Application of Lactobacillus coryniformis subsp. coryniformis in highly efficient degradation of aflatoxin

Abstract

The present invention discloses an application of Lactobacillus coryniformis subsp. coryniformis in efficiently degrading aflatoxin. The application method is as follows: Inoculate Lactobacillus coryniformis subsp. coryniformis 523L5 into an LB liquid medium containing aflatoxin B1, and carry out a light-avoiding reaction at 25-55 °C and pH 3-9 to degrade aflatoxin B1. Lactobacillus coryniformis subsp. coryniformis 523L5 of the present invention has good growth performance, relatively high acid-producing ability, strong salt tolerance and acid tolerance, and can tolerate a salinity of about 7%. It can grow under the condition of pH 4.5 and has good fermentation performance. Its fermentation broth can efficiently degrade aflatoxin B1 under the conditions of slightly acidic (pH 5-7) and relatively high temperature (50-55 °C), and the highest degradation rate can reach 88.13% at 40 °C and pH 7.0.

Description

(1) Technical Field

[0001] The present invention relates to the field of microbial detoxification, and in particular to the application of Lactobacillus coryniformis subsp. coryniformis in the efficient degradation of aflatoxin. (2) Background Art

[0002] Aflatoxins are secondary metabolites with polyketide-like biological activities produced by Aspergillus flavus, Aspergillus parasiticus and other Aspergillus species. As one of the five major mycotoxins, aflatoxins are the main source of food and feed contamination, and are widely present in food crops such as wheat, corn, sorghum, peanuts, soybeans, cassava, as well as spices, fruits, milk, meat, fermented products and feeds. Among them, aflatoxin B1 (AFB1) is the most widely contaminated and most toxic toxin, with nephrotoxicity, hepatotoxicity and immunotoxicity. At the same time, it is also carcinogenic and teratogenic, and is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization. The wide contamination and toxicity of AFB1 pose a serious threat to the health and safety of humans and animals and the development of agriculture. Therefore, there is an urgent need for strategies to remove AFB1.

[0003] Traditional physical methods such as high temperature, non-thermal methods like non-thermal plasma, electron beam radiation, and pulsed light, as well as physical adsorption methods are safe and reliable but have poor degradation effects on toxins (Yousefi M, Mohammadi MA, Khajavi MZ et al., Application of novel non-thermal physical technologies to degrade mycotoxins, Journal of fungi (Basel), 2021.). Chemical methods convert toxins through chemical reactions. Common methods include hydrolysis, ammoniation, oxidation-reduction, etc. (Pankaj SK, Shi H, Keener KM, A review of novel physical and chemical decontamination technologies for aflatoxin in food. Trends in Food Science & Technology, 2018.), but chemical substances will remain, affecting the nutritional value of food and even causing secondary toxic effects. Biodegradation refers to the ability of microorganisms and certain substances they produce to interact with toxins, change their original structures, and convert them into low-toxic or non-toxic substances (Guan Y, Chen J, Nepovimova E et al., Aflatoxin Detoxification Using Microorganisms and Enzymes. Toxins, 2021.). In recent years, the technology of biological detoxification has gradually become popular. Compared with physical and chemical detoxification methods, biological detoxification is safer and more effective and can also retain the nutritional value and flavor quality of food.

