CN102031235A - Enterococcus faecium ANSE228 and application thereof - Google Patents
Enterococcus faecium ANSE228 and application thereof Download PDFInfo
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Abstract
The invention provides an Enterococcus faecium ANSE228 of which the collection number is CGMCC No.4082. The invention also provides application of the Enterococcus faecium ANSE228 to inhibition of salmonella pullorum and/or Escherichia coli and/or Staphylococcus aureus. The Enterococcus faecium ANSE228 is obtained by processes of repeated separation, purification, rejuvenation and the like, and has high biological activity, obvious probiotic property, high adversity resistance and the like. The invention also provides a microecological agent which contains the Enterococcus faecium ANSE228. When the microecological agent is added into drinking water and/or feeds for breeding animals, the Enterococcus faecium ANSE228 can be quickly activated and reproduced and a dominant beneficial flora can be formed after the Enterococcus faecium ANSE228 is fed into intestinal canals of the animals, and the Enterococcus faecium ANSE228 has the effects of reducing a harmful flora in the intestinal canals, adjusting microecological balance of the intestinal canals, substituting for medicaments such as antibiotic and the like, and improving weight increment of the animals and the utilization rate of the feeds.
Description
Technical field
The present invention relates to a kind of enterococcus faecalis and application thereof.
Background technology
Along with society and the reach of science, food safety is subjected to people's attention day by day, because dangerous food not only is detrimental to health and life, even also may have a negative impact to the descendants.Green, safe food is appealed by society, and the safety of feed is exactly the safety of food.
Microbiotic has vital role as fodder additives for aspects such as promoting animal growth, raising disease resistance.But its a large amount of for a long time uses have caused the generation of some negative effects, have for example destroyed the microecological balance of animal self, have increased the disease of alimentary tract of livestock husbandry and birds odds, and can make pathogenic bacteria sport Resistant strain etc.In animal body residual of what is more important, microbiotic passed to the mankind by food chain, brings hidden danger to health of people.Given this, a lot of countries all forbid in feed and limit the use of microbiotic as fodder additives.Therefore, the exploitation of microbiotic substitute products has also become the emphasis of countries in the world researchs.Wherein probiotics is because its special advantages, as the non-specific antibacterial and germicidal action of wide spectrum, environmental protection, noresidue etc., using the alternative field of microbiotic just more and more to be paid attention to.
This research with enterococcus faecalis (Enterococcus faecium) as research object, integrated existing theory and practice understanding of benefit being given birth to enterococcus faecalis, filter out have anti-microbial activity, the benefit of resistance and security gives birth to enterococcus faecalis, the zymotechnique of optimizing this bacterium also, study it as the influence of probiotics to breeding performonce fo animals, the application and the practice of giving birth to enterococcus faecalis for benefit lay the foundation.
Summary of the invention
The object of the present invention is to provide a kind of enterococcus faecalis.
Another object of the present invention is to provide the application of above-mentioned enterococcus faecalis.
The present invention also aims to provide a kind of probiotics.
A further object of the present invention is to provide the application of above-mentioned probiotics.
In order to realize purpose of the present invention, the invention provides a kind of enterococcus faecalis (Enterococcusfaecium) ANSE228, its deposit number is CGMCC No.4082, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on August 16th, 2010.Described enterococcus faecalis ANSE228 obtains by technologies such as separation repeatedly, purifying, rejuvenation, advantage such as the remarkable and anti-adversity of strong, the beneficial natural disposition of biologically active is good.
The cultural method of the ANSE228 of enterococcus faecalis described in the present invention is as follows: (seed liquor is that the single bacterium colony of picking is cultivated in the seed liquor substratum, and its viable bacteria concentration is with 10 to get enterococcus faecalis ANSE228 seed liquor 1-5ml
9The CFU/ml meter; The culture of seed liquid base is identical with the substratum and the culture condition of fermentation with culture condition), be inoculated into and carry out the shake flask fermentation cultivation in the 50-100ml substratum.Described substratum is made up of following component: Semen Maydis powder 10-25g/L, dregs of beans 10-30g/L, glucose 0-15g/L, dipotassium hydrogen phosphate 0.1-0.2g, potassium primary phosphate 1.0-2.0g, manganese sulfate monohydrate 0.5-2.0g, magnesium sulfate heptahydrate 0.5-2.0g, distilled water 800-1200mL, the pH value is 6.5-7.5.
Wherein, be preferably: Semen Maydis powder 15g, dregs of beans 20g, glucose 5g, dipotassium hydrogen phosphate 0.15g, potassium primary phosphate 1.5g, manganese sulfate monohydrate 1.0g, magnesium sulfate heptahydrate 1.5g, distilled water 1000mL, the pH value is 7.0.
The condition of shake flask fermentation described in the present invention is: leavening temperature 25-45 ℃, and fermentation time 18-36h, pH value 6.5-7.5 leaves standstill cultivation; Be preferably: 37 ℃ of leavening temperatures, fermentation time 24h, pH value 7.0.
10L fermentor tank pilot scale substratum is identical with the shake flask fermentation substratum.10L fermentor tank pilot scale fermentation condition is: liquid amount is the 4-7L substratum, and inoculum size is the 30-100ml shake-flask seed liquid, and leavening temperature is 25-45 ℃, fermentation time 18-36h, pH value 6.5-7.5, mixing speed 200-400r/min.Wherein, be preferably: liquid amount is the 5L substratum, and inoculum size is that 50ml shake-flask seed liquid leavening temperature is 37 ℃, fermentation time 24h, pH value 7.0, mixing speed 300r/min.
The present invention also provides enterococcus faecalis ANSE228 application in suppressing white dysentery Salmonellas, intestinal bacteria and/or streptococcus aureus.Described bacterial strain has significant beneficial natural disposition, can significantly suppress the growth and breeding of pathogenic bacterium such as white dysentery Salmonellas, intestinal bacteria and streptococcus aureus.
The present invention also provides described enterococcus faecalis ANSE228 application in the hot environment of simulated gastric fluid and/or simulation cholate and/or 75-90 ℃.Described bacterial strain has stronger resistance, can tolerate simulation hydrochloric acid in gastric juice, simulation cholate and hot environment, and can keep viable bacteria survival rate higher more than 80%.
The method of inspection of the beneficial natural disposition of enterococcus faecalis ANSE228 of the present invention is as follows:
On the aseptic technique platform, preparation thickness is the MRS flat board about 4mm, is 10 with pathogenic bacterium (intestinal bacteria, streptococcus aureus and Salmonellas) