A method for cultivating a strain of Bacillus coagulans capable of degrading zearalenone

Abstract

The present invention relates to a method for taming and cultivating a strain of Bacillus coagulans capable of degrading zearalenone. The strain is based on strains of Bacillus coagulans preserved by a company, and the strain is preserved in the Guangdong Provincial Microbial Culture Collection Center with a preservation number of GDMCC.GIM1.645. The strain is a well-known and public strain and is easy to obtain. The strain is inoculated in an MRS culture solution containing different concentrations of zearalenone for step-by-step subculture; the obtained new Bacillus coagulans AMNJ008 has the characteristics of high fermentation level, high spore production rate, high temperature resistance, strong stress resistance, high degradation rate of zearalenone, etc.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Bacillus coagulans capable of degrading zearalenone and a domestication and cultivation method thereof. Background Art

[0002] Zearalenone (Zearalenone, ZEN) is a fungal toxin produced by a variety of Fusarium and released into the environment. It has an estrogen-like effect, mainly manifested as chronic toxicity, including toxicity to the reproductive system and genotoxicity and the impact of the endocrine system. Animals eating feed contaminated by zearalenone are prone to cause animal reproduction cycle disorders, and even cause reproductive disorders, affecting the body of animal production and development, especially in the fetal and neonatal period, some protective barriers and physiological functions such as metabolism and excretion in the body are not yet perfect, and are more susceptible to zearalenone. Zearalenone can stimulate estrogen receptor transcription at 1~3mg / kg, leading to reproductive disorders. Most animals are sensitive to zearalenone, such as pigs, cattle, sheep, turkeys and chickens. Zearalenone can cause poisoning to the reproductive system of female animals, and is transmitted from the mother to the fetus through absorption of the placenta, damaging the health of the young and even causing miscarriage. Among livestock and poultry, pigs, especially gilts, are the most sensitive to zearalenone. When the feed contains more than 1 mg / kg of zearalenone, it can cause estrogen poisoning in pigs. Since zearalenone is produced by Fusarium during the growth of crops in the field, it is difficult to prevent it effectively, and effective detoxification technology must be adopted to eliminate the contamination of zearalenone.

[0003] At present, the methods for controlling zearalenone mainly include physical methods, chemical methods and biological methods. Commonly used physical methods for controlling zearalenone include removal, washing, shelling, grinding, heat treatment, pressure cooking, irradiation and adsorbent adsorption. For grains slightly contaminated with zearalenone, some toxins can be removed by means of removal, washing, shelling and grinding, which are generally suitable for the removal of zearalenone in a small amount of grains. Heat treatment and pressure cooking methods are used to sterilize fungi that produce zearalenone, but since the nature of zearalenone is very stable, these two methods have little effect on zearalenone, and high temperature treatment will destroy the nutritional value of food or feed. Irradiation technology can not only effectively remove toxins, but also kill Fusarium at the same time, but the composition, properties and toxicology of the products after irradiation degradation are still lacking. Adsorbent adsorption is also a method that can effectively remove zearalenone pollution, but the adsorption method will also adsorb nutrients in food while adsorbing toxins, and since the toxins are not degraded, it will cause pollution to the environment. Commonly used chemical methods for removing zearalenone include ozone treatment, hydrogen peroxide treatment and sodium carbonate immersion, but the addition of chemical reagents will introduce many uncertainties, and the potential toxicity of new products also needs further study.

