CN102373172B - Enterococcus faecium and application thereof - Google Patents
Enterococcus faecium and application thereof Download PDFInfo
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Abstract
The invention discloses an enterococcus faecium and application thereof. An enterococcus faecium strain provided by the invention is enterococcus faecium suppressant 80, and the collection number is CGMCC No.5058. As proved by experiments, the enterococcus faecium suppressant 80 is obtained by separating, identifying and screening; and the enterococcus faecium suppressant 80 has high resistance, high bacterial resistance and probiotic characteristics, and can be taken as an additive for preparing animal feeds; and the animals include but is not limited to various animals such as pigs, cows, sheep, chicken and the like. The feeds have similar functions to antibiotic feeds, but do not have the side effects of the antibiotic feeds.
Description
Technical field
The present invention relates to biological technical field, relate in particular to an Enterococcus faecalis and application thereof.
Background technology
When adding microbiotic in the feed intensive animal husbandry development is made major contribution, residual etc. in the destruction of the side effect of its generation such as autogenous infection and superinfection, chemical sproof generation, normal intestinal flora and livestock product and the environment brings safely serious threat to aquaculture, animal and human food prods.Probiotics is exactly as main take microorganism alive, after the animal absorption is a certain amount of, the gi tract privileged site have the bacterial strain of some amount can adhesion, field planting and growth, main by regulating intestinal canal microbial bacteria group structure peace weighing apparatus, to growth of animal and the healthy preparation that plays beneficial effect, it is a kind of novel green safe additive of good substitute antibiotics.
Faecium (Enterococcus Faecium) extensively is present in nature and the gastrointestinal tract of livestock and fowls, is a kind of beneficial microorganism, and common faecium is relatively poor because of characteristics such as it are acidproof, high temperature resistant, fails widespread use in fodder additives.Therefore, according to the demand of present livestock industry, screen the faecium that a strain acid-resistant and anti-high-temperature has probiotic properties and have vast potential for future development.
Summary of the invention
An object of the present invention is to provide faecium (Enterococcus Faecium) health 80.
This bacterial strain provided by the invention, its preserving number are CGMCC No.5058.
Another object of the present invention provides a kind of animal feedstuff additive.
Above-mentioned animal feedstuff additive, its activeconstituents are described faecium (Enterococcus Faecium) health 80CGMCC No.5058.
The animal-feed that contains above-mentioned animal feedstuff additive also is the scope of protection of the invention.
In above-mentioned fodder additives or the animal-feed, animal wherein is specially pig or chicken.
The 3rd purpose of the present invention provides a kind of antibacterial product.
Antibacterial product provided by the invention, its activeconstituents are above-mentioned faecium (Enterococcus Faecium) health 80 CGMCC No.5058.
In above-mentioned antibacterial product, the said products is medicine or microbial inoculum;
In above-mentioned antibacterial product, above-mentioned antibacterial for suppressing salmonella typhi (Salmonella typhi) and/or intestinal bacteria (Escherichia Coli), be specially and suppress salmonella typhi (Salmonella typhi), intestinal bacteria (Escherichia Coli) K88 and/or intestinal bacteria (Escherichia Coli) O157.
Above-mentioned faecium (Enterococcus Faecium) health 80CGMCC No.5058 is at preparation animal feedstuff additive, animal-feed, antibacterial product and/or promote that the application in the growth of animal also is the scope of protection of the invention.
In the above-mentioned application, described antibacterial for suppressing salmonella typhi (Salmonella typhi) and/or intestinal bacteria (Escherichia Coli), be specially and suppress salmonella typhi (Salmonella typhi), intestinal bacteria (Escherichia Coli) K88 and/or intestinal bacteria (Escherichia Coli) O 157;
Described promotion growth of animal is embodied in following 1) or 2):
1) improves pig average daily gain, the average daily ingestion amount of raising pig, raising pigskin hair index, reduction pig feedstuff-meat ratio and/or reduction diarrhea of pigs rate;
2) improve chicken average daily gain and/or improve average daily ingestion amount.
The 4th purpose of the present invention provides a kind of preparation method of animal feedstuff additive.
Method provided by the invention comprises the steps:
Above-mentioned faecium (Enterococcus Faecium) health of fermenting 80 CGMCC No.5058 collect tunning, namely obtain animal feedstuff additive.
In above-mentioned animal feedstuff additive preparation method, the temperature of described fermentation is 35 ℃-37 ℃, is specially 37 ℃ in the embodiments of the invention;
In above-mentioned animal feedstuff additive preparation method, described fermentation time is 15h-80h, is specially 20h in the embodiments of the invention;
In above-mentioned animal feedstuff additive preparation method, the substratum of described fermentation is the MRS substratum.
