CN114874932A - Lactobacillus johnsonii and application thereof in degrading deoxynivalenol and inhibiting pathogenic bacteria - Google Patents
Lactobacillus johnsonii and application thereof in degrading deoxynivalenol and inhibiting pathogenic bacteria Download PDFInfo
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Abstract
The invention discloses Lactobacillus johnsonii and application thereof in degrading deoxynivalenol and inhibiting pathogenic bacteria, wherein the Lactobacillus johnsonii is NJD412 which is classified and named as Lactobacillus johnsonii, and the preservation number is CGMCC No. 23553. The lactobacillus johnsonii NJD412 is obtained by separating and purifying dog excrement, and the MRS plate bacterial colony is semitransparent and circular, has the diameter of about 1-2mm, and has irregular edge. Experiments prove that the lactobacillus johnsonii NJD412 can effectively degrade deoxynivalenol in vitro and in vivo and has obvious inhibiting effect on enterotoxigenic escherichia coli and salmonella typhimurium. The lactobacillus johnsonii NJD412 and the fermentation liquor thereof can be used for relieving animal poisoning caused by deoxynivalenol pollution and preventing and treating animal diseases induced by other pathogenic bacteria, are safe to use and have good market application prospects.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus johnsonii and application thereof in degrading deoxynivalenol and inhibiting pathogenic bacteria.
Background
At present, animal husbandry production faces a plurality of challenges, the problems of environmental pollution, feed pollution, pathogenic microorganism invasion and the like are particularly prominent, economic benefits of animal husbandry are seriously influenced, and sustainable development of animal husbandry is hindered. The feed is easily polluted by environmental factors due to the fact that the feed relates to multiple links of raw material supply, processing flow, transportation and storage and the like, the main symptoms are that the feed is polluted by raw materials, improper in processing, bad and inferior in transportation and storage conditions and the like, the feed is polluted by fungi, bacteria and various toxic metabolites in the environment, diseases of digestive systems, immune systems, reproductive systems and the like of the livestock are caused or aggravated, the livestock breeding burden is increased, and losses are caused to the breeding industry.
Mycotoxins are small molecular secondary metabolites produced by fungi, have wide pollution and great harm, seriously harm the health of animals, and are one of the most serious problems in the feed industry and the breeding industry at present. The most seriously polluted mycotoxins mainly comprise aflatoxin B1, fumonisin B1, zearalenone, deoxynivalenol and the like. The deoxynivalenol is the most serious in pollution and shows a trend of increasing year by year, and the detection rate of the deoxynivalenol in feed raw materials such as corn, wheat and bran is 100 percent, the detection rate of complete feed exceeds 95 percent and the pollution is extremely serious according to the 2020 investigation result. The ingestion of deoxynivalenol has intestinal toxicity, immunotoxicity, reproductive toxicity and potential genetic toxicity, can cause the symptoms of anorexia, weight loss, metabolic disturbance, vomiting, diarrhea and the like of animals, seriously damages the hematopoietic system to cause death, and causes huge economic loss.
Colibacillosis is the most common bacterial infectious disease in breeding management, is caused by pathogenic escherichia coli, seriously harms animals such as pigs, chickens, geese, sheep and the like, particularly has great influence on young animals, causes symptoms such as mental depression, feed intake reduction, diarrhea and the like of the animals, and is very easy to cause death of the animals. The disease can be transmitted by intake of polluted drinking water and feed, respiratory tract and mating activities, is easy to cause large-scale infection, and is one of the most serious bacterial diseases faced by the breeding industry. Among them, enterotoxigenic escherichia coli is the most common one of pathogenic escherichia coli, is one of the main pathogens of diarrhea of piglets, calves, lambs and the like, causes acute diarrhea to occur in young animals to cause sudden death, and seriously hinders the benign development of the breeding industry.
At present, the mycotoxin prevention and control mainly comprises a physical method, a chemical method and a biological method, and the specific methods comprise biodegradation, adsorption, irradiation, ozone detoxification and the like. Compared with other methods, biodegradation is a research hotspot in the degradation direction of mycotoxins as a safe, efficient and environment-friendly detoxification technology. However, vaccination is one of the effective means for preventing and treating the young animals from being attacked by pathogenic escherichia coli, but the vaccines have obvious geographic limitations and safety problems, so more and more researches begin to pay attention to biological prevention and treatment technologies, and researches show that probiotics have great potential in preventing and treating bacterial diseases. The strengthening of the mycotoxin biodegradation technology and the practical research of probiotics on the pathogenic force of pathogenic bacteria prevention and control are carried out, and new breakthrough points and innovation points are searched, so that the development trend of the mycotoxin and pathogenic bacteria prevention and control is realized. At present, although the application of lactobacillus johnsonii in preparing animal feed is researched, the application of lactobacillus johnsonii in degrading deoxynivalenol and inhibiting pathogenic bacteria is rarely reported.
Disclosure of Invention
In order to overcome at least one problem in the prior art, the invention provides a lactobacillus johnsonii NJD412 with the capacity of degrading deoxynivalenol and the bacteriostatic action and application thereof, wherein the lactobacillus johnsonii NJD412 can obviously degrade the deoxynivalenol, has the action of obviously inhibiting enterotoxigenic escherichia coli and salmonella typhimurium, is proved to be safe in organisms and can play the role, and can be used for preparing feed additives and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a Lactobacillus johnsonii strain, wherein the 16s rDNA sequence is shown in SEQ ID No. 3.
