Stenotrophomonas maltophilia and its application

Abstract

The present invention discloses the use of Stenotrophomonas maltophilia Sarfraz and Stenotrophomonas maltophilia and / or bacterial agents and / or mycotoxin degrading agents in inhibiting fungal growth and / or degrading mycotoxin, wherein the bacterial agent contains Stenotrophomonas maltophilia, and the active ingredients of the mycotoxin degrading agent include one or more of the Stenotrophomonas maltophilia, the fermentation broth of the Stenotrophomonas maltophilia, or the metabolites of the Stenotrophomonas maltophilia. The Stenotrophomonas maltophilia Sarfraz of the present invention can effectively inhibit fungal growth and degrade mycotoxin, and has a good application prospect in developing microbial preparations for inhibiting the growth of mycotoxin-producing bacteria.

Description

Technical Field

[0001] The present invention relates to the fields of microbiology and biodegradation, and more particularly to Stenotrophomonas maltophilia and its application. Background Art

[0002] Fungal contamination can cause food and feed to become moldy and deteriorate, significantly reduce nutritional quality and processing quality, and cause huge economic losses. In addition, the secondary metabolites mycotoxins produced by fungi are highly carcinogenic, teratogenic, and mutagenic. Therefore, screening and developing safe and non-toxic microorganisms that can effectively inhibit fungal growth and degrade mycotoxins, and strengthening fungal prevention and control research have become urgent needs to ensure my country's food security and food safety.

[0003] Deoxynivalenol (DON) belongs to the B-group trichothecenes, mainly produced by Fusarium graminearum and Fusarium luteum, and is one of the most widely distributed mycotoxins in the world. DON toxins have multiple toxic effects such as acute toxicity, chronic toxicity, cytotoxicity, and immunotoxicity, and have attracted widespread attention from researchers around the world. Therefore, preventing and reducing the entry of DON toxins into the food chain of humans and animals is the main research content to prevent its harm. Among them, using reasonable food processing to reduce the content of DON toxins plays an important role in reducing the health risks of DON toxins to the human body. However, due to the stable chemical properties and high temperature resistance of DON toxins, that is, DON is quite stable between 170℃ and 350℃, will not degrade when heated at 170℃ for 30min, and is still very stable under boiling, baking, and even steam conditions at 135℃, a large amount of DON toxins will still remain after the processing of contaminated food raw materials, and ordinary food processing such as bread, cakes and beer is not effective in removing DON toxins.

[0004] Ochratoxins are a group of toxic metabolites with similar structures produced by toxin-producing strains such as Aspergillus and Penicillium, which are widely present in various foods, feeds and other agricultural and sideline products. Ochratoxins include 7 compounds with similar chemical structures, among which ochratoxin A (OTA) is the most widely distributed in nature, the most toxic, and has the greatest impact on humans, animals and plants. OTA has multiple toxicities such as renal toxicity, hepatotoxicity, immunotoxicity, as well as teratogenicity, carcinogenicity and mutagenicity, and has great potential harm to animal and human health. Therefore, countries around the world attach importance to the detection and control of OTA and have formulated relevant limit standards to ensure food safety and eliminate technical barriers in international trade. Therefore, conducting research on the inhibition and removal technology of ochratoxins is of great significance for protecting human and animal health and ensuring national food safety and grain security.

[0005] Zearalenone (ZEN) is an estrogenic fungal toxin produced by Fusarium during secondary metabolism. ZEN contaminates a wide range of grains, such as wheat, barley, corn, oats, sorghum, rye, millet and related products of these grains. It is one of the most widely contaminated Fusarium toxins in the world. ZEN is toxic to both animals and humans, mainly affecting the reproductive function of animals, reducing the embryo survival rate of pregnant animals and the birth weight of newborns, and causing reproductive disorders in animals. Eating food contaminated by it can cause diseases such as liver cancer, testicular cancer, esophageal cancer and precocious puberty. In addition, ZEN also has immunotoxicity, hepatotoxicity, and genotoxicity, and has a certain impact on tumor occurrence.

[0006] Aflatoxins (AFs) are a class of toxic secondary metabolites mainly produced by fungi such as Aspergillus flavus and A. parasiticus. They have carcinogenic, teratogenic and mutagenic effects. Only a dose of 0.294 mg / kg can cause acute poisoning and death in sensitive animals. Its main target is the liver. According to epidemiological surveys, up to 28% of primary hepatocellular carcinomas worldwide are caused by AFs. In addition, they can also cause acute lesions in the kidneys and adrenal glands. AFs are compounds with similar molecular structures and physical and chemical properties. They all contain a bifuran ring and a oxazinone (also called coumarin) in their structures. They are the most stable fungal toxins in physical and chemical properties discovered so far. At present, more than 20 types have been isolated and identified, mainly those that emit blue light (Blue) under ultraviolet light are group B (aflatoxin B1 and B2) and those that emit green light (Green) are group G (aflatoxin G1 and G2). Mainly divided into AFB l (C 17 H 12 O6), AFB2(C 17 H 14 O6), AFG1(C 17 H 12 O7) and AFG2(C 17 H 14 O7).

[0007] Mycotoxin detoxification methods can be divided into three categories: physical, chemical and biological, including ammoniation, alkali, high temperature, irradiation and ultrafiltration-diafiltration. These methods have the disadvantages of unstable effect, large loss of nutrients, complex degradation products, difficult to determine the toxicity of degradation products, and difficulty in large-scale production. In addition, there is adsorption, which adsorbs nutrients while adsorbing toxins. Among them, biological methods have become a hot topic in the research of mycotoxin degradation and detoxification in recent years because of their high efficiency, strong specificity and no pollution to food, feed and the environment. However, most of the microbial detoxification strains reported so far only have an effect on one or two mycotoxins in practical applications, and their broad spectrum is not strong, and their practical significance and application value are not great. Therefore, it is necessary to find strains that can degrade mycotoxins with a broad spectrum and high efficiency, and truly apply them to the fields of agricultural production and food safety. Biological control is mainly microbial antagonism of fungi, which is a relatively green, environmentally friendly and effective way. Summary of the invention

[0008] The purpose of the present invention is to provide Stenotrophomonas maltophilia and its application. Stenotrophomonas maltophilia Sarfraz has a broad-spectrum antibacterial activity and the function of degrading fungal toxins, and has a good application prospect in the development of microbial preparations for inhibiting the growth of fungal toxin-producing bacteria.

