CN107254424A - A kind of livestock and poultry new liquid compound micro-ecological preparation and preparation method thereof - Google Patents
A kind of livestock and poultry new liquid compound micro-ecological preparation and preparation method thereof Download PDFInfo
- Publication number
- CN107254424A CN107254424A CN201710569185.1A CN201710569185A CN107254424A CN 107254424 A CN107254424 A CN 107254424A CN 201710569185 A CN201710569185 A CN 201710569185A CN 107254424 A CN107254424 A CN 107254424A
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- China
- Prior art keywords
- extract
- liquid
- fermentation
- rcm
- radix pseudostellariae
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- Granted
Links
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- 244000144977 poultry Species 0.000 title claims abstract description 19
- 150000001875 compounds Chemical class 0.000 title claims abstract description 14
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- 235000018291 probiotics Nutrition 0.000 abstract description 24
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 11
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- 150000003839 salts Chemical class 0.000 description 3
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical group C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
- A61K31/10—Sulfides; Sulfoxides; Sulfones
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/74—Bacteria
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- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A61K36/752—Citrus, e.g. lime, orange or lemon
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
-
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Abstract
The invention belongs to probiotics technology for producing field, and in particular to a kind of livestock and poultry new liquid compound micro-ecological preparation and preparation method thereof.Including step：（1）Two kinds of fermentation strain activation；（2）It is prepared by fermentation seed liquid；（3）Two plants of bacterium mix common fermentation；（4）Liquid is mixed admittedly；（5）Bottling.The method that the present invention is provided can speed up liquid state fermentation speed, prepared beneficial to high concentration bacterium solution, gained probiotics can effectively prevent and treat the disease caused by staphylococcus aureus, micrococcus luteus and ETEC, animal and bird intestines probiotics is supplemented, can assisting in preventing and treating livestock birds heat irritability syndrome；Equally, method provided by the present invention can also be compounded effectively in vitro with Chinese herbal medicine medicinal extract builds new liquid preparation, and beneficial to multiple diversity operation, technique is simple；It is finally that the inventive method does not need stringent sterilization operation, significantly reduces work difficulty, reduce energy waste, it is cost-effective.
Description
Technical field
The invention belongs to probiotics technology for producing field, and in particular to a kind of livestock and poultry are combined micro- with new liquid
Ecological agent and preparation method thereof.
Background technology
China is large agricultural country, and animal husbandry development occupies important proportion, and according to country, supply side is reformed in recent years, whole nation poultry
Herding scale and its development will greatly drive the industry development, therefore, from the point of view of the overall situation, the industrialization of China's animal husbandry, scale, standard
The development of change will be promoted further, and development potentiality of China's livestock and poultry breeding industry during following " 13 " is huge, and space is huge,
It is significant.However, China's aquaculture has the extremely serious phenomenon of abuse of antibiotics phenomenon, country had increased the country in recent years
The management work of livestock and poultry breeding industry abuse of antibiotics, Minister Agriculture of China put into effect part antibiotic respectively at 2016 and 2017 and prohibited
Bulletin.Meanwhile, the cry that the domestic or even whole world is cultivated to nonreactive is increasing, different antibiotic substitutes meet the tendency of and
Raw, current scientist has found that probiotics prepared by probiotics fermention is a wherein most potential class, solid-state and liquid
Probiotics is quickly grown.Present scientific research proves that probiotics can not only improve drug effect, nontoxic residue-free, moreover it is possible to
Actively supplement beneficial bacteria of intestinal tract, reparation and releveling beneficial to gut flora.
Currently, it is better with fermented type probiotics product in probiotics formulation, fermented type probiotics
It can be divided into liquid probiotics, semisolid probiotics and the class of solid-state probiotics three according to formulation.Liquid Tiny ecosystem
Preparation is to utilize the synergy between microorganism, has played the synergy advantage of colony, the stabilization of thalline is improved well
Property, bioactivity is maintained, to adjust animal physiological activation plays important function, and preparation technology is simple, be effectively reduced
Cost, can produce huge economic benefit.Comprehensive available data, which can sum up liquid probiotics, can carry out drinking-water use, mouth
Take, spice is used and four class occupation modes of sprinkling, but used with drinking water, for oral use and spice uses slightly many.However, liquid is micro- at present
Ecological agent product, which is appointed, so has urgent problem：1. stringent sterilization is needed before liquid state fermentation, complex operation and time-consuming；②
There is swollen bottle phenomenon in bottled liquid probiotics product section, reduces shelf life；3. liquid state fermentation lacks outstanding association
With fermentation hybrid bacterial strain；4. fermentation accelerant species is few in liquid state fermentation, more lacks integration of drinking and medicinal herbs fermentation accelerant.
The content of the invention
To overcome problem existing during liquid probiotics preparation in the prior art, it is an object of the invention to provide
A kind of livestock and poultry new liquid compound micro-ecological preparation and preparation method thereof.
To achieve the above object, the technical scheme that the present invention takes is as follows：
The preparation method of the new liquid compound micro-ecological preparation of a kind of livestock and poultry, it is characterised in that step is as follows：
（1）, two kinds of fermentation strains activation：
Enterococcus faecalis（Enterococcus faecalis）BD-1 activation：By enterococcus faecalis BD-1 test tube slants strain in nothing
Rinsed under collarium border with sterile saline, be then seeded into the MRS fluid nutrient mediums of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae
In, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/mL
Meter（I.e. 100mL MRS fluid nutrient mediums must 0.2 ~ 0.5g of extract containing radix pseudostellariae）；Enterococcus faecalis BD-1 is new strains, applicant
The bacterial strain is preserved in China typical culture collection center, address：Wuchang, wuhan Luo Jia Shan, preservation date：2017 7
The moon 7；Deposit number：CCTCC NO：M2017416；
Clostridium butyricum（Clostridium butyricum）RCM-2 activation：Clostridium butyricum RCM-2 test tube slants strain is existed
Rinsed under gnotobasis with sterile saline, be then seeded into the MRS Liquid Cultures of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae
In base, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/
ML is counted（I.e. 100mL MRS fluid nutrient mediums must 0.2 ~ 0.5g of extract containing radix pseudostellariae）；Clostridium butyricum RCM-2 is new strains, Shen
Ask someone that the bacterial strain is preserved in into China typical culture collection center, address：Wuchang, wuhan Luo Jia Shan, preservation date：
On June 29th, 2017；Deposit number：CCTCC NO M2017396；
（2）, prepare enterococcus faecalis BD-1 and clostridium butyricum RCM-2 fermentation seed liquid respectively：
Enterococcus faecalis BD-1 fermentation seed liquid：Under gnotobasis, by step（1）Gained enterococcus faecalis BD-1 activating solution presses body
Product percentage 3 ~ 5% is inoculated into the MRS fluid nutrient mediums containing mass percent for 0.2 ~ 0.5% radix pseudostellariae palpus extract, 35 ~
42 ~ 48h of static fermentation at 37 DEG C；Wherein, the percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL（I.e.