Further, the Lactobacillus johnsonii is named as NJD412, is classified and named as Lactobacillus johnsonii, has the preservation number of CGMCC No.23553, has the preservation date of 2021, 10 and 09 months, has the preservation unit of China general microbiological culture Collection center (CGMCC), and has the preservation unit address of China institute of microbiology, institute of sciences, No.3, West Lu 1, North Chen, and Yangyang, Beijing.
Further, the lactobacillus johnsonii NJD412 was isolated from dog faeces.
Further, the colony of lactobacillus johnsonii NJD412 is translucent, circular, about 1-2mm in diameter, wet in surface, and irregular in edge.
Further, the lactobacillus johnsonii NJD412 reached logarithmic growth phase after 3h of culture, showing good growth activity; the lactobacillus johnsonii NJD412 has good acid production capacity; the lactobacillus johnsonii NJD412 has certain tolerance to acidic environments with different pH values; the lactobacillus johnsonii NJD412 has certain tolerance to different concentrations of bile salt environments; the lactobacillus johnsonii nqd 412 has good biological safety.
Further, the lactobacillus johnsonii NJD412 has bacteriostatic ability; in one embodiment, lactobacillus johnsonii NJD412 has a significant inhibitory effect on enterotoxigenic e.coli and salmonella typhimurium, which significantly inhibits the growth of enterotoxigenic e.coli and salmonella typhimurium.
Further, the lactobacillus johnsonii NJD412 has the ability to degrade deoxynivalenol. In a specific embodiment, lactobacillus johnsonii NJD412 is inoculated into a culture medium containing deoxynivalenol, the in-vitro degradation efficiency of the deoxynivalenol is 52.49 +/-2.45%, and the content of the deoxynivalenol can be effectively degraded; in a specific embodiment, oral administration of lactobacillus johnsonii NJD412 reduces the amount of the toxin in animals fed with a feed contaminated with deoxynivalenol, and lactobacillus johnsonii NJD412 has a good in vivo deoxynivalenol degrading ability and is capable of reducing the serum deoxynivalenol concentration.
Further, the lactobacillus johnsonii NJD412 has a protective effect on pathogenic bacteria, deoxynivalenol or a combined effect of the pathogenic bacteria, the deoxynivalenol or the deoxynivalenol. In one embodiment, lactobacillus johnsonii NJD412 is effective in reducing weight loss in mice resulting from combination, in reducing organ index changes resulting from infection or combination, and in reducing intestinal histology and intestinal inflammation. For example, it can specifically prevent the toxic action of enterotoxigenic Escherichia coli to animals, and relieve animal poisoning caused by deoxynivalenol contamination.
In a second aspect of the invention, there is provided a microbial inoculant comprising lactobacillus johnsonii as described in any one of the first aspects of the invention; or prepared by culturing Lactobacillus johnsonii according to any of the first aspects of the invention.
Furthermore, the viable bacteria concentration of the microbial inoculum is 2 multiplied by 10 7 ～2×10 9 CFU/mL; preferably 5X 10 7 ～5×10 8 CFU/mL; more preferably 1X 10 8 CFU/mL. In practical applications, the microbial agent may be prepared to a predetermined concentration for use.
Further, the preparation steps of the microbial agent comprise: activating lactobacillus johnsonii NJD412, subculturing the activated lactobacillus johnsonii liquid into an MRS liquid culture medium (purchased from Qingdao Haibo biology, HB0384-1), placing the MRS liquid culture medium into a constant-temperature incubator at 37 ℃, standing and culturing for 16-24 hours, and adjusting fermentation liquor CFU to obtain the microbial agent. Specifically, subculturing to 100mL of MRS liquid culture medium according to the volume ratio of 5-10% (preferably 5%) for culture.
Further, after the microbial agent (fermentation liquid) is centrifuged and filtered by a bacterial filter, a thallus-free lactobacillus johnsonii NJD412 fermentation liquid can be obtained. Specifically, the mixture was centrifuged at 4000rpm for 10min and filtered through a bacterial filter having a diameter of 0.22. mu.m.
A third aspect of the invention provides a use of a lactobacillus johnsonii according to any of the first aspects of the invention, or a microbial inoculant according to any of the second aspects of the invention, the use comprising at least one of the following uses: the application in degrading deoxynivalenol and inhibiting pathogenic bacteria.
Further, in the above application, the pathogenic bacteria include at least one of enterotoxigenic escherichia coli and salmonella typhimurium.
Further, in the above use, the use comprises using the lactobacillus johnsonii or the microbial agent for the preparation of a product having at least one of the following functions: the deoxynivalenol is degraded in vitro, the absorption of animals on the deoxynivalenol is reduced, the concentration of the deoxynivalenol in animal serum is reduced, and the growth of pathogenic bacteria is inhibited.
Further, in the above application, the application comprises the use of the lactobacillus johnsonii or the microbial agent for preparing a product for preventing or treating animal damage caused by pathogenic bacteria, deoxynivalenol or a combination of the pathogenic bacteria and the deoxynivalenol.
Further, the product comprises feed, food, medicine and additives.
