CN114181860A - A method of domestication and cultivation of a strain of Bacillus coagulans that degrades zearalenone - Google Patents
A method of domestication and cultivation of a strain of Bacillus coagulans that degrades zearalenone Download PDFInfo
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Abstract
本发明涉及一种降解玉米赤霉烯酮的凝结芽孢杆菌菌株的驯化培育方法，是以公司保存的各株凝结芽孢杆菌为基础菌种，菌种保存于广东省微生物菌种保藏中心，保藏编号GDMCC.GIM1.645。该菌种是公知公用菌种，方便获得。将其接种于含不同玉米赤霉烯酮浓度的MRS培养液中进行逐级传代培养；得到的新凝结芽孢杆菌AMNJ008，菌株AMNJ008具有发酵水平高、产芽孢率高、耐高温、抗逆性强、降解玉米赤霉烯酮降解率高等特点。The invention relates to a method for domesticating and cultivating Bacillus coagulans strains that degrade zearalenone, which is based on each strain of Bacillus coagulans preserved by the company as the basic strains, and the strains are preserved in the Guangdong Provincial Microbial Culture Collection Center with a preservation number. GDMCC.GIM1.645. The strain is a well-known public strain and is easy to obtain. It was inoculated into MRS medium containing different zearalenone concentrations for subculture; the obtained new Bacillus coagulans AMNJ008, strain AMNJ008 has high fermentation level, high sporulation rate, high temperature resistance and strong stress resistance. , Degradation of zearalenone with high degradation rate.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to bacillus coagulans for degrading zearalenone and a domestication and cultivation method thereof.
Background
Zearalenone (ZEN), a mycotoxin produced by a variety of fusarium species and released into the environment, has estrogen-like effects, mainly manifested as chronic toxicity, including toxicity to the reproductive system as well as genotoxicity and endocrine system effects. When animals eat feed polluted by zearalenone, the breeding cycle of the animals is easy to be disordered, even breeding obstacles are caused, organisms in the production and development of the animals are affected, especially in the fetal and neonatal periods, some protective barriers in vivo and physiological functions such as metabolism and excretion are not complete, and the feed is more susceptible to the zearalenone. The zearalenone can stimulate the transcription of an estrogen receptor at 1-3 mg/kg, so that the propagation disorder is caused. Most animals are sensitive to zearalenone, such as pigs, cattle, sheep, turkeys and chickens. Zearalenone can cause toxicity to the reproductive system of female animals and can be transmitted from the mother to the fetus by absorption from the placenta, impairing the health of the fetus and even causing abortion. In livestock and poultry, pigs, especially replacement gilts, are most sensitive to zearalenone, and when the feed contains more than 1mg/kg of zearalenone, estrogen poisoning of the pigs can be caused. Because zearalenone is produced by fusarium during field growth of crops, it is difficult to have an effect on its prevention, and an effective detoxification technology must be adopted to eliminate zearalenone contamination.
The existing methods for controlling zearalenone mainly comprise physical methods, chemical methods and biological methods. The physical methods commonly used for zearalenone control include rejecting, water washing, shelling, milling, heat treating, autoclaving, irradiating, and adsorbent adsorption. For the grains slightly polluting the zearalenone, partial toxins can be removed by means of removing, washing, shelling, grinding and the like, and the method is generally suitable for removing the zearalenone from a small amount of grains. The heat treatment and pressure cooking methods have a bactericidal effect on the zearalenone-producing fungi, but the zearalenone is not greatly affected by the two methods due to the stable properties of the zearalenone, and the high-temperature treatment can destroy the nutritional value of food or feed. The treatment of mycotoxin by irradiation technology can not only effectively remove toxin, but also kill fusarium, but the composition, properties, toxicology and the like of products after irradiation degradation are still lack of research. The adsorption of the adsorbent is also a method capable of effectively removing zearalenone pollution, but the adsorption method can adsorb nutrients in food while adsorbing toxins, and the toxins are not degraded, so that the environmental pollution is caused. Common chemical methods for removing zearalenone comprise ozone treatment, hydrogen peroxide treatment, sodium carbonate soaking and the like, but the addition of chemical agents introduces a plurality of uncertain factors, and the potential toxicity of the generated new product needs to be further researched.
