric fluid complies with the Chinese Pharmacopoeia). After anaerobic culture for 12 hours, the bacteria were counted on MRS plates after gradient dilution to calculate the number of colonies.

[0070] Preparation method of artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, stir well and add water to make up to 1000 mL.

[0071] Preparation method of artificial intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate and add 500 mL of water. Use 0.4% sodium hydroxide solution to adjust the pH to 6.8; take 10 g of pancreatic enzyme and add appropriate amount of water to dissolve, mix the two liquids, and add water to make up to 1000 mL. The so-called dilute hydrochloric acid is 1 mol / L hydrochloric acid.

[0072] 2.5 Comparison of copper ion adsorption

[0073] The adsorption and degradation of copper ions is an important indicator for the strain to eliminate copper ion microecological agents.4 The amount of bacteria was added to MRS liquid culture medium respectively, and then the MRS was placed in a 37°C shaker and cultured for 12 hours. After adjusting to the same OD value, the bacteria were centrifuged, the precipitate was separated, added to 50 mg / L copper ion solution, cultured for 1 hour, centrifuged again, and the supernatant was separated to measure the copper ion concentration and statistical analysis of the data. This experiment was repeated three times.

[0074] 3 Results

[0075] 3.1 Gram staining results

[0076] Gram staining of the isolated strain revealed that the strain was purple and was a Gram-positive bacterium. Figure 1 .

[0077] 3.2 Bacterial 16S rDNA results

[0078] The genome of the strain was extracted and the product was sent to the company for sequencing after PCR. The sequencing results were confirmed to be Bacillus amyloliquefaciens after BLAST comparison on NCBI. The PCR results were as follows Figure 2 The strain was named Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Wang, hereinafter referred to as Bacillus amyloliquefaciens Wang. The nucleotide sequence of 16SrDNA of the strain is shown in SEQ ID NO: 3.

[0079] 3.3 Plotting the growth curve

[0080] Take Bacillus amyloliquefaciens Wang and measure the OD every 2 hours until 18 hours. Then draw a growth curve from 0 to 15 hours. The results are as follows: Figure 3 The strain is in the logarithmic growth phase from 4 to 10 hours, and reaches the plateau phase from 10 to 15 hours.

[0081] 3.4 Results of resistance to artificial intestinal fluid and artificial gastric fluid

[0082] The strain's tolerance to artificial intestinal fluid and artificial gastric fluid was tested by simulating artificial intestinal fluid and gastric fluid in vitro. Bacillus amyloliquefaciens Wang showed strong tolerance to artificial intestinal fluid, with a survival rate of up to 92%. It also showed tolerance to artificial gastric fluid, with a survival rate of 85%. Figure 4 The survival rate of the present invention refers to the ratio of the amount of bacteria after treatment to the amount of bacteria without treatment, multiplied by 100%.

[0083] 3.5 Results of strain adsorption of copper ions

[0084] In order to compare the adsorption capacity of Bacillus amyloliquefaciens Wang on copper ions, this experiment selected multiple strains for parallel comparison, including Lactobacillus salivarius, Lactobacillus rumenicus, Lactobacillus plantarum WSCFI, Lactobacillus rhamnosus, Bacillus licheniformis, Lactobacillus reuteri LAC, and Clostridium parafermentans para, and tested the content of copper ions in the culture medium supernatant. Figure 5 The mean of three repetitions in each group is shown. Among them, after the same copper ion concentration solution was cultured for the same time, the less copper ion content in the supernatant after centrifugation, the stronger the adsorption capacity. The results showed that the copper ion content in the supernatant of the Bacillus amyloliquefaciens group was the lowest, which was 4.65 mg / L, indicating that the copper ions in the culture medium were adsorbed by Bacillus amyloliquefaciens, and Bacillus amyloliquefaciens had the strongest adsorption capacity for copper ions.

[0085] Example 2 Study on the effect of biological elimination of copper ions

[0086] 2.1 Experimental animals: 20-day-old weaned piglets

[0087] MRS medium: glucose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.2g / L, ammonium citrate 2g / L, sodium acetate 5g / L, Tween-80 1ml / L, pH 6.0±0.05, sterilized at 121℃ for 20min. Solid medium needs to add 1.2% agar.

