Aflatoxin-degrading bacterial strain and application thereof

Abstract

The application discloses a strain for degrading aflatoxin and application thereof, and belongs to the technical field of microorganisms. The application screens a strain for efficiently degrading aflatoxin from a sea cucumber intestinal flora, and names the strain as DLWHY-1. The strain is preserved in the China General Microbiological Culture Collection Center on July 27, 2023, and the strain preservation number is CGMCC No. 28025. The strain is used for degrading aflatoxin in materials, such as grains, dairy products, bean products, nuts such as peanuts, traditional Chinese medicinal materials and feed, etc. After the supernatant fermented for 5 days is used for degrading aflatoxin, the toxin residual rate is reduced by nearly 90% compared with a blank control group, and the toxin residual rate is only about 10%. Compared with a non-microbial method, the process is green and environmentally friendly, the product is stable, and the product is non-toxic.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain that degrades aflatoxin and its applications. Background Technology

[0002] Aflatoxin (AFT) is often produced by Aspergillus flavus (… Aspergillus flavus Aflatoxin (AFT) is a highly toxic secondary metabolite produced in moldy grains and other contaminated materials. The main molecular forms of AFT include B, G, and M, with B being the most toxic. In 1993, the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) classified AFT as a Group 1 carcinogen. AFT has extremely strong carcinogenic effects, seriously threatening the health of humans, animals, and poultry. Research reports indicate a close relationship between AFT and liver cancer, and it can also cause symptoms such as tissue blood loss and anorexia.

[0003] Traditional AFT (Aflatoxin Free) detoxification methods include alkali treatment, oxidation treatment, antioxidants, filtration detoxification, high-temperature treatment, dilution of qualified raw materials, and chemical detoxification using Bata. These traditional methods have many problems, such as damaging feed nutrients, causing toxicity to animals, low detoxification efficiency, high cost, and secondary environmental pollution. Biological control technology is the most promising method for controlling aflatoxin contamination. In recent years, scholars have focused on researching biological control methods; therefore, screening for functional strains that inhibit aflatoxin growth and degrade and remove aflatoxins is particularly important. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a strain that degrades aflatoxin and its application. This invention enriches the members of aflatoxin-degrading bacteria, optimizes the fermentation conditions of aflatoxin-degrading strains, and can significantly reduce the aflatoxin residue rate in materials that are prone to Aspergillus growth, such as grains, dairy products, soy products, nuts such as peanuts, Chinese medicinal materials, and feed.

[0005] The objective of this invention is achieved through the following means:

[0006] This invention provides a strain of Bacillus laterosporus that degrades aflatoxin (Bacillus lateralis). Brevibacillus laterite DLWHY-1 was deposited on July 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28025; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In another aspect, the present invention provides an aflatoxin degrading agent, which is prepared by fermenting the above-mentioned Bacillus lateralis DLWHY-1.

[0008] Based on the above technical solution, the preparation process of the aflatoxin degrading agent further includes the following steps: dispensing the *Bacillus laterosporus* DLWHY-1 at a concentration of 1×10⁻⁶... 3 -1×10 10 Inoculate the culture medium with a concentration of 100 cells / mL and incubate aerobically at 25-35℃ and pH 5-7 for 24-120 h. Centrifuge the fermentation broth, discard the precipitate, and obtain the fermentation supernatant.

[0009] Based on the above technical solution, the carbon source in the fermentation medium further includes maltose, lactose, glucose, sucrose and fructose, and the nitrogen source includes yeast extract, peptone and beef extract.

[0010] Based on the above technical solution, the fermentation culture medium further comprises: 1-3% maltose, 0.5-3% yeast extract, and 0.5-1.5% sodium chloride.

[0011] Based on the above technical solution, further, the centrifugation conditions are 1-5℃, 6000-8000 rpm, and centrifugation for 5-30 minutes.

[0012] In another aspect, the present invention provides the application of the above-mentioned Bacillus lateralis DLWHY-1 and aflatoxin degrading agent in reducing aflatoxin in materials prone to Aspergillus flavus growth, wherein the materials prone to Aspergillus flavus growth include grains, dairy products, soy products, nuts, Chinese medicinal materials and feed.

