CN107338203A - A kind of feeding enterococcus faecalis, the probiotics for preventing and treating fowl and animal thermal stress and preparation method thereof - Google Patents
A kind of feeding enterococcus faecalis, the probiotics for preventing and treating fowl and animal thermal stress and preparation method thereof Download PDFInfo
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- CN107338203A CN107338203A CN201710559483.2A CN201710559483A CN107338203A CN 107338203 A CN107338203 A CN 107338203A CN 201710559483 A CN201710559483 A CN 201710559483A CN 107338203 A CN107338203 A CN 107338203A
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- enterococcus faecalis
- fermentation
- probiotics
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- radix pseudostellariae
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
-
- 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
- C12N1/205—Bacterial isolates
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to Chinese herbal microecological preparation field, discloses a kind of feeding enterococcus faecalis, the probiotics for preventing and treating fowl and animal thermal stress and preparation method thereof.Enterococcus faecalis BD 1 of the present invention, deposit number are CCTCC NO：M2017416.Formed by the ferment substrates mass percents of the probiotics product of preparation of enterococcus faecalis BD 1 in the present invention and be：Green tea 25 ~ 30%, green tea extraction residue 10 ~ 20%, radix pseudostellariae palpus 10 ~ 15%, dried orange peel 10 ~ 15%, hawthorn 10 ~ 20%, the dry jujube 10 ~ 15% of stoning, allicin 0.05 ~ 0.1%.Enterococcus faecalis BD 1 of the present invention has the ability of good resistance to Tea Polyphenols and allicin, in addition, radix pseudostellariae palpus extract can remarkably promote the strain growth.Probiotics of the present invention not only can also significantly reduce damage rate of goods beneficial to the preservation of finished product active ingredient, extend the shelf time.
Description
Technical field
The invention belongs to Chinese herbal microecological preparation field, and in particular to a kind of feeding enterococcus faecalis, preventing and treating livestock and poultry heat should
Sharp probiotics and preparation method thereof.
Background technology
Heat stress is the principal element for influenceing livestock and poultry summer growth performance, while is also luring for the outburst various infectious diseases of livestock and poultry
Cause.In today of intensive culture, with the increase of stocking density and the trend of global warming, heat stress turns into shadow
One of an important factor for ringing aquaculture development.Wherein in Swine Production, lose as caused by high temperature and more protrude, under force of labor
Drop, disease increase, mortality and the death rate rise, and production cost improves, it has also become pig industry, especially large-scale pig farm is in the summer
The huge difficult problem faced in season.Equally, poultry table feather is abundant, is metabolized vigorous, no sweat gland, more sensitive to high temperature, in heat stress
During intestines and stomach be subject to due to ischemic injuries enteron aisle, amount of blood supply is reduced during ischemic and oxygen supply reduces, enteron aisle intake and profit
Improved with the ability compensatory of oxygen, cause Intestinal epithelial cells to be damaged oedema, cell membrane and Cell tracking broken cells are bad
Extremely, the health of poultry intestinal tract is largely affected, and is easy to cause intestinal immunity to reduce, environment in intestinal microecology
By disorder, promote the generation of other relevance diseases, endanger performance of poultry.
At present, for preventing, the approach of fowl and animal thermal stress is concentrated mainly on feeding management, reducing agent adds（As vitamin,
Tea Polyphenols etc.）, enhancing nutrition, invigorating spleen and reinforcing stomach, supplement beneficial bacteria of intestinal tract.Wherein, southern livestock and poultry breeding industry is by tea dust or tea
Slag is applied in the work of prevention fowl and animal thermal stress, and is studied the Tea Polyphenols found in cheap tea dust or tea grounds and played important go back
Originality antioxidation.But do not have the report of high-efficiency fermenting type tealeaves probiotics product also on the market at present, it is main former
Because being the absence of being resistant to the tea leaf fermentation probiotics strain of Tea Polyphenols, next to that even if generality is prepared for such product but because of tea
Polyphenol dissolution rate is small, Tea Polyphenols oxidational losses is serious and can not reach preferable application effect.In addition, current fermented type Tiny ecosystem
Preparation（Water content 10 ~ 40%）It is have phenomena such as being polluted in shelf life by mould more, how to solve the problem to improving finished product shelf
Phase is significant.