[0009] In order to achieve these objectives and other advantages of the present invention, a Stenotrophomonas maltophilia Sarfraz is provided. The Stenotrophomonas maltophilia strain has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 20, 2021, with a deposit number of CGMCC No. 22572.

[0010] The invention relates to the use of Stenotrophomonas maltophilia and / or a bacterial agent and / or a mycotoxin degrading agent in inhibiting fungal growth and / or degrading mycotoxins. The bacterial agent contains Stenotrophomonas maltophilia, and the active ingredient of the mycotoxin degrading agent includes one or more of the Stenotrophomonas maltophilia, the fermentation broth of the Stenotrophomonas maltophilia, or the metabolites of the Stenotrophomonas maltophilia.

[0011] Preferably, in the application of the Stenotrophomonas maltophilia, the Stenotrophomonas maltophilia is used to inhibit the growth of Fusarium oxysporum, Fusarium graminearum, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae and Aspergillus ochraceus.

[0012] Preferably, in the application of the Stenotrophomonas maltophilia, the mycotoxins are deoxynivalenol, ochratoxin A, zearalenone, group B aflatoxins and group G aflatoxins.

[0013] Preferably, in the application of the Stenotrophomonas maltophilia, the group B aflatoxins are aflatoxins B1 and B2, and the group G aflatoxins are aflatoxins G1 and G2.

[0014] A bacterial agent containing Stenotrophomonas maltophilia.

[0015] A mycotoxin degrading agent, wherein the active ingredient of the mycotoxin degrading agent includes one or more of the Stenotrophomonas maltophilia, the fermentation broth of the Stenotrophomonas maltophilia, or the metabolites of the Stenotrophomonas maltophilia.

[0016] A method for inhibiting fungal growth, comprising using Stenotrophomonas maltophilia or a bacterial agent to inhibit fungi, wherein the bacterial agent contains Stenotrophomonas maltophilia.

[0017] A method for degrading fungal toxins, comprising: using one or more of Stenotrophomonas maltophilia, a bacterial agent or a fungal toxin degrading agent to perform biodegradation treatment on the fungal toxins, wherein the bacterial agent contains Stenotrophomonas maltophilia, and the active ingredient of the fungal toxin degrading agent includes one or more of the Stenotrophomonas maltophilia, a fermentation broth of the Stenotrophomonas maltophilia or a metabolite of the Stenotrophomonas maltophilia.

[0018] The present invention has at least the following beneficial effects:

[0019] The Stenotrophomonas maltophilia Sarfraz of the present invention can effectively inhibit the growth of fungi and degrade mycotoxins, and has a good application prospect in the development of microbial preparations for inhibiting the growth of mycotoxin-producing bacteria.

[0020] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The Stenotrophomonas maltophilia involved in the present invention is classified and named Stenotrophomonas maltophilia, and the strain name is Sarfraz. The Stenotrophomonas maltophilia strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on May 20, 2021, and the deposit number is CGMCC No.22572.

[0022] Figure 1 This is a plate confrontation diagram of the antagonism of Stenotrophomonas maltophilia against Fusarium oxysporum of the present invention;

[0023] Figure 2This is a plate confrontation diagram of the antagonism of Stenotrophomonas maltophilia against Fusarium graminearum of the present invention;

[0024] Figure 3 It is a plate confrontation diagram of the antagonism of the present invention's Stenotrophomonas maltophilia against parasitic Aspergillus;

[0025] Figure 4 This is a plate confrontation diagram of the antagonism of the present invention's Stenotrophomonas maltophilia against Aspergillus fumigatus;

[0026] Figure 5 This is a plate confrontation diagram of the antagonism of Aspergillus flavus by the Stenotrophomonas maltophilia of the present invention;

[0027] Figure 6 This is a plate confrontation diagram of the antagonism of Aspergillus oryzae by the Stenotrophomonas maltophilia of the present invention;

[0028] Figure 7 This is a plate confrontation diagram of the antagonism of Aspergillus ochraceus by the Stenotrophomonas maltophilia of the present invention;

[0029] Figure 8 This is a liquid chromatogram showing the degradation effect of deoxynivalenol;

[0030] Fig. 9 It is the liquid chromatography diagram of the degradation effect of ochratoxin A;

[0031] Fig.10 This is the liquid chromatography diagram of the degradation effect of zearalenone;

[0032] Fig.11 It is the liquid chromatography diagram of the degradation effect of aflatoxin B1;

[0033] Fig.12 It is the liquid chromatography diagram of the degradation effect of aflatoxin B2;

[0034] Fig.13 It is the liquid chromatography diagram of the degradation effect of aflatoxin G1;

[0035] Fig.14 This is the liquid chromatogram of the degradation effect of aflatoxin G2. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0037] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0038] Stenotrophomonas maltophilia Sarfraz, the Stenotrophomonas maltophilia strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on May 20, 2021, with the deposit number CGMCC No.22572.

[0039] The invention relates to the use of Stenotrophomonas maltophilia and / or a bacterial agent and / or a mycotoxin degrading agent in inhibiting fungal growth and / or degrading mycotoxins. The bacterial agent contains Stenotrophomonas maltophilia, and the active ingredient of the mycotoxin degrading agent includes one or more of the Stenotrophomonas maltophilia, the fermentation broth of the Stenotrophomonas maltophilia, or the metabolites of the Stenotrophomonas maltophilia.

