CN110791460A

CN110791460A - Strain for inhibiting growth of aspergillus flavus and generation of toxin

Info

Publication number: CN110791460A
Application number: CN201911232297.3A
Authority: CN
Inventors: 朱英莲; 杨庆利
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2020-02-14
Anticipated expiration: 2039-12-04
Also published as: CN110791460B

Abstract

The invention provides a lactobacillus plantarum strain for inhibiting growth of aspergillus flavus and production of toxin simultaneously, and the preservation number of the strain is CGMCC No 18655. The lactobacillus plantarum provided by the invention is applied to the field of feed processing or food processing. In another aspect, the invention provides an aspergillus flavus growth inhibitor or aflatoxin remover, which is obtained by freeze-drying the supernatant produced by fermenting the screened strain FJS 003. The strain provided by the invention can effectively inhibit the growth of aspergillus flavus and the generation of toxin, the inhibition rate of the strain fermentation supernatant on the growth of aspergillus flavus spores is as high as 99%, and the removal rate on the aflatoxin is as high as more than 70%; the obvious beneficial effect is produced.

Description

Strain for inhibiting growth of aspergillus flavus and generation of toxin

Technical Field

The invention belongs to the technical field of food microorganism screening, and particularly relates to a strain for inhibiting growth of aspergillus flavus and production of toxins.

Background

Aspergillus flavus (Aspergillus flavus) is a pathogenic fungus, and is mostly present in moldy crops, Aspergillus colonies grow and reproduce quickly, the initial Aspergillus flavus colonies are yellow, and the colonies turn grey green by the end. The thallus has branched hyphae and aerial hyphae, small peduncles are on the surface of the aerial hyphae, and the aspergillus flavus is spread in the air through conidium. Aflatoxins, which are toxins with acute and chronic toxicity, are produced by aspergillus flavus and are highly toxic. Aflatoxin B1 is a class 1 carcinogen because of its strong carcinogenic and teratogenic properties. The aspergillus flavus is not sensitive to temperature and can still survive at high temperature; it is stable in neutral environment and weak acidic environment, can be partially decomposed in strong acid, and can be quickly decomposed in strong alkali to produce non-toxic salt, but said reaction is reversible, and when it meets acid, its toxicity can be recovered.

The maximum limit standard of aflatoxin B1(AFB1) in grains and products thereof specified in China is 20 mug/kg. Aflatoxins affect humans primarily through contaminated grains, corn, peanuts, legumes, fruits and milk, and pose a serious threat to human health. If the feed of the dairy cow is mainly prepared by mixing corn, wheat and grains, if the feed pollutes AFB1, AFB1 is hydroxylated in vivo and converted into aflatoxin M1(AFM1) after the dairy cow eats the feed, and AFM1 mainly exists in milk, kidney, liver and mammary gland. People who eat the polluted dairy products can be poisoned and are particularly dangerous to infants, so that many researches focus on solving and controlling the pollution of aflatoxin in feed and grains.

At present, the method for removing aflatoxin comprises physical, biological and chemical methods. Physical and chemical methods can cause problems of residue, secondary pollution and the like. Physical methods are energy intensive, while the use of chemical agents can cause strains to become resistant and can increase the production of toxins if improperly used.

The biological detoxification effect of aflatoxin is mainly shown in the following two points: firstly, the removal of toxin is realized by utilizing the combination or adsorption effect of microbial cells or cell wall extracts on the toxin; secondly, the toxin is converted into low-toxicity or non-toxic substances by using enzyme or metabolite generated in the growth and metabolism process of the microorganism, so as to achieve the aim of detoxification. Therefore, the biological control technology is the most promising method for controlling aspergillus flavus pollution. Therefore, in recent years, researchers have been dedicated to research on biological control methods, and it is necessary to screen and obtain functional strains having an inhibiting effect on the growth of aspergillus flavus and a toxin removing effect.

Disclosure of Invention

The invention aims to provide a Lactobacillus plantarum strain for inhibiting growth of Aspergillus flavus and simultaneously inhibiting generation of toxins and application thereof.

