Application of Acidobacterium Gottfriedii and its bacterial agent in reducing drug residues in feces and sewage

Abstract

The invention relates to an acid-fast-growing Gottfriedia and an application of a bacterial agent thereof in reducing drug residues in manure and sewage, belonging to the technical field of functional microbial screening. The acid-fast-growing Gottfriedia TW007 is classified and named as: Gottfriedia acidiceleris TW007, screened in the soil of a Yantai orchard, and the strain was preserved in the China Center for Type Culture Collection on June 1, 2022, with a preservation number of: CCTCC NO: M 2022769; also provided is an application of a bacterial agent containing acid-fast-growing Gottfriedia TW007 in reducing drug residues in manure and sewage, the bacterial agent can effectively reduce antibiotic residues in livestock and poultry manure and sewage, reduce environmental pollution and the resulting human health risks, and has a stable effect, and can colonize in the soil for a long time to maintain good antibiotic degradation ability.

Description

Technical Field

[0001] The invention relates to an acid-fast-growing Gottfriedii bacterium and an application of a bacterial agent thereof in reducing drug residues in excrement, and in particular to an acid-fast-growing Gottfriedii bacterium and an application of a bacterial agent thereof in reducing antibiotic residues in livestock and poultry excrement treatment, and belongs to the technical field of functional microorganism screening. Background Art

[0002] In recent years, the total amount of livestock and poultry breeding in my country has continued to increase, producing about 3.8 billion tons of livestock and poultry manure every year. Many large and medium-sized livestock and poultry farms do not treat livestock and poultry manure harmlessly or as a resource, but directly apply it to farmland or enter water bodies with sewage, which pollutes the surrounding environment on the one hand, and on the other hand, the various antibiotic residues in livestock and poultry manure are gradually destroying the ecological balance of farmland, causing potential harm to the quality and safety of agricultural products and affecting human health.

[0003] The problem of antibiotic residues in livestock and poultry manure cannot be ignored. Since the US FDA first approved the use of antibiotics as feed additives in the 1950s, countries around the world have successively used antibiotics in livestock and poultry farming; veterinary antibiotics can not only treat and prevent animal diseases, but can also be added to feed as growth promoters. In recent years, with the improvement of people's living standards, the demand for animal food has increased, and the production scale of feed mills and farms has continued to expand. Driven by economic interests, antibiotics are widely used to prevent disease outbreaks and promote animal growth.

[0004] A study published by the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, shows that in 2013, the total amount of antibiotics used in China was about 162,000 tons, of which 84,000 tons were used in livestock and poultry farming. Tetracyclines, quinolones, macrolides, sulfonamides, etc. are widely used. When antibiotics enter the animal body, only a small part of them participate in the body's metabolism to exert their efficacy, and the remaining 30%-90% are excreted from the body in the form of parent or metabolites with feces and urine. When livestock and poultry manure is applied to the soil as fertilizer, the antibiotics in it can be adsorbed and accumulated by the soil, killing or inhibiting important indigenous microorganisms in the soil ecosystem, such as nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and actinomycetes, reducing the efficiency of soil nutrient circulation, and also producing varying degrees of ecotoxicological effects on some plants, affecting their growth. For example, oxytetracycline can significantly inhibit the growth of alfalfa by affecting the physiological processes of its roots.

[0005] In order to ensure efficient and safe utilization of livestock and poultry manure, an effective way is to compost it before agricultural use, and use microbial fermentation to decompose the complex organic matter in livestock and poultry manure into humus, kill pathogenic microorganisms, and reduce some antibiotic residues. The microbial agents used in composting directly affect the reduction rate of antibiotic residues.

[0006] Chinese patent application CN111154675 reported a strain of acid-fast-growing Bacillus SYY15 and its application. The bacterium can not only synthesize ACC deaminase to enhance the stress resistance of crops, but also degrade a large amount of insoluble organic phosphorus and inorganic phosphorus, and produce IAA, protease and other growth-promoting substances, but it cannot reduce antibiotic residues.

[0007] The existing microbial products on the market are of varying quality and unstable. It is necessary to further develop microbial strains and preparations that can effectively and quickly reduce antibiotic residues. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a fast-growing Gottfriediella and its bacterial agent for use in reducing drug residues in livestock and poultry manure treatment.

