Arthrobacter citratus, preparation for degrading AFB1 and method for degrading AFB1

Abstract

The invention discloses arthrobacter citratus, a preparation for degrading AFB1 and a method for degrading AFB1, and belongs to the technical field of microorganisms. The arthrobacter citreum provided by the invention is an arthrobacter citreum soilh02 strain, the strain is preserved in the China General Microbiological Culture Collection Center on June 6, 2025, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 34782. The strain soilh02 disclosed by the invention has a relatively good degradation effect on AFB1, and active ingredients for degrading the AFB1 are located in fermentation supernate of the strain soilh02; in addition, under the condition that metal ions are added, the degradation effect of the fermentation liquor of the strain soilh02 on AFB1 can be further improved. Therefore, the strain soilh02 has an important application prospect in degradation of the AFB1.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a strain of Arthrobacter citreus, a preparation for degrading AFB1 and a method for degrading AFB1. BACKGROUND

[0002] In recent years, food safety problems have attracted widespread attention, and mycotoxin pollution has become an important factor threatening human health. Among the many mycotoxins, aflatoxin B1 is of great concern due to its strong toxicity and high contamination rate. As a secondary metabolite produced by Aspergillus and Fusarium fungi, aflatoxin B1 not only has strong carcinogenicity, but also can cause genetic mutations. According to statistics, about 25% of food crops and feed worldwide are contaminated with this toxin, causing huge waste of food resources.

[0003] In the existing aflatoxin degradation technology, adsorption method is widely used in the removal of aflatoxin B1 in food and environment, but it has significant limitations: first, this method only fixes the toxin by using adsorbents such as activated carbon and clay, without destroying the molecular structure of the toxin, and there is a risk of re-releasing the toxin due to changes in environmental conditions; second, the adsorption process lacks selectivity, and while removing the toxin, it may also lose nutrients such as vitamins and minerals in the feed; third, some mineral adsorbents may contain heavy metal impurities, and long-term use may have the risk of accumulating toxicity. In addition, this method also has the problems of limited adsorption capacity, the need for regular replacement of adsorbents, better effect on liquid systems than on solid substrates, and the possibility of the used adsorbents becoming toxic waste. Microbial method stands out due to its unique advantages. Compared with traditional physical and chemical methods, microbial degradation has the characteristics of high detoxification efficiency, strong specificity, low cost, environmental friendliness and little effect on food quality. Therefore, it is urgent to screen new aflatoxin B1 degrading bacteria, enrich the types of microorganisms for degrading aflatoxin B1, and lay a foundation for developing aflatoxin B1 degradation methods with high detoxification efficiency, strong specificity, low cost and environmental friendliness. SUMMARY

[0004] In view of the problems in the prior art, the present application aims to provide a strain of Arthrobacter citreus, a preparation for degrading AFB1 and a method for degrading AFB1.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The strain of Arthrobacter citreus is Arthrobacter citreus soilh02 strain, which was preserved in China General Microbiological Culture Collection Center on June 6, 2025, and the preservation number is CGMCC No.34782.

[0006] The application of the above-mentioned Arthrobacter citreus in degrading AFB1.

[0007] A preparation for degrading AFB1, wherein the effective component comprises a fermentation broth or a fermentation supernatant of Arthrobacter citreus with a preservation number of CGMCC No.34782.

[0008] On the basis of the above-mentioned scheme, the preparation method of the fermentation broth of Arthrobacter citreus with the preservation number of CGMCC No.34782 is as follows: The seed liquid of Arthrobacter citreus with the preservation number of CGMCC No.34782 is inoculated into a fermentation medium at an inoculation amount of 1%-5%, the initial pH of the medium is 5-9, and the fermentation culture is carried out at 22-42°C and 180 rpm for 12-54 h; and then the fermentation broth is obtained.

[0009] On the basis of the above-mentioned scheme, the inoculation amount of the seed liquid is 2%, the initial pH of the medium is 8, the fermentation temperature is 37°C, and the fermentation time is 48 h.

