Strains isolated from the intestines of Northeast Min pigs and their application in degrading vomitoxin

Abstract

This invention relates to the field of microbiology, specifically to a strain isolated from the intestines of Northeast China wild pigs and its application in degrading vomitoxin. The strain isolated from the intestines of Northeast China wild pigs is a strain of *Bacillus amyloliquefaciens*, named *Bacillus amyloliquefaciens* (…). Bacillus amylolyticus The strain NM1101 was deposited on May 20, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241003. This strain exhibits significant in vitro and in vivo degradation of vomitoxin. Furthermore, it enhances the growth performance of piglets, demonstrating peroxidase and other enzyme activities, strong growth capacity, and high tolerance to acidic environments. *Bacillus amyloliquefaciens* can significantly reduce the damage of vomitoxin to intestinal villi and various organs in piglets, particularly playing a potential protective role in maintaining intestinal nutrient transport function.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain separated from the intestine of a Northeastern pig and application of the strain in degrading vomitoxin. 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] Deoxynivalenol (DON), also known as deoxynivalenol, belongs to the trichothecenes. It is mainly a secondary metabolite produced by plant pathogens Fusarium graminearum and Fusarium fungi, and cereal crops are most often contaminated. According to a survey conducted in 2016, feed and feed samples collected worldwide (a total of 11,275 samples were tested for DON content) were tested for DON content, and it was found that 71% of the samples were found to contain DON, and the measured DON content ranged from 0.3-49 mg / kg. In order to regulate the DON content in crops, my country's national standard GB2761-2011 stipulates that the limit standard for DON in cereals and cereal products (including corn, cornmeal, barley, wheat, oatmeal and wheat flour) is 1000 μg / kg.

[0004] The epoxide group of DON can bind to the ribosome to inhibit protein synthesis, thereby inducing certain protein kinases and regulating downstream genes, affecting the expression of proteins involved in immune regulation and cell apoptosis. When animals eat contaminated crops or foods, their immune systems will be damaged and unable to respond normally. The structures of organs such as the liver, kidneys and spleen will be damaged, and severe cases may lead to apoptosis of organ cells. In order to reduce the intake of these contaminated crops by animals, it is of great practical significance to develop products that can degrade vomitoxin. Traditional physical and chemical degradation methods have many defects compared to biological degradation. Although they can degrade contaminated crops or food, they destroy their nutrients. Generally, large amounts will cause secondary pollution. The biological method uses the characteristics of microorganisms to degrade toxins. It is a safe and effective method. Therefore, it is particularly important to develop or discover strains that can safely and effectively degrade vomitoxin. Summary of the invention

[0005] The present invention isolates a strain of Bacillus amyloliquefaciens from the intestine of Northeast Min pigs. The strain has fast growth ability, acid resistance, high peroxidase activity and other enzyme capabilities, and has obvious in vitro and in vivo degrading function of vomitoxin. In addition, the strain can be well colonized when used in vivo, and can significantly alleviate the growth problems, intestinal damage, and organ damage caused by vomitoxin while degrading vomitoxin and avoiding the accumulation of vomitoxin in the body, and plays an important role in maintaining the intestinal nutrient transport function.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In a first aspect of the present invention, a strain of Bacillus amyloliquefaciens is provided, which is named Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101, which was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on May 20, 2024, with the deposit number CCTCC NO: M 20241003.

[0008] In a second aspect of the present invention, there is provided the Bacillus amyloliquefaciens described in the first aspect ( Bacillus amylolyticus ) Fermentation product of NM1101.

[0009] In the third aspect of the present invention, there is provided the Bacillus amyloliquefaciens described in the first aspect above ( Bacillus amylolyticus ) Use of NM1101 or its fermentation product in the preparation of a product with the function of degrading vomitoxin in vitro.

[0010] The in vitro degradation of vomitoxin in the present invention refers to the use of microorganisms, and in the present invention, it refers to the use of Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101 or its fermentation products degrade vomitoxin in an in vitro environment and break it down into non-toxic or low-toxic substances. This process does not occur in vivo, but is carried out in a laboratory or various specific application environments.

[0011] The function of degrading vomitoxin in vitro can be used to degrade vomitoxin in feed, grain and agricultural products, reduce toxin accumulation, and ensure food and feed safety. For example, the Bacillus amyloliquefaciens described in the first aspect of the present invention is used in the process of feed production and storage to pre-degrade vomitoxin in the feed, ensure that the toxin content in the feed entering the animal is low, and further ensure the health of the animal. At the same time, it can also be applied to the storage and processing of agricultural products and grains to reduce toxin pollution and ensure food safety.

[0012] In addition, the function of degrading vomitoxin in vitro can also be used to reduce the content of vomitoxin in the environment, such as reducing the vomitoxin in soil and water bodies, or to treat the vomitoxin in agricultural waste (such as straw and animal manure), promote degradation and harmless treatment, and reduce environmental pollution.

[0013] In some embodiments of the present invention, the product is in the form of a biodegradable agent (such as a soil degradation and repair agent, a water purification biological agent, an agricultural waste degrading agent, etc.) and a biological detoxifier (such as a feed detoxifier, a grain detoxifier, a soil detoxifier, etc.). Specific applications include but are not limited to feed processing to ensure the safety of feed and protect animal health; grain processing to reduce vomitoxin in grain and ensure food safety; soil remediation to improve soil health and restore its productivity; water purification to purify polluted water sources and protect aquatic ecosystems, etc.

[0014] In a fourth aspect of the present invention, there is provided the Bacillus amyloliquefaciens described in the first aspect above ( Bacillus amylolyticus ) Use of NM1101 or its fermentation product in the preparation of a product having the function of degrading vomitoxin in vivo.