[0040] The application of the Stenotrophomonas maltophilia is to inhibit the growth of Fusarium oxysporum, Fusarium graminearum, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae and Aspergillus ochraceus.

[0041] In the application of the Stenotrophomonas maltophilia, the mycotoxins are deoxynivalenol, ochratoxin A, zearalenone, group B aflatoxins and group G aflatoxins.

[0042] In the application of the Stenotrophomonas maltophilia, the group B aflatoxins are aflatoxins B1 and B2, and the group G aflatoxins are aflatoxins G1 and G2.

[0043] A bacterial agent containing Stenotrophomonas maltophilia.

[0044] A mycotoxin degrading agent, wherein the active ingredient of the mycotoxin degrading agent includes one or more of the Stenotrophomonas maltophilia, the fermentation broth of the Stenotrophomonas maltophilia, or the metabolites of the Stenotrophomonas maltophilia.

[0045] A method for inhibiting fungal growth, comprising using Stenotrophomonas maltophilia or a bacterial agent to inhibit fungi, wherein the bacterial agent contains Stenotrophomonas maltophilia.

[0046] A method for degrading fungal toxins, comprising: using one or more of Stenotrophomonas maltophilia, a bacterial agent or a fungal toxin degrading agent to perform biodegradation treatment on the fungal toxins, wherein the bacterial agent contains Stenotrophomonas maltophilia, and the active ingredient of the fungal toxin degrading agent includes one or more of the Stenotrophomonas maltophilia, a fermentation broth of the Stenotrophomonas maltophilia or a metabolite of the Stenotrophomonas maltophilia.

[0047] In the present invention, ppm: mg / L, ppb: μg / L.

[0048] Fusarium oxysporum f.sp lycopersici was obtained from the Grain and Oil Loss Reduction and Mycotoxin Control Innovation Team of the Institute of Agro-product Processing, Chinese Academy of Agricultural Sciences.

[0049] Fusarium graminearum, numbered PH-1, was obtained from the Grain and Oil Loss Reduction and Mycotoxin Control Innovation Team of the Institute of Agro-product Processing, Chinese Academy of Agricultural Sciences.

[0050] Aspergillus parasiticus, numbered CGMCC No.3.6155, was purchased from the Microbial Culture Collection Center of the Chinese Academy of Sciences.

[0051] Aspergillus fumigatus, numbered CGMCC No.3.11434, was purchased from the Microbial Culture Collection Center, Chinese Academy of Sciences.

[0052] Aspergillus flavus, numbered NRRL3357, was obtained from the Grain and Oil Loss Reduction and Mycotoxin Control Innovation Team of the Institute of Agro-product Processing, Chinese Academy of Agricultural Sciences.

[0053] Aspergillus oryzae, numbered CGMCC No.3.13905, was purchased from the Microbial Culture Collection Center of the Chinese Academy of Sciences.

[0054] Aspergillus ochraceous, numbered NRRL3174, was obtained from the Grain and Oil Loss Reduction and Mycotoxin Control Innovation Team, Institute of Agro-product Processing, Chinese Academy of Agricultural Sciences.

[0055] Deoxynivalenol (DON) standard: China MZ standard, product number M26001.

[0056] Ochratoxin A (OTA) standard: China MZ standard, product number M44001.

[0057] Zearalenone (ZEN) standard: China MZ standard, product number M51001.

[0058] Aflatoxin B1 standard: China MZ standard, product number AF031.

[0059] Aflatoxin B2 standard solution in methanol: China MZ standard, product number MSL019.

[0060] Aflatoxin G1 standard solution in methanol: China MZ standard, product number MSL020.

[0061] Aflatoxin G2 standard solution in methanol: China MZ standard, product number MSL021.

[0062] Deoxynivalenol toxin immunoaffinity column: China MZ standard, product number HCM0625B.

[0063] Ochratoxin A immunoaffinity column: China MZ standard, product number HCM0725.

[0064] Zearalenone immunoaffinity column: China MZ standard, product number HCM0525B.

[0065] Aflatoxin B1 immunoaffinity column: China MZ standard, product number HCM0350A.

[0066] Aflatoxin B1 / B2 / G1 / G2 immunoaffinity column: China MZ standard, product number HCM0125.

[0067] 1×0.1% Tween PBS buffer: VICAM, USA, product number G1112.

[0068] Experiment 1: Screening and identification of Stenotrophomonas maltophilia Sarfraz

[0069] Using natural soil as the bacterial source, 5 g of soil sample was placed in a 50 mL centrifuge tube, 45 mL of deionized water was added, and the tube was shaken on a shaker for 30 min and allowed to stand for 30 s to make 10 -1 g / mL; take 100μL 10 -1 Add 900 μL of deionized water to the dilution solution in a 1 mL EP tube and vortex for 3-5 seconds to make 10 -2 g / mL, take 100μL 10 -2 Add 900 μL of deionized water to the dilution solution in a 1 mL EP tube and vortex for 3-5 seconds to make 10 -3 g / mL, and so on to 10 -6 g / mL; NA plates were used to spread 300 μL of the dilution solution and cultured at 37°C for 24 h; single colonies (with different shapes, sizes, and viscosities) were picked from the NA plates using the four-zone streak method, and the single colonies were added to LB liquid culture medium and cultured at 37°C, 180 rpm (revolutions / minute) for 48 h to obtain bacterial culture for later use.

[0070] Use the pour plate method to pour the prepared sterile supernatant of different bacteria into the unsolidified PDA medium at about 45°C, mix well, and quickly pour the plate. After the PDA medium solidifies, drop 10 μL of a 1×10 7 CFU / mL of Fusarium oxysporum spore suspension was used, and sterile blank culture medium was used instead of supernatant as control. It was incubated upside down in a constant temperature incubator at 28°C for 5 days. 10 μL of LB liquid culture medium was dropped in one place as a blank control group. After the liquid was dried, it was sealed and incubated upside down at 37°C for 5 days. After observation, the strain with better inhibition of Fusarium oxysporum growth was selected and stored in glycerol at -80°C for later use.