[0009] The technical solution of the present invention to solve the above technical problems is as follows:

[0010] A fast-growing Gottfriedia acidiceleris TW007, classified and named: Gottfriedia acidiceleris TW007, screened from the soil of a Yantai orchard, the strain was deposited in the China Center for Type Culture Collection on June 1, 2022, and the deposit number is: CCTCC NO: M 2022769; the 16S rDNA of the fast-growing Gottfriedia acidiceleris TW007 is shown in SEQ NO.1.

[0011] A bacterial agent containing the acid-fast Gottfriedii bacteria TW007.

[0012] The bacterial agent is used to reduce drug residues in livestock and poultry manure.

[0013] The preparation method of the bacterial agent comprises the following specific steps:

[0014] (1) Strain activation

[0015] Pick out the strain of Acidovorax Gottfriedii TW007 and streak it and inoculate it into the solid slant medium, and culture it in a constant temperature incubator at 30°C for 18 hours until a large number of bacterial cells grow on the slant for later use;

[0016] (2) Liquid seed preparation

[0017] The activated acid-fast Gottfriedii bacteria TW007 in step (1) was selected and inoculated into the seed culture medium, and the medium was sealed with gauze, and cultured in a shaking incubator at 30° C. and 180 rpm for 18 hours to grow a large number of bacteria to obtain liquid seeds;

[0018] (3) inoculating the liquid seeds prepared in step (2) into the fermentation medium at an inoculum rate of 5% by volume, controlling the dissolved oxygen to 30%, the pH to 7.2, the temperature to 30° C., and aerating and culturing for 22 hours. When the amount of live bacteria of acid-fast Gottfriedii sp. TW007 in the fermentation broth reaches the target value, collecting the fermentation broth, which is the bacterial agent containing acid-fast Gottfriedii sp. TW007.

[0019] Furthermore, the amount of live bacteria of Gottfriedii acidophilus TW007 in the bacterial agent is not less than 10 9 cfu / mL or 10 9 cfu / g.

[0020] Furthermore, the solid slant culture medium in step (1) is: 10.0-15.0 g of tryptone, 1.0-5.0 g of soy peptone, 1.0-5.0 g of NaCl, 15.0-20.0 g of agar powder, dissolved in distilled water to a volume of 1000 mL, pH 6.0, and sterilized at 121° C. for 15-20 min.

[0021] Furthermore, the seed culture medium in step (2) is: tryptone 10.0-15.0 g / L, soy peptone 1.0-5.0 g / L, yeast extract 0.1-0.5 g / L, NaCl 1.0-5.0 g / L, KH2PO4 1.0-5.0 g / L, dissolved in distilled water to make up to volume, pH 6.0, and sterilized by high-pressure steam at 121°C for 15-20 min.

[0022] Furthermore, the fermentation medium in step (3) is: tryptone 10.0-15.0 g / L, soy peptone 1.0-5.0 g / L, glucose 1.0-5.0 g / L, yeast extract 1.0-5.0 g / L, MgSO4 0.1-0.5 g / L, KH2PO4 1.0-5.0 g / L, NaCl 1.0-5.0 g / L, dissolved in distilled water to make up to volume, pH 6.0, and sterilized by high-pressure steam at 121°C for 15-20 min.

[0023] Furthermore, the bacterial agent also contains one or a combination of two or more of yeast, lactic acid bacteria, bacillus, and photosynthetic bacteria; the bacterial agent can be concentrated to form a liquid bacterial agent or freeze-dried to form a solid bacterial powder.

[0024] The beneficial effects of the present invention are:

[0025] (1) Acidovorax Gottfriedii TW007 was screened from the soil of Yantai. It is a non-pathogenic bacterium that is harmless to humans and animals, does not pollute the environment, does not endanger the ecological balance, and complies with biosafety regulations;

[0026] (2) The strain can be used for harmless treatment of livestock and poultry manure, effectively reducing antibiotic residues in livestock and poultry manure, reducing environmental pollution and the resulting human health risks, and has a stable effect. It can colonize in the soil for a long time and maintain a good antibiotic degradation ability;

[0027] (3) The strain can be used alone or mixed with other bacteria such as yeast, lactic acid bacteria, bacillus, photosynthetic bacteria, etc., and can be used in situ or ex situ. The bacterial solution or powder can be directly sprayed on livestock and poultry manure at an inoculation rate of 0.1% to 1.0%. Due to its strong resistance to stress, it can grow well in livestock and poultry manure, occupy a favorable ecological niche, inhibit the growth and reproduction of pathogenic microorganisms, and degrade antibiotic residues. Spraying once can exert long-term effects.