[0013] This invention also provides a method for degrading aflatoxin in materials susceptible to Aspergillus flavus growth using the aforementioned Bacillus laterosporus DLWHY-1, which mainly includes the following steps:

[0014] (1) Inoculate the aforementioned Bacillus lateralis DLWHY-1 into the seed culture medium and culture for 5-12 h;

[0015] (2) Inoculate the bacterial culture obtained in step (1) into the fermentation medium at an inoculation volume ratio of 10-20% and culture for 3-5 days;

[0016] (3) Centrifuge the fermentation culture obtained in step (2) and collect the supernatant;

[0017] (4) Spray the supernatant obtained in step (3) or the fermentation liquid obtained in step (2) onto the material that is prone to Aspergillus flavus growth to degrade the aflatoxin in the material.

[0018] Based on the above technical solution, the materials that are prone to Aspergillus flavus growth include grains, dairy products, soy products, nuts, Chinese medicinal herbs, and feed.

[0019] Based on the above technical solution, the seed culture medium mentioned in step (1) further includes: yeast extract 0.5-2%, peptone 0.5-2%, and NaCl 0.5-1.5%.

[0020] Based on the above technical solution, the culture conditions in step (1) are further as follows: cultured in a shaker at 25-35℃ and 100-200rpm.

[0021] Based on the above technical solution, the culture conditions in step (2) are further as follows: cultured at 25-35℃, pH 5-7, and 100-200rpm in a shaker.

[0022] Based on the above technical solution, further, the centrifugation conditions in step (3) are 1-5℃, 6000-8000 rpm, and centrifugation for 5-30 min.

[0023] Based on the above technical solution, further, the specific process of step (4) is as follows: spray the supernatant obtained in step (3) or the fermentation liquid obtained in step (2) onto the silage material that is prone to aflatoxin growth, the spraying amount is 10-50 mL / kg of the material (wet weight) that is prone to aflatoxin growth, seal, and culture at 25-33℃ and pH 6-8 for 5-7 days to degrade the aflatoxin in the material that is prone to aflatoxin growth.

[0024] The advantages of this invention over the prior art are as follows:

[0025] This invention uses aflatoxin degradation rate as the detection standard, and optimizes screening and fermentation conditions to better suit application, resulting in a strain capable of efficiently degrading aflatoxin. This strain is then used to degrade aflatoxin in materials prone to Aspergillus growth, such as grains, dairy products, soy products, nuts like peanuts, medicinal herbs, and animal feed. The supernatant from 5 days of fermentation showed a nearly 90% reduction in aflatoxin residue compared to the control group, with a residue rate of only about 10%. Compared to non-microbial methods, this approach offers advantages such as a green and environmentally friendly process, stable products, and non-toxicity. Attached Figure Description

[0026] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0027] Figure 1 Image of plate culture of strain DLWHY-1.

[0028] Figure 2Images showing the morphological characteristics of strain DLWHY-1. Detailed Implementation

[0029] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and all materials and reagents used can be purchased from biological or chemical reagent companies. "Natural pH" means that the pH value of the culture medium is not adjusted during preparation.

[0031] 1. Culture medium

[0032] ① Basic culture medium: Each liter of distilled water contains 5g of yeast extract, 10g of NaCl, and 10g of peptone, with a natural pH; sterilize at 121℃ for 15 min.

[0033] ② Initial screening culture medium: 2g coumarin, 0.3g potassium dihydrogen phosphate, 1g calcium chloride, 0.2g MgSO4·7H2O, 1g ammonium sulfate, 0.01g ferrous sulfate heptahydrate per liter of distilled water, pH natural, sterilized at 121℃ for 15 min;

[0034] ③ Seed culture medium: Each liter of distilled water contains 10g of yeast extract, 10g of peptone, and 10g of NaCl, pH 7.0, sterilized at 121℃ for 15 min.

[0035] ④ Fermentation medium: Each liter of distilled water contains 20g of maltose, 15g of yeast extract, and 10g of sodium chloride. The pH is 7.0. Sterilize at 121℃ for 15 min.

[0036] 2. Methods for calculating viable cell count

[0037] Take 200 μL of bacterial culture suspension into an EP tube containing 800 μL of sterile water, mix thoroughly, and dilute 6 or 7 times in sequence. Then, take 200 μL of each of the different dilutions into a culture dish for solidified bacterial counting medium, spread evenly with a triangular rake, and incubate at 30°C for 24 h. Count the number of colonies and calculate the number of microorganisms in the bacterial solution.

