Prosteria aryabhattai BQS-1 and application thereof

Abstract

The invention discloses a Prussia adamsii BQS-1 (Bacillus amyloliquefaciens BQS-1) and an application thereof. Relates to the technical field of microorganisms. The strain provided by the invention can be used for degrading zearalenone in feed. In a zearalenone solution with the initial concentration of 2 mg / L, the degradation rate can reach 94.9 + / -0.5% after the strain acts for 1 d. The corn flour and the microbial inoculum are uniformly mixed, and the ZEN content is reduced by 61.8 + / -6.8% within 24 hours. If the pH value of the corn flour is adjusted to be neutral and 4 g / L glucose is added into a fermentation culture medium, the degradation effect can be further improved, and the degradation rate is increased to 75.8 + / -3.5%. Therefore, the prelisteria aryabhattai BQS-1 has a wide application prospect in the aspect of developing the ZEN degrading bacterial agent.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a strain of *Primatellorum argentea* BQS-1 and its applications. Background Technology

[0002] Zearalenone (ZEN) is a polyketide fungal toxin produced by Fusarium wilt. ZEN possesses estrogen-like activity and is highly toxic to the reproductive system of mammals. Long-term intake of ZEN through the food chain may lead to estrogen effect syndrome in humans, manifesting as abnormal breast development, infertility or miscarriage, and precocious puberty. Furthermore, ZEN exhibits various toxic effects, including genotoxicity, carcinogenicity, cytotoxicity, and immunotoxicity. ZEN primarily contaminates corn, wheat, and their products, with particularly high detection rates and severe contamination in feed ingredients and products, often exceeding safe levels, posing a significant threat to agricultural product safety and the economy. Moreover, agricultural products are often simultaneously affected by multiple toxin contaminations and pesticide residues, and their potential synergistic effects further increase the threat to human and animal health. Given ZEN's high toxicity, wide range of contamination, and high risk of human exposure, developing efficient and practical ZEN removal technologies is of urgent and significant practical importance for reducing agricultural product losses and mitigating ZEN hazards.

[0003] Highly efficient ZEN removal technologies from corn, wheat, and their products have broad application prospects and significant economic and social benefits. Currently, ZEN detoxification methods mainly include physical methods (such as high temperature, irradiation, and physical adsorption), chemical methods (such as ozone, hydrogen peroxide, and sodium carbonate), and biological methods (such as biodegradation, enzymatic degradation, and microbial cell structure adsorption). Physicochemical detoxification methods, such as irradiation, physical adsorbents, and ozone treatment, have poor specificity and may alter the nutritional structure of the product, causing nutrient loss and even secondary pollution. Biological detoxification methods have advantages such as high efficiency, high specificity, environmental friendliness, and no impact on the nutritional structure of agricultural products. They represent an important research direction for addressing ZEN contamination in agricultural products and mainly include biodegradation and bioadsorption. Biodegradation primarily utilizes microorganisms and their produced degrading enzymes to catalyze the decomposition of toxins.

[0004] Domestic and international scholars have screened several microbial strains capable of efficiently degrading ZEN from samples such as soil, animal feces, grains, and feed, but no strains of *Priscilla argentea* have been found. Priestia aryabhattai Systematic reports on the degradation of ZEN toxins in food and feed.

[0005] Therefore, whether or not a *Primatellorum argentea* BQS-1 and its application can be provided to overcome the above-mentioned technical deficiencies is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides *Primatechisella argentea* BQS-1 and its applications. The aim is to rapidly screen new, highly efficient ZEN-degrading strains from natural environmental samples such as soil, in order to solve the problem of ZEN contamination in agricultural products and protect human and animal health.

[0007] Preservation information: *Primatellis aureus* ( Priestia aryabhattai BQS-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC: NO.32710 and deposit date November 19, 2024.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A type of Primate bacterium ( Priestia aryabhattai BQS-1, with accession number CGMCC No.32710.

[0009] The present invention also provides a method for culturing the above-mentioned *Primatellorum aestivum*, wherein the bacteria are inoculated into a culture medium and cultured.

[0010] Preferred method: The strain is streaked on LB solid medium for isolation and activated culture at 37±1 ℃; a single colony is picked and inoculated into LB liquid medium and cultured at 37±1 ℃ and 200 r / min for 18–24 h.

[0011] The present invention also provides a microbial inoculum containing the above-mentioned *Primatellorum aspergillus* and / or fermentation products of *Primatellorum aspergillus*.

[0012] The present invention also provides the application of the above-mentioned *Priscilla argentea* or the above-mentioned microbial agents in the processing of agricultural products.

[0013] Preferred: Degraded zearalenone.

