CN105368755A - Acid-yielding Enterococcus faecium, bacteriostatic microecological preparation and application thereof - Google Patents
Acid-yielding Enterococcus faecium, bacteriostatic microecological preparation and application thereof Download PDFInfo
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- CN105368755A CN105368755A CN201510917814.6A CN201510917814A CN105368755A CN 105368755 A CN105368755 A CN 105368755A CN 201510917814 A CN201510917814 A CN 201510917814A CN 105368755 A CN105368755 A CN 105368755A
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Abstract
The invention relates to the field of microorganisms, in particular to an acid-yielding Enterococcus faecium, a bacteriostatic microecological preparation and application thereof. A lactic acid bacterium used is high-acid-yield Enterococcus faecium screened from an animal intestine, and the high-acid-yield Enterococcus faecium is preserved under CGMCC No.9666. The invention provides a method of using bacteriocin formed by combining the Enterococcus faecium with Bacillus subtilis to produce the efficiently-bacteriostatic microecological preparation. the Enterococcus faecium quickly proliferates in the animal intestine and generates organic acid to lower pH value of the animal intestine. Bacteriostatic effect of the bacteriocin used in a low-pH-value environment is improved remarkably, and the bacteriocin plays a role in inhibiting and killing harmful bacteria in the intestine, so that planting of probiotics such as the Enterococcus faecium in the animal intestine is promoted. The efficiently-bacteriostatic microecological preparation organically combines the bacteriocin generated by the Bacillus subtilis with the Enterococcus faecium to jointly play a bacteriostatic role, improves organism immunity, promotes productivity of bred animals and has a wide application prospect.
Description
Technical field
The present invention relates to microorganism field, particularly, the present invention relates to the sour faecium of a kind of product, antibacterial probiotics and application thereof.
Background technology
Along with China's agricultural breeding structural adjustment; mass-producing, grouping of the world economy cultivation become development trend; raiser, in order to pursue maximization of economic benefit, adds feeding antibiotic, hormone and probiotics preparation etc. in feed, to reach the object promoting animal weightening finish and growth fast.But the medicines such as interpolation growth promotion feeding antibiotic can make, and the resistance of pathogenic bacterium is more and more general, Antibiotic Resistance is more and more wider, animal epidemic is caused more and more to be difficult to control thus, form vicious cycle, not only affect the Sustainable development of livestock breeding industry, also can cause the severe overweight of residual antibiotic and hormone in fowl poultry kind product, directly have influence on the healthy of the mankind.
Bacteriocin be by some bacterium by genes encoding, Ribosome biogenesis there is antibacterial bioactive albumen or peptide class.Bacteriocin is obviously different in biosynthesizing, mechanism of action, antimicrobial spectrum etc. from microbiotic, and bacteriocin can use the object reaching the growth of control objectives pathogenic micro-organism safely, effectively.Milk-acid bacteria is the mankind and animal intestinal flora, can secrete lactic acid in animal intestinal, reduces the pH value of animal intestinal, is usually used in humans and animals probiotic products.
Subtilis is not the permanent bacterium of enteron aisle, only belongs to the bacterium that passes by one's way, can not long-term field planting and growth in enteron aisle, and its can enter gi tract along with food, finally excretes together with ight soil again.If directly add by subtilis, because its residence time in animal intestinal is short, its meta-bolites--bacteriocin also do not synthesize or resultant quantity less, cannot bacteriostatic action be given full play to.
Subtilis is carried out large scale fermentation and produces bacteriocin by the present invention in vitro, and itself and milk-acid bacteria are carried out synergistic combination, prepares the probiotics of Efficient antibacterial.This probiotics enters after animal gastrointestinal tract can fast onset bacteriostatic action, effectively regulates GI microecological balance, safeguards animal health.
Summary of the invention
The object of the present invention is to provide the sour faecium of a kind of product and antibacterial probiotics thereof, it has the ability of Efficient antibacterial.
Another object of the present invention provides the application of above-mentioned product acid faecium and antibacterial probiotics thereof.
The sour faecium CE001 (Enterococcusfaecium) of product provided by the invention, its culture presevation is numbered: CGMCCNO.9666.Faecium of the present invention, has good product acid activity.