[0004] Lactic Acid Bacteria (LAB) are a type of microorganisms widely existing in nature, and they have important applications in the food industry, especially in the production of fermented foods. In recent years, studies have found that certain lactic acid bacteria (including Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus kefiri, etc.) have the ability to remove aflatoxins. The main mechanisms of action include cell wall adsorption and degradation of metabolites. Among them, cell wall adsorption refers to the interaction between components such as peptidoglycan, carbohydrates, and phosphates on the cell wall and the functional groups of aflatoxin B1, and the toxin is bound through physical adsorption, ion exchange, and complexation (Asurmendi P, Gerbaldo G, Pascual L, Barberis L., Lactic acid bacteria with promising AFB1 binding properties as an alternative strategy to mitigate contamination on brewers’ grains. Journal of Environmental Science and Health Part B - Pesticides Food Contamin, 2020.). Organic acids are metabolites produced by lactic acid bacteria, and they achieve antifungal effects by hindering fungal metabolic activity (Saelim K, Jampaphaeng K, Maneerat S., Functional properties of Lactobacillus plantarum S0 / 7 isolated fermented stinky bean (Sa Taw Dong) and its use as a starter culture. Journal of Functional Foods, 2017.). In addition to organic acids, lactic acid bacteria also produce antimicrobial peptides, which destroy the integrity of the cell membrane by interacting with lipids on the cell membrane, achieving the effect of inhibiting bacteria (Muhialdin BJ, Algboory HL, Kadum H, et al., Antifungal activity determination for the peptides generated by Lactobacillus plantarum TE10 against Aspergillus flavus in maize seeds. Food Control, 2020.).In addition, the detoxification activity of the fermentation supernatant of some lactic acid bacteria was significantly reduced after treatment with proteinase K, indicating that there is a certain protein or enzyme in the fermentation supernatant that can degrade aflatoxin B1 into non-toxic substances (Yingchao Z, Peng W, Qing K, Peter J, Biotransformation of aflatoxin B1 by Lactobacillus helviticus FAM22155 in wheat bran by solid-state fermentation. Food Chemistry, 2021.).

[0005] In the patent "A Strain S262 Degrading Aflatoxin B1 and Its Application" (CN113430128A) by Liu Na et al., a Bacillus sonorensis S262 isolated from pig feces was provided. The degradation rate of aflatoxin B1 after 24 h was 45.5%, and after 72 h it was 84.32%. A better degradation effect needed to be achieved by increasing the temperature or prolonging the degradation time. In the patent "A Strain Degrading Aflatoxin and Its Cultivation and Application" (CN117004504A) by Zhang Xiaojing et al., a Bacillus amyloliquefaciens ZJSY6 isolated from soil was screened. The degradation rate of aflatoxin B1 after 24 h was 55.87%, and after 72 h it was 87.72%. This strain showed a positive result in the pH 5.7 positive experiment, but a better degradation effect needed to be achieved by prolonging the degradation time. The strains mentioned in the above patents could not efficiently degrade aflatoxin B1, and their applicable ranges were limited.

[0006] Therefore, it is necessary to screen a microbial strain with a wider applicable range of temperature and pH conditions and more capable of efficiently degrading aflatoxin B1. (III) Summary of the Invention

[0007] The object of the present invention is to provide an application of Lactobacillus coryniformis subsp. coryniformis in the efficient degradation of aflatoxin. The fermentation supernatant of Lactobacillus coryniformis subsp. coryniformis can effectively degrade aflatoxin B1, and the highest degradation rate can reach 88.13%. Moreover, this strain has good acid resistance and can play a better degradation role under the conditions of pH 5 - 7. At the same time, it has good heat resistance and can still play a degradation role at 50 - 55°C, solving the problem that other microorganisms cannot efficiently play a degradation role under acidic and relatively high temperature conditions.

[0008] The technical solution adopted by the present invention is as follows:

[0009] The present invention provides an application of Lactobacillus coryniformis subsp. coryniformis in the efficient degradation of aflatoxin.

[0010] Furthermore, the Loigolactobacillus coryniformis subsp. coryniformis is Loigolactobacillus coryniformis subsp. coryniformis 523L5, which is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 31070, the deposit date is June 24, 2024, and the deposit location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] Furthermore, the method of the application is as follows: inoculate Loigolactobacillus coryniformis subsp. coryniformis 523L5 into an LB liquid medium containing aflatoxin B1, and carry out a light-avoiding reaction at 25 - 55 °C and pH 3 - 9 to degrade aflatoxin B1.

[0012] Furthermore, the concentration of aflatoxin B1 in the LB liquid medium is 0.05 - 2 μg / mL.

[0013] Furthermore, the conditions of the light-avoiding reaction are 37 °C, pH 7, and reacting for 48 h.

[0014] Furthermore, before inoculating Loigolactobacillus coryniformis subsp. coryniformis 523L5, it is first inoculated into an LB liquid medium and cultured in a shaker at 37 °C for 24 h for activation, and then inoculated into an LB liquid medium containing aflatoxin B1 at an inoculation amount of 1% (v / v).