The content of the invention
Lack the tea leaf fermentation probiotics strain for being resistant to Tea Polyphenols in the prior art to overcome and be difficult to prepare efficient hair
The short problem of ferment type tealeaves probiotics, probiotics finished product shelf life, it is an object of the invention to provide a kind of feeding
Enterococcus faecalis, the probiotics for preventing and treating fowl and animal thermal stress and preparation method thereof.
To achieve the above object, the technical scheme that the present invention takes is as follows：
A kind of feeding enterococcus faecalis（Enterococcus faecalis）The bacterial strain is preserved in Chinese allusion quotation by BD-1, applicant
Type culture collection, address：Wuchang, wuhan Luo Jia Shan, preservation date：On July 7th, 2017；Deposit number：CCTCC
NO：M2017416.
The method that the probiotics of preventing and treating fowl and animal thermal stress is prepared using the feeding enterococcus faecalis BD-1, step is such as
Under：
（1）, enterococcus faecalis BD-1 activation：
Enterococcus faecalis BD-1 test tube slants strain is rinsed with sterile saline in an aseptic environment, is then seeded into containing crown prince
In the MRS fluid nutrient mediums of ginseng palpus extract 0.2 ~ 0.5%, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the radix pseudostellariae must carry
The percentage of thing is taken in terms of quality percent by volume g/mL（I.e. 100mL MRS fluid nutrient mediums containing radix pseudostellariae must extract 0.2 ~
0.5g）；
（2）, prepare enterococcus faecalis BD-1 fermentation seed liquids：
Under gnotobasis, by step（1）Gained actication of culture liquid is inoculated into by percent by volume 3 ~ 5% must extract containing radix pseudostellariae
In 0.2 ~ 0.5% MRS fluid nutrient mediums, 42 ~ 48h of static fermentation at 35 ~ 37 DEG C, enterococcus faecalis BD-1 fermentation seed liquids are made；
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 contain
Radix pseudostellariae must 0.2 ~ 0.5g of extract）；
（3）, prepare fermentation substrate matrix：
First, raw material is prepared by mass percentage：Green tea 25 ~ 30%, green tea extraction residue 10 ~ 20%, radix pseudostellariae must 10 ~ 15%, it is old
Skin 10 ~ 15%, hawthorn 10 ~ 20%, the dry jujube 10 ~ 15% of stoning, allicin 0.05 ~ 0.1%；Then, first crush in addition to allicin
Raw material, then gained raw material powder will be crushed and be well mixed with allicin, fermentation substrate matrix is made；
（4）, fermentation：
By step（2）Gained fermentation seed liquid adds step（3）Gained fermentation substrate Medium Culture, is stirred and evenly mixed, and loads fermentation bag
And seal, then by fermentation bag under the conditions of lucifuge in 30 ~ 37 DEG C ferment 14 ~ 21d, be made probiotics；Wherein, with volume matter
Percentage L/kg meters are measured, the percentage that fermentation seed liquid accounts for fermentation substrate matrix is 30 ~ 40%.
Preferably, step（3）In, grinding particle size is preferably 60 ~ 80 mesh.
The probiotics of the preventing and treating fowl and animal thermal stress prepared using the preparation method.
In the present invention, green tea extraction residue（Refer to green tea in left residue after extraction）It must be extracted with radix pseudostellariae
Thing can be obtained by prior art, for example green tea extracting method can refer to but be not limited to existing water extracting method, and radix pseudostellariae must carry
Take thing can refer to but be not limited to it is entitled filed in applicant's August in 2016 30 days " a kind of radix pseudostellariae must extract preparation
The patent of method "（Application number 201610758305.8）Disclosed in method obtain.