In above-mentioned animal feedstuff additive preparation method, described animal is pig or chicken.
The 5th purpose of the present invention provides a kind of preparation method of antibacterial product.
Method provided by the invention, above-mentioned faecium (Enterococcus Faecium) the health 80CGMCC No.5058 that comprises the steps: to ferment collects tunning, namely obtains antibacterial product;
In the preparation method of above-mentioned antibacterial product, the temperature of described fermentation is 35 ℃-37 ℃, is specially 37 ℃ in the embodiments of the invention;
In the preparation method of above-mentioned antibacterial product, described fermentation time is 15h-80h, is specially 20h in the embodiments of the invention;
In the preparation method of above-mentioned antibacterial product, the substratum of described fermentation is the MRS substratum; The pH value of this substratum is 6-7, and the pH value of this substratum is specially 7.
In the preparation method of above-mentioned antibacterial product, described product is medicine or microbial inoculum;
In the preparation method of above-mentioned antibacterial product, above-mentioned antibacterial for suppressing salmonella typhi (Salmonella typhi) and/or intestinal bacteria (Escherichia Coli), be specially and suppress salmonella typhi (Salmonella typhi), intestinal bacteria (Escherichia Coli) K88 and/or intestinal bacteria (Escherichia Coli) O 157.
Of the present invention experimental results show that, the present invention by separate, identify, screening, obtain faecium health 80, its strong stress resistance, anti-miscellaneous bacteria ability be strong, have probiotic properties, can be used as additive for the preparation of animal-feed, animal wherein includes but not limited to the various animals such as pig, ox, sheep, chicken.This feed has and the similar function of antibiotic feed, but the side effect of antibiotic-free feed.Faecium of the present invention is mainly as the additive of animal-feed, microbiotic in the alternative existing animal diets, regulate microecological balance in the animal intestine, thereby have the prophylactic effect of the non-specific immune function of enhancing, nutritional factor can also be provided simultaneously, promote the nutraceutical production performance of digesting and assimilating, reducing diarrhoea, promotion growth of animal and improve food conversion ratio, improve weanling pig and growing-finishing pig.
Faecium of the present invention plays a role in health care to control animal digestive system disease, can stimulate its gastrointestinal development to growing animal simultaneously, so it is applied in the effect that can play disease-resistant growth-promoting in the feed as fodder additives.Simultaneously, faecium of the present invention has no drug resistance residual in animal product with medicine, can not produce potential harm to the mankind's health, be a kind of promising green feed additive.
The above-mentioned bacterial strain health 80 of mentioning is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 14th, 2011 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5058, and its Classification And Nomenclature is faecium (Enterococcus Faecium).
Description of drawings
Fig. 1 is the growth curve of faecium (Enterococcus Faecium) health 80
Fig. 2 is milk-acid bacteria 16S rRNA gene order phylogenetic tree in the Feed Sample
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Culture medium prescription used among the following embodiment is as follows:
1, MRS broth culture
Peptone 10g, beef powder 5g, glucose 20g, tween 80 1ml, dipotassium hydrogen phosphate 2g, sodium acetate 5g, citric acid tri-amonia 2g, magnesium sulfate heptahydrate 0.2g, four water manganous sulfate 0.05g, yeast powder 4g is settled to 1L with distilled water.
2, MRS nutrient agar
Add agar 15g in the 1L MRS broth culture.
3, Mai Kangkai substratum
The moon shows peptone 3g, pig cholate 5g, and toluylene red 0.025g, peptone 17g, lactose 10g, Viola crystallina 0.001g, sodium-chlor 5g, agar 15g is settled to 1L with distilled water.
4, M17 substratum
Soy peptone 5.0g, yeast extract 5.0g, casein peptone 5g, xitix 0.5g, beef extract 2.5g, β-Phosphoric acid glycerol esters disodium 19g, MgSO47H
2O 0.25g, agar 15g is settled to 1L with distilled water.
The Isolation and Identification of embodiment 1, faecium (Enterococcus Faecium) health 80
Faecium of the present invention extraction from Feed Sample, separation, screening, purifying obtain.
