A Lysine-containing Bacillus that degrades Alternaria toxins and its application

Abstract

This invention discloses a lysinibacillus macroides bacterium that degrades Alternaria alternata toxins and its applications, belonging to the field of microbial technology. The lysinibacillus macroides is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Hubei Province, with accession number CCTCC No. M20241816. This invention is the first to obtain a lysinibacillus macroides bacterium that can effectively reduce and degrade Alternaria alternata toxins, which helps improve the quality of agricultural products (including aquatic products).

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a lysine-containing Bacillus that degrades Alternaria toxins and its applications. Background Technology

[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. Extensive research indicates that alternaria toxins possess carcinogenic, teratogenic, cytotoxic, genotoxic, and acute toxicity, posing various adverse effects on human health. Alternaria toxins can contaminate fruits, vegetables, and field crops, rendering them devoid of nutritional and economic value, causing significant losses. Alternaria methyl ether (AME) and alternaria hydroxyl alcohol (AOH) have high detection rates in various fruits and their products, while *TeA* (te-acid) contributes the most to dietary exposure, reaching 42.6%. Furthermore, *TeA* has the highest content in aquatic samples, averaging 58.91 μg / kg. Their chemical structures are as follows:

[0003]

[0004] Currently, methods for degrading toxins mainly include physical, chemical, and biological degradation methods. The most common physical method is heat treatment, which is highly efficient 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 pressure (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 have adverse effects on food quality and nutritional value; the use of fungicides can cause various plant diseases and enhance cross-resistance (Carrascal-Hernández et al., 2022).

[0005] People tend to use more environmentally friendly alternatives to eliminate toxins. Currently, there are relatively few studies on the stability and degradation of Alternaria alternifolia toxins, both domestically and internationally, and even fewer reports on the degradation methods using microorganisms. Ge Na et al. (Optimization of the process for removing Alternaria alternifolia 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, mainly studying the process conditions for its adsorption and removal of TeA from citrus juice. Under the conditions of 1.20 g / 20 mL inactivated lactic acid bacteria powder, 11.43 h adsorption time, 250 μg / L initial TeA concentration, and 3.15 pH of orange juice, the TeA removal rate was 86.98%.

[0006] While various methods for controlling or eliminating Alternaria toxins have been proposed, each method 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, this invention proposes a lysine-containing Bacillus that degrades Alternaria toxin and its application, which has achieved very beneficial effects in reducing Alternaria toxin. Specifically, to achieve the objectives of this invention, the following technical solution is proposed:

[0008] One aspect of this invention relates to a Lysinibacillus macroides, which is deposited at the China Center for Type Microbiology, located at Wuhan University, Hubei Province, with accession number CCTCC No. M20241816.

[0009] Another aspect of the present invention relates to the use of the aforementioned lysine-containing Bacillus in the degradation of Alternaria toxins.

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

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

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

[0013] Beneficial effects

[0014] The beneficial effects of this invention are: this invention is the first to obtain a lysinibacillus macroides that can effectively reduce and degrade Alternaria toxins, which helps to improve the quality of agricultural products (including aquatic products). Detailed Implementation

[0015] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise specified, all reagents involved in the embodiments of this 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 discovered that Alternaria toxin was never detected in the intestines of some turbot from the Yantai Laizhou Aquaculture Experimental Farm. Through further research, a strain of Lysinibacillus macroides was isolated from the intestines of the aforementioned crucian carp and named LY015. This microbial strain is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Hubei Province, with accession number CCTCCNo.M20241816 and deposit date of August 19, 2024.