[0088] 2.2 In vivo copper ion bioelimination experiment

[0089] Experimental groups: 4 groups in total, 6 mice in each group.

[0090] (1) Negative control group: The mice were fed only low-copper feed (20 mg / kg) for 104 days.

[0091] (2) Positive control group: fed with high copper feed (1300 mg / kg) for 104 days.

[0092] (3) Starch-dissolving group: The mice were fed with high copper feed (1300 mg / kg) for 104 days and fed with bacteria (Bacillus amyloliquefaciens Wang) at a strain concentration of 2×10 10 CFU / mL.

[0093] (4) Treatment group: The mice were first fed with high copper feed (1300 mg / kg) for 104 days, and then fed with bacteria (Bacillus amyloliquefaciens Wang) for 28 days, with a daily bacterial dose of 2×10 10 CFU.

[0094] 2.2.1 Detection of copper content in serum and copper content in feces

[0095] In the animal experiment, serum was collected from weaned piglets before and after feeding bacteria, and the copper content in serum and copper content in feces were tested respectively.

[0096] The copper content was determined by flame atomic absorption spectrometry.

[0097] 2.2.2 Relative quantification of copper ion transporters

[0098] The ileum and colon of the same weight of weaned piglets in each group were collected, and the intestinal segments were frozen and ground with liquid nitrogen. Tissue RNA was extracted using a tissue RNA extraction kit and reverse transcribed into cDNA. ATP7A, ATP7B, SOD1, SOD3, and SLC31A1 in the tissues were measured, and the statistics were calculated and the columns were drawn.

[0099] 2.2.3 Detect the copper content in the ileum and colon trunk tissues.

[0100] The ileum and colon tissues of different groups were placed in an oven at 80°C and dried for 10 hours, and the copper content in the dried tissues was detected.

[0101] 3. Results

[0102] 3.1 In vivo copper ion bioelimination experiment

[0103] 3.1.1 Detection of copper content in serum and copper content in feces

[0104] The copper content was determined by flame atomic absorption spectrometry.

[0105] Among them, at 8 and 30 days of the experiment, the concentration of copper ions in the serum of the starch-dissolving group, the positive control group and the negative control group was Figure 6 .

[0106] Result analysis: According to the results of the 8th day of the experiment ( Figure 6 , left), there was no significant difference in serum copper ion concentration between the starch-dissolving group and the positive control group on the 8th day. This indicates that in the early stage of the experiment, the effect of Bacillus amyloliquefaciens on copper ion concentration has not yet been clearly manifested. The serum copper ion concentration of the positive control group was significantly higher than that of the negative control group (** P <0.01), which indicates that the high copper feed has begun to accumulate copper ions in the pigs, while the low copper feed group maintains a lower copper ion level. According to the results of the 30th day of the experiment ( Figure 6 , right picture): By the 30th day, the serum copper ion concentration of each group decreased. This may be because the body gradually adapted to the copper content in the feed and cleared some of the excess copper through its own metabolic regulation mechanism. Among them, the serum copper ion concentration of the starch-dissolving group decreased most significantly, and there was a significant difference compared with the positive control group (**** P<0.0001). This indicates that Bacillus amyloliquefaciens effectively promotes the removal of copper ions or reduces the absorption of copper.

[0107] The comparison of serum copper ion content between the treatment group and the negative control group and the positive control group on the 17th day (left figure, i.e. the 121st day of the experiment) and the 28th day (right figure, i.e. the 132nd day of the experiment) of the feeding bacteria treatment is shown in the figure below. Figure 7 shown.