[0004] Defects and shortcomings of existing technologies: Due to the constraints of strain sources and fermentation technology, the stability of mycotoxin-degrading bacteria products on the market is uneven, the degradation rate is not high, and the number of live bacteria, metabolites and stress resistance of the strains cannot be kept stable, which makes it difficult to meet the application in feed processing;

[0005] The technical problem to be solved by the present invention is: how to obtain mycotoxin-degrading bacteria with high efficiency and strong stability is imminent, and the zearalenone-degrading bacteria, Bacillus coagulans, with high degradation rate, high yield and strong stability are screened through domestication and breeding. Therefore, domestication and breeding to screen Bacillus coagulans with high degradation rate and stable succession is an important way to solve the current problem of mycotoxins plaguing the feed and livestock and poultry farming industries. Summary of the invention

[0006] In order to solve the above problems, the present invention discloses a Bacillus coagulans capable of degrading zearalenone and a method for acclimating and cultivating the same.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] A strain of Bacillus coagulans capable of degrading zearalenone of zearalenone is numbered AMNJ008. The Bacillus coagulans is used for feed processing and production, is a strain included in the feed additive catalog, and has a promoting effect on the health of farmed animals.

[0009] The strain is stored in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC.GIM1.645. The strain is a well-known and publicly used strain and is easy to obtain.

[0010] A method for acclimating and cultivating Bacillus coagulans for degrading zearalenone comprises the following steps:

[0011] Step 1: provide 16 MRS liquid culture media containing the same concentration of zearalenone, respectively inoculate them into 16 Bacillus coagulans strains stored in the company's strain bank, and then carry out mixed culture at 37°C for 24 hours, respectively detect the zearalenone concentration of the supernatant in the 16 mixed strain liquids, select the mixed strain liquid with the highest zearalenone concentration degradation rate for streaking amplification culture on the MRS plate, and select a single bacterium to obtain the target strain;

[0012] Step 2, providing N portions of MRS liquid culture medium and the above-mentioned Bacillus coagulans seed solution; adding zearalenone methanol stock solution to the N portions of the MRS liquid culture medium, respectively, to prepare N portions of acclimation culture medium with different zearalenone concentrations;

[0013] Step 3, inoculating the Bacillus coagulans seed liquid into N acclimation culture media with different zearalenone concentrations with the same inoculation amount for cultivation, to obtain N portions of primary induced mixed strain liquid; detecting the zearalenone concentration of the supernatant in the N portions of the primary induced mixed strain liquid, respectively, selecting the primary induced mixed strain liquid with the largest zearalenone concentration degradation rate for streaking amplification culture on an MRS culture plate, to obtain a circulating starting strain;

[0014] Step 4, sequentially using N portions of the acclimated culture medium containing zearalenone in increasing concentrations from low to high to perform step-by-step subculture on the circulating strain, and when the zearalenone concentration in the supernatant after step-by-step subculture remains unchanged, retaining this strain as a Bacillus coagulans production strain for degrading zearalenone, wherein N is a natural number greater than or equal to 3.

[0015] Furthermore, in the step one, the preparation method of the MRS liquid culture 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 diammonium citrate, 1.0g of Tween-80, 0.4g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.29 of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to make up to 100mL, adjusting the pH to 6.3, 121°C, 0.1Mpa, and sterilizing for 20min to obtain the strain preservation culture medium.

[0016] Furthermore, in the step 2, the preparation method of the Bacillus coagulans seed solution includes: inoculating a single Bacillus coagulans bacterium into the MRS liquid culture medium at an inoculation rate of 0.5% to 5%, and culturing it at a constant temperature of 37° C. for 24 h to 36 h to obtain the Bacillus coagulans seed solution.

[0017] Furthermore, it also includes providing a strain preservation medium, and the coagulant Bacillus production strain that degrades zearalenone is preserved in the strain preservation medium.

[0018] Furthermore, the step of preparing the strain preservation medium includes: adding 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 diammonium citrate, 1.0g of Tween-80, 0.4g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.29 of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, and distilled water to make up to 100mL, adjusting the pH to 6.3, 121°C, 0.1Mpa, and sterilizing for 20min to obtain the strain preservation medium.