(1) isolation and purification of faecium health 80 (NFER-5)
1.1 the separation and Culture of bacterial strain
Get the healthy weanling pig ight soil of 1g in the test tube that the 9mL stroke-physiological saline solution is housed, whirlpool device concussion mixing, be 1: 10 diluent, getting diluent carries out ten times and increases progressively dilution again, then select each 1mL of diluent of 3 suitable gradients to coat MRS nutrient agar and the M17 nutrient agar that contains the 10ppm cycloheximide, 37 ℃ of anaerobism are cultivated 48～72h, observe and the record colonial morphology, the single bacterium colony of feature that picking grows fine in the MRS liquid nutrient medium, the separation and purification of ruling.Observe meat soup and whether become muddy, have 4 ℃ of refrigerator storages of muddy placement for subsequent use.
1.2 gramstaining
Draw a small amount of MRS broth culture with asepsis injector, drop on the slide glass, oven dry is fixing gently on spirit lamp flame.Drip violet staining liquid, dye 1min, washing; Drip the Gram's iodine solution mordant dyeing, effect 1min, washing; Drip acetone ethanol mixed solution (acetone: 95% ethanol=3: 7) decolouring 30s, washing; Drip husky yellow staining fluid and redye 1min, washing is waited to do, and observes at ordinary optical microscope, and thalline takes on a red color negative, purple positive.Be the consistent bacillus of Gram-positive form, further carry out catalase test.
1.3 catalase test
Do the MRS slant medium, get the about 0.2mL injection of culture MRS nutrient agar inclined-plane, 5%CO2 incubator are housed, cultivate 24h for 37 ℃, grow bacterium colony after, 3% superoxol is added drop-wise on the bacterium colony, if it is negative not having the Bubble formation explanation, be positive if the Bubble formation explanation is arranged.Can tentatively think genus lactubacillus (Lactobacillus) by the culture that MRS substratum anaerobism is cultivated through Gram-positive and catalase test feminine gender.
3 parts of Feed Samples in conjunction with gramstaining and catalase test, are isolated 5 strains of lactic acid bacteria through separation and purification altogether, wherein bacillus 3 strains, and coccus 2 strains, and distinguish called after NFER-1, NFER-2, NFER-3, NFER-4, NFER-5.
(2), resistance seed selection and biological characteristic research
1.1 resistance toheat seed selection
5 strains of above-mentioned screening bacterium NFER-1 to be measured, NFER-2, NFER-3, NFER-4, NFER-5 strain are inoculated in the MRS liquid nutrient medium by the inoculum size of 2% (v/v), after processing 10min in 60 ℃, 70 ℃, the 80 ℃ water-baths, measure its viable count, observations behind 37 ℃ of constant temperature culture 24h, relatively its before processing at each temperature with processing after viable count.
The calculation formula of survival rate is:
S heat=n
1/ n
0
S heat is respectively through the faecium survival rate after 60 ℃, 70 ℃, the 80 ℃ processing; n
0Be every milliliter of viable count before the heat treated; n
1Be every milliliter of viable count behind the heat treated 10min.
The result is as shown in table 1, as can be seen from Table 1, NFER-2, NFER-4, NFER-5 also have certain viable count behind 70 ℃ of thermal treatment 10min, and NFER-5 concentration is up to 1.72 * 10
5, and NFER-5 still has higher viable count at 80 ℃ after processing 10min, and concentration is 1.00 * 10
4And NFER-1, NFER-3 without viable bacteria, proves that this two strains bacterial strain is thermo-labile after 70 ℃ are processed 10min.The pelleting temperature of general pig starter feed is between 70 ℃～85 ℃, high temperature resistant after survival rate low also be milk-acid bacteria as one of major limitation sexual factor of fodder additives.From heat-resisting survival rate, the fermentation lactobacillus of this test seed selection can tolerate the high temperature when granulating, and will have preferably future as fodder additives.Choose NFER-2, NFER-4, NFER-5 carries out next step screening.
Table 1 oven test result
1.2 acid resistance seed selection
With the bacterial strain NFER-2 preferably of thermotolerance in the above-mentioned test, NFER-4, it is in 3.0 the MRS liquid nutrient medium that NFER-5 is inoculated into the pH value by the inoculum size of 2% (v/v), adopt dull and stereotyped tilt-pour process to measure its viable count at 0h, 1h, 2h, 3h respectively, 37 ℃ of constant temperature culture 24h observationss, record its viable count, calculate the survival rate of each bacterial strain.
The calculation formula of survival rate is:
S
Acid=n
x/ n
0
S
AcidFaecium survival rate for different time after processing through pH3.0; n
0For pH3.0 processes front every milliliter of viable count; n
xBe every milliliter of viable count behind pH3.0 processing 0h, 1h, 2h, the 3h.