Further, the product also comprises agriculturally and pharmaceutically acceptable auxiliaries, including but not limited to: dispersants, stabilizers, carriers, and the like. Dosage forms for the above products include, but are not limited to: liquid preparations, solid preparations (such as powders), and the like.
Further, the animal injury comprises at least one of: deoxynivalenol poisoning, animal injury induced by pathogenic bacteria; specifically, the method can comprise the following steps: weight loss, infection, organ index change, intestinal histological changes, intestinal inflammation, etc.
In a fourth aspect of the present invention, there is provided a method of degrading deoxynivalenol and/or suppressing pathogenic bacteria, comprising the steps of: activating and culturing lactobacillus johnsonii as defined in any one of the first aspects of the invention to obtain a microbial agent, and applying the microbial agent to an object to be treated in a predetermined dose to degrade deoxynivalenol and/or repress pathogenic bacteria.
Further, the preparation steps of the microbial agent comprise: activating lactobacillus johnsonii NJD412, subculturing the activated bacterium liquid into an MRS liquid culture medium, placing the MRS liquid culture medium in a constant-temperature incubator at 37 ℃, standing and culturing for 16-24 hours, and adjusting fermentation liquor CFU to obtain the microbial agent. Specifically, subculturing to 100mL of MRS liquid culture medium according to the volume ratio of 5-10% (preferably 5%) for culture.
Furthermore, the viable bacteria concentration of the microbial inoculum is 2 multiplied by 10 7 ～2×10 9 CFU/mL; preferably 5X 10 7 ～5×10 8 CFU/mL; more preferably 1X 10 8 CFU/mL. In practical applications, the microbial agent may be prepared to a predetermined concentration for use.
Further, the object to be treated is an animal, and the animal includes, but is not limited to, pigs, chickens, geese, sheep, ducks, rats and the like.
Further, in a specific embodiment, the subject to be treated is a mouse, and the dose of the microbial agent is 1 × 10 6 ～1×10 10 CFU/machine, the frequency of application is 4-14 days of continuous drenching. More specifically, in the safety evaluation, the administration doses thereof were respectively: 1X 10 6 CFU/only, 1 × 10 8 CFU/only, 1 × 10 10 Continuously drenching CFU/CFU for 7 days; in the evaluation of the in vivo degradation activity of the deoxynivalenol, the application dosage is as follows: 1 x 10 8 Continuously drenching CFU/CFU for 10 days; in the verification of the protection of the combined action of pathogenic bacteria and deoxynivalenol, the application dosage is as follows: 1X 10 8 CFU/mouse, drench continuously for 14 days.
Compared with the prior art, the technical scheme adopted by the invention has the following beneficial effects:
the lactobacillus johnsonii NJD412 which can degrade deoxynivalenol and has a remarkable inhibiting effect on pathogenic bacteria such as enterotoxigenic escherichia coli, salmonella typhimurium and the like common in animal production is screened out from dog excrement. Specifically, lactobacillus johnsonii NJD412 can degrade deoxynivalenol in vitro, reduce the absorption of animals on the deoxynivalenol, inhibit the growth of enterotoxigenic escherichia coli and salmonella typhimurium, prevent and relieve the harm of the enterotoxigenic escherichia coli to the growth performance and digestive system of the animals, and further effectively improve the resistance of the animals to pathogenic bacteria such as the deoxynivalenol, the enterotoxigenic escherichia coli and the like. Therefore, the lactobacillus johnsonii NJD412 can be used for preventing and treating the harm of deoxynivalenol and other pathogenic bacteria to animals, and the bacterium is simple to use, reliable in safety, good in effect and good in market application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a colony morphology of Lactobacillus johnsonii NJD412 in an embodiment of the present invention.
FIG. 2 is a graph showing the growth of Lactobacillus johnsonii NJD412 in accordance with one embodiment of the present invention.
FIG. 3 is a diagram illustrating the results of an acid resistance test on Lactobacillus johnsonii NJD412 in accordance with an embodiment of the present invention.
FIG. 4 is a graph showing the results of the bile salt resistance test of Lactobacillus johnsonii NJD412 in accordance with one embodiment of the present invention.
FIG. 5 is a schematic diagram showing the inhibitory effect of Lactobacillus johnsonii NJD412 on enterotoxigenic Escherichia coli and Salmonella typhimurium in one embodiment of the present invention.
FIG. 6 is a schematic diagram showing the effect of Lactobacillus johnsonii NJD412 on the weight change of normal mice in accordance with one embodiment of the present invention.
FIG. 7 is a graph showing the effect of Lactobacillus johnsonii NJD412 on the change in body weight of mice which produce a combination of enterotoxigenic Escherichia coli and deoxynivalenol in accordance with one embodiment of the present invention.
FIG. 8 is a graph showing the effect of Lactobacillus johnsonii NJD412 on the expression of mRNA for jejunal inflammatory factor in mice with combined action of enterotoxigenic Escherichia coli and deoxynivalenol in accordance with one embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. The experimental materials not shown in the following examples are all commercially available materials. The equipment used in each step in the following examples is conventional. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless otherwise defined or indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
In the following examples, MRS plates were purchased from Qingdao Haibo Bio Inc., HB 0384; MRS liquid medium purchased from Qingdao Haibobo, HB 0384-1; LB plates were purchased from Qingdao haibo bio, HB 0129; SPF male C57BL/6 mice were purchased from Nanjing Qinglongshan breeding farms, qls 02-0102; styrene oxide is available from carbofuran technologies ltd, 994540; deoxynivalenol is available from sigma aldrich trade ltd D0156.