Antibacterial probiotics of the present invention, comprise subtilis institute bacteriocinogeny and the present invention above-mentioned product acid faecium; Described bacteriocin and the ratio of faecium be 1g bacteriocin than 2 ~ 500,000,000 faecium viable counts.
Subtilis used in the present invention can be any bacterial strain of existing bacteriocin, especially preferred, and subtilis of the present invention is selected purchased from Chinese agriculture Microbiological Culture Collection administrative center, and its preserving number is the bacterial strain of ACCC10619.
The preparation method of above-mentioned antibacterial probiotics of the present invention, comprises the following steps:
1) subtilis of preparation institute bacteriocinogeny is joined in proportion in the faecium fermented liquid in fermentation later stage, be uniformly mixed;
2) mixed fermented liquid solid-liquid separation is obtained bacterium mud; drying protectant is added in bacterium mud; the mass volume ratio (g/L) of described bacterium mud and drying protectant is 5% ~ 25%, adopts granulation, freeze-drying or spraying dry to make antibacterial probiotics.
Further, preparation in accordance with the present invention, the preparation of described bacteriocin comprises the following steps:
1) bacillus subtilis strain is inoculated in fermentation nutrient solution, under 37 ~ 39 DEG C of conditions, cultivates 48 ~ 50h, obtain fermented liquid;
2) supernatant liquor is got by centrifugal for fermentation liquor 5000 ~ 12000r/m 5-30min, in supernatant liquor, slowly add ammonium sulfate solids powder saltout, limit edged stirs, until saturation ratio reaches 20 ~ 80%, then under 4 ~ 5 DEG C of conditions, 4 ~ 2h is left standstill, again under 2 ~ 10 DEG C of conditions, with the centrifugal 10 ~ 30min of 5000 ~ 12000r/m rotating speed, abandoning supernatant obtains bacteriocin.
Bacillus subtilis strain fermentation nutrient solution of the present invention can use well known in the art arbitrarily for the nutrient solution of subtilis, preferably, nutrient solution used comprises (weight ratio): Semen Maydis powder 0.5% ~ 3.5%, soybean cake powder 1.0% ~ 5.0%, fish meal 0.01% ~ 0.5%, glucose 0.2% ~ 2.0%, calcium carbonate 0.5% ~ 5.0%, ammonium sulfate 0.001% ~ 0.2%, dipotassium hydrogen phosphate 0.001% ~ 0.2%, magnesium sulfate 0.001% ~ 0.2%, manganous sulfate 0.001% ~ 0.2%, pure water 1 liter, pH is 7.0 ~ 7.5.
The unit of activity of described bacteriocin can reach 17361.68U/mg, can suppress multiple Gram-negative and Gram-positive pathogens bacterium.
Preparation in accordance with the present invention, the preparation method of described faecium fermented liquid comprises the following steps:
1) in seed culture medium, cultivate faecium 18 ~ 24 hours under 35 ~ 45 DEG C of aerobics or facultative conditions, become first order seed;
2) primary seed solution is seeded in seeding tank carries out enlarged culturing, cultivate 18 ~ 24 hours under 35 ~ 45 DEG C of aerobics or facultative conditions, become secondary seed;
3) secondary seed solution of acquisition is seeded in fermentor tank carries out fermentation culture, under 30 ~ 45 DEG C of aerobics or facultative conditions, adopt timing or continuous feeding batch fermentation manner to cultivate 18 ~ 36 hours;
As preferably, step 1) and 2) use MRS substratum to be seed culture medium.
As preferably, step 3) fermentation adopts the MRS substratum of improvement, and the MRS substratum of described improvement is added with soy peptone 5 ~ 30g in often liter of MRS substratum, glucose 1 ~ 10g, yeast powder 1 ~ 10g, sodium acetate 1 ~ 10g, citric acid diamines 0.1 ~ 8g, Tween800.1 ~ 5g, dipotassium hydrogen phosphate 0.1 ~ 5g, magnesium sulfate 0.05 ~ lg, manganous sulfate 0.001 ~ 0.2g, calcium carbonate 1 ~ 30g, pure water 1 liter, adjust ph to 5.5 ~ 7.5.
Present invention also offers the application of the sour faecium of described product and antibacterial probiotics, the application especially in antibacterial and fodder additives.
