Fermentation probiotic lactobacillus plantarum for reducing aflatoxin B1 and application of fermentation probiotic lactobacillus plantarum

Abstract

The invention belongs to the field of microorganisms, and particularly discloses a fermentation probiotic lactobacillus plantarum strain for reducing aflatoxin B1 and application of the fermentation probiotic lactobacillus plantarum strain. The lactobacillus plantarum YS-718 is preserved in the China Center for Type Culture Collection on November 15, 2023, the preservation number is CCTCC NO.M 20232226, and the lactobacillus plantarum YS-718 is preserved in the China Center for Type Culture Collection (CCTCC) for short. The lactobacillus plantarum YS-718 is screened and separated from traditional fermented pickled Chinese cabbages in Liuzhou, aflatoxin B1 can be effectively reduced, and the lactobacillus plantarum YS-718 has a good reduction effect on aflatoxin B1 in peanut meal. The separated microorganisms belong to a probiotic microorganism catalog allowed to be added into feed in China, and can be applied to detoxification of aflatoxin B1 in feed.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a fermented probiotic Lactobacillus plantarum capable of reducing aflatoxin B1 and an application thereof. Background Art

[0002] Aflatoxins are mainly toxic secondary metabolites produced by Aspergillus fungi such as Aspergillus flavus and Aspergillus parasiticus. They are teratogenic, mutagenic and carcinogenic. The main common types are B1, B2, M1, M2, G1, G2, etc. Among these toxins, aflatoxin B1 (AFB1) is considered to be the most common, most toxic and most harmful toxin. Its toxicity is about 10 times that of potassium cyanide and 68 times that of arsenic. It can easily contaminate food crops such as peanuts and corn, seriously threatening the health of humans and animals, and causing great economic losses in grain and oil. Fermented feed is centered on microbial fermentation, which transforms and decomposes anti-nutritional factors and macromolecules in feed raw materials to produce feed raw materials with good palatability and high nutritional value. Peanut meal is also an important fermented feed raw material. Through microbial fermentation, it has the following advantages: 1) Degrading large molecular weight proteins in peanut meal to obtain rich small peptides, thereby improving its digestion and absorption rate in animals; 2) Effectively reducing or removing antigenic components and trypsin inhibitors in peanut meal; 3) Fermented peanut meal has a unique aroma, which can greatly improve the flavor and quality of feed.

[0003] Screening of fermented probiotics that have aflatoxin B1-reducing effect is of great significance for the safe and efficient degradation of aflatoxins and the high-quality utilization of peanut meal. Summary of the invention

[0004] The present invention mainly aims at the lack of fermented probiotic strains for efficiently and safely reducing aflatoxin B1 in feed in the prior art, and provides a fermented probiotic Lactobacillus plantarum for reducing aflatoxin B1 and its application. The Lactobacillus plantarum YS-718 of the present invention can effectively reduce aflatoxin B1 and has a good reduction effect on aflatoxin B1 in peanut meal.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides Lactobacillus plantarum YS-718 (Lactobacillus plantarum), which was deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on November 15, 2023, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO.M 20232226.

[0007] The plant lactobacillus YS-718 of the present invention is isolated from Liuzhou traditional fermented pickled cabbage. Screening method: take an appropriate amount of 5.00g of Liuzhou traditional pickled cabbage in 50mL of MRS liquid culture medium, shake and culture on a shaker at 37°C and 200rpm / min; after the solution becomes turbid, collect the supernatant and store it.

[0008] Take 1 mL of the supernatant from the above culture and prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The dilution was taken, 100 μL of the dilution was taken, and spread on the MRS agar medium, and cultured at 37°C for 12-24h (3 parallels for each dilution), and the growth of the colonies on the plate was observed. The single colonies that were clearly visible were selected, and the inoculation loop was used to pick out and transferred to the primary screening medium containing coumarin for plate streaking. The strains with better growth were picked out and transferred to the MRS agar medium, and separated and purified many times, and a total of 10 pure strains with aflatoxin reduction were obtained, of which 3 were plant lactobacillus. By comparing their effects on degrading toxins, and referring to the catalog of probiotics allowed to be added to feed, a plant lactobacillus strain with the best effect (plant lactobacillus YS-718) was finally screened out and preserved in the China Center for Type Culture Collection, referred to as CCTCC, with a preservation number of CCTCCNO.M 20232226, and classified as: Lactobacillus plantarum YS-718.