[0038] Calculation formula: Viable bacteria count (original bacterial solution / mL) = Colony count per dilution plate × Dilution factor × 5

[0039] Culture medium for bacterial counting: 3g glucose, 9g yeast extract, 5g sodium chloride, and 2% agar per liter of distilled water, sterilized at 121℃ for 15 min.

[0040] 3. Determination of aflatoxin B1

[0041] The kit employs a one-step sandwich enzyme-linked immunosorbent assay (ELISA) using a double antibody. Sample / standard and HRP-labeled detection antibody are added sequentially to microwells pre-coated with aflatoxin B1 antibody. After incubation and thorough washing, the sample is developed with the substrate TMB. TMB is converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The intensity of the color is positively correlated with the aflatoxin B1 concentration in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the sample concentration.

[0042] Operating steps:

[0043] (1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a self-sealing bag and put them back at 4℃.

[0044] (2) Set up standard wells and sample wells, and add 50 μL of standard at different concentrations to each standard well;

[0045] (3) Add 10 μL of the sample to be tested to the sample well first, and then add 40 μL of sample diluent; do not add to the blank well.

[0046] (4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60 min.

[0047] (5) Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 5 times (or use a plate washer).

[0048] (6) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0049] (7) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0050] Result judgment:

[0051] Plotting the standard curve: In an Excel worksheet, plot the standard concentration on the x-axis and the corresponding OD value on the y-axis to create a linear regression curve for the standard. Calculate the concentration value of each sample according to the curve equation.

[0052] Example 1: Screening, isolation and identification of strain DLWHY-1

[0053] I. Isolation and Screening of Strains DLWHY-1

[0054] This invention isolates and screens bacterial strains from the intestinal flora of sea cucumbers, wherein the sea cucumbers are *Stichopus japonicus* (spiny sea cucumbers from Dalian). Apostichopus Japanese Live animals, purchased from Dalian Changxing Aquatic Products Wholesale Market.

[0055] (1) Preparation of mixed bacterial culture in sea cucumber intestine

[0056] The live sea cucumbers were washed with sterile water, and their intestines were dissected and removed in a sterile room. A portion of the intestines was mixed with a certain volume of sterile water, shaken thoroughly, centrifuged, and the supernatant was inoculated into 10 times the amount of basal culture medium. The mixture was then cultured in a shake flask at 35°C and 170 rpm for 48 hours to obtain a mixed bacterial solution of sea cucumber intestines. This solution was stored at 4°C for later use.

[0057] (2) Initial screening

[0058] The mixed bacterial culture of sea cucumber intestines prepared in step (1) was serially diluted and spread onto a primary screening medium plate. It was incubated upside down at 30°C until single colonies were formed. The bacteria that could grow on the primary screening plate were aflatoxin-degrading bacteria. Single colonies were observed and selected, transferred to another coumarin plate, and incubated at 35°C for 5 days. The colony morphology was then observed.

[0059] (3) Secondary screening

[0060] Select colonies from the initial screening plate, inoculate them into seed culture medium, and culture them in shake flasks at 35℃ and 180 rpm for 24 h. Then, inoculate them into fermentation culture medium at a 2% (v / v) inoculation rate and culture them in shake flasks at 30℃ and 180 rpm for 24 h to obtain bacterial culture broth. Centrifuge the bacterial culture broth and collect the supernatant.

[0061] The viable cell count in the culture medium was determined using the above method, and a strain with a good viable cell count was selected and named DLWHY-1.

[0062] (4) Preservation of microbial strains

[0063] The strain DLWHY-1 obtained in step (3) was cultured in seed culture medium, and the culture was made into 30% glycerol tubes and stored in a -80℃ refrigerator for later use.

[0064] II. Identification of strain DLWHY-1

[0065] (1) Morphological characteristics

[0066] like Figure 1-2As shown, the bacteria are Gram-positive, with slender rod-shaped cells, and occasionally oval spores can be observed; during the logarithmic growth phase, the cells become short and thick rod-shaped; peritrichous flagella are approximately 0.88 μm × 2.2 μm in size.

