CN112680372B - Enterococcus faecalis and application thereof in broiler feed - Google Patents
Enterococcus faecalis and application thereof in broiler feed Download PDFInfo
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- CN112680372B CN112680372B CN202011146965.3A CN202011146965A CN112680372B CN 112680372 B CN112680372 B CN 112680372B CN 202011146965 A CN202011146965 A CN 202011146965A CN 112680372 B CN112680372 B CN 112680372B
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Abstract
The invention is suitable for the technical field of microbial breeding, and provides a strain of Enterococcus faecalis, wherein the Enterococcus faecalis is PLBS014(Enterococcus faecalis PLBS014), which is preserved in the China general microbiological culture Collection center (CGMCC) for 12-17 th in 2018, and the preservation number is CGMCC NO. 16954; the invention also provides application of the enterococcus faecalis strain in broiler feed. Therefore, the enterococcus faecalis PLBS014 disclosed by the invention has no pathogenicity, has good drug resistance, can improve the production performance and the immunity of broiler chickens, inhibit the reproduction of pathogenic bacteria, promote the growth of beneficial bacteria, enhance the disease resistance of broiler chickens, and is beneficial to increasing the economic benefit of broiler chicken breeding.
Description
Technical Field
The invention relates to the technical field of microbial breeding, in particular to enterococcus faecalis and application thereof in broiler feed.
Background
The production of commercial broilers is one of the important industries for the development of livestock husbandry in China and is also an important support for earning foreign exchange for the export of livestock products in China. For a long time, the broiler production in China is mainly based on vaccine and environmental control and is matched with a veterinary epidemic prevention system of chemical drugs and antibiotics, and as a result, chemical drugs, hormones, antibiotics and the like are remained, so that animals generate drug resistance, and carcinogenesis, teratogenesis and mutation are caused, and the broiler production can harm human health through a food chain, thereby forming a great threat to human health and public health.
The probiotics is a novel feed additive which appears in recent years, can inhibit the adhesion and growth and reproduction of pathogenic bacteria, keep the micro-ecological balance of the gastrointestinal tract of animals, and the probiotics generated by the metabolism can be used as a nutrient source of a host or help the digestion and absorption of the animals.
Enterococcus faecalis is used as one of probiotics, is clearly marked as one of probiotics capable of being directly applied to animals by the U.S. food and drug administration since 1984, is a facultative anaerobic gram-positive bacterium existing in intestinal tracts of human beings and animals, can soften fibers in feed and improve the conversion rate of the feed; enterococcus faecalis can generate a plurality of antibacterial substances which have good inhibitory action on pathogenic bacteria such as salmonella, escherichia coli, staphylococcus aureus and the like; research shows that enterococcus faecalis can produce Volgamycin and can effectively inhibit growth and reproduction of Listeria monocytogenes, staphylococcus aureus and putrefying microorganisms.
However, the application research of enterococcus faecalis in broiler diets is rarely reported, the addition amount and the effect of enterococcus faecalis are not the same, and the research on whether enterococcus faecalis can be applied to broiler diets instead of antibiotics is not reported.
In view of the above, the prior art is obviously inconvenient and disadvantageous in practical use, and needs to be improved.
Disclosure of Invention
In view of the defects, the invention aims to provide enterococcus faecalis and application thereof in broiler feed, wherein the enterococcus faecalis PLBS014 has no pathogenicity and good drug resistance, can improve the production performance and the immunity of broiler chickens, inhibit the reproduction of pathogenic bacteria, promote the growth of beneficial bacteria, enhance the disease resistance of broiler chickens, and is beneficial to increase the economic benefit of broiler chicken breeding.
In order to achieve the purpose, the invention provides a strain of Enterococcus faecalis, wherein the Enterococcus faecalis is PLBS014(Enterococcus faecalis PLBS014) which is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 12 and 17 days, and the preservation number is CGMCC NO. 16954.
Enterococcus faecalis according to the present invention, said enterococcus faecalis is taken from cow dung.
According to the enterococcus faecalis disclosed by the invention, the addition amount of the enterococcus faecalis in the basic ration of the broiler is 50-1000 mg/kg.
According to the enterococcus faecalis disclosed by the invention, the addition amount of the enterococcus faecalis in the basic ration of the broiler is 446-651 mg/kg.
