Application of Ougan seed oil in remediation of soil contaminated with antibiotic resistance genes

Abstract

This invention relates to the application of Ougan mandarin seed oil in the remediation of soil contaminated with antibiotic resistance genes. The Ougan mandarin seed oil is obtained using common physical pressing methods. Depending on the actual soil conditions, an appropriate proportion of Ougan mandarin seed oil is directly added to the soil, and cultivation in the dark significantly reduces the abundance of antibiotic resistance genes in the soil. The seed oil used in this invention has limited market application, turning waste into treasure, is low-cost, safe, and non-toxic. The application method is simple to operate and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of environmental engineering technology, specifically to the application of Ougan seed oil in the remediation of soil contaminated with antibiotic resistance genes. Background Technology

[0002] Soil microbial communities are incredibly rich, containing a large number of antibiotic resistance genes with diverse structures and functions. These genes are transferred horizontally among soil microorganisms, accelerating their spread and allowing microorganisms to rapidly adapt to antibiotic stress. These genes can be transmitted to humans through the food chain or direct contact, posing a risk to human health. The spread of antibiotic resistance genes also presents greater challenges in treating infections, as commonly used antibiotics may not effectively inhibit the growth of antibiotic-resistant strains. Currently, methods such as the rational use of antibiotics, bioremediation, phytoremediation, heat treatment, biochar application, and gene editing technologies can effectively control and reduce antibiotic resistance gene contamination in soil.

[0003] Okangan seed oil is a plant oil extracted from the seeds of the Okangan fruit (a type of citrus). It is rich in various nutrients, including unsaturated fatty acids (such as linoleic acid and linolenic acid), vitamin E, and flavonoids. Currently, the production volume of Okangan seed oil on the market is relatively small, and there are no reports on its use for controlling antibiotic resistance gene (ARG) pollution. Whether it can reduce ARG pollution in soil and ensure positive effects is not addressed in existing technologies. Summary of the Invention

[0004] The purpose of this invention is to provide an application of Ougan seed oil in remediating soil contaminated with antibiotic resistance genes, in order to solve the problem of the proliferation of antibiotic resistance genes in existing soils.

[0005] This invention provides the application of Oogan seed oil in the remediation of soil contaminated with antibiotic resistance genes;

[0006] Preferably, the antibiotic resistance genes include chloramphenicol, multidrugs, sulfonamides, tetracyclines, aminoglycosides, β-lactams, and MLS.

[0007] Preferably, the method for extracting Oolong mandarin seed oil includes the following steps:

[0008] (1) Soak fresh Ougan oranges in 95℃ hot water for 30-60 seconds, peel off the peel and separate the segments while hot, pick out the Ougan orange seeds and rinse them several times with clean water, dry them at (65±5)℃ and then put them into a pulverizer for pulverization, and dry them through a 40-mesh sieve.

[0009] (2) Wrap the Ougan seed powder with filter paper and place it in a Soxhlet extraction tube. Add n-hexane to the extraction flask and extract at 80°C for 8-12 hours. Then, use a rotary evaporator to evaporate the extracted sample solution under vacuum at 40°C to obtain Ougan seed oil.

[0010] The method of using Oogan seed oil to remediate soil contaminated with antibiotic resistance genes is as follows:

[0011] Add Ougan seed oil to the soil, stir well, adjust the soil moisture content to 50-60% of the maximum water holding capacity, and cultivate in the dark for 30-60 days, replenishing water regularly.

[0012] Preferably, the amount of Ougan seed oil added is 40-60 ml / kg of soil dry weight.

[0013] Preferably, the culture conditions are light-protected culture.

[0014] Preferably, the culture time is 30 days.

[0015] Antibiotic resistance gene abundance refers to the copy number of various antibiotic resistance genes in the genome. The higher the abundance, the greater the number of that gene. Abundance is divided into absolute abundance and relative abundance, where relative abundance describes the percentage of a single gene out of the total number of genes.