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0016] Loigolactobacillus coryniformis subsp. coryniformis 523L5 of the present invention has good growth performance, high acid production ability, strong salt tolerance ability and acid tolerance ability, and can tolerate a salinity of about 7%. It can grow under the condition of pH 4.5 and has good fermentation performance. Its fermentation broth can efficiently degrade aflatoxin B1 under the conditions of slightly acidic (pH 5 - 7) and relatively high temperature (50 - 55 °C), and the highest degradation rate can reach 88.13% at 40 °C and pH 7.0. (IV) Description of the Drawings

[0017] Figure 1 Colony (a) and cell morphology diagram (b) of strain 523L5.

[0018] Figure 2 Phylogenetic tree of strain 523L5.

[0019] Figure 3 Growth curves of strain 523L5 in LB medium and LB medium supplemented with aflatoxin B1.

[0020] Figure 4, Growth curve of strain 523L5 under different pH conditions.

[0021] Figure 5 , Single colony of strain 523L5 on solid medium with coumarin as the sole carbon source.

[0022] Figure 6 , HPLC chromatograms of strain 523L5 before and after removing aflatoxin B1. The peak at 20 min is the peak of aflatoxin B1.

[0023] Figure 7 , Degradation rate of aflatoxin B1 by strain 523L5 under different pH conditions.

[0024] Figure 8 , Degradation rate of aflatoxin B1 by strain 523L5 under different temperature conditions.

[0025] Figure 9 , Degradation rate of aflatoxin B1 by strain 523L5 over time.

[0026] Figure 10 , Degradation rate of aflatoxin B1 with different concentrations by strain 523L5.

[0027] Figure 11 , Degradation rate of aflatoxin B1 by the fermentation supernatant of strain 523L5 and its treatment with heat and proteinase K. (V) Specific implementation modes

[0028] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all obtained from commercial channels unless otherwise specified.

[0030] The media used are as follows:

[0031] 1) MRS solid medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 5.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, ammonium citrate hydrogen 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 mL, agar 14.0 g, pH 6.5 ± 0.2, dissolved in 1000 mL distilled water, sterilized at 121 °C for 15 min.

[0032] 2) Coumarin solid medium: 0.25 g of coumarin, 0.25 g of ammonium sulfate, 0.625 g of potassium dihydrogen phosphate, 0.0625 g of magnesium sulfate heptahydrate, 0.125 g of disodium hydrogen phosphate, 0.025 g of calcium chloride, 0.25 mg of ferrous sulfate heptahydrate, 5 g of agar, dissolved in 250 mL of distilled water, autoclaved at 121 °C for 15 min, and poured into plates and cooled.

[0033] 3) LB liquid medium: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, dissolved in 1000 mL of distilled water, autoclaved at 121 °C for 15 min.

[0034] 4) Nitrite degradation test medium: Add 2 mL of 5 mg / mL NaNO2 aqueous solution to 50 mL of MRS liquid medium.

[0035] Example 1: Screening and identification of Lactobacillus coryniformis subsp. coryniformis 523L5

[0036] 1. Preparation of "Fragrant throughout" pickled vegetables

[0037] Wash fresh mustard stems in running tap water, drain, cut into appropriate sized pieces, blanch by boiling, and after cooling, weigh 1.2 kg of mustard stems and put them into a 3 L pickling jar. At the same time, add 1% chili and 1% garlic by mass fraction (based on the weight of the mustard stems). Add 3% brine solution to cover the mustard stems, seal, and place at room temperature (25 - 30 °C) for fermentation for 3 months. After fermentation is completed, trained team members score the color, texture, aroma, and taste of the fermented pickled vegetables according to Table 1. Pickled vegetables with a total score greater than 70 are defined as excellent fermented pickled vegetables and used as samples for isolating strains in the following steps.

[0038] Table 1 Scoring criteria

[0039]

[0040] 2. Primary screening:

[0041] Serial dilute the "Fragrant throughout" pickled vegetable brine from step 1 with sterile normal saline by 10 - fold gradients. Pipette 100 μL of 10 0 (i.e., the original solution), 10 -3 , 10 -4 samples of two dilution degrees, and evenly spread them on the MRS solid medium plate with a sterile spreading rod. After culturing at 37 °C for 48 h, pick colonies with a calcium - dissolving circle and different morphologies onto the MRS solid medium plate and streak repeatedly to finally obtain single colonies, denoted as strains 523L1, 523L5, and 523L12. Store the purified strains in 20% glycerol.