[0067] (2) Physiological and biochemical characteristics

[0068] Its physiological and biochemical characteristics include: glucose and mannitol produce acid, while arabinose and xylose do not produce acid or hydrolyze starch; it uses citrate and propionate to reduce nitrate to nitrite; it decomposes casein; it is stable at pH 5.0-8.0 and is not sensitive to light.

[0069] (3) Molecular identification results

[0070] 16S rDNA sequencing revealed a 16S rDNA sequence length of 2162 bp (see sequence listing for details). BLAST analysis showed that it is related to *Bacillus laterosporus* (…). Brevibacillus laterosporus The strain (E7593-50) showed the highest similarity, exceeding 99%. This strain is *Bacillus laterosporus*.

[0071] III. Preservation of strain DLWHY-1

[0072] Based on the above isolation, screening, and strain identification results, strain DLWHY-1 was confirmed to belong to the genus *Bacillus laterosporus*. Brevibacillus laterosporus) The novel strain, named DLWHY-1, was deposited on July 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28025; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0073] Example 2: Optimization of fermentation conditions for strain DLWHY-1

[0074] (1) Determination of culture medium composition

[0075] strain DLWHY-1 was used at 2×10 6 The inoculum concentration of 100 cells / mL was inoculated into fermentation media with different carbon sources, nitrogen sources, and inorganic salt types and contents. The media were cultured aerobically at 30℃ and pH 7.5 for 24 h. The viable cell count in the culture medium was determined according to the above method. The results are shown in Tables 1-4.

[0076] Table 1. Effects of different carbon sources on viable bacterial count

[0077]

[0078] Table 2. Effects of different nitrogen sources on viable bacterial count

[0079]

[0080] Table 3. Effect of carbon source content on viable bacterial count

[0081]

[0082] Table 4. Effect of nitrogen source content on viable bacterial count

[0083]

[0084] The strain DLWHY-1 described in this invention exhibits the highest viable cell count when the fermentation medium composition is: 3% maltose, 1.5% yeast extract, and 1% sodium chloride, indicating that this is the optimal culture medium composition for strain DLWHY-1.

[0085] (2) Determination of the optimal pH of the culture medium

[0086] strain DLWHY-1 was used at 2×10 6 The inoculum was inoculated into fermentation media with initial pH values ​​of 5.5, 6.5, 7.5, 8.5, and 9.5, respectively. After culturing in shake flasks at 30℃ and 170 rpm for 24 h, the viable cell count in the culture medium was compared and measured. The results are shown in Table 5.

[0087] Table 5. Effect of initial pH of culture medium on viable cell count

[0088]

[0089] As shown in Table 5, when the pH is 4-7, the number of viable bacteria in the culture medium increases slowly with the increase of pH value. When the pH is 8, the number of viable bacteria of this strain begins to decrease. Based on the above characteristics, the initial pH should be controlled at 4-7, preferably pH 7.

[0090] (3) Determination of the shaking speed

[0091] When culturing in 250 mL Erlenmeyer flasks, the strain DLWHY-1 was rotated at speeds of 90, 120, 150, 180, and 210 rpm / min, respectively, at a speed of 2×10⁻⁶ rpm. 6 The culture medium was inoculated at a concentration of 100 cells / mL, and after incubation in shake flasks at 35°C for 24 h, the number of viable bacteria in the culture medium was compared and measured. The results are shown in Table 6.

[0092] Table 6. Effect of shaker speed on viable cell count

[0093]

[0094] Due to the low solubility of oxygen in liquids, the shaking speed determines the dissolved oxygen level. This strain is clearly an aerobic bacterium; the number of viable bacteria is lower when the shaking speed is below 100 rpm compared to higher speeds. Simultaneously, the number of viable bacteria is highest when the flask volume is 180 rpm / min. Therefore, it can be determined that the optimal dissolved oxygen level for culturing strain DLWHY-1 in a 250 mL Erlenmeyer flask is 180 rpm / min.

[0095] (4) Determination of the optimal culture temperature

[0096] strain DLWHY-1 was used at 2×10 6 The concentration of bacteria / mL in culture medium was inoculated into the fermentation medium and cultured in shake flasks at 25℃, 30℃, 35℃, 40℃ and 45℃ for 24 h at 180 rpm. The viable bacteria count in the culture medium and the collagenase activity in the centrifuged supernatant were compared and measured. The results are shown in Table 7.

