CN116555078B - A Bacillus subtilis strain capable of effectively degrading cypermethrin and its application - Google Patents

Abstract

The present invention relates to the field of using microorganisms to reduce pollutants in the environment or food, and specifically to a strain of Bacillus subtilis isolated from healthy broiler feces with a high ability to degrade cypermethrin. The Bacillus subtilis strain that can effectively degrade cypermethrin is numbered J6 and is registered and preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration; the deposit number is CGMCC No.26229, and the deposit date is December 27, 2022. In addition to being able to degrade cypermethrin, the strain also has a good degradation effect on other pyrethroid pesticides and their main degradation product-3-phenoxybenzoic acid. Bacillus subtilis J6 of the present invention can be used to develop corresponding biological preparations for the reduction of pyrethroids in the environment, feed and food; or the strain can be developed into health foods or medicines to reduce the hazards of pyrethroid pesticides to human exposure.

Description

A Bacillus subtilis strain capable of effectively degrading cypermethrin and its application

Technical Field

The invention relates to the field of using microorganisms to remove pollutants in the environment or feed and food, and in particular to a strain of Bacillus subtilis separated and screened from the cecal contents of healthy broiler chickens and having good degradation ability for cypermethrin.

Background technique

Pyrethroid pesticides are widely used in pesticide formulations and insecticidal aerosols because they are stable to light and heat, have a broad insecticidal spectrum, have ovicidal activity, and have extremely high stomach toxicity and contact killing effects on agricultural and forestry pests such as Lepidoptera on cereal crops, fruits and vegetables, and sanitary pests such as mosquitoes, flies, and cockroaches.

In the past, pyrethroid pesticides have been considered to be low-toxic and safe to use. However, as the use of pyrethroids continues to increase, the various environmental pollution, food safety, adverse effects on human health and other issues caused by them have become increasingly prominent, attracting widespread attention from countries around the world. Due to the good stability and persistence of pyrethroid pesticides in soil and water, their large-scale use has led to the continuous accumulation of background residues in the environment, which directly affects the quality of agricultural products and food safety, and threatens human health.

Most of the pyrethroid-degrading bacteria currently available are isolated from soil, sludge and other environments contaminated by pesticides. They cannot be used to remove pyrethroid pesticides from food, and are difficult to add to feed and food to alleviate the chronic damage caused by pesticides to animals and humans. Pesticide residues are a long-standing and unavoidable safety hazard, but there is currently no effective removal method or approach that can be used to reduce pesticide residues in the food, animal husbandry and human body. Therefore, it is necessary to develop probiotic strains with the ability to reduce pesticides.

Summary of the invention

In view of the problems existing in the prior art, the present invention aims to provide a strain of Bacillus subtilis J6 that can effectively degrade cypermethrin and its application.

The strain provided by the present invention is Bacillus subtilis, strain number J6, deposited in the General Microbiology Center of the China Culture Collection Administration, the storage location is No. 1 Beichen West Road, Chaoyang District, Beijing, the storage number is CGMCC No.26229, and the storage date is December 27, 2022.

The invention provides separation, screening and identification of animal-derived pesticide-degrading strains.

Take 0.1-0.5 g of cecal contents from healthy animals, add 2 mL of modified basal medium (cypermethrin concentration is 10-50 mg/L), vortex thoroughly, transfer to a vial, and place at 37°C, 160 rpm shaking culture for 2-5 days. After the culture is completed, add 2 mL of acetonitrile, ultrasonic extraction for 30 min, mix well and transfer 1.5 mL to an EP tube, centrifuge at 12000 rpm for 10 min, take the supernatant and filter through a 0.22 μm organic filter membrane, collect the filtrate and use high performance liquid chromatography (HPLC) to determine the cypermethrin concentration, and calculate the degradation rate of cypermethrin by cecal contents. The culture medium without cecal contents was used as the control.

Weigh 0.1-1 g of the cecal content sample with cypermethrin degradation effect obtained above, add 9-9.9 mL of physiological saline, vortex mix and perform gradient dilution, take the dilution solution and spread it on the modified basal medium solid culture medium, and culture it aerobically or anaerobically at 37°C for 2-4 days. Select strains with different colony morphology and perform multiple streaking purification until the colony morphology on the same plate is uniform, and then store them on slant. Take a loop of the slant strain and inoculate it into 5 mL of LB liquid culture medium, culture it at 37°C for 2-4 days, inoculate it into 30 mL of LB liquid culture medium at a 2% inoculum, and culture it at 37°C and 160 rpm for 1-5 days.