[0019] The beneficial effects of the present invention are as follows: the zearalenone-degrading Bacillus coagulans obtained by the domestication and cultivation method provided by the present invention can achieve a degradation capacity of 35% for zearalenone, and the new zearalenone-degrading Bacillus coagulans culture fluid prepared after amplification and cultivation of the zearalenone-degrading Bacillus coagulans producing strain can achieve a degradation capacity of 65.2% for zearalenone; the degradation rate of zearalenone by the zearalenone-degrading Bacillus coagulans and its culture fluid obtained after 10 generations of cultivation can reach more than 80%. At the same time, the first generation of zearalenone-degrading Bacillus coagulans after domestication does not degenerate after more than 10 subcultures, and has genetic stability, and the degradation capacity of the zearalenone-degrading Bacillus coagulans culture fluid is much higher than that of the strain itself, which is conducive to adding it to feed. In addition, the domestication and cultivation method of the Bacillus coagulans strain provided by the present invention is simple in steps and easy to operate. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific examples. It should be understood that the following specific implementation methods are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0021] Example 1

[0022] Isolation of Bacillus coagulans AMNJ008:

[0023] The stock culture (16 strains of Bacillus coagulans) was taken out and streaked on a plate and inoculated into MRS solid medium and incubated at 37°C for 24 hours for activation. Then, a single strain was selected and inoculated into MRS liquid medium and incubated at 37°C for 24 hours for expansion to obtain the primary screening seed solution.

[0024] The inoculum was inoculated at a 1% (V / V) inoculation volume into MRS primary screening medium supplemented with 5ug / mL ZEN for enrichment culture. The remaining ZEN in the culture was extracted by ultrasonic using an equal volume of methanol, and the residual ZEN was quantitatively analyzed by HPLC-PAD.

[0025] The concentrations of zearalenone in the supernatant of the primary screening culture medium of 16 strains of Bacillus coagulans were tested respectively. When the concentration of zearalenone in the supernatant remained unchanged, it was considered that the strain had reached its maximum tolerance to zearalenone. The Bacillus coagulans with the highest degradation rate was calculated and numbered AMNJ008. This strain was retained as the basic mother strain for the domestication of Bacillus coagulans that degrades zearalenone.

[0026] Example 2

[0027] This embodiment provides a Bacillus coagulans strain that degrades zearalenone. The strain number of the Bacillus coagulans strain is AMNJ008.

[0028] This embodiment also provides a method for acclimating and cultivating Bacillus coagulans for degrading zearalenone, which specifically comprises the following steps:

[0029] (1) First, weigh 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 1.0 g of Tween-80, 0.4 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.29 of manganese sulfate, 20.0 g of calcium carbonate, and 15-20 g of agar, add distilled water to make up to 100 mL, adjust the pH to 6.3, sterilize at 121°C, 0.1 MPa for 20 min, and prepare MRS liquid medium;

[0030] Then, the MRS liquid culture medium is divided into 10 portions, and the mother solution of Bacillus coagulans is inoculated into one of the MRS liquid culture medium at an inoculum amount of 1% (V / V), and the culture is statically cultured at 37° C. for 24 hours to obtain a seed solution of Bacillus coagulans; zearalenone methanol stock solution is added to the remaining 9 portions of the MRS liquid culture medium respectively to prepare 9 acclimated culture media with different zearalenone concentrations of 3.0 ug / mL, 3.5 ug / mL, 4.0 ug / mL, 4.5 ug / mL, 5.0 ug / mL, 5.5 ug / mL, 6.0 ug / mL, 6.5 ug / mL and 7.0 ug / mL, respectively, and the acclimated culture media are respectively filled into 9 conical flasks;

[0031] (2) The Bacillus coagulans seed liquid is inoculated into 9 acclimation culture medium flasks containing different zearalenone concentrations prepared in the above step (1); the 9 inoculated flasks are placed at 37°C for 24 hours to obtain 9 portions of primary induced mixed strain liquid; the zearalenone concentrations of the supernatants in the 9 portions of the primary induced mixed strain liquid are detected respectively, and the primary induced mixed strain liquid with the highest zearalenone concentration degradation rate in the supernatant is streaked and amplified on an MRS culture plate to obtain a circulating starting strain;