[0071] After identification, the strain is Stenotrophomonas maltophilia Sarfraz. The Stenotrophomonas maltophilia is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration. The preservation date is May 20, 2021, the preservation number is CGMCC No.22572, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0072] in,

[0073] NA (Beijing beef extract peptone agar medium): purchased from Luqiao Company, prepared immediately before use, weigh 33g into 1L distilled water, heat and boil until completely dissolved, sterilize at 121℃ for 15min, cool to 46℃ and pour onto a plate.

[0074] LB liquid culture medium: composed of a solvent and a solute; the solutes are peptone, beef extract and NaCl, and the solvent is water; the concentration of peptone in the LB liquid culture medium is 1 g / 100 mL, the concentration of beef extract in the LB liquid culture medium is 0.3 g / 100 mL, the concentration of NaCl in the LB liquid culture medium is 0.5 g / 100 mL, and the pH of the culture medium is adjusted to 7.4 with NaOH.

[0075] PDA (potato dextrose agar medium): purchased from Beijing Luqiao Company, prepared immediately before use, weigh 33 g into 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121°C under high pressure for 15 min, cool to 46°C and pour onto a plate.

[0076] Experiment 2: Antifungal test of Stenotrophomonas maltophilia

[0077] The plate confrontation culture method was used for screening. 10 μL of 1×10 7 CFU / mL of the test fungal spore suspension was dropped in the center of the PDA plate, and 10 μL of Stenotrophomonas maltophilia (OD 600=0.8) suspension was dripped around, and 10 μL of LB liquid culture medium was dripped in one place as a blank control group. After the liquid dried on the PDA culture medium at the alcohol lamp of the clean bench, it was placed in a constant temperature incubator at 28°C for incubation. It was inverted and cultured for about 6 days. The growth of mycelium was observed every other day until the mycelium covered the plate. Each test was repeated 3 times. The diameter of the fungus to be tested was measured using a vernier caliper using the cross method, and the average value was taken as the measurement result. The inhibition rate was calculated according to the following formula:

[0078] Inhibition rate (%) = (colony growth diameter of control group - colony growth diameter of treatment group) / colony growth diameter of control group × 100%.

[0079] The fungus to be tested was Fusarium oxysporum. Figure 1 As shown, Figure 1 The left side is the treatment group, and the right side is the control group, indicating that Stenotrophomonas maltophilia has an inhibitory effect on Fusarium oxysporum, with an inhibition rate of 76%.

[0080] The fungus to be tested was Fusarium graminearum. Figure 2 As shown, Figure 2 The left side is the treatment group, and the right side is the control group, indicating that Stenotrophomonas maltophilia has an inhibitory effect on Fusarium graminearum, with an inhibition rate of 79%.

[0081] The fungus to be tested is Aspergillus parasiticus. Figure 3 As shown, Figure 3 The left side is the treatment group, and the right side is the control group, indicating that Stenotrophomonas maltophilia has an inhibitory effect on parasitic Aspergillus, with an inhibition rate of 71%.

[0082] The fungus to be tested is Aspergillus fumigatus. Figure 4 As shown, Figure 4 The left side is the treatment group, and the right side is the control group, indicating that Stenotrophomonas maltophilia has an inhibitory effect on Aspergillus fumigatus, with an inhibition rate of 61%.

[0083] The fungus to be tested is Aspergillus flavus. Figure 5 As shown, Figure 5 The left side is the treatment group, and the right side is the control group, indicating that Stenotrophomonas maltophilia has an inhibitory effect on Aspergillus flavus, with an inhibition rate of 59%.

[0084] The fungus to be tested was Aspergillus oryzae. Figure 6 As shown, Figure 6 The left side is the treatment group, and the right side is the control group, indicating that Stenotrophomonas maltophilia has an inhibitory effect on Aspergillus oryzae, with an inhibition rate of 56%.

[0085] The fungus to be tested is Aspergillus ochraceus. Figure 7 As shown, Figure 7The left side is the treatment group, and the right side is the control group, indicating that Stenotrophomonas maltophilia has an inhibitory effect on Aspergillus ochraceus, with an inhibition rate of 37%.

[0086] Experiment 3: Degradation of deoxynivalenol by Stenotrophomonas maltophilia

[0087] 1. Inoculate the maltophilia bacteria into LB liquid medium until the initial OD 600 =0.05, cultured at 37°C with shaking at 200 rpm (rotation radius 20 mm) for 24 h, and centrifuged at 10 000 r / min for 10 min to collect the supernatant.

[0088] 2. Dissolve 1 mg of deoxynivalenol (DON) standard (MZ standard, catalog number M26001) in 10 mL of chromatographic grade methanol to obtain a DON solution with a concentration of 100 ppm.

[0089] 3. Prepare experimental group solution:

[0090] Take 5 mL of the supernatant collected in step 1 and place it in a 10 mL centrifuge tube. Add 5 μL of the DON solution obtained in step 2 to the centrifuge tube. Mix thoroughly and let stand at 37 °C for 72 h. Then centrifuge at 10 000 g for 10 min and collect the supernatant to obtain the experimental group solution.

[0091] 4. Prepare the control group solution:

[0092] According to the method of step 3, 5 mL of LB liquid culture medium was taken to replace 5 mL of the supernatant collected in step 1, and the other operations remained unchanged to obtain a control group solution.

[0093] 5. Effect detection:

[0094] The experimental group solution and the control group solution were used as test solutions, and the following steps were performed:

[0095] 1) Add 6 volumes of anhydrous methanol to 4 volumes of the test solution, extract at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant for the next purification operation.