The defects and shortcomings of the prior art are as follows: the stability of the mycotoxin degrading bacteria in the current market is not uniform in level and high in degradation rate due to the restriction of a strain source and a fermentation technology, and the viable count, metabolites and stress resistance of the strain cannot be kept stable, so that the application in feed processing is difficult to meet;
the technical problems to be solved by the invention are as follows: how to obtain the mycotoxin degrading bacteria with high efficiency and strong stability is very urgent, and the bacillus coagulans which is the zearalenone degrading bacteria with high degradation rate, high yield and strong stability is selected through domestication breeding, so that the domestication breeding, the selection and the selection of the bacillus coagulans with high degradation rate and stable passage is an important way for solving the problems of the feed and livestock and poultry breeding puzzled by mycotoxin at present.
Disclosure of Invention
In order to solve the problems, the invention discloses bacillus coagulans capable of degrading zearalenone and a domestication and cultivation method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the bacillus coagulans strain is AMNJ008, is used for feed processing production, belongs to a feed additive catalogue, includes strains and has a promoting effect on the health of cultured animals.
The strain is preserved in Guangdong province microorganism strain preservation center with the preservation number GDMCC. GIM1.645. The strain is a known public strain and is convenient to obtain.
A domestication and cultivation method of bacillus coagulans for degrading zearalenone comprises the following steps:
step one, providing 16 parts of MRS liquid culture medium containing zearalenone with the same concentration, respectively accessing 16 parts of bacillus coagulans stored in a strain library of a company, then performing mixed culture for 24H at 37 ℃, respectively detecting the zearalenone concentration of supernatant liquid in 16 parts of mixed strain liquid, selecting the mixed strain liquid with the highest zearalenone concentration degradation rate, performing streak amplification culture on an MRS plate, and selecting a single bacterium to obtain a target strain;
step two, providing N parts of MRS liquid culture medium and the bacillus coagulans seed liquid; respectively adding zearalenone methanol stock solutions into N parts of the MRS liquid culture medium to prepare N parts of acclimatized culture media with different zearalenone concentrations;
respectively inoculating the bacillus coagulans seed solution into N domesticated culture media with different zearalenone concentrations by the same inoculation amount for culture to obtain N parts of primary induced mixed strain solution; respectively detecting the zearalenone concentration of the supernatant in N parts of the primary induced mixed strain liquid, and selecting the primary induced mixed strain liquid with the highest zearalenone concentration degradation rate to perform streak amplification culture on an MRS culture plate to obtain a circulating starting strain;
and step four, sequentially adopting N parts of acclimation culture medium containing zearalenone with the concentration from low to high to carry out step-by-step subculture on the circulating strain, and keeping the strain as a bacillus coagulans production strain for degrading zearalenone when the zearalenone concentration in the supernatant after the step-by-step subculture is unchanged, wherein N is a natural number more than or equal to 3.
Further, in the first step, the preparation method of the MRS liquid medium includes: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of diamine citrate, 801.0 g of tween-801.0 g, 0.4g of dipotassium phosphate, 0.58g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to a constant volume of 100mL, adjusting the pH value to 6.3, adjusting the temperature to 121 ℃, and sterilizing for 20min to obtain the strain preservation culture medium.
Further, in the second step, the preparation method of the bacillus coagulans seed solution comprises the following steps: inoculating a single strain of Bacillus coagulans to the MRS liquid medium in an inoculation amount of 0.5% -5%, and performing constant-temperature stationary culture at 37 deg.C for 24 h-36 h to obtain the Bacillus coagulans seed liquid.
Further, the method also comprises the step of providing a strain preservation culture medium, and preserving the bacillus coagulans producing strain for degrading the zearalenone in the strain preservation culture medium.