[0010] On the basis of the above-mentioned scheme, the fermentation medium comprises beef extract 5 g / L, peptone 10 g / L, sodium chloride 3 g / L, sodium phosphate dibasic 2 g / L, and glucose 1 g / L.

[0011] On the basis of the above-mentioned scheme, the preparation method of the seed liquid is as follows: the single colony of Arthrobacter citreus with the preservation number of CGMCC No.34782 is inoculated into LB medium with pH 7.4, and then the medium is cultured at 37°C and 170 rpm for overnight, and then the seed liquid is mixed with 40% sterile glycerol at a ratio of 1:1 and stored at -20°C.

[0012] On the basis of the above-mentioned scheme, the preparation method of the fermentation supernatant of Arthrobacter citreus with the preservation number of CGMCC No.34782 is as follows: the fermentation broth of Arthrobacter citreus with the preservation number of CGMCC No.34782 is centrifuged at 10000 rpm for 2 min to remove the bacterial precipitate, and then the cell-free supernatant is obtained.

[0013] On the basis of the above-mentioned scheme, further comprising a metal ion, and the metal ion is Cu 2+ .

[0014] On the basis of the above-mentioned scheme, the final concentration of the metal ion is 10 mM.

[0015] A method for degrading AFB1, wherein the above-mentioned preparation for degrading AFB1 is mixed with a sample to be degraded at a volume ratio of 4:1, and then the mixture is cultured at 37°C and 180 rpm for 24-120 h in dark and constant temperature shaking, and then the degradation is completed.

[0016] On the basis of the above-mentioned scheme, the degradation time is 72 h.

[0017] On the basis of the above scheme, the sample to be degraded is peanut cake, corn yellow water.

[0018] Advantages of the technical scheme of the present application The present application separates and obtains a strain of Arthrobacter citreus, which has a good degradation effect on AFB1. By optimizing the fermentation conditions of the strain, the degradation effect of the fermentation liquor on AFB1 can be improved. Through detection, the active ingredient for degrading AFB1 is located in the fermentation supernatant of the strain soilh02. Moreover, under the condition of adding Cu 2+ , the degradation effect of the fermentation liquor of the strain soilh02 on AFB1 can be further improved. Therefore, the fermentation liquor or fermentation supernatant of the strain soilh02 has a good degradation effect on AFB1, and the degradation method is mild, green, safe, pollution-free and low in cost, and has an important application prospect in the degradation of AFB1. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Morphological observation of the strain soilh02 (wherein A is a colony morphology diagram; B is a scanning electron microscope diagram); Figure 2 Gram staining result diagram of the strain soilh02; Figure 3 Phylogenetic tree of the strain soilh02; Figure 4 AFB1 standard curve diagram; Figure 5 Effect of different inoculation amounts on the degradation effect of the fermentation liquor of the strain soilh02 on AFB1; Figure 6 Effect of different initial pH values on the degradation effect of the fermentation liquor of the strain soilh02 on AFB1; Figure 7 Effect of different fermentation temperatures on the degradation effect of the fermentation liquor of the strain soilh02 on AFB1 Figure 8 Effect of different fermentation times on the degradation effect of the fermentation liquor of the strain soilh02 on AFB1; Figure 9 Effect of different degradation times on the degradation effect of the fermentation liquor of the strain soilh02 on AFB1; Figure 10 Effect of metal ions on the degradation effect of the fermentation liquor of the strain soilh02 on AFB1; Figure 11 Effect of different components of the fermentation liquor of the strain soilh02 on the degradation of AFB1. DETAILED DESCRIPTION

[0020] The terms used in the present application have meanings ordinarily understood by those of ordinary skill in the art, unless otherwise explicitly provided. The present application is described in further detail below in connection with specific examples and with reference to the data. The following examples are merely for the purpose of illustration of the present application and are not intended to limit the scope of the present application in any way.

[0021] The experimental methods in the following examples are all conventional methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The experimental materials, reagents, and drugs used in the following examples, unless otherwise specified, can be purchased through general channels.