[0032] (3) The circulating starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 3.0 ug / mL at an inoculation rate of 1% and placed at 37°C for 24 hours to obtain a second-generation circulating starting strain. The second-generation circulating starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 3.5 ug / mL at an inoculation rate of 1% and placed at 37°C for 24 hours to obtain a third-generation circulating starting strain. The starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 4.0 ug / mL and placed at 37°C for 24 hours to obtain a fourth-generation circulating starting strain. The fourth-generation circulating starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 4.5 ug / mL at an inoculation rate of 1% and placed at 37°C for 24 hours to obtain a fifth-generation circulating starting strain. The fifth-generation circulating starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 5.0 ug / mL at an inoculation rate of 1%. / mL of acclimation medium and placed at 37°C for 24 hours to obtain the sixth generation of circulating starting strains, which were inoculated with the sixth generation of circulating starting strains into the acclimation medium with a corn zearalenone concentration of 5.5ug / mL at an inoculation rate of 1% and placed at 37°C for 24 hours to obtain the seventh generation of circulating starting strains, which were inoculated with the seventh generation of circulating starting strains into the acclimation medium with a corn zearalenone concentration of 6.0ug / mL at an inoculation rate of 1% and placed at 37°C for 24 hours. After static culture for 24 hours, the eighth-generation cycle starting strain was obtained. The eighth-generation cycle starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 6.5 ug / mL at an inoculation size of 1% and placed at 37°C for static culture for 24 hours to obtain a ninth-generation cycle starting strain. The ninth-generation cycle starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 7.0 ug / mL at an inoculation size of 1% and placed at 37°C for static culture for 24 hours to obtain a tenth-generation cycle starting strain.

[0033] After each generation of culture, the concentration of zearalenone in the supernatant of each generation was tested. When the concentration of zearalenone in the supernatant remained unchanged, it was considered that the strain had reached its maximum tolerance to zearalenone, and the strain was retained as a Bacillus coagulans production strain for degrading zearalenone.

[0034] The method for acclimating and cultivating the Bacillus coagulans strain that degrades zearalenone also includes preparing a strain preservation medium, and preserving the Bacillus coagulans strain that degrades zearalenone in the strain preservation medium. The step of preparing the strain preservation medium includes: first 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, 1.0g of Tween-80, 0.4g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.29g of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to 100mL, adjusting the pH to 6.3, 121°C, 0.1Mpa, sterilizing for 20min, and preparing the strain preservation medium.

[0035] Performance Testing

[0036] Toxin degradation test:

[0037] In the method for acclimating and cultivating a strain of Bacillus coagulans capable of degrading zearalenone provided in this embodiment, in step (2), there are five culture media with reduced zearalenone concentrations, which are named T1, T2, T3, T4, and T5, respectively. The zearalenone degradation rates in the three culture media with reduced zearalenone concentrations are 5.12%, 7.68%, 22.08%, 29.80%, and 35.0%, respectively.

[0038] Then, T5 is selected as the breeding object and is cultured step by step according to the step (3), and a first-generation Bacillus coagulans producing strain capable of stably degrading zearalenone is domesticated and bred. The degradation rate of zearalenone by the first-generation Bacillus coagulans producing strain is stably maintained at more than 35.0% under the condition of 30°C. The degradation effect of the first-generation Bacillus coagulans producing strain on zearalenone at different co-culture times is measured, and the concentration of zearalenone can be reduced from 6.0 ug / mL to 3.128 ug / mL, and the degradation rate is 47.87%;

[0039] Finally, the first generation of Bacillus coagulans production strain was cultured for 10 generations using the MRS liquid culture medium. After 10 generations of culture, the degradation rate of zearalenone by the tenth generation of Bacillus coagulans production strain and its culture solution obtained reached more than 80.0%.