The invention also provides a method for screening enterococcus faecalis, which comprises the following steps:
step one
Collecting excrement of growing and fattening cattle, and performing gradient dilution on the excrement to obtain diluents with different concentration gradients;
step two
Respectively taking the diluent, inoculating the diluent into a Columbia sheep blood agar plate, culturing and observing the morphology of a bacterial colony;
step three
Picking a single colony, and carrying out pure culture on the single colony with the colony characteristics of enterococcus faecalis;
the pure culture colonies were subjected to gram staining and microscopic examination.
According to the method for screening enterococcus faecalis of the present invention, in the step one, the dilution gradient of the excrement is 10, 1 × 102，1×103，1×104，1×105，1×106，1×107。
According to the method for screening enterococcus faecalis, in the second step, the culture condition of the bacterial colony is culture at 37 ℃ for 20-30 h.
Enterococcus faecalis according to the present invention, use of said enterococcus faecalis in broiler feed.
The enterococcus faecalis PLBS014 provided by the invention has no pathogenicity and good drug resistance, and the enterococcus faecalis PLBS014 with an appropriate level is added into the daily ration of the broiler chicken, so that the feed utilization rate and the nutrient absorption rate of the broiler chicken can be improved, the production performance of the broiler chicken can be improved, the immunity of the broiler chicken body can be improved, the reproduction of pathogenic bacteria can be inhibited, the growth of beneficial bacteria can be promoted, the disease resistance of the broiler chicken body can be enhanced, and the economic benefit of broiler chicken breeding can be increased.
Drawings
FIG. 1 is a colony morphology of enterococcus faecalis PLBS014 of the present invention;
FIG. 2 is a graph showing the growth of a enterococcus faecalis PLBS014 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides enterococcus faecalis, which has the scientific name of Latin: enterococcus faecalis; the Enterococcus faecalis is Enterococcus faecalis PLBS014(Enterococcus faecalis PLBS014), which is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 of Xilu 1 of Beijing Kogyo-oriented north Chen) in 17 days 12 and 8 years 2018, and the preservation number is CGMCC NO. 16954.
The enterococcus faecalis is taken from cow dung, and the invention provides a screening method of enterococcus faecalis PLBS014, which comprises the following steps:
step one
Collecting feces of growing and fattening cattle from cattle farm in Qingdao city, and subjecting the feces to gradient (10, 1 × 10)2，1×103，1×104，1×105，1×106，1×107) Diluting to obtain the diluent with different concentration gradients.
Step two
And respectively taking the diluent, inoculating the diluent into a Columbia sheep blood agar plate, culturing at 37 ℃ for 20-30 h, and observing colony morphology.
Step three
Picking a single colony, carrying out pure culture on the single colony with the colony characteristics of the enterococcus faecalis, and carrying out gram staining and microscopic examination on the pure culture colony.
The colony morphology observed by using an optical microscope and a stereoscope is as follows: the bacterial colony is gray white, opaque and smooth in surface, and the diameter of the bacterial colony is 0.5-1 mm. The liquid culture is carried out by adopting common broth, the culture solution is uniform and turbid, and thallus precipitates are arranged at the bottom, which indicates that the cells are anaerobic or facultative anaerobic. The thalli is gram-positive cocci observed under an optical microscope and is arranged in a single, paired or short chain shape. (see FIG. 1)
After the strains are screened by the method, the strains are identified, and the invention provides an identification method of enterococcus faecalis strains, which comprises the following steps:
identification of a biochemical test
Performing biochemical test according to conventional bacterial inoculation method, inoculating the isolated strain into 6.5% NaCl, sodium equate, esculin, mannitol, sorbitol, lactose, inulin, raffinose, trehalose, and salicin biochemical reaction tube, performing anaerobic culture at 37 deg.C for 24 hr, and observing the result.
The isolated strain can grow in 6.5% NaCl, hydrolyze esculin in the presence of 40% bile, hydrolyze sodium equate, mannose, sorbitol, arginine, lactose and salicin, and does not decompose inulin, sorbose, raffinose and arabinose. The biochemical characteristics of the strain are consistent with those of enterococcus.