Further, the step of preparing the strain preservation medium comprises: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of diamine citrate, 801.0 g of tween-801.0 g, 0.4g of dipotassium phosphate, 0.58g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to a constant volume of 100mL, adjusting the pH to 6.3, controlling the temperature to 121 ℃, and sterilizing for 20min to obtain the strain preservation culture medium.
The invention has the beneficial effects that: the degradation capability of the bacillus coagulans producing strain for degrading zearalenone obtained by the domestication and cultivation method provided by the invention on zearalenone can reach 35%, and the degradation capability of the bacillus coagulans culture solution prepared by performing amplification culture on the bacillus coagulans producing strain for degrading zearalenone can reach 65.2%; the degradation rate of the bacillus coagulans and the culture solution thereof on zearalenone obtained after 10 generations of culture can reach more than 80%. Meanwhile, the domesticated first generation of the bacillus coagulans is not degenerated after more than 10 times of subculture, has genetic stability, and the degradation capability of the bacillus coagulans culture solution is far higher than that of the strain, thereby being beneficial to being added into feed for use. In addition, the bacillus coagulans strain domestication and cultivation method provided by the invention has simple steps and is easy to operate.
Detailed Description
The present invention will be further illustrated by the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1
Isolation of bacillus coagulans AMNJ 008:
taking out a plate of stock preservation strains (16 bacillus coagulans), streaking and inoculating the plate into an MRS solid culture medium, standing and culturing for 24h at 37 ℃, activating, then selecting a single strain to inoculate MRS liquid culture medium, standing and culturing for 24h at 37 ℃, and expanding and culturing to obtain a primary screening seed solution;
inoculating the strain into MRS primary screening culture medium added with 5ug/mLZEN according to the inoculation amount of 1% (V/V) for enrichment culture, performing ultrasonic extraction on the residual ZEN in the culture by using equal volume of methanol, and performing quantitative analysis on the residual ZEN by using HPLC-PAD.
Respectively detecting the concentration of the zearalenone in the supernatant of the 16 strains of bacillus coagulans primary screening culture medium, considering that the maximum tolerance capability of the strain to the zearalenone is achieved when the concentration of the zearalenone in the supernatant is unchanged, calculating the bacillus coagulans with the largest degradation rate, wherein the bacillus coagulans is numbered AMNJ008, and keeping the strain as a bacillus coagulans domestication basic mother bacterium for degrading the zearalenone.
Example 2
This example provides a bacillus coagulans that degrades zearalenone, this bacillus coagulans strain, number: AMNJ 008.
The embodiment also provides a domestication and cultivation method of bacillus coagulans for degrading zearalenone, which specifically comprises the following steps:
(1) firstly, weighing 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of diamine citrate, 801.0 g of tween-tween, 0.4g of dipotassium phosphate, 0.58g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to a constant volume of 100mL, adjusting the pH value to 6.3, controlling the temperature to 121 ℃, 0.1Mpa, and sterilizing for 20min to prepare the MRS liquid culture medium;
then dividing the MRS liquid culture medium into 10 parts, inoculating a bacillus coagulans mother solution into one part of the MRS liquid culture medium according to the standard that the inoculation amount is 1% (V/V), and performing standing culture at 37 ℃ for 24 hours to obtain a bacillus coagulans seed solution; respectively adding zearalenone methanol stock solutions into the remaining 9 parts of MRS liquid culture medium to prepare 9 domesticated culture media with different zearalenone concentrations, wherein the zearalenone concentrations are respectively 3.0ug/mL, 3.5ug/mL, 4.0ug/mL, 4.5ug/mL, 5.0ug/mL, 5.5ug/mL, 6.0ug/mL, 6.5ug/mL and 7.0ug/mL, and respectively filling the domesticated culture media into 9 triangular flasks;