[0040] Implementation Case 3

[0041] This embodiment provides a method for acclimating and cultivating Bacillus coagulans that degrades zearalenone. The specific steps are substantially the same as those in Example 1, except that:

[0042] In the step (1), adding the zearalenone methanol stock solution to the remaining 9 portions of the MRS liquid culture medium to prepare 9 acclimation culture media with different zearalenone concentrations of 3.0 ug / mL, 3.5 ug / mL, 4.0 ug / mL, 4.5 ug / mL, 5.0 ug / mL, 5.5 ug / mL, 6.0 ug / mL, 6.5 ug / mL and 7.0 ug / mL, and respectively loading them into 9 conical flasks;

[0043] The step (3) is as follows: inoculating the circulating starting strain into an acclimation medium with a corn zearalenone concentration of 3.0 ug / mL at an inoculation amount of 3% and placing it at 37°C for static culture for 24 hours to obtain a second-generation circulating starting strain; inoculating the second-generation circulating starting strain into an acclimation medium with a corn zearalenone concentration of 3.5 ug / mL at an inoculation amount of 3% and placing it at 37°C for static culture for 24 hours to obtain a third-generation circulating starting strain; The third generation cycle starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 4.0 ug / mL and placed at 37°C for 24 hours to obtain a fourth generation cycle starting strain. The fourth generation cycle starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 4.5 ug / mL at an inoculation rate of 3% and placed at 37°C for 24 hours to obtain a fifth generation cycle starting strain. The fifth generation cycle starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 4.5 ug / mL at an inoculation rate of 3%. The six-generation circulating starting strain was inoculated into the acclimation medium with a concentration of 5.0 ug / mL and placed at 37°C for 24 hours to obtain the six-generation circulating starting strain. The six-generation circulating starting strain was inoculated into the acclimation medium with a concentration of 5.5 ug / mL of zearalenone at an inoculation rate of 3% and placed at 37°C for 24 hours to obtain the seven-generation circulating starting strain. The seven-generation circulating starting strain was inoculated into the acclimation medium with a concentration of 6.0 ug / mL of zearalenone at an inoculation rate of 3% and placed at 37°C for 24 hours to obtain the seven-generation circulating starting strain. After static culture at 37°C for 24 hours, an eighth-generation circulating starting strain was obtained. The eighth-generation circulating starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 6.5 ug / mL at an inoculation size of 3% and placed at 37°C for static culture for 24 hours to obtain a ninth-generation circulating starting strain. The ninth-generation circulating starting strain was inoculated into an acclimated medium with a corn zearalenone concentration of 7.0 ug / mL at an inoculation size of 3% and placed at 37°C for static culture for 24 hours to obtain a tenth-generation circulating starting strain.

[0044] After each generation of culture, the concentration of zearalenone in the supernatant of each generation was tested. When the concentration of zearalenone in the supernatant remained unchanged, it was considered that the strain had reached its maximum tolerance to zearalenone, and the strain was retained as a production strain of Bacillus coagulans for degrading zearalenone.

[0045] Performance Testing

[0046] Toxin degradation test:

[0047] In the method for acclimating and cultivating a strain of Bacillus coagulans capable of degrading zearalenone provided in this embodiment, in step (2), there are five culture media with reduced zearalenone concentrations, which are named T1, T2, T3, T4, and T5, respectively. The zearalenone degradation rates in the three culture media with reduced zearalenone concentrations are 5.37%, 6.98%, 21.56%, 30.13%, and 35.4%, respectively.

[0048] Then, T5 is selected as the breeding object and is cultured step by step according to the step (3), and a first-generation Bacillus coagulans producing strain capable of stably degrading zearalenone is domesticated and bred. The degradation rate of zearalenone by the first-generation Bacillus coagulans producing strain is stably maintained at more than 35.0% under the condition of 30°C. The degradation effect of the first-generation Bacillus coagulans producing strain on zearalenone at different co-culture times is measured, and the concentration of zearalenone can be reduced from 6.0 ug / mL to 2.512 ug / mL, and the degradation rate is 58.13%;

[0049] Finally, the first generation of Bacillus coagulans production strain was cultured for 10 generations using the MRS liquid culture medium. After 10 generations of culture, the degradation rate of zearalenone by the tenth generation of Bacillus coagulans production strain and its culture solution obtained reached more than 80.0%.