[0096] 2) Take the supernatant obtained in step 1) and use a vomitoxin immunoaffinity column to remove impurities. The specific operation is as follows:

[0097] Take the supernatant obtained in step 1) and pass it through the DON toxin immunoaffinity column, adjust the flow rate to 1-2 drops / s, until the air completely passes through the immunoaffinity column. Use 10mL of pure water to pass through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column. Finally, use 1mL of anhydrous methanol to elute the affinity column at a flow rate of 1-2 drops / s, collect the eluate in a 1.5mL centrifuge tube, filter with a 0.22μm organic phase nylon membrane, and then load it into a 2mL chromatographic injection vial to obtain a sample solution.

[0098] 3) Take the sample solution obtained in step 2) and use HPLC to detect the sample obtained by purification and extraction.

[0099] The HPLC detection conditions were as follows: mobile phase acetonitrile: water = 2:8; flow rate 1 mL / min; chromatographic column C18 150 mm × 4.6 mm, 5 μm; UV detector 218 nm; column temperature 30°C; injection volume 20 μL.

[0100] The degradation rate of deoxynivalenol (DON) was calculated as follows:

[0101] DON degradation rate (%) = (residual DON content in the control group - residual DON content in the experimental group) / residual DON content in the control group × 100.

[0102] The experiment was repeated five times and the results were averaged.

[0103] Test results such as Figure 8 As shown, A is the deoxynivalenol standard (the retention time of DON toxin is 4.777min); B is the control group (the retention time of DON toxin is 4.782min); C is the experimental group (the retention time of DON toxin is 4.779min).

[0104] The residual DON content in the control group was 99.75 ± 0.98 μg / L;

[0105] The residual DON content in the experimental group was 31.92 ± 1.60 μg / L;

[0106] The results showed that Stenotrophomonas maltophilia had a certain degradation effect on DON toxin, with a degradation rate of 67.83%.

[0107] Experiment 4: Degradation of ochratoxin A by Stenotrophomonas maltophilia

[0108] 1. Inoculate the maltophilia bacteria into LB liquid medium until the initial OD 600 =0.05, cultured at 37°C with shaking at 200 rpm (rotation radius 20 mm) for 24 h, and centrifuged at 10 000 r / min for 10 min to collect the supernatant.

[0109] 2. Dissolve 1 mg of OTA standard (MZ standard, catalog number M44001) in 10 mL of chromatographic grade methanol to obtain a 100 ppm OTA standard solution.

[0110] 3. Prepare experimental group solution:

[0111] Take 5 mL of the supernatant collected in step 1 and place it in a 10 mL centrifuge tube. Add 5 μL of the OTA solution obtained in step 2 to the centrifuge tube. After thorough mixing, let it stand at 37 °C for 72 h. Then, centrifuge at 10,000 g for 10 min and collect the supernatant to obtain the experimental group solution.

[0112] 4. Prepare the control group solution:

[0113] According to the method of step 3, 5 mL of LB liquid culture medium was taken to replace 5 mL of the supernatant collected in step 1, and the other operations remained unchanged to obtain a control group solution.

[0114] 5. Effect detection:

[0115] The experimental group solution and the control group solution were used as test solutions, and the following steps were performed:

[0116] 1) Add 6 volumes of anhydrous methanol to 4 volumes of the test solution, extract at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant for the next purification operation.

[0117] 2) Take the supernatant obtained in step 1) and use an OTA immunoaffinity column to remove impurities. The specific operation is as follows:

[0118] Take the supernatant obtained in step 1) and pass it through the OTA immunoaffinity column, adjusting the flow rate to 1-2 drops / s until the air completely passes through the immunoaffinity column. Use 10mL of pure water to pass through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column. Finally, use 1mL of anhydrous methanol to elute the affinity column at a flow rate of 1-2 drops / s, collect the eluate in a 1.5mL centrifuge tube, filter with a 0.22μm organic phase nylon membrane, and then load it into a 2mL chromatographic injection vial to obtain a sample solution.

[0119] 3) Take the sample solution obtained in step 2) and use HPLC (post-column photochemical derivatization) to detect the sample obtained by purification and extraction.

[0120] HPLC detection conditions are as follows: mobile phase acetonitrile: water: acetic acid = 99:99:2; flow rate 1 mL / min; chromatographic column C18 150 mm × 4.6 mm, 5 μm; excitation wavelength 333 nm, detection wavelength 460 nm; column temperature 30°C; injection volume 20 μL.

[0121] Calculate the degradation rate of ochratoxin A (OTA) by:

[0122] OTA degradation rate (%) = (residual OTA content in the control group - residual OTA content in the experimental group) / residual OTA content in the control group × 100.

[0123] The experiment was repeated five times and the results were averaged.

[0124] Test results such as Fig. 9 As shown, A is the ochratoxin A standard (the retention time of OTA is 13.965 min); B is the control group (the retention time of OTA is 13.952 min); C is the experimental group (the retention time of OTA is 13.977 min).

[0125] The residual OTA content in the control group was 99.75±0.98μg / L;

[0126] The residual OTA content in the experimental group was 38.21±1.60μg / L;

[0127] The results showed that Stenotrophomonas maltophilia had a certain degradation effect on ochratoxin A (OTA), with a degradation rate of 61.54%.

[0128] Experiment 5: Degradation of zearalenone by Stenotrophomonas maltophilia

[0129] 1. Inoculate the maltophilia bacteria into LB liquid medium until the initial OD 600 =0.05, cultured at 37°C with shaking at 200 rpm (rotation radius 20 mm) for 24 h, and centrifuged at 10 000 r / min for 10 min to collect the supernatant.

[0130] 2. Dissolve 1 mg of zearalenone (ZEN) standard (MZ standard, catalog number M51001) in 10 mL of chromatographic grade methanol to obtain a ZEN standard solution with a concentration of 100 ppm.

[0131] 3. Prepare experimental group solution:

[0132] Take 5 mL of the supernatant collected in step 1 and place it in a 10 mL centrifuge tube. Add 10 μL of the ZEN solution obtained in step 2 to the centrifuge tube. Mix thoroughly and let stand at 37 °C for 72 h. Then centrifuge at 10 000 g for 10 min and collect the supernatant to obtain the experimental group solution.