[0097] Table 7. Effect of culture temperature on viable cell count

[0098]

[0099] As shown in Table 7, the number of viable bacteria varies greatly between 25℃ and 35℃, reaching its highest level at 35℃ and then decreasing at 40℃. Based on this result, the optimal culture temperature for strain DLWHY-1 is determined to be 30-35℃, preferably 35℃.

[0100] (5) Determination of the optimal culture time

[0101] strain DLWHY-1 was used at 2×10 6 The culture medium was inoculated with a concentration of 1000 cells / mL and cultured in shake flasks at 35°C. The OD of the bacterial culture was measured after 12, 24, 36, 48 and 72 h. The results are shown in Table 8.

[0102] Table 8 Effect of incubation time on viable cell count

[0103]

[0104] As shown in Table 8, the number of viable bacteria in the culture medium reached stability between 12 and 24 hours. Based on the above results, it was determined that strain DLWHY-1 reached the stable period after 24 hours of culture.

[0105] Example 3: Experiment on the degradation of aflatoxin by strain DLWHY-1

[0106] Aflatoxin degradation test: strain DLWHY-1 was subjected to a degradation rate of 2 × 10⁻⁶. 6Inoculate the culture medium with a concentration of 100 cells / mL and incubate aerobically at 30°C and pH 7 for 48 h. Centrifuge the fermentation broth at 4°C and 8000 rpm for 10 min, discard the precipitate, and obtain the fermentation supernatant.

[0107] Take 1 mL of sterile fermentation supernatant containing aflatoxin (aflatoxin B1 content of 32.11 ppb), add 1 mL of fermentation supernatant of strain DLWHY-1, and react in a shaker at 25℃ and pH 5 for 48 h. Degrade the aflatoxin in the sample; refer to the aflatoxin B1 determination method for specific detection methods. 1 mL of sterile water served as a blank control. The effects of varying reaction temperatures, pH values, reaction times, and the fermentation time of strain DLWHY-1 on the aflatoxin degradation rate were investigated. The results are shown in Tables 9-12.

[0108] Table 9. Effects of fermentation broth at different reaction temperatures on aflatoxin degradation rate.

[0109]

[0110] Table 10 Effect of fermentation broth at different reaction pH on aflatoxin degradation rate

[0111]

[0112] Table 11 Effect of fermentation broth at different reaction times on aflatoxin degradation rate

[0113]

[0114] Table 12 Effect of fermentation time of strain DLWHY-1 on aflatoxin degradation rate

[0115]

[0116] The table above shows that the reaction conditions between the fermentation broth and the toxin have a significant impact on the degradation of aflatoxin. The highest degradation rate was achieved when the fermentation time was 5 days, the supernatant reacted with aflatoxin for 5 days, the temperature was 30℃, and the pH was 7. Under the combined conditions, the degradation rate of aflatoxin in the fermentation broth reached 88.23%.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Bacillus laterosporus that degrades aflatoxin ( Brevibacillus laterosporus DLWHY-1, characterized in that, It was deposited on July 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28025; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. An aflatoxin degrading agent, characterized in that, The aflatoxin degrading agent is prepared by fermentation of Bacillus lateralis DLWHY-1 as described in claim 1.

3. The aflatoxin degrading agent according to claim 2, characterized in that, The preparation process of the aflatoxin degrading agent includes the following steps: Bacillus laterosporus DLWHY-1 is added at a concentration of 1×10⁻⁶... 3 -1×10 10 Inoculate the culture medium with a concentration of 100 cells / mL, and culture aerobically at 25-35℃ and pH 5-7 for 24-120 h. Centrifuge the fermentation broth, discard the precipitate, and obtain the fermentation supernatant.

4. The aflatoxin degrading agent according to claim 3, characterized in that, The carbon sources in the fermentation medium include maltose, lactose, glucose, sucrose, and fructose, while the nitrogen sources include yeast extract, peptone, and beef extract.

5. The aflatoxin degrading agent according to claim 3, characterized in that, Centrifugation conditions: 1-5℃, 6000-8000 rpm, centrifugation for 5-30 min.

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