[0016] The data measured in this invention were obtained by screening representative antibiotic resistance genes based on gene abundance. Among them, chloramphenicol antibiotic resistance genes include catl, fexB, cmlA9, etc.; multidrug antibiotic resistance genes include AcrF, sdrM, MexK, etc.; sulfonamide antibiotic resistance genes include sul1, sul2, etc.; tetracycline antibiotic resistance genes include tet(36), tet(K), tet(X), etc.; aminoglycoside antibiotic resistance genes include AAC(3)-IId, aadA8, apmA, etc.; β-lactam antibiotic resistance genes include OXA-681, BJP-1, IDC-1, etc.; and MLS antibiotic resistance genes include oleB, carA, LnuH, etc.

[0017] The combined effects of this invention include: this invention broadens the application field of Ougan seed oil, and for the first time applies it to the prevention and control of antibiotic resistance genes;

[0018] Using Ougan seed oil to reduce antibiotic resistance genes is a simple method. The application method and dosage can be freely adjusted according to the actual situation. It is easy to apply, saves time, and reduces workload. Attached Figure Description

[0019] Figure 1 The effect of Ougan seed oil on the total abundance of antibiotic resistance genes in soil;

[0020] Figure 2 The reduction rate of antibiotic resistance genes in soil by Ougan seed oil. Detailed Implementation

[0021] Example 1

[0022] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments below are the preferred embodiments of this patent. However, this patent is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the scope of this patent technology. Unless otherwise specified, the following components are calculated by weight.

[0023] The soil used in this experiment was collected from Aksu Prefecture, Xinjiang Uygur Autonomous Region. Topsoil samples (0-20 cm) were collected using a five-point sampling method. After being transported back to the laboratory, the soil was air-dried to remove impurities such as weeds, stones, and dead leaves, and then sieved through a 2 mm sieve for later use. The physicochemical properties of the soil were determined by the Zhejiang Academy of Agricultural Sciences and are shown in Table S1.

[0024] Table S1 Soil physicochemical properties

[0025]

[0026] It should be noted that metagenomic sequencing counts a wide variety of antibiotic resistance genes in the soil. The data measured in this embodiment were obtained by screening representative antibiotic resistance gene types based on gene abundance. Among them, chloramphenicol antibiotic resistance genes include catl, fexB, cmlA9, etc.; multidrug antibiotic resistance genes include AcrF, sdrM, MexK, etc.; sulfonamide antibiotic resistance genes include sul1, sul2, etc.; tetracycline antibiotic resistance genes include tet(36), tet(K), tet(X), etc.; aminoglycoside antibiotic resistance genes include AAC(3)-IId, aadA8, apmA, etc.; β-lactam antibiotic resistance genes include OXA-681, BJP-1, IDC-1, etc.; and MLS antibiotic resistance genes include oleB, carA, LnuH, etc.

[0027] This invention verifies and evaluates the application effect of Oogan seed oil in remediating soil contaminated with antibiotic resistance genes through laboratory simulation experiments. The specific operation steps are as follows:

[0028] S1. Soil pretreatment:

[0029] Weigh 3000g of soil (dry weight), add 6% chicken manure fertilizer, adjust the soil moisture content to 60% of maximum water holding capacity, mix well, and pass through a 2mm sieve. Place the mixed soil in a black plastic bag to reduce moisture evaporation, and incubate at room temperature for 3 days to ensure that multiple antibiotic resistance genes are incorporated into the soil.

[0030] S2. Preparation of Oolong mandarin seed oil:

[0031] Soak fresh Ougan oranges in 95℃ hot water for 60 seconds, peel off the peel and separate the segments while hot, pick out the Ougan orange seeds, rinse several times with clean water, dry at 65℃, then put them into a grinder for grinding, and dry through a 40-mesh sieve.

[0032] Wrap the Ougan seed powder in filter paper and place it in a Soxhlet extraction tube. Add n-hexane to the extraction flask and extract at 80°C for 10 hours. The extracted sample solution is then evaporated under vacuum at 40°C using a rotary evaporator to obtain Ougan seed oil, which is then stored in a refrigerator for later use.