(2) respectively inoculating the bacillus coagulans seed liquid into 9 acclimation culture medium triangular flasks filled with different zearalenone concentrations, which are prepared in the step (1); placing the inoculated 9 triangular flasks at 37 ℃ for standing culture for 24 hours to obtain 9 parts of primary induction mixed strain liquid; respectively detecting the zearalenone concentration of the supernatant in 9 parts of the primary induced mixed strain liquid, and carrying out streak amplification culture on the primary induced mixed strain liquid with the maximum zearalenone concentration degradation rate value in the supernatant on an MRS culture plate to obtain a circulating starting strain;
(3) inoculating the circulating initial strain into a domesticated culture medium with zearalenone concentration of 3.0ug/mL according to the standard of inoculation amount of 1%, standing and culturing at 37 ℃ for 24h to obtain a second-generation circulating initial strain, inoculating the second-generation circulating initial strain into a domesticated culture medium with zearalenone concentration of 3.5ug/mL according to the standard of inoculation amount of 1%, standing and culturing at 37 ℃ for 24h to obtain a third-generation circulating initial strain, inoculating the third-generation circulating initial strain into a domesticated culture medium with zearalenone concentration of 4.0ug/mL according to the standard of inoculation amount of 1%, standing and culturing at 37 ℃ for 24h to obtain a fourth-generation circulating initial strain, inoculating the fourth-generation circulating initial strain into a domesticated culture medium with zearalenone concentration of 4.5ug/mL according to the standard of inoculation amount of 1%, standing and culturing at 37 ℃ for 24h, obtaining a five-generation circulating starting strain, inoculating the five-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 5.0ug/mL according to the standard of an inoculum size of 1%, standing and culturing at 37 ℃ for 24h to obtain a six-generation circulating starting strain, inoculating the six-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 5.5ug/mL according to the standard of the inoculum size of 1%, standing and culturing at 37 ℃ for 24h to obtain a seven-generation circulating starting strain, inoculating the seven-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 6.0ug/mL according to the standard of the inoculum size of 1%, standing and culturing at 37 ℃ for 24h to obtain an eight-generation circulating starting strain, inoculating the eight-generation circulating starting strain into the acclimatization culture medium with zearalenone concentration of 6.5ug/mL according to the standard of the inoculum size of 1%, standing and culturing at 37 ℃ for 24h, obtaining nine-generation circulating starting strains, inoculating the nine-generation circulating starting strains into an acclimatization culture medium with zearalenone concentration of 7.0ug/mL according to the standard that the inoculation amount is 1%, and standing and culturing at 37 ℃ for 24h to obtain ten-generation circulating starting strains.
After each generation of culture is finished, the concentration of the zearalenone in the supernatant of each generation is detected respectively, when the concentration of the zearalenone in the supernatant is unchanged, the maximum tolerance of the strain to the zearalenone is considered to be achieved, and the strain is reserved as a bacillus coagulans production strain for degrading the zearalenone.
The acclimatization and cultivation method of the bacillus coagulans strain for degrading the zearalenone further comprises the steps of preparing a strain preservation culture medium, and preserving the bacillus coagulans production strain for degrading the zearalenone in the strain preservation culture medium. The preparation method of the strain preservation culture medium comprises the steps of weighing 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of diamine citrate, 801.0 g of tween-801.0 g, 0.4g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0g of calcium carbonate and 15-20g of agar, adding distilled water to a constant volume of 100mL, adjusting the pH value to 6.3, adjusting the temperature to 121 ℃, keeping the pressure at 0.1MPa, and sterilizing for 20min to obtain the strain preservation culture medium.
Performance testing
Toxin degradation test:
in the acclimatization and cultivation method of bacillus coagulans for degrading zearalenone provided by the embodiment, in the step (2), five zearalenone concentration-reducing culture media named as T1, T2, T3, T4 and T5 appear, and zearalenone degradation rates in three zearalenone concentration-reducing culture media are 5.12%, 7.68%, 22.08%, 29.80% and 35.0%, respectively.
