Lysinibacillus capable of degrading alternaria alternata toxin and application of lysinibacillus capable of degrading alternaria alternata toxin

Abstract

The invention discloses lysinibacillus sp. For degrading alternaria alternata toxins and application of the lysinibacillus sp., and belongs to the technical field of microorganisms, the lysinibacillus sp. Is preserved in China Center for Type Microbiological Culture Collection, the preservation address is Wuhan University, Hubei Province, and the preservation number is CCTCC No.M20241816. The invention further discloses a preparation method of the lysinibacillus sp. According to the invention, the Lysinibacillus macroides capable of effectively reducing and degrading alternaria alternata toxin is obtained for the first time, and the quality of agricultural products (including aquatic products) is favorably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a lysinic bacillus capable of degrading Alternaria toxin and an application thereof. Background Art

[0002] Alternaria toxins are secondary metabolites produced by fungi in agricultural products, primarily from the genus Alternaria. Other molds, such as Pyricularia oryzae, Penicillium chrysogenum, and Penicillium aurantiogriseum, can also produce Alternaria toxins. Numerous studies have shown that Alternaria toxins exhibit carcinogenic, teratogenic, cytotoxic, genotoxic, and acute toxicity, with multiple adverse effects on human health. Alternaria toxins can contaminate fruits, vegetables, and field crops, depleting their nutritional and economic value and causing significant losses. Among them, Alternaria methyl ether (AME) and Alternaria ol (AOH) are found at high rates in various fruits and their products, while Alternaria tenuisic acid (TeA) contributes the most to dietary exposure, reaching 42.6%. TeA is also found at the highest levels in aquatic samples, averaging 58.91 μg / kg. Their chemical structures are as follows:

[0003]

[0004] Currently, the main methods for degrading mycotoxins include physical, chemical, and biological degradation. The most common physical method is heat treatment, which is highly effective but reduces the sensory and nutritional properties of food (Farooq et al., 2023). Non-thermal methods include pulsed electric field treatment, cold plasma, ultraviolet radiation, and high voltage (Mukhtar et al., 2022; Singh et al., 2022; Singh et al., 2014; Suman, 2021). Chemical methods include various fungicides with dicarboximide groups, phenylboronic acid and boric acid, and ozone (Martinko et al., 2022, Sujayasree et al., 2022). Although chemical techniques can reduce the content of mycotoxins, they may negatively affect food quality and nutritional value. The use of fungicides can also lead to the development of various plant diseases and cross-resistance (Carrascal-Hernández et al., 2022).

[0005] People tend to use more environmentally friendly alternatives to eliminate toxins. Currently, there are relatively few reports on the stability and degradation of Alternaria toxins, and even fewer on microbial degradation methods. Ge Na et al. (Optimization of the process for removing Alternaria toxin TeA from citrus juice using inactivated lactic acid bacteria, Food Science, 2017, Vol. 38, No. 14) used inactivated lactic acid bacteria cells as an adsorbent to investigate the adsorption conditions for TeA removal from citrus juice. Under these conditions, the inactivated lactic acid bacteria powder dosage was 1.20 g / 20 mL, the adsorption time was 11.43 h, the initial TeA concentration was 250 μg / L, and the orange juice pH was 3.15. Under these conditions, the TeA removal rate was 86.98%.

[0006] While various methods have been proposed to control or eliminate Alternaria toxins, each has its limitations. Therefore, exploring more efficient, convenient, and economical methods is crucial. Microbial degradation technology is still in its early stages of research, and the screening of highly efficient degrading bacteria is urgently needed for food safety control. Summary of the Invention

[0007] Based on the above reasons, the present invention proposes a lysinophilic Bacillus for degrading Alternaria toxins and its application, which has achieved very beneficial effects in reducing Alternaria toxins. Specifically, to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

[0008] One aspect of the present invention relates to a lysinibacillus macroides. The lysinibacillus macroides is deposited in the China Collection of Typical Microorganisms, with the deposit address being Wuhan University in Hubei Province, and the deposit number being CCTCC No. M20241816.

[0009] Another aspect of the present invention relates to the use of the above-mentioned lysinic Bacillus in degrading Alternaria toxins.

[0010] Another aspect of the present invention relates to the use of Bacillus lysinin in reducing the levels of Alternaria toxins in muscle and / or fish skin.

[0011] In a preferred embodiment of the present invention, the muscles and skin are the muscles and skin of turbot.

[0012] In a preferred embodiment of the present invention, the Alternaria toxin is selected from Alternaria solani (AOH) and / or Alternaria solani methyl ether (AME) and / or Alternaria tenuisic acid (TeA).

[0013] Beneficial effects

[0014] The beneficial effects of the present invention are: the present invention obtains for the first time a Lysinibacillus macroides capable of effectively reducing and degrading Alternaria toxins, which helps to improve the quality of agricultural products (including aquatic products). DETAILED DESCRIPTION

[0015] To further understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0016] Unless otherwise specified, all reagents involved in the embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0017] Example 1:

[0018] 1. Isolation and preservation of Lysinibacillus macroides:

[0019] The inventors of this application found that the Alternaria toxin in the intestines of some turbot in the Yantai Laizhou breeding test field had never been detected. Through further research, a strain of Lysinibacillus macroides was isolated from the intestines of the above-mentioned crucian carp and named LY015. The microbial strain was deposited in the China Center for Type Microorganisms Collection, with the preservation address being Wuhan University, Hubei Province. The preservation number is: CCTCCNo.M20241816, and the preservation date is August 19, 2024.