100mL MRS fluid nutrient mediums must 0.2 ~ 0.5g of extract containing radix pseudostellariae）；
Clostridium butyricum RCM-2 fermentation seed liquid：Under gnotobasis, by step（1）Gained clostridium butyricum RCM-2 activating solution is pressed
Percent by volume 3 ~ 5% is inoculated into the MRS fluid nutrient mediums containing mass percent for 0.2 ~ 0.5% radix pseudostellariae palpus extract,
42 ~ 48h of static fermentation at 35 ~ 37 DEG C；Wherein, the percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL
（I.e. 100mL MRS fluid nutrient mediums must 0.2 ~ 0.5g of extract containing radix pseudostellariae）；
（3）, two plants of bacterium mix common fermentation：
The hot water of 50 ~ 60 DEG C of injection first into fermentation tank of the top with air bleeding valve, now air bleeding valve is in completely closed state,
Then nutritional ingredient is added, is stirred, sealed shell of tank adds Tea Polyphenols when water temperature in fermentation tank is down to below 40 DEG C
Powder, allicin powder, stir, and sealed shell of tank preserves 30min ~ 1h, then accesses enterococcus faecalis BD-1 fermentation seed liquid and fourth
Sour clostridium RCM-2 fermentation seed liquid by volume 1: 1 ~ 5: 1 composition seed mixture liquid, lid lid sealing, 32 ~ 37 DEG C of static hairs
During 6 ~ 8h of ferment, half-open air bleeding valve then proceedes to close air bleeding valve during 36 ~ 40h of fermentation, terminates fermentation；
The nutritional ingredient is that radix pseudostellariae must extract, brown sugar, yeast extract, vitamin C, CMC and brewer's wort；To account for hot water
Percentages, radix pseudostellariae must extract account for 0.2 ~ 0.5%, brown sugar account for 10 ~ 15%, yeast extract account for 5 ~ 8%, vitamin C account for 1 ~ 2%,
CMC, which accounts for 0.5 ~ 1.5%, brewer's wort and accounts for 1 ~ 5%, tea polyphenol powder and account for 0.1 ~ 0.25%, allicin powder, accounts for 0.01 ~ 0.025%, seed mixture
Liquid accounts for 5 ~ 10%, and the wherein percentage of brewer's wort and seed mixture liquid is volume ratio, and remaining is mass volume ratio g/mL；
（4）, liquid mixes admittedly：
" liquid " refers to step（3）Gained zymotic fluid, it is described " Gu " refer to following solids 1. or solid 2.：
The composition of solid 1. is：Tea polyphenol powder, allicin powder and prebiotics, wherein prebiotics are oligoisomaltose, oligomeric fruit
The one or two of sugar, to account in terms of the mass percent of liquid-solid mixture total amount, tea polyphenol powder accounts for 0.25 ~ 1.5%, allicin powder
Account for 0.05 ~ 0.1%, prebiotics and account for 1 ~ 5%；
The composition of solid 2. is：Tea polyphenol powder, allicin powder and the Chinese herbal medicine medicinal extract that immunity of livestock can be improved；Mixed admittedly with accounting for liquid
The mass percent meter of compound total amount, tea polyphenol powder, which accounts for 0.1 ~ 0.8%, allicin powder and accounts for 0.02 ~ 0.05%, Chinese herbal medicine medicinal extract, accounts for 10
~20%；
（5）, treat step（4）After end, it is again sealed off tank body and preserves 1 ~ 3h, produce livestock and poultry with new liquid compound microecological system
Agent.
Preferably, in terms of mass fraction, the Chinese herbal medicine medicinal extract is by each bulk drug of following proportionings through alcohol extracting or/and water
Carry and obtain：10 ~ 30 parts of 20 ~ 60 parts of radix pseudostellariae palpus, 10 ~ 30 parts of dried orange peel, 10 ~ 30 parts of hawthorn and Medicated Leaven.
Further, the extraction step of the Chinese herbal medicine medicinal extract is：
（i）Each bulk drug is weighed by formula, plus 75% alcohol reflux is extracted 1 ~ 3 hour, after cooling, slag-liquid separation obtains the dregs of a decoction and second
Alcohol extract, the dregs of a decoction add water to cook extraction 1 ~ 2 time, merge ethanol extract and aqueous extract；
（ii）By step（i）The thick paste that it is 1.2 ~ 1.35 to 28 DEG C of relative densities that the extract solution of merging, which is concentrated under reduced pressure, produces medium-height grass
Medicine medicinal extract.
The livestock and poultry prepared using the preparation method are with new liquid compound micro-ecological preparation.
In the present invention, radix pseudostellariae palpus extract can be obtained by prior art, be such as but not limited to applicant in August, 2016
The patent of " a kind of preparation method of radix pseudostellariae palpus extract " entitled filed in 30 days（Application number 201610758305.8）In
Disclosed method.
Beneficial effect：
1st, enterococcus faecalis BD-1 is located away from peasant household of village under Zherong County in the present invention（Coordinate：119。51 ' 59.7 〞 E, 27。13′34.0〞
N）48h bacterium are cultivated in autotrophy healthy adult pig caecum mucous layer, gained enterococcus faecalis BD-1 safety non-toxics, 37 DEG C of MRS culture mediums
Fall surface smooth bumps, periphery is neat, and colony colour canescence, 0.5 ~ 1.2mm of colony diameter, microscopy G+, no gemma, thalline is long
About 0.5 ~ 1 μm of degree；Clostridium butyricum RCM-2 of the present invention is located away from peasant household of village under Zherong County（Coordinate：119。52 ' 04.1 〞 E, 27。13′
32.7〞N）The caecum mucous membrane section of the healthy adult cock cultivated from circle mountain forest, gained clostridium butyricum RCM-2 safety non-toxics, 37
Culture 48h bacterium colonies surface smooth bumps in DEG C RCM culture mediums, periphery is neat, and colony colour milk yellow is opaque, and slightly acid is smelly
Taste, 0.5 ~ 1.5cm of colony diameter, microscopy G+ have gemma, gemma oval, the life of gemma end or the life of secondary end, the thalline for spore of sprouting is in shuttle
Shape, about 3.5 ~ 7.0 μm of thalline length；
2nd, fermentation strain enterococcus faecalis BD-1 of the invention and clostridium butyricum RCM-2 is with excellent resistance to Tea Polyphenols and allicin
The new strains of ability, in addition, with preferable cooperative fermentation ability, described radix pseudostellariae palpus extract is even more there is provided a kind of new
The integration of drinking and medicinal herbs fermentation accelerant of type, can remarkably promote the cooperative fermentation speed, save fermentation time；Meanwhile, fermentation strain
Enterococcus faecalis BD-1 and clostridium butyricum RCM-2 can effectively prevent and treat staphylococcus aureus, micrococcus luteus and ETEC
Caused disease, and animal and bird intestines probiotics can be supplemented, can assisting in preventing and treating poultry beneficial to impaired intestine microenvironment is repaired
Fowl heat irritability syndrome；
3rd, the fermentation leading portion work of preparation method of the present invention be use generality hot water normality cool and add percentage for 0.1 ~
0.25% tea polyphenol powder and 0.01 ~ 0.025% allicin powder are used for suppressing varied bacteria growing, and non-selection traditional high temperature is high
Pressure sterilizing（121 DEG C, 20 ~ 30min）, it is not necessary to stringent sterilization is operated, and significantly reduces work difficulty, saves energy consumption, saves the time,
Simplify operation difficulty.Meanwhile, add Tea Polyphenols, allicin after fermentation again, acid zymotic fluid environment can pole remarkably promote
The stability of Tea Polyphenols, allicin, not only beneficial to the extension of validity of Tea Polyphenols, allicin, moreover it is possible to which zymotic fluid suppression is promoted in collaboration
Bacterium ability and the phenomenon for reducing swollen bottle in the fluid-like state probiotics shelf life；In addition, Tea Polyphenols, allicin are also widely used in
Antioxidant and phagostimulant in livestock and poultry cultivation, being capable of assisting in preventing and treating livestock birds heat irritability syndrome；Equally, it is provided by the present invention
Method can also be compounded effectively in vitro with Chinese herbal medicine medicinal extract builds new liquid probiotics, beneficial to multiple diversity operation, technique letter
It is single.
