A strain of Penicillium hoeksii MD1 and its application

Abstract

The present invention relates to a strain of Penicillium herquei MD1, whose strain collection number is CGMCC No. 40418. The present invention also relates to the application of the strain in degrading aflatoxin B1. The Penicillium herquei MD1 screened out by the present invention can efficiently degrade aflatoxin B1, and the strain can biodegrade food crops and feed contaminated by aflatoxin B1, which is greener and more environmentally friendly than physical and chemical degradation, and is easy to cultivate, simple to operate, and easy to implement.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to a strain of Penicillium hoeksei MD1 and application thereof. Background Art

[0002] Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic fungal toxin. Studies have shown that AFB1 contamination is widely present in moldy and spoiled feed, food and grain crops, and the scope of contamination is still expanding, causing serious harm and huge economic losses to the food industry and livestock and poultry farming. Therefore, it is crucial to find a method that can degrade or even eliminate AFB1. There are many methods for aflatoxin removal, which are mainly divided into physical, chemical and biological methods. Physical methods include heating, radiation, and the use of adsorbents; chemical methods include adding food preservatives and fumigating with strong oxidants such as chlorine dioxide. However, the above methods will cause food to deteriorate and produce chemical residues during the food processing process, which will cause a series of safety problems, making it difficult to implement and promote these technologies. In order to reduce AFB1, not produce residual toxicity, ensure nutritional value, and not change the characteristics of food or feed, researchers began to explore biological detoxification methods. Using microorganisms, especially microorganisms with probiotic properties, to detoxify AFB1 is a green, efficient, environmentally friendly and safe method. Therefore, finding a biodegradable bacterium that can degrade AFB1 is of great significance for the biological control of aflatoxin contamination in cereals and animal feed. Summary of the invention

[0003] The first object of the present invention is to provide a strain of Penicillium herquei MD1 to solve the problem that there is currently no microbial strain that can effectively degrade aflatoxin B1.

[0004] The second object of the present invention is to provide the use of the above-mentioned Penicillium herquei MD1.

[0005] The present invention is achieved through the following technical solutions:

[0006] 1. A strain of Penicillium herquei MD1, whose strain accession number is CGMCC No.40418.

[0007] 2. Application of the above-mentioned Penicillium herquei MD1 in the degradation of aflatoxin B1.

[0008] The present invention screened out a strain that can efficiently degrade AFB1 from 62 fungal strains preserved in the laboratory, and the degradation rate of AFB1 is as high as 99.2%. Through morphological and ITS rDNA gene identification, the strain MD1 is determined to be Penicillium herquei. The components of Penicillium herquei MD1 that can effectively degrade AFB1 are located in the bacterial body, and the degradation active components can be separated after the bacterial body is ground and crushed at 5000r / min.

[0009] The positive effects of adopting the above technical scheme: Penicillium herquei MD1 screened out by the present invention can efficiently degrade aflatoxin B1. The strain can biodegrade food crops and feed contaminated by aflatoxin B1, which is greener and more environmentally friendly than physical and chemical degradation, and is easy to cultivate, simple to operate and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Colony morphology (A) and characteristics under microscope (B) and scanning electron microscope (C) of strain MD1;

[0011] Figure 2 Electrophoresis pattern of ITS rDNA gene PCR products of strain MD1;

[0012] Figure 3 Phylogenetic tree of strain MD1;

[0013] Figure 4 Degradation of AFB1 by Penicillium hoeksii MD1 suspension (A), bacterial cells (B), spores (C), and fermentation liquid (D);

[0014] Figure 5 The degradation effect of broken cells of Penicillium hoeksii MD1 on AFB1;

[0015] Figure 6 Degradation of AFB1 by Penicillium hoeksii MD1 at different rotation speeds.

[0016] The Penicillium herquei MD1 involved in the present invention was deposited with a patent procedure on November 10, 2022 in a deposit center recognized by the China Patent Office or the International Patent Organization. The full name of the deposit unit is the General Microbiology Center of the China Microorganism Culture Collection Administration, abbreviated as CGMCC. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Deposit Number: CGMCC No.40418. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below in conjunction with specific embodiments, but it should not be construed as limiting the present invention:

[0018] Example 1

[0019] This example illustrates the screening of Penicillium hoeksii MD1.