Example 1 isolation, screening and characterization of Lactobacillus johnsonii (Lactobacillus johnsonii) NJD412
The lactobacillus johnsonii NJD412 was obtained in this example using the following method:
(1) isolation and screening of lactic acid bacteria
Collecting 10 parts of different dog excrement samples in the basalt area of Nanjing, weighing 0.1g of excrement, and diluting with sterile normal saline to 10% -4 And spreading on MRS plate (purchased from HB0384, Haibo biology, Qingdao), standing at 37 deg.C for 48h, and preliminarily observing plate colonies. A proper single colony is aseptically picked and inoculated in an MRS liquid culture medium (purchased from Qingdao Haibo biology company, HB0384-1), and after static culture for 48h at 37 ℃, 10 mu L of bacterial liquid MRS plate is taken and streaked. This procedure was repeated three times to obtain a purified culture.
Purification of the bacterial strains according to the bacterial DON extraction kit (Beijing Soilebao science and technology limited) operation instructions extraction of purified bacterial strains of DNA.
Identifying all the obtained purified culture strains by adopting a 16s rDNA molecular characteristic identification method, wherein the 16s rDNA primers are as follows: 16S rDNA 27F: AGAGTTTGATCCTGGCTCAG (SEQ ID No. 1); 16S rDNA 1492R: GGTTACCTTTGTTACGACTT (SEQ ID No. 2). PCR reaction (50. mu.L): 2X Phanta Max Master Mix 25. mu.L, 2. mu.L each of upstream and downstream primers (10. mu.M), template DNA 150ng, plus ddH 2 O to a total volume of 50. mu.L. PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 90s, for 35 cycles; extension was complete for 5min at 72 ℃. And (3) sending the PCR product to a sequencing company for sequencing, comparing a sequencing result by an NCBI database, and preliminarily screening 27 strains of lactic acid bacteria.
(2) Screening of lactic acid bacteria with deoxynivalenol degradation capability
Preparing an inorganic salt culture medium: (NH) 4 ) 2 SO 4 2.0g，MgSO 4 ·7H 2 O 0.2g，CaCl 2 0.01g，FeSO 4 ·7H 2 O0.001g，Na 2 HPO 4 ·12H 2 O 1.5g，KH 2 PO 4 1.5 g. Adding distilled water to volume of 1L, adjusting pH to 7.0, and autoclaving at 121 deg.C for 20 min.
Preparing an inorganic salt culture medium containing styrene oxide: 114.00 μ L of the filtered styrene oxide was added to 100mL of the sterilized inorganic salt medium, and the mixture was mixed to prepare 10mM of the styrene oxide-containing inorganic salt medium. Styrene oxide is available from Bailingwei technologies, Inc., 994540.
Preparing an inorganic salt culture medium containing deoxynivalenol: adding 5mg of deoxynivalenol into 5mL of sterilized inorganic salt culture medium, uniformly mixing, and preparing into 1mg/mL of inorganic salt culture medium containing deoxynivalenol. Deoxynivalenol is available from sigma aldrich trade ltd D0156.
Respectively sucking primary-screened probiotics, inoculating the probiotics to an inorganic salt culture solution containing styrene oxide according to a proportion of 5%, carrying out primary screening, and carrying out static culture at 37 ℃ for 5d to observe the growth condition. Inoculating a strain which can grow in an inorganic salt culture solution containing styrene oxide into an MRS liquid culture medium again for activation, inoculating the activated strain into an inorganic salt culture medium containing a certain amount of deoxynivalenol, standing and culturing for 5d at 37 ℃, and detecting the content of the deoxynivalenol in the culture solution by using an ELISA method so as to screen out the canine probiotics capable of degrading the deoxynivalenol. Through the tests, a lactic acid bacterium with the capacity of degrading deoxynivalenol is screened out and named as NJD 412. Through molecular identification and sequence comparison, the strain is Lactobacillus johnsonii, the length of the 16s rDNA of the strain is 1474bp, and the sequence is shown as SEQ ID No. 3.
The colony morphology of the MRS plate of Lactobacillus johnsonii NJD412 is shown in FIG. 1, the colony is semitransparent, circular, about 1-2mm in diameter, wet in surface and irregular in edge; in the screening process, the degradation efficiency of the lactobacillus johnsonii NJD412 to the deoxynivalenol is 52.49 +/-2.45%.
The Lactobacillus johnsonii (Lactobacillus johnsonii) NJD412 screened in this example has been strain-preserved with the preservation number of CGMCC No.23553, the preservation date of 2021 year, 10 month and 09 days, the preservation unit is the common microorganism center of the china committee for culture collection of microorganisms (CGMCC), and the preservation unit address is the institute of microbiology of china academy of sciences No.3, west lou 1 north chen of the sunward area, beijing.