[0020] 2. Strain identification

[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 products were detected by 1% agarose gel electrophoresis, and the PCR stock solution was sent to Bomeide Biotechnology Co., Ltd. for sequencing. 16S rRNA gene sequence analysis, after sequence comparison and identification, confirmed that strain LY015 is *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 Alternaria alternifolia (AOH) / Alternaria alternifolia methyl ether (AME) / Alternaria alternifolia ketoxin (TeA) degradation

[0025] The degradation system consisted of 500 μl of enrichment medium (1 L containing 10 g peptone, 1 g NaCl, and 5 g glucose), with 5 μl of Lysinibacillus macroides LY015 suspension added and cultured overnight at 37°C. Then, 5 μl of 100 ppm Alternaria alternatasol propionate (AOH), Alternaria alternatasol propionate methyl ether (AME), or Alternaria alternatasol propionate ketone acid (TeA) solution were added. After shaking at 220 rpm and 37°C for 24 h, the mixture was acidified with 1 M hydrochloric acid, and the reaction was terminated by adding 1 mL of ethyl acetate. Following vortex extraction, the mixture was centrifuged at 13400 g for 5 min, and 800 μl of the organic phase was collected, dried under nitrogen, and redissolved in 800 μl of methanol. The solution was filtered through a 0.22 μm filter and then analyzed by high-performance liquid chromatography (HPLC).

[0026] UV-HPLC (Thermo Fisher, Waltham, MA, USA) was used for detection and separation on a C18 reversed-phase column (4.6 × 250 mm, 5 μm) (Thermo Fisher). Mobile phase A was 0.2% formic acid aqueous solution, and mobile phase B was acetonitrile. The injection ratio was A phase:B phase = 30:70, and the flow rate was 0.5 mL / min. The column pressure remained stable at approximately 36.8 bar throughout the process. The detection wavelength for AOH and AME was 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 results showed that 96.08% of AOH, 75.10% of AME, and 93.12% of TeA were degraded.

[0027] 4. The effect of Bacillus lysine-containing bacteria LY015 on mitigating the toxicity of turbot caused by toxins.

[0028] In the toxin contamination experiment, a basal diet for juvenile turbot was formulated using fishmeal, fish oil, and soybean meal as the main ingredients, with a crude protein level of approximately 42% and a crude fat level of approximately 8%. Experimental diets were prepared by adding 2.5 mg / kg of AOH, AME, and TeA toxins to the basal diet, and the nutrient composition of the diets was determined according to the AOAC method. One hundred and fifty healthy juvenile turbot were randomly divided into three groups and cultured for eight weeks. The actual toxin content in the skin and muscle of the turbot was determined using liquid chromatography-tandem mass spectrometry.

[0029] Experimental grouping and treatment

[0030] Treatment Group 1 Basic Feed (BD, Control)

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

[0032] Treatment group 3: Basic feed (BD) + toxin + Lysine-containing Bacillus LY015 (10) 8 CFU / kg

[0033] The skin and muscle of turbot were separated, weighed, and then extracted with acetonitrile (1.5% formic acid). Precipitation was performed using anhydrous MgSO4 and NaCl. An ACQUITY UPLC BEH C18 column was used for separation, with gradient elution using acetonitrile and 0.1% formic acid aqueous solution. Electrospray ionization (ESI+) in positive ion (ESI+) multiple reaction mode was used for monitoring. The actual toxin content in the turbot skin and muscle was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The experimental 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 indicate that Bacillus lysine LY015 can reduce the deposition of Alternaria alternata (AOH) / Alternaria alternata methyl ether (AME) / Alternaria alternata ketone acid (TeA) in the muscle and skin tissue of turbot.

[0037] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A Bacillus lysinoformis (Bacillus lysinoformis) characterized by: Lysinibacillus macroides The Lysinibacillus macroides is preserved in China Center for Type Culture Collection, Wuhan University, Hubei Province, and the preservation number is CCTCC No. M20241816. ​ 2. The Lysinibacillus macroides of claim 1 is used in degrading Alternaria toxins, and the Alternaria toxins are selected from the group consisting of alternariol (AOH) and / or alternariol monomethyl ether (AME) and / or tenuazonic acid (TeA).

3. The Lysinibacillus macroides of claim 1 is used in reducing Alternaria toxins in muscle and / or fish skin, and the Alternaria toxins are selected from the group consisting of alternariol (AOH) and / or alternariol monomethyl ether (AME) and / or tenuazonic acid (TeA).

4. Use according to claim 3, characterized in that: The muscle and fish skin are muscle and fish skin of Scophthalmus maximus.

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