[0020] 2. Identification of bacterial species

[0021] Genomic DNA was extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit. Using the genomic DNA of the test strain as a template, the target fragment was amplified using the universal primers 27F / 1492R for the bacterial 16S rRNA gene. The PCR product was analyzed by 1% agarose gel electrophoresis, and the PCR stock solution was sent to Biomed Biotechnology Co., Ltd. for sequencing. 16S rRNA gene sequence analysis confirmed that the lysinibacillus strain LY015 was identified as Lysinibacillus macroides using MK757660.1 (NCBI) as the standard. The 16S rDNA gene sequence is shown in SEQ ID NO. 1.

[0022]

[0023] 3. Quantitative analysis of degradation activity

[0024] Determination of the degradation of Alternaria solani (AOH) / Alternaria solani methyl ether (AME) / TeA toxin

[0025] The degradation system consisted of 500 μl of enrichment medium (1 L containing 10 g peptone, 1 g NaCl, and 5 g glucose) and 5 μl of a Lysinibacillus macroides LY015 suspension. The cells were incubated overnight at 37°C, followed by the addition of 5 μl of a 100 ppm solution of alternator hydroxylamine (AOH), alternator methyl ether (AME), or alternator acetic acid (TeA). The mixture was shaken at 220 rpm and 37°C for 24 hours, acidified with 1 M hydrochloric acid, and terminated with 1 ml of ethyl acetate. After vortex extraction and centrifugation at 13,400 g for 5 minutes, 800 μl of the organic phase was collected, dried under nitrogen, redissolved in 800 μl of methanol, filtered through a 0.22 μm filter, and analyzed by high-pressure liquid chromatography.

[0026] UV-HPLC (Thermo Fisher, Waltham, MA, USA) was used for detection and separation on a C18 reverse-phase column (4.6×250 mm, 5 μm) (Thermo Fisher). Mobile phase A was 0.2% formic acid in water, and mobile phase B was acetonitrile. The inlet ratio was phase A:phase B = 30:70, and the flow rate was 0.5 ml / min. Throughout the process, the column pressure remained stable at around 36.8 bar. The detection wavelengths for AOH and AME were both 256 nm, with retention times of 7.02 min and 12.03 min, respectively; the detection wavelength for TeA was 280 nm, with a retention time of 5.18 min. The test results showed that 96.08% of AOH was degraded, 75.10% of AME was degraded, and 93.12% of TeA was degraded.

[0027] 4. Improvement of the toxicity of turbot induced by toxins by Bacillus lysininii LY015

[0028] For the toxin infection experiment, a basic feed for turbot juveniles was prepared using fish meal, fish oil, and soybean meal as the primary ingredients, with a crude protein level of approximately 42% and a crude fat level of approximately 8%. AOH, AME, and TeA toxins were added to the basic feed at 2.5 mg / kg. Nutrient composition of the feed was determined using AOAC methods. 150 healthy turbot juveniles were randomly divided into three groups for an eight-week culture experiment. The actual toxin levels in the turbot skin and muscle were determined using liquid chromatography-tandem mass spectrometry.

[0029] Experimental group treatment

[0030] Treatment group 1 basal feed (BD, Control)

[0031] Treatment group 2 basic feed (BD) + toxin

[0032] Treatment group 3 basic feed (BD) + toxin + lysinophil LY015 (10 8 CFU / kg)

[0033] Turbot skin and muscle were separated and weighed separately. The samples were then extracted with acetonitrile (1.5% formic acid) and salted out with anhydrous MgSO4 and NaCl. The toxin content in the skin and muscle of turbot was determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) using an ACQUITY UPLC BEH C18 column with a gradient elution of acetonitrile and 0.1% formic acid in water. Electrospray ionization (ESI+) multi-reaction mode monitoring was used. The results are shown in the table below.

[0034] Table 1: Results of toxin detection in turbot skin and muscle

[0035]

[0036] The above experimental results show that Bacillus lysinic acid LY015 can reduce the deposition of alternator ol (AOH) / alternator methyl ether (AME) / tenocysteine ​​acid (TeA) in the muscle and skin tissues of turbot.

[0037] The preferred embodiments of the present invention are described above, but they are not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A lysinibacillus, characterized in that: The Lysinibacillus macroides is deposited in the China Center for Type Microorganism Collection, Wuhan University, Hubei Province, with a deposit number of CCTCC No. M20241816.

2. Use of the lysinibacillus according to claim 1 in degrading Alternaria toxin.

3. The use according to claim 2, characterized in that: The Alternaria toxin is selected from the group consisting of Alternaria solani (AOH) and / or Alternaria solani methyl ether (AME) and / or Tetrasodium tenuifolinic acid (TeA).

4. Use of the lysinibacillus according to claim 1 in reducing the levels of Alternaria toxins in muscle and / or fish skin.

5. The use according to claim 4, characterized in that: The muscles and fish skin are those of turbot.

6. The use according to claim 4 or 5, characterized in that: The Alternaria toxin is selected from the group consisting of Alternaria solani (AOH) and / or Alternaria solani methyl ether (AME) and / or Tetrasodium tenuifolinic acid (TeA).

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