Identification of two Gene sequences
The rescreened strain is sent to the company Limited in the Biotechnology engineering (Shanghai) to be sequenced, the obtained 16S r RNA sequence information is compared with the 16S database of NCBI, and the parameter identification is set to be more than 95. And then selecting the first 30 RNA sequences with the highest identification (if the sequence is not sufficient, all the RNA sequences are taken), performing sequence multiple comparison by utilizing a muscle software, and constructing a phylogenetic tree by adopting a FastTree software component. The results show that the rescreened strain is closest to the published Enterococcus faecalis faecalium 505. Thus, the rescreened strain was classified as enterococcus faecalis, designated enterococcus faecalis PLBS 014.
In order to verify the strain properties of enterococcus faecalis PLBS014 of the present invention, strain property tests including growth curve, pathogenicity and drug sensitivity tests of the strain were performed.
Growth curve of the Strain
Inoculating the strain into a sterilized MRS agar culture medium, carrying out shake culture at a constant temperature of 35-39 ℃, observing the production condition of the strain for 48 hours, sampling at an interval of 2 hours at the early stage, sampling at intervals of 4, 6 and 12 hours at the later stage, measuring the absorbance of the strain at a wavelength of 560nm by using a visible spectrophotometer, repeating for 5 times, and drawing the change trend of a strain growth curve (see figure 2).
According to the growth curve, the strain grows rapidly and exponentially between 0 and 8 hours of culture, grows slowly between 8 and 48 hours and is basically in a stable state.
Pathogenicity test
Dividing mice into test group and control group, selecting single colony after separation and purification, inoculating in THB culture solution, shaking overnight at 37 deg.CCulturing, diluting to 1 × 10 with 0.15mol/L PBS8cfu/mL, intraperitoneal injection of test mice and control mice 0.15mol/mL PBS, and the dosage is 0.2 mL/mouse. Mice were scored for morbidity every 2h observation. Immediately dissecting dead white mice, observing the pathological change condition of tissues/organs, and aseptically collecting lung, kidney and other pathological materials for pathogen separation and identification.
The test result shows that no symptom is seen in the test group white mouse and the control group white mouse within 1 week, no pathological change is seen after the autopsy, and the bacterium is not cultured from each organ. Indicating that the enterococcus faecalis strain PLBS014 was not pathogenic.
Susceptibility test
Selecting drug sensitive paper sheets of 16 clinically common anti-positive cocci drugs such as oxacillin, tetracycline, penicillin G, vancomycin, erythromycin, chloramphenicol, levofloxacin and the like, attaching the drug sensitive paper sheets to a plate culture medium which is uniformly coated with bacterial liquid, culturing for 16-18 h at 35 ℃, measuring the diameter of a bacteriostatic zone, and judging the drug sensitive condition. The results of the drug sensitivity test are shown in the table I.
Test results show that the enterococcus faecalis PLBS014 strain has drug resistance to beta-lactam antibiotics, including oxacillin, macrolide antibiotics, erythromycin, clarithromycin, polypeptide antibiotics, polymyxin B, lincomycin, sulfonamide antibiotics, compound sulfamethoxazole, quinolone antibiotics, norfloxacin, ciprofloxacin, levofloxacin, tetracycline antibiotics, spectinomycin, and the like; enterococcus faecalis PLBS014 strain sensitive to beta-lactam antibiotic penicillin G, synthetic antibiotic nitrofurantoin; enterococcus faecalis strain PLBS014 was moderately resistant to the polypeptide antibiotics vancomycin, the tetracycline antibiotic minocycline.
The enterococcus faecalis PLBS014 strain has no pathogenicity and has better drug resistance, so the enterococcus faecalis PLBS014 is applied to the broiler feed, and in order to verify the application value of the enterococcus faecalis PLBS014 in the broiler feed, the invention also provides an application method of the enterococcus faecalis in the broiler feed.
First, experimental animal and raising management
The test adopts completely random grouping design, 1-day-old AA broiler chicken with similar weight and good health condition is selected and randomly divided into 4 groups (a control group and three test groups). The control group is fed with basic diet, the experimental group is added with enterococcus faecalis PLBS014 on the basis of the basic diet, and the viable count of the enterococcus faecalis PLBS014 is 2 × 108CFU/kg. The broiler chickens had free access to food and water for a total of 42 days.
The basal diet was formulated with reference to broiler nutrition recommendations in NRC (2012) poultry nutrition requirements.
Second, feed formula
The enterococcus faecalis PLBS014 is added into the basic daily ration of the broiler chicken, and the addition amount is 50-1000 mg/kg.