[0022] Example 1 Isolation, purification and identification of the strain Isolation and purification: soil collected in peanut field in Qingdao, Shandong Province in September 2023 was used as the source of bacteria, 10 g of soil sample was added to 100 mL of sterile water, mixed to obtain a microbial suspension, and then placed for 30 s. The microbial suspension was inoculated into LB broth medium at an inoculation amount of 10%, and cultured at 37°C with constant temperature shaking at 180 rpm for 12-16 h for strain enrichment, and the enrichment liquid was obtained. The enrichment liquid was inoculated into the primary screening medium with coumarin as the sole carbon source at an inoculation amount of 5%, and cultured at 37°C with constant temperature shaking at 180 rpm for 7 days for the primary screening of AFB1-degrading bacteria. The strains obtained by primary screening were purified by streaking on plates to obtain strain soilh02.

[0023] Identification of the strain: (1) Morphological identification: Strain soilh02 was repeatedly purified by streaking on plates until independent single colonies with uniform morphology were obtained. The morphological characteristics of the colonies on the plate culture medium were observed.

[0024] The results are shown in Figure 1 Strain soilh02 was a yellow round colony in LB solid medium, the edge of the colony was neat, the central part of the colony was convex, and the surface of the colony was smooth and moist Figure 1 (central A). Scanning electron microscopy Figure 1 (central B) showed that the strain soilh02 was uniform rod-shaped.

[0025] (2) Physiological and biochemical characteristics identification Strain soilh02 was subjected to Gram staining, catalase test, methyl red test, V-P test, oxidase test, starch hydrolysis, sugar fermentation test and acid-base test; the physiological and biochemical characteristics of strain soilh02 were determined. The specific test methods are as follows: ① Gram staining Bacterial suspension preparation: using a sterile inoculation ring to take a small amount of bacteria to be tested, and mix it with physiological saline previously added on the slide to make a uniform bacterial suspension. The prepared smear is fixed under the flame of an alcohol lamp.

[0026] Primary staining: add a suitable amount of crystal violet dye to cover the smear position, act for 1 minute, then pour off the dye and rinse with distilled water.

[0027] Mordanting: evenly add a suitable amount of iodine solution and act for 1 minute, then remove the dye and rinse with distilled water.

[0028] Decolorization: use 95% ethanol to decolorize the bacterial bodies, gently shake the slide for 20 seconds until no purple dye is eluted, and then terminate the decolorization with distilled water.

[0029] Counterstaining: apply saffron dye for counterstaining for 1 minute, and finally rinse thoroughly with distilled water.

[0030] Microscopic examination: wait for the test liquid on the slide to dry completely, then observe the Gram staining under an oil immersion lens. If the bacterial bodies appear blue, the test bacteria are considered to be Gram-positive bacteria (G+); if the bacterial bodies appear light red or purple, the test bacteria are considered to be Gram-negative bacteria (G-).

[0031] ②Catalase experiment First, inoculate the test strain on LB solid medium and incubate in a constant temperature incubator at 37°C for 48 h. Then, add a small amount of 3% hydrogen peroxide solution on the surface of the slide, use an inoculation loop to pick a small amount of bacterial bodies of the test bacteria, and smear them in the hydrogen peroxide solution. Observe whether bubbles are produced in the slide. If bubbles appear, the test bacteria are considered to be catalase experiment positive (+), otherwise negative (-).

[0032] ③Methyl red experiment Inoculate the test strain in a liquid medium containing glucose and proteose peptone, and incubate in a constant temperature incubator at 37°C for 2-3 days. After incubation, add methyl red indicator for color development. The result determination standard is: if the culture solution appears obvious red color, it is determined as methyl red test positive (+), if it remains yellow, it is determined as negative (-).

[0033] ④V-P experiment Inoculate the test strain in a liquid medium containing glucose and proteose peptone, and incubate in a constant temperature incubator at 37°C for 2-6 days. Then, mix equal volumes of the culture liquid and 40% sodium hydroxide solution uniformly, and add a suitable amount of creatine to react for 10 minutes. If red color appears, it is V-P test positive (+), but some color development changes need to be observed for a longer time, and no color development is determined as negative (-).