Brief description of the drawings
Fig. 1：Enterococcus faecalis BD-1 systematic evolution tree.
Fig. 2：Clostridium butyricum RCM-2 systematic evolution tree.
Embodiment
In following examples, it by number of patent application is that embodiment 1 is public in CN201610758305.8 that Radix Pseudostellariae extract, which is,
The method opened is prepared；The formula of each culture medium is：
MRS fluid nutrient mediums (1L)：Peptone 10g, powdered beef 5g, glucose 20g, dusty yeast 4g, sodium acetate 5g, phosphoric acid hydrogen two
Potassium 2g, magnesium sulfate 0.2g, Triammonium citrate 2g, manganese sulfate 0.05g, Tween 80 1mL, purified water 1L, pH value 6.2 ± 0.2；
MRS agar plates or MRS slant mediums（1L）：Peptone 10g, powdered beef 5g, glucose 20g, dusty yeast 4g, acetic acid
Sodium 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.2g, Triammonium citrate 2g, manganese sulfate 0.05g, Tween 80 1mL, agar 15g, purifying
Water 1L, pH value 6.2 ± 0.2；
RCM agar plates or RCM slant mediums（1L）：Yeast extract 3g, beef extract 10g, tryptone 10g, glucose
5g, soluble starch 1g, sodium chloride 5g, sodium acetate trihydrate 3g, cysteine hydrochloride 0.15g, agar 15g, purified water 1L,
PH value 6.8 ± 0.2, sterilizes standby；
Starch culture-medium（1L）：Beef extract 5g, peptone 5g, sodium chloride 0.5g, soluble starch 20g, agar 18g, purified water
1L, pH=7.2 ± 0.1, sterilize standby；
Wherein, the MRS fluid nutrient mediums containing Radix Pseudostellariae extract, Tea Polyphenols or allicin are each meant in above-mentioned MRS Liquid Cultures
On the basis of based formulas, addition Radix Pseudostellariae extract, Tea Polyphenols or allicin are still further corresponded to；It is many containing Bromocresol purple, tea
Each mean on the basis of above-mentioned MRS/RCM agar plates formula, still further correspond in the MRS/RCM agar plates of phenol or allicin
Add Bromocresol purple, Tea Polyphenols or allicin.
Embodiment 1-- enterococcus faecalis BD-1 separation and identification
1st, enterococcus faecalis BD-1 separation, enrichment, purifying
Under aseptic technique, peasant household of village under Fujian Province Ningde City Zherong County is taken（Coordinate：119。51 ' 59.7 〞 E, 27。13′
34.0〞N）Autotrophy healthy adult pig caecum mucous membrane section, scrapes mucous membrane mucus with the slide of sterilizing and is managed in EP, use sterile physiological salt
Water dilution 102Times, take 0.1mL to be coated on the MRS agar plates containing Bromocresol purple (0.01%, g/mL), after coating
MRS agar plates are placed in anaerobic culture box, 37 DEG C of culture 48h, select the bacterium colony line purifying of culture medium flavescence, choose mirror
Gram-positive, nonspore-bearing bacterial strain are examined in containing bromocresol purple (0.01%, g/mL), Tea Polyphenols（0.01%, g/mL）With
Allicin（0.01%, g/mL）MRS agar plates on secondary screening, it is final choose bacterium colony yellow color substantially, bacterium colony surface it is smooth convex
Rise, periphery it is neat as object bacterial strain, Strain Designation is BD-1, and carries out slant preservation to strain with MRS slant mediums.
2nd, morphological observation
Observe bacterial strain BD-1 colony morphology characteristic, it is known that：Bacterium colony surface smooth bumps on MRS agar plates, periphery is neat, bacterium
Fall color canescence, 0.5 ~ 1.2mm of colony diameter, microscopy G+, no gemma, about 0.5 ~ 1 μm of thalline length.
3rd, physiological and biochemical property
Bacterial strain BD-1 is identified by the biochemical identification experiment of lactic acid bacteria, motility, oxidase test etc. have been carried out respectively raw
Change CHARACTERISTICS IDENTIFICATION, result of the test control《Primary Jie Shi Bacteria Identifications handbook》Carry out synthetic determination.It the results are shown in Table 1.
The physiology seriation index determining result of isolated strains shows to be consistent with lactic acid producing enterococcus strain result.
4th, the 16S rDNA sequencings of BD-1 bacterial strains are compared
Utilize bacterial genomes DNA extraction agent boxes（DP302-02, TIANGEN）Bacterial strain BD-1 STb gene is extracted, with bacterial strain
BD-1 STb gene is template, primer P0 (5 '-AGAGTTTGATCCTGGCTCAG -3 ') and PC3 (5 ' -
GGTTACCTTGTTACGACTT -3 ') guiding under PCR expand the 16S rDNA sequences of the bacterial strain, PCR reaction systems are：10
5.0 2.0 μ L, Primer 1 (20 μm of ol/L) of μ L, dNTP (10mmol/L) of × Buffer 1.0 μ L, Primer2 (20 μm of ol/L)
1.0 μ L, the μ L of template DNA 2.5, Taq enzyme (5.0U/ μ L) 0.5 μ L, ultra-pure water 38 μ L, total 50 μ L.PCR reaction conditions are：First 94
℃5min；Then 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations；Last 72 DEG C of extensions 8min.After reaction terminates,
1% agarose gel electrophoresis detection is carried out to pcr amplification product, marker is compared and obtains purpose band.With purchased from Axygen public affairs
The Ago-Gel QIAquick Gel Extraction Kit of department reclaims and purifies the purpose band, send raw work bioengineering（Shanghai）Limited company
Determined dna sequence is carried out, sequencing result is shown in SEQ ID No.1.