[0028] (4) The bacterial agent has the characteristics of simple and efficient preparation process, stable and reliable products, and has obvious technical advantages that can be easily commercialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The microscopic morphology of the acid-fast Gottfriedii bacteria TW007 of the present invention;

[0030] Figure 2 This is a phylogenetic tree of the acid-fast Gottfriedii sp. TW007 of the present invention based on the 16S rDNA sequence. DETAILED DESCRIPTION

[0031] The following is combined with Figure 1 and attached Figure 2 The principles and features of the present invention are described, and the examples given are only used to explain the present invention, rather than to limit the scope of the present invention.

[0032] Example 1: Isolation, screening and identification of Gottfriedia acidophila TW007

[0033] Initial screening: Take 10g of farmland soil, dissolve it in 90mL of sterile water, shake at 200rpm for 30min, take 1mL of bacterial suspension for gradient dilution, take 10 -3 , 10 -4 , 10 -5 , 10 -6 Four gradients, treated in boiling water bath for 2 min, spread on LB solid plate medium, and cultured at 30°C for 72 hours. Pick single colonies from the plate for microscopic examination, record their microscopic morphology, and transfer single colonies with different colony morphology and microscopic morphology to slant LB medium, culture at 30°C for 48 hours, and store at 4°C for later use.

[0034] Rescreening: Prepare 5 mg / mL amoxicillin stock solution, add it to LB medium to a final amoxicillin concentration of 50 μg / mL, pour plates, inoculate the primary screened strains on LB plate medium containing amoxicillin, select colonies that can grow normally, inoculate them into liquid LB medium containing 50 μg / mL amoxicillin, and culture them at 30°C for 48 h. Detect the change in amoxicillin concentration by HPLC, pick the bacterial solution with significantly reduced amoxicillin, purify and culture it by streaking three times, and store it in glycerol tubes at -80°C.

[0035] Identification: The colony morphology was observed on LB medium plates after 48 hours of culture. The strain TW007 was observed microscopically by Gram staining. The attachment of bacterial flagella and pili was observed under an electron microscope. The physiological and biochemical characteristics of the strain were studied using the BioMérieux VITEK 2Compact fully automatic bacterial identification and drug sensitivity analysis system. The strain TW007 was classified based on the phylogenetic analysis of the 16S rDNA sequence.

[0036] The results showed that the colonies of strain TW007 were round, opaque white, smooth, with regular edges and a translucent halo; Gram staining was purple, indicating that it was a Gram-positive bacterium with a rod-shaped body and blunt ends. Figure 1 shown.

[0037] The results of physiological and biochemical tests showed that strain TW007 could not grow normally in 6.5% sodium chloride, and the resistance reactions of L-aspartate arylaminease, leucine arylaminease, phenylalanine arylaminease, proline arylaminease, alanine arylaminease, tyrosine arylaminease, alanine-phenylalanine-proline arylaminease, ELLMAN, α-glucosidase and polymyxin B were positive.

[0038] The specific physiological and biochemical characteristics are shown in Table 1:

[0039] Table 1 Physiological and biochemical characteristics of strain TW007

[0040]

[0041]

[0042] Note: “+” indicates a positive reaction, and “-” indicates a negative reaction.

[0043] The genomic DNA of strain TW007 was extracted for 16S rDNA gene amplification and sequencing. The Blast system was used to compare with the known gene sequences in GenBank, and the relevant sequence information of homologous strains was screened for phylogenetic analysis. The Neighbor-Joining method in the software MEGA7.0 was used to construct a phylogenetic tree. The results showed that strain TW007 was most closely related to Gottfriedia acidiceleris NR_043774, with a 16S rDNA similarity of 99.7%. Combined with the morphological characteristics of the strain, it was finally identified as Gottfriedia acidiceleris. The strain was deposited in the China Center for Type Culture Collection on June 1, 2022, and the classification name was Gottfriediaacidiceleris TW007, the deposit number was CCTCC NO:M 2022769, and the deposit address was Wuhan, China.

[0044] The 16S rDNA sequence of the acid-fast Gottfriedii bacteria TW007 is:

[0045]

[0046] Example 2: Preparation method of bacterial agent 1 containing Acidovorax Gottfriedii TW007

[0047] (1) Strain activation

[0048] Pick the strain of Acidovorax Gottfriedii TW007 and streak it onto a solid slant medium (tryptone 15.0 g, soy peptone 5.0 g, NaCl 5.0 g, agar powder 15.0 g, distilled water to 1000 mL, pH 6.0, sterilized at 121°C for 20 min), and culture it in a constant temperature incubator at 30°C for 18 hours. When a large number of bacterial cells grow on the slant, they are used to transfer liquid seeds.