Take 1 mL of culture medium and add 4 mL of acetonitrile, extract by ultrasonic for 30 min, then mix the extract, take 1.5 mL of liquid and centrifuge (12000 rpm, 10 min), take the supernatant and pass it through a 0.22 μm organic filter membrane, collect the filtrate and use high performance liquid chromatography (HPLC) to determine the concentration of cypermethrin, and calculate the degradation rate of cypermethrin by the strain.

The HPLC detection method is as follows: Gemini 100Å C18 column (4.6 mm×150 mm, 5 μm), acetonitrile: ultrapure water = 83:17 as the mobile phase, flow rate of 1.0 mL/min, column temperature of 25°C, injection volume of 10 μL, detector is ultraviolet detector, detection wavelength of 210 nm.

The calculation formula of cypermethrin degradation rate is:

( C ₀ - C )/ C ₀ *100%

Where: C0 is _the concentration of cypermethrin in the control (mg/L); C is the concentration of cypermethrin remaining after adding cecal contents/strain (mg/L).

Morphological identification of strains: The colonies on the plate are round or oval, off-white with a slight yellow tint, convex in the center, wrinkled on the surface, and moist inside. Gram staining is positive and rod-shaped.

Physiological and biochemical identification of the strain: The strain can ferment D-glucose, D-fructose, D-mannose, D-cellobiose, sucrose, trehalose, xylose, and gentiobiose; it can hydrolyze starch; it can assimilate D-mannitol and N-acetylglucosamine; it can grow at 7% NaCl and pH 5; the V-P test is positive; the catalase is positive; it can utilize citrate and hydrolyze gelatin, but cannot utilize propionate.

Molecular genetic identification of strain J6.

The strain J6 was sequenced by 16S rDNA and the following sequence was obtained:

CCCTTGGCCGTATTCTAATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGG AGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACG CTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTG ATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGAACCGGG.

The 16S rDNA gene sequencing results of the strain were compared with the sequences in the GenBank database by Blast homology. The strain sequence was highly homologous to the Bacillus subtilis gene sequence and the accession number was OQ195114. Combined with morphological and physiological and biochemical indicators, strain J6 was finally identified as Bacillus subtilis.

The strain with high efficiency in adsorbing cypermethrin provided by the present invention is isolated and screened from the cecal contents of healthy broiler chickens. The strain also has the ability to degrade cypermethrin, deltamethrin, cypermethrin and 3-phenoxybenzoic acid, the main degradation product of pyrethroid pesticides, and can be used to reduce residual cypermethrin in the environment and food.

The culture medium used in the present invention is: improved basic culture medium: inulin 4g, glucose 4g, beef extract 2g, peptone 2g, yeast powder 2g, NaCl ₂ 2g, K ₂ HPO ₄ 0.04g, KH ₂ PO ₄ 0.04g, MgSO ₄ ·4H ₂ O 0.01g, CaCl ₂ 0.01g, NaHCO ₃ 2g, L-cysteine 0.5g, Tween-80 2g, bile salt 0.5g, distilled water 1000mL, sterilized at 121℃ for 15min. LB culture medium: tryptone 10g, yeast extract powder 5g, NaCl 10g, Tween-80 2g, distilled water to 1000mL, sterilized at 121℃ for 20min. Add 2% agar to obtain the corresponding solid culture medium.

Compared with the prior art, the present invention has the following positive effects: for the first time, Bacillus subtilis with the ability to degrade pyrethroid pesticides such as cypermethrin and the intermediate 3-phenoxybenzoic acid is obtained from the cecal contents of healthy animals. The strain is safe in source and can provide a good bacterial source for the removal of residual cypermethrin in the environment and food, as well as in animals and humans.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of culture temperature on the degradation of cypermethrin by the strain.

Figure 2 shows the effect of substrate concentration on the degradation of cypermethrin by the strain.

Figure 3 shows the effect of metal ions on the degradation of cypermethrin by the strain.

Implementation

Example 1: Degradation characteristics of cypermethrin by Bacillus subtilis J6.

1) Effect of culture temperature on degradation of cypermethrin by J6 The seed solution of strain J6 was inoculated at 2% ( v/v ) into LB medium with a cypermethrin concentration of 50 mg/L and cultured at 20, 25, 30, 37 and 45°C with shaking at 160 rpm. The degradation rate of cypermethrin by J6 was determined using the methods described in [0010] and [0011], with cypermethrin working solution without bacteria at different temperatures as the positive control.

2) Effect of substrate concentration on degradation of cypermethrin by J6 The seed solution of strain J6 was inoculated at a 2% ( v/v ) inoculum into LB medium containing 20, 50, 80, 100, and 150 mg/L cypermethrin, respectively, and cultured at 37°C and 160 rpm for 3 days. The degradation rate of cypermethrin by J6 was determined using the methods described in [0010] and [0011].