[0015] In the embodiments of the present invention, experiments have verified that vomitoxin has many adverse effects on animals and even seriously endangers the health of animals, including but not limited to:

[0016] (1) It can affect the growth performance of animals, leading to slow weight gain and growth retardation;

[0017] (2) Deoxynivalenol can damage intestinal health in animals, including but not limited to destroying the intestinal villi structure, resulting in decreased villi height, irregular surface or partial loss, thus affecting the normal function of the intestine;

[0018] (3) Deoxynivalenol causes intestinal absorption disorders, including but not limited to inhibiting the expression of intestinal nutrient transport genes such as SGLT1, PePT1, and ASCT2, affecting the absorption of glucose, amino acids, and peptides, leading to malnutrition.

[0019] (4) Deoxynivalenol can cause multiple organ damage, including but not limited to: Deoxynivalenol accumulates in the liver, leading to liver hypertrophy and pathological changes (such as liver cell degeneration and necrosis, and in severe cases, liver failure). Deoxynivalenol has a toxic effect on the kidneys, which may cause kidney enlargement and pathological changes (such as renal damage, etc.), affecting the excretion of waste in the body. Deoxynivalenol may cause pathological changes in the heart and lungs, such as bleeding, congestion and edema, affecting cardiopulmonary function. In addition, deoxynivalenol can cause spleen enlargement and affect the function of the immune system.

[0020] In an embodiment of the present invention, the in vivo degradation of vomitoxin in the present invention refers to the use of specific microorganisms, mainly Bacillus amyloliquefaciens ( Bacillus amylolyticus )NM1101 or its fermentation products directly degrade or transform the ingested vomitoxin in animals or humans or promote the elimination of vomitoxin, thereby reducing the accumulation and toxic effects of vomitoxin in the body. This process significantly reduces the toxicity of vomitoxin and protects the health of the host by taking place in the biological system in the body.

[0021] In some embodiments of the present invention, the Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101 or its fermented product can alleviate and improve health problems caused by the accumulation of vomitoxin in the body. The health problems include but are not limited to intestinal damage, organ damage, immunosuppression and nutrient absorption disorders.

[0022] In some embodiments of the present invention, the reducing and improving health problems caused by the accumulation of vomitoxin in the body includes protecting the integrity of the intestinal villus structure.

[0023] In some embodiments of the present invention, the reducing and improving health problems caused by the accumulation of vomitoxin in the body includes reducing stress response and damage to internal organs, including the heart, liver, lungs, spleen and kidneys.

[0024] In some embodiments of the present invention, the reduction and improvement of health problems caused by the accumulation of vomitoxin in the body includes relieving the inhibition of vomitoxin on the expression of intestinal nutrient transport genes, thereby helping the intestine to restore normal nutrient (such as monosaccharides such as glucose, galactose and fructose, dipeptides and tripeptides) absorption function, and the intestinal nutrient transport gene is SGLT1, PePT1 or ASCT2 gene.

[0025] In some embodiments of the present invention, the method of reducing and improving health problems caused by the accumulation of vomitoxin in the body includes increasing the intestinal absorption of monosaccharides (such as glucose, galactose and fructose, etc.) by promoting the expression of the intestinal nutrient transport gene GLUT2 in the presence of vomitoxin.

[0026] In the fifth aspect of the present invention, there is provided a use of the Bacillus amyloliquefaciens strain or a fermentation product thereof described in the first aspect in the preparation of a product having the function of inhibiting the expression of intestinal nutrient transport genes SGLT1 and PePT1.

[0027] In an embodiment of the present invention, Bacillus amyloliquefaciens ( Bacillus amylolyticus) The use of NM1101 or its fermentation products can inhibit the expression of PePT1 gene and SGLT1 gene. Among them, PePT1 protein absorbs these substances by co-transporting protons and peptides. In the small intestine, GLUT2 protein is a key protein involved in the facilitated diffusion transport of glucose, fructose and galactose, mainly on the basement membrane of small intestinal epithelial cells. This transport is independent of sodium ions and energy and is responsible for transporting absorbed monosaccharides from the cell to the blood. This shows that Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101 or its fermented products can significantly affect the intestinal absorption of peptides and glucose by inhibiting the expression of PePT1 and SGLT1 genes. This effect not only helps regulate metabolic function, but may also play a key role in intestinal health management and the treatment of certain specific diseases.

[0028] Specifically, 1) regulating intestinal nutrient absorption by inhibiting the expression of PePT1 and SGLT1 genes, Bacillus amyloliquefaciens ( Bacillus amylolyticus 1) NM1101 or its fermentation products can reduce the intestinal absorption of glucose and peptide nutrients. This may be beneficial to some people who need to control nutrient absorption, such as patients with metabolic disorders or obesity. 2) Potential metabolic regulation, such as in cases where it is necessary to reduce the absorption of carbohydrates and peptides, such as diabetes management or weight control, inhibiting the expression of these genes may help lower blood sugar levels and control weight, and improve metabolic health. 3) Impact on intestinal health, such as this inhibition may reduce the burden on the intestine by reducing excessive absorption of nutrients, especially in the face of intestinal stress or excessive nutrient intake, which helps maintain intestinal health. 4) Specific disease management, such as for certain diseases that rely on specific nutrient absorption pathways, such as specific types of malabsorption syndrome or metabolic diseases, this inhibition of gene expression may provide new strategies for treatment and management. Of course, such effects can also be used for scientific research, such as research on nutrient absorption mechanisms, research on metabolic diseases, drug screening, research on the regulation of gene expression, research on the interaction between intestinal microorganisms and hosts, research on model microorganisms, and so on.