[0042] 3. Re - screening:

[0043] Use a sterile inoculation loop to pick up a loop of the bacterial liquid stored in the glycerol tube in step 2 and streak it on an MRS solid medium plate. After culturing at 37 °C for 48 h, pick up a loop of single colonies and transfer them into a test tube containing MRS liquid medium. Mix well and culture at 37 °C for 24 h. Measure the bacterial density (OD600nm) and pH value of the samples at 12 h and 24 h respectively. The results are shown in Table 2.

[0044] Take 2 mL of the activated culture solution (OD 600nm = 1) and inoculate it into 50 mL of the sterilized nitrite degradation test medium. After culturing at 37 °C for 70 h, use the N-(1-Naphthyl)ethylenediamine dihydrochloride method to measure the nitrite content, and calculate the nitrite degradation rate of each strain. The results are shown in Table 2.

[0045] At the same time, spread the activated strains on MRS solid media containing different mass concentrations (3%, 4%, 5%) of NaCl and 2% mass concentration of calcium carbonate. Culture at 37 °C for 48 h. The appearance of a calcium dissolution zone around the colonies indicates that the strain has salt tolerance. The results of the NaCl concentration that the strain can tolerate are shown in Table 2.

[0046] Table 2 Determination results of the fermentation performance of each strain

[0047]

[0048]

[0049] Among them, strain 523L5 has good growth ability, strong acid production ability, salt tolerance ability, and strong nitrite degradation ability.

[0050] 4. Identification of strain 523L5

[0051] Morphological identification: Pipette 100 μL of the bacterial liquid stored in the glycerol tube and streak it on an MRS solid medium plate. After culturing at 37 °C for 48 h, observe the colony morphology with the naked eye. After Gram staining, observe the cell morphology under an optical microscope. The colony and cell morphology of strain 523L5 are as Figure 1 shown. The colonies are observed to be round, semi-transparent, white, moist and smooth, with neat edges with the naked eye. Under the optical microscope, the strain appears blue-violet and belongs to Gram-positive bacteria, rod-shaped.

[0052] Molecular identification: The selected strains were inoculated into test tubes containing MRS liquid medium and cultured in a shaker at 37 °C for 16 h to harvest the bacteria. DNA was extracted according to the bacterial genomic DNA extraction kit. Using this DNA as a template, the full-length 16S rDNA sequence was amplified with universal bacterial primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'). After the PCR products passed the inspection, they were sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. Homology alignment was performed by BLAST, and a phylogenetic tree was constructed using MEGA 10.0, as Figure 2 shown.

[0053] The 16S rDNA sequence (SEQ ID NO.1) was imported into NCBI for BLAST homology alignment. The results showed that the strain 523L5 had 100% similarity with Loigolactobacillus coryniformis subsp. coryniformis. Combining the morphological characteristics, the strain 523L5 was identified as Loigolactobacillus coryniformis subsp. Coryniformis, and was deposited in the China General Microbiological Culture Collection Center at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 31070 and the deposit date of June 24, 2024.

[0054] Example 2: Fermentation performance test of Loigolactobacillus coryniformis subsp. 523L5

[0055] 1. Growth curve determination

[0056] Growth ability is an important parameter for evaluating starter cultures, and the growth curve can intuitively understand the growth pattern of bacteria.

[0057] Loigolactobacillus coryniformis subsp. 523L5 was inoculated into LB liquid medium and cultured in a shaker at 37 °C for 24 h for activation. The activated bacterial solution was inoculated into LB liquid medium containing 1 μg / mL aflatoxin B1 at an inoculation amount of 1% (V / V) and cultured statically at 37 °C for 72 h. Samples were taken every 1 h within 0 - 7 h and 25 - 48 h, and every 15 min during the middle logarithmic growth phase (i.e., 7 - 25 h) to measure the bacterial density (OD 600nm ). Under the same conditions, the LB liquid medium containing 1 μg / mL aflatoxin B1 was changed to LB liquid medium, and other operations were the same.