Beneficial effect：
1st, the feeding enterococcus faecalis BD-1 of the present invention is located away from peasant household of village under Zherong County（Coordinate：119。51 ' 59.7 〞 E, 27。13′
34.0〞N ）Autotrophy healthy adult pig caecum mucous layer, gained enterococcus faecalis BD-1 is safe and non-toxic, is trained in 37 DEG C of MRS culture mediums
48h bacterium colonies surface smooth bumps are supported, periphery is neat, colony colour canescence, 0.5 ~ 1.2mm of colony diameter, microscopy G+, no bud
Spore, about 0.5 ~ 1 μm of thalline length.Enterococcus faecalis BD-1 well-grown, viable count under Anaerobic culturel and aerobic culture reach respectively
To 2.82 × 109CFU/mL、2.04×109CFU/mL, the bacterial strain have well acidproof, bile tolerance and heat-resisting ability, simultaneously
The bacterial strain has the ability of good resistance to Tea Polyphenols and allicin, and the bacterial strain can tolerate Tea Polyphenols and the concentration of allicin is respectively
1.0%th, 0.1%, the concentration hypothallus survival rate is respectively 70.26% and 70.43%, in Tea Polyphenols（2%）And allicin（0.1%）Connection
Cooperation with its lower survival rate up to 53.72%, beyond 50%；In addition, radix pseudostellariae palpus extract can remarkably promote the strain growth；Excrement
Enterococcus BD-1 can significantly inhibit the growth of pathogenic bacteria staphylococcus aureus, micrococcus luteus and ETEC；
2nd, the preparation method of probiotics of the present invention can be beneficial to the dissolution of Tea Polyphenols active ingredient, protection Tea Polyphenols and reduction tea
Polyphenol loses, and can effectively improve the polyphenol content that dissociates in fermented product, moreover it is possible to extend the shelf life time of product, and can reach
To the effect for preventing mould contamination very well；
3rd, enterococcus faecalis BD-1 of the present invention is beneficial to environment in the Tiny ecosystem of intestinal disorder and repaired and releveling, utilizes its preparation
Probiotics product there is the effect of invigorating spleen and reinforcing stomach, can be used in prevent and treat livestock and poultry summer intensive cultivation caused by heat stress it is comprehensive
Disease is closed, summer livestock and poultry cultivation production capacity is improved, reduces disease, is a kind of preferable Tiny ecosystem hair of potential anti-heat stress
Ferment preparation.
Brief description of the drawings
Fig. 1：Enterococcus faecalis BD-1 systematic evolution tree.
Embodiment
In following examples, the green tea extraction residue refers to that green tea is stayed after the extraction of existing water extracting method
Under residue；The radix pseudostellariae must extract method as disclosed in number of patent application is embodiment 1 in CN201610758305.8
It 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；
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 more containing Bromocresol purple, tea
Each meant in the MRS agar plates of phenol or allicin on the basis of above-mentioned MRS agar plates formula, still further correspond to addition bromine first
Phenol violet indicator, 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, scrape mucous membrane mucus with the slide of sterilizing and managed in EP, with 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 to choose that bacterium colony yellow color is obvious, bacterium colony surface is smooth convex
Rise, the neat conduct object bacterial strain in periphery, Strain Designation BD-1, and slant preservation is carried out 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, it is raw to have carried out motility, oxidase test etc. respectively
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 compare
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.
Compared with measured sequence is carried out into Blast similarity analysis with the 16S rDNA sequences in Genbank, as a result bacterium
Strain BD-1 withEnterococcus faecium strain KCI1、Enterococcus lactisStrain K12419C's
The homology of 16S rDNA sequences has 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.
Embodiment 2-- enterococcus faecalis BD-1 anaerobism and aerobic fermentation in MRS fluid nutrient mediums
（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 rinses, and washes lower media surface lawn, is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）Enterococcus faecalis BD-1 static fermentations of 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, zymotic fluid viable count and pH value are determined when terminal is cultivated；
（3）Enterococcus faecalis BD-1 aerobic fermentations in MRS fluid nutrient mediums：
By inoculum concentration 3%（Volume ratio）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, zymotic fluid viable count and pH are determined when terminal is cultivated
Value.
It the results are shown in Table 2.