Measured sequence and the 16S rDNA sequences in Genbank are carried out into Blastn similarity analysis to be compared, as a result
Bacterial strain BD-1 withEnterococcus faecium strain KCI1、Enterococcus lactis strain K12419C
The homologys of 16S rDNA sequences reached 100%, therefore BD-1 is accredited as enterococcus faecalis, its systematic evolution tree such as Fig. 1.
With reference to above-mentioned morphology, physiological and biochemical property and sequencing and chadogram result, bacterial strain BD-1 of the present invention is identified simultaneously
Classification And Nomenclature is enterococcus faecalis（Enterococcus faecalis）BD-1.
5th, Tea Polyphenols is to enterococcus faecalis BD-1 growths, pH influence
（1）Enterococcus faecalis BD-1 activation：Enterococcus faecalis BD-1 test tube slants strain is used into sterile physiological salt in an aseptic environment
Water is rinsed, and is washed lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is seeded in anaerobic box to be contained
The gradient of Tea Polyphenols is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle
It is set to（g/mL）：0%th, 0.10%, 0.25%, 0.50%, 1.00%, zymotic fluid pH value and viable count, knot are determined when terminal is cultivated
Fruit is shown in Table 2.
As seen from the results in Table 2：The concentration that enterococcus faecalis BD-1 of the present invention can tolerate Tea Polyphenols is 1.0%, the concentration hypothallus
Survival rate is 70.26%.
6th, allicin is to enterococcus faecalis BD-1 growths, pH influence
（1）Enterococcus faecalis BD-1 activation：With the step under the present embodiment the 5th（1）；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is seeded in anaerobic box to be contained
The gradient of allicin is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle
It is set to（g/mL）：0%th, 0.010%, 0.025%, 0.050%, 0.10%, determine zymotic fluid pH value and viable bacteria when terminal is cultivated
Number, the results are shown in Table 3；
As seen from the results in Table 3：The concentration that enterococcus faecalis BD-1 of the present invention can tolerate allicin is 0.1%, concentration hypothallus survival
Rate is 70.43%；
（3）Enterococcus faecalis is resistant to Tea Polyphenols and allicin growth contrast：
By inoculum concentration 3%（Volume ratio）Respectively by step（1）Gained enterococcus faecalis BD-1（It is calculated as A）Activating solution and control bacterium B,
C, D activating solution are seeded in anaerobic box in the anaerobism serum bottle of the fluid nutrient medium containing MRS, wherein in anaerobism serum bottle
In MRS fluid nutrient mediums 1. the gradient of Tea Polyphenols set gradually for（g/mL）：0%th, 1.00%, 2.00%, 2. the gradient of allicin according to
It is secondary to be set to（g/mL）：0%th, 0.05%, 0.1%, 48h is continuously cultivated, control is used as by 0%+0%（Viable count is defined as X0）, pass through
Formula：Thalline loss late=（X0-X）/ X0* 100% calculates enterococcus faecalis thalline loss late, the results are shown in Table 4.
As seen from the results in Table 4：In Tea Polyphenols（2%）And allicin（0.1%）Enterococcus faecalis BD-1 of the present invention under synergy
Survival rate is up to 53.7%, beyond 50%, far above the similar bacterial strain in market.
7th, enterococcus faecalis bacteriostatic experiment
（1）Enterococcus faecalis BD-1 activation：With the step under the present embodiment the 5th（1）；
（2）The static fermentation of enterococcus faecalis BD-1 anaerobism in MRS fluid nutrient mediums：
By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is seeded to containing MRS in anaerobic box
In the anaerobism serum bottle of fluid nutrient medium, 37 DEG C of static gas wave refrigerator 48h take zymotic fluid to be detected using Odontothrips loti when fermentation termination
To the fungistatic effect of staphylococcus aureus, micrococcus luteus and ETEC, using nonvaccinated MRS fluid nutrient mediums as
Control；
（3）Enterococcus faecalis BD-1 aerobic fermentations in MRS fluid nutrient mediums：
By inoculum concentration（Volume ratio）3% by step（1）Gained enterococcus faecalis BD-1 activating solution access is equipped with 100 mL MRS liquid
In 500 mL triangular flasks of culture medium, 37 DEG C, 48h is cultivated under 150rpm, takes zymotic fluid to utilize Odontothrips loti when terminal is cultivated
The fungistatic effect to staphylococcus aureus, micrococcus luteus and ETEC is detected, with nonvaccinated MRS Liquid Cultures
Base is control.
Antibacterial circle diameter size is shown in Table 5.
As seen from the results in Table 5：It is micro- that enterococcus faecalis BD-1 of the present invention can significantly inhibit pathogenic bacteria staphylococcus aureus, gamboge
Coccus and the growth of ETEC, and anaerobic cultures are relatively better than aerobic culture.
Embodiment 2-- clostridium butyricums RCM-2 separation and identification
1st, clostridium butyricum RCM-2 separation, enrichment, purifying
Under aseptic technique, peasant household of village under Fujian Province Ningde City Zherong County is taken（Coordinate：119。52 ' 04.1 〞 E, 27。13′
32.7〞N）The caecum mucous membrane section of the healthy adult cock cultivated from circle mountain forest, with the slide of sterilizing scrape mucous membrane mucus in
EP is managed, and 10 are diluted with sterile saline2Times, 60 DEG C, 30min are then heated to, takes 0.1mL to be coated on and is indicated containing bromocresol purple
On the RCM agar plates of agent (0.01%, g/mL), RCM agar plates are placed in anaerobic culture box after coating, 37 DEG C of cultures
48h, selects the bacterium colony line purifying of culture medium flavescence, choose microscopy Gram-positive, have the shaft-like bacterial strain of gemma in containing
Bromocresol purple (0.01%, g/mL), Tea Polyphenols（0.01%, g/mL）And allicin（0.01%, g/mL）RCM agar plates on it is multiple
Sieve, finally choose periphery of bacterial colonies yellow color substantially, bacterium colony surface smooth bumps, milk yellow is opaque, there is the conduct of acid smell
Preparation bacterial strain, the maximum conduct pair of transparent circle is selected after preparation bacterial strain then is coated on into starch culture-medium, 37 DEG C of culture 48h again
As bacterial strain, Strain Designation is RCM-2, and carries out slant preservation to strain with RCM slant mediums.
2nd, morphological observation
Observe bacterial strain RCM-2 colony morphology characteristic, it is known that：Bacterium colony surface smooth bumps on RCM agar plates, bacterium colony face yellow fraction
Color is opaque, slightly acid smell, 0.5 ~ 1.5cm of colony diameter, microscopy G+, there is gemma, gemma oval, the life of gemma end or secondary end
Raw, the thalline for spore of sprouting is in fusiform, about 3.5 ~ 7.0 μm of thalline length.