[0020] (1) The strain was streaked onto a PDA plate and cultured in a biochemical incubator. When the mycelium grew well and produced a large number of spores, the spores were washed off and the concentration of the spore solution was adjusted to 10 6 CFU / mL;

[0021] (2) The spore suspension in step (1) was inoculated into a sterile PDB medium containing AFB1 at an inoculum volume of 2% (volume fraction) (the final concentration of AFB1 was 100 ng / mL), and a sterile PDB solution containing the same AFB1 mass concentration was used as a blank control. The culture was carried out at 28°C and 160 rpm for 72 h. Each treatment group was replicated three times.

[0022] (3) detecting the AFB1 content of the sample in step (2), and selecting the strain with the highest AFB1 degradation rate as the target strain;

[0023] (4) observing the bacterial strains screened in step (3) by colony morphology, microscopy and scanning electron microscopy, and performing molecular biological identification by PCR amplification;

[0024] (5) Isolating bacterial suspensions, bacterial cells, spore suspensions, and fermentation broths of degrading bacteria, and testing their degradation effects on AFB1;

[0025] (6) breaking the degradable bacterial cells and detecting the degradation effects of the broken bacterial cells and intracellular fluid on AFB1;

[0026] (7) Detect the degradation effect of broken bacteria and intracellular fluid at different rotation speeds on AFB1.

[0027] The results are shown in Table 1.

[0028] Table 1 Screening results of aflatoxin B1-degrading strains

[0029]

[0030] As shown in Table 3, the degradation rate of AFB1 by MD1-degrading bacteria was the highest, reaching more than 99%.

[0031] Example 2

[0032] This example illustrates strain identification as well as morphological identification.

[0033] (1) The strain was inoculated on a PDA plate and cultured at 28°C for 5 days. The spores were washed with saline and the concentration of the spore solution was adjusted to 106 CFU / mL. 2% of the spore solution was inoculated into PDB medium containing AFB1 (AFB1-PDB). The final concentration of AFB1 in AFB1-PDB was 100 ng / mL. AFB1-PDB without spore solution was used as a blank control. After culturing at 0 h and 28°C, 160 r / min for 72 h, samples were taken to detect the AFB1 content (ng / mL);

[0034] (2) Take 1 mL of the liquid in (1) and filter it through a 0.22 μm filter membrane and store it at -20°C for testing. The LC-MS-MS conditions are: WATERS H-Class / Xevo TQ-S tandem quadrupole LC / MS, C-18 chromatographic column (2.1 mm×100 mm, 1.7 μm), mobile phase 0.1% formic acid water: methanol = 1:1 (V / V); injection volume 0.5 μL, flow rate 0.4 mL / min, column temperature 40°C, gradient elution as shown in Table 2; mass spectrometry conditions are ESI+ ionization mode, capillary voltage 3 KV, temperature 500°C, nebulizer gas flow rate 1000 L / Hr, acquisition mode MRM (multiple reaction monitoring mode). The mass spectrum information of AFB1 is shown in Table 3.

[0035] Table 2 Mobile phase gradient elution program

[0036]

[0037] Table 3 Retention time, monitoring ions and mass spectrometry acquisition parameters of AFB1

[0038]

[0039] (3) Prepare a series of AFB1 standard working solutions with concentrations of 5, 25, 50, 100, 200, and 300 ng / mL using methanol. The standard curve equation obtained by testing is y=2940.82x+51823.3, R 2 =0.9979, indicating that AFB1 has good linearity in the range of 5-300 ng / mL.

[0040] The degradation rate of AFB1 was calculated using the following formula:

[0041] Degradation rate (%) = (1-AFB1 concentration after reaction / initial AFB1 concentration) × 100%

[0042] The residual rate of AFB1 is calculated using the following formula:

[0043] Residual rate (%) = AFB1 concentration after reaction / initial AFB1 concentration × 100%

[0044] (4) Purify the target strain and inoculate the purified strain on the PDA plate. After culturing at 28°C for 3 days, observe the colony morphology. Insert the cover glass at a 45-degree angle into the PDA plate, inoculate the spore solution between the cover glass and the culture medium, and after culturing at 28°C for 3 days, remove the cover glass and observe the morphology of hyphae and sporangium using a microscope and scanning electron microscope.