Then, the T5 is used as a breeding object, and is subjected to step-by-step subculture by the step (3), a first generation bacillus coagulans production strain capable of stably degrading zearalenone is domesticated and bred, the degradation rate of the first generation bacillus coagulans production strain on zearalenone is stably kept to be more than 35.0% at 30 ℃, the degradation effect of the first generation bacillus coagulans production strain on zearalenone is determined at different co-culture time, the zearalenone concentration can be reduced to 3.128 ug/mL from 6.0ug/mL, and the degradation rate is 47.87%;
and finally, performing 10-generation culture on the first-generation bacillus coagulans producing strain by using the MRS liquid culture medium, wherein the degradation rate of the tenth-generation bacillus coagulans producing strain obtained after 10-generation culture and the culture solution thereof on zearalenone is more than 80.0%.
Embodiment 3
The present example provides a method for acclimatizing and culturing bacillus coagulans for degrading zearalenone, which comprises the following steps in substantially the same way as those in example 1, except that:
in the step (1), adding zearalenone methanol stock solution to the remaining 9 parts of MRS liquid culture medium to prepare acclimatized culture media with zearalenone concentrations of 3.0ug/mL, 3.5ug/mL, 4.0ug/mL, 4.5ug/mL, 5.0ug/mL, 5.5ug/mL, 6.0ug/mL, 6.5ug/mL and 7.0ug/mL respectively, and filling the acclimatized culture media with 9 zearalenone concentrations into 9 triangular flasks respectively;
the step (3) is as follows: inoculating the circulating initial strain into a domesticated culture medium with zearalenone concentration of 3.0ug/mL according to the standard of inoculation amount of 3%, standing and culturing at 37 ℃ for 24h to obtain a second-generation circulating initial strain, inoculating the second-generation circulating initial strain into a domesticated culture medium with zearalenone concentration of 3.5ug/mL according to the standard of inoculation amount of 3%, standing and culturing at 37 ℃ for 24h to obtain a third-generation circulating initial strain, inoculating the third-generation circulating initial strain into a domesticated culture medium with zearalenone concentration of 4.0ug/mL according to the standard of inoculation amount of 3%, standing and culturing at 37 ℃ for 24h to obtain a fourth-generation circulating initial strain, inoculating the fourth-generation circulating initial strain into a domesticated culture medium with zearalenone concentration of 4.5ug/mL according to the standard of inoculation amount of 3%, standing and culturing at 37 ℃ for 24h, obtaining a five-generation circulating starting strain, inoculating the five-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 5.0ug/mL according to the standard of 3% of the inoculum size, standing and culturing at 37 ℃ for 24h to obtain a six-generation circulating starting strain, inoculating the six-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 5.5ug/mL according to the standard of 3% of the inoculum size, standing and culturing at 37 ℃ for 24h to obtain a seven-generation circulating starting strain, inoculating the seven-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 6.0ug/mL according to the standard of 3% of the inoculum size, standing and culturing at 37 ℃ for 24h to obtain an eight-generation circulating starting strain, inoculating the eight-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 6.5ug/mL according to the standard of 3% of the inoculum size, standing and culturing at 37 ℃ for 24h, obtaining nine-generation circulating starting strains, inoculating the nine-generation circulating starting strains into an acclimatization culture medium with zearalenone concentration of 7.0ug/mL according to the standard of 3% of the inoculation amount, and standing and culturing at 37 ℃ for 24h to obtain ten-generation circulating starting strains.
After each generation of culture is finished, the concentration of the zearalenone in the supernatant of each generation is detected respectively, when the concentration of the zearalenone in the supernatant is unchanged, the maximum tolerance of the strain to the zearalenone is considered to be achieved, and the strain is reserved as a bacillus coagulans producing strain for degrading the zearalenone.
Performance testing
Toxin degradation test:
in the acclimatization and cultivation method of bacillus coagulans for degrading zearalenone provided by the embodiment, five zearalenone concentration-reducing culture media named as T1, T2, T3, T4 and T5 appear in the step (2), and the zearalenone degradation rates in the three zearalenone concentration-reducing culture media are 5.37%, 6.98%, 21.56%, 30.13% and 35.4%, respectively.