[0029] In a sixth aspect of the present invention, a product is provided, comprising the Bacillus amyloliquefaciens strain or a fermentation product thereof as described in the first aspect.

[0030] Compared with the prior art, the advantages of the present invention include: the present invention provides a strain of Bacillus amyloliquefaciens ( Bacillus amylolyticus) NM1101, this strain has excellent growth ability, acid resistance, high peroxidase activity, etc., can degrade vomitoxin in vivo and in vitro, can colonize well in animals, and can significantly alleviate growth problems, intestinal damage, and organ damage caused by vomitoxin while degrading vomitoxin and avoiding its accumulation in the body. In addition, it plays an important role in maintaining and regulating intestinal nutrient transport. The strain has multiple functionalities and a wide range of application potentials, giving it great commercial prospects in markets such as animal feed, and can meet the growing health and environmental protection needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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:

[0032] Figure 1 The present invention shows that Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Isolation (left) and Gram staining (right) results of NM1101.

[0033] Figure 2 The groups (A) and experimental setup (B) of co-fermentation of different strains with corn and DON are shown.

[0034] Figure 3 The results of screening for strains with strong oxidizing ability using oxidized methylene blue medium are shown, wherein strains with strong oxidizing ability appear yellow.

[0035] Figure 4 The results of enzyme activity detection of each strain are shown.

[0036] Figure 5 The figure shows the degradation ability of different strains of vomitoxin in different time periods after co-fermentation with corn and vomitoxin. The left figure shows the degradation rate after 12 hours of co-fermentation, and the right figure shows the degradation rate after 24 hours of co-fermentation.

[0037] Figure 6 The present invention shows that Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Growth curve of NM1101.

[0038] Figure 7 The present invention shows that Bacillus amyloliquefaciens ( Bacillus amylolyticus ) Acid resistance of NM1101.

[0039] Figure 8 The changes in body weight (A) and body weight growth rate (B) of piglets in each group are shown.

[0040] Fig. 9The figure shows the comparison of the content of DON in feces of piglets in each group on days 1, 5, 10, 15, 20 and 24 of DON feeding.

[0041] Fig.10 The electron microscopic observation results of the duodenal villi of piglets in each group are shown.

[0042] Fig.11 A general intuitive comparison of each organ in each group is shown, and the organs in each row from top to bottom are liver, heart, lung, spleen, and kidney.

[0043] Fig.12 Comparison of each organ as a percentage of body weight in each group is shown.

[0044] Fig.13 Comparison of DON levels in different intestinal tissue segments (duodenum, jejunum, ileum, cecum, colon) and liver among the groups is shown.

[0045] Fig.14 The expression of nutrient transport gene mRNA (SGLT1, PePT1, GLUT2, ASCT2) in the intestine of each group of piglets is shown.

[0046] In the above figures, ns means not significant. p >0.05; * p <0.05; ** p <0.01; *** p <0.001. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] Example 1 Isolation and identification of Bacillus amyloliquefaciens and its ability to degrade vomitoxin in vitro

[0050] 1. Materials: phosphate buffer, MRS agar plate, anaerobic jar, 1.5 mL centrifuge tube, biochemical fermentation tube, PCR enzyme, primer, deionized water, oxidized methylene blue reagent, vomitoxin ELISA test kit, enzyme activity test kit.

[0051] 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.

[0052] Identification medium: 100 mL of water, 1 g of beef extract, 1 g of peptone, 1 g of yeast extract, 20 g of tomato juice, 1 g of glucose, 0.05 mL of Tween, 1.7 g of calcium carbonate, 0.01 g of bromocresol green, and 2 g of agar. Sterilize at 121°C for 20 min.

[0053] Reagent preparation:

[0054] Preparation of TAE (50×TAE): Tris 242g, Na2EDTA·2H2O 37.2g, add 800mL of deionized water to a beaker and stir thoroughly. Then add 57.1mL of acetic acid and stir thoroughly. Add deionized water to make the volume 1L, and dilute 50 times when used.

[0055] Loading Buffer 6× Loading Buffer: EDTA: 4.4 g, bromophenol blue: 250 mg, xylene cyanol blue FF: 250 mg. Add 200 mL of deionized water, heat and stir thoroughly, add 180 mL of glycerol, adjust the pH to 7.0 with 2 M NaOH, and dilute to 500 mL with deionized water.

[0056] Preparation of 1% agarose gel: weigh 1g of agarose, add 100mL of TAE solution, beat in a microwave until clear, let it cool, add 5μL of EB, and it can be used after solidification.

[0057] The strains to be screened include Clostridium butyricum, Lactobacillus salivarius, Ruminococcus, Lactobacillus thermophilus, Bacillus licheniformis, Clostridium bifermentative (para), Lactobacillus rhamnosus, and strains isolated from the intestines of Northeast Min pigs. All of the above strains are stored in the Animal Microecological Preparation Engineering Research Center of Jilin Agricultural University.

[0058] Among them, the isolation method of the strain isolated from the intestine of the Northeast Min pig is as follows: take fresh feces of the Northeast Min pig, use the gradient dilution method, dilute the feces gradiently, and spread them on MRS culture medium and place them in a 37°C shaker for 18 hours. After a single colony grows on the culture medium, pick a single colony in liquid MRS, culture it, perform Gram staining, and identify it. Gram staining microscopy shows that the strain isolated from the intestine of the Northeast Min pig is purple short rod, which grows as small white colonies in solid culture medium. The results are shown in Figure 1 .