See Table 2 by each the bacterial strain survival rate after the pH3.0 processing.Only have as can be seen from the table NFER-5 to show stronger tolerance, survival rate still reached 80% after 3h processed, this survival rate should be comparatively desirable for its anti-restraining effect or killing action of crossing hydrochloric acid in gastric juice, but is subject to strong inhibition under this condition of NFER-2 and NFER-4.Carry out next step test so choose NFER-5.
Table 2 different time acid resistance test result
1.3 bile tolerance seed selection
The bacterial strain that activation is good is done doubling dilution with stroke-physiological saline solution, choose suitable dilution gradient and draw the 1mL diluent and be put in the plate of sterilizing, do repetition, then with the MRS solid medium pour plate that contains 0.30% and 1.0% Glycocholate sodium, cultivate 48h for 37 ℃, carry out enumeration, as test group; With the MRS solid medium pour plate that does not contain Glycocholate sodium, cultivate 48h for 37 ℃ simultaneously, enumeration, in contrast group.Calculate the survival rate of bacterial strain.
The calculation formula of survival rate is:
S
Acid=n
x/ n
0
S
AcidBe the faecium survival rate of processing through 0.30% and 1.0% Glycocholate sodium; n
0Be every milliliter of viable count processing with the MRS solid medium that does not contain Glycocholate sodium; n
xFor processing rear every milliliter of viable count with containing 0.30% and 1.0% Glycocholate sodium.
Survival results after the various biliary salt concn is processed sees Table 3.As seen from table, NFER-5 has high tolerance under 0.3% the condition, and 1.0% cholate is also had certain tolerance, and survival rate has reached 47.39%.Studies show that cholate is the more disadvantageous factor of ratio hydrochloric acid in gastric juice that the intestines milk-acid bacteria runs in animal gastrointestinal tract.Chou and Weimer research finds that taming milk-acid bacteria with the cholate selectivity is effective to the tolerance of cholate, the energy force rate parental generation milk-acid bacteria of the milk-acid bacteria bile tolerance that domestication obtains afterwards through the several generations cholate is strong, milk-acid bacteria produces tolerance to the cholate performance easily, and have certain heredity, the faecium that selects bile tolerance has great importance aborning.
Table 3 various biliary salt concn tolerance test result
1.4 antibiotics resistance seed selection
Various microbiotic are pressed the listed dissolution with solvents of table 4, behind the bacteriological filtration by in the aseptic MRS substratum that joins sterilization of test institute expense, with seed selection in the above-mentioned test good bacterial strain NFER-5 be inoculated in the MRS liquid nutrient medium that contains different content microbiotic (table 4) by the inoculum size of 2% (v/v), cultivate 24h, observe its colony growth situation for 37 ℃.
The various Antibiotics of table 4, consumption and test-results
Annotate :+, thalli growth;-, thalline is not long
The result is as shown in table 5, is the Antibiotics of using in the test, each microbiotic usage quantity and test-results.
The various Antibiotics of table 5, consumption and test-results
Annotate :+, thalli growth;-, thalline is not long
NFER-5 grows in the substratum that contains olaquindox, wild marjoram oil, roxarsone, Zinc-bacitracin, Pro-gen 90, duomycin and colistin normally as can be seen from the table; In the substratum that contains mequindox, the large fertilizer of speed, tylosin, sulphamethazine, trimethoprim, kitasamycin, do not grow.Illustrate that NFER-5 is to olaquindox, wild marjoram oil, roxarsone, Zinc-bacitracin, Pro-gen 90, duomycin and the resistance that have against the enemy; To other antibiotic sensitive.
1.5, antibiotic activity
Behind 37 ℃ of cultivations of NFER-5 (called after health 80) usefulness MRS broth culture 24h, get 5mL culture (8.2 * 10
9CFU/mL) respectively with 5,10, the 15mL nutrient broth mixes, make 3 nutrient broths (1/2 that contain different bacterium culture concentration, 1/3,1/4), inoculate respectively salmonella typhi (Salmonella typhi, CVCC2212, veterinary microorganism DSMZ of country), streptococcus aureus (CVCC1882, veterinary microorganism DSMZ of country) intestinal bacteria K88 (Escherichia Coli, CMCC44742, Chinese medicine bacterium preservation administrative center) and 0157 (Escherichia Coli, available from China Veterinary Drugs Supervisory Inst.), inoculum size is 10% of nutrient solution, puts 37 ℃, 5%CO
2Incubator is cultivated 24h.
Detect the colony number of every group of culture, the results are shown in Table 6.