TABLE-drug sensitivity test results
"-" indicates no zone of inhibition, "S" indicates sensitivity, "I" indicates moderate sensitivity, and "R" indicates resistance.
Third, measuring the index
Measurement of production Properties
On the 21 st day and the 42 th day, the broilers are weighed on an empty stomach, and the broilers are forbidden for 12 hours before being weighed, and water is freely drunk. And calculating the average daily gain, the average daily feed intake and the feed weight ratio of the broiler chickens at each stage according to the records during the test.
Serum immunoglobulin content
Placing the blood collected in the test in a procoagulant tube, standing at room temperature for 20min, centrifuging at 3000r/min for 15min, taking the upper layer serum, and determining the content of IgA, IgG and IgM.
Intestinal flora determination
Dissecting the slaughtered broiler chickens, taking cecal contents in a sterile environment, and adding normal saline for dilution; and respectively culturing the diluent in a specific culture medium, wherein escherichia coli and salmonella are subjected to aerobic culture for 24 hours in a 37 ℃ biochemical incubator and then subjected to colony counting, and lactobacillus is subjected to anaerobic culture for 48 hours in the 37 ℃ biochemical incubator and then subjected to colony counting.
In order to further verify the application value of the enterococcus faecalis PLBS014 in the invention in broiler feed, the invention feeds broiler chickens by the application method, sets the following several examples, and measures the related indexes of the broiler chickens in each example. The broiler chicken feeding management method in each example was the same, and only the addition amount of enterococcus faecalis PLBS014 in each example was changed, and therefore, the same contents in each example are not listed.
The results of the related indexes of the broiler chickens in each embodiment are shown in the table II, the table III and the table IV; the amount of enterococcus faecalis PLBS014 added in each example is shown in Table five.
Comparative example
The enterococcus faecalis PLBS014 of the present invention was added to a basal diet of broiler chicken at an amount of 0.
After the completion of the breeding, the average daily gain of the broiler chicken is 43.08g, the average daily feed intake is 84.66g, and the feed-weight ratio is 1.96; measuring the content of immunoglobulin A in the serum of the broiler chicken to be 0.92ng/mL, the content of immunoglobulin G in the serum of the broiler chicken to be 7.45ng/mL and the content of immunoglobulin M in the serum of the broiler chicken to be 1.26 ng/mL; it was found that the intestinal flora of broiler chickens had an Escherichia coli content of 7.51lgCFU/g, a Salmonella content of 6.45lgCFU/g, and a Lactobacillus content of 7.85 lgCFU/g.
Example 1
The enterococcus faecalis PLBS014 of the invention was added to the basal diet of broiler chickens at an amount of 446 mg/kg.
After the completion of the breeding, the average daily gain of the broiler chicken is 47.16g, the average daily feed intake is 89.45g, and the feed-weight ratio is 1.89; measuring the content of immunoglobulin A in the serum of the broiler chicken to be 1.17ng/mL, the content of immunoglobulin G in the serum of the broiler chicken to be 8.91ng/mL and the content of immunoglobulin M in the serum of the broiler chicken to be 2.09 ng/mL; it was found that the intestinal flora of broiler chickens had an Escherichia coli content of 7.10lgCFU/g, a Salmonella content of 6.10lgCFU/g, and a Lactobacillus content of 9.05 lgCFU/g.
Example 2
The enterococcus faecalis PLBS014 of the invention was added to the basal diet of broiler chickens in an amount of 519 mg/kg.
After the completion of the breeding, the average daily gain of the broiler chicken is 47.32g, the average daily feed intake is 89.07g, and the feed-weight ratio is 1.88; measuring the content of immunoglobulin A in the serum of the broiler chicken to be 1.15ng/mL, the content of immunoglobulin G in the serum of the broiler chicken to be 8.75ng/mL and the content of immunoglobulin M in the serum of the broiler chicken to be 1.94 ng/mL; it was found that the intestinal flora of broiler chickens had an Escherichia coli content of 7.16lgCFU/g, a Salmonella content of 6.12lgCFU/g, and a Lactobacillus content of 9.11 lgCFU/g.
Example 3
The enterococcus faecalis PLBS014 of the invention was added to the basal diet of broiler chickens in an amount of 651 mg/kg.