[0133] 4. Prepare the control group solution:

[0134] According to the method of step 3, 5 mL of LB liquid culture medium was taken to replace 5 mL of the supernatant collected in step 1, and the other operations remained unchanged to obtain a control group solution.

[0135] 5. Effect detection:

[0136] The experimental group solution and the control group solution were used as test solutions, and the following steps were performed:

[0137] 1) Add 6 volumes of anhydrous methanol to 4 volumes of the test solution, extract at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant for the next purification operation.

[0138] 2) Take the supernatant obtained in step 1) and use a zearalenone immunoaffinity column to remove impurities. The specific operation is as follows:

[0139] Take the supernatant obtained in step 1) and pass it through the ZEN immunoaffinity column, adjusting the flow rate to 1-2 drops / s until the air completely passes through the immunoaffinity column. Use 10mL of pure water to pass through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column. Finally, use 1mL of anhydrous methanol to elute the affinity column at a flow rate of 1-2 drops / s, collect the eluate in a 1.5mL centrifuge tube, filter it with a 0.22μm organic phase nylon membrane, and then put it into a 2mL chromatographic injection vial to obtain a sample solution.

[0140] 3) Take the sample solution obtained in step 2) and use HPLC (post-column photochemical derivatization) to detect the sample obtained by purification and extraction.

[0141] HPLC detection conditions were as follows: mobile phase acetonitrile: water = 7:3; flow rate 1 mL / min; chromatographic column C18 150 mm × 4.6 mm, 5 μm; excitation wavelength 274 nm, detection wavelength 440 nm; column temperature 30°C; injection volume 20 μL.

[0142] The degradation rate of zearalenone (ZEN) was calculated as follows:

[0143] ZEN degradation rate (%) = (residual ZEN content in the control group - residual ZEN content in the experimental group) / residual ZEN content in the control group × 100.

[0144] The experiment was repeated five times and the results were averaged.

[0145] Test results such as Fig.10 As shown, A is the zearalenone standard (the retention time of ZEN is 4.142min); B is the control group (the retention time of ZEN is 4.127min); and C is the experimental group (the retention time of ZEN is 4.191min).

[0146] The residual ZEN content in the control group was 199.75 ± 0.98 μg / L;

[0147] The residual ZEN content in the experimental group was 91.20 ± 1.60 μg / L;

[0148] The results showed that Stenotrophomonas maltophilia had a degradation effect on ZEN, with a degradation rate of 54.30%.

[0149] Experiment 6: Degradation of aflatoxin B1 by Stenotrophomonas maltophilia

[0150] 1. Inoculate the maltophilia bacteria into LB liquid medium until the initial OD 600 =0.05, culture at 37°C, 200 rpm (rotation radius 20 mm) with shaking for 24 h, centrifuge at 10 000 r / min for 10 min, and collect the supernatant.

[0151] 2. Dissolve 1 mg of AFB1 standard (MZ standard, catalog number AF031) in 10 mL of chromatographic grade methanol to obtain an AFB1 solution with a concentration of 100 ppm.

[0152] 3. Prepare experimental group solution:

[0153] Take 5 mL of the supernatant collected in step 1 and place it in a 10 mL centrifuge tube. Add 15 μL of the AFB1 solution obtained in step 2 to the centrifuge tube. Mix thoroughly and let stand at 37 °C for 72 h. Then centrifuge at 10 000 g for 10 min and collect the supernatant to obtain the experimental group solution.

[0154] 4. Prepare the control group solution:

[0155] According to the method of step 3, 5 mL of LB liquid culture medium was taken to replace 5 mL of the supernatant collected in step 1, and the other operations remained unchanged to obtain a control group solution.

[0156] 5. Effect detection:

[0157] The experimental group solution and the control group solution were used as test solutions, and the following steps were performed:

[0158] 1) Add 6 volumes of anhydrous methanol to 4 volumes of the test solution, extract at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant for the next purification operation.

[0159] 2) Take the supernatant obtained in step 1) and use AFB1 immunoaffinity column to remove impurities. The specific operation is as follows:

[0160] Take the supernatant obtained in step 1) and pass it through the AFB1 immunoaffinity column, adjusting the flow rate to 1-2 drops / s until the air completely passes through the immunoaffinity column. Use 10mL of pure water to pass through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column. Finally, use 1mL of anhydrous methanol to elute the affinity column at a flow rate of 1-2 drops / s, collect the eluate in a 1.5mL centrifuge tube, filter it with a 0.22μm organic phase nylon membrane, and then put it into a 2mL chromatographic injection vial to obtain a sample solution.

[0161] 3) Take the sample solution obtained in step 2) and use HPLC (post-column photochemical derivatization) to detect the sample obtained by purification and extraction.

[0162] HPLC detection conditions were as follows: mobile phase methanol: water = 7:3; flow rate 1 mL / min; chromatographic column C18 150 mm × 4.6 mm, 5 μm; excitation wavelength 350 nm, detection wavelength 450 nm; column temperature 30°C; injection volume 20 μL.

[0163] Calculate the degradation rate of aflatoxin B1 (AFB1) by:

[0164] AFB1 degradation rate (%) = (residual AFB1 content of the control group - residual AFB1 content of the experimental group) / residual AFB1 content of the control group × 100.

[0165] The experiment was repeated five times and the results were averaged.

[0166] Test results such as Fig.11 As shown, A is the aflatoxin standard (the retention time of AFB1 is 5.152 min); B is the control group (the retention time of AFB1 is 5.171 min); and C is the experimental group (the retention time of AFB1 is 5.185 min).

[0167] The residual AFB1 content in the control group was 299.75±0.98μg / L;

[0168] The residual AFB1 content in the experimental group was 119.85 ± 1.30 μg / L;

[0169] The results showed that Stenotrophomonas maltophilia had a good degradation effect on AFB1, with a degradation rate of 60.15%.