Table 6 mixed culture test-results
As can be seen from the table, 80 pairs of salmonella typhis of health all have preferably inhibition under different concns, can make 5-6 order of magnitude of its viable count decline, and the growth of self are unaffected, and bacteria concentration all remains on 1 * 10
8More than; And intestinal bacteria K88 and O157 are had certain restraining effect, can make 2-3 order of magnitude of its decline, but effect is unobvious to salmonella typhi, but the viable count of NFER-5 remains unchanged all basically; NFER-5 does not have inhibition to streptococcus aureus, and the growth of himself is not affected by streptococcus aureus yet.
1.6 storage tolerance survival rate
The MRS broth culture, regulating pH is 6.7, place Hungates to roll pipe, every pipe dress 20mL MRS broth culture, after making the aseptic meat soup of anaerobism, add 1mL NFER-5 in every pipe, be placed in 37 ℃ of incubators and cultivate, carry out live bacterial count respectively at 0h, 24h, 72h, 104h, 128h, 176h sampling 1mL.After method of counting carries out gradient dilution with asepsis injector taking-up 1mL, 10
-4～10
-7Extent of dilution is got the 0.3mL diluent in the upper evenly coating of MRS nutrient agar (pH5.2), and each gradient is done 3 Duplicate Samples, and plate is placed on 37 ℃, 5%CO
2Incubator in, cultivate 24h, get colony number and be 50～150 plate count, represent the result with mean value.
The calculation formula of survival rate is:
S
Storage=n
1/ n
0
S
StorageBe the NFER-5 survival rate after the process storage; n
0For preserving front every milliliter of viable count; n
1For preserving respectively every milliliter of viable count behind 0h, 24h, 72h, 104h, 128h, the 176h.
The result is as shown in table 7,
Table 7 is viable count and the survival results of test strain different storage time.
As can be seen from the table, the initial live bacteria concentration of NFER-5 is 2.4 * 10
10Cfu/mL, NFER-5 still keeps higher number of viable behind 37 ℃ of constant temperature culture 7d, reaches 6.8 * 10
9Cfu/mL.Illustrate that this bacterium has preferably stability.
1.7 growth curve is measured
Dress 300mL MRS broth culture in the 500mL Erlenmeyer flask.By 1% inoculum size inoculation NFER-5 culture, cultivate 18h for 37 ℃, not add MRS liquid nutrient medium for examination bacterium liquid as blank, measured its OD600 every one hour.Record data are also drawn growth curve.
Growth curve mainly reflects a kind of microbial growth characteristic, and microbial growth generally experiences lag period, logarithmic phase, stationary phase and decline phase four-stage, and this is a kind of typical growth curves model.Be that microorganism is to the adaptive process of new growing environment lag period, in this course, microorganism shows as the constant or decline of quantity, and himself macromole and micromolecular composition are adjusted, and also can produce specific material such as enzyme etc. simultaneously and adapt to new environment.Logarithmic phase be microorganism to after the new environmental adaptation, growth and breeding speed is the stage of geometricprogression, is a fastest stage of quantity growth, shows as the increase of thalline quantity and weight.But arrived the latter stage of logarithmic phase, because the thalli growth metabolism is to the consumption of nutritive substance and the accumulation of toxic products, the growth and breeding speed of bacterium descends.Be the stage that rate of bacterial growth and rate of death tend to balance stationary phase.The decline phase bacterial number obviously descends.Measure growth curve and have vital role for definite suitable fermentation time.
The growth curve of NFER-5 as shown in Figure 1.As can be seen from the figure, NFER-5 enters logarithmic phase after cultivating 2 hours, and the OD600 value is risen rapidly by 0.18 beginning, enters stationary phase in 10 hours, and the OD600 value reaches 1.41, and this bacteria growing speed is described.Can find out that from growth curve be 10-18h after cultivation the best harvesting time of NFER-5, at this section period results thalline, can reduce the cost that obtains the unit viable bacteria.
1.8NFER-5 extracting genome DNA and 16S rRNA order-checking
The 16SDNA sequential analysis: the extraction of above-mentioned NFER-5 bacteria total DNA adopts bacterial genomes DNA extraction test kit (biochemical (Beijing) Science and Technology Ltd. of day root, Tiangen DP302-02) to extract.16S rDNA amplimer adopts the bacterium universal primer, and its primer sequence is: forward primer is 27f (corresponding to Escherichia coil 8-27 bit base): 5 '-AGAGTTTGATCCTGGCTC AG-3 '; Reverse primer is 1495r (corresponding to Escherichia coil 1495-1515 bit base): 5 '-CTACGGCTACCTTGTTACGA-3 '.(50 μ L) is as shown in table 7 for reaction system:
Table 8PCR amplification system
The pcr amplification program is: 95 ℃ of denaturation 5min; 94 ℃ of sex change 1min; 58 ℃ of annealing 1min; 72 ℃ are extended 2min, carry out 30 circulations, and last 72 ℃ are extended 10min.PCR product utilization 1% agarose gel electrophoresis detects, send the calm and peaceful biotechnology of Sino-U.S. (Beijing) company limited to carry out sequencing after the positive products of the about 1500bp of fragment length is purified, wherein the nucleotides sequence of the encoding gene of the 16S rRNA of NFER-5 bacterium is classified the sequence 1 in the sequence table as.