[0009] The strain was identified using 16S rDNA specific amplification technology, and the results showed that it was a strain of Lactobacillus plantarum, which belongs to the family of Lactobacillaceae and is included in the national list of probiotics allowed to be added to feed.

[0010] The second aspect of the present invention provides an aflatoxin biotreatment agent, comprising the fermentation broth of the above-mentioned Lactobacillus plantarum YS-718.

[0011] The third aspect of the present invention provides the application of the above-mentioned Lactobacillus plantarum YS-718 in reducing aflatoxin. The specific application method is: contacting the fermentation liquid of Lactobacillus plantarum YS-718 with a biological sample to reduce aflatoxin B1 in the biological sample.

[0012] According to the above scheme, the application method is: adding the fermentation liquid of Lactobacillus plantarum YS-718 to sterilized peanut meal to ferment and reduce aflatoxin. The fermentation conditions are 37±1°C and fermentation for 48-72h.

[0013] According to the above scheme, the fermentation liquid of Lactobacillus plantarum YS-718 may or may not be heat-treated before being added to the sample to be treated. The heat treatment is carried out at 98±1°C.

[0014] According to the above scheme, the final concentration of the Lactobacillus plantarum YS-718 fermentation broth is (1-9)×108 CFU / mL.

[0015] According to the above scheme, the preparation method of the Lactobacillus plantarum YS-718 fermentation broth is as follows: Lactobacillus plantarum YS-718 is activated in MRS agar, cultured in a 37° C. incubator for 48 hours, a single colony of Lactobacillus plantarum YS-718 is picked up with an inoculation needle, transferred to MRS broth (MRS liquid culture medium, used for selective enrichment of lactic acid bacteria in food), and cultured with shaking for 12 hours to 24 hours to obtain the fermentation broth of the fermented probiotic Lactobacillus plantarum YS-718.

[0016] Beneficial effects of the present invention:

[0017] The plant lactobacillus YS-718 of the present invention can effectively reduce aflatoxin B1 and has a good reducing effect on aflatoxin B1 in peanut meal.

[0018] The microorganism plant lactobacillus separated by the present invention belongs to the catalog of probiotic microorganisms allowed to be added to feed by the state, and can be applied to peanut meal fermentation to detoxify aflatoxin B1. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0020] Figure 1 The colony morphology of Lactobacillus plantarum YS-718 of the present invention is shown. Figure 1 The middle left picture shows the single bacterial morphology of Lactobacillus plantarum YS-718, and the right picture shows its enlarged colony.

[0021] Figure 2 The figure shows the reduction effect of Lactobacillus plantarum YS-718 on AFB1 in the blank spiked experiment of peanut meal in Example 2 (the spiked concentration of aflatoxin B1 is 20 ng / g). DETAILED DESCRIPTION

[0022] Preferred embodiments of the present invention will be described in more detail below.

[0023] The present invention adopts a high performance liquid chromatography analysis method to measure the AFB1 content.

[0024] Example 1 Isolation and Identification of Lactobacillus plantarum YS-718

[0025] (1) Isolation of Lactobacillus plantarum

[0026] Lactobacillus plantarum was isolated from traditional pickled cabbage in Liuzhou

[0027] Primary screening culture medium containing coumarin: (NH4)2SO4 0.5g, KH2PO4 0.5g, KNO3 0.5g, MgSO4·7H2O0.25g, FeCl3·6H2O 0.003g, CaCl2·2H2O 0.005g, agar 20g, coumarin 1.0g, pH 7.2, sterilization at 121℃ for 20min.