Embodiment 3-- radix pseudostellariaes palpus extract is to enterococcus faecalis BD-1 growths, pH influence
（1）Enterococcus faecalis BD-1 activation：With the step of embodiment 2（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
In the anaerobism serum bottle of MRS fluid nutrient mediums, radix pseudostellariae palpus extract in the MRS fluid nutrient mediums wherein in anaerobism serum bottle
Gradient set gradually for（g/mL）：0%th, 0.10%, 0.25%, 0.5%, 37 DEG C of static gas wave refrigerator 48h, fermentation is determined when terminal is cultivated
Liquid pH value and viable count, the results are shown in Table 3；
（3）By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is inoculated into equipped with 100 mLMRS
In 500 mL triangular flasks of fluid nutrient medium, in the MRS fluid nutrient mediums wherein in bottle radix pseudostellariae must extract gradient successively
It is arranged to（g/mL）：0%th, 0.10%, 0.25%, 0.5%, 37 DEG C, 48h is cultivated under 150rpm, zymotic fluid is determined when terminal is cultivated
PH value and viable count, the results are shown in Table 4；
From table 3-4 results：Radix pseudostellariae palpus extract can remarkably promote enterococcus faecalis BD-1 growths of the present invention.
Embodiment 4-- Tea Polyphenols grows to enterococcus faecalis BD-1, pH influence
（1）Enterococcus faecalis BD-1 activation：With the step of embodiment 2（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 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 arranged 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 5.
As seen from the results in Table 5：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%.
Embodiment 5-- allicins grow to enterococcus faecalis BD-1, pH influence
（1）Enterococcus faecalis BD-1 activation：With the step of embodiment 2（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 arranged to（g/mL）：0%th, 0.010%, 0.025%, 0.050%, 0.10%, zymotic fluid pH value and viable bacteria are determined when terminal is cultivated
Number, the results are shown in Table 6；
As seen from the results in Table 6：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 is 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 arranged to（g/mL）：0%th, 0.05%, 0.1%, 48h is continuously cultivated, by 0%+0% as control（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 7.
As seen from the results in Table 7：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.
Embodiment 6-- enterococcus faecalis bacteriostatic experiments
（1）Enterococcus faecalis BD-1 activation：With the step of embodiment 2（1）；
（2）Enterococcus faecalis BD-1 static fermentations of 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, zymotic fluid is taken 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, 48h is cultivated under 150rpm, takes zymotic fluid to utilize Odontothrips loti when terminal is cultivated by 37 DEG C
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 8.
As seen from the results in Table 8：Enterococcus faecalis BD-1 of the present invention can significantly inhibit pathogenic bacteria staphylococcus aureus, micrococcus luteus
With the growth of ETEC, and anaerobic cultures are relatively better than aerobic culture.
Embodiment 7-- enterococcus faecalis BD-1 is acidproof, the experiment of bile tolerance, high temperature resistant
（1）Enterococcus faecalis BD-1 activation：With the step of embodiment 2（1）；
（2）Acid resistance test：By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is in anaerobic box
It is seeded in the anaerobism serum bottle of the fluid nutrient medium containing MRS, 37 DEG C of static gas wave refrigerator 32h, as acid resistance strain liquid；Then 1M is used
Hydrochloric acid solution adjusts the initial pH value of MRS fluid nutrient mediums, the initial pH value gradients of setting MRS fluid nutrient mediums is respectively 2.0,
3.0th, 4.0,5.0,6.0,7.0,115 DEG C of sterilizing 20min, cool down room temperature；Then acid resistance strain liquid is pressed 3%（Volume ratio）Connect
Kind amount is inoculated in the MRS fluid nutrient mediums of each gradient, is mixed, the static 48h at 37 DEG C, measures each pH ladders at the end for the treatment of respectively
The mixed liquor 1mL of degree, carry out 25 times of normal saline dilutions, with each gradient not to be inoculated with MRS fluid nutrient medium physiological saline dilute