[0170] Experiment 7: Degradation of aflatoxin B2 by Stenotrophomonas maltophilia

[0171] 1. Inoculate the maltophilia bacteria into LB liquid medium until the initial OD 600 =0.05, cultured at 37°C with shaking at 200 rpm (rotation radius 20 mm) for 24 h, and centrifuged at 10 000 r / min for 10 min to collect the supernatant.

[0172] 2. Prepare experimental group solution:

[0173] Take 5 mL of the supernatant collected in step 1 and place it in a 10 mL centrifuge tube. Add 15 μL of 100 ppm aflatoxin B2 standard solution in methanol to the centrifuge tube. Mix thoroughly and let stand at 37 °C for 72 h. Then centrifuge at 10 000 g for 10 min and collect the supernatant to obtain the experimental group solution.

[0174] 3. Prepare the control group solution:

[0175] According to the method of step 2, 5 mL of LB liquid culture medium was taken to replace 5 mL of the supernatant collected in step 1, and the other operations remained unchanged to obtain a control group solution.

[0176] 4. Effect detection:

[0177] The experimental group solution and the control group solution were used as test solutions, and the following steps were performed:

[0178] 1) Add 6 volumes of anhydrous methanol to 4 volumes of the test solution, extract at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant for the next purification operation.

[0179] 2) Take the supernatant obtained in step 1) and use an aflatoxin B1 / B2 / G1 / G2 immunoaffinity column to remove impurities. The specific operation is as follows:

[0180] Take the supernatant obtained in step 1) and pass it through the aflatoxin B1 / B2 / G1 / G2 immunoaffinity column, adjust the flow rate to 1-2 drops / s, until the air completely passes through the immunoaffinity column. Use 10mL of pure water to pass through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column. Finally, use 1mL of anhydrous methanol to elute the affinity column at a flow rate of 1-2 drops / s, collect the eluate in a 1.5mL centrifuge tube, filter with a 0.22μm organic phase nylon membrane, and then load it into a 2mL chromatographic injection vial to obtain a sample solution.

[0181] 3) Take the sample solution obtained in step 2) and use HPLC (post-column photochemical derivatization) to detect the sample obtained by purification and extraction.

[0182] HPLC detection conditions were as follows: mobile phase methanol: water = 1:1; flow rate 1 mL / min; chromatographic column C18 150 mm × 4.6 mm, 5 μm; excitation wavelength 350 nm, detection wavelength 450 nm; column temperature 30°C; injection volume 20 μL.

[0183] Calculate the degradation rate of aflatoxin B2 (AFB2) by:

[0184] AFB2 degradation rate (%) = (residual AFB2 content of the control group - residual AFB2 content of the experimental group) / residual AFB2 content of the control group × 100.

[0185] The experiment was repeated five times and the results were averaged.

[0186] Test results such as Fig.12 As shown, A is the aflatoxin B2 standard (the retention time of AFB2 is 4.453 min); B is the control group (the retention time of AFB2 is 4.464 min); C is the experimental group (the retention time of AFB2 is 4.457 min).

[0187] The residual AFB2 content in the control group was 299.75 ± 0.98 μg / L;

[0188] The residual AFB2 content in the experimental group was 164.85 ± 1.20 μg / L;

[0189] The results showed that Stenotrophomonas maltophilia had a certain degradation effect on AFB2, with a degradation rate of 45.13%.

[0190] Experiment 8: Degradation of aflatoxin G1 by Stenotrophomonas maltophilia

[0191] 1. Inoculate the maltophilia bacteria into LB liquid medium until the initial OD 600 =0.05, cultured at 37°C with shaking at 200 rpm (rotation radius 20 mm) for 24 h, and centrifuged at 10 000 r / min for 10 min to collect the supernatant.

[0192] 2. Prepare experimental group solution:

[0193] Take 5 mL of the supernatant collected in step 1 and place it in a 10 mL centrifuge tube. Add 15 μL of 100 ppm aflatoxin G1 standard solution in methanol to the centrifuge tube. Mix thoroughly and let stand at 37 °C for 72 h. Then centrifuge at 10 000 g for 10 min and collect the supernatant to obtain the experimental group solution.

[0194] 3. Prepare the control group solution:

[0195] According to the method of step 2, 5 mL of LB liquid culture medium was taken to replace 5 mL of the supernatant collected in step 1, and the other operations remained unchanged to obtain a control group solution.

[0196] 4. Effect detection:

[0197] The experimental group solution and the control group solution were used as test solutions, and the following steps were performed:

[0198] 1) Add 6 volumes of anhydrous methanol to 4 volumes of the test solution, extract at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant for the next purification operation.

[0199] 2) Take the supernatant obtained in step 1 and use an aflatoxin B1 / B2 / G1 / G2 immunoaffinity column to remove impurities. The specific operation is as follows:

[0200] Take the supernatant obtained in step 1) and pass it through the aflatoxin B1 / B2 / G1 / G2 immunoaffinity column, adjust the flow rate to 1-2 drops / s, until the air completely passes through the immunoaffinity column. Use 10mL of pure water to pass through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column. Finally, use 1mL of anhydrous methanol to elute the affinity column at a flow rate of 1-2 drops / s, collect the eluate in a 1.5mL centrifuge tube, filter with a 0.22μm organic phase nylon membrane, and then load it into a 2mL chromatographic injection vial to obtain a sample solution.

[0201] 3) Take the sample solution obtained in step 2) and use HPLC (post-column photochemical derivatization) to detect the sample obtained by purification and extraction.

[0202] HPLC detection conditions were as follows: mobile phase methanol: water = 1:1; flow rate 1 mL / min; chromatographic column C18 150 mm × 4.6 mm, 5 μm; excitation wavelength 350 nm, detection wavelength 450 nm; column temperature 30°C; injection volume 20 μL.