[0028] (2) Isolation and purification of strains

[0029] Take an appropriate amount of 5.00 g of Liuzhou traditional pickled cabbage in 50 mL of MRS liquid culture medium, and culture it on a shaker at 37°C and 200 r / min; after the solution changes from clear to turbid, collect the supernatant and save it.

[0030] Take 1 mL of the supernatant from the above culture and prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Take 100 μL of the dilution and spread it on MRS agar medium. Culture it at 37°C for 12-24 hours (three parallels for each dilution), and observe the growth of the colonies on the plate. Select single colonies with good growth, pick them out with an inoculation loop and transfer them to the primary screening medium containing coumarin for plate streaking. Pick out single colonies with good growth and transfer them to MRS agar medium. Separate and purify them multiple times to obtain pure strains.

[0031] The single colonies on the above plates were picked up and inoculated into MRS liquid culture medium, and cultured on a shaker at 37°C and 200 r / min for 12 h, and 100 μL of 200 ng / mL AFB1 standard solution was added to make the final concentration of AFB1 in the fermentation broth 20 ng / mL, and enriched and cultured on a constant temperature shaker at 37°C and 200 r / min for 96 h, and the supernatant was taken to detect the corresponding AFB1 content. A total of 10 strains with the potential to reduce aflatoxin were obtained. After repeated tests on the reduction performance of these 10 strains, a strain with the best reduction performance was obtained, named YS-718.

[0032] Example 2 Identification of Lactobacillus plantarum YS-718

[0033] (1) Colony morphology of Lactobacillus plantarum

[0034] Bacterial morphology study: Colony morphology Figure 1The main biological characteristics of the strain are: after growing on MRS agar medium for 48 hours, the colonies are round, moist, milky white, and the surface of the colonies is smooth and easy to pick up.

[0035] The edges are neat.

[0036] (2) Molecular biological identification of Lactobacillus plantarum YS-718

[0037] The total DNA of the YS-718 strain was extracted, and PCR amplification was performed using bacterial 16S rDNA universal primers, and the amplified product was sequenced, as shown in SEQ ID: NO 1. According to the homologous sequence comparison analysis of the NCBI database, the homology between the YS-718 strain and Lactobacillus plantarum (KR153313.1) was 100%, and the strain was identified as Lactobacillus plantarum and named Lactobacillus plantarum YS-718.

[0038] SEQ ID: NO 1 (5'→3')

[0039] CCTGTACGACTTCACCCTAATCATCTGTCCCACCTTAGGCGGCTGGTTCCTAAAAGGTT

[0040] ACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGG

[0041] CCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCA

[0042] TGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTAC

[0043] TCCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCA

[0044] TAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGGTTTGTCACCGGCAGTCT

[0045] CACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGG

[0046] ACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCAT

[0047] GTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAG

[0048] GTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTC

[0049] AATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTA

[0050] GCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATG

[0051] GACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTA

[0052] CAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGC

[0053] TACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTT

[0054] CTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTA

[0055] CGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGT

[0056] AGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATA

[0057] TGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTT

[0058] GCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTT

[0059] TGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACTACGTATCATT

[0060] GCCATGGGTGAGCCGTTACCCCACCCATCTAGCTAATACGCCGCGGGACCATCCAAAAGT

[0061] GATAGCCGAAGCCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATT

[0062] AGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCAC

[0063] CAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTT

[0064] CGTTCGACTTGCATGTATTAGGCACGCCGCA

[0065] Example 3 Method for detecting AFB1 in peanut meal

[0066] The detection method of AFB1 in the present invention is as follows: weigh 5.00g sterilized peanut meal into a 50mL centrifuge tube, add 25mL acetonitrile-water solution (V / V=84:16), shake at 2500r / min for 1h, centrifuge at 5000r / min for 10min, take the supernatant and filter it with double-layer filter paper for use. Take 1mL of filtrate and add 5mL PBS solution to mix. The mixed solution passes through the immunoaffinity column at a natural flow rate, and finally elutes the toxin into a test tube with 1mL methanol, and the filtrate is filtered through a 0.22μm organic ultrafiltration membrane. The content of AFB1 in the filtrate is determined by high performance liquid chromatography-post-column photochemical derivatization method.