[0034] ⑤Oxidase experiment Oxidase test: 10-20 μL of distilled water was added to the oxidase test paper, and a small amount of the test bacteria was added to the oxidase test paper with a sterile glass rod or a sterile inoculation loop. If the test paper turned blue or blue-purple within 30 seconds, the oxidase test was positive (+), otherwise, the oxidase test was negative (-).

[0035] ⑥Starch hydrolysis Starch hydrolysis medium 63 g was dissolved in 1 L of distilled water, and sterilized at 121°C for 20 minutes. When the medium was cooled to about 60°C, it was poured into a plate. The test bacteria were inoculated into the starch-containing medium, and incubated at 37°C for 72 hours. Then, a small amount of Lugol's iodine solution was added to the colonies, and the plate was gently shaken. The color change of the iodine solution was observed. If the iodine solution did not turn blue, the test was positive (+), otherwise, the test was negative (-).

[0036] ⑦Sugar fermentation test The test bacteria were inoculated into media containing glucose, lactose, fructose, maltose, mannitol, and sucrose (the content of the sugar was kept at 0.5%), and incubated at 37°C for 24 hours. Then, a small amount of bromocresol purple indicator was added, and the color change was observed and recorded. If the test bacteria could ferment a certain sugar or alcohol, acid would be produced in the system, causing the color of the medium to change from purple to yellow, which was considered positive (+). If the test bacteria did not ferment sugar, no acid would be produced, and the system would remain purple, which was considered negative (-).

[0037] ⑧pH test The test bacteria were inoculated into LB broth with initial pH values of 4, 5, 6, 7, 8, and 9, and incubated at 37°C with shaking at 180 rpm for 24 hours. The growth of the test bacteria was observed. If the bacteria grew, the test was positive (+), otherwise, the test was negative (-).

[0038] The results are shown in Table 1 and Figure 1. Figure 2 After Gram staining, the bacteria of strain soilh02 appeared blue ( Figure 2 ). After adding a small amount of 3% H2O2, a large amount of bubbles was produced. After adding methyl red reagent, the culture solution turned red. In the v-p test, the culture solution did not turn red. In the carbon source utilization test, the results for lactose, glucose, fructose, maltose, and sucrose were positive, and the result for mannitol was negative. In the oxidase test and starch hydrolysis test, the oxidase test paper turned blue, and the iodine solution did not turn blue.

[0039] Table 1 Physiological and biochemical identification of strain soilh02 (3) Molecular biology identification The purified strain soilh02 was inoculated in LB broth medium, incubated at 37°C, 160 rpm for 12 h. The DNA was lysed by lysis solution using T5 Direct PCR Kit Plant, and the bacterial 16S rRNA sequence was amplified by selecting universal primer PNR (using 27F and 1492R), and the specific fragment amplified was sequenced, and the 16S rRNA sequence of the strain soilh02 was as shown in SEQ ID NO: 1.

[0040] SEQ ID NO: 1 (5'→3') The PCR reaction system was as follows: 27F 1 μL, 1492R 1 μL, Tamplate 2 μL, 2x T5 Direct PCR Mix (containing Taq enzyme, buffer, dNTP) 10 μL, Nuclease-Free Water 6 μL; The PCR amplification conditions were as follows: 98 ℃ 3 min; 98 ℃ 10 s, 55 ℃ 16 s, 72 ℃ 20 s, 35 cycles were set; 72 ℃ 5 min.

[0041] The amplified primers were as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2); 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 3); The 16S rRNA sequence of the strain soilh02 obtained by sequencing was subjected to comparative analysis of results in the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the sequence results with a homology higher than 97% were selected to determine the genus of the strain soilh02. After multiple sequence comparisons, the results were obtained using MEGA11.0 software, the maximum likelihood method was selected, and the phylogenetic tree of the strain soilh02 was constructed.