Applicant studies bacteriocin its bacteriostasis meeting significantly lifting in acid condition finding that producing bacillus subtilis is raw.Bacteriocin coordinates with the faecium of high-yield lactic acid is organic by the present invention, makes it play synergy.Faecium generation lactic acid causes the sour environment in animal intestinal, bacteriocin bacteriostasis in this sour environment significantly promotes, kill pathogenic bacteria, the field planting of faecium in animal intestinal can be promoted again conversely, supplement normal intestinal flora, improve immunological competence, reduce antibiotic use in livestock and poultry cultivation, promote livestock industry sustainable health development.
Accompanying drawing explanation
Fig. 1 is the bacterium colony that faecium of the present invention (Enterococcusfaecium) bacterial strain is formed on MRS nutrient agar.
Fig. 2 is the dull and stereotyped schematic diagram of Oxford cup that bacteriocin bacteriostatic activity of the present invention detects, and wherein SH represents high dosage sample solution, and SL represents low-dosage sample solution, and BH represents high dosage standardized solution, and BL represents Low-dose Standard solution.
Fig. 3 is that the bacteriostatic activity of bacteriocin of the present invention under different pH condition compares (embodiment 3 table 6 group 1).
Fig. 4 is that probiotics of the present invention compares with the bacteriostatic activity of compound micro-ecological preparation under same concentrations of faecium with single bacteriocin, single subtilis and subtilis.
Faecium CE001 (Enterococcusfaecium) of the present invention is preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 16th, 2014, Institute of Microorganism, Academia Sinica, 100101), deposit number is CGMCCNO.9666.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Substratum used in following embodiment is filled a prescription as follows if no special instructions:
MRS substratum:
Peptone 10g, beef powder 5g, glucose 20g, tween 80 ml, dipotassium hydrogen phosphate 2g, sodium acetate 5g, citric acid tri-amonia 2g, magnesium sulfate heptahydrate 0.2g, manganese sulfate monohydrate 0.05g, yeast powder 4g, is settled to 1L with distilled water.
MRS nutrient agar:
Agar 15g is added in 1LMRS substratum.
The Isolation and Identification of embodiment 1 faecium (Enterococcusfaecium)
(1) isolation and purification of faecium (Enterococcusfaecium)
The separation and Culture of 1.1 bacterial strains
Healthy chicken is lethal, dissect after, get small intestinal mucosa and each 1g of intestinal contents with aseptic procedure respectively, add in the bottle that 9mL sterile saline is housed, carry out doubling dilution after mixing, get 10
-4, 10
-5, 10
-6dilution suspension 100mL, is added drop-wise on 3 MRS solid mediums respectively, smears evenly with glass stick, then in 37 DEG C of anaerobism and aerobic cultivation 48h, observes colonial morphology and whether has molten calcium ring.The milk-acid bacteria colony inoculation selecting molten calcium ring, in MRS liquid nutrient medium, carries out line separation and purification.Observe meat soup and whether become muddy, have muddy placement 4 DEG C of refrigerator storages for subsequent use.
1.2 gramstaining
Draw a small amount of MRS broth culture with asepsis injector, drop on slide glass, spirit lamp flame is dried gently fixing.Drip violet staining liquid, dye 30s, washing; Drip Gram's iodine solution mordant dyeing, effect 1min, washing; Decolour 30s to drip acetone ethanol mixed solution (acetone: 95% ethanol=3:7), washing; Drip husky yellow staining fluid and redye 1min, washing, wait to do, observe under ordinary optical microscope, thalline takes on a red color as feminine gender, purple be the positive.In the coccus that Gram-positive form is consistent, carry out catalase experiment further.
1.3 catalase experiments
Do MRS slant medium, get culture 0.2mL, inject and MRS nutrient agar inclined-plane is housed, 5%CO
2incubator, cultivates 24h, after growing bacterium colony, is added drop-wise on bacterium colony by 3% superoxol for 37 DEG C, if it is negative for not having bubble to produce explanation, if it is positive for having bubble to produce explanation.Genus lactubacillus (Lactobacillus) tentatively can be thought through Gram-positive and catalase experiment feminine gender by the culture of MRS substratum Anaerobic culturel.
