CN117821329A - Enterococcus faecium and application thereof in preparing feed additive - Google Patents
Enterococcus faecium and application thereof in preparing feed additive Download PDFInfo
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- 241000194031 Enterococcus faecium Species 0.000 title claims abstract description 52
- 239000003674 animal food additive Substances 0.000 title claims abstract description 14
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to enterococcus faecium and application thereof in preparing feed additives. The enterococcus faecium AK-GRw2 has the preservation number of: cctccc No. M20231981. The enterococcus faecium AK-GRw2 has excellent trypsin resistance and acid resistance, has good inhibition performance on escherichia coli and staphylococcus aureus, can promote the growth of geese, establish good intestinal flora ecology, improve immunity, influence and improve animal production and health by being added into goose feed, and provides a better reference basis for microecologics for expanding goose-derived lactobacillus resource libraries and application.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to enterococcus faecium and application thereof in preparing feed additives.
Background
Lactic acid bacteria are novel microecological preparations which are widely applied, are mature in application and good in effect on a plurality of animals at present, become a focus of research due to the characteristics of green, safety, environmental protection and the like, can improve the intestinal functions of organisms through substances such as bacteriocins and the like generated by the lactic acid bacteria, and are one of common strains widely applied to treatment and prevention of digestive tract and genital tract diseases of people and animals, silage, dairy products and the like. Meanwhile, the lactobacillus can decompose toxic and harmful substances generated in the culture environment, inhibit the growth of harmful bacteria in the water body, and play a role in purifying the water quality; has stronger protease, lipase and amylase activities, and promotes the absorption and utilization efficiency of feed; can stimulate the development of immune organs, and enhance immunity and resistance of organism.
Enterococcus faecium (enterococcus faecium) is an anaerobic, non-spore-forming gram-positive bacterium belonging to one type of lactic acid bacteria. The thalli are round or elliptic and are arranged in a chain shape, and no spores are produced. Round colonies with a diameter of 0.5-1 mm, which are off-white, opaque, smooth in surface, can be formed on the blood plates. Enterococcus faecium exists widely in the environment, belongs to intestinal symbiotic bacteria, and is widely applied to food fermentation, livestock and poultry production and medical care at present.
Along with the breeding industry of geese, the development is rapid. However, the goose breeding technology is relatively lagged relative to the mature chicken breeding mode, and the problems of scattered breeding, irregular breeding, low efficiency, frequent occurrence of bacterial diseases and the like appear. Nowadays, antibiotic-free cultivation becomes an inevitable trend of the development of the animal husbandry in the future, and people increasingly pursue safe and green foods. Accordingly, efforts are underway to find feed additives that can replace antibiotics. The lactobacillus strains of the existing lactobacillus preparations in the market are mostly screened and separated from natural environment or intestinal tracts of original animals, the strains separated from the environment are mainly used for fermentation, the tolerance capacity of gastric acid bile salts and the like is not strong, and the safety performance is also uneven. Because the physiological characteristics of geese are greatly different from those of domestic animals, the strain obtained by separating and screening other mammal intestinal tracts is directly applied to the production of geese, and the application effect is not ideal. But the goose-derived lactobacillus strain screening and separating resources are few at present, and the method can be truly applied to less actual production. Therefore, the development of a new goose-source lactobacillus strain with beneficial characteristics has wide prospect.
Disclosure of Invention
The invention aims to overcome the defect that goose-source lactobacillus strains are few in screening and separating resources in the prior art, and provides enterococcus faecium and application thereof in preparing feed additives.
In order to solve the technical problems, the invention adopts the following technical scheme.
In a first aspect the invention provides an enterococcus faecium AK-GRw strain having the accession number: cctccc No. M20231981.
In a second aspect, the invention provides the use of enterococcus faecium AK-GRw2 in the preparation of a feed additive for promoting goose growth.
In some embodiments of the invention, the feed additive is used to promote intestinal mucosal development.