3rd, physiological and biochemical property
Reference《The outstanding Bacteria Identification handbook of uncle》Physiology and biochemistry identification is carried out to bacterial strain, respectively with catalase, amylase, nitrate also
Former and sugar etc. carries out physiological and biochemical test, the results are shown in Table 6.
By its with《The outstanding Bacteria Identification handbook of uncle》Physiology and biochemistry with clostridium butyricum in common bacteria system identification handbook is special
Property be compared, as a result find that bacterial strain RCM-2 is consistent substantially with the physio-biochemical characteristics of clostridium butyricum, therefore Preliminary Identification bacterial strain
RCM-2 is clostridium butyricum.
4th, sequencing and chadogram
Utilize bacterial genomes DNA extraction agent boxes（DP302-02, TIANGEN）Bacterial strain RCM-2 STb gene is extracted, with bacterial strain
RCM-2 STb gene is template, primer P0 (5 '-AGAGTTTGATCCTGGCTCAG -3 ') and PC3 (5 ' -
GGTTACCTTGTTACGACTT -3 ') guiding under PCR expand the 16S rDNA sequences of the bacterial strain, PCR reaction systems are：10
5.0 2.0 μ L, Primer 1 (20 μm of ol/L) of μ L, dNTP (10mmol/L) of × Buffer 1.0 μ L, Primer2 (20 μm of ol/L)
1.0 μ L, the μ L of template DNA 2.5, Taq enzyme (5.0U/ μ L) 0.5 μ L, ultra-pure water 38 μ L, total 50 μ L.PCR reaction conditions are：First 94
℃5min；Then 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, totally 30 circulations；Last 72 DEG C of extensions 8min.After reaction terminates,
1% agarose gel electrophoresis detection is carried out to pcr amplification product, marker is compared and obtains purpose band.With purchased from Axygen public affairs
The Ago-Gel QIAquick Gel Extraction Kit of department reclaims and purifies the purpose band, send raw work bioengineering（Shanghai）Limited company
Determined dna sequence is carried out, sequencing result is shown in SEQ ID No.2.
Measured sequence and the 16S rDNA sequences in Genbank are carried out into Blastn similarity analysis to be compared, as a result
The homology of bacterial strain RCM-2 and Clostridium butyricum strain 1005-15098 16S rDNA sequences reaches
98%, therefore RCM-2 is accredited as clostridium butyricum, its systematic evolution tree such as Fig. 2.
With reference to above-mentioned morphology, physiological and biochemical property and sequencing and chadogram result, bacterial strain RCM-2 of the present invention is identified simultaneously
Classification And Nomenclature is clostridium butyricum（Clostridium butyricum）RCM-2.
5th, Tea Polyphenols is to clostridium butyricum RCM-2 growths, pH influence
（1）Clostridium butyricum RCM-2 activation：Clostridium butyricum RCM-2 test tube slants strain is used into sterile physiological in an aseptic environment
Normal saline washing, washes lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2 activating solution is seeded in anaerobic box to be contained
The gradient of Tea Polyphenols is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle
It is set to（g/mL）：0%th, 0.10%, 0.25%, 0.50%, 1.00%, zymotic fluid pH value and viable count, knot are determined when terminal is cultivated
Fruit is shown in Table 7.
As seen from the results in Table 7：The concentration that clostridium butyricum RCM-2 of the present invention can tolerate Tea Polyphenols is bacterium under 1.0%, the concentration
Body survival rate is 87.3%.
6th, allicin is to clostridium butyricum RCM-2 growths, pH influence
（1）Clostridium butyricum RCM-2 activation：With the step under the present embodiment the 5th（1）；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2 activating solution is seeded in anaerobic box to be contained
The gradient of allicin is successively in the anaerobism serum bottle of MRS fluid nutrient mediums, in the MRS fluid nutrient mediums wherein in anaerobism serum bottle
It is set to（g/mL）：0%th, 0.010%, 0.025%, 0.050%, 0.10%, determine zymotic fluid pH value and viable bacteria when terminal is cultivated
Number, the results are shown in Table 8；
As seen from the results in Table 8：The concentration that clostridium butyricum RCM-2 of the present invention can tolerate allicin is 0.1%, and the concentration hypothallus is deposited
Motility rate is 85.5%；
（3）Clostridium butyricum is resistant to Tea Polyphenols and allicin growth contrast：
By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2（It is calculated as A）Activating solution be inoculated with anaerobic box
Into the anaerobism serum bottle of the fluid nutrient medium containing MRS, the 1. ladder of Tea Polyphenols in the MRS fluid nutrient mediums wherein in anaerobism serum bottle
Degree set gradually for（g/mL）：0%th, 1.00%, 2.00%, 2. the gradient of allicin set gradually for（g/mL）：0%、0.05%、
0.1%, 48h is continuously cultivated, control is used as by 0%+0%（Viable count is defined as X0）, pass through formula：Thalline loss late=（X0-X）/
X0* 100% calculates clostridium butyricum thalline loss late, the results are shown in Table 9.
As seen from the results in Table 9：In Tea Polyphenols（1%）And allicin（0.05%）Clostridium butyricum RCM- of the present invention under synergy
2 survival rates are up to 88.5%, beyond 50%, far above the similar bacterial strain in market.
7th, clostridium butyricum bacteriostatic experiment
（1）Clostridium butyricum RCM-2 activation：With the step under the present embodiment the 5th（1）；
（2）By inoculum concentration 3%（Volume ratio）By step（1）Gained clostridium butyricum RCM-2 activating solution is seeded in anaerobic box to be contained
In the anaerobism serum bottle of MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h take zymotic fluid to utilize Odontothrips loti when fermentation termination
The fungistatic effect to staphylococcus aureus, micrococcus luteus and ETEC is detected, with nonvaccinated MRS Liquid Cultures
Base is control；Antibacterial circle diameter size is shown in Table 10.
As seen from the results in Table 10：Clostridium butyricum RCM-2 of the present invention can significantly inhibit pathogenic bacteria staphylococcus aureus, gamboge
Micrococcus luteus and the growth of ETEC.
Embodiment 3-- enterococcus faecalis BD-1 and clostridium butyricum RCM-2 symbiosis are long
（1）The activation of bacterial strain
Enterococcus faecalis BD-1 activation：Enterococcus faecalis BD-1 test tube slant strain is used into sterile saline in an aseptic environment
Rinse, wash lower media surface lawn, be then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
Clostridium butyricum RCM-2 activation：Clostridium butyricum RCM-2 test tube slant strain is used into sterile physiological salt in an aseptic environment
Water is rinsed, and is washed lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）Then by enterococcus faecalis BD-1 activating solution, clostridium butyricum RCM-2 activating solution and both by volume 1:1 answers
The activating solution of conjunction is respectively according to inoculum concentration 3%（Volume ratio）It is inoculated in 200 mL anaerobism serum bottles of MRS fluid nutrient mediums, its
In MRS fluid nutrient mediums in middle anaerobism serum bottle radix pseudostellariae must extract gradient set gradually for（Mass ratio）：0%、
0.25%, 37 DEG C of static gas wave refrigerator 48h, determine zymotic fluid viable count when terminal is cultivated, clostridium butyricum RCM-2 bud are determined in addition
Spore rate；It the results are shown in Table 11.