[0058] As Figure 3As shown in the figure, from 0 to 4 h, Lactobacillus coryniformis subsp. coryniformis 523L5 was in the growth lag phase and grew slowly; after about 6 h, it entered the logarithmic growth phase and the number of bacterial cells increased rapidly; after about 15 h, the growth slowed down and it began to enter the stationary phase. The growth status in the medium containing aflatoxin B1 was similar to that in the medium without the toxin added.

[0059] Therefore, Lactobacillus coryniformis subsp. coryniformis 523L5 has good growth ability, and the presence of aflatoxin B1 does not affect its growth.

[0060] 2. Effect of pH on the growth of strain 523L5

[0061] The strain 523L5 was activated by culturing in LB liquid medium at 37 °C for 24 h, and then inoculated into LB liquid media with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 at an inoculation amount of 1% (V / V), and statically cultured at 37 °C for 72 h. Samples were taken every 1 h within 0 - 7 h and 25 - 48 h, and every 15 min during the middle logarithmic growth phase (i.e., 7 - 25 h), and the bacterial density (OD 600nm ) of the samples was measured.

[0062] As Figure 4 shown, when the pH is less than 4.0, Lactobacillus coryniformis subsp. coryniformis 523L5 can hardly grow normally; when the pH rises to 5, Lactobacillus coryniformis subsp. coryniformis 523L5 is less inhibited; when the pH rises above 6.0, Lactobacillus coryniformis subsp. coryniformis 523L5 can grow normally, and when the pH reaches above 8 (i.e., under alkaline conditions), its growth is significantly lower than that at pH 6 and 7.

[0063] This indicates that strain 523L5 has good acid tolerance and its growth status is better under acidic to slightly neutral conditions than under alkaline conditions.

[0064] Example 3. Application of Lactobacillus coryniformis subsp. coryniformis 523L5 in degrading aflatoxin B1

[0065] 1. Growth ability of strain 523L5 on coumarin medium

[0066] The single colony of Lactobacillus coryniformis subsp. coryniformis 523L5 screened and identified in Example 1 was inoculated into LB liquid medium and cultured on a shaker at 37 °C for 48 h. After sufficient enrichment, 200 μL of the enriched bacterial liquid was spread on a coumarin solid medium with a coumarin concentration of 1 g / L and cultured at 37 °C for 3 - 7 days until visible colonies appeared. Single colonies were isolated and then transferred to fresh coumarin solid medium plates and cultured repeatedly 3 times to obtain single colonies that could grow using coumarin as a carbon source, as Figure 5 shown.

[0067] 2. Identification of the Degradation Ability of Lactobacillus coryniformis subsp. coryniformis 523L5 to Aflatoxin B1

[0068] Lactobacillus coryniformis subsp. coryniformis 523L5 was inoculated into LB liquid medium and cultured in a shaker at 37 °C for 24 h for activation. Then it was inoculated into LB medium containing 1 μg / mL aflatoxin B1 and pH 7.0 at an inoculation amount of 1% (V / V). The group without inoculating bacteria was used as the control group. They were reacted in the dark in a shaker at 37 °C for 36 h, and a total of five parallel groups were made. After the reaction, the supernatant was collected by centrifugation, vortexed with an equal volume of methanol for 60 s, filtered through a 0.22 μm filter membrane, and the content of aflatoxin B1 in the filtrate was detected by high performance liquid chromatography (HPLC).

[0069] The conditions of high performance liquid chromatography (HPLC) were as follows: mobile phase A was pure water, mobile phase B was a mixture of methanol and acetonitrile with a volume ratio of 1:1, and the volume ratio of phase A to phase B was 50:50; the chromatographic column was a C18 column; the flow rate was 0.8 mL / min; the injection volume was 50 μL; the column temperature was 40 °C, the excitation wavelength was 360 nm, the emission wavelength was 440 nm, and post-column derivation was carried out.

[0070]

[0071] In the formula, A0 is the content of aflatoxin B1 in the control group, and A1 is the content of aflatoxin B1 in the experimental group

[0072] Figure 6 The high performance liquid chromatography diagrams before and after the degradation of aflatoxin B1 by strain 523L5 are shown. The peak time of aflatoxin B1 is about 20 min. After 24 h, the degradation rate of aflatoxin B1 reached 52.20%.