[0045] like Figure 1 As shown, the degrading bacteria grow on potato dextrose agar medium as yellow-green circular colonies with a small amount of radial grooves that make the colonies appear petal-shaped, with a velvety and slightly flocculent texture, green conidia, and yellow hyphae ( Figure 1 A). Observed under a microscope, conidiophores are generated in the matrix and aerial hyphae. The broom branches are typically arranged in two whorls, occasionally in a single whorl or irregularly. There are 5-8 or more conidiophores in each whorl, which are closely attached to each other and are basically the same length ( Figure 1 B). Under a scanning electron microscope, conidia are oval in shape, or nearly spindle-shaped, with smooth or slightly rough walls, and loose and irregularly entangled conidia chains ( Figure 1 C).

[0046] Example 3

[0047] This example illustrates the molecular biological identification of the strain.

[0048] PCR amplification was performed using the universal primers for fungi ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′, ITS4: 5′-TCCTCCGCTTATTGATATGC-3′. The PCR reaction system was 25 μL: 2×Taq Mix 12.5 μL, primers ITS1 and ITS4 1 μL each, a small amount of PDA plate MD1 mycelium, and ddH2O 10.5 μL. PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ pre-denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, 30 cycles; 72℃ extension for 10 min. 1.5% agarose gel electrophoresis was used to detect the PCR amplification product. The 600 bp PCR amplification product was purified using a DNA purification kit, connected to the T Vector vector, and transformed into competent cells (DH5α Competent Cells). Primer synthesis and sequencing of the T vector-connected PCR product were completed by Shanghai Sangon Biotechnology Co., Ltd. The sequencing results were compared using BLAST in the NCBI system to construct a phylogenetic tree.

[0049] like Figure 2 As shown in FIG. 1 , the length of the target fragment of the degrading bacteria measured by agarose gel electrophoresis of the PCR amplification product is 579 bp; Figure 3As shown, MEGA7 was used to construct a phylogenetic tree, and the analysis results showed that the degradation bacteria had the highest homology with Penicillium hoeksii, which was 99%. Combined with the morphological characteristics of the degradation bacteria, it can be determined that it is Penicillium hoeksii, belonging to the genus Penicillium.

[0050] Example 4

[0051] 2% spore suspension was inoculated into PDB medium, cultured at 28℃, 160r / min for 3 days, and the cells and fermentation liquid were collected by filtration every 12h. The cells were rinsed with PBS (0.01mol / L pH 7.2-7.4) 3 times, and the fermentation liquid was filtered through a 0.22μm filter for use. The cells were resuspended in AFB1-PDB, 990μL of fermentation liquid was added with 10μL 100ng / mL AFB1 standard stock solution, and sampled after culturing at 28℃, 160r / min for 24h; 2% spore liquid was inoculated into AFB1-PDB, cultured at 28℃, 160r / min for 3 days, and sampled every 12h; a total of 10mL of physiological saline containing different volumes of spore liquid (0, 1, 3, 5, 7, 9mL) was added with AFB1 to make the final concentration 100ng / mL. After culturing at 28°C and 160 r / min for 24 h, samples were taken; AFB1-PDB was used as a blank control, the AFB1 content was detected, and the AFB1 residual rate was calculated.

