CN113278550B

CN113278550B - Bacillus coagulans for improving intestinal degradation of aflatoxin zearalenone

Info

Publication number: CN113278550B
Application number: CN202110547346.3A
Authority: CN
Inventors: 贾佩峤; 万俊康; 戎晓平; 徐洁; 张辉
Original assignee: Ke Run Sheng Technology Development Co ltd
Current assignee: Ke Run Sheng Technology Development Co ltd
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-07-01
Anticipated expiration: 2041-05-19
Also published as: CN113278550A

Abstract

The invention discloses bacillus coagulans for improving intestinal degradation of aflatoxin zearalenone, wherein the preservation number of the bacillus coagulans is CGMCC NO. 20893; the preparation contains an extract of a culture of the bacillus coagulans; the invention has the advantages that: can inhibit intestinal pathogenic bacteria, can efficiently degrade aflatoxin and zearalenone, has simple action condition, does not destroy nutrient components in the feed, and can be added into the feed for a long time.

Description

Bacillus coagulans for improving intestinal degradation of aflatoxin zearalenone

Technical Field

The invention relates to bacillus coagulans, in particular to bacillus coagulans for improving the degradation of aflatoxin and zearalenone in animal intestinal tractsBacillus coagulansB4.0120), belonging to the field of agricultural biology.

Background

The use of antibiotics has been a great progress in human history. The use of antibiotics greatly improves the disease resistance of animals, promotes the growth of the animals and improves the breeding level of livestock and poultry. However, with the use of a large amount of antibiotics, people find that the problems of bacterial drug resistance, environmental pollution and the like caused by the abuse of the antibiotics are gradually highlighted. Therefore, from 7 months and 1 day in 2020, China stops producing commercial feeds containing growth-promoting drug feed additives. The banning of antibiotics is a great impact on livestock and poultry breeding, and a product capable of efficiently replacing antibiotics is urgently needed. In addition, mycotoxins are a class of toxic metabolites produced by fungi during their growth and reproduction. It is a great hazard to animals and humans. Common mycotoxins include Aflatoxin (AF), Zearalenone (ZEA), vomitoxin (DON), Ochratoxin (OT), and Fumonisin (FB), and the toxic effects of various mycotoxins on animals are different. In actual production, the simultaneous contamination of several mycotoxins often appears, and most of the mycotoxins are in a synergistic relationship, so that the search for microorganisms capable of degrading multiple mycotoxins simultaneously is an effective means for solving the current mycotoxin contamination problem.

Disclosure of Invention

The invention aims to provide bacillus coagulans which can improve animal intestinal tracts and degrade aflatoxin and zearalenone, inhibit intestinal pathogenic bacteria, efficiently degrade aflatoxin and zearalenone, have simple action conditions, and a fermentation product of the bacillus coagulans does not destroy nutrient components in feed and can be added into the feed for a long time.

The technical scheme of the invention is as follows:

the bacillus coagulans for improving the intestinal degradation of aflatoxin zearalenone is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, the preservation address is the microbiological research institute of China academy of sciences No. 3, West Lu No. 1 Hospital, Kyoho, Beijing, and the preservation number is CGMCC NO. 20893.

The liquid culture medium of the bacillus coagulans comprises the following components in percentage by weight: 10 per mill of corn flour, 10 per mill of peptone and 10 per mill of yeast powder, and the culture conditions are as follows: maintaining pH at 5.8-6.5 at 40 deg.C, and fermenting under aeration to obtain Bacillus coagulans liquid culture; the formula weight fraction of the solid culture medium is as follows: 90% of fine bran, 5% of peptone, 5% of yeast extract powder, 50% of water, and the culture conditions are as follows: and performing microaerophilic fermentation at 40 ℃ to obtain a bacillus coagulans solid culture.

A biological agent comprising an extract of the above Bacillus coagulans culture.

The preparation method of the extract comprises the following steps: inoculating Bacillus coagulans in liquid fermentation tank, aerobically fermenting at 40 deg.C for 24hr, or culturing solid culture at 40 deg.C for 3 days, centrifuging, spray drying the fermented liquid to obtain solid fermented material, and rapidly drying; finally mixing the mixture with a carrier according to a certain proportion.

The carrier comprises one or more of wheat bran, corncob powder, corn starch, rice bran, glucose, zeolite powder and bentonite.

The content of the extract in the biological preparation is 20-60% by weight.