The Lactobacillus plantarum FJS003 strain provided by the invention is named Lactobacillus sp, which is preserved in China general microbiological culture Collection center of China microbiological culture Collection center No. 3 of West Lu No. 1 Hospital, Tokyo, Asahori, Navigo, Beijing, 10 months and 10 days in 2019, and the preservation number is CGMCC No. 18655.

The lactobacillus plantarum provided by the invention is applied to the field of feed processing or food processing.

In another aspect, the invention provides an aspergillus flavus growth inhibitor and an aflatoxin remover, which are obtained by freeze-drying supernatant produced by fermenting the screened strain FJS 003.

The strain provided by the invention can effectively inhibit the growth of aspergillus flavus and the generation of toxin, the inhibition rate of the strain fermentation supernatant on the growth of aspergillus flavus spores is as high as 99%, and the removal rate on the aflatoxin is as high as more than 70%; the obvious beneficial effect is produced.

Drawings

FIG. 1: screening a result graph by a double-layer plate method;

FIG. 2: phylogenetic tree diagram of strain FJS 003;

FIG. 3: influence graph of the treatment time on the growth inhibition of the strain by the aspergillus flavus;

FIG. 4: influence diagram of pH on growth inhibition of aspergillus flavus by the strain;

FIG. 5: a technical result graph of a blood counting chamber;

FIG. 6: the concentration of the experimental group and the control group is compared.

Detailed Description

The invention screens and obtains the plant lactobacillus strain which has the inhibition effect on the growth and the spore growth of the aspergillus flavus and the removal effect on the aflatoxin, and the strain is fermented to prepare the aspergillus flavus growth inhibitor and the aflatoxin remover, and the produced preparation has good effect and effect.

The invention is explained in detail below with reference to the figures and examples.

Example 1 isolation and screening of lactic acid bacteria

1) Separation and screening of lactic acid bacteria

Fermented foods such as sausage, yoghourt, pickle, fermented soybean meal, fermented corn meal and the like which are easy to pollute the aspergillus flavus are collected from a plurality of food enterprises. Selecting batch food with stable quality, good color, long storage time and no Aspergillus flavus detection for strain screening. A sample of 25g (25mL) was taken under sterile conditions and homogenized in 225mL sterile physiological saline to prepare a 1:10 sample homogenate. Then 10 times serial dilution is carried out, MRS containing calcium carbonate is coated on sample homogeneous liquid with proper dilution, and bacterial colony with calcium dissolving ring is separated after culture, thus obtaining pure bacterial strain to be screened.

2) Screening of strain with inhibiting effect on growth of aspergillus flavus and removing effect on aflatoxin

(1) Preliminary screening

Inoculating the strain with the calcium-solubilizing ring into a rescreened inorganic salt culture medium containing 0.1% coumarin, standing and culturing at 37 ℃ for 48h, observing the growth condition of the strain, if the culture medium becomes turbid, dipping the rescreened lactobacillus by an inoculating loop, streaking and culturing on a culture medium plate, picking a single colony by the inoculating loop for preservation after the colony grows out, and then carrying out further rescreening.

The primary screening result shows that the culture medium containing coumarin can become turbid after the three strains are cultured in the primary screening culture medium for 48 hours, and the three strains can grow by taking coumarin as a carbon source. And the aflatoxin is a structural analogue of dihydrofurocoumarin, so the three strains are preliminarily judged to be capable of decomposing and utilizing the aflatoxin and are respectively named as a strain b, a strain e and a strain h.

(2) Double sieve

And further screening the strains with the inhibition effect on the aspergillus flavus by using a double-layer plate method aiming at the three strains of the strain b, the strain e and the strain h.

Wherein the lower flat plate is MRS solid culture medium, strains b, e and h are picked by inoculating loops and drawn into two parallel straight lines of about 3cm on the flat plate respectively, and the flat plate is cultured for 24h at constant temperature of 37 ℃. Then pouring the PDA culture medium containing 0.2mL of aspergillus flavus spore liquid into a culture dish as an upper culture medium, culturing at the constant temperature of 28 ℃ for 72h, and observing whether a bacteriostatic zone appears around a growth strip of the screened strain. The results show that the strains b, e and h have certain inhibition effect on the growth of the aspergillus flavus, so that the re-screening is continuously carried out by the effect of removing the aflatoxin.