[0073] 3. Effects of Different pH Conditions on the Degradation of Aflatoxin B1 by Strain 523L5

[0074] Strain 523L5 was inoculated into LB liquid medium and cultured in a shaker at 37 °C for 24 h for activation. Then it was inoculated into LB medium containing 1 μg / mL aflatoxin B1 and pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 at an inoculation amount of 1% (v / v). The group without inoculating bacteria was used as the control group. They were reacted in the dark in a shaker at 37 °C for 24 h. After the reaction, the supernatant was collected by centrifugation, vortexed with an equal volume of methanol for 60 s, filtered through a 0.22 μm filter membrane, and the content and degradation rate of aflatoxin B1 in the filtrate were detected by liquid chromatography.

[0075] As Figure 7As shown, when the pH is less than 4.0, there is almost no degradation effect on aflatoxin B1; after the pH is greater than 5.0, as the pH value increases, the degradation rate of aflatoxin B1 shows an upward trend, reaching a maximum of 54.84% at pH 7.0; while after the pH is greater than 8, the degradation effect on aflatoxin B1 shows an obvious downward trend.

[0076] 4. Effects of different temperature conditions on the degradation of aflatoxin B1 by strain 523L5

[0077] Inoculate strain 523L5 into LB liquid medium and culture it in a shaker at 37°C for 24 h for activation. Then inoculate it into LB medium containing 1 μg / mL aflatoxin B1 and pH 7.0 at an inoculation amount of 1% (v / v). The group without inoculating bacteria serves as the control group. React in the dark in a shaker at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 55°C for 48 h respectively. After the reaction, centrifuge to collect the supernatant, vortex it with an equal volume of methanol for 60 s, filter it through a 0.22 μm filter membrane, and then detect the content of aflatoxin B1 in the filtrate by liquid chromatography and calculate the degradation rate.

[0078] As Figure 8 shown, when the temperature is lower than 40°C, the degradation rate of aflatoxin B1 shows an upward trend, reaching a maximum of 88.13% at 40°C. After the temperature is higher than 50°C, the growth of strain 523L5 is inhibited, but it can still exert a degradation effect of 76.07%. This indicates that a certain heat-resistant active substance in the fermentation broth plays a major degradation role.

[0079] 5. Effects of different incubation times on the degradation of aflatoxin B1 by strain 523L5

[0080] Inoculate strain 523L5 into LB liquid medium and culture it in a shaker at 37°C for 24 h for activation. Then inoculate it into LB medium containing 1 μg / mL aflatoxin B1 and pH 7.0 at an inoculation amount of 1% (v / v). The group without inoculating bacteria serves as the control group. React in the dark in a shaker at 37°C for 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h respectively. After the reaction, centrifuge to collect the supernatant, vortex it with an equal volume of methanol for 60 s, filter it through a 0.22 μm filter membrane, and then detect the content of aflatoxin B1 in the filtrate by liquid chromatography and calculate the degradation rate.

[0081] As Figure 9 shown, as the incubation time increases, the degradation rate of aflatoxin B1 by strain 523L5 shows an upward trend, reaching a maximum of 74.21% at 48 h. After 60 h, the degradation rate of aflatoxin B1 shows a slightly decreasing trend, which may be due to the inhibition of the growth of strain 523L5, and part of the degradation effect comes from the adsorption of the bacterial cells.

[0082] The reaction solutions after reacting for 24 h, 48 h, and 72 h were sampled and centrifuged respectively. The precipitate was resuspended with methanol, sonicated at 300 W for 10 min, and centrifuged again. Then, the supernatant was taken to detect the content of aflatoxin B1. The results are shown in Table 3.

[0083] Table 3 Content of aflatoxin B1 in the precipitate of 523L5 bacteria after 24 h, 48 h, and 72 h

[0084]

[0085] As shown in Table 3, 523L5 bacteria had a small amount of adsorption effect on bacteria, and it decreased significantly at 72 h.