Then, the T5 is used as a breeding object, and is subjected to step-by-step subculture by the step (3), a first-generation bacillus coagulans production strain capable of stably degrading zearalenone is domesticated and bred, the degradation rate of the first-generation bacillus coagulans production strain to zearalenone is stably kept to be more than 35.0% at 30 ℃, the degradation effect of the first-generation bacillus coagulans production strain to zearalenone is determined at different co-culture time, the zearalenone concentration can be reduced to 2.512 ug/mL from 6.0ug/mL, and the degradation rate is 58.13%;
and finally, performing 10-generation culture on the first-generation bacillus coagulans producing strain by using the MRS liquid culture medium, wherein the degradation rate of the tenth-generation bacillus coagulans producing strain obtained after 10-generation culture and the culture solution thereof on zearalenone is more than 80.0%.
Example 4
The present example provides a method for domesticating and cultivating bacillus coagulans strain for degrading zearalenone, which comprises the following steps in substantially the same way as in example 1, except that:
dividing the MRS liquid culture medium into 6 parts, inoculating bacillus coagulans into one part of the MRS liquid culture medium according to the standard that the inoculation amount is 3%, and performing static culture on the bacillus coagulans at a constant temperature of 37 ℃ for 24 hours to obtain bacillus coagulans seed liquid. Respectively adding zearalenone methanol stock solutions into the remaining 5 parts of MRS liquid culture medium to prepare 5 acclimatized culture media with zearalenone concentrations of 4.0ug/mL, 4.5ug/mL, 5.0ug/mL, 5.5ug/mL and 6.0ug/mL, and respectively filling the acclimatized culture media into 5 triangular flasks to prepare acclimatized culture media;
the step (3) is as follows: inoculating the circulating initial strain into an acclimatization culture medium with zearalenone concentration of 4.0ug/mL according to the standard of inoculation amount of 3%, standing at 37 ℃ for 24h to obtain a second-generation circulating initial strain, inoculating the second-generation circulating initial strain into an acclimatization culture medium with zearalenone concentration of 4.5ug/mL according to the standard of inoculation amount of 3%, standing at 37 ℃ for 24h to obtain a third-generation circulating initial strain, inoculating the third-generation circulating initial strain into an acclimatization culture medium with zearalenone concentration of 5.0ug/mL according to the standard of inoculation amount of 3%, standing at 37 ℃ for 24h to obtain a fourth-generation circulating initial strain, inoculating the fourth-generation circulating initial strain into an acclimatization culture medium with zearalenone concentration of 5.5ug/mL according to the standard of inoculation amount of 3%, standing at 37 ℃ for 24h to obtain a fourth-generation circulating initial strain, and (3) obtaining a five-generation circulating starting strain, inoculating the five-generation circulating starting strain into an acclimatization culture medium with zearalenone concentration of 6.0ug/mL according to the standard of the inoculation amount of 3%, and standing and culturing the five-generation circulating starting strain at a constant temperature of 37 ℃ for 24 hours to obtain a six-generation circulating starting strain. After each generation of culture is finished, the concentration of the zearalenone in the supernatant of each generation is detected respectively, when the concentration of the zearalenone in the supernatant is unchanged, the maximum tolerance of the strain to the zearalenone is considered to be achieved, and the strain is reserved as a production strain for degrading the zearalenone.
Performance testing
Toxin degradation test:
in the acclimatization and cultivation method of bacillus coagulans for degrading zearalenone provided by the embodiment, five zearalenone concentration-reducing culture media named as T1, T2, T3, T4 and T5 appear in the step (1), and the zearalenone degradation rates in the three zearalenone concentration-reducing culture media are 5.45%, 7.01%, 21.36%, 30.05% and 34.9%, respectively.