(3) Further rescreening

Transferring the primarily screened strain into 50mL of MRS broth with the inoculation amount of 5%, culturing at 37 ℃ for 24h, and sucking 1000 mu L of bacterial liquid and 25 mu L of AFB1 standard substance into a sterilized centrifuge tube; draw 1000 μ L of MRS broth with 25 μ L of AFB1 standard in a sterilized centrifuge tube as a blank. Then culturing at 37 deg.C in dark for 72h, centrifuging at 8000r/min for 10min to remove thallus, and collecting supernatant to measure its removal rate of AFB 1. The removal rate of AFB1 was determined using 96-well plates according to the ELISA kit instructions and then determining the OD of each well at a wavelength of 450 nm. And 3 times of parallel tests are carried out, and the measured data are subjected to computational analysis.

The result shows that the strain e and the strain h have no removal effect on the aflatoxin; the strain b has obvious inhibition effect on aflatoxin, and the removal rate of aflatoxin is up to 77.36%. Therefore, the strain b is subsequently further studied as the best strain for inhibiting the growth of aspergillus flavus and removing aflatoxin, and is renamed to FJS 003.

(4) 16SrDNA identification of strain FJS003

Placing 1.5mL of bacterial liquid in a sterile centrifuge tube, centrifuging for 5min at 10000r/min, removing supernatant to obtain thalli, adding 500 mu LTE buffer solution and 50 mu L of lysozyme, reacting for 60min at 37 ℃, adding 30 mu L of 10% SDS and 15 mu L of proteinase K, fully mixing uniformly, incubating for 60min at 37 ℃, adding 100 mu L of NaCl solution with the concentration of 5mol/L, mixing uniformly, adding 80 mu L of CTAB/NaCl (5% w/v) solution, reacting for 1-2 h at 65 ℃, and adding phenol with the same volume: chloroform: and (3) uniformly mixing isoamyl alcohol 25:24:1, centrifuging at 10000r/min for 5min to remove thalli, taking supernatant for later use, adding isopropanol with the volume of 0.6-0.8 times of that of the supernatant, centrifuging to obtain a precipitate, washing the precipitate with 1mL of 70% ethanol, centrifuging, performing sterile drying on the precipitate, and re-suspending to obtain template DNA.

The genomic DNA of the lactic acid bacterium strain was used as a template, and PCR amplification was carried out using bacterial universal primers 27f (5 ': AGAGTTTGATCCTGGCTC AG-3') and 1495r (5'-CTACGGCTACCTTGTTACGA-3'). The PCR products were detected and sequenced using 1.0% agarose gel electrophoresis. The determined sequences were compared for homology using the nucleic acid Blast technique at the NCBI website GenBank database. The result of PCR amplification and agarose gel electrophoresis detection shows that the PCR product of the strain is 1.474kb, and can be used for DNA sequencing, and the sequence of 6SrDNA of the screened strain is SEQID NO. 1. Homology analysis shows that the 16S rDNA gene sequence of the tested strain has the highest homology with lactobacillus plantarum; a phylogenetic tree was constructed as shown in figure 2. The sequencing result of the strain is shown in SEQ ID NO. 1.

Example 2: inhibition of strain FJS003 on growth of aspergillus flavus thallus

1) Influence of treatment time on growth inhibition of aspergillus flavus by strain

Setting an experimental group and a blank control group, wherein:

the experimental group is that 500 mu L of FJS003 bacteria fermentation liquor is evenly coated on a PDA culture medium, and 10 mu L of aspergillus flavus mould spore liquid is inoculated in the center of the culture medium after the liquid is completely absorbed;

blank control group: the PDA medium center which was not coated with FJS003 strain was inoculated with 10. mu.L of Aspergillus flavus mold spore liquid.

The experimental group and the blank control group were placed in a constant temperature incubator at 28 ℃, the colony diameters were measured at 48h, 72h, and 96h of incubation time, respectively, and the inhibition ratios were calculated according to formula 1, with the results shown in fig. 3.