Example 2 growth Curve, in vitro acid production, acid resistance, bile salt resistance assay of Lactobacillus johnsonii NJD412
(1) Determination of growth curves
Activating the Lactobacillus johnsonii NJD412 with the deoxynivalenol degradation capability in example 1, passaging the activated bacterial liquid to 100mL of MRS liquid culture medium according to 5%, placing the MRS liquid culture medium in a constant-temperature incubator at 37 ℃, standing and culturing for 24h, sucking 1mL of bacterial liquid every 3h for continuous 10-fold dilution, wherein the concentration of the dilution is adjusted to 10 -1 To 10 -7 And respectively sucking 100 mu L of diluent, coating the diluent on an MRS plate culture medium, placing the MRS plate culture medium in a constant-temperature incubator at 37 ℃ for standing culture for 24 hours, and counting. And calculating the concentration of the bacterial liquid, recording, and repeatedly taking an average value for 3 times. The growth curve of Lactobacillus johnsonii NJD412 is plotted with the cultivation time (hours) as the abscissa and the bacterial liquid concentration as the ordinate.
The results are shown in fig. 2, and it is known that lactobacillus johnsonii nqd 412 having deoxynivalenol degradation ability reaches logarithmic growth phase after 3h of culture, growth rate is extremely fast, and good growth activity is shown.
(2) Acid production test
Activating the Lactobacillus johnsonii NJD412 with the deoxynivalenol degrading capability in example 1, transferring the activated bacterial liquid to 100mL of MRS liquid culture medium by 5%, placing the MRS liquid culture medium in a 37 ℃ constant-temperature incubator, standing and culturing for 12 hours, sucking 10mL of bacterial liquid into an EP tube, centrifuging for 10min at 4000r/min, sucking the supernatant, measuring and recording the pH value of the supernatant by using a pH meter, and repeating three times of tests to obtain an average value.
The result shows that the pH value of the supernatant of lactobacillus johnsonii NJD412 is 4.38 +/-0.11 after 12 hours and is obviously lower than the pH value (6.8) of an MRS culture medium, so that the lactobacillus johnsonii NJD412 has good acid production capability, can reduce the pH value of an intestinal tract and create an environment which is not beneficial to the growth and propagation of other pathogenic bacteria, thereby inhibiting the growth of the pathogenic bacteria and preventing the intestinal tract diseases caused by infection factors.
(3) Acid resistance test
Preparation of MRS media of different pH values required for the test: preparing MRS liquid culture medium with 1M hydrochloric acid solution, adjusting pH value of MRS liquid culture medium to 2.0, 3.0, 4.0, 5.0, and 6.0 with pH meter, subpackaging, labeling, and autoclaving.
Activating the lactobacillus johnsonii NJD412 with the deoxynivalenol degradation capability in example 1, passaging the activated bacterial liquid to MRS liquid culture media with different pH values according to 5%, placing the activated bacterial liquid into a 37 ℃ constant temperature incubator for static culture by taking the MRS culture media without the adjusted pH value as a control, sucking 1mL of bacterial liquid every 1h for continuous 10-fold dilution, sucking 100 mu L of diluted liquid respectively, coating the diluted liquid on an MRS plate culture medium, placing the MRS plate culture medium in the 37 ℃ constant temperature incubator for static culture for 24h, and counting. And calculating the number of live bacteria in the bacterial liquid, recording, and repeating the test for three times to obtain an average value.
As shown in FIG. 3, it was found that Lactobacillus johnsonii NJD412 having the ability to degrade deoxynivalenol was inoculated into a medium having a pH of 6.0 and maintained the same growth rate as the control; inoculating into culture medium with pH of 5.0, 4.0, and 3.0, and the viable count is 8.10 × 10 after 6 hr 6 CFU/mL、5.07×10 6 CFU/mL、2.73×10 6 CFU/mL; inoculating into culture medium with pH of 2.0, and viable count after 2 hr is 1.33 × 10 4 CFU/mL. The bacteria have certain tolerance to acid environments with different pH values.
(4) Bile salt resistance test
Preparation of MRS liquid culture media with different cholate concentrations required by the test: adding 0.1, 0.2, 0.3 and 0.4g pig bile salt into 100mL MRS liquid culture medium respectively to prepare MRS culture medium with bile salt concentration of 0.1%, 0.2%, 0.3% and 0.4%, subpackaging, labeling, and autoclaving for use.
Activating the lactobacillus johnsonii NJD412 with the deoxynivalenol degradation capability in example 1, passaging the activated bacterial liquid to MRS liquid culture mediums with different cholate concentrations according to 10%, placing the MRS liquid culture mediums in a constant-temperature incubator at 37 ℃ for static culture, sucking 1mL of bacterial liquid every 2h for continuous 10-fold dilution, sucking 100 mu L of diluted liquid respectively, coating the diluted liquid on an MRS plate culture medium, placing the MRS plate culture medium in the constant-temperature incubator at 37 ℃ for static culture for 24h, and counting. And calculating the number of live bacteria in the bacterial liquid, recording, and repeating the test for three times to obtain an average value.
As shown in FIG. 4, it was found that Lactobacillus johnsonii NJD412 having deoxynivalenol degradation ability was inoculated in MRS medium having a bile salt concentration of 0.1%, and the viable cell count after 6 hours was 1.21X 10 9 CFU/mL; inoculating in MRS culture medium with bile salt concentration of 0.2%, and viable count after 6 hr is 3.53 × 10 7 CFU/mL; inoculating in MRS culture medium with bile salt concentration of 0.3%, and viable count after 6 hr is 1.34 × 10 5 CFU/mL; inoculating in MRS culture medium with bile salt concentration of 0.4%, and viable count after 2 hr is 1.63 × 10 4 CFU/mL. The bacteria have certain tolerance to different concentrations of bile salt environments.