[0014] As can be seen from the above technical solution, compared with the prior art, this invention discloses a *Primatechisella esculenta* BQS-1 and its application, achieving the following technical effects: The strain provided by this invention can be used to degrade zearalenone in feed. In a zearalenone solution with an initial concentration of 2 mg / L, the degradation rate of this strain after 1 day of action can reach 94.9±0.5%. When corn flour and the bacterial agent are mixed evenly, the ZEN content decreases by 61.8±6.8% within 24 hours. If the pH of the corn flour is adjusted to neutral and 4 g / L glucose is added to the fermentation medium, the degradation effect can be further improved, and the degradation rate can be increased to 75.8±3.5%. Therefore, *Primatechisella esculenta* BQS-1 has broad application prospects in the development of ZEN-degrading bacterial agents. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The attached figure shows the colony morphology of the strain provided by this invention after 1 day of culture on LB solid medium.

[0017] Figure 2 The attached figure is a Gram-stained microscopic morphology image of the strain provided by the present invention.

[0018] Figure 3 The attached figure is a phylogenetic tree diagram of the 16S rDNA strain provided by the present invention.

[0019] Figure 4 The attached figure shows the effect of different initial cell concentrations of the strain provided by the present invention on the degradation efficiency of zearalenone in zearalenone.

[0020] Figure 5 The attached figure shows the degradation efficiency of the strain provided by this invention for different concentrations of zearalenone.

[0021] Figure 6 The attached figure shows the degradation efficiency of zearalenone by the strain provided by this invention under different pH conditions.

[0022] Figure 7 The attached figure shows the degradation efficiency of the microbial agent provided by this invention on zearalenone contaminating corn flour. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention discloses a strain of *Primatellorum argentea* BQS-1 and its applications.

[0025] Unless otherwise specified, the technical means not detailed in the following embodiments are conventional methods in the art, and the materials and reagents are all commercially available.

[0026] Example 1 Screening and identification of *Primatellorum auriculi* BQS-1 1. Screening and isolation of zearalenone-degrading bacteria Soil samples were collected from the Pinggu Ecological Park in Beijing at a depth of 5–15 cm, placed in resealable bags, and quickly brought back to the laboratory for use.

[0027] Weigh 2 g of soil sample into a sterile 50 mL Erlenmeyer flask, add 20 mL of sterile deionized water, and shake at 150 r / min for 1 h. Filter through sterile triple-layer gauze to remove soil particles and collect the filtrate. Dilute the filtrate to 10⁻¹⁰ using a 10-fold serial dilution method. -6 Take 10 respectively -4 10 -5 10 -6 200 µL of the dilution solution was spread onto LB agar plates, air-dried, and incubated at 37 °C. Single colonies of various morphologies were picked from the plates and inoculated into LB liquid medium containing ZEN, and incubated at 37 °C and 120 r / min for 24 h. Half of the bacterial culture was stored for later use, and the other half was used to extract residual ZEN and calculate the degradation rate. Strains with a degradation rate greater than 90% were screened for secondary validation, and the highly efficient degrading bacterium BQS-1 was finally obtained.

[0028] 2. Identification of strain BQS-1 (1) Morphological identification Strawberry strain BQS-1 was streaked on LB agar plates and incubated upside down at 37 °C for 24 h. Colony morphology was observed: colonies were round, slightly convex, white, opaque, and smooth. Figure 1 Gram staining followed by microscopic observation revealed it to be a Gram-positive bacillus. Figure 2 ).

[0029] (2) Molecular identification Genomic DNA was extracted from the bacterial strain using a bacterial genomic DNA extraction kit. Using gDNA as a template, 16S rDNA was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The PCR system (50 µL) included: 25 µL high-fidelity DNA polymerase premix, 22 µL deionized water, 1 µL each of forward and reverse primers, and 1 µL template. Reaction conditions: 98 ℃ pre-denaturation for 3 min; 35 cycles of 98 ℃ for 10 s, 55 ℃ for 10 s, and 72 ℃ for 10 s. The amplified products were verified by agarose gel electrophoresis and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA sequence was BLAST aligned to the NCBI database, and a phylogenetic tree was constructed using MEGA12 software. Figure 3The method employed a maximum likelihood tree and bootstrap testing, with 1000 replicate samplings, constructed using the Tamura-Nei model. Strain BQS-1 and *Primatellella sesquiterpenes* (…) Priestia aryabhattai B8W22 showed the highest homology (99.3%) and the closest evolutionary distance. Based on morphological characteristics and molecular identification results, this strain was identified as *Priscilla auriculata*. Priestia aryabhattai ). To preserve the patent through patent procedures.

[0030] Example 2 Degradation experiment of zearalenone by strain BQS-1 Strain BQS-1 was activated by streaking on LB agar plates and incubated at 37±1 ℃. Single colonies were picked and inoculated into LB liquid medium and incubated at 37±1 ℃ and 200 r / min for 18–24 h, with a seed culture concentration of 10. 6 -10 7 CFU / mL. Inoculate the seed culture into LB medium containing 2 mg / L ZEN at a 2% inoculum and incubate at 37±1 ℃ and 200 r / min for 24 h.