[0108] Results: On the 17th day of the treatment, the serum copper concentration of the positive control group was the highest, close to 15 mg / L, reflecting that the copper accumulated by the high-copper diet in the first 104 days still maintained a high level in the body. The copper concentration in the treatment group was significantly lower than that in the positive control group, about 7 mg / L. This shows that after 17 days of Bacillus amyloliquefaciens treatment, the copper concentration in the treatment group has begun to decrease significantly, and there is a significant difference in the serum copper concentration between the treatment group and the positive control group (** P <0.01), the serum copper ion concentration in the treatment group was significantly lower than that in the positive control group. The copper ion concentration in the negative control group was slightly higher than that in the treatment group but significantly lower than that in the positive control group. Due to the use of low-copper feed in the early stage, the copper content in this group remained at a low level. After 28 days of feeding, the serum copper ion concentration of the positive control group was observed to increase slightly compared with that on the 17th day, indicating that even after the high-copper diet was stopped, the copper ion concentration in the body continued to increase. It is speculated that this increase can be attributed to the accumulation effect of the high-copper feed in the previous 104 days. Copper in the tissues may continue to be released into the blood after the high-copper diet is stopped, resulting in the increase of serum copper ion concentration after the high-copper diet is stopped. The copper ion concentration in the treatment group continued to decrease, and there was still a significant difference compared with the positive control group. The copper ion concentration in the negative control group was the lowest, with basically no significant changes. Since the negative control group used low-copper feed throughout the whole process, the accumulation of copper in the body was very small, and the serum copper ion concentration remained at a low level throughout the experiment, and did not change much. The above results show that Bacillus amyloliquefaciens Wang effectively promotes the removal of copper ions or reduces the absorption of copper, and this strain has an effective regulatory and mitigating effect on copper accumulation caused by a high-copper diet.

[0109] Among them, the copper ion content in the feces of piglets in the starch decomposition group, positive control group and negative control group on the 13th day of the experiment was as follows: Figure 8 shown.

[0110] according to Figure 8 It can be seen that the copper ion content in the feces of piglets in each group is different, among which the copper ion content in the feces of piglets in the Bacillus amyloliquefaciens group is significantly higher than that in other groups (** P <0.01; **** P<0.0001), indicating that copper ions, under the action of Bacillus amyloliquefaciens, can significantly enhance the clearance of copper ions by piglets or promote copper excretion, and be excreted from the body through feces.

[0111] 3.1.2 Relative quantification results of copper ion transporters and antioxidant proteins

[0112] The relative quantitative method was used to explore the expression levels of ATP7A (ATPase Copper Transporting Alpha), ATP7B (ATPase Copper Transporting Beta), SLC31A1 (Solute Carrier Family 31 Member 1), SOD1 (Superoxide Dismutase 1), and SOD3 (Superoxide Dismutase 3) genes in each group of tissues (ileum, colon, or liver). The results are as follows: Fig. 9 shown.

[0113] Among them, the ATP7A gene encodes a copper-transporting ATPase, which is involved in transporting copper ions from the cell to the outside of the cell or to specific organelles to maintain the balance of copper in the body. It is mainly expressed in tissues such as the intestine, blood vessel wall and brain. The ATP7B gene encodes another copper-transporting ATPase, which is mainly expressed in stem cells and acts in the liver to help excrete copper ions from the body, usually through bile excretion. The SLC31A1 gene encodes a copper transporter, which is responsible for the uptake and absorption of copper ions and is the main way for copper to enter cells. It is widely distributed in intestinal epithelial cells, hepatocytes and other tissues. The SOD1 gene encodes an antioxidant enzyme responsible for converting superoxide free radicals into hydrogen peroxide and oxygen to protect cells from oxidative damage. It is mainly expressed in the cytoplasm and mitochondria. The SOD3 gene encodes another antioxidant enzyme, which is mainly present in the extracellular matrix and is responsible for scavenging superoxide free radicals outside the cell to prevent tissue damage. It is mainly expressed in the extracellular matrix and plasma.

[0114] according to Fig. 9 The results showed that compared with the positive control group, the SOD1 (* P <0.05)、SOD3(* P <0.05)、ATP7A(** P <0.01) and ATP7B(* P<0.05) gene expression was significantly higher and had no significant difference with the negative control group. This indicates that Bacillus amyloliquefaciens can antagonize the decrease in SOD1, SOD3, ATP7A and ATP7B gene expression caused by a high copper diet, enhance the antioxidant defense capacity and copper excretion of piglets, and reduce copper absorption. Compared with the high copper feed (positive control group), the expression level of SLC31A1 in the amylolytic group was significantly lower (** P <0.01), and there was no significant difference with the negative control group. This shows that Bacillus amyloliquefaciens can antagonize the increase in SLC31A1 gene expression caused by a high-copper diet, reduce the absorption of copper, and avoid copper accumulation.