Animal intestinal sample, through separation and purification, isolates 5 strain Lactococcus called after YB-1, YB-2, YB-3, YB-4, YB-5 respectively altogether in conjunction with gramstaining and catalase experiment.
(2) acid-producing bacteria strain screening study
5 strain test strains of above-mentioned screening are inoculated in 5mLMRS liquid nutrient medium by the inoculum size of 2% (v/v), after 37 DEG C of anaerobically fermenting 24h, detect pH value and the lactic acid content of the centrifugal supernatant of fermented liquid.The results are shown in Table 1.The product acid amount of YB-5 lactobacillus strain, higher than all the other bacterial strains, shows that this strains of lactic acid bacteria has good product acid activity.
The pH of table 1 streptococcus acidi lactici fermented solution and Lactic Acid from Fermentation Broth content (mg/L) thereof
| Strain number | PH value | Lactic acid content |
| YB-1 | 4.4 | 3637.05 |
| YB-2 | 4.8 | 3152.51 |
| YB-3 | 4.2 | 4082.13 |
| YB-4 | 4.2 | 4141.62 |
| YB-5 | 3.6 | 5092.51 |
(3) extracting genome DNA and 16SrRNA order-checking
16SrRNA gene sequencing: the extraction of above-mentioned YB-5 bacteria total DNA adopts bacterial genomes DNA extraction kit (sky root biochemistry (Beijing) Science and Technology Ltd., TiangenDP302-02) to extract.16SrRNA gene amplification primer adopts bacterial universal primers, and its primer sequence is: forward primer is 27F (corresponding to Escherichiacoil8-27 bit base): 5'-AGAGTTTGATCCTGGCTCAG-3'; Reverse primer is 1495R (corresponding to Escherichiacoil1495-1515 bit base): 5'-CTACGGCTACCTTGTTACGA-3'.Reaction system (50 μ L) is as shown in table 2:
Table 2PCR amplification system
Pcr amplification program is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min; 58 DEG C of annealing 1min; 72 DEG C extend 2min, carry out 30 circulations, and last 72 DEG C extend 10min.PCR primer utilizes 1% agarose gel electrophoresis to detect, biotechnology (Beijing) company limited of positive products purified Hou Songrui Boxing section that fragment length is about 1500bp carries out sequencing, and the nucleotide sequence of the encoding gene of the 16SrRNA of YB-5 bacterium is as shown in SEQIDNo.1.
The gene order obtained carries out BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) sequence analysis in GenBank database.Qualification result finds that the homology of YB-5 and faecium (Enterococcusfaecium) is 99%, is defined as Enterococcusfaecium.Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 16th, 2014 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.9666, and its Classification And Nomenclature is faecium CE001 (Enterococcusfaecium).
The cell of faecium CE001 (Enterococcusfaecium) (CGMCCNo.9666) is spherical, and Gram-positive, other biological characteristics is as shown in table 3.This faecium (Enterococcusfaecium) is the one of enterococcus spp (Enterococcussp.).
The biological characteristics of table 3 faecium
The preparation of embodiment 2 producing bacillus subtilis bacteriocin
(1) with the inoculum size of 0.5%, Bacillus subtilis strain is inoculated in fermentor tank, adopts ordinary method under the condition of 37 DEG C, 150 revs/min, to cultivate 50h on substratum, obtain fermented liquid.Described substratum comprises (weight ratio): Semen Maydis powder 0.5%, soybean cake powder 3%, fish meal 0.1%, glucose 1.0%, calcium carbonate 1.59%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.01%, manganous sulfate 0.01%, pure water 1 liter, pH is 7.0 ~ 7.5;
(2) supernatant liquor is got after the centrifugal 20min of fermentation liquor 10000r/m, in supernatant liquor, slowly add ammonium sulfate solids powder saltout, limit edged stirs, until saturation ratio reaches 80%, then under 4 DEG C of conditions, 12h is left standstill, again under 5 DEG C of conditions, 12000r/m, centrifugal 30min, abandoning supernatant obtains bacteriocin.