3) Effect of metal ions on the degradation of cypermethrin by J6 The seed solution of strain J6 was inoculated at a 2% ( v/v ) inoculum into LB medium containing 50 mg/L cypermethrin and 0.01% ( w/v ) of different metal salts (CuSO ₄ , MnSO ₄ , ZnSO ₄ , CdCl ₂ , Pb (CH ₃ COO) ₂ ) and cultured at 37°C and 160 rpm for 3 days. The degradation rate of cypermethrin by J6 was determined using the methods described in [0010] and [0011].

The degradation characteristics of strain J6 on cypermethrin are as follows: the degradation of cypermethrin by strain J6 is not greatly affected by temperature, with a degradation rate of more than 40% in the range of 20-45℃, and the highest degradation rate at 37℃; in the concentration range of 20-150 mg/L, the degradation rate decreases with the increase of substrate concentration; Cd ²⁺ , Zn ²⁺ and Cu ²⁺ have different degrees of inhibition on the degradation of cypermethrin by strain J6, and Mn ²⁺ and Pb ²⁺ have no effect on the degradation effect. This research result provides a data reference for the application of strain J6 in the degradation of residual cypermethrin in the environment and food.

Example 2: Degradation effect of Bacillus subtilis J6 on other pyrethroid pesticides.

The strain J6 seed liquid was inoculated at a 2% inoculum into LB liquid culture medium containing 50 mg/L deltamethrin and cypermethrin, and cultured at 37°C with shaking for 3 days. The degradation rates of deltamethrin and cypermethrin were determined using the methods described in [0010] and [0011].

Strain J6 showed good degradation ability for both deltamethrin and cypermethrin, with degradation rates of 65.48% and 14.68%, respectively.

Example 3: Degradation effect of Bacillus subtilis J6 on 3-phenoxybenzoic acid, the main intermediate of pyrethroid pesticides.

The strain was inoculated into LB liquid medium containing 50 mg/L 3-phenoxybenzoic acid at a 2% inoculum, and cultured at 37℃ for 24 h. 1 mL of culture solution was taken at 0 h and 24 h, 4 mL of acetonitrile was added, and ultrasonic extraction was performed for 30 min. After mixing, 1.5 mL was transferred and centrifuged at 12000 r/min for 10 min. The supernatant was taken and filtered through a 0.22 μm organic filter membrane. The filtrate was collected to determine its degradation rate of 3-phenoxybenzoic acid. Chromatographic conditions: Gemini 100Å C18 column (4.6 mm×150 mm, 5 μm), mobile phase was acetonitrile: pH 2.5 phosphoric acid water = 55:45, flow rate was 0.7 mL/min, column temperature was 25℃, injection volume was 10 μL, detector was UV detector, and detection wavelength was 210 nm.

The results showed that the degradation rate of strain J6 for 50 mg/L 3-phenoxybenzoic acid was 76.12%.

Example 4: Degradation effect of Bacillus subtilis J6 on cypermethrin in milk.

Milk preparation: Dissolve 20 g skim milk powder in 1000 mL distilled water and sterilize at 115°C for 15 min. Add a certain volume of cypermethrin to make the final concentration of cypermethrin 50 mg/L.

The J6 strain seed solution was inoculated at a 2% ( v/v ) inoculum into milk containing 50 mg/L cypermethrin and cultured at 37°C and 160 rpm. Samples were taken at 0 h, 1 d, 2 d, and 3 d, and the cypermethrin degradation rate was determined using the method described in [0010] and [0011].

The degradation rates of cypermethrin by strain J6 were 26.73%, 36.41% and 52.56% in 1d, 2d and 3d, respectively, indicating that J6 had a good degradation effect on cypermethrin in milk.

The above examples are merely descriptions of the embodiments of the present invention, and are not intended to limit the scope of the present invention. For those skilled in the art, the above descriptions may be improved or modified, but all such improvements or modifications shall fall within the protection scope determined by the claims of the present invention. In view of the problems existing in the prior art, the present invention aims to provide a strain of Bacillus subtilis J6 that can effectively degrade cypermethrin and its application.

Claims (3)

1. A bacillus subtilis (Bacillus subtilis) J6 for effectively degrading cypermethrin is characterized by being preserved in China general microbiological culture Collection center, wherein the preservation place is North Star Xiyu No.1 institute of the Korean area of Beijing city, the preservation number is CGMCC No. 26129, and the preservation date is 2022, 12 and 27.

2. The use of bacillus subtilis (Bacillus subtilis) J6 according to claim 1, characterized in that: degradation of cypermethrin, fenvalerate, deltamethrin and 3-phenoxybenzoic acid.

3. The use of bacillus subtilis (Bacillus subtilis) J6 according to claim 1, characterized in that: is used for degrading residual cypermethrin in cow milk.