25 times are released as control, is measured OD600Value.It the results are shown in Table 9；
（3）Bile tolerance is tested：By inoculum concentration 3%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is in anaerobic box
In be seeded in the anaerobism serum bottle of the fluid nutrient medium containing MRS, 37 DEG C of static gas wave refrigerator 32h, as bile tolerance strain liquid；Then to
Cholate is added in MRS fluid nutrient mediums, sets the initial cholate gradient of MRS fluid nutrient mediums（g/mL）Respectively 0.1%, 0.2%,
0.3%th, 0.4%, 0.5%, 1.0%, 2.0%, then divide and take 9mL to go in test tube, lid test tube plug, 115 DEG C of sterilizing 20min, cooling chamber
Temperature；Then each 1mL of bile tolerance strain liquid is added to the test tube of 9mL MRS fluid nutrient mediums in an aseptic environment, mixed simultaneously
Sealing, the static 2h at 37 DEG C, while acid resistance strain liquid 1mL is measured in the MRS fluid nutrient mediums added containing 9mL without cholate
In test tube, method is same as above, as control；Measure the mixed liquor 1mL of each cholate gradient and control group respectively when 2h, carry out 108
Normal saline dilution, and be coated on the MRS agar plates of pH6.2 ± 0.2,37 DEG C are inverted culture 48h, are counted, are calculated respectively
Survival rate；Survival rate=（Control group viable count-test group viable count）/ control group viable count * 100%.It the results are shown in Table 10；
（4）High temperature resistant is tested：
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 32h, as high temperature resistant strain liquid；Then dividing takes 4.5mL to go to
In 5mL EP pipes, lid lid sealing, then 37 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, static 0.5h in 70 DEG C of hot bath are respectively implanted, wherein
37 DEG C, as control, finally take the high temperature resistant strain liquid 1mL after treatment of different temperature, carry out 10 respectively8Normal saline dilution, and
It is coated on the MRS agar plates of pH6.2 ± 0.2,37 DEG C are inverted culture 48h, are counted respectively, are calculated survival rate.Survival rate=
（Control group viable count-test group viable count）/ control group viable count * 100%；It the results are shown in Table 11；
From table 9-11 results：Enterococcus faecalis BD-1 of the present invention has well acidproof, alkaline-resisting, bile tolerance and high temperature resistant energy
Power.
Embodiment 8-- safety testings
Experimental animal：KM kind small white mouses, SPF levels, body weight 18-22g, male and female half and half, are purchased from institute of Chinese materia medica of Chongqing City.
Experiment：Choose healthy mice 10, male and female half and half, average weight：It is female to be（21.90±0.5）G, hero are（22.00
±0.5）g；Mouse fasting（It can't help water）After 12h, by enterococcus faecalis BD-1 suspension（About 5.0 × 108CFU/ml）Gavage is given
Medicine, 3 times a day equivalent administration, administration accumulated dose is 1.5mL；Wherein, the process for preparation of enterococcus faecalis BD-1 suspension is：
（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 rinses, and is then seeded into MRS fluid nutrient mediums, 37 DEG C of static gas wave refrigerator 48h；
（2）, expand culture：By inoculum concentration 5%（Volume ratio）By step（1）Gained enterococcus faecalis BD-1 activating solution is in anaerobic box
In be seeded in the anaerobism serum bottle of the fluid nutrient medium containing MRS, 37 DEG C of static gas wave refrigerator 48h；
（3）Dilution：Determination step（2）Total viable count of gained nutrient solution, sterile saline is added to enter it according to total viable count
Row, which is diluted to, requires concentration.
Mouse is normally raised in cleaning grade receptacle, room temperature after administration（21±0.5）DEG C, relative humidity 45% ~ 60%, observation
The activity of animal, fur, diet, excrement whether there is abnormal change, and observe and whether there is poisoning symptom and death, Continuous Observation three weeks,
It the results are shown in Table 12.
From the result of table 12：Enterococcus faecalis BD-1 of the present invention is safe and non-toxic.