[0067] Example 4 Application of direct solid-state fermentation of microorganisms of the present invention in reducing AFB1 in peanut meal

[0068] Blank peanut meal sample spiked: Grind the peanut meal, pass it through a 40 mesh sieve, weigh 5.00g of peanut meal and sterilize it in a 50mL centrifuge tube at 121℃ for 20min. Add 100μL or 150μL of 1000ng / mL AFB1 standard solution to the peanut meal to obtain peanut meal samples with spiked concentrations of 20ng / g and 30ng / g, respectively. Pick a single colony on the MRS solid medium and inoculate it into MRS liquid medium and culture it at 37℃ and 200r / min for 12h. Take 1mL of fermentation broth (concentration of 1-9×10 8 CFU / mL) was added to the sterilized peanut meal sample, and 3 mL of sterile water was added, and fermented at 37°C for 48 hours. The experiment was repeated three times. At the same time, the experiment without adding Lactobacillus plantarum YS-718 fermentation liquid was set as the control group, and the AFB1 content in the samples before and after treatment was measured. The results showed that the aflatoxin B1 content in the control group was basically unchanged (see Figure 2 CK). The fermentation broth (concentration of 1-9×10 8 CFU / mL) added to peanut meal for 48 hours showed excellent aflatoxin B1 reduction rate. The reduction rate when the initial concentration of AFB1 in peanut meal was low (20ng / g) was higher than the reduction rate when the initial concentration of AFB1 in peanut meal was high (30ng / g). The reduction rate of AFB1 in peanut meal by Lactobacillus plantarum was related to the initial concentration of AFB1 in peanut meal. The excellent reduction effect after heat treatment may be due to the metabolites such as protein produced after the strain fermentation.

[0069] Table 1 spiked concentration is 30ng / g

[0070]

[0071] Table 2: spiked concentration is 20 ng / g

[0072]

[0073] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. Lactobacillus plantarum YS-718, characterized in that: It was deposited in the China Center for Type Culture Collection on November 15, 2023, with the deposit number being CCTCC NO.M 20232226.

2. An aflatoxin biotreatment agent comprising the fermentation broth of Lactobacillus plantarum YS-718 according to claim 1.

3. Use of the Lactobacillus plantarum YS-718 according to claim 1 or the aflatoxin biotreatment agent according to claim 2 in reducing aflatoxin B1.

4. The use according to claim 3, characterized in that: The specific application method is: bringing the fermentation liquid of Lactobacillus plantarum YS-718 into contact with the biological sample to reduce the aflatoxin contamination of the biological sample.

5. The use according to claim 4, characterized in that: The fermentation liquid of Lactobacillus plantarum YS-718 is added into the sterilized peanut meal to ferment and eliminate aflatoxin.

6. The use according to claim 5, characterized in that: The fermentation conditions are as follows: fermentation at 37±1°C for 48-72h.

7. The use according to claim 5, characterized in that: The fermentation liquid of Lactobacillus plantarum YS-718 is added to the sample to be processed, and then added to the sterilized peanut meal after heat treatment, and processed at 37±1°C for 48-72h to reduce aflatoxin.

8. The use according to claim 4, characterized in that: The final concentration of the Lactobacillus plantarum YS-718 fermentation broth is (1-9)×10 8 CFU / mL.

9. The use according to claim 4, characterized in that: The preparation method of the Lactobacillus plantarum YS-718 fermentation liquid is as follows: Lactobacillus plantarum YS-718 is activated in MRS agar, cultured in a 37° C. incubator for 48 hours, a single colony of Lactobacillus plantarum YS-718 is picked up with an inoculation needle, transferred to an MRS liquid culture medium, and cultured with shaking for 12 hours to 24 hours to obtain the fermentation liquid of the probiotic Lactobacillus plantarum YS-718.

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