[0059] The strain isolated from the intestine of the Northeast Min pig was sequenced, and the sequencing results were compared with the NCBI database. The 16S rRNA gene sequence of the strain is shown in SEQ ID NO: 1. The results showed that the strain was closely homologous to the two strains of Bacillus amyloliquefaciens HQ853016.1 and MW165777.1. The identification results showed that the strain isolated from the intestine of the Northeast Min pig was Bacillus amyloliquefaciens, and it was named Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101. The strain was deposited in the China Center for Type Culture Collection at Wuhan University, Wuhan, China on May 20, 2024, with the deposit number CCTCC NO: M 20241003. Unless otherwise specified, the Bacillus amyloliquefaciens appearing in the following embodiments or drawings of the present invention refers to Bacillus amyloliquefaciens ( Bacillus amylolyticus )NM1101.

[0060] 2. Methods

[0061] 2.1 Screening of strains with high peroxide activity using methylene blue oxidation

[0062] Method: When preparing MRS medium, add peroxymethylene blue, which is blue, add the isolated strain, and culture it anaerobically at 37°C for 18 hours. Observe its color.

[0063] Methylene Blue is a redox indicator that is blue in an oxidized state and colorless or yellow in a reduced state. After adding peroxymethylene blue to the MRS medium, if the cultured strain has a high peroxidase activity, they will decompose peroxides such as hydrogen peroxide and reduce oxidized methylene blue. If the culture medium changes from blue to yellow, it means that the strain has peroxidase activity and can reduce oxidized methylene blue. The depth of the color change is related to the strength of the peroxidase activity of the strain: the more obvious the color change, the stronger the peroxidase activity.

[0064] 2.2 Enzyme activity detection

[0065] Potassium permanganate titration method Take 0.2 mL of enzyme solution diluted to about 0.1 mg / mL, add 20 mL of water and 3 mL of 2% hydrogen peroxide solution, and place at 25 ℃ for 15 min; add 10 mL of 20% sulfuric acid solution to terminate the reaction, and titrate with potassium permanganate standard solution until it turns slightly red and does not fade for 30 seconds, and record the volume of potassium permanganate standard solution consumed (V1). At the same time, perform a blank test, record the volume of potassium permanganate standard solution consumed (V0), and calculate the enzyme activity according to the following formula. Perform a total of 6 parallel tests.

[0066] Enzyme activity (U / mL) = [(V0-V1) × C × (5 / 2) × df ] / (T×0.2);

[0067] Wherein, (V0-V1) represents the volume difference of potassium permanganate standard solution consumed by hydrogen peroxide with and without enzyme, reflecting the decomposition amount of hydrogen peroxide. C represents the molar concentration of potassium permanganate standard solution, which is used to calculate the molar amount of potassium permanganate consumed in the reaction. (5 / 2) is a constant, which is used to convert the amount of potassium permanganate consumed to the amount of hydrogen peroxide decomposed, and is obtained according to the stoichiometric relationship between potassium permanganate and hydrogen peroxide in the reaction. df is the dilution factor, used to convert the measured enzyme activity to the concentration of the original enzyme solution. T is the reaction time, used to standardize the unit of enzyme activity. 0.2 is the volume of enzyme solution used in the reaction, used to convert the enzyme activity per unit amount of enzyme solution.

[0068] 2.3 Screening of strains capable of degrading vomitoxin

[0069] Each strain to be screened was added at a concentration of 1×10 6 CFU / mL was added to corn without DON, and the same dose of 0.5 mg DON was added thereto and co-fermented at 37 °C for 12 h and 24 h (experimental groups and apparatus see Figure 2 ), using the vomitoxin detection kit to detect the vomitoxin content at two time points.

[0070] 2.4 Growth characteristics detection

[0071] The growth curve of the strain with the most obvious effect of degrading vomitoxin was drawn. And the survival of the strain in different acidic environments (pH range from 2.5 to 7.0) was analyzed through acid resistance experiments. Culture conditions: MRS medium, pH 6.2, growth temperature 37℃, anaerobic environment, culture for 25 hours.

[0072] 3. Results

[0073] 3.1 Results of screening strains with high peroxide activity using methylene blue oxidative medium

[0074] The results showed that all strains to be screened showed peroxide activity. Compared with the control group without added strains, all strains could change the color of the oxidized methylene blue medium. Among them, the peroxide activity of Bacillus amyloliquefaciens NM1101 was the strongest, which made the oxidized methylene blue medium turn into a lighter yellow. The results are shown in Figure 3 .

[0075] 3.2 Enzyme activity test results

[0076] The enzyme activity of each strain was tested using an enzyme activity detection kit. Compared with the control group without added strains, the enzyme activities of Lactobacillus rhamnosus, Bacillus amyloliquefaciens NM1101, and Clostridium bifermentans were higher, all significantly higher than the control group. Among them, the enzyme activity of Bacillus amyloliquefaciens NM1101 was the most significant, and the results are shown in Table 1. Figure 4 The average of multiple measurements for each group is shown.

[0077] Table 1 Results of enzyme activity determination of different strains (potassium permanganate titration method, unit U / mL)

[0078]

[0079] 3.3 Screening of strains capable of degrading vomitoxin

[0080] Different strains were co-fermented with corn and vomitoxin, and the degradation efficiency was compared to screen the strains with the highest degradation efficiency at different times. The results showed that at 12 hours of co-fermentation, the degradation efficiency from high to low was Bacillus amyloliquefaciens NM1101>Streptococcus thermophilus>Lactobacillus salivarius>Clostridium butyricum>Lactobacillus rhamnosus. At 24 hours of co-fermentation, the degradation efficiency from high to low was Bacillus amyloliquefaciens NM1101>Lactobacillus salivarius>Streptococcus thermophilus>Clostridium butyricum>Lactobacillus rhamnosus. The results are shown in Tables 2 and 3, where Figure 5 The mean degradation rates of different strains at 12 h and 24 h of co-fermentation are shown.