The preparation of embodiment 9-- probioticses
Preparation process is as follows：
（1）, enterococcus faecalis BD-1 activation：
Enterococcus faecalis BD-1 test tube slants strain is rinsed with sterile saline in an aseptic environment, washes lower media surface bacterium
Tongue, it is then seeded into 500 mL anaerobism serum bottles of the MRS fluid nutrient mediums equipped with the palpus extract 0.25% containing radix pseudostellariae, 37
DEG C static gas wave refrigerator 48h；The percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL（That is 100mL MRS liquid
Body culture medium must extract 0.25g containing radix pseudostellariae）；
（2）, prepare enterococcus faecalis BD-1 fermentation seed liquids：
Under gnotobasis, by step（1）Gained enterococcus faecalis BD-1 activating solution is inoculated into equipped with containing too by percent by volume 5%
In the fermentation tank of the MRS fluid nutrient mediums of son ginseng palpus extract 0.25%, static fermentation 48h at 37 DEG C, enterococcus faecalis BD-1 is made
Fermentation seed liquid；The percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL（I.e. 100mL MRS liquid is trained
Supporting base must extract 0.25g containing radix pseudostellariae）；
（3）, prepare fermentation substrate matrix：
First, raw material is prepared by mass percentage：Green tea 25%, green tea extraction residue 15%, radix pseudostellariae must 14.94%, dried orange peel 15%,
Hawthorn 15%, the dry jujube 15% of stoning, allicin 0.06%；Then, the raw material in addition to allicin is first crushed, granular size is 80 mesh,
Recycle and authorize the utility model patent equipment (patent No.：201620106247.6) mix crushing gained raw material powder with allicin
Uniformly, fermentation substrate matrix is made；
（4）, fermentation：
First by step（3）The fermentation substrate matrix of preparation is dosed in mixing plant（Equipment uses utility model patent equipment,
The patent No.：201620053478.5）, then by step（2）Gained fermentation seed liquid is added in equipment, stirring, and speed of agitator is
100 rpm, retention time 25min, wherein, in terms of volume mass percentage L/kg, fermentation seed liquid accounts for fermentation substrate matrix
Percentage be 35%；Compound is loaded into fermentation bag rapidly after stirring（Fermentation bag uses utility model patent bag, patent
Number：201620018088.4）It is interior, sealing, end to be packed, fermentation bag is directly transported to fermentation plant, fermentation plant requirement
Constant temperature（35℃）And lucifuge, after fermentation bag will arrange stacking in fermentation plant, fermentation time 21d, probiotics is made.
Compare the preparation of probiotics：Market is regard as control strain, use and sheet by the use of similar bacterial strain B, C, D for totally 3 kinds
Invention enterococcus faecalis BD-1 same ways and condition carry out activation and the preparation of fermentation seed liquid, then with identical fermentation substrate matrix
Carry out fermentation 21d.
The thalline viable count of the detection present invention and control finished product probiotics, free Tea Polyphenols amount, mould contamination respectively
3 indexs of rate；Then it is again that 1 year thalline survival rate for detecting finished product again of probiotics finished product shelf storage, free tea is more
3 phenol amount, mould contamination rate indexs.It the results are shown in Table 13.
As shown in Table 13：Enterococcus faecalis BD-1 of the present invention has viable count height compared to the similar bacterial strain in other markets, and significantly
Increase the content of the free Tea Polyphenols of finished product and extend the shelf life time of product, and the effect for preventing mould contamination very well can be reached
Fruit.
Influence of the embodiment 10-- finished products probiotics to piglet growth performance
Finished product probiotics prepared by enterococcus faecalis BD-1 of the present invention is used to be tested as research object in 2016 using embodiment 9
On July 1, was to August 1 day（Summer）, from 60 miscellaneous weanling pigs of age in days ternary of health 30 (Du × length × big) as dynamic for examination
Thing, three groups are randomly divided into, every group of repetition five：1. control group, i.e. Basic drawing group；2. Basic drawing+micro- the life of finished product of the present invention
State preparation（By quality ratio, based on addition diet 5%）Group；3. Basic drawing+antibiotic（Sulfate colistin, purity
For 10%, by quality ratio, the 0.05% of diet based on addition）Group.Feeding piglet is in open pig housing during experiment.From
By searching for food, each column circle is provided with free water device and supplies piglet free water.By the immune programme for children of pig farm routine veterinary infectious disease
The immune of piglet common transmittable disease is carried out, its daily management mode is pressed in pig farm management.It is empty by head in on-test 0d, 30d early morning
Abdomen claims piglet individual weight and feed dosage, and diarrhoea situation of the record per column piglet, calculates weanling pig average daily gain daily
(kg/d), feed dosage (kg), feedstuff-meat ratio, diarrhea disease percentage, the death rate, test period 30d.