The gene order that obtains is carried out BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) sequence analysis in the GenBank database, and utilizes local software Mega4.0 and type culture to carry out the research of phyletic evolution sibship.Utilize the cluster W constructing system evo-devo tree in the software, plant as the boundary threshold value of planting arrives identification of strains to be measured greater than 99% take homology.
Equally above-mentioned NFER-1, NFER-2, NFER-3 and NFER-4 are also carried out the evaluation of 16S rRNA, 16S rRNA gene order and the drawing system evolutionary tree of milk-acid bacteria and 4 strain type strains see Fig. 2 in the employing MEGA4.0 software analysis feed.Can get from systematic evolution tree: NFER-1 and NFER-4 and Lactobacillus parabuchneriJCM12493 sibship are nearer, the homology of 16S rRNA gene order and Lactobacillus parabuchneri is more than 99%, thereby this 2 strain bacterium is attributed to Lactobacillus parabuchneri; NFER-3 and Lactobacillus plantarum NCDO1752 are in same subgroup, are accredited as Lactobacillus plantarum in conjunction with the homology result; The homology of NFER-2 and Streptococcus thermophilus is 99%, also is in same branch with Streptococcus thermophilusATCC19258, so be accredited as Streptococcus thermophilus; NFER-5 and Enterococcus faecium LMG11423 are in same branch, are defined as Enterococcus faecium, called after faecium (Enterococcus Faecium) health 80.
Therefore, according to bacteriostatic test, strain separating position and biological characteristics seed selection, filter out health 80 as the production bacterial classification of probiotic bacterium.Bacterial strain health 80 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 14th, 2011 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5058, and its Classification And Nomenclature is faecium (Enterococcus Faecium).
The cell of faecium (Enterococcus Faecium) health 80CGMCC No.5058 is spherical, Gram-positive, and other biological characteristics is as shown in table 9.
The biological characteristics of table 9 health 80
This faecium (Enterococcus Faecium) is a kind of of enterococcus spp (Enterococcus).
The application of experimental example 2, faecium (Enterococcus Faecium) health 80CGMCC No.5058
One, on the impact of weaned piglets
1.1 materials and methods:
Experimental animal
Choose 100 of the DLY ternary hybrid piglets of 28 ± 2d wean, mean body weight 9.0 ± 0.32kg, distinguishing at random component by body weight is 2 treatment group, every group of 5 repetitions, each repeats 10 piglets.
1.2 the cultivation of faecium health 80
Faecium (Enterococcus Faecium) health 80CGMCC No.5058 is inoculated in the MRS substratum by 1% inoculum size, under 37 ℃ of conditions, cultivates 20h, obtain health 80 cultures.
1.3 test grouping
It is two processing that test is divided into, and processing 1 is test group, and basal diet adds health 80 cultures of 100g/t; Process 2 and be control group (basal diet is only arranged).Composition and the nutritive ingredient of basal diet see Table 10.
The basal diet of table 10 growth test forms and trophic level
Annotate:
1. crude protein, Methionin, methionine(Met), Gelucystine, Threonine, calcium and phosphorus are measured value.
2. per kilogram Preblend provides: vitamin A, 11,000IU; Vitamin D3 500,000 I.U/GM, 1503IU; Vitamin-E, 44.1IU; Vitamin K, 4.0mg; Riboflavin, 5.22mg; Pantothenic acid, 20.0mg; Nicotinic acid, 26.0mg; Vitamin B12,0.01mg; Manganese, 35.0mg; Iron, 100.0mg; Zinc, 90.0mg; Copper, 16.5mg; Iodine, 0.30mg; Selenium, 0.30mg.
1.4 feeding and management
Test in Ruzhou City that three source animal husbandry pig houses carry out.45 days trial periods.The duration of test feeding piglet is in fully closed child care piglet house, and the temperature in the house remains on 24～27 ℃.Free choice feeding, each hurdle circle are equipped with Duckbill-type drinking trough and freely drink water for piglet.1% Preblend autogamy of basal diet does not contain any microbiotic.The immunity of piglet is undertaken by the immune programme for children of pig routine veterinary transmissible disease, feeding piglet control measures strict implement health and epidemic prevention system.