[0049] (2) Liquid seed preparation

[0050] Pick out the activated strain and inoculate it into a medium containing seed culture medium (tryptone 15.0 g / L, soy peptone 5.0 g / L, yeast extract powder 0.3 g / L, NaCl 5.0 g / L, KH2PO4 2.5 g / L, dissolved in distilled water to 1000 mL, pH 6.0, sterilized by high pressure steam at 121°C for 20 min), seal with gauze, and culture at 30°C, 180 r / min shaking table for 18 hours to grow a large number of bacteria to obtain liquid seeds;

[0051] (3) Liquid fermentation

[0052] The liquid seeds prepared in step (2) were inoculated into a fermentation medium (tryptone 10.0 g / L, soy peptone 5.0 g / L, glucose 2.5 g / L, yeast extract 5.0 g / L, MgSO4 0.5 g / L, KH2PO4 2.5 g / L, NaCl 5.0 g / L, dissolved in distilled water to 1000 mL, pH 6.0, sterilized by high pressure steam at 121°C for 20 min) at a volume ratio of 5%, the dissolved oxygen was controlled at 30%, the pH was 7.2, the temperature was 30°C, and the culture was aerated for 22 hours. When the viable bacteria of Gottfriedii acidophilus TW007 in the fermentation broth reached 10 9 cfu / mL, the fermentation broth is collected and is the bacterial agent containing acid-fast Gottfriedii TW007.

[0053] The acid-fast-growing Gottfriediella TW007 can be used alone or in combination with other bacteria such as yeast, lactic acid bacteria, bacillus, photosynthetic bacteria, or one or more of the above to prepare a mixed bacterial agent for use. The amount of live bacteria of the acid-fast-growing Gottfriediella TW007 in the bacterial agent is not less than 10 9 cfu / mL or 10 9 cfu / g; when in use, the bacterial agent can be concentrated into liquid bacterial agent or freeze-dried into solid bacterial powder according to customer needs.

[0054] Example 3: Application of a bacterial agent containing Gottfriediella acidophila TW007 in the treatment of livestock and poultry manure to reduce drug residues in manure

[0055] 10 mL of the bacterial agent containing acid-fast Gottfriedii bacteria TW007 was inoculated into 1 kg of chicken manure and 1 L of pig sewage containing a certain concentration of antibiotic residues, and the mixture was evenly mixed. The chicken manure was shallowly buried in the soil, and the pig sewage was left to stand at room temperature, respectively, as test group A and test group B. 10 mL of sterile water was added to the control group instead of the bacterial solution. Three replicates were set for each group, and samples were taken at regular intervals to determine the antibiotic concentration. The results are shown in Table 3 below. After the livestock and poultry manure was inoculated with strain TW007, the degradation rate of amoxicillin in chicken manure was 73% after 21 days, and the degradation rate of cefquinome sulfate in pig sewage was 89% after 7 days, which were higher than 57% and 52% of the control group, respectively, showing a good effect of reducing antibiotics.

[0056] Table 2 Effect of strain TW007 on the reduction of antibiotic residues in livestock and poultry manure

[0057]

[0058] Example 4: Application of a bacterial agent containing Acidovorax Gottfriedii TW007 in livestock and poultry manure composting

[0059] 2000kg of fresh chicken manure was added with an appropriate amount of sawdust and mixed well, and the water content was adjusted to about 60%, and the mixture was divided into two parts, chicken manure A and chicken manure B. The inoculum containing acid-fast Gottfriedii TW007 was evenly sprayed into chicken manure B at a 0.1% inoculation rate, and an equal amount of sterile water was added to chicken manure A as a blank control. The compost was turned twice on the 2nd and 4th days, and the compost was fermented for 10 days. The concentration of amoxicillin in the chicken manure was detected on the 0th, 3rd, 6th and 10th days. The results are shown in Table 3. In the test group with the inoculum containing acid-fast Gottfriedii TW007, the residual amoxicillin in the chicken manure after composting was significantly lower than that in the control group, indicating that the inoculum containing acid-fast Gottfriedii TW007 had a significant effect on the reduction of amoxicillin in chicken manure compost.