The composition of Basic drawing is shown in Table 14.
It the results are shown in Table 15.
As shown in Table 15：Compared to control group and antibiotic group, finished product probiotics group of the present invention have significant weightening,
Increase efficiency of feed utilization, reduce bacterial diarrhea and reduce the death rate, ensure piglet healthy growth.
Influence of the embodiment 11-- finished products probiotics to the production performance of laying hen
Finished product probiotics prepared by enterococcus faecalis BD-1 of the present invention is used to be tested as research object in 2016 using embodiment 9
On July 1, was to August 1 day（Summer）, close from 300 health, 28 weeks laying rate and overall chicken group mean laying rate 85% with
The uniform healthy blue brown laying hen in 28 week old sea of upper and body weight, is randomly divided into three groups, every group of repetition 100：1. control group, i.e. base
Plinth diet group；2. basal diet+finished product of the present invention probiotics（By quality ratio, based on addition diet 3%）Group；
3. basal diet+antibiotic（Sulfate colistin, purity 10%, by quality ratio, the 0.05% of diet based on addition）
Group.Layer breeding is in Laying House during experiment.Free choice feeding, 30 DEG C of the temperature on average ＞ of hen house, humidity 60 ~ 90%, each
Hen house is provided with free drip water fountain and supplies laying hen free water, and chicken farm management presses its daily management mode.It is every in on-test
Its early morning by egg production, material quantity is only calculated, records the diarrhoea situation of laying hen daily, calculates feedstuff-egg ratio, the diarrhoea morbidity of laying hen
Rate, the death rate, and randomly select 5 laying hens every group of last day and carry out blood drawing detection SOD enzymes（Animal body under normal circumstances
The interior active material that can be constantly be generated free radical and endogenous Green Tea Extract, generation and the body anti-oxidative defense system of free radical
It is between system in a kind of good dynamic equilibrium, leading indicator of the SOD enzymes as oxidation resistance）Content（U/mg）, experiment week
Phase 30d.
The composition of basal diet is shown in Table 16.
It the results are shown in Table 17.
As shown in Table 17：Compared to control group and antibiotic group, finished product probiotics group of the present invention has significant increase
The anti-oxidant anti-heat stress ability of laying hen, increase efficiency of feed utilization, reduce bacterial diarrhea and reduce the death rate, ensure laying hen health
Production performance.
SEQUENCE LISTING
<110>Fujian Bei Di pharmaceutcal corporation, Ltds
<120>A kind of feeding enterococcus faecalis, the probiotics for preventing and treating fowl and animal thermal stress and preparation method thereof
<130>
<160> 1
<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
Claims (4)
- A kind of 1. feeding enterococcus faecalis（Enterococcus faecalis）BD-1, it is characterised in that：Deposit number is CCTCC NO：M2017416.