[0081] Table 2 Degradation rate of different strains after 12 h of co-fermentation (%)

[0082]

[0083] Table 3 Degradation rate of different strains after 24h co-fermentation (%)

[0084]

[0085] 3.4 Results of the growth characteristics test of Bacillus amyloliquefaciens

[0086] The growth curve of Bacillus amyloliquefaciens NM1101 is as follows Figure 6As shown in the figure, according to the growth curve, the OD value of the strain began to rise rapidly after the 3rd hour of cultivation, indicating that the strain had a short lag period and could quickly adapt to the environment and enter a rapid growth period. After the 15th hour, the OD value reached a platform, close to the maximum value of about 1.7, which indicated that the growth rate and death rate of the strain were roughly balanced and entered a stable period.

[0087] Acid resistance Figure 7 This indicates that the strain has good acid resistance and can grow well in the pH range of 2.5-7.0. When the pH is as low as 2.5, its survival rate is still as high as about 55%.

[0088] Example 2 Study on the effect of Bacillus amyloliquefaciens on degrading vomitoxin in vivo

[0089] 1. Experimental strain: Bacillus amyloliquefaciens ( Bacillus amylolyticus )NM1101

[0090] 2. Main reagents and consumables: phosphate buffer, MRS agar plate, anaerobic tank, 1.5mL centrifuge tube, biochemical fermentation tube, PCR enzyme, primer, deionized water, oxidized methylene blue reagent, vomitoxin ELISA test kit, enzyme activity test kit.

[0091] 3. Main instruments: ABI Prism7500QT-qPCR instrument (ABI, USA); heparin sodium anticoagulant tube and procoagulant tube purchased from (Jiangsu) Kangjian Medical Supplies Co., Ltd.; tissue embedding machine, paraffin slicer, DMi8 fluorescence inverted microscope purchased from (Germany) Leica Instrument Co., Ltd.; tissue homogenizer domestically produced.

[0092] 4. Reagents required for the experiment

[0093] MRS culture medium: same as in Example 1.

[0094] Reagent preparation

[0095] Preparation of TAE (50×TAE): Same as Example 1.

[0096] Preparation of 1% agarose gel: same as in Example 1.

[0097] Table 4 Primer list

[0098]

[0099] 5. Experimental animals: 12 healthy Landrace piglets aged 4 weeks (negative for porcine reproductive and respiratory syndrome virus (PRRSV) antigen and antibody) were purchased from a private pig farm in Changchun City, Jilin Province.

[0100] 6. Test methods

[0101] 1) Experimental plan

[0102] Experimental animal grouping: divided into 4 groups, 3 animals in each group (n=3), male and female were randomly assigned, and the diet was corn feed, which may contain vomitoxin not exceeding the standard limit.

[0103] The groups, strain feeding and virus attack conditions are as follows:

[0104] Healthy group: After the experimental piglets adapted to feeding for 2 days, they ate and drank normal food and water, but were not fed with Bacillus amyloliquefaciens and vomitoxin for a total of 30 days.

[0105] Deoxynivalenol group (DON group): After the experimental piglets adapted to feeding for 2 days, they ate and drank normal food and water, and were fed 0.3 mg of deoxynivalenol every day for 30 days.

[0106] Bacillus amyloliquefaciens group (Ba group): After the experimental piglets were adapted to feeding for 2 days, they were given normal diet and water, and were gavaged with Bacillus amyloliquefaciens (1×10 10 CFU / mL, fed 10 mL) for a total of 30 days.

[0107] Vomitoxin + Bacillus amyloliquefaciens group (Don+Ba group): After the experimental piglets were adapted to feeding for 2 days, they were fed with normal diet and water, and fed with 0.3 mg of Vomitoxin per day. At the same time, Bacillus amyloliquefaciens (1×10 10 CFU / mL, fed 10 mL) for a total of 30 days.

[0108] During the experiment, each group of piglets was weighed every day, the weight changes of the piglets were recorded, and the weight change curve was drawn. Feces of each group of piglets at different time points were collected, the content of vomitoxin in each feces was tested, and a bar graph was drawn.

[0109] All piglets were slaughtered on the 30th day of the experiment, and the following procedures were performed: (i) The jejunal intestinal segments of each group of piglets were collected for scanning electron microscopy to observe the integrity of the surface of the jejunal villi of the piglets. (ii) The heart, liver, spleen, kidney, and lung samples of each group of piglets were collected for gross observation and comparison of the degree of damage. (iii) The weight of each organ was weighed, and the ratio of organ weight to body weight was calculated. (iv) The content of vomitoxin in each intestinal segment of each group of piglets was detected using a vomitoxin detection kit and a bar graph was drawn. (v) RNA was extracted from the small intestine of each group of piglets, and the relative expression levels of nutrient transport genes SGLT1, PePT1, GLUT2, and ASCT2 in the intestine of each group of piglets were statistically analyzed by fluorescence quantitative method to explore the effect of Bacillus amyloliquefaciens on nutrient transport genes in the intestine of piglets.

[0110] 2) Data statistical analysis: Graphpad Prism8 software was used for data statistics, and one-way analysis of variance was used for statistical analysis of data differences.