1.5 sample collection and processing
Test claims to calculate day weight gain and feed food consumption by the piglet individual weight in when beginning and when finishing.In the whole feeding experiment stage, every morning 9:00 point is observed swine excrement situation (every diarrhoea pig was only added up once the same day), morbidity and death condition.During off-test the outward appearance of piglet is pursued the head scoring, concrete grammar sees Table 11
The ruddy degree of fur index=skin+hair color brightness+hair is along degree
Table 11 piglet outward appearance standards of grading
| Mark | The ruddy degree of skin | Hair color brightness | Hair is along |
| 1 | Pale | Matt | Obviously in disorder |
| 2 | Little red | Faint gloss | Faint in |
| 3 | Ruddy | Obvious gloss | Soft |
1.6 data statistics
Test the independent sample t check of all the data SPSS 12.0 (SPSS Inc., USA) statistical software and process statistics.
2, results and analysis
2.1 faecium on weaned piglet after the impact of growth performance
Statistic analysis result sees Table 12, and average daily ingestion amount between test group, average daily gain difference are not remarkable, but compare significant difference with control group, show and add daily ingestion amount and the day weight gain that faecium can increase weanling pig in the daily ration.The feedstuff-meat ratio of test group is lower than control group, and significant difference, shows that adding faecium in the daily ration can reduce the weanling pig feedstuff-meat ratio.Therefore, add the growth performance that faecium, cecropin can the Effective Raise weanling pigs in the daily ration.
Add faecium in table 12 daily ration to the impact of Production Performance of Weaning Pigs
2.2 add faecium in the daily ration to the impact of diarrhea of weaned piglets
Each is organized the diarrhoea situation and sees Table 13, and test group has reduced by 77.20% compared with the control, shows to add the diarrhoea that faecium can effectively reduce weanling pig in the daily ration.
Table 13 diarrhea of weaned piglets situation
2.3 add faecium in the daily ration to the impact of weanling pig fur index
Found out that by table 14 the fur index of test group significantly is better than control group, show and add the fur that faecium can be improved pig in the daily ration.
Add faecium in table 14 daily ration to the impact of piglet fur index
| Pig number | Test group | Control group | Pig number | Test | Control group | |
| 1 | 333 | 233 | 26 | 333 | 233 | |
| 2 | 322 | 333 | 27 | 333 | 232 |
| 3 | 333 | 223 | 28 | 333 | 233 |
| 4 | 322 | 322 | 29 | 332 | 223 |
| 5 | 322 | 233 | 30 | 333 | 333 |
| 6 | 333 | 323 | 31 | 322 | 232 |
| 7 | 333 | 333 | 32 | 223 | 233 |
| 8 | 333 | 322 | 33 | 322 | 233 |
| 9 | 333 | 333 | 34 | 333 | 233 |
| 10 | 333 | 233 | 35 | 333 | 233 |
| 11 | 232 | 212 | 36 | 333 | 322 |
| 12 | 322 | 222 | 37 | 333 | 333 |
| 13 | 322 | 322 | 38 | 333 | 322 |
| 14 | 323 | 322 | 39 | 333 | 333 |
| 15 | 333 | 233 | 40 | 333 | 233 |
| 16 | 333 | 223 | 41 | 322 | 222 |
| 17 | 323 | 322 | 42 | 233 | 322 |
| 18 | 333 | 233 | 43 | 323 | 223 |
| 19 | 232 | 333 | 44 | 333 | 333 |
| 20 | 333 | 322 | 45 | 333 | 322 |
| 21 | 322 | 221 | 46 | 332 | 231 |
| 22 | 323 | 222 | 47 | 333 | 233 |
| 23 | 333 | 223 | 48 | 333 | 233 |
| 24 | 322 | 223 | 49 | 333 | 121 |
| 25 | 333 | 233 | 50 | 333 | 221 |
2.4 Economic and Efficiency Analysis
The child care pig is by 18 yuan/kg, and feed is by 6.15 yuan/kg, and faecium is pressed 200 yuan/kg,, then respectively organize income and see Table 15, compare with control group, add every pig of faecium in the daily ration and can increase income 13.61 yuan.
Add faecium in table 15 daily ration to the impact of piglet economic benefit
Therefore, add the production performance that faecium can significantly improve the sour milk piglet in the daily ration, reduce the grice diarrhoea rate, improve the pig fur, increase the economic benefit of pig.