[0086] 6. Degradation effect of 523L5 strain on aflatoxin B1 with different concentrations

[0087] The 523L5 strain was inoculated into LB liquid medium and cultured in a shaker at 37 °C for 24 h for activation. Then, it was inoculated into LB medium containing 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, and 2 μg / mL aflatoxin B1 and pH 7.0 at an inoculation amount of 1% (v / v). The group without inoculating bacteria was used as the control group, and they were reacted in the dark in a shaker at 37 °C for 24 h. After the reaction, the supernatant was collected by centrifugation, vortexed with an equal volume of methanol for 60 s, filtered through a 0.22 μm filter membrane, and the content of aflatoxin B1 in the filtrate was detected by liquid chromatography to calculate the degradation rate.

[0088] As Figure 10 shown, with the increase in the concentration of aflatoxin B1, the ability of 523L5 strain to degrade aflatoxin B1 showed an upward trend, and the degradation rate of 2 μg / mL aflatoxin B1 reached the maximum value of 59.82%.

[0089] 7. Degradation effect of the fermentation supernatant of 523L5 strain and its treatment with proteinase K on aflatoxin B1

[0090] Inoculate strain 523L5 into LB liquid medium and culture it in a shaker at 37°C for 24 h for activation. Then inoculate it into LB medium containing 1 μg / mL aflatoxin B1 and with a pH of 7.0 at an inoculation amount of 1% (v / v), and react in the dark in a shaker at 37°C for 48 h. After centrifuging at 10,000 rpm for 10 min, collect the fermentation supernatant and cell precipitate respectively. Resuspend the precipitate with 5 volumes of 1 M PBS buffer. Take out 2 mL of the fermentation supernatant and place it in a 5 mL centrifuge tube, and keep it in a boiling water bath for 20 min to obtain a heat-denatured supernatant. Then take another 2 mL of the fermentation supernatant and place it in a 5 mL centrifuge tube, add 1 μg / mL proteinase K, mix well, and let it stand at room temperature for 20 min to obtain a proteinase K-treated supernatant.

[0091] Add 1 μg / mL aflatoxin B1 to 1 mL of PBS buffer, and then add 50 μL of the fermentation supernatant (denoted as FS), the heat-denatured supernatant (denoted as FS-T), the proteinase K-treated supernatant (denoted as FS-PrK), and the resuspended cell precipitate (denoted as cells) respectively. After mixing well, react in the dark in a shaker at 37°C for 24 h. After the reaction, centrifuge to collect the supernatant, vortex it with an equal volume of methanol for 60 s, filter it through a 0.22 μm filter membrane, and detect the content of aflatoxin B1 in the filtrate by liquid chromatography to calculate the degradation rate.

[0092] As Figure 11 shown, the main active component for the degradation of aflatoxin B1 by strain 523L5 is in the fermentation supernatant, and the degradation rate is as high as 69.29%. After heat denaturation treatment and proteinase K treatment, the degradation rate decreases significantly, indicating that the substance playing a degradation role in the fermentation supernatant is a certain proteinaceous substance or a certain enzyme.

Claims

1. An application of Lactobacillus coryneformis subspecies coryneformis in the efficient degradation of aflatoxin, characterized in that: The Lactobacillus coryneformis subspecies is Lactobacillus coryneformis subspecies ( Loigolactobacillus coryniformis subsp. coryniformis )523L5, the deposit number is CGMCC No.31070.

2. The use according to claim 1, characterized in that The application method is: inoculating Lactobacillus coryneformis subspecies 523L5 into LB liquid culture medium containing aflatoxin B1, and performing light-proof reaction under the conditions of 25-55° C. and pH 5.0-9.0 to degrade aflatoxin B1.

3. The use according to claim 2, characterized in that The concentration of aflatoxin B1 in the LB liquid culture medium is 0.05-2 μg / mL.

4. The use according to claim 2, characterized in that The light-proof reaction conditions are 37° C., pH 7.0 and reaction time for 48 hours.

5. The use according to claim 2, characterized in that The Lactobacillus coryneformis subspecies 523L5 was first inoculated into LB liquid culture medium before inoculation, cultured in a shaking incubator at 37° C. for 24 h for activation, and then inoculated into LB liquid culture medium containing aflatoxin B1 at an inoculation volume concentration of 1%.

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