In some embodiments of the invention, the feed additive comprises a culture of the enterococcus faecium AK-GRw2 or enterococcus faecium AK-GRw2.
In some embodiments of the present invention, the method for preparing a culture of enterococcus faecium AK-GRw2 comprises the steps of: inoculating the enterococcus faecium AK-GRw2 into a broth culture medium containing MRS, shake culturing for 20-26h, and removing thalli to obtain a culture of enterococcus faecium AK-GRw2.
The third aspect of the invention provides application of enterococcus faecium AK-GRw2 in preparation of antibacterial preparations, wherein antibacterial types of the antibacterial preparations comprise staphylococcus aureus and/or escherichia coli.
In a fourth aspect, the invention provides the use of enterococcus faecium AK-GRw2 in the manufacture of a medicament for the treatment of a disease caused by infection with staphylococcus aureus and/or escherichia coli.
In a fifth aspect, the present invention provides a feed additive, the active ingredient of which is enterococcus faecium AK-GRw2 or a culture thereof according to claim 1.
In a sixth aspect, the present invention provides a bacteriostatic agent comprising enterococcus faecium AK-GRw2 or a culture thereof as claimed in claim 1 as an active ingredient.
In a seventh aspect, the present invention provides a medicament comprising enterococcus faecium AK-GRw2 or a culture thereof as claimed in claim 1 as an active ingredient.
Compared with the prior art, the invention has the following beneficial effects: the invention screens out the goose-source lactobacillus faecium enterococcus AK-GRw2 with specific probiotics from the intestinal tracts of healthy geese. The goose-source lactobacillus strain with the characteristics of inhibiting the growth and the colonization of pathogenic bacteria, having strong adhesion colonization capacity and the like is screened from a large number of strains, and the method provides a better reference basis for a microecological preparation for expanding a goose-source lactobacillus resource library and application.
The enterococcus faecium AK-GRw2 provided by the invention has the survival rate of 92.9% in the pH=2 period, the survival rate of 35.3% when the concentration of bile salt is 0.2%, the survival rate of 276.5% under 1.2% trypsin, the bacteria inhibition zone of the culture of the enterococcus faecium AK-GRw can reach 26mm for staphylococcus aureus, and the bacteria inhibition zone of the culture of the enterococcus faecium can also reach more than 12mm for escherichia coli.
Preservation of biological materials
Enterococcus faecium Enteromorpha K-GRw, china Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M20231981 in 10 month 23 of 2023, and eight paths of Lopa nationality in Wuchang district of Wuhan, hubei province with the preservation address of Lopa nationality.
Drawings
FIG. 1 is a graph showing the characteristics of enterococcus faecium AK-GRw2 MRS agar culture.
FIG. 2 is a graph showing the results of enterococcus faecium AK-GRw2 MRS broth culture.
FIG. 3 is a graph showing the results of gram staining of enterococcus faecium AK-GRw2.
FIG. 4 is a graph showing the results of the enterococcus faecium AK-GRw2 indole formation assay, nitrate reduction assay, VP assay, and methyl red assay.
FIG. 5 is a graph showing the results of measurement of sucrose, glucose, maltose and lactose in enterococcus faecium AK-GRw2.
FIG. 6 is a graph showing the results of 16S rRNA sequencing of enterococcus faecium AK-GRw2.
FIG. 7 is a graph showing the results of E.coli inhibition by enterococcus faecium AK-GRw2.
FIG. 8 is a graph showing the results of the inhibition of enterococcus faecium AK-GRw2 against Staphylococcus aureus.
FIG. 9 is a diagram showing the result of the electrophoresis of the virulence gene of enterococcus faecium AK-GRw 2; wherein M is MakerDL2000;1-3 are respectively a gelatinase virulence gene gelE, a glial protein adhesion virulence gene ace and an endocardial antigen virulence gene efaA.
FIG. 10 is a 26h growth plot of enterococcus faecium AK-GRw2.