The Performance Detection that embodiment 3 bacteriocin is antibacterial
(1) antimicrobial spectrum of bacteriocin
The bacteriocin precipitation of getting 1g obtained adds 5 respectively, 10, in 20mL nutrient broth, make 3 nutrient broths (20% containing different bacterium element concentration, 10%, 5%), with the nutrient broth not containing bacteriocin in contrast, inoculate salmonella typhi (Salmonellatyphi respectively, CVCC2212, National Veterinary Culture Collection), streptococcus aureus (Staphylococcusaureus, CVCC1882, National Veterinary Culture Collection), E.coli K88 (EscherichiaColi, CMCC44742, Chinese medicine bacterium preservation administrative center) and colon bacillus 0157 (EscherichiaColi, purchased from China Veterinary Drugs Supervisory Inst.), inoculum size is 5% of nutrient solution, put 37 DEG C, 5%CO
224h cultivated by incubator.
Detect the colony number often organizing culture, the results are shown in Table 4.
Table 4 micro-organisms test-results
As can be seen from the table, the bacteriocin that producing bacillus subtilis is raw under different concns all has good inhibition to salmonella typhi, streptococcus aureus and two strain intestinal bacteria, can make its viable count decline 4-5 order of magnitude.
(2) bacteriostatic activity of bacteriocin detects
Get the sodium acetate buffer (pH6.5) that 1g bacteriocin precipitation is dissolved in 20mL0.02mol/L.Odontothrips loti is adopted to detect its bacteriostatic activity.Bacteriostatic activity analytical procedure:
(1) colistine sulfate standard substance 0.0360g is accurately taken, be settled to 10mL with phosphoric acid buffer, 2000rpm, after centrifugal 10min, redilution 10 is doubly as high dosage concentration, and by high dosage concentration redilution 1 times, make it to be made into high low dosage two concentration gradient solution;
(2) 10mL element agar is laid in culture dish (diameter 90mm, dark 20mm), is placed in standing on level table solidifying;
(3) get and be diluted to about 10
7the indicator of the fresh culture of cfu/mL--intestinal bacteria (Escherichiacoli) 100 μ L and thawing also mix to the LB solid medium (about 10mL) be incubated to 55 DEG C, are poured in flat board.Put down gently to cooling on levels operation platform, ensure the planeness of agar gelling wild Oryza species plane;
(4) Oxford cup aseptic nipper is positioned on flat board gently, after 200 μ L bacteriocin solution are added Oxford cup, 12h is left standstill under 4 ~ 5 DEG C of conditions, after 37 DEG C of cultivation 24h, flat board is taken out subsequently, pour out Oxford cup, flat board is placed on the platform of a black background, by the size of each inhibition zone of vernier caliper measurement, get its mean value, calculate by formula (1) and tire.
In formula:
C
sH---tiring of sample solution, unit is U/mg;
C
bH---tiring of standardized solution, unit is U/mg;
X
sH---the antibacterial circle diameter caused by high dosage sample solution, unit is millimeter (mm);
X
sL---the antibacterial circle diameter caused by low-dosage sample solution, unit is millimeter (mm);
X
bH---the antibacterial circle diameter caused by high dosage standardized solution, unit is millimeter (mm);
X
bL---the antibacterial circle diameter caused by Low-dose Standard solution, unit is millimeter (mm);
K---the ratio of high dosage multiple and low dosage multiple.
C in the present embodiment
bHfor 22782U/mg, experimental result is as table 5, and the bacteriocin that producing bacillus subtilis is raw is after testing tired as 17361.68U/mg.
Table 5 inhibition zone measurement result (mm)
(3) bacteriostatic activity of bacteriocin is with pH value changing conditions
Get the sodium acetate buffer that 1g bacteriocin precipitation is dissolved in 20mL0.02mol/L, pH value of solution is adjusted to 4.0,5.0,6.0,7.0 respectively.Getting 1g bacteriocin precipitation is again dissolved in 20mL faecium fermented liquid (pH4.0), adopts Odontothrips loti to detect its bacteriostasis property.Bacteriostasis property analytical procedure:
(1) 10mL element agar is laid in culture dish (diameter 90mm, dark 20mm), is placed in standing on level table solidifying;
(2) get and be diluted to about 10
7the indicator of the fresh culture of cfu/mL--intestinal bacteria (Escherichiacoli) 100 μ L and thawing also mix to the LB solid medium (about 10mL) be incubated to 55 DEG C, are poured in flat board.Put down gently to cooling on levels operation platform, ensure the planeness of agar gelling wild Oryza species plane;
(3) Oxford cup aseptic nipper is positioned on flat board gently, after each solution of 200 μ L is added Oxford cup, 12h is left standstill under 4-5 DEG C of condition, after 37 DEG C of cultivation 24h, flat board is taken out subsequently, pour out Oxford cup, flat board is placed on the platform of a black background, by the size of each inhibition zone of vernier caliper measurement.Setup Experiments three groups is parallel.Result is as shown in table 6.