Then, the T5 is used as a breeding object, and is subjected to step-by-step subculture by the step (3), a first generation bacillus coagulans production strain capable of stably degrading zearalenone is domesticated and bred, the degradation rate of the first generation bacillus coagulans production strain on zearalenone is stably kept to be more than 35.0% at 30 ℃, the degradation effect of the first generation bacillus coagulans production strain on zearalenone is determined at different co-culture time, the zearalenone concentration can be reduced to 2.778 ug/mL from 6.0ug/mL, and the degradation rate is 53.7%;
and finally, performing 10-generation culture on the first-generation bacillus coagulans producing strain by using the MRS liquid culture medium, wherein the degradation rate of the tenth-generation bacillus coagulans producing strain obtained after 10-generation culture and the culture solution thereof on zearalenone is more than 80.0%.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. A acclimatization and cultivation method of bacillus coagulans for degrading zearalenone is characterized by comprising the following steps:
step one, providing 16 parts of MRS liquid culture medium containing zearalenone with the same concentration, respectively accessing 16 parts of bacillus coagulans stored in a strain library of a company, then performing mixed culture for 24H at 37 ℃, respectively detecting the zearalenone concentration of supernatant liquid in 16 parts of mixed strain liquid, selecting the mixed strain liquid with the highest zearalenone concentration degradation rate, performing streak amplification culture on an MRS plate, and selecting a single bacterium to obtain a target strain;
step two, providing N parts of MRS liquid culture medium and the bacillus coagulans seed liquid; respectively adding zearalenone methanol stock solutions into N parts of the MRS liquid culture medium to prepare N parts of acclimatized culture media with different zearalenone concentrations;
respectively inoculating the bacillus coagulans seed solution into N domesticated culture media with different zearalenone concentrations by the same inoculation amount for culture to obtain N parts of primary induced mixed strain solution; respectively detecting the zearalenone concentration of the supernatant in N parts of the primary induced mixed strain liquid, and selecting the primary induced mixed strain liquid with the highest zearalenone concentration degradation rate to perform streak amplification culture on an MRS culture plate to obtain a circulating starting strain;
and step four, sequentially adopting N parts of acclimation culture medium containing zearalenone with the concentration from low to high to carry out step-by-step subculture on the circulating strain, and keeping the strain as a bacillus coagulans production strain for degrading zearalenone when the zearalenone concentration in the supernatant after the step-by-step subculture is unchanged, wherein N is a natural number more than or equal to 3.
2. The acclimatization and cultivation method of bacillus coagulans strain for degrading zearalenone according to claim 1, wherein in the first step, the preparation method of the MRS liquid medium comprises: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of diamine citrate, 801.0 g of tween-801.0 g, 0.4g of dipotassium phosphate, 0.58g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to a constant volume of 100mL, adjusting the pH value to 6.3, adjusting the temperature to 121 ℃, and sterilizing for 20min to obtain the strain preservation culture medium.
3. The method for acclimatizing and cultivating a bacillus coagulans strain for degrading zearalenone according to claim 2, wherein the preparation method of the bacillus coagulans seed solution in the second step comprises: inoculating a single strain of Bacillus coagulans to the MRS liquid medium in an inoculation amount of 0.5% -5%, and performing constant-temperature stationary culture at 37 deg.C for 24 h-36 h to obtain the Bacillus coagulans seed liquid.
4. The method as claimed in claim 3, further comprising providing a strain preservation medium, and preserving the strain produced by Bacillus coagulans for degrading zearalenone in the strain preservation medium.
5. The acclimatization and cultivation method of bacillus coagulans strain for degrading zearalenone according to claim 4, wherein the step of preparing the strain preservation medium comprises: 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of diamine citrate, 801.0 g of tween-801.0 g, 0.4g of dipotassium phosphate, 0.58g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to a constant volume of 100mL, adjusting the pH to 6.3, controlling the temperature to 121 ℃, and sterilizing for 20min to obtain the strain preservation culture medium.
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