FIG. 11 is a graph showing the 26. 26hpH variation of enterococcus faecium AK-GRw2.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
EXAMPLE 1 isolation culture and purification of seed
Scraping mucosa of intestinal segment of healthy goose with a sterile scalpel in an ultra-clean workbench, placing into a sterile test tube containing normal saline, shaking, immediately diluting with raw ripple water, and collecting dilution gradient of 10 -4 ，10 -5 ，10 -6 100. Mu.L of dilution of (B) into MRS-CaCO 3 Plating the plates on agar medium. Oxygen in the tank is consumed by a glowing jar method, anaerobic culture is carried out overnight, and plates with good growth vigor and not crowded colonies are selected for further screening. Colony selection criteria were: the colony has good growth vigor, white color, raised round surface, regular moist edge, and large calcium dissolving ring. Streaking of conditioned colonies with an inoculating loop onto MRS-CaCO 3 Is cultured in an agar medium of (2) and circulated at least 5 times. Therefore, a plurality of pure strain can be primarily screened, and the strain is named AK-GRw2. In MRS-CaCO 3 Colonies on agar medium of (2) are shown in FIG. 1. Growth after overnight incubation in MRS broth was shown by cloudiness of the white viscous precipitant at the bottom as shown in FIG. 2.
2. Smear dyeing inspection
The single purified colonies were picked and smeared, stained and observed under a microscope with an oil microscope to find that AK-GRw bacteria were single or multiple, spherical blue-violet gram-positive bacteria (the result is also found for other bacteria), and the results are shown in FIG. 3.
3. Biochemical test
Glucose, lactose, maltose, sucrose fermentation test, methyl red test, VP test, nitrate reduction test and indole formation test were performed separately: the bacteria to be detected are inoculated into peptone water and glucose peptone water respectively by an inoculating loop or an inoculating needle, and sugar fermentation tubes (glucose, lactose, maltose and sucrose) are cultivated for 24-48h at 37 ℃.
4. And (3) PCR identification: PCR amplification was performed using the universal primers 27F and 1525R of the bacterial 16S rRNA gene.
(1) Nucleic acid extraction: the individual colonies of the bacteria were picked with an inoculating loop and inoculated into MRS broth, cultured with shaking at 37℃and 120rpm for 24 hours. Then extracting nucleic acid by using Ezup column type bacterial genome DNA extraction kit of Shanghai biological engineering Co., ltd
(2) PCR amplification and sequencing analysis of 16 SrRNA: the PCR reaction system was 50. Mu.L: PCRMaster Mix 25. Mu.L, upper primer 2. Mu.L, lower primer 2. Mu.L, DNA template 5. Mu.L, sterile water 16. Mu.L. PCR reaction conditions: 95 ℃ for 5min;95℃30s,55℃30s,72℃1min,30 cycles; and at 72℃for 10min. The PCR product is sent to Shanghai engineering bioengineering Co., ltd for sequencing after agarose gel electrophoresis detection, the sequence is shown as SEQ ID NO:1, the sequencing result is shown in figure 6, the sequencing result is compared and analyzed in a nucleic acid database on NCBI website, the 16S rRNA genes of AK-GRw bacteria and enterococcus faecium S98b strain are found, genBank accession number: KC692195 has 100.00% homology, and the test bacteria are confirmed to be enterococcus faecium, and the results are shown in Table 1.
TABLE 1 results of comparative analysis in NCBI nucleic acid database
5. Acid and bile salt resistance and digestive enzyme resistance test
Adding 100 mu L of AK-GRw bacterial liquid into 10mLMRS broth, and carrying out shaking culture at 37 ℃ and 120rpm for 24 hours; centrifuging at 8000r/min for 10min to collect thalli; the cells were suspended in MRS broth having pH values of 2 and 3, bile salt concentrations of 0.2% and 0.6%, and trypsin concentrations of 0.8% and 1.2%, respectively, and incubated at 37℃for 0 and 4 hours, and absorbance was sampled and measured, respectively, to calculate survival rate. The calculation formula is as follows: survival = X2/X1X 100%. Wherein: x1 is the number of viable bacteria in MRS broth with different pH values, bile salts and trypsin concentrations when cultured for 0 hour, and X2 is the number of viable bacteria in cfu/mL when cultured for 4 hours. As shown in table 2.