Table 6 inhibition zone measurement result (mm)
The result of table 6 shows that the biocidal property of bacteriocin can promote along with the reduction of pH value, and in the faecium fermented liquid of pH4.0, the biocidal property of bacteriocin significantly improves.
The preparation of the probiotics of embodiment 4 Efficient antibacterial
Probiotics 1:
(1) be seed culture medium with MRS substratum, under 37 DEG C of aerobics or facultative conditions, cultivate faecium 18 hours, become first order seed;
(2) by primary seed solution by 0.5% inoculum size (volume ratio) be seeded in seeding tank and carry out enlarged culturing, adopt MRS substratum to be seed culture medium, cultivate 18 hours under 37 DEG C of aerobics or facultative conditions, become secondary seed;
(3) secondary seed solution that step (2) obtains is seeded in fermentor tank carries out fermentation culture, the MRS substratum adopting improvement is fermention medium, under 37 DEG C of aerobics or facultative conditions, timing or continuous feeding batch fermentation manner is adopted to cultivate 36 hours; The MRS substratum of described improvement is added with soy peptone 10g in often liter of MRS substratum, glucose 10g, yeast powder 5g, sodium acetate 1g, citric acid diamines 0.8g, Tween801g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.08g, manganous sulfate 0.1g, calcium carbonate 20g, pure water 1 liter, adjust ph 7.0.
(4) bacteriocin prepared is joined faecium fermented liquid according to 1g bacteriocin than the ratio of 500,000,000 faecium viable counts, be uniformly mixed.Under 1000rpm condition, zymocyte liquid is carried out collected by centrifugation thalline.
(5) after centrifugal complete bacterium mud is weighed; bacterium mud is mixed according to mass volume ratio (g/L) 1:5 with protective material, in-70 DEG C of pre-freeze 2 ~ 3h, in advance open cold lyophilizer; temperature to be frozen is down to-40 DEG C, rapidly the thalline that pre-freeze is good is put into freezing 8h.Wherein said protective material formula is: skim-milk 14%, sucrose 10%, oligofructose 16% and Sodium Glutamate 10%, sterilized water 1L.
Probiotics 2:
Except the bacteriocin prepared joins except faecium fermented liquid according to 1g bacteriocin than the ratio of 200,000,000 faecium viable counts by step (4), other preparation method is with sample 1.
The fungistatic effect research of embodiment 5 probiotics
Odontothrips loti is adopted to be contrasted by the bacteriostasis property of prepare 1% (g/mL) concentration probiotics and the single bacteriocin of 1% (g/mL) concentration, 1% (g/mL) single subtilis solution and the subtilis of collecting from market and faecium compound micro-ecological preparation 10,000,000,000 (ratio of subtilis and faecium is 8:2) product 1% (g/mL) solution.Bacteriostasis property analytical procedure:
(1) 10mL element agar is laid in culture dish (diameter 90mm, dark 20mm), is placed in standing on level table solidifying;
(2) get and be diluted to about 10
7the indicator of the fresh culture of cfu/mL--intestinal bacteria (Escherichiacoli) 100 μ L and thawing also mix to the LB solid medium (about 10mL) be incubated to 55 DEG C, are poured in flat board.Put down gently to cooling on levels operation platform, ensure the planeness of agar gelling wild Oryza species plane;
(3) Oxford cup aseptic nipper is positioned on flat board gently, after each solution of 200 μ L is added Oxford cup, under 4-5 DEG C of condition, leave standstill 12h, after 37 DEG C of cultivation 24h, flat board is taken out subsequently, pour out Oxford cup, flat board is placed on the platform of a black background.Result as shown in Figure 4.
Under result shows same concentrations, the biocidal property of probiotics of the present invention is significantly higher than single bacteriocin.Bacillus subtilis formulation and subtilis and faecium compound formulation produce without obvious inhibition zone.