[0050] Implementation Case 4

[0051] This embodiment provides a method for acclimating and cultivating a Bacillus coagulans strain that degrades zearalenone. The specific steps are substantially the same as those in Implementation Example 1, except that:

[0052] In the step (1), the MRS liquid culture medium is divided into 6 portions, Bacillus coagulans is inoculated into one of the MRS liquid culture medium at a standard inoculation amount of 3%, and the medium is kept statically cultured at 37°C for 24 hours to obtain a Bacillus coagulans seed solution. Zearalenone methanol stock solution is added to the remaining 5 portions of the MRS liquid culture medium to prepare acclimation culture media with 5 different zearalenone concentrations of 4.0ug / mL, 4.5ug / mL, 5.0ug / mL, 5.5ug / mL, and 6.0ug / mL, respectively, and the acclimation culture media are respectively filled into 5 triangular flasks to prepare acclimation culture media;

[0053] The step (3) is as follows: inoculating the circulating starting strain into an acclimation medium with a corn zearalenone concentration of 4.0 ug / mL at an inoculation amount of 3% and placing it at a constant temperature and culturing it for 24 hours at 37°C to obtain a second-generation circulating starting strain; inoculating the second-generation circulating starting strain into an acclimation medium with a corn zearalenone concentration of 4.5 ug / mL at an inoculation amount of 3% and placing it at a constant temperature and culturing it for 24 hours at 37°C to obtain a third-generation circulating starting strain; inoculating the third-generation circulating starting strain into an acclimation medium with a corn zearalenone concentration of 5.0 ug / mL at an inoculation amount of 3% and placing it at a constant temperature and culturing it for 24 hours at 37°C to obtain a third-generation circulating starting strain; ug / mL of acclimation medium and placed at 37°C for constant temperature static culture for 24h to obtain the fourth generation of circulating starting strains, the fourth generation of circulating starting strains were inoculated into the acclimation medium with a concentration of 5.5ug / mL of zearalenone at an inoculation amount of 3% and placed at 37°C for constant temperature static culture for 24h to obtain the fifth generation of circulating starting strains, the fifth generation of circulating starting strains were inoculated into the acclimation medium with a concentration of 6.0ug / mL of zearalenone at an inoculation amount of 3% and placed at 37°C for constant temperature static culture for 24h to obtain the sixth generation of circulating starting strains. After each generation of culture, the concentration of zearalenone in the supernatant of each generation was detected respectively. When the concentration of zearalenone in the supernatant remained unchanged, it was considered that the strain had reached the maximum tolerance of zearalenone, and this strain was retained as the production strain for degrading zearalenone.

[0054] Performance Testing

[0055] Toxin degradation test:

[0056] In the method for acclimating and cultivating a strain of Bacillus coagulans capable of degrading zearalenone provided in this embodiment, in step (1), there are five culture media with reduced zearalenone concentrations, which are named T1, T2, T3, T4, and T5, respectively. The zearalenone degradation rates in the three culture media with reduced zearalenone concentrations are 5.45%, 7.01%, 21.36%, 30.05%, and 34.9%, respectively.