TABLE 2 acid and bile salt resistance and digestive enzyme resistance test results
6. Oxford cup plate method bacteriostasis test
(1) Preparing a nutrient agar plate and a MAIKAI agar plate, and placing the plates into a refrigerator at 4 ℃ for later use;
(2) Culturing the indicator bacteria in a proper liquid culture medium and under proper culture conditions overnight;
(3) The AK-GRw2 strain is inoculated into a test tube containing 10mL of MRS broth, the culture is carried out at 37 ℃ under 120rpm shaking for 24h, centrifugation is carried out at 5000rpm for 15min, the thalli are removed, and the supernatant is remained as AK-GRw2 cell-free fermentation supernatant.
(4) The method comprises the steps of absorbing a certain concentration of the indicator fungus suspension on a flat plate in one step, uniformly coating by using a sterile glass rake, slightly placing sterile oxford cups into a culture dish by using tweezers, and uniformly placing 3 or 4 oxford cups into a horizontally placed flat plate.
(5) AK-GRw2 cell-free fermentation supernatant was added to the well of oxford cup and spread for 12h in a refrigerator at 4 ℃.
(6) After culturing at 37 ℃ for 24 hours, the diameter of the inhibition zone is measured by a vernier caliper.
1 goose-derived pathogenic escherichia coli and 1 staphylococcus aureus which are separately stored in the laboratory are respectively selected as indicator bacteria, and antibacterial tests are respectively carried out. The results show that the specific inhibition zone size separated in the invention is shown in Table 3, and the phenomenon is shown in figures 8-9.
TABLE 3 antibacterial test results of AK-GRw Strain against pathogenic bacteria
7. Virulence gene detection
(1) Nucleic acid extraction: the individual colonies of the bacteria were picked with an inoculating loop and inoculated into MRS broth, cultured with shaking at 37℃and 120rpm for 24 hours. Then extracting nucleic acid by using an Ezup column type bacterial genome DNA extraction kit of Shanghai biological engineering Co., ltd.
(2) Primer synthesis: the sequence of the designed 4 virulence gene primer against enterococcus faecalis is shown in Table 4 and is synthesized by Shanghai Biotechnology Co., ltd.
(3) PCR amplification and analysis of primers: PCR reaction system: 2. Mu.L of bacterial DNA template, 10. Mu. Mol/L of each of the upstream and downstream primers.
The PCR reaction conditions are as follows: pre-denaturation at 94℃for 2min, denaturation at 94℃for 1min, annealing at 56℃for 1min, extension at 72℃for 1min,30 cycles total. The results of 1% agarose electrophoresis and observation and recording are shown in FIG. 9. The results show that the strain has no corresponding virulence genes.
TABLE 4 virulence gene primer sequences
8. Analysis of growth Properties and lactic acid production Capacity
The AK-GRw strain was inoculated into MRS broth, cultured with shaking at 37℃and 120rpm, sampled 1 time every 2 hours, and OD and pH values at 600nm were measured with respect to the sterile MRS broth, and corresponding curves were drawn with respect to the culture time on the abscissa and the OD and pH values on the ordinate, respectively. See fig. 10 and 11. From the graph, AK-GRw bacteria start to rapidly increase in the logarithmic phase at 6h, and the concentration changes little in the plateau phase at about 20 h. The acid yield of AK-GRw bacteria is rapidly increased at 6h, and the acid yield is stabilized at 18h, so that the pH value in the fermentation liquor is close to 4.