Certainly; the present invention can also have various embodiments; when not deviating from the present invention's spirit and essence thereof; those of ordinary skill in the art can openly make various corresponding change and modification according to of the present invention, but these change accordingly and are out of shape the protection domain that all should belong to the claim appended by the present invention.
Claims (9)
1. one kind is produced sour faecium CE001 (Enterococcusfaecium), and it is characterized in that, the culture presevation of described faecium is numbered: CGMCCNO.9666.
2. an antibacterial probiotics, is characterized in that, described antibacterial probiotics comprises subtilis institute bacteriocinogeny and product according to claim 1 acid faecium; Described bacteriocin and the ratio of faecium be 1g bacteriocin than 2 ~ 500,000,000 faecium viable counts.
3. a preparation method for antibacterial probiotics described in claim 2, comprises the following steps:
1) subtilis of preparation institute bacteriocinogeny is joined in proportion in the faecium fermented liquid in fermentation later stage, be uniformly mixed;
2) mixed fermented liquid solid-liquid separation is obtained bacterium mud; drying protectant is added in bacterium mud; the mass volume ratio (g/L) of described bacterium mud and drying protectant is 5% ~ 25%, adopts granulation, freeze-drying or spraying dry to make antibacterial probiotics.
4. preparation method according to claim 3, is characterized in that, the preparation of described bacteriocin comprises the following steps:
1) bacillus subtilis strain is inoculated in fermentation nutrient solution, under 30 ~ 39 DEG C of conditions, cultivates 40 ~ 50h, obtain fermented liquid;
2) supernatant liquor is got by centrifugal for fermentation liquor 5000 ~ 12000r/m 5-30min, in supernatant liquor, slowly add ammonium sulfate solids powder saltout, limit edged stirs, until saturation ratio reaches 20% ~ 80%, then under 4 ~ 5 DEG C of conditions, 4 ~ 12h is left standstill, again under 4 ~ 5 DEG C of conditions, with the centrifugal 10 ~ 30min of 5000 ~ 12000r/m rotating speed, abandoning supernatant obtains bacteriocin.
5. the preparation method according to claim 3 or 4, is characterized in that, the preparation method of described faecium fermented liquid comprises the following steps:
1) in seed culture medium, cultivate faecium 18 ~ 24 hours under 35 ~ 45 DEG C of aerobics or facultative conditions, become first order seed;
2) primary seed solution is seeded in seeding tank carries out enlarged culturing, cultivate 18 ~ 24 hours under 35 ~ 45 DEG C of aerobics or facultative conditions, become secondary seed;
3) secondary seed solution of acquisition is seeded in fermentor tank carries out fermentation culture, under 30 ~ 45 DEG C of aerobics or facultative conditions, adopt timing or continuous feeding batch fermentation manner to cultivate 18 ~ 36 hours.
6. preparation method according to claim 5, is characterized in that, step 1) and 2) use MRS substratum to be seed culture medium.
7. preparation method according to claim 5, it is characterized in that, step 3) fermenting adopts the MRS substratum of improvement, the MRS substratum of described improvement is added with soy peptone 5 ~ 30g in often liter of MRS substratum, glucose 1 ~ 10g, yeast powder 1 ~ 10g, sodium acetate 1 ~ 10g, citric acid diamines 0.1 ~ 8g, Tween800.1 ~ 5g, dipotassium hydrogen phosphate 0.1 ~ 5g, magnesium sulfate 0.05 ~ lg, manganous sulfate 0.001 ~ 0.2g, calcium carbonate 1 ~ 30g, pure water 1 liter, adjust ph to 5.5 ~ 7.5.
8. described in claim 1, produce the application of sour faecium in antibacterial and fodder additives.
9. the application of antibacterial probiotics described in claim 2 in antibacterial and fodder additives.