As can be seen from FIG. 3, the inhibition effect of the strain FJS003 on the growth of Aspergillus flavus is significant depending on the time. When the culture time is 36h, the growth inhibition rate of the aspergillus flavus is 42.80 percent; when the culture time is 48h, the growth inhibition rate of aspergillus flavus is obviously increased to 42.65 percent; when the culture time is 60 hours, the growth inhibition rate of the aspergillus flavus is 41.82 percent, and the inhibition rate has no significant difference. The result shows that the secretion of the substances with the inhibiting effect on the aflatoxin reaches a high peak in 36 hours, and the inhibiting effect can reach the highest in a short time. On the basis of saving time and resources, the aspergillus flavus growth inhibition effect of the strain is only cultured for 36 hours, the bacteriostasis efficiency is high, and the growth of the aspergillus flavus can be inhibited in a short time.

3) Influence of pH on growth inhibition of aspergillus flavus by strain

The experimental group and the blank control group were set. Experimental groups: adjusting the pH values of FJS003 bacteria fermentation liquor to be 4, 6 and 8 respectively by using 1mol/L NaOH solution and HCl solution, and using a blank control group as MRS liquid culture medium with the pH value of 7. Respectively sucking 500 mu L of lactic acid bacteria culture solution after different treatments, uniformly coating the lactic acid bacteria culture solution on a PDA culture medium, after the liquid is completely absorbed, inoculating 10 mu L of aspergillus flavus mold spore solution in the center of the culture medium, culturing for 72h at 28 ℃, measuring the diameter of a bacterial colony, and calculating the inhibition rate according to a formula 1, as shown in figure 4. As shown in FIG. 4, the FJS003 bacteria has better inhibition effect on Aspergillus flavus at pH 4-6, and the pH range is wider than that of the previously reported bacteriostasis.

Example 3: effect of strain FJS003 on AFB1 removal

1) Culture of lactic acid bacteria

And (3) selecting the screened excellent strain FJS003 by using an inoculating loop, inoculating the excellent strain FJS003 into an MRS broth culture medium in an inoculation amount of 1%, culturing at the constant temperature of 37 ℃ for 72h to obtain a lactic acid bacteria culture solution, and measuring and recording the pH value of the lactic acid bacteria culture solution.

2) Preparation of supernatant, suspension and intracellular liquid of strain fermentation

Preparation of fermentation supernatant: centrifuging 10mL of lactic acid bacteria culture solution at 4 deg.C at 10000r/min for 15min to obtain supernatant;

preparation of bacterial body weight suspension: washing the centrifuged thallus with phosphate buffer solution, centrifuging, repeating for 3 times, and adding 10mL of sterile phosphate buffer solution to prepare a bacterial suspension;

preparation of intracellular fluid: and washing the centrifuged thallus by using a phosphate buffer solution, centrifuging, repeating for 3 times, adding 10mL of sterile phosphate buffer solution, mixing uniformly, carrying out ultrasonic crushing for 30min, centrifuging, and preparing intracellular fluid.

3) Removing effect of strain fermentation supernatant, strain weight suspension and intracellular fluid on AFB1

Experimental groups: adding 25 mu L of AFB1 standard substance into 10mL of culture solution, 10mL of supernatant, 10mL of bacterial suspension and 10mL of intracellular fluid respectively; blank control group: to 10mL of sterile phosphate buffer was added 25. mu.L of AFB1 standard.

The experimental group and the blank control group are placed in a dark place at 37 ℃ for culturing for 72h, and then the removal rate of each component to AFB1 is detected, the result shows that the thallus heavy suspension and the intracellular fluid have no obvious removal effect on aflatoxin, but the removal rate of the fermentation supernatant to the aflatoxin is up to 75.23%, and the removal rate has no significant difference compared with the removal rate 77.36 of the strain FJS003 fermentation liquor to the aflatoxin during re-screening. Therefore, it was preliminarily judged that a certain fermentation product in the supernatant from the fermentation of the strain had an effect of removing aflatoxin.

Example 4: inhibition of aspergillus flavus spore growth by strain fermentation supernatant

The experimental group and the blank control group were set. Experimental groups: adding 500 mu L of aspergillus flavus spore liquid into 5mL of lactobacillus supernatant; blank control group: 500 μ L of Aspergillus flavus spore liquid was added to 5mL of MRS liquid medium. Culturing at 28 deg.C for 72h, counting spores with a blood counting plate, and calculating spore growth inhibition rate according to formula 2, with the counting result shown in FIG. 5.