As shown in Table 11：Radix pseudostellariae palpus extract can remarkably promote enterococcus faecalis BD-1 and clostridium butyricum RCM-2 symbiosis
It is long, and promote degree to be more than two plants of single strain quantity sums two plants of bacterium symbiosis under comparable sodium；In addition, under 0.25% concentration
RCM-2 gemma rate can be respectively facilitated, the gemma rate of symbiosis culture is further improved.
The preparation of embodiment 4-- probioticses
Preparation process is as follows：
（1）, two kinds of fermentation strains activation：
Enterococcus faecalis BD-1 test tube slants strain is rinsed with sterile saline in an aseptic environment, lower media surface bacterium is washed
In tongue, the 500 mL anaerobism serum bottles for being then seeded into the MRS fluid nutrient mediums equipped with the palpus extract 0.25% containing radix pseudostellariae, 37
DEG C static gas wave refrigerator 48h；Wherein, the percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；
Clostridium butyricum RCM-2 test tube slants strain is rinsed with sterile saline in an aseptic environment, lower media surface is washed
In lawn, the 500 mL anaerobism serum bottles for being then seeded into the MRS fluid nutrient mediums equipped with the palpus extract 0.25% containing radix pseudostellariae,
37 DEG C of static gas wave refrigerator 48h；Wherein, the percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；
（2）, prepare enterococcus faecalis BD-1 and clostridium butyricum RCM-2 fermentation seed liquid respectively：
Enterococcus faecalis BD-1 fermentation seed liquid：Under gnotobasis, by step（1）Gained enterococcus faecalis BD-1 activating solutions press volume
Percentage 3% is inoculated into the 1L anaerobism serum bottles of the MRS fluid nutrient mediums equipped with the palpus extract 0.25% containing radix pseudostellariae, at 37 DEG C
Static fermentation 36h, is made enterococcus faecalis BD-1 fermentation seed liquids；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality
Percent by volume g/mL is counted；
Clostridium butyricum RCM-2 fermentation seed liquid：Under gnotobasis, by step（1）Gained clostridium butyricum RCM-2 activating solutions press body
Product percentage 3% is inoculated into the 1L anaerobism serum bottles of the MRS fluid nutrient mediums equipped with the palpus extract 0.25% containing radix pseudostellariae, 37 DEG C
Under static fermentation 36h, clostridium butyricum RCM-2 fermentation seed liquids are made；Wherein, the percentage of the radix pseudostellariae palpus extract is with matter
Measure percent by volume g/mL meters；
（3）, two plants of bacterium mix common fermentation：
The hot water of 55 DEG C of injection first into fermentation tank of the top with air bleeding valve, now air bleeding valve is in completely closed state, so
After add nutritional ingredient, 90rpm stirring 5min, sealed shell of tank, added when water temperature in fermentation tank is down to 37 DEG C tea polyphenol powder,
Allicin powder, stirs, and sealed shell of tank preserves 45min, then accesses enterococcus faecalis BD-1 fermentation seed liquid and clostridium butyricum
1: the 1 seed mixture liquid constituted, the lid lid sealing by volume of RCM-2 fermentation seed liquid, during 37 DEG C of static fermentation 6h, partly begins to rehearse
Air valve, then proceedes to close air bleeding valve during fermentation 38h, terminates fermentation；
The nutritional ingredient is that radix pseudostellariae must extract, brown sugar, yeast extract, vitamin C, CMC and brewer's wort；To account for hot water
Percentages, radix pseudostellariae must extract account for 0.25%, brown sugar account for 15%, yeast extract account for 8%, vitamin C account for 2%, CMC account for 1.0%,
Brewer's wort, which accounts for 3%, tea polyphenol powder and accounts for 0.15%, allicin powder and account for 0.02%, seed mixture liquid, accounts for 7%, wherein brewer's wort and hybrid
The percentage of sub- liquid is volume ratio, and remaining is mass volume ratio g/mL；
（4）, liquid mix admittedly, mixing speed of agitator be 100rpm, the time is 20min：
" liquid " refers to step（3）Gained zymotic fluid, described " Gu " refer to tea polyphenol powder, allicin powder and prebiotics, wherein
Prebiotics is oligoisomaltose, to account in terms of the mass percent of liquid-solid mixture total amount, and tea polyphenol powder accounts for 0.5%, allicin powder
Account for 0.1%, prebiotics and account for 3%；
（5）, treat step（4）After end, it is again sealed off tank body and preserves 2h, produce livestock and poultry with new liquid compound micro-ecological preparation,
Ponderable quantity and bottled packing, bottle use 250mL brown plastic bottles, are 200mL per bottled liquid measure.
10 bottles are taken respectively, and equivalent is divided into 2 groups of A and B, A group scene are uncapped and samples detection miscellaneous bacteria rate, pathogenic bacteria respectively（It is golden yellow
Color staphylococcus, micrococcus luteus and ETEC）Antibacterial circle diameter；B groups are then in 35 DEG C of air-conditioning temperature control room internal memories of temperature
Put 6 months, then detection miscellaneous bacteria rate, pathogenic bacteria respectively（Staphylococcus aureus, micrococcus luteus and ETEC）It is antibacterial
Loop diameter and swollen bottle rate.It the results are shown in Table 12.
As shown in Table 12：Liquid probiotics manufactured in the present embodiment extends its shelf life without swollen bottle phenomenon.
Using product manufactured in the present embodiment as experimental pharmacy, with staphylococcus aureus: micrococcus luteus: E
Bacterium=1: 1: 1 pathogenic bacteria complex liquid（Staphylococcus aureus, micrococcus luteus, three kinds of pathogenic bacteria of ETEC are individually
Nutrient broth activation is carried out, the bacterium solution after activation 3% is added to three of the Nutrient broth containing 100mL by volume
Angle medicine bottle carries out 100rpm medicine bottle culture, and 24h obtains bacterium solution, pathogenic bacteria complex liquid finally is made according to volume ratio 1: 1: 1, its
Middle 1L Nutrient broths composition is：Peptone 10g, beef extract powder 3g, sodium chloride 5g, final pH 7.3 ± 0.2）It is continuous to fill
Feeding healthy chicken 2d makes the artificial Infective of test chicken to build animal pattern, and the wherein sum of chicken model is 200, is divided into two
Group, one group is treated using the embodiment product, and one group without treatment（Control group）.The utilization treated using the embodiment product
Treated using the mode of feeding, three times per day, only, drinking-water, feeding and freedom of movement treat 3d to each 2mL/.Check and control after three days
More rate（Healing refers to that the clinical symptoms of chicken are wholly absent, and mental status also recovers normal completely；Cure rate is the chicken number cured
Account for the percentage of this group of chicken sum）, the death rate（The death rate is the percentage that dead chicken number accounts for this group of chicken sum）, chicken
Fresh excreta situation, as a result see the table below 13.