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| CN108753650A (en) * | 2018-06-13 | 2018-11-06 | 青岛蔚蓝生物股份有限公司 | Enterococcus faecium and compound micro-ecological preparation prepared therefrom |
| CN109423466A (en) * | 2017-09-04 | 2019-03-05 | 北京科为博生物科技有限公司 | A kind of composite fermentation microbial inoculum and its application |
| CN110903961A (en) * | 2019-12-16 | 2020-03-24 | 陕西麦可罗生物科技有限公司 | Improved device and method for determining biological potency of a large number of antibiotic samples |
| CN111109433A (en) * | 2018-10-31 | 2020-05-08 | 北京科为博生物科技有限公司 | Compound biological preparation for regulating intestinal health of sows and preparation method and application thereof |
| CN113475622A (en) * | 2021-06-29 | 2021-10-08 | 湖北华扬科技发展有限公司 | Biological feed additive, preparation method and application thereof |
| CN113583905A (en) * | 2019-11-29 | 2021-11-02 | 天津市天合力药物研发有限公司 | Preparation method and application of enterococcus faecium microbial inoculum |
| CN113717899A (en) * | 2021-10-09 | 2021-11-30 | 广州金水动物保健品有限公司 | Preparation method of feeding enterococcus faecium raw powder |
| CN114317344A (en) * | 2021-12-28 | 2022-04-12 | 武汉科缘生物发展有限责任公司 | Enterococcus faecium, application thereof, composition thereof and fermentation culture method of enterococcus faecium |
| CN116200302A (en) * | 2023-01-10 | 2023-06-02 | 太原市威尔潞威科技发展有限公司 | Enterococcus faecium LV-434, microbial agent, and preparation methods and applications thereof |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109423466A (en) * | 2017-09-04 | 2019-03-05 | 北京科为博生物科技有限公司 | A kind of composite fermentation microbial inoculum and its application |
| CN109423466B (en) * | 2017-09-04 | 2020-12-15 | 北京科为博生物科技有限公司 | Compound fermentation inoculant and application thereof |
| CN108641979A (en) * | 2018-04-22 | 2018-10-12 | 青岛蔚蓝生物股份有限公司 | A kind of enterococcus faecium, its high density fermentation cultural method and probiotics prepared therefrom |
| CN108641979B (en) * | 2018-04-22 | 2020-02-14 | 青岛蔚蓝生物股份有限公司 | Enterococcus faecium, high-density fermentation culture method thereof and microecological preparation prepared from enterococcus faecium |
| CN108753650A (en) * | 2018-06-13 | 2018-11-06 | 青岛蔚蓝生物股份有限公司 | Enterococcus faecium and compound micro-ecological preparation prepared therefrom |
| CN108753650B (en) * | 2018-06-13 | 2020-02-14 | 青岛蔚蓝生物股份有限公司 | Enterococcus faecium and composite microecological preparation prepared from enterococcus faecium |
| CN111109433A (en) * | 2018-10-31 | 2020-05-08 | 北京科为博生物科技有限公司 | Compound biological preparation for regulating intestinal health of sows and preparation method and application thereof |
| CN113583905A (en) * | 2019-11-29 | 2021-11-02 | 天津市天合力药物研发有限公司 | Preparation method and application of enterococcus faecium microbial inoculum |
| CN110903961A (en) * | 2019-12-16 | 2020-03-24 | 陕西麦可罗生物科技有限公司 | Improved device and method for determining biological potency of a large number of antibiotic samples |
| CN113475622A (en) * | 2021-06-29 | 2021-10-08 | 湖北华扬科技发展有限公司 | Biological feed additive, preparation method and application thereof |
| CN113717899A (en) * | 2021-10-09 | 2021-11-30 | 广州金水动物保健品有限公司 | Preparation method of feeding enterococcus faecium raw powder |
| CN114317344A (en) * | 2021-12-28 | 2022-04-12 | 武汉科缘生物发展有限责任公司 | Enterococcus faecium, application thereof, composition thereof and fermentation culture method of enterococcus faecium |
| CN114317344B (en) * | 2021-12-28 | 2023-11-07 | 武汉科缘生物发展有限责任公司 | Enterococcus faecium and application thereof, composition and fermentation culture method of enterococcus faecium |
| CN116200302A (en) * | 2023-01-10 | 2023-06-02 | 太原市威尔潞威科技发展有限公司 | Enterococcus faecium LV-434, microbial agent, and preparation methods and applications thereof |
| CN116200302B (en) * | 2023-01-10 | 2023-10-27 | 太原市威尔潞威科技发展有限公司 | Enterococcus faecium LV-434, microbial agent, and preparation methods and applications thereof |
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