- 2. the side of the probiotics of preventing and treating fowl and animal thermal stress is prepared using feeding enterococcus faecalis BD-1 as claimed in claim 1 Method, it is characterised in that step is as follows：（1）, enterococcus faecalis BD-1 activation：Enterococcus faecalis BD-1 test tube slants strain is rinsed with sterile saline in an aseptic environment, is then seeded into containing crown prince In the MRS fluid nutrient mediums of ginseng palpus extract 0.2 ~ 0.5%, 35 ~ 37 DEG C of 42 ~ 48h of static gas wave refrigerator；Wherein, the radix pseudostellariae must carry The percentage of thing is taken in terms of quality percent by volume g/mL；（2）, prepare enterococcus faecalis BD-1 fermentation seed liquids：Under gnotobasis, by step（1）Gained actication of culture liquid is inoculated into by percent by volume 3 ~ 5% must extract containing radix pseudostellariae In 0.2 ~ 0.5% MRS fluid nutrient mediums, 42 ~ 48h of static fermentation at 35 ~ 37 DEG C, enterococcus faecalis BD-1 fermentation seed liquids are made； Wherein, the percentage of the radix pseudostellariae palpus extract is in terms of quality percent by volume g/mL；（3）, prepare fermentation substrate matrix：First, raw material is prepared by mass percentage：Green tea 25 ~ 30%, green tea extraction residue 10 ~ 20%, radix pseudostellariae must 10 ~ 15%, it is old Skin 10 ~ 15%, hawthorn 10 ~ 20%, the dry jujube 10 ~ 15% of stoning, allicin 0.05 ~ 0.1%；Then, first crush in addition to allicin Raw material, then gained raw material powder will be crushed and be well mixed with allicin, fermentation substrate matrix is made；（4）, fermentation：By step（2）Gained fermentation seed liquid adds step（3）Gained fermentation substrate Medium Culture, is stirred and evenly mixed, and loads fermentation bag And seal, then by fermentation bag under the conditions of lucifuge in 30 ~ 37 DEG C ferment 14 ~ 21d, be made probiotics；Wherein, with volume matter Percentage L/kg meters are measured, the percentage that fermentation seed liquid accounts for fermentation substrate matrix is 30 ~ 40%.
- 3. preparation method as claimed in claim 2, it is characterised in that：Step（3）In, grinding particle size is 60 ~ 80 mesh.
- A kind of 4. probiotics of the preventing and treating fowl and animal thermal stress prepared using the preparation method as described in Claims 2 or 3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108935962A (en) * | 2018-09-30 | 2018-12-07 | 福建贝迪药业有限公司 | A kind of pulvis and preparation method thereof taking off mould protect liver for livestock and poultry |
| CN111944771A (en) * | 2019-10-29 | 2020-11-17 | 华南农业大学 | Application of tea polyphenol or components thereof in preparation of enterococcus faecalis phage passivator |
| CN115011739A (en) * | 2022-08-03 | 2022-09-06 | 南京邦康生物技术有限公司 | Probiotics production control method and system |
| CN115804796A (en) * | 2022-09-13 | 2023-03-17 | 西北农林科技大学 | Application of enterococcus faecium phosphate buffer solution diluent in improving heat stress testis spermatogenic ability |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031235B (en) * | 2010-11-09 | 2012-07-25 | 中国农业大学 | Enterococcus faecium ANSE228 and application thereof |
| CN103333830B (en) * | 2013-06-28 | 2016-08-10 | 中山市微科生物技术有限公司 | Pig source enterococcus faecalis, microbial ecological agent and application thereof |
| CN106344761B (en) * | 2016-08-30 | 2019-07-05 | 福建贝迪药业有限公司 | A kind of fermented type Chinese herbal and crude drugs preparations and preparation method thereof improving immunity of livestock |
-
2017
- 2017-07-11 CN CN201710559483.2A patent/CN107338203B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108935962A (en) * | 2018-09-30 | 2018-12-07 | 福建贝迪药业有限公司 | A kind of pulvis and preparation method thereof taking off mould protect liver for livestock and poultry |
| CN111944771A (en) * | 2019-10-29 | 2020-11-17 | 华南农业大学 | Application of tea polyphenol or components thereof in preparation of enterococcus faecalis phage passivator |
| CN111944771B (en) * | 2019-10-29 | 2022-06-14 | 华南农业大学 | Application of tea polyphenol or components thereof in preparation of enterococcus faecalis phage deactivator |
| CN115011739A (en) * | 2022-08-03 | 2022-09-06 | 南京邦康生物技术有限公司 | Probiotics production control method and system |
| CN115011739B (en) * | 2022-08-03 | 2022-11-01 | 南京邦康生物技术有限公司 | Probiotics production control method and system |
| CN115804796A (en) * | 2022-09-13 | 2023-03-17 | 西北农林科技大学 | Application of enterococcus faecium phosphate buffer solution diluent in improving heat stress testis spermatogenic ability |
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