[0042] After the 16S rRNA sequence (1402 bp) of the strain soilh02 was subjected to Blast online comparison in the NCBI website, the results were as shown in Figure 3 The strain soilh02 had very high similarity with Arthrobacter citreus strain DSM 15046 strain. Through the constructed phylogenetic tree, the similarity of the strain soilh02 and Arthrobacter citreus strain DSM 15046 strain could reach 100%. Combined with the morphological and physiological and biochemical identification results, the strain soilh02 was identified as Arthrobacter citreus, and was preserved in the China General Microbiological Culture Collection Center on June 6, 2025, with a preservation number of CGMCC No. 34782 and a preservation address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard.

[0043] Example 2 The preparation method of the fermentation bacterial liquid with high efficiency of degrading AFB1 was as follows: (1) The preparation method of the seed culture solution of the strain soilh02 is as follows: a single colony of the strain soilh02 is inoculated into LB medium (10 g / L of tryptone, 10 g / L of sodium chloride, and 5 g / L of yeast extract) with pH 7.4, and is cultured at 37°C and 170 rpm for overnight, and is mixed with 40% sterile glycerol at a ratio of 1:1, and is stored at -20°C.

[0044] (2) The seed solution prepared above is inoculated into fermentation medium (5 g / L of beef extract, 10 g / L of protein peptone, 3 g / L of sodium chloride, 2 g / L of sodium phosphate dibasic, and 1 g / L of glucose) at an inoculation amount of 2%, and is cultured at 37°C and 180 rpm for 48 h, and the fermentation supernatant is obtained.

[0045] Example 3 The preparation method of the fermentation supernatant for degrading AFB1 is as follows: (1) The preparation method of the seed culture solution of the strain soilh02 is as follows: a single colony of the strain soilh02 is inoculated into LB medium (10 g / L of tryptone, 10 g / L of sodium chloride, and 5 g / L of yeast extract) with pH 7.4, and is cultured at 37°C and 170 rpm for overnight, and is mixed with 40% sterile glycerol at a ratio of 1:1, and is stored at -20°C.

[0046] (2) The seed solution prepared above is inoculated into fermentation medium (5 g / L of beef extract, 10 g / L of protein peptone, 3 g / L of sodium chloride, 2 g / L of sodium phosphate dibasic, and 1 g / L of glucose) with pH 7 at an inoculation amount of 2%, and is cultured at 37°C and 180 rpm for 48 h, and the fermentation supernatant is obtained.

[0047] Example 4 A method for degrading AFB1 is as follows: The fermentation supernatant of the strain soilh02 is mixed with a sample to be degraded at a volume ratio of 4:1 (if the fermentation supernatant of the strain soilh02 has been subjected to concentration, the volume ratio can be adjusted according to the concentration ratio), and is cultured at 37°C and 180 rpm for 72 h.

[0048] Effect of different inoculation amounts on the degradation of AFB1 by the fermentation supernatant of the strain soilh02 Based on the method in Example 2, five different inoculum concentration gradients (1%-5%, in 1% increments) were set up. The culture medium of strain soilh02 was sequentially inoculated into fermentation medium at pH 7 at the aforementioned five inoculum amounts. All cultures were incubated at 37°C and 180 rpm for 24 h. The degradation rate of AFB1 by the fermentation broth was determined using the following method: 800 μL of fermentation broth was mixed with 200 μL of AFB1 working solution (500 μg / L) and added to a 20 mL sterile EP tube to obtain a mixed bacterial solution with a final concentration of 100 μg / L AFB1. The mixture was incubated at 37℃ with shaking at 180 rpm in the dark for 72 h. The residual AFB1 concentration was determined using ELISA. 800 μL of sterile liquid fermentation medium and 200 μL of AFB1 working solution served as a control group. The residual concentration of AFB1 in the culture system was analyzed using ELISA.

[0049] Establishment of the standard curve: The logarithmic concentration of the AFB1 standard solution (log Cstandard) is set as the independent variable, and the percentage of the absorbance value of each standard well to the absorbance value of the zero-concentration well (Bstandard / B0) is used as the dependent variable to plot the standard curve. By measuring the absorbance values ​​of the test sample (Bexperimental group) and the blank test solution (Bcontrol group), the ratio of the absorbance to B0 is calculated, and the actual concentration (C) of AFB1 in the test sample can be found from the standard curve. The resulting standard curve ( Figure 4 The slope of the curve is -20.1075, and the intercept is 17.7585. Therefore, the standard curve is y = -20.1075x + 17.7585, R0. 2 The value is 0.9994.