The mass ratio of the bacillus coagulans fermentation extract to the carrier is as follows: 20-60: 80-40.

The action objects of the biological preparation comprise feed, raw materials for preparing the feed and the like.

The bacillus coagulans strain 16SrDNA gene sequence determination:

ACATCTGTCACTTCGGCGGCTGGCTCCGTAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGCAGGCGGGTTGCAGCCTGCAATCCGAACTGGGAATGGTTTTCTGGGATTGGCTTAACCTCGCGGTCTCGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGGTCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGGAAGGCCCTGTCTCCAGGGAGGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGCCTCCCAGTTTCCAATGACCGCTTGCGGTTGAGCCGCAAGATTTCACATCAGACTTAAGAAGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGCCGGGTACCGTCAAGGCGCCGCCCTGTTCGAACGGCACTTGTTCTTCCCCGGCAACAGAGTTTTACGACCCGAAGGCCTTCTTCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCCCACCAACTAGCTAATGCGCCGCGGGCCCATCTGTAAGTGACAGCAGAAGCCGTCTTTCCTTTTTCCTCCATGCGGAGGAAAAAACTATCCGGTATTAGCCCCGGTTTCCCGGCGTTATCCCGATCTTACAGGCAGGTTGCCCACGTGTTACTCACCCGTCCGCCGCTAACCTTTTAAAAGCAAGCTTTTAAAAGGTCCGCACGACTTGCATGTATTAGGCAGCCCCATGGCAC。

the bacillus coagulans belongs to facultative anaerobe, consumes oxygen in intestinal tracts in the early stage after entering an animal body, creates an anaerobic environment, inhibits the growth of aerobic bacteria such as escherichia coli and the like, generates organic acid in the later stage, is beneficial to the growth and the propagation of lactobacillus which is beneficial to the intestinal tracts, and improves the micro-ecological environment of the intestinal tracts. Meanwhile, the invention can efficiently degrade aflatoxin and zearalenone, so that the invention has wide application prospect in the field of livestock and poultry breeding.

The invention has the beneficial effects that: can inhibit intestinal pathogenic bacteria, can efficiently degrade aflatoxin and zearalenone, has simple action condition, does not destroy nutrient components in the feed, and can be added into the feed for a long time.

The invention is further illustrated by the following figures and examples.

Drawings

FIG. 1 is a diagram showing the cells of Bacillus coagulans under a 1600X microscope in accordance with an embodiment of the present invention;

FIG. 2 is a spore map of Bacillus coagulans under a 1600 Xmicroscope according to an embodiment of the present invention;

FIG. 3 is a graph showing the antagonistic effect of the strain of the present invention on Escherichia coli;

FIG. 4 is a graph of the antagonistic effect of the strains of the example of the invention on Staphylococcus aureus;

FIG. 5 is a graph of Salmonella antagonism of the strains of the examples of the present invention;

FIG. 6 shows a chromatogram of aflatoxin B1 content of a sample taken at 0 hr;

FIG. 7 shows the chromatogram of aflatoxin B1 content in a sample after 24hr degradation by the strain of the invention;

FIG. 8 shows a chromatogram of zearalenone content of a sample taken at 0 hr;

FIG. 9 shows a chromatogram of zearalenone content in a sample after 24hr degradation by the strain of the present invention.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

Example 1

The bacillus coagulans for improving intestinal degradation of aflatoxin zearalenone is characterized in that the liquid culture medium formula of the bacillus coagulans comprises the following components in parts by weight: 10 per mill of corn flour, 10 per mill of peptone and 10 per mill of yeast powder, and the culture conditions are as follows: maintaining pH at 5.8-6.5 at 40 deg.C, and fermenting under aeration to obtain Bacillus coagulans liquid culture; the formula weight fraction of the solid culture medium is as follows: 90% of fine bran, 5% of peptone, 5% of yeast extract powder, 50% of water, and the culture conditions are as follows: and performing microaerophilic fermentation at 40 ℃ to obtain a bacillus coagulans solid culture.

The content of the extract in the biological preparation is 20-60% by weight.

The mass ratio of the bacillus coagulans fermentation extract to the carrier is as follows: 20: 80 or 30: 70 or 40: 60 or 50: 60 or 60: 40.

the Bacillus coagulans strain is isolated from rancid milk.

The putrefactive milk is collected for a strain screening test. Spreading rancid milk on YPD plate, culturing at 40 deg.C for 48hr, and transferring each single colony on the plate to blank YPD plate to obtain purified strain. Culturing the bacterial colony with acid production capacity in a liquid PCY culture medium for 24hr, and measuring the antibacterial capacity and the degradation capacity of aflatoxin and zearalenone.