After the completion of the breeding, the average daily gain of the broiler chicken is 47.25g, the average daily feed intake is 88.89g, and the feed weight ratio is 1.88; measuring the content of immunoglobulin A in the serum of the broiler chicken to be 1.13ng/mL, the content of immunoglobulin G in the serum of the broiler chicken to be 8.83ng/mL and the content of immunoglobulin M in the serum of the broiler chicken to be 2.03 ng/mL; it was found that the intestinal flora of broiler chickens had an Escherichia coli content of 7.20lgCFU/g, a Salmonella content of 5.79lgCFU/g, and a Lactobacillus content of 9.10 lgCFU/g.
Because the implementation processes of the embodiments are the same, the specific processes of other embodiments are not listed, and only the three embodiments with better effects are listed.
Growth performance of the broiler chicken
Serum immunoglobulin content ng/mL of table three broiler chicken
Intestinal flora content lgCFU/g of broiler chicken in Table IV
Table five examples the addition amount of enterococcus faecalis PLBS014 was mg/kg
The examples show that the addition of the enterococcus faecalis PLBS014 of the invention to the basal diet of broiler can improve the average daily gain and average daily feed intake of broiler, reduce the feed weight ratio of broiler, and indicate that the enterococcus faecalis PLBS014 promotes the growth and development of broiler, effectively degrades the anti-nutritional factors in the feed, eliminates the anti-nutritional effect, promotes the digestion and decomposition of organism on nutrients, and thus improves the feed utilization rate. Enterococcus faecalis PLBS014 used as a feed additive is added into livestock and poultry feed, can adjust intestinal flora and enhance the digestion and absorption functions of gastrointestinal tract, and further improves the growth performance and feed conversion rate of animals.
The addition of enterococcus faecalis PLBS014 of the invention to the basal diet of broiler can increase the content of immunoglobulin A, immunoglobulin M and immunoglobulin G in the serum of broiler, which shows that enterococcus faecalis PLBS014 can increase the secretion of immunoglobulin in broiler body, and the disease resistance of broiler body is enhanced due to the antibacterial and antiviral functions of immunoglobulin. Enterococcus faecalis PLBS014 used as a feed additive is added into livestock and poultry feed, and can be used as a non-specific immune factor to activate the immune system of the organism and regulate the immune function of the organism, thereby maintaining the health of animals.
The enterococcus faecalis PLBS014 disclosed by the invention is added into the basic daily ration of the broiler chicken, so that the quantity of harmful intestinal bacteria such as escherichia coli and salmonella can be reduced, the quantity of beneficial intestinal bacteria such as lactobacillus can be increased, and the enterococcus faecalis PLBS014 can regulate intestinal flora of the broiler chicken body. The enterococcus faecalis PLBS014 used as a feed additive is added into livestock and poultry feed, can generate various antibacterial substances, inhibit the reproduction of pathogenic bacteria, promote the growth of beneficial bacteria and reduce the morbidity of livestock and poultry.
In addition, it can be seen from the above examples that when the addition amount of the enterococcus faecalis PLBS014 to the basal diet of broiler is 446 to 651mg/kg, the broiler has the best productivity and the organism immunoglobulin content, and therefore, the optimum addition amount of the enterococcus faecalis PLBS014 to the basal diet of broiler is 446 to 651 mg/kg.
In conclusion, the enterococcus faecalis PLBS014 provided by the invention has no pathogenicity and good drug resistance, and the enterococcus faecalis PLBS014 with an appropriate level is added into the daily ration of the broiler chicken, so that the feed utilization rate and nutrient absorption rate of the broiler chicken can be improved, the production performance of the broiler chicken can be improved, the immunity of the broiler chicken body can be improved, the propagation of pathogenic bacteria can be inhibited, the growth of beneficial bacteria can be promoted, the disease resistance of the broiler chicken body can be enhanced, and the economic benefit of broiler chicken breeding can be increased.
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (2)
1. The Enterococcus faecalis is strain is Enterococcus faecalis PLBS014(Enterococcus faecalis PLBS014), and is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 12 months and 17 days, wherein the preservation number is CGMCC NO. 16954;
the enterococcus faecalis is obtained from cow dung.
2. Use of enterococcus faecalis according to claim 1 in broiler feed.
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| CN104293696B (en) * | 2014-09-16 | 2016-08-17 | 李雪平 | One strain enterococcus faecalis HEW-A131 and application thereof |
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