[0111] 7. Results

[0112] 1) Weight change results: The weight of each group of piglets was measured, and the growth rate of each group of piglets was calculated. The weight gain curve was drawn. The results are shown in Figure 8 The results showed that the piglets in the healthy group and the Bacillus amyloliquefaciens group showed significant weight gain, indicating that normal diet and the addition of Bacillus amyloliquefaciens helped the growth of piglets. The weight gain of the DON group was inhibited, indicating that DON had an inhibitory effect on the growth of piglets. The weight gain of the DON + Bacillus amyloliquefaciens group was improved compared with the DON group alone, suggesting that Bacillus amyloliquefaciens may have a certain effect on alleviating the negative effects of DON.

[0113] 2) Results of the content of vomitoxin in pig feces at different time points: The content of vomitoxin in the feces of piglets in each group was tested on the 1st, 5th, 10th, 15th, 20th and 24th days after feeding vomitoxin. The content of vomitoxin in the feces of piglets fed with vomitoxin was significantly different from that of piglets fed with vomitoxin and Bacillus amyloliquefaciens at the same time after 15th, 20th and 24th days, indicating that Bacillus amyloliquefaciens has a degrading effect on vomitoxin after colonization in the intestine. Fig. 9 .

[0114] 3) Comparison of the integrity of intestinal villi: The jejunum of each group of piglets was collected and the intestinal villi were scanned by scanning electron microscopy. The results showed that the intestinal villi of the piglets in the DON group were significantly damaged, as shown by decreased villus height, irregular surface or partial loss, while the intestinal villi of the piglets in the Bacillus amyloliquefaciens group were relatively complete and regular in shape, with higher villus height and obvious villus protrusions on the surface. Although the villus structure of the DON+Bacillus amyloliquefaciens group was improved compared with the DON group, some signs of damage were still visible, and the villus height and density were slightly decreased compared with the healthy group and the Bacillus amyloliquefaciens group. Fig.10 , indicating that vomitoxin has a significant destructive effect on the intestinal villi of piglets, while Bacillus amyloliquefaciens can protect the integrity of the intestinal villi structure to a certain extent. Bacillus amyloliquefaciens not only helps maintain intestinal health, but may also play a certain protective and repair role when attacked by toxins, reducing the damage of vomitoxin to the intestine.

[0115] 4) Results of damage to various organs of pigs

[0116] In order to understand the protective effect of Bacillus amyloliquefaciens on the organs of piglets fed with vomitoxin, the piglets in each group were autopsied. Fig.11. According to the results, the liver, heart, lungs, spleen and kidneys of the DON group showed obvious pathological changes, especially bleeding. This shows that DON has obvious toxic effects on these organs, causing tissue damage and bleeding. The organ tissues of these piglets in the Bacillus amyloliquefaciens group were similar to those in the healthy group, and no obvious pathological changes were observed. The organ surface was smooth and the color was normal, and there was no bleeding or other lesions. The conditions of the organs in the DON + Bacillus amyloliquefaciens group were similar to those in the healthy group, and no obvious pathological changes were observed, indicating that Bacillus amyloliquefaciens has a significant protective effect against the toxicity of DON. The organs of the healthy group were normal, and no abnormal pathological phenomena were observed, which served as a control for the healthy state.

[0117] These results show the severe toxic effects of DON on the major organs of piglets, leading to multi-organ hemorrhage and tissue damage. However, piglets fed with Bacillus amyloliquefaciens did not show obvious lesions in their organs when exposed to DON, indicating that Bacillus amyloliquefaciens can significantly reduce the toxic effects of DON and protect organ health.

[0118] At the same time, the weight of each organ was weighed, and the ratio of the weight of each organ to body weight in each group was calculated. The comparison results are shown in Fig.12 .

[0119] According to the results, heart / body weight percentage: the cardiac index of the DON group was the highest and significantly higher than that of the other groups, which may be due to the enlargement or edema of the heart caused by DON. The cardiac index of the DON+Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group was lower and close to the level of the healthy group, indicating that Bacillus amyloliquefaciens may have a protective effect on the heart and alleviate the negative effects of DON.

[0120] Liver / body weight percentage: The liver index of the DON group was the highest, which may reflect liver hypertrophy or lesions caused by DON. The liver index of the DON+Bacillus amyloliquefaciens group was significantly lower than that of the DON group, indicating that Bacillus amyloliquefaciens may have the effect of reducing liver burden. The liver index of the Bacillus amyloliquefaciens group and the healthy group was similar, indicating that Bacillus amyloliquefaciens did not cause abnormal liver enlargement.

[0121] Spleen / body weight percentage: The spleen index of the DON group was the highest, which may be due to splenomegaly caused by immune response. The spleen index of the DON+Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group was lower and close to the healthy group, indicating that Bacillus amyloliquefaciens may have a regulatory effect on the immune system and alleviate the excessive immune response caused by DON.

[0122] Lung / body weight percentage: The lung index of the DON group was the highest, which may reflect pathological changes such as pulmonary edema or congestion. The lung index of the DON+Bacillus amyloliquefaciens group was lower, close to the level of the Bacillus amyloliquefaciens group and the healthy group, indicating that Bacillus amyloliquefaciens may have alleviated the damage of DON to the lungs.

[0123] Kidney / body weight percentage: The kidney index in the DON group was the highest, which may indicate increased burden or pathological changes in the kidney. The kidney index in the DON+Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group was similar to that in the healthy group, indicating that Bacillus amyloliquefaciens may have a protective effect on the kidney.

[0124] These results suggest that Bacillus amyloliquefaciens may have a protective effect on multiple organs of piglets and reduce the toxic damage caused by DON.