Example 2 antagonistic action of the strains of the invention against intestinal harmful bacteria

The strain culture solution is activated: and (3) culturing the purified strain in a liquid PCY culture medium, and activating for 24 hours to obtain a culture solution of the strain.

Activation of strain culture solution: culturing purified Escherichia coli, Salmonella and Staphylococcus aureus in LB liquid culture medium for 24hr to obtain strain culture solution. The viable count was estimated using a hemocytometer and diluted to a concentration of 10-3/mL with sterile physiological saline.

Preparing a strain-containing culture solution plate: preparing LB solid culture medium, subpackaging in 250mL conical flask, sterilizing at 121 deg.C for 20min, cooling to about 40 deg.C, adding 1mL of the diluted strain culture solution before the culture medium solidifies, mixing well, and pouring into plate.

After the plates have solidified, they are punched with a 6mm diameter sterile punch, 4 holes per plate being made as parallel. The cultured Bacillus coagulans culture medium of the present invention was then added to the punched wells, and 50. mu.L of the culture medium was added to each well. Culturing at 40 deg.C for 24hr, observing and measuring the diameter of the zone.

As can be seen from figures 3, 4 and 5, the bacillus coagulans has a remarkable antagonistic effect on common harmful bacteria in animal intestinal tracts. Wherein the diameter of the inhibition zone for escherichia coli is 14-15 mm, the diameter of the inhibition zone for staphylococcus aureus is 14-16 mm, and the diameter of the inhibition zone for salmonella is 14-15 mm.

Example 3 degradation rates of the strains of the invention for aflatoxin and zearalenone

Firstly, sample treatment:

the strain purified in example 1 was activated in PCY broth for 24hr to obtain a culture of the strain of the present invention. Under the premise of aseptic operation, 50mL of the culture solution is accurately measured and placed in a 250mL triangular flask, and if the culture solution is a solid sample, 0.1g of solid material is added into 50mL of sterile culture medium (the culture medium comprises 1% of corn flour, 1% of peptone and 1% of yeast powder). Then 1mL of aflatoxin B1 or 1mL of zearalenone standard substance is added to make the concentration of aflatoxin B1 in the final reaction system be 50 μ g/L and the concentration of zearalenone be 500 μ g/L, the mixture is uniformly mixed, and the mixture is cultured under the conditions of 40 ℃ and 150 rpm. Meanwhile, a blank control without adding any culture solution or solid material is made, and other treatments are the same.

1mL of the culture solution was sampled at 0h and 24h, and 4mL of chromatographic grade methanol was added and mixed well. Shaking and extracting in shaking table for 30 min. After extraction, centrifuging at 4000rpm for 5min, filtering with glass fiber filter paper, collecting filtrate, purifying with immunoaffinity column, and detecting with high performance liquid chromatography.

Secondly, purification:

5mL of filtrate passing through the glass fiber filter paper is taken, 20mL of PBS buffer solution is added, and the mixture is uniformly mixed and is to be detected.

A10 mL glass syringe was attached to the immunoaffinity column. And (3) removing the plug below the immunoaffinity column, removing the protective solution in the column, and accurately transferring 20mL of the diluted solution to be detected into an injector (which can be transferred for 2 times, and each time is 10 mL). An air pressure pump was connected to the syringe and the pressure was adjusted to allow the solution to pass through the immunoaffinity column at a flow rate of about 1mL/min until air entered the column. Adding 10mL double distilled water to wash for 2 times, adjusting the flow rate to be about 3mL/min until air enters the affinity column, and discarding all effluent liquid.

Finally, accurately adding 0.5 mL (total volume is 1 mL) of chromatographically pure methanol in 2 times, incubating for 2min each time,

the flow rate was around 1mL/min and the eluate was collected in a brown vial for HPLC analysis.

Drawing a standard curve:

according to the use requirement, accurately sucking a certain amount of aflatoxin standard stock solution (B1100 mug/L) or zearalenone standard stock solution (ZEA 1000 mug/L), diluting with chromatographic grade methanol, and respectively preparing a series of standard working solutions with concentration: the concentration of AFB1 is 0. mu.g/L, 1. mu.g/L, 5. mu.g/L, 25. mu.g/L and 50. mu.g/L respectively; the concentrations of ZEA were: 0. mu.g/L, 100. mu.g/L, 200. mu.g/L, 500. mu.g/L, 1000. mu.g/L. And (3) drawing a standard working curve by taking the concentration of the standard working solution as a horizontal coordinate and the peak area as a vertical coordinate, and quantifying the sample by using the standard curve.