[0052] like Figure 4 As shown in the figure, the degradation bacteria can significantly degrade AFB1 when growing in PDB medium. The mass concentration of AFB1 begins to decrease after 36 hours. The degradation rate is fastest at 36-48 hours, and the residual rate of AFB1 decreases from 83.2% to 21.6%. At 72 hours, the residual rate of AFB1 is only 1.7%. The results show that the degradation bacteria can effectively degrade AFB1 during the growth process ( Figure 4 a). The growth of the mycelial pellets of the degradation bacteria increased with the extension of the culture time, and the residual rate of AFB1 decreased from 90.1% at the beginning to 28.27%. The results showed that the mycelial pellets of the degradation bacteria can effectively degrade AFB1 ( Figure 4 b). The degradation effect of the spore suspension of degrading bacteria on AFB1 was not significantly different from that of the untreated group, and the mass concentration of AFB1 did not change significantly with the increase of the spore suspension content, indicating that the spore suspension had no degradation effect on AFB1 ( Figure 4 c). The degradation effect of the fermentation broth of the degrading bacteria on AFB1 showed a trend of increasing first and then decreasing over time, but there was no significant difference compared with the untreated group, indicating that the fermentation broth had no degradation effect on AFB1 ( Figure 4 d).

[0053] Example 5

[0054] 2% spore solution was inoculated into PDB, and then divided into 4 parts after shaking, numbered A, B, C, and D, and cultured at 28°C and 160r / min for 3 days, and centrifuged at 10000r / min for 10min to obtain a precipitate, and rinsed with PBS 3 times. A and B were not crushed, and C and D were ground and crushed in a mortar and resuspended in PBS containing AFB1 (AFB1-PBS). The final concentration of AFB1 in AFB1-PBS was 100ng / mL. A and C were taken 0h, and B and D were taken 24h later. 10mL of the extractant was added, and the extractant was acetonitrile: water: acetic acid = 80:19:1 (V / V / V), shaken for 2min, and placed at 4°C for 15min. 3mL of the supernatant was taken and added with 0.5g of dispersed solid phase extraction filler, shaken for 2min, centrifuged at 10000r / min for 1min, and 1mL of the supernatant was taken and filtered through a 0.22μm filter membrane and stored at -20°C for testing. The AFB1 content detection method is the same as above.

[0055] like Figure 5 As shown in the figure, the AFB1 content of the unbroken system decreased by 55.25 ng / mL after 24 hours, and the AFB1 content of the broken system decreased by 73.25 ng / mL after 24 hours. The AFB1 content of the unbroken system decreased by 18 ng / mL more than that of the broken system after 24 hours. It can be inferred that strain MD1 may have two degradation mechanisms: one is that there are enzymes with degradation activity in the bacterial cell wall; the other is that two or more enzymes in the bacterial cell wall and intracellular fluid degrade AFB1 through combined action.

[0056] Example 6

[0057] Inoculate 2% spore suspension into PDB medium, culture at 28℃, 160r / min for 3 days, centrifuge at 10000r / min for 10min to obtain a precipitate, rinse 3 times with PBS. Grind the precipitate in a mortar containing quartz sand, add PBS to make the volume 20mL, pipette 5mL into 3 centrifuge tubes, centrifuge at 1000, 5000, and 10000r / min for 10min, take the precipitate and resuspend it in PBS-AFB1, and take samples for testing after 24h.

[0058] like Figure 6As shown in the figure, the broken bacteria were dissolved in PBS and separated at 1000r / min, 5000r / min, and 10000r / min. The precipitates obtained were redissolved in PBS. After 24h of reaction, the AFB1 contents were 88.0ng / mL, 81.2ng / mL, and 81.4ng / mL, respectively. It can be seen that with the increase of the rotation speed, the active ingredients in the precipitate increased. The AFB1 content of the 1000r / min system decreased significantly compared with the 5000r / min system, but the AFB1 content of the 5000r / min system was not much different from that of the 10000r / min system. The results show that the active ingredients that effectively degrade AFB1 in strain MD1 can be separated at 5000r / min.

[0059] The Penicillium herquei MD1 screened out by the present invention can efficiently degrade aflatoxin B1. The strain can biodegrade food crops and feed contaminated by aflatoxin B1, is greener and more environmentally friendly than physical and chemical degradation, and is easy to cultivate, simple to operate, and easy to implement.

Claims

1. A strain of Penicillium herquei MD1, whose strain collection number is CGMCC No.40418.

2. Use of Penicillium herquei MD1 according to claim 1 in degrading aflatoxin B1.

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