[0033] S3, Microcosm Experiment:

[0034] Weigh 100g (dry weight) of soil into a white porcelain dish, add 6ml of Okan tangerine seed oil, mix well, and adjust the soil moisture content to 60% of its maximum water holding capacity using deionized water. Transfer the soil to a beaker, cover the mouth of the beaker with aluminum foil, and poke small holes in it. Incubate the beaker in the dark for 30 days, weighing it every 3 days, adding deionized water, and stirring well to ensure the moisture content is 60% of the maximum water holding capacity. A control treatment without Okan tangerine seed oil was also included. Each group had three replicates.

[0035] S4. Soil DNA extraction and metagenomic sequencing:

[0036] Weigh 0.5g of soil sample and use FastDNA. TMTotal DNA was extracted from soil using the Spin Kit for Soil, and metagenomic sequencing was performed on an Illumina Novaseq 6000 sequencing platform using the PE150 sequencing strategy. The raw sequencing data was quality controlled using FASTP under default settings to remove low-quality sequences and obscure bases, resulting in clean reads. Subsequently, the clean reads of each sample were aligned to the Comprehensive Antibiotic Resistance Database (CARD) using DIAMOND (blastx,--evalue1e-5,--id 90,--max-target-seqs 1). Sequences with a copula length less than 25 amino acids were removed from the alignment results. The types, subtypes, and resistance mechanisms of ARGs were classified. ARG abundance units were converted to copy number / 16S rRNA gene copy number for standardization.

[0037] The formula for calculating the relative abundance of ARGs (copy number / 16S rRNA gene copy number) is as follows:

[0038]

[0039] R iARG-likereads : The sum of the lengths of all sequences aligned to the i-th ARG reference sequence in the ARG database; L iARGreferencesequence : The nucleotide length of the i-th reference sequence in the ARG database; R j16S-likereads The sum of the lengths of all sequences aligned to the j-th sequence in the 16S rRNA gene database; L j16Sreferencesequence : The nucleotide length of the j-th 16S rRNA reference sequence in the 16S rRNA gene database.

[0040] The effect of Ougan seed oil on the total abundance of antibiotic resistance genes in soil, such as Figure 1 As shown. By Figure 1 It was found that after 30 days of treatment, the total abundance of antibiotic resistance genes in the soil of the control treatment and the Ougan seed oil treatment were 0.37-0.45 and 0.29-0.31 copies / 16S rRNA gene copies, respectively. Ougan seed oil significantly reduced the total abundance of antibiotic resistance genes in the soil (p<0.05). Compared with the control treatment, the total abundance of antibiotic resistance genes in the Ougan seed oil treatment decreased by 24.08%-29.29%.

[0041] In-depth analysis of soil samples yielded the following results: Figure 2As shown, Ougan seed oil can significantly reduce the number of resistance genes for chloramphenicol, multidrug, sulfonamide, and tetracycline antibiotics in the soil (p<0.05), and can also reduce the abundance of resistance genes for aminoglycoside, MLS, and β-lactam antibiotics (p>0.05). After 30 days of soil treatment with Ougan seed oil, the reduction rates of resistance genes for chloramphenicol (CAT1, FexB), multidrug (AcrF, sdrM), tetracycline (Tet(36), Tet(K), aminoglycoside (AAC(3)-IId, aadA8), β-lactam (OXA-681, BJP-1), and MLS (oleB, carA) were all 100%.

[0042] The specific embodiments are merely explanations of this application and are not intended to limit it. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they are within the scope of the claims of this application.

Claims

1. Use of Citrus aurantium L. seed oil for remediation of soils contaminated with antibiotic resistance genes, characterized in that, Add the Wukuan seed oil to the soil, stir well, adjust the soil water content to 50-60% of the maximum water holding capacity, and place in the dark, and supplement water regularly; The addition amount of the Wukuan seed oil is 40-60 ml / kg of dry soil; The culture condition is light avoidance; The culture time is 30 days; The antibiotic resistance genes include chloramphenicol, tetracycline, aminoglycoside, and MLS.

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