Thirdly, detection:

detecting conditions of aflatoxin:

a chromatographic column: c18 column, 4.6 mm × 150 mm, 5 μm;

mobile phase: methanol: water =45: 55;

flow rate: 1 mL/min;

fluorescence detector wavelength: excitation wavelength λ ex 360 nm, emission wavelength λ em 440nm (post-open-column derivatization pool);

sample introduction amount: 20 mu L of the solution;

aflatoxin B1 peak off time: 17-19 min.

And (3) detecting conditions of zearalenone:

a chromatographic column: c18, 150 mm. times.4.6 mm. times.5 μm;

mobile phase: acetonitrile: water: methanol =46:46: 8;

flow rate: 1 ml/min;

detection wavelength: an excitation wavelength λ ex 274nm and an emission wavelength λ em 440 nm;

sample loading amount: 20 mu L of the solution;

time to peak for zearalenone: 11-13 min.

Fourthly, obtaining a result:

the results show that: according to high performance liquid chromatography detection data, the 0h sample AFB1 content is 48.90 mug/L, the 24h sample AFB1 content is 4.73 mug/L, the degradation rate of the strain to AFB1 is 90.33%, the 0h sample ZEA content is 864.91 mug/L, and the 24h sample ZEA content is 169.579 mug/L, so the degradation rate of the strain to ZEA is 80.39%.

Example 4 Effect of the strains of the invention on growth Performance and intestinal health of weaned piglets

120 healthy 21-day-old ternary-hybrid weaned piglets (Du multiplied by long multiplied by big ternary-hybrid) with similar body weight and consistent body type are selected and randomly divided into 4 treatments, each treatment is 5 times repeated, and each treatment is 6 times repeated. Wherein the C0 group is control group, and is fed with corn-soybean meal type basic ration; feeding the T1 group with 100g/T daily ration of the preparation on the basis of the basic daily ration; the T2 group is fed with 300g/T daily ration of the preparation of the invention on the basis of the basic daily ration; the T3 group is fed with 500g/T daily ration of the preparation on the basis of the basic daily ration.

During the test period, the test animals freely feed and drink water, and carry out immunization and parasite expelling according to the routine management program of a pig farm. The pre-feeding period is 5 days, and the test period is 21 days. Feeding is stopped at 20:00 night before the test is finished, water is normally drunk, pigs are weighed one by one on an empty stomach in the morning of the next day, the material remaining amount and the material consumption are recorded by taking the repetition as a unit, and the Average Daily Gain (ADG), the Average Daily Feed Intake (ADFI) and the material weight ratio in the test period are calculated. After slaughtering, the abdominal cavity is opened rapidly, the two ends of the cecum are ligated rapidly, part of the cecum content is taken out in sterile operation, stored at 4 ℃ and subjected to intestinal microorganism detection.

TABLE 1 design of the experiments

Note: actually measuring the viable count of the bacillus coagulans in the bacillus coagulans biological preparation to be 1.03 multiplied by 10¹⁰CFU/g。

TABLE 2 influence of Bacillus coagulans biologics on growth performance of weaned piglets

The data in the same column are marked with different lower case letters, which indicates that the difference is significant.

As can be seen from the data in Table 2, the addition of the bacillus coagulans biological agent can obviously increase the weight and average daily gain of weaned pigs, obviously reduce the feed-weight ratio of the weaned pigs and improve the production performance of the weaned pigs, which indicates that the bacterial strain can improve the growth performance of animals and improve the utilization rate of feed.

TABLE 3 influence of Bacillus coagulans biologics on intestinal microorganisms of weaned piglets

As can be seen from Table 3, the addition of the strain can obviously reduce the number of harmful bacteria Escherichia coli in the intestinal tract of the weaned pig and obviously increase the number of beneficial bacteria bifidobacteria and lactobacilli in the intestinal tract. The bacterial strain can adjust the micro-ecological balance of animal intestinal tracts and maintain the health of organisms.

In conclusion, laboratory experiments and animal experiments fully show that the strain can regulate the intestinal health of animals, simultaneously efficiently degrade aflatoxin and zearalenone, and has a great application value in actual production.