[0050] The degradation rate of AFB1 can be obtained using the following formula: The effects of different inoculum amounts on the degradation of AFB1 by the fermentation broth of strain soilh02 are as follows: Figure 5 As shown, with the increase of inoculum amount, the degradation of AFB1 by the fermentation broth of strain soilh02 first increased and then gradually decreased. The degradation effect of AFB1 was the best when the inoculum amount was 2%, which was 81.42%.

[0051] Effect of different initial pH on the degradation of AFB1 by fermentation broth of strain soilh02 On the basis of the method of Example 2, seed liquid of strain soilh02 was inoculated into fermentation medium at an inoculation amount of 1%. All treatment groups were cultured at 37°C under constant temperature conditions at a shaking speed of 180 rpm for 24 h, and the initial pH of the culture medium was adjusted to 5, 6, 7, 8, and 9, respectively. After fermentation, the AFB1 degradation rate of the fermentation liquor (72 h) was determined. The determination method was the same as above. The results are shown in Figure 6 As the initial pH increased, the AFB1 degradation rate of the fermentation liquor of strain soilh02 first increased and then decreased, and the removal rate of AFB1 was the highest at a pH of about 8, which was 85.1%.

[0052] Effect of different fermentation temperatures on the AFB1 degradation effect of the fermentation liquor of strain soilh02 On the basis of the method of Example 2, seed liquid of strain soilh02 was inoculated into fermentation medium at an inoculation amount of 1%. All treatment groups were cultured at 37°C under constant temperature conditions at a shaking speed of 180 rpm for 24 h, and the initial pH of the culture medium was adjusted to 5, 6, 7, 8, and 9, respectively. After fermentation, the AFB1 degradation rate of the fermentation liquor (72 h) was determined. The determination method was the same as above. The results are shown in Figure 7 As the initial pH increased, the AFB1 degradation rate of the fermentation liquor of strain soilh02 first increased and then decreased, and the removal rate of AFB1 was the highest at a pH of about 8, which was 85.1%.

[0053] Effect of different fermentation times on the AFB1 degradation effect of the fermentation liquor of strain soilh02 On the basis of the method of Example 2, seed liquid of strain soilh02 was inoculated into fermentation medium at an inoculation amount of 1%. All treatment groups were cultured at 37°C under constant temperature conditions at a shaking speed of 180 rpm for 24 h, and the initial pH of the culture medium was adjusted to 5, 6, 7, 8, and 9, respectively. After fermentation, the AFB1 degradation rate of the fermentation liquor (72 h) was determined. The determination method was the same as above. The results are shown in Figure 8 As the initial pH increased, the AFB1 degradation rate of the fermentation liquor of strain soilh02 first increased and then decreased, and the removal rate of AFB1 was the highest at a pH of about 8, which was 85.1%.

[0054] Effect of different degradation times on the AFB1 degradation effect of the fermentation liquor of strain soilh02 On the basis of the method of embodiment 2, seed bacteria of strain soilh02 were inoculated into fermentation medium at pH 7 at an inoculation amount of 1%. The culture was carried out at 37°C under constant temperature conditions at a shaking speed of 180 rpm for 24 h to obtain the fermentation liquor of strain soilh02.

[0055] Take 800 μL of the fermentation bacteria and 200 μL of AFB1 working solution with a concentration of 500 μg / L, mix well, and add to a 20 mL sterile EP tube to obtain a mixed bacteria solution with a final concentration of 100 μg / L AFB1. Incubate at 37°C, 180 rpm, in the dark for 120 h. Test the degradation efficiency of AFB1 at different degradation times every 24 h until 120 h. A total of 5 samples were taken, and the determination method is as above.