[0060] Table 3 Effect of strain TW007 on the reduction of amoxicillin in chicken manure compost

[0061] time Control group (mg / kg) Test group (mg / kg) Day 0 6.08 6.08 3 days 4.82 3.50 6 days 2.15 0.71 10 days 1.71 0.23 Degradation rate (%) 72 96

[0062] The results of Example 3 and Example 4 show that the bacterial agent prepared by the strain can be applied to the harmless treatment of livestock and poultry manure, including fresh manure, manure composting process, garbage or sewage mixed with livestock and poultry manure, and can effectively reduce antibiotic residues in livestock and poultry manure, reduce environmental pollution and the resulting human health risks, and the effect is stable, and can colonize in the soil for a long time to maintain a good antibiotic degradation ability;

[0063] This strain can be used alone or made into a mixed bacterial agent with other bacteria such as yeast, lactic acid bacteria, bacillus, photosynthetic bacteria, etc. It can be used for in-situ treatment or ex-situ treatment. The bacterial liquid or powder can be directly sprayed on livestock and poultry manure at an inoculation rate of 0.1% to 1.0%. Due to its strong resistance to stress, it can grow well in livestock and poultry manure, occupy a favorable ecological niche, inhibit the growth and reproduction of pathogenic microorganisms, and degrade antibiotic residues. Spraying once can exert a long-term effect.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Acidophilus Gottfriedii Gottfriedia acidiceleris ) TW007, the acid-fast Gottfriedii bacteria TW007 was deposited in the China Center for Type Culture Collection on June 1, 2022, with the deposit number: CCTCC NO: M2022769; the 16S rDNA of the acid-fast Gottfriedii bacteria TW007 is shown in SEQ NO.

1.

2. A bacterial agent containing the acid-fast Gottfriedii bacteria TW007 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent further comprises one or a combination of two or more of yeast, lactic acid bacteria, bacillus, and photosynthetic bacteria; the bacterial agent can be concentrated to form a liquid bacterial agent or freeze-dried to form a solid bacterial powder.

4. Use of the microbial agent according to claim 2 in reducing amoxicillin and / or cefquinome sulfate drug residues in livestock and poultry manure.

5. A method for preparing the bacterial agent according to claim 2, characterized in that: The specific steps are as follows: (1) Strain activation Pick the strain of Acidovorax Gottfriedii TW007 and inoculate it into the solid slant medium, and culture it in a constant temperature incubator at 30°C for 18 hours until a large number of bacterial cells grow on the slant for later use; (2) Liquid seed preparation The activated acid-fast Gottfriedii bacteria TW007 in step (1) is selected and inoculated into the seed culture medium, and the medium is sealed with gauze, and cultured in a shaking incubator at 30° C. and 180 rpm for 18 hours to grow a large number of bacteria, thereby obtaining liquid seeds; (3) The liquid seeds prepared in step (2) are inoculated into the fermentation medium at an inoculum rate of 5% by volume, the dissolved oxygen is controlled at 30%, the pH value is 7.2, the temperature is 30° C., and the culture is carried out under aeration for 22 hours. When the amount of live bacteria of Acidophilus Gottfriedii TW007 in the fermentation broth reaches the target value, the fermentation broth is collected to obtain the bacterial agent containing Acidophilus Gottfriedii TW007.

6. The preparation method according to claim 5, characterized in that: The amount of live bacteria of the acid-fast Gottfriedii bacteria TW007 in the bacterial agent is not less than 10 9 cfu / mL or 10 9 cfu / g.

7. The preparation method according to claim 5, characterized in that: The solid slant culture medium in step (1) is: 10.0-15.0 g of tryptone, 1.0-5.0 g of soy peptone, 1.0-5.0 g of NaCl, and 15.0-20.0 g of agar powder, dissolved in distilled water to a volume of 1000 mL, pH 6.0, and sterilized at 121°C for 15-20 min.

8. The preparation method according to claim 5, characterized in that: The seed culture medium in step (2) is: 10.0-15.0 g / L tryptone, 1.0-5.0 g / L soy peptone, 0.1-0.5 g / L yeast extract, 1.0-5.0 g / L NaCl, and 1.0-5.0 g / L KH2PO4, dissolved in distilled water to a constant volume, pH 6.0, and sterilized by high-pressure steam at 121°C for 15-20 min.

9. The preparation method according to claim 5, characterized in that: The fermentation medium in step (3) is: 10.0-15.0 g / L tryptone, 1.0-5.0 g / L soy peptone, 1.0-5.0 g / L glucose, 1.0-5.0 g / L yeast extract, 0.1-0.5 g / L MgSO4, 1.0-5.0 g / L KH2PO4, and 1.0-5.0 g / L NaCl, dissolved in distilled water to a constant volume, pH 6.0, and sterilized by high-pressure steam at 121°C for 15-20 min.

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