Two, faecium (Enterococcus Faecium) health 80CGMCC No.5058 is on the impact of growth of meat chicken performance
1.1 materials and methods:
Experimental animal
Test selects healthy love to pull out increasingly 3000 of (Arbor Acres, AA) broiler chicks, and mean body weight is about 49.1g, and distinguishing at random component by body weight is 2 treatment group, every group of 5 repetitions, and each repeats 300 chickens.
1.2 the cultivation of faecium health 80
Faecium (Enterococcus Faecium) health 80CGMCC No.5058 is inoculated in the MRS substratum by 1% inoculum size, under 37 ℃ of conditions, cultivates 20h, obtain health 80 cultures.
1.3 test grouping
It is two processing that test is divided into, and processing 1 is test group, and basal diet adds health 80 cultures of 100g/t; Processing 2 is control group, and composition and the nutritive ingredient of basal diet see Table 16.Whole trial period is 54 days.
Table 16 Diet Formula and trophic level (%)
1.4 feeding and management
Broiler chicken field feeding manner is semi-enclosed online flat supporting, and front 15 days particulate material of feeding, 15 days are fed to dry mash to delivering for sale; Free choice feeding and drinking-water; Sterilize weekly twice, disinfection way band chicken spraying disinfection; Temperature is controlled by temperature regulator; Type of heating adopts the gas blower blowing hot-air; Longitudinal ventilation; The illumination incandescent light; Observe chicken group's healthy state every day.
1.5 sample collection and processing
Test claims chicken in when beginning and when finishing, and calculates day weight gain, feed food consumption and feed efficiency.Morning every day, 9:00 observed the chicken group, the record death toll.
1.6 statistical study
Statistical study is carried out in the independent sample t of statistical study employing SPSS12.0 (SPSSInc., the USA) software of data-check.
2, result and discussion
The statistics of the growth performance index of duration of test broiler chicken sees Table 17.
Faecium is on the impact of growth of meat chicken performance in table 17 daily ration
As can be seen from Table 17, add day weight gain (P＜0.01), daily ingestion amount (P＜0.05) that faecium can broiler chicken in the daily ration, reduce feed conversion rate (P＜0.05).Although the survival rate of test group chicken descends slightly, difference is significantly (P＞0.05) not, and this may to get proventriculitis relevant with chick in the process of the test.
Therefore, adding the growth performance that faecium can significantly improve broiler chicken in the daily ration, is a kind of useful bacterial classification.
Claims (11)
1. animal feedstuff additive, its activeconstituents is faecium (Enterococcus Faecium) health 80 CGMCC No.5058; Described animal is pig or chicken.
2. the animal-feed that contains animal feedstuff additive claimed in claim 1; Described animal is pig or chicken.
3. antibacterial product, its activeconstituents is faecium (Enterococcus Faecium) health 80 CGMCCNo.5058.
4. antibacterial product according to claim 3 is characterized in that: described antibacterial for suppressing salmonella typhi (Salmonella typhi) and/or intestinal bacteria (Escherichia Coli).
5. the application of faecium (Enterococcus Faecium) health 80 CGMCC No.5058 in preparation animal feedstuff additive, animal-feed, antibacterial product and/or promotion growth of animal; Described animal is pig or chicken.
6. application according to claim 5 is characterized in that: described antibacterial for suppressing salmonella typhi (Salmonella typhi) and/or intestinal bacteria (Escherichia Coli).
7. a method for preparing animal feedstuff additive comprises the steps:
Fermentation faecium (Enterococcus Faecium) health 80 CGMCC No.5058 collect tunning, namely obtain animal feedstuff additive.
8. method according to claim 7 is characterized in that:
The temperature of described fermentation is 35 ℃-37 ℃;
Described fermentation time is 15h-80h;
The substratum of described fermentation is the MRS substratum.
9. method according to claim 8 is characterized in that:
The temperature of described fermentation is 37 ℃;
Described fermentation time is 20h.
10. the preparation method of an antibacterial product comprises the steps:
Fermentation faecium (Enterococcus Faecium) health 80 CGMCC No.5058 collect tunning, namely obtain antibacterial product;
The temperature of described fermentation is 35 ℃-37 ℃;
Described fermentation time is 15h-80h;
The substratum of described fermentation is the MRS substratum;
Described antibacterial for suppressing salmonella typhi (Salmonella typhi) and/or intestinal bacteria (Escherichia Coli).
11. method according to claim 10 is characterized in that: the temperature of described fermentation is 37 ℃; Described fermentation time is 20h.
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