[0115] In summary, the use of Bacillus amyloliquefaciens can simultaneously antagonize the decrease in SOD1, SOD3, ATP7A and ATP7B gene expression and the increase in SLC31A1 gene expression caused by a high-copper diet, which not only increases the excretion of copper in piglets, enhances the ability to clear copper, reduces the absorption of copper, but also enhances the antioxidant defense ability of piglets.

[0116] 3.1.3 Copper content in ileum and colon trunk tissue

[0117] The copper content in ileal and colonic trunk tissues is shown in Fig.10 .

[0118] Positive control group: The highest copper content was found in the ileum and colon, indicating that the high copper diet resulted in significant copper accumulation.

[0119] Amylolytic group: The copper content was significantly lower than that of the positive control group, but higher than that of the negative control group, which indicates that although the use of Bacillus amylolyticus failed to completely restore the level of low-copper feed, it could significantly reduce the copper accumulation caused by high-copper feed.

[0120] The above results further support the conclusion that Bacillus amyloliquefaciens can enhance the scavenging effect of copper by regulating the expression of copper metabolism genes. Bacillus amyloliquefaciens can effectively alleviate the problem of copper accumulation caused by high-copper feed and reduce the negative impact of high-copper feed, which is of great significance for the regulation of copper metabolism.

[0121] 3.1.4 Liver function of pigs in different groups

[0122] Comparison of enzyme activities (alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase) in the liver of piglets in each group Fig.11 shown.

[0123] The activities of alanine aminotransferase, γ-glutamyl transferase, and alkaline phosphatase in the positive control group were significantly higher, indicating that high-copper diets have adverse effects on internal organs, especially the liver, such as causing significant stress reactions and damage. The starch-dissolving group was able to significantly reduce the activities of alanine aminotransferase, γ-glutamyl transferase, and alkaline phosphatase (**) compared with the positive control group. P <0.01;*** P <0.001), indicating that Bacillus amyloliquefaciens effectively alleviated the stress response and damage of visceral organs caused by high copper diet.

[0124] 4 Conclusion

[0125] Bacillus amyloliquefaciens has an antioxidant defense effect, which is manifested by enhancing the expression of antioxidant genes (SOD1 and SOD3 genes) and reducing oxidative stress. Bacillus amyloliquefaciens has a bioremoval effect on copper ions, which is manifested by promoting the expression of ATP7A and ATP7B genes and reducing the accumulation of copper in the body. Bacillus amyloliquefaciens inhibits the expression of SLC31A1 gene and reduces copper absorption. In addition, Bacillus amyloliquefaciens has a protective effect on visceral organs, which is manifested by reducing the activity of visceral organ enzymes (alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase) and reducing visceral organ stress response and damage. These results show that starch treatment effectively alleviates the negative effects caused by high-copper feed through multiple mechanisms and has an important protective effect on the health of piglets.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens, which is named Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Wang, was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on May 20, 2024, with the deposit number CCTCC NO: M 20241004.

2. Use of the Bacillus amyloliquefaciens according to claim 1 in the preparation of a product for environmental remediation, wherein the product is a bioremediation agent for adsorbing copper ions in the environment.

3. Use of the Bacillus amyloliquefaciens according to claim 1 in the preparation of a feed additive for enhancing antioxidant defense, reducing copper ion absorption and promoting copper excretion.

4. Use of the Bacillus amyloliquefaciens according to claim 1 in the preparation of a medicine for protecting internal organs, wherein: Protecting the internal organs refers to reducing the stress response and damage of the internal organs by reducing the activity of the internal organ enzymes; wherein the internal organ is the liver, and the enzymes are alanine aminotransferase, γ-glutamyl transferase and alkaline phosphatase.

5. A product comprising the Bacillus amyloliquefaciens according to claim 1, wherein: The product is a feed additive, a medicine for protecting internal organs, or a product for environmental restoration; the feed additive is used to enhance antioxidant defense, reduce copper ion absorption, and promote copper excretion.

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