Sequence listing

<110> science and technology development Co., Ltd

<120> bacillus coagulans for improving intestinal degradation of aflatoxin zearalenone

<141> 2021-05-19

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<170> SIPOSequenceListing 1.0

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<213> Bacillus coagulans (Bacillus coagulans)

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acatctgtca cttcggcggc tggctccgta aggttacctc accgacttcg ggtgttacaa 60

actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc 120

tgatccgcga ttactagcga ttccggcttc atgcaggcgg gttgcagcct gcaatccgaa 180

ctgggaatgg ttttctggga ttggcttaac ctcgcggtct cgcagccctt tgtaccatcc 240

attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc 300

ttcctccggt ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag 360

gtcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 420

accatgcacc acctgtcact ctgtcccccg aaggggaagg ccctgtctcc agggaggtca 480

gaggatgtca agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac 540

cgcttgtgcg ggcccccgtc aattcctttg agtttcagcc ttgcggccgt actccccagg 600

cggagtgctt aatgcgttag ctgcagcact aaagggcgga aaccctctaa cacttagcac 660

tcatcgttta cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc 720

gcctcagcgt cagttacaga ccagagagcc gccttcgcca ctggtgttcc tccacatctc 780

tacgcatttc accgctacac gtggaattcc actctcctct tctgcactca agcctcccag 840

tttccaatga ccgcttgcgg ttgagccgca agatttcaca tcagacttaa gaagccgcct 900

gcgcgcgctt tacgcccaat aattccggac aacgcttgcc acctacgtat taccgcggct 960

gctggcacgt agttagccgt ggctttctgg ccgggtaccg tcaaggcgcc gccctgttcg 1020

aacggcactt gttcttcccc ggcaacagag ttttacgacc cgaaggcctt cttcactcac 1080

gcggcgttgc tccgtcagac tttcgtccat tgcggaagat tccctactgc tgcctcccgt 1140

aggagtttgg gccgtgtctc agtcccaatg tggccgatca ccctctcagg tcggctacgc 1200

atcgttgcct tggtgagccg ttaccccacc aactagctaa tgcgccgcgg gcccatctgt 1260

aagtgacagc agaagccgtc tttccttttt cctccatgcg gaggaaaaaa ctatccggta 1320

ttagccccgg tttcccggcg ttatcccgat cttacaggca ggttgcccac gtgttactca 1380

cccgtccgcc gctaaccttt taaaagcaag cttttaaaag gtccgcacga cttgcatgta 1440

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Claims

1. Aspergillus flavus for improving intestinal degradationBacillus coagulans toxin zearalenone, characterized in that: the Bacillus coagulans bacterium (A), (B), (C), (B), (C), (B), (C)Bacillus coagulans) The preservation number of (2) is CGMCC NO. 20893.

2. The method for preparing bacillus coagulans for improving the intestinal degradation of aflatoxin zearalenone according to claim 1 comprises the following steps:

the liquid culture medium comprises the following components in percentage by weight: 10 per mill of corn flour, 10 per mill of peptone and 10 per mill of yeast powder, and the culture conditions are as follows: maintaining pH at 5.8-6.5 at 40 deg.C, and fermenting under aeration to obtain Bacillus coagulans liquid culture;

the formula weight fraction of the solid culture medium is as follows: 90% of fine bran, 5% of peptone, 5% of yeast extract powder, 50% of water, and the culture conditions are as follows: and performing microaerophilic fermentation at 40 ℃ to obtain a bacillus coagulans solid culture.

3. A biological agent characterized by: the preparation contains an extract of a culture of Bacillus coagulans as described in claim 1 or 2, the extract being prepared by: inoculating Bacillus coagulans in liquid fermentation tank, aerobically fermenting at 40 deg.C for 24hr, or culturing solid culture at 40 deg.C for 3 days, centrifuging, spray drying the fermented liquid to obtain solid fermented material, and rapidly drying; and finally mixing with a carrier.

4. The biological agent according to claim 3, characterized in that: the carrier comprises one or more of wheat bran, corncob powder, corn starch, rice bran, glucose, zeolite powder and bentonite.

5. The biological agent according to claim 3, characterized in that: the content of the extract in the biological preparation is 20-60% by weight.

6. The biological agent according to claim 3, characterized in that: the mass ratio of the bacillus coagulans fermentation extract to the carrier is 20: 80 or 30: 70 or 40: 60 or 50: 60 or 60: 40.