The result shows that the fermentation supernatant of the strain FJS003 has strong inhibition effect on the growth of aspergillus flavus spores. After the experimental group is cultured for 72 hours, the experimental group still clarifies; the control group had very high turbidity. Upon observation and counting on the hemacytometer, as shown in fig. 5 and 6, the number of spores in the experimental group was significantly lower than that in the control group, and many mycelia resulted from spore germination were found in the control group. Counting shows that the inhibition rate of fermentation supernatant liquid of the strain FJS003 for culturing 72h on the growth of aspergillus flavus spores is as high as 99.97%.

Example 5: preparation of aspergillus flavus growth inhibitor and aflatoxin remover

Preparation of an aspergillus flavus growth inhibitor: the selected strain FJS003 is selected by an inoculating loop and inoculated into an MRS broth culture medium with the inoculation amount of 1 percent, and the strain FJS003 is cultured for 24 to 72 hours at the constant temperature of 37 ℃ to obtain the lactobacillus culture fermentation liquor with certain pH. Concentrating the fermentation broth, and directly refrigerating or freeze drying (adding cryoprotectant) to obtain active bacteria-containing Aspergillus flavus growth inhibitor and aflatoxin remover with viable bacteria count of 1.0 × 10⁹～1.0×10¹²cfu/(mL/g). Or centrifuging the fermentation liquid at 4 deg.C at 10000r/min for 15min to obtain supernatant, concentrating the supernatant, and directly refrigerating or concentrating and freeze drying to obtain live bacteria-free Aspergillus flavus growth inhibitor and aflatoxin remover.

The Aspergillus flavus growth inhibitor and aflatoxin remover can be used in fermented food and feed. The application method of the aspergillus flavus growth inhibitor and the aflatoxin remover in fermented feed and fermented food comprises the application of the aspergillus flavus growth inhibitor containing live bacteria and the aflatoxin remover, and the inoculation amount is 1 multiplied by 10⁶～1×10⁹cfu/g (mL) feed or food is prepared by inoculating lactobacillus plantarum powder or fermented concentrated solution into feed or food raw materials, fully mixing uniformly, and fermenting; the use of the aspergillus flavus growth inhibitor and the aflatoxin remover without live bacteria comprises inoculating lactobacillus plantarum fermented supernatant concentrated solution or fermented supernatant concentrated freeze-dried powder into feed or food raw materials by 5-15 percent of addition amount, fully mixing and fermenting.

Sequence listing

<110> Qingdao agricultural university

<120> strain for inhibiting growth of aspergillus flavus and production of toxin

<160>1

<170>SIPOSequenceListing 1.0

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<211>1475

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<213> Artificial Sequence (Artificial Sequence)