[0031] Residual ZEN was extracted with 2 times its volume of dichloromethane. The organic phase was dried under nitrogen at 35 °C, reconstituted with acetonitrile, and the ZEN concentration was determined by HPLC-FLD and LC-MS / MS. Using untreated LB medium as a control, the degradation rate was calculated according to the following formula: ZEN degradation rate = (ZEN content in control group - ZEN content in treatment group) / ZEN content in control group × 100%.

[0032] During the screening period, the degradation rate of this strain reached 94.9±0.5% after 1 day of treatment with an initial concentration of 2 mg / L zearalenone solution.

[0033] Example 3 Effect of initial strain concentration on zearalenone degradation The seed culture obtained in Example 2 was diluted 10 times and 100 times to obtain the original solution and 10 times, respectively. -1 and 10 -2 Three concentration gradients were used. Each culture was inoculated at a 2% inoculum into LB medium containing 2 mg / L ZEN and incubated at 37±1 ℃ and 200 r / min for 24 h. ZEN residues were extracted and detected according to the method in Example 2, and the degradation rate was calculated. Figure 4 ).

[0034] The results showed that even when the seed culture was diluted 100 times, it had no significant effect on the ZEN degradation efficiency, indicating that the strain has a strong proliferation ability and is suitable for development as a degradation agent.

[0035] Example 4 Degradation capacity of strain BQS-1 for different concentrations of ZEN The seed culture prepared in Example 2 was inoculated at a rate of 2% into LB medium containing 0.5, 1, 2, 5, and 50 mg / L ZEN, and cultured at 37±1 °C and 200 r / min for 24 h. ZEN residues were detected and degradation rates were calculated according to the method in Example 2. Figure 5 The results showed that the strain maintained a ZEN degradation rate of over 95% at concentrations of 0.5–5 mg / L, while the degradation rate decreased to 42.3 ± 1.3% at a high concentration of 50 mg / L. This indicates that the strain has a stable degradation effect when the ZEN concentration does not exceed 10 times the national standard limit (0.5 mg / L).

[0036] Example 5 Degradation capacity of strain BQS-1 at different pH levels LB medium containing 2 mg / L ZEN was prepared at pH 4, 5, 6, 7, and 8. Seed culture prepared in Example 2 was inoculated at a 2% inoculum rate and cultured at 37±1 °C and 200 r / min for 24 h. ZEN residues were detected and degradation rate was calculated according to the method in Example 2. Figure 6 The results showed that the degradation rate remained above 70% at pH 6–8, but dropped below 30% at pH below 5.

[0037] Example 6 Experiment on the application of microbial agents in the degradation of zearalenone in corn flour The bacterial strain was cultured and expanded according to the method described in Example 2 above. The bacterial cells were collected by centrifugation and resuspended in 1 / 10 volume of sterile water to concentrate the cells to a concentration 10 times that of the original fermentation broth, yielding the bacterial agent. The bacterial suspension was then thoroughly mixed with water-treated corn flour at a volume ratio of 1:9. Three experimental groups were set up: ① Corn flour + bacterial suspension; ② Corn flour + bacterial suspension adjusted to neutral pH; ③ Corn flour + bacterial suspension adjusted to neutral pH + 4 g / L glucose. The final ZEN concentration in each group of corn flour was 2 mg / kg. Treatment was carried out at 37±1 ℃ and 200 r / min for 24 h. 5 mL of sample was taken, 20 mL of acetonitrile was added, and the mixture was vortexed for 30 min. After adding salt and separating the layers, 2 mL of the organic phase was taken and dried under nitrogen at 65 ℃. The solution was then reconstituted with 1 mL of 30% acetonitrile aqueous solution, filtered through a 0.2 μm PTFE syringe filter, and the ZEN concentration was detected by LC-MS / MS. Corn flour contaminated with ZEN and treated with sterile water was used as a control. The degradation rate was calculated (…). Figure 7 ).

[0038] The results showed that under natural conditions, the degradation rate of ZEN by the bacterial agent was 61.8±6.8%; after adjusting the pH to neutral, it increased to 65.1±1.1%; and after supplementing with 4 g / L glucose, it could be further increased to 75.8±3.5%, reducing the ZEN concentration from 2 mg / kg to about 0.5 mg / kg, which is close to the national standard limit.

[0039] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0040] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of Priestia aryabhattai BQS-1, characterized in that, The accession number is CGMCC No. 32710.

2. The method for culturing *Primatellorum aestivum* according to claim 1, characterized in that, The bacteria are inoculated into a culture medium and cultured.

3. The cultivation method as described in claim 2, characterized in that, The strain was streaked on LB solid medium for isolation and activated at 37±1 ℃; single colonies were picked and inoculated into LB liquid medium and cultured at 37±1 ℃ and 200 r / min for 18–24 h.

4. A microbial inoculant, characterized in that, Fermentation products containing *Primatex auriculi* as described in claim 1 and / or *Primatex auriculi*.

5. The application of the *Priscilla argentea* of claim 1, or the microbial agent of claim 4, in the processing of agricultural products.

6. The application as described in claim 5, characterized in that, Degrades zearalenone.

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