9. Animal safety test
8 healthy unvaccinated Wanxi white geese with similar body weight and 5 days old incubated in the laboratory were randomly selected, and were divided into a control group and a test group, each of which was 4. The test group is irrigated once daily for culturing 1mL of an overnight AK-GRw strain culture, and the control group is irrigated with distilled water with the same amount every day, and the feeding conditions are as follows: feeding on the net, adding heat preservation lamp at 1-7 days old, keeping warm for the rest time, feeding at room temperature, and freely feeding with 1 time of daily grain change. The feed is a compound feed for commercial meat geese in the early stage. The vitality and health state of two groups of geese are observed every day, the geese are sacrificed by adopting neck vein bloodletting after 14 days, and the visceral organs are observed to be diseased or not by section inspection. As a result, the two groups of gosling do not have diarrhea, death and other symptoms, and obvious lesions are not seen in the section examination of the two groups of viscera.
Thus, the safety performance is good. Experiments show that the AK-GRw strain provided by the invention has good viability in the intestinal environment of geese, can adapt to the gastrointestinal environment, has good pathogen resistance, is safe to use, can be used in a brooding stage, establishes good intestinal flora ecology of geese, enhances the disease resistance, promotes growth and intestinal mucosa development, builds the flora ecology of a relatively healthy cultivation environment, and has a great application prospect and development potential.
The lactobacillus AK-GRw2 strain is separated from the cecum of the goose, and identified as enterococcus faecium. Experiments show that the AK-GRw2 strain obtained by separation has good acid resistance, general bile salt resistance and excellent trypsin resistance, the survival rate of AK-GRw2 under 1.2% trypsin can reach 276.5%, the AK-GRw strain has good antibacterial performance on escherichia coli and staphylococcus aureus, the antibacterial circle of the culture of the strain can reach 26mm for staphylococcus aureus, and the antibacterial circle of the strain can also reach more than 12mm for escherichia coli. The gosling is fed with AK-GRw2 strain bacteria liquid for 14 days, obvious morbidity and death are not seen, viscera are not diseased after section inspection, common virulence genes are not detected, and therefore the safety performance is good. Experiments show that the AK-GRw strain provided by the invention has good viability in the intestinal environment of geese, can adapt to the gastrointestinal environment, has good pathogen resistance, is safe to use, can be used in a brooding stage, establishes good intestinal flora ecology of geese, enhances the disease resistance, promotes growth and intestinal mucosa development, builds the flora ecology of a relatively healthy cultivation environment, and has a great application prospect and development potential.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. Enterococcus faecium AK-GRw2, characterized by the deposit number: cctccc No. M20231981.
2. Use of enterococcus faecium AK-GRw2 according to claim 1 for the preparation of a feed additive for promoting goose growth.
3. The use according to claim 2, wherein the feed additive is for promoting intestinal mucosal development.
4. The use according to claim 2, wherein the feed additive comprises a culture of said enterococcus faecium AK-GRw2 or enterococcus faecium AK-GRw.
5. The use according to claim 2, wherein the preparation method of the culture of enterococcus faecium AK-GRw2 comprises the following steps: inoculating the enterococcus faecium AK-GRw2 into a broth culture medium containing MRS, shake culturing for 20-26h, and removing thalli to obtain a culture of enterococcus faecium AK-GRw2.
6. Use of enterococcus faecium AK-GRw2 according to claim 1 for the preparation of a bacteriostatic formulation, characterized in that the bacteriostatic species of the bacteriostatic formulation comprise staphylococcus aureus and/or escherichia coli.
7. Use of enterococcus faecium AK-GRw2 according to claim 1 for the manufacture of a medicament for the treatment of diseases caused by infection with staphylococcus aureus and/or escherichia coli.
8. A feed additive contains enterococcus faecium AK-GRw2 or its culture as claimed in claim 1 as effective ingredient.
9. An antibacterial preparation, characterized in that the effective component is enterococcus faecium AK-GRw2 or a culture thereof according to claim 1.
10. A medicament, wherein the active ingredient is enterococcus faecium AK-GRw2 or a culture thereof according to claim 1.
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