[0203] The degradation rate of aflatoxin G1 (AFG1) was calculated as follows:

[0204] AFG1 degradation rate (%) = (residual AFG1 content in the control group - residual AFG1 content in the experimental group) / residual AFG1 content in the control group × 100.

[0205] The experiment was repeated five times and the results were averaged.

[0206] Test results such as Fig.13 As shown, A is the aflatoxin G1 standard (the retention time of AFG1 is 4.815min); B is the control group (the retention time of AFG1 is 4.897min); and C is the experimental group (the retention time of AFG1 is 4.821min).

[0207] The residual AFG1 content in the control group was 299.75 ± 0.98 μg / L;

[0208] The residual AFG1 content in the experimental group was 141.80 ± 1.20 μg / L;

[0209] The results showed that Stenotrophomonas maltophilia had a very high degradation effect on AFG1, with a degradation rate of 52.70%.

[0210] Experiment 9: Degradation of aflatoxin G2 by Stenotrophomonas maltophilia

[0211] 1. Inoculate the maltophilia bacteria into LB liquid medium until the initial OD 600 =0.05, cultured at 37°C with shaking at 200 rpm (rotation radius 20 mm) for 24 h, and centrifuged at 10 000 r / min for 10 min to collect the supernatant.

[0212] 2. Prepare experimental group solution:

[0213] Take 5 mL of the supernatant collected in step 1 and place it in a 10 mL centrifuge tube. Add 15 μL of 100 ppm aflatoxin G2 standard solution in methanol to the centrifuge tube. Mix thoroughly and let stand at 37 °C for 72 h. Then centrifuge at 10 000 g for 10 min and collect the supernatant to obtain the experimental group solution.

[0214] 3. Prepare the control group solution:

[0215] According to the method of step 2, 5 mL of LB liquid culture medium was taken to replace 5 mL of the supernatant collected in step 1, and the other operations remained unchanged to obtain a control group solution.

[0216] 4. Effect detection:

[0217] The experimental group solution and the control group solution were used as test solutions, and the following steps were performed:

[0218] 1) Add 6 volumes of anhydrous methanol to 4 volumes of the test solution, extract at room temperature for 5 minutes, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant for the next purification operation.

[0219] 2) Take the supernatant obtained in step 1) and use an aflatoxin B1 / B2 / G1 / G2 immunoaffinity column to remove impurities. The specific operation is as follows:

[0220] Take the supernatant obtained in step 1) and pass it through the aflatoxin B1 / B2 / G1 / G2 immunoaffinity column, adjust the flow rate to 1-2 drops / s, until the air completely passes through the immunoaffinity column. Use 10mL of pure water to pass through the affinity column at a flow rate of 1-2 drops / s to clean the affinity column. Finally, use 1mL of anhydrous methanol to elute the affinity column at a flow rate of 1-2 drops / s, collect the eluate in a 1.5mL centrifuge tube, filter with a 0.22μm organic phase nylon membrane, and then load it into a 2mL chromatographic injection vial to obtain a sample solution.

[0221] 3) Take the sample solution obtained in step 2) and use HPLC (post-column photochemical derivatization) to detect the sample obtained by purification and extraction.

[0222] HPLC detection conditions were as follows: mobile phase methanol: water = 1:1; flow rate 1 mL / min; chromatographic column C18 150 mm × 4.6 mm, 5 μm; excitation wavelength 350 nm, detection wavelength 450 nm; column temperature 30°C; injection volume 20 μL.

[0223] Calculate the degradation rate of aflatoxin G2 (AFG2) by:

[0224] AFG2 degradation rate (%) = (residual AFG2 content in the control group - residual AFG2 content in the experimental group) / residual AFG2 content in the control group × 100.

[0225] The experiment was repeated five times and the results were averaged.

[0226] Test results such as Fig.14 As shown, A is the aflatoxin standard (the retention time of AFG2 is 4.948 min); B is the control group (the retention time of AFG2 is 4.914 min); and C is the experimental group (the retention time of AFG2 is 4.909 min).

[0227] The residual AFG2 content in the control group was 299.75 ± 0.98 μg / L;

[0228] The residual AFG2 content in the experimental group was 183.75 ± 1.20 μg / L;

[0229] The results showed that Stenotrophomonas maltophilia had a very good degradation effect on AFG2, with a degradation rate of 38.69%.

[0230] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ) Sarfraz, characterized in that The Stenotrophomonas maltophilia strain was deposited in the General Microbiology Center of the China Culture Collection Administration on May 20, 2021, with the deposit number CGMCC No. 22572.

2. A bacterial agent, characterized in that: The bacterial agent contains the Stenotrophomonas maltophilia according to claim 1.

3. Use of the Stenotrophomonas maltophilia according to claim 1 or the bacterial agent according to claim 2 in inhibiting the growth of Fusarium oxysporum, Fusarium graminearum, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus fumigatus, Aspergillus oryzae and Aspergillus ochraceus.

4. Use of the Stenotrophomonas maltophilia according to claim 1 or the fermentation broth of the Stenotrophomonas maltophilia according to claim 1 as an active ingredient in degrading fungal toxins, wherein: The fungal toxins are deoxynivalenol, ochratoxin A, zearalenone, group B aflatoxins and group G aflatoxins.

5. The use according to claim 4, characterized in that The group B aflatoxins are aflatoxins B1 and B2, and the group G aflatoxins are aflatoxins G1 and G2.

6. A method for inhibiting fungal growth, characterized in that: The Stenotrophomonas maltophilia described in claim 1 or the bacterial agent described in claim 2 is used to inhibit Fusarium oxysporum, Fusarium graminearum, Aspergillus parasiticus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae and Aspergillus ochraceus.

7. A method for degrading mycotoxins, characterized in that: Deoxynivalenol, ochratoxin A, zearalenone, group B aflatoxins and group G aflatoxins are biodegraded using the Stenotrophomonas maltophilia described in claim 1 or the fermentation broth of the Stenotrophomonas maltophilia described in claim 1 as an active ingredient.

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