[0125] 5) Results of toxin content in each intestinal segment: In order to explore the ability of Bacillus amyloliquefaciens to degrade vomitoxin accumulated in the intestine of piglets, the content of vomitoxin in the duodenum, jejunum, ileum, cecum, colon and liver of each group of piglets was tested. The results showed that the content of vomitoxin accumulated in the duodenum was the highest, and the content of vomitoxin in the jejunum, ileum, liver, cecum and colon gradually decreased. Among the various intestinal segments, the content of vomitoxin in the intestinal segments of piglets fed with Bacillus amyloliquefaciens was significantly lower than that of the group fed with vomitoxin. Comparison results can be seen in Fig.13 .

[0126] Content of DON in the duodenum: The content of DON in the DON group was significantly higher than that in the healthy group, indicating that DON had a higher accumulation in the duodenum. The toxin content in the DON+Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group was significantly lower than that in the DON group and was close to that in the healthy group, indicating that Bacillus amyloliquefaciens significantly reduced the content of DON in the duodenum.

[0127] Content of vomitoxin in the jejunum: Although the content of vomitoxin in the Bacillus amyloliquefaciens group was the lowest, there was no significant difference between the vomitoxin group, the vomitoxin + Bacillus amyloliquefaciens group, and the Bacillus amyloliquefaciens group, indicating that the distribution of vomitoxin in the jejunum was relatively uniform, and the effect of Bacillus amyloliquefaciens on the toxin content in the jejunum was not significant.

[0128] DON content in the ileum: The toxin content in the DON group was significantly higher than that in the healthy group, while the content in the DON+Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group was significantly reduced, close to that in the healthy group, indicating that Bacillus amyloliquefaciens helps reduce toxin accumulation in the ileum.

[0129] The content of vomitoxin in the cecum: The toxin content of the vomitoxin group was significantly higher than that of the other groups, indicating that vomitoxin had significant accumulation in the cecum. The vomitoxin + Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group significantly reduced the toxin content in the cecum, approaching the level of the healthy group.

[0130] DON content in the colon: The toxin content in the DON group was significantly higher than that in the other groups, but the toxin content in the DON+Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group was not significantly different from that in the healthy group, indicating that Bacillus amyloliquefaciens also has a similar protective effect in the colon.

[0131] DON content in the liver: The DON group had significantly higher toxin content than the other groups, indicating that DON accumulated in the liver. The DON+Bacillus amyloliquefaciens group and the Bacillus amyloliquefaciens group significantly reduced toxin content in the liver, approaching that of the healthy group, indicating that Bacillus amyloliquefaciens helps reduce toxin accumulation in the liver.

[0132] Overall, Bacillus amyloliquefaciens significantly reduced the content of DON in multiple organs, especially in the duodenum, ileum, cecum, colon and liver. This suggests that Bacillus amyloliquefaciens has a potential protective effect in reducing the accumulation of DON in the body, which may be achieved through mechanisms such as degrading toxins, promoting excretion or preventing toxin absorption. Comparing the data of the healthy group with those of the other experimental groups, the use of Bacillus amyloliquefaciens significantly improved the health status of piglets exposed to DON.

[0133] DON + Bacillus amyloliquefaciens group: From the first to the tenth day of feeding DON, the DON content in the feces was similar to that of the DON group, with no significant difference between the two, and the degradation effect of Bacillus amyloliquefaciens was not apparent. From the 15th day, the DON content in the feces began to be significantly lower than that of the DON group, indicating that the degradation effect of Bacillus amyloliquefaciens began to appear. The data on the 20th and 24th days further confirmed this, and the DON content in the DON + Bacillus amyloliquefaciens group continued to be significantly lower than that of the DON group, indicating that the degradation effect of Bacillus amyloliquefaciens on DON continued.

[0134] 6) Effects of Bacillus amyloliquefaciens on the expression of nutrient transport genes in the intestine of piglets

[0135] The expression of nutrient transport genes SGLT1, PePT1, GLUT2, and ASCT2 in the small intestine of piglets was detected. Fig.14 .

[0136] Among them, the protein encoded by SGLT1 is sodium-glucose co-transporter 1, which is mainly located on the brush border membrane of the small intestinal epithelial cells. This protein is responsible for the active transport of sodium-dependent glucose and galactose, transporting glucose and sodium ions from the intestinal cavity to the intestinal epithelial cells and then into the blood. This is the main pathway for the absorption of glucose and galactose. According to the experimental results, vomitoxin significantly inhibited the expression of the SGLT1 gene (** p <0.01), which may affect the absorption of glucose and galactose in the small intestine, thereby affecting energy acquisition. Bacillus amyloliquefaciens itself has no significant promoting effect on the expression of SGLT1 in the absence of vomitoxin, and even has a certain inhibitory effect (* p <0.05); When fed with vomitoxin and Bacillus amyloliquefaciens at the same time, although the expression of SGLT1 was restored, it was still significantly lower than that of the healthy group (** p <0.01), indicating that Bacillus amyloliquefaciens had limited effect on the restoration of SGLT1 expression.

[0137] The protein encoded by PePT1 (also known as SLC15A1) is a peptide transporter 1 located on the brush border membrane of the small intestinal epithelial cells. PePT1 is responsible for the proton-dependent absorption of peptides, mainly dipeptides and tripeptides. These peptides are composed of protein digestion products and certain drugs. PePT1 absorbs these substances by co-transporting protons and peptides. According to the experimental results, DON significantly inhibited the expression of the PePT1 gene (**** p <0.0001), indicating that vomitoxin has a strong inhibitory effect on the gene, which may lead to a reduction in intestinal absorption of peptides and affect protein nutrition. In the Bacillus amyloliquefaciens group, the expression level of the PePT1 gene was significantly lower than that in the healthy group (**** p <0.0001), but higher than the DON group (* p <0.05), indicating that Bacillus amyloliquefaciens has an inhibitory effect on PePT1 gene expression in the absence of DON. In the DON+Bacillus amyloliquefaciens group, the expression level of PePT1 gene was significantly higher than that in the DON group (*** p <0.001), but there was still a significant difference compared with the healthy group (* p <0.05). This indicates that Bacillus amyloliquefaciens can alleviate the inhibitory effect of DON on PePT1 to a certain extent in the presence of DON, but it cannot completely restore to normal levels.