[0056] The results are shown in Figure 9 As the degradation time increases, the degradation effect of strain soilh02 fermentation liquor on AFB1 shows a certain upward trend. When the degradation time reaches 72 h, the degradation rate of AFB1 of strain soilh02 fermentation liquor increases significantly, reaching 70.76%. However, after 72 h, with the extension of the degradation time, the degradation rate of AFB1 of strain soilh02 fermentation liquor no longer shows a significant increase. When the degradation time reaches 96 h and 120 h, the degradation rate of AFB1 has no significant difference (p>0.05). Therefore, after the degradation time exceeds 72 h, the degradation rate of AFB1 of strain soilh02 cannot be increased. Considering the time cost and the degradation effect, the optimal degradation time of strain soilh02 fermentation liquor for degrading AFB1 is determined to be 72 h.

[0057] In summary, the optimal fermentation conditions of strain soilh02 fermentation liquor are as follows: the degradation conditions are 2% inoculation amount, initial pH 8, fermentation temperature 37°C, and fermentation time 48 h. The optimal degradation time of AFB1 is 72 h. Under these conditions, the degradation rate of AFB1 can reach 87.63%.

[0058] Effect of metal ions on the degradation of AFB1 by strain soilh02 fermentation liquor On the basis of the method of Example 2, seed bacteria liquid of strain soilh02 was inoculated into fermentation medium with pH 8 at an inoculation amount of 2%. The culture was carried out at 37°C with a shaking speed of 180 rpm for 24 h. CaCl2, MgCl2, CuSO4·5H2O, FeCl3, and ZnSO4·7H2O were weighed and added into the fermentation liquid, respectively, and after being mixed thoroughly, the final concentration of metal ions reached 10 mM (the weight of CaCl2, MgCl2, CuSO4·5H2O, FeCl3, and ZnSO4·7H2O was 0.044 g, 0.038 g, 0.1 g, 0.065 g, and 0.115 g, respectively). The degradation rate of AFB1 in the fermentation liquid with added metal ions was determined by the same method as above.

[0059] The results are shown in Table 1. Figure 10 Compared with the degradation rate of 87.63% under the optimal degradation condition, the degradation rate of AFB1 changed significantly (p<0.05) after the addition of different metal ions. After the addition of Cu 2+ , the degradation rate of AFB1 increased significantly to 93.96%. After the addition of Ca 2+ and Fe 3+ , the degradation rate of AFB1 was low. After the addition of Mg 2+ and Zn 2+ , the degradation effect of AFB1 was slightly reduced. Among them, Ca 2+ had a strong inhibitory effect on the degradation of AFB1 by the fermentation liquid, and Cu 2+ had a significant promoting effect on the degradation of AFB1 by the fermentation liquid. Therefore, it is believed that Cu 2+ can be used as the optimal activator for the degradation of AFB1 by strain soilh02.

[0060] Effect of different components of fermentation liquid of strain soilh02 on the degradation of AFB1 Fermentation supernatant, inactivated cells, and cell lysate of the fermentation liquid of strain soilh02 were prepared, respectively, for determining the degradation effect of different components on AFB1.

[0061] The preparation method of fermentation supernatant was as follows: the fermentation bacteria liquid prepared by the method of Example 2 was centrifuged at 10000 rpm for 2 min to remove the bacterial precipitate, and the fermentation supernatant was obtained.

[0062] Preparation of inactivated cells: the fermentation bacteria liquid prepared by the method of Example 2 was centrifuged at 10000 rpm for 2 min to remove the supernatant, and the bacterial precipitate was obtained. Then, the bacterial precipitate was washed with sterile PBS for 2-3 times, resuspended in sterile PBS, and then the bacterial suspension was boiled at 100°C for 10 min to obtain the inactivated cell suspension.

[0063] Cell lysate preparation: the fermentation broth prepared by the method of Example 2 was centrifuged at 10000 rpm for 2 min to remove the supernatant, and the bacterial precipitate was washed with sterile PBS for 2-3 times, resuspended in sterile PBS, and then the bacterial cells were broken by ultrasonic cell disrupter. The broken solution was immediately centrifuged at 5000 rpm for 10 min, and the supernatant after centrifugation was the cell lysate.