Example 3 inhibition of pathogenic bacteria by Lactobacillus johnsonii NJD412 assay
In this example, Escherichia coli and Salmonella typhimurium were used as enterotoxigenic bacteria, and the bacteriostatic ability of Lactobacillus johnsonii NJD412 was tested.
Inoculating Lactobacillus johnsonii NJD412 in MRS liquid culture medium at a ratio of 5%, standing at 37 deg.C for 24 hr, and adjusting the concentration of the liquid to 1 × 10 8 CFU/mL. And (3) centrifuging the bacterial liquid at 4000rpm for 10min, and filtering the bacterial liquid by using a bacterial filter with the diameter of 0.22 mu m to obtain the lactobacillus johnsonii NJD412 fermentation liquid without thalli.
Respectively and uniformly inoculating activated enterotoxigenic escherichia coli and salmonella typhimurium on an LB plate (purchased from Qingdao Haibo biology company, HB0129), carrying out inverted culture at 37 ℃ for 1h, taking out, punching a round hole on the LB plate inoculated with the pathogenic bacteria by using a puncher, respectively adding 100 mu L of bacterial liquid and 100 mu L of bacteria-free fermentation liquid into the round hole, and adding 100 mu L of sterile physiological saline to prepare a blank control. Then, the plate is placed in an incubator at 37 ℃ for culture, and the bacteriostasis condition is observed after 12 hours, wherein the bacteriostasis effect is shown in figure 5. The results show that the lactobacillus johnsonii NJD412 has obvious inhibition effect on enterotoxigenic escherichia coli and salmonella typhimurium.
Example 4 safety test of Lactobacillus johnsonii NJD412
This example uses normal mice as subjects to evaluate the safety of Lactobacillus johnsonii NJD412 by feeding.
To test the safety of the strains of this example, a mouse safety test was performed. Selecting 48 healthy SPF (specific pathogen free) grade male C57BL/6 mice (purchased from Nanjing Qinglongshan animal breeding farm, qls02-0102) with the age of 6 weeks, dividing the mice into a control group, a low-dose group, a medium-dose group and a high-dose group, respectively feeding the mice in a sterilized mouse cage, filling equal amount of sawdust, feeding equal amount of sawdust, ensuring sufficient drinking water and proper environmental temperature and humidity, keeping environmental sanitation, and ensuring that all the mice are healthy and have no stress phenomenon after being adaptively fed for one week.
The lactobacillus johnsonii NJD412 with the deoxynivalenol degradation capability in example 1 is activated, the activated bacterial liquid is passaged to 100mL of MRS liquid culture medium according to 5 percent, and the MRS liquid culture medium is placed in a constant temperature incubator at 37 ℃ for static culture for 16h, and then colony counting is carried out. Adjusting the concentration of the bacterial liquid to 1 × 10 6 CFU/only (low dose group), 1X 10 8 CFU/body (middle dose group), 1X 10 10 The mice were gavaged with CFU/mouse (high dose group) and were continuously gavaged for 7d, using gavage equivalent of sterile MRS medium as control. And observing and recording the weight, the feeding, the behavior, the physical characteristics, the death condition and the like of the mice.
All mice have no uncomfortable symptoms after gastric lavage, the mice move normally, the mental state is good, the hair color is smooth and clean, the movement is free, the breathing is uniform, abnormal secretion does not exist, and all mice eat normal drinking water, normal excrement and normal urine; the body weight change of the mice is shown in fig. 6, and the body weight of the mice in the test period has no significant difference compared with the control; after the experiment, the mice were euthanized and all the experimental mice were necropsied, and no abnormality was found in organs such as heart, liver, spleen, lung and kidney. The results indicate that lactobacillus johnsonii NJD412 has good biological safety.
Example 5 in vivo Deoxynivalenol degrading Activity of Lactobacillus johnsonii NJD412
To verify the in vivo degradability of deoxynivalenol of lactobacillus johnsonii NJD412, a mouse test was performed.
Selecting healthy 48 SPF (specific pathogen free) grade male C57BL/6 mice with the age of 6 weeks, dividing the mice into a control group, a toxicity attacking + NJD412 group and a toxicity attacking + other lactobacillus group (lactobacillus johnsonii SHBCC D51535, Shanghai preservation biotechnology center), respectively feeding the mice in a sterilized mouse cage, filling equal amount of sawdust, feeding equal amount of sawdust, ensuring sufficient drinking water and proper environmental temperature and humidity, keeping environmental sanitation, and ensuring that all the mice are healthy and have no stress phenomenon after adaptive feeding for one week.