[0138] The protein encoded by GLUT2 (also known as SLC2A2) is glucose transporter 2, which is mainly located in the liver, pancreatic beta cells, small intestine and kidney. Among them, in the small intestine, GLUT2 is a key protein involved in the facilitated diffusion transport of glucose, fructose and galactose, mainly on the basement membrane of small intestinal epithelial cells. This transport is independent of sodium ions and energy, and is responsible for transporting absorbed monosaccharides from the cell to the blood. According to the experimental results, the expression level of the GLUT2 gene did not change much in different experimental groups, and there was no significant difference in most cases. This may indicate that the expression of GLUT2 is relatively stable under these experimental conditions and is not easily affected by external factors. Although in most cases, there was no significant difference in the expression of the GLUT2 gene, the expression level of the GLUT2 gene in the DON + Bacillus amyloliquefaciens group was slightly higher than that in the Bacillus amyloliquefaciens group, reaching a statistically significant level (* p <0.05). This may suggest that in the presence of DON, B. amyloliquefaciens may partially compensate for the negative effects of DON through some mechanisms and maintain or promote the expression of GLUT2.

[0139] The protein encoded by ASCT2 (also known as SLC1A5) is a neutral amino acid transporter 2, which is widely distributed in various tissues, including the small intestine. In the small intestine, ASCT2 is responsible for the sodium-dependent transport of neutral amino acids (such as alanine, serine, and cysteine), and is one of the important pathways for amino acid transport across cell membranes. According to the experimental results, the expression level of the ASCT2 gene in the DON group was significantly higher than that in the healthy group (* p <0.05), indicating that DON significantly upregulated the expression of ASCT2. This may be part of the intestinal cell response to DON stress, attempting to increase the absorption of amino acids to compensate for the damage. The expression of ASCT2 gene in the Bacillus amyloliquefaciens group was lower than that in the DON group (* p <0.05), but there was no significant difference compared with the healthy group (ns). This indicates that Bacillus amyloliquefaciens has no significant effect on the expression of the ASCT2 gene in the absence of DON. The expression of the ASCT2 gene in the DON+Bacillus amyloliquefaciens group was significantly lower than that in the DON group (** p <0.01), which was not significantly different from the healthy group and the Bacillus amyloliquefaciens group (ns). This suggests that Bacillus amyloliquefaciens can effectively counteract the upregulation of ASCT2 gene expression by DON and restore the expression level to a near-normal state. Overall, Bacillus amyloliquefaciens showed a certain protective effect in alleviating the effects of DON on ASCT2 gene expression, possibly by regulating the intestinal stress response to maintain normal amino acid absorption. This provides further support for the potential application of Bacillus amyloliquefaciens in improving intestinal health, especially in the presence of harmful substances.

[0140] Deoxynivalenol significantly affected the expression of SGLT1, PePT1, and ASCT2 genes, while Bacillus amyloliquefaciens alone also significantly affected the expression of SGLT1 and PePT1 genes, and had little or no significant effect on GLUT2 and ASCT2. Bacillus amyloliquefaciens can significantly alleviate the negative effects of deoxynivalenol in some cases, especially in the expression of PePT1 and ASCT2. Specifically, Bacillus amyloliquefaciens can significantly alleviate the inhibitory effect of deoxynivalenol on PePT1 gene expression, which helps to restore the intestinal transport function of peptide nutrients. In addition, when Bacillus amyloliquefaciens is used in combination with deoxynivalenol, it has a positive regulatory effect on the expression of GLUT2 gene, which may help maintain the glucose transport function. Bacillus amyloliquefaciens can also partially alleviate the upregulation of ASCT2 gene expression caused by deoxynivalenol, helping to maintain the balance and transport function of neutral amino acids in the intestine. Overall, Bacillus amyloliquefaciens shows a potential protective effect in alleviating the toxic effects of deoxynivalenol, especially in maintaining the intestinal nutrient transport function. These results provide theoretical support for the application of Bacillus amyloliquefaciens as a feed additive, especially in the case of improving toxin contamination in feed.

[0141] This example proves that the Bacillus amyloliquefaciens from the intestines of Northeast Min pigs has obvious functions of degrading vomitoxin in vivo and in vitro. At the same time, the strain can also improve the growth performance of piglets, showing peroxidase activity and other enzyme activities, strong growth ability and strong tolerance in acidic environment. Bacillus amyloliquefaciens can significantly reduce the damage of vomitoxin to the intestinal villi of piglets and the damage to various organs, especially playing a potential protective role in maintaining the intestinal nutrient transport function.

[0142] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Although the present application 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 principles of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Bacillus amyloliquefaciens, which is named Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101, which was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on May 20, 2024, with the deposit number CCTCC NO: M 20241003.

2. Use of the Bacillus amyloliquefaciens according to claim 1 in preparing a product having at least one of the following functions: (1) Degradation of vomitoxin in vitro; (2) Degradation of vomitoxin in the body.

3. The use according to claim 2, characterized in that: The products include pharmaceuticals, probiotic preparations and biodetoxifiers.

4. A product comprising the Bacillus amyloliquefaciens strain according to claim 1, wherein: The products include pharmaceuticals, probiotic preparations and biodetoxifiers.

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