[0064] 800 μL of fermentation supernatant, inactivated cell suspension or cell lysate was taken respectively, mixed with 200 μL of AFB1 working solution, incubated at 37℃, 180 rpm, and avoided light for 72 h, and then the AFB1 degradation rate was determined.

[0065] The results are shown in Table 1. Figure 11 As shown in Table 1, the AFB1 degradation rate of the supernatant of strain soilh02 was 67.56%, while the AFB1 degradation rates of the inactivated cells and the cell lysate were only 9.37% and 13.85%, respectively, so the active ingredient exists in the supernatant, which is an extracellular product of bacteria.

[0066] Degradation effect of fermentation broth of strain soilh02 in peanut meal The peanut cake was ground and dried at 65℃ overnight. After being fully dried, it was stored at room temperature. 5 g of peanut meal powder was taken and autoclaved at 121℃ for 20 min. After sterilization, 1 mL of AFB1 solution with a concentration of 100 ppb was added, mixed well, and then dried at 65℃ overnight. The fermentation broth prepared by the method of Example 2 was centrifuged at 10000 rpm for 2 min at 4℃, 4 mL of supernatant was taken, filtered through a sterile 0.22 μm filter, and then transferred to a new sterile EP tube for standby. The supernatant was added to the AFB1-containing peanut meal powder, incubated at 37℃, 160 rpm, and avoided light for 5 d. The fermented peanut was centrifuged at 5000 rpm for 15 min, dried at 65℃, and then weighed. 10 g of crushed sample was added to 20 mL of methanol solution, shaken for 3 min, centrifuged at 4000 rpm, filtered with filter paper, and then the supernatant was taken. The control group was mixed with 4 mL of fermentation medium and 5 g of AFB1-containing peanut meal powder, and then the same treatment was performed as above. The AFB1 degradation rate in the fermented peanut meal was 82.62% as determined by using the EILSA kit.

[0067] The above description is only a preferred embodiment of the present application, and does not limit the present application in other forms. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application, without departing from the technical solution of the present application, still falls within the protection scope of the present application.

Claims

1. A strain of *Arthrobacter citrinum*, characterized in that, The Arthrobactercitreus strain is Arthrobactercitreus soilh02, which was deposited on June 6, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34782.

2. The application of the Lemonobacterium citrinum according to claim 1 in the degradation of AFB1.

3. A formulation for degrading AFB1, characterized in that, The active ingredient contains fermentation broth or fermentation supernatant of Bacillus citrinum with preservation number CGMCC No. 34782.

4. The formulation for degrading AFB1 according to claim 3, characterized in that, The method for preparing the fermentation broth of *Arthrobacter citrinum* with accession number CGMCC No. 34782 is as follows: Seed culture of *Arthrobacter citrinum* with preservation number CGMCC No. 34782 was inoculated into fermentation medium at an inoculation rate of 1%-5%, with an initial pH of 5-9. Fermentation was carried out at 22℃-42℃ and 180 rpm for 12-54 hours.

5. The formulation for degrading AFB1 according to claim 4, characterized in that, The inoculum size of the seed culture was 2%; the initial pH of the culture medium was 8; the fermentation temperature was 37℃; and the fermentation time was 48 h.

6. The formulation for degrading AFB1 according to claim 4, characterized in that, The fermentation medium consisted of 5 g / L beef extract, 10 g / L peptone, 3 g / L sodium chloride, 2 g / L disodium hydrogen phosphate, and 1 g / L glucose.

7. The formulation for degrading AFB1 according to any one of claims 3-6, characterized in that, It further contains metal ions, wherein the metal ions are Cu. 2+ .

8. The formulation for degrading AFB1 according to claim 7, characterized in that, The final concentration of the metal ions is 10 mM.

9. A method for degrading AFB1, characterized in that, Mix the AFB1 degradation formulation according to any one of claims 3-6 with the sample to be degraded at a volume ratio of 4:1, and incubate at 37°C and 180 rpm in the dark for 24-120 hours to degrade.

10. The method for degrading AFB1 according to claim 9, characterized in that, The samples to be degraded were peanut meal and corn syrup.

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