The lactobacillus johnsonii NJD412 with the deoxynivalenol degradation capability in example 1 and other lactic acid bacteria used for control are activated, the activated bacteria liquid is passaged to 100mL of MRS liquid culture medium according to 5 percent, the MRS liquid culture medium is placed in a constant temperature incubator at 37 ℃, and the colony counting is carried out after the static culture is carried out for 16 h. Respectively performing intragastric administration on the group of counteracting toxic substance + NJD412 and the group of counteracting toxic substance + other lactobacillus at a rate of 1 × 10 per day 8 CFU/only. Beginning on day four, each mouse in each group except the control group was gavaged with 3mg/kg body weight deoxynivalenol. The control group was gavaged with the same dose of MRS medium or saline over the test period. The test period was 10 days total, and after the test was completed, blood was collected from the eyeballs and the mice were euthanized.
Experiments show that the concentrations of the deoxynivalenol in the sera of the mice in the challenge group and the challenge + other lactobacillus group are 103.17 +/-18.08 ng/mL and 105.48 +/-31.17 ng/mL respectively, and the concentration of the deoxynivalenol in the sera of the mice in the challenge + NJD412 group is 56.14 +/-19.26 ng/mL. This indicates that Lactobacillus johnsonii NJD412 has good in vivo degradability of deoxynivalenol.
Example 6 protection test of Lactobacillus johnsonii NJD412 on the Combined action of pathogenic bacteria and deoxynivalenol
In this example, enterotoxigenic escherichia coli is taken as an example, and a mouse test is performed to verify the protective effect of lactobacillus johnsonii nqd 412 on harm of enterotoxigenic escherichia coli and deoxynivalenol in vivo.
Selecting healthy 96 SPF male C57BL/6 mice with age of 6 weeks, dividing the mice into a Control group (Control), an NJD412 group (NJD412), an infection group (ETEC, enterotoxigenic Escherichia coli infection), an attack group (DON, deoxynivalenol exposure), an attack + infection group (ETEC + DON), an infection + NJD412 group (ETEC + NJD412), an attack + NJD412 group (DON + NJD412), and an infection + attack + NJD412 group (ETEC + DON + NJD412), respectively feeding the mice in an equal amount of wood chips in a sterilized mouse cage to ensure sufficient drinking water and proper environmental temperature and humidity, keeping environmental sanitation, and ensuring the health and no stress phenomenon of all the mice after adaptive feeding for one week.
The lactobacillus johnsonii NJD412 with the deoxynivalenol degradation capability in example 1 is activated, the activated bacterial liquid is passaged to 100mL of MRS liquid culture medium according to 5 percent, and the MRS liquid culture medium is placed in a constant temperature incubator at 37 ℃ for static culture for 16h, and then colony counting is carried out. NJD412 group, infection + NJD412 group, challenge + NJD412 group, infection + challenge + NJD412 group, gavage 1X 10 per day 8 CFU/NJD 412 strain; on the 6 th to 8 th days of the experiment, mice in an infection group, a challenge group and an infection group, an infection group and an NJD412 group, and mice in an infection group, a challenge group and an NJD412 group are gavaged with enterotoxigenic escherichia coli and are multiplied by 1 multiplied by 10 8 CFU/only; then, mice in a toxin attacking group, a toxin attacking and infecting group, a toxin attacking and NJD412 group and a group of infecting, attacking and NJD412 are subjected to gastric lavage with deoxynivalenol every day, wherein the dosage is 1mg/kg of body weight. The control group was gavaged with the same dose of MRS medium or saline over the test period. The test period is 14 days, and the weight, feeding, behavior, physical characteristics, fatality and the like of the mice are observed and recorded.
Experiments show that the NJD412 can effectively relieve the weight loss of mice caused by infection and reduce the weight loss of mice caused by combined action, as shown in FIG. 7. Meanwhile, the NJD412 for early gavage can effectively reduce organ index change (Table 1) and intestinal inflammation (FIG. 8) caused by infection or combination. This shows that NJD412 can effectively prevent pathogenic bacteria enterotoxigenic Escherichia coli and animal injury caused by combined action of pathogenic bacteria enterotoxigenic Escherichia coli and deoxynivalenol, and has very good practical effect.