<400>1

agcacttggc ggcgtgctat acatgcaagt cgaacgaact ctggtattga ttggtgcttg 60

catcatgatt tacatttgag tgagtggcga actggtgagt aacacgtggg aaacctgccc 120

agaagcgggg gataacacct ggaaacagtt tgctaatacc gcataacaac ttggaccgca 180

tggtccgagt ttgaaagatg gcttcggcta tcacttttgg atggtcccgc agcgtattag 240

ctagatggtg gggtaacggc tcaccatggc aatgatacgt agccgacctg agagggtaat 300

cggccacatt gggactgaga cacggcccaa actcctacgg gaggcagcag tagggaatct 360

tccacaatgg acgaaagtct gatggagcaa cgccgcgtga gtgaagaagg gtttcggctc 420

gtaaaactct gttgttaaag aagaacatat ctgagagtaa ctgttcaggt attgacggta 480

tttaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa 540

gcgttgtccg gatttattgg gcgtaaagcg agcgcaggcg gttttttaag tctgatgtga 600

aacccttcgg ctcaaccgaa gaagtgcatc ggaaactggg aaacttgagt gcagaagagg 660

acagtggaac tccatgtgta gcggtgaaat gcgtagatat atggaagaac accagtggcg 720

aaggcggctg tctggtctgt atctgacgct gaggctcgaa agtatgggta gcaaacagga 780

ttagataccc tggtagtcca taccgtaaac gatgaatgct aagtgttgga gggtttccgc 840

ccttcagtgc tgcagctaac gcattaagca ttccgcctgg ggagtacggc cgcaaggctg 900

aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 960

ctacgcgaag aaccttacca ggtcttgaca tactatgcaa atctaagaga ttagacgtac 1020

ccttcgggga catggataca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1080

gggttaagtc ccgctacgag cgcaaccctt attatcagtt gccagcatta agttgggcac 1140

tctggtgaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc 1200

cccttatgac ctgggctaca cacgtgctac aatggatggt acaacgagtt gcgaactcgc 1260

gagagtaagc taatctctta aagccattct cagttcggat tgtaggctgc aactcgccta 1320

catgaaggcg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg 1380

ccttgtacac accgcccgtc acaccatgag agtttgtaac acccaaagtc ggtggggtaa 1440

ccttttagga accagccgcc taaggtgaca gattg 1475

Claims

1. A Lactobacillus plantarum strain is characterized in that the preservation number of the Lactobacillus strain is CGMCC No 18655.

2. Use of a strain of Lactobacillus plantarum according to claim 1 in the field of food processing or in feed processing.

3. A method for inhibiting growth of Aspergillus flavus thallus and spore growth is characterized in that the method comprises the step of fermenting the Lactobacillus plantarum strain disclosed by claim 1 at 30-37 ℃ for 36-72 hours, the pH of fermentation liquor is less than 7, the fermentation liquor or a freeze-dried preparation thereof is used for treating the Aspergillus flavus at 25-30 ℃ for 36-72 hours, and the fermentation liquor cannot be subjected to heat treatment before use.

4. A method for removing aflatoxin B1, which is characterized in that the method comprises the steps of fermenting the Lactobacillus plantarum strain disclosed by claim 1 at 30-37 ℃ for 36-72 hours, wherein the pH of the fermentation liquor is less than 7, and culturing the fermentation liquor or a freeze-dried preparation thereof, a fermentation supernatant or a freeze-dried preparation thereof at 30-37 ℃ for 36-72 hours in the dark.

5. An Aspergillus flavus growth inhibitor characterized in that the Aspergillus flavus growth inhibitor is produced using the Lactobacillus plantarum strain of claim 1.

6. The Aspergillus flavus growth inhibitor according to claim 5, wherein the Aspergillus flavus growth inhibitor is obtained by freeze-drying a supernatant produced by fermenting a Lactobacillus plantarum strain.

7. The Aspergillus flavus growth inhibitor as set forth in claim 5, wherein the preparation method of the Aspergillus flavus growth inhibitor is as follows: fermenting the strain at 30-37 ℃ for 36-72 h, wherein the pH of the fermentation liquor is less than 7, concentrating the fermentation liquor, and directly refrigerating or freeze-drying to obtain the aspergillus flavus growth inhibitor and the aflatoxin remover containing viable bacteria, wherein the viable bacteria number is 1.0 multiplied by 10⁹～1.0×10¹²cfu。

8. The Aspergillus flavus growth inhibitor according to claim 7, wherein the preparation method of the Aspergillus flavus growth inhibitor comprises the steps of centrifuging the fermentation liquor at 4 ℃ for 15min at 10000r/min to obtain a supernatant, and directly refrigerating the supernatant after concentration or freeze-drying the supernatant after concentration to obtain the live bacteria-free Aspergillus flavus growth inhibitor and the aflatoxin remover.

9. Use of the Aspergillus flavus growth inhibitor of any one of claims 5-8 in fermented feed and fermented food products.

10. The use of the Aspergillus flavus growth inhibitor in fermented feed and fermented food as claimed in any one of claims 5-8, wherein the method comprises the use of the Aspergillus flavus growth inhibitor containing live bacteria and the aflatoxin-removing agent in an inoculation amount of 1 x 10⁶～1×10⁹Inoculating plant lactobacillus powder or fermented concentrated solution into feed or food raw material cfu/g feed or food, and mixing wellFermenting; the use of the aspergillus flavus growth inhibitor and the aflatoxin remover which do not contain live bacteria comprises inoculating lactobacillus plantarum fermented supernatant concentrated solution or fermented supernatant concentrated freeze-dried powder into feed or food raw materials by 5-15 percent of addition amount, and fully mixing and fermenting.