The preparation of embodiment 5-- probioticses
It is with the difference of embodiment 4：Step（4）In, described " Gu " refer to tea polyphenol powder, allicin powder and poultry can be improved
The Chinese herbal medicine medicinal extract of immunity of fowl, to account in terms of the mass percent of liquid-solid mixture total amount, tea polyphenol powder accounts for 0.20%, allicin
Powder accounts for 0.04%, Chinese herbal medicine medicinal extract and accounts for 15%；The Chinese herbal medicine medicinal extract is extracted by each bulk drug of following proportionings and obtained：Radix pseudostellariae palpus
20 parts of 40 parts, 20 parts of dried orange peel, 20 parts of hawthorn and Medicated Leaven；Extraction step is：
（i）Each bulk drug is weighed by formula, plus 75% alcohol reflux is extracted 2 hours, after cooling, slag-liquid separation obtains the dregs of a decoction and ethanol
Extract solution, the dregs of a decoction add water to cook extraction 2 times, merge ethanol extract and aqueous extract；
（ii）By step（i）The thick paste that it is 1.2 to 28 DEG C of relative densities that the extract solution of merging, which is concentrated under reduced pressure, produces Chinese herbal medicine leaching
Cream.
10 bottles are taken respectively, and equivalent is divided into 2 groups of A and B, A group scene are uncapped and samples detection miscellaneous bacteria rate, pathogenic bacteria respectively（It is golden yellow
Color staphylococcus, micrococcus luteus and ETEC）Antibacterial circle diameter；B groups are then in 35 DEG C of air-conditioning temperature control room internal memories of temperature
Put 6 months, then detection miscellaneous bacteria rate, pathogenic bacteria respectively（Staphylococcus aureus, micrococcus luteus and ETEC）It is antibacterial
Loop diameter and swollen bottle rate.It the results are shown in Table 14.
As shown in Table 14：Liquid probiotics manufactured in the present embodiment extends its shelf life without swollen bottle phenomenon.
Using product manufactured in the present embodiment as experimental pharmacy, with staphylococcus aureus: micrococcus luteus: E
Bacterium=1: 1: 1 pathogenic bacteria complex liquid feeds healthy chicken and builds animal pattern（Method ibid embodiment）Continuously feeding healthy chicken 2d makes
The artificial Infective of test chicken builds animal pattern, and wherein the sum of chicken model is 200, is divided into two groups, and one group using should
Embodiment product is treated, and one group using Chinese herbal medicine medicinal extract as control.The utilization treated using the embodiment product and Chinese herbal medicine medicinal extract
Treated using the mode of feeding, three times per day, both each dosages are respectively 2mL/, 0.3g/, drinking-water, feeding and activity
Freely, 7d is treated.Experiment checks cure rate after 3 days（Healing refers to that the clinical symptoms of chicken are wholly absent, and mental status is also completely extensive
It is multiple normal；Cure rate is the percentage that the chicken number cured accounts for this group of chicken sum）, the death rate（The death rate is dead chicken
Number accounts for the percentage of this group of chicken sum）, chicken fresh excreta situation.Record the 1st day and the 7th day every survival test chicken in addition
Body weight calculates chicken average weight gain amount by the chicken average weight of the 1st day and the 7th day again（G/ is only）, as a result see the table below 15.
SEQUENCE LISTING
<110>Fujian Bei Di pharmaceutcal corporation, Ltds
<120>A kind of livestock and poultry new liquid compound micro-ecological preparation and preparation method thereof
<130>
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 1461
<212> DNA
<213>It is unknown
<400> 1
cgcaggcgcg tactatacat gcagtcgtac gcttcttttt ccaccggagc ttgctccacc 60
ggaaaaagag gagtggcgaa cgggtgagta acacgtgggt aacctgccca tcagaagggg 120
ataacacttg gaaacaggtg ctaataccgt ataacaatcg aaaccgcatg gttttgattt 180
gaaaggcgct ttcgggtgtc gctgatggat ggacccgcgg tgcattagct agttggtgag 240
gtaacggctc accaaggcca cgatgcatag ccgacctgag agggtgatcg gccacattgg 300
gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc ggcaatggac 360
gaaagtctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaaactctgt 420
tgttagagaa gaacaaggat gagagtaact gttcatccct tgacggtatc taaccagaaa 480
gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540
tttattgggc gtaaagcgag cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct 600
caaccgggga gggtcattgg aaactgggag acttgagtgc agaagaggag agtggaattc 660
catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cagtggcgaa ggcggctctc 720
tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt agataccctg 780
gtagtccacg ccgtaaacga tgagtgctaa gtgttggagg gtttccgccc ttcagtgctg 840
cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 900
attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960
ccttaccagg tcttgacatc ctttgaccac tctagagata gagcttcccc ttcgggggca 1020
aagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080
gcaacgagcg caacccttat tgttagttgc catcattcag ttgggcactc tagcaagact 1140
gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1200
gggctacaca cgtgctacaa tgggaagtac aacgagttgc gaagtcgcga ggctaagcta 1260
atctcttaaa gcttctctca gttcggattg caggctgcaa ctcgcctgca tgaagccgga 1320
atcgctagta atcgcggatc agcacgccgc ggtgaatacg ttcccgggcc ttgtacacac 1380
cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tgaggtaacc tttttggagc 1440
cagccgccta agtgattgat t 1461
<210> 2
<211> 1415
<212> DNA
<213>It is unknown
<400> 2
ggcagtggcg gcatcttacc atgcagtcga gcgatgaagc tccttcggga gtggattagc 60
ggcggacggg tgagtaacac gtgggtaacc tgcctcatag aggggaatag cctttcgaaa 120
ggaagattaa taccgcataa gattgtagta ccgcatggta cagcaattaa aggagtaatc 180
cgctatgaga tggacccgcg tcgcattagc tagttggtga ggtaacggct caccaaggcg 240
acgatgcgta gccgacctga gagggtgatc ggccacattg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt ggggaatatt gcacaatggg ggaaaccctg atgcagcaac 360
gccgcgtgag tgatgacggt cttcggattg taaagctctg tctttaggga cgataatgac 420
ggtacctaag gaggaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 480
gcaagcgttg tccggattta ctgggcgtaa agggagcgta ggtggatatt taagtgggat 540
gtgaaatacc cgggcttaac ctgggtgctg cattccaaac tggatatcta gagtgcagga 600
gaggaaagga gaattcctag tgtagcggtg aaatgcgtag agattaggaa gaataccagt 660
ggcgaaggcg cctttctgga ctgtaactga cactgaggct cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgaa tactaggtgt aggggttgtc 780
atgacctctg tgccgccgct aacgcattaa gtattccgcc tggggagtac ggtcgcaaga 840
ttaaaactca aaggaattga cgggggcccg cacaagcagc ggagcatgtg gtttaattag 900