[0057] Then, T5 is selected as the breeding object and is cultured step by step according to the step (3), and a first-generation Bacillus coagulans producing strain capable of stably degrading zearalenone is domesticated and bred. The degradation rate of zearalenone by the first-generation Bacillus coagulans producing strain is stably maintained at more than 35.0% under the condition of 30°C. The degradation effect of the first-generation Bacillus coagulans producing strain on zearalenone at different co-culture times is measured, and the concentration of zearalenone can be reduced from 6.0 ug / mL to 2.778 ug / mL, and the degradation rate is 53.7%;

[0058] Finally, the first generation of Bacillus coagulans production strain was cultured for 10 generations using the MRS liquid culture medium. After 10 generations of culture, the degradation rate of zearalenone by the tenth generation of Bacillus coagulans production strain and its culture solution obtained reached more than 80.0%.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A method for cultivating a strain of Bacillus coagulans capable of degrading zearalenone. It is characterized in that The following steps are involved: Step 1: provide 16 MRS liquid culture media containing the same concentration of zearalenone, respectively inoculate them into 16 Bacillus coagulans strains stored in the company's strain bank, and then carry out mixed culture at 37°C for 24 hours, respectively detect the zearalenone concentration of the supernatant in the 16 mixed strain liquids, select the mixed strain liquid with the highest zearalenone concentration degradation rate for streaking amplification culture on the MRS plate, and select a single bacterium to obtain the target strain; Step 2, providing N portions of MRS liquid culture medium and the above-mentioned Bacillus coagulans seed solution; adding zearalenone methanol stock solution to the N portions of the MRS liquid culture medium, respectively, to prepare N portions of acclimation culture medium with different zearalenone concentrations; Step 3, inoculating the Bacillus coagulans seed liquid into N acclimation culture media with different zearalenone concentrations with the same inoculation amount for cultivation, to obtain N portions of primary induced mixed strain liquid; detecting the zearalenone concentration of the supernatant in the N portions of the primary induced mixed strain liquid, respectively, selecting the primary induced mixed strain liquid with the largest zearalenone concentration degradation rate for streaking amplification culture on an MRS culture plate, to obtain a circulating starting strain; Step 4, sequentially using N portions of the acclimation culture medium containing zearalenone in increasing concentrations to perform step-by-step subculture on the circulating strain, and when the zearalenone concentration in the supernatant after the step-by-step subculture remains unchanged, retaining the strain as a Bacillus coagulans production strain for degrading zearalenone, wherein N is a natural number greater than or equal to 3; The Bacillus coagulans strain is numbered AMNJ008 and is stored in Guangdong Microbiological Culture Collection Center with a storage number of GDMCC.GIM1.

645.

2. The method for acclimating and cultivating the Bacillus coagulans strain capable of degrading zearalenone according to claim 1, It is characterized in that In the step 1, the preparation method of the MRS liquid culture 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 diammonium citrate, 1.0g of Tween-80, 0.4g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.29 of manganese sulfate, 20.0g of calcium carbonate, 15-20g of agar, adding distilled water to make up to 100mL, adjusting the pH to 6.3, 121°C, 0.1Mpa, sterilizing for 20min, and obtaining the strain preservation culture medium.

3. The method for acclimating and cultivating the Bacillus coagulans strain capable of degrading zearalenone according to claim 2, It is characterized in that In the step 2, the preparation method of the Bacillus coagulans seed liquid includes: inoculating a single Bacillus coagulans bacterium into the MRS liquid culture medium at an inoculation rate of 0.5% to 5%, and culturing it at a constant temperature of 37° C. for 24 h to 36 h to obtain the Bacillus coagulans seed liquid.

4. The method for acclimating and cultivating the Bacillus coagulans strain for degrading zearalenone according to claim 3, It is characterized in that The method also includes providing a strain preservation medium, in which the Bacillus coagulans strain producing strain for degrading zearalenone is preserved.

5. The method for acclimating and cultivating the Bacillus coagulans strain for degrading zearalenone according to claim 4, It is characterized in that The step of preparing the strain preservation medium comprises: adding 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of diamine citrate, 1.0 g of Tween-80, 0.4 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate, 0.29 g of manganese sulfate, 20.0 g of calcium carbonate, 15-20 g of agar, distilled water to make the volume to 100 mL, adjusting the pH to 6.3, 121° C., 0.1 Mpa, and sterilizing for 20 min to obtain the strain preservation medium.