TABLE 1 index changes in the organs of mice that are affected by the combination of Enterobacter johnsonii NJD412 and deoxynivalenol
From the above embodiments, it can be known that lactobacillus johnsonii nqd 412 of the present invention can effectively degrade deoxynivalenol in vitro and in vivo, and has significant inhibitory effect on enterotoxigenic escherichia coli and salmonella typhimurium; and the lactobacillus johnsonii NJD412 and the fermentation liquor thereof can be used for relieving animal poisoning caused by deoxynivalenol pollution and preventing and treating animal diseases induced by other pathogenic bacteria, and are safe to use and have good market application prospects.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110> Nanjing university of agriculture
<120> Lactobacillus johnsonii and application thereof in degrading deoxynivalenol and inhibiting pathogenic bacteria
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 27F primer (Artificial Sequence)
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<213> 1492R primer (Artificial Sequence)
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<211> 1474
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<213> 16srDNA Sequence (Artificial Sequence)
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gcagtgcggg tgctatacat gcagtcgagc gagcttgcct agatgatttt agtgcttgca 60
ctaaatgaaa ctagatacaa gcgagcggcg gacgggtgag taacacgtgg gtaacctgcc 120
caagagactg ggataacacc tggaaacaga tgctaatacc ggataacaac actagacgca 180
tgtctagagt ttgaaagatg gttctgctat cactcttgga tggacctgcg gtgcattagc 240
tagttggtaa ggtaacggct taccaaggca atgatgcata gccgagttga gagactgatc 300
ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360
ccacaatgga cgaaagtctg atggagcaac gccgcgtgag tgaagaaggg tttcggctcg 420
taaagctctg ttggtagtga agaaagatag aggtagtaac tggcctttat ttgacggtaa 480
ttacttagaa agtcacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540
cgttgtccgg atttattggg cgtaaagcga gtgcaggcgg ttcaataagt ctgatgtgaa 600
agccttcggc tcaaccggag aattgcatca gaaactgttg aacttgagtg cagaagagga 660
gagtggaact ccatgtgtag cggtggaatg cgtagatata tggaagaaca ccagtggcga 720
aggcggctct ctggtctgca actgacgctg aggctcgaaa gcatgggtag cgaacaggat 780
tagataccct ggtagtccat gccgtaaacg atgagtgcta agtgttggga ggtttccgcc 840
tctcagtgct gcagctaacg cattaagcac tccgcctggg gagtacgacc gcaaggttga 900
aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960
aacgcgaaga accttaccag gtcttgacat ccagtgcaaa cctaagagat taggtgttcc 1020
cttcggggac gctgagacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaacccttg tcattagttg ccatcattaa gttgggcact 1140
ctaatgagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatgcc 1200
ccttatgacc tgggctacac acgtgctaca atggacggta caacgagaag cgaacctgcg 1260
aaggcaagcg gatctcttaa agccgttctc agttcggact gtaggctgca actcgcctac 1320
acgaagctgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc 1380
cttgtacaca ccgcccgtca caccatgaga gtctgtaaca cccaaagccg gtgggataac 1440
ctttatagga gtcagccgtc taagtagaca atgg 1474
Claims (10)
1. Lactobacillus johnsonii, wherein the 16s rDNA sequence of Lactobacillus johnsonii is shown in SEQ ID No. 3.
2. The Lactobacillus johnsonii according to claim 1, wherein said Lactobacillus johnsonii has the name NJD412 and the classification name Lactobacillus johnsonii and the preservation number CGMCC No. 23553.
3. A microbial agent, comprising the lactobacillus johnsonii of claim 1 or 2; or prepared by culturing the Lactobacillus johnsonii strain of claim 1 or 2.
4. The microbial inoculant according to claim 3, wherein the viable bacteria concentration of the microbial inoculant is 2 x 10 7 ～2×10 9 CFU/mL。
5. Use of lactobacillus johnsonii according to claim 1 or 2 or a microbial agent according to claim 3 or 4, wherein said use comprises at least one of the following applications: the application in degrading deoxynivalenol and inhibiting pathogenic bacteria.
6. The use of claim 5, wherein the pathogenic bacteria comprise at least one of enterotoxigenic Escherichia coli, Salmonella typhimurium.
7. Use according to claim 5 or 6, comprising the use of said Lactobacillus johnsonii or said microbial agent for the preparation of a product having at least one of the following functions: degrading deoxynivalenol in vitro; reducing the absorption of the deoxynivalenol by animals; reducing the concentration of deoxynivalenol in animal serum; and, inhibiting the growth of pathogenic bacteria.
8. The use according to claim 5 or 6, characterized in that it comprises the use of said Lactobacillus johnsonii or said microbial agent for the preparation of a product for the prevention or treatment of animal damage caused by pathogenic bacteria, deoxynivalenol or a combination of both.
9. A method of degrading deoxynivalenol and/or suppressing pathogenic bacteria comprising the steps of: activating and culturing lactobacillus johnsonii according to claim 1 or 2 to obtain a microbial agent, and applying the microbial agent to an object to be treated in a predetermined dosage to degrade deoxynivalenol and/or repress pathogenic bacteria.
10. The method according to claim 9, wherein the subject to be treated is a mouse, and the microbial agent is administered at a dose of 1X 10 6 ～1×10 10 CFU/machine, the frequency of application is 4-14 days of continuous drenching.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102178128A (en) * | 2011-03-24 | 2011-09-14 | 南昌大学 | Application of bacillus subtilis ZDY1982 to degradation of mycotoxin deoxynivalenol |
| WO2013026033A2 (en) * | 2011-08-18 | 2013-02-21 | Dupont Nutrition Biosciences Aps | Strains and methods useful for mycotoxins |
| CN112126607A (en) * | 2020-11-26 | 2020-12-25 | 中国农业科学院北京畜牧兽医研究所 | Bacterial strain for degrading deoxynivalenol and application |
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|---|---|---|---|---|
| CN102178128A (en) * | 2011-03-24 | 2011-09-14 | 南昌大学 | Application of bacillus subtilis ZDY1982 to degradation of mycotoxin deoxynivalenol |
| WO2013026033A2 (en) * | 2011-08-18 | 2013-02-21 | Dupont Nutrition Biosciences Aps | Strains and methods useful for mycotoxins |
| CN112126607A (en) * | 2020-11-26 | 2020-12-25 | 中国农业科学院北京畜牧兽医研究所 | Bacterial strain for degrading deoxynivalenol and application |
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| SHENGRU WU 等: "Intestinal toxicity of deoxynivalenol is limited by supplementation with Lactobacillus plantarum JM113 and consequentially altered gut microbiota in broiler chickens", 《JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY》 * |
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