aagcaacgag aagcacctta cctagacttg acatctcctg aatttctgtg taatggagga 960
agccatttcg gtcgcaggaa cacaggtggc gcatggttgt tgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgcaa cgagcgcacc ctttattttt agttgctccc atttagttga 1080
gcactctagc gagactcccg gggttaaccg ggaggaaggt ggggatgacg tcaaatcctc 1140
atccccctta tgtatagggc cacacacgtg ctacaatggt cggtacaatg agacgcaccc 1200
tcgcgagagt gagcaaaact ataaaaccga tctcatttcg gattgtaggc tgaatctcgc 1260
ccacaaaaag ctggagtttc tagtactcgc gactcaaaat gtcgcggaga atacgttccc 1320
gcgccttgca cacacccccc ctcaccccat gagagttcgc aatacccaaa gttcgtgagc 1380
taacctcaag gaggcagcga cctaagtagt agcgt 1415
Claims (4)
1. a kind of livestock and poultry preparation method of new liquid compound micro-ecological preparation, it is characterised in that step is as follows：
（1）, two kinds of fermentation strains activation：
Enterococcus faecalis（Enterococcus faecalis）BD-1 activation：By enterococcus faecalis BD-1 test tube slants strain in nothing
Rinsed under collarium border with sterile saline, be then seeded into the MRS fluid nutrient mediums of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae
In, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/mL
Meter；Enterococcus faecalis BD-1 is new strains, and deposit number is CCTCC NO：M2017416；
Clostridium butyricum（Clostridium butyricum）RCM-2 activation：Clostridium butyricum RCM-2 test tube slants strain is existed
Rinsed under gnotobasis with sterile saline, be then seeded into the MRS Liquid Cultures of the palpus extract 0.2 ~ 0.5% containing radix pseudostellariae
In base, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/
ML is counted；Clostridium butyricum RCM-2 is new strains, and deposit number is CCTCC NO M2017396；
（2）, prepare enterococcus faecalis BD-1 and clostridium butyricum RCM-2 fermentation seed liquid respectively：
Enterococcus faecalis BD-1 fermentation seed liquid：Under gnotobasis, by step（1）Gained enterococcus faecalis BD-1 activating solution presses body
Product percentage 3 ~ 5% is inoculated into the MRS fluid nutrient mediums containing mass percent for 0.2 ~ 0.5% radix pseudostellariae palpus extract, 35 ~
42 ~ 48h of static fermentation at 37 DEG C；Wherein, the percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；
Clostridium butyricum RCM-2 fermentation seed liquid：Under gnotobasis, by step（1）Gained clostridium butyricum RCM-2 activating solution is pressed
Percent by volume 3 ~ 5% is inoculated into the MRS fluid nutrient mediums containing mass percent for 0.2 ~ 0.5% radix pseudostellariae palpus extract,
42 ~ 48h of static fermentation at 35 ~ 37 DEG C；Wherein, the percentage of the radix pseudostellariae palpus extract is with quality percent by volume g/mL
Meter；
（3）, two plants of bacterium mix common fermentation：
The hot water of 50 ~ 60 DEG C of injection first into fermentation tank of the top with air bleeding valve, now air bleeding valve is in completely closed state,
Then nutritional ingredient is added, is stirred, sealed shell of tank adds Tea Polyphenols when water temperature in fermentation tank is down to below 40 DEG C
Powder, allicin powder, stir, and sealed shell of tank preserves 30min ~ 1h, then accesses enterococcus faecalis BD-1 fermentation seed liquid and fourth
Sour clostridium RCM-2 fermentation seed liquid by volume 1: 1 ~ 5: 1 composition seed mixture liquid, lid lid sealing, 32 ~ 37 DEG C of static hairs
During 6 ~ 8h of ferment, half-open air bleeding valve then proceedes to close air bleeding valve during 36 ~ 40h of fermentation, terminates fermentation；
The nutritional ingredient is that radix pseudostellariae must extract, brown sugar, yeast extract, vitamin C, CMC and brewer's wort；To account for hot water
Percentages, radix pseudostellariae must extract account for 0.2 ~ 0.5%, brown sugar account for 10 ~ 15%, yeast extract account for 5 ~ 8%, vitamin C account for 1 ~ 2%,
CMC, which accounts for 0.5 ~ 1.5%, brewer's wort and accounts for 1 ~ 5%, tea polyphenol powder and account for 0.1 ~ 0.25%, allicin powder, accounts for 0.01 ~ 0.025%, seed mixture
Liquid accounts for 5 ~ 10%, and the wherein percentage of brewer's wort and seed mixture liquid is volume ratio, and remaining is mass volume ratio g/mL；
（4）, liquid mixes admittedly：
" liquid " refers to step（3）Gained zymotic fluid, it is described " Gu " refer to following solids 1. or solid 2.：
The composition of solid 1. is：Tea polyphenol powder, allicin powder and prebiotics, wherein prebiotics are oligoisomaltose, oligomeric fruit
The one or two of sugar, to account in terms of the mass percent of liquid-solid mixture total amount, tea polyphenol powder accounts for 0.25 ~ 1.5%, allicin powder
Account for 0.05 ~ 0.1%, prebiotics and account for 1 ~ 5%；
The composition of solid 2. is：Tea polyphenol powder, allicin powder and the Chinese herbal medicine medicinal extract that immunity of livestock can be improved；Mixed admittedly with accounting for liquid
The mass percent meter of compound total amount, tea polyphenol powder, which accounts for 0.1 ~ 0.8%, allicin powder and accounts for 0.02 ~ 0.05%, Chinese herbal medicine medicinal extract, accounts for 10
~20%；
（5）, treat step（4）After end, it is again sealed off tank body and preserves 1 ~ 3h, produce livestock and poultry with new liquid compound microecological system
Agent.
2. preparation method as claimed in claim 1, it is characterised in that：In terms of mass fraction, the Chinese herbal medicine medicinal extract is by following
Each bulk drug of proportioning is obtained through alcohol extracting and/or water extraction：Radix pseudostellariae must 20 ~ 60 parts, 10 ~ 30 parts of dried orange peel, 10 ~ 30 parts of hawthorn and six
10 ~ 30 parts of Divine Comedy.
3. preparation method as claimed in claim 2, it is characterised in that：The extraction step of the Chinese herbal medicine medicinal extract is：
（i）Each bulk drug is weighed by formula, plus 75% alcohol reflux is extracted 1 ~ 3 hour, after cooling, slag-liquid separation obtains the dregs of a decoction and second
Alcohol extract, the dregs of a decoction add water to cook extraction 1 ~ 2 time, merge ethanol extract and aqueous extract；
（ii）By step（i）The thick paste that it is 1.2 ~ 1.35 to 28 DEG C of relative densities that the extract solution of merging, which is concentrated under reduced pressure, produces medium-height grass
Medicine medicinal extract.
4. one kind utilizes livestock and poultry prepared by any preparation method of such as claim 1 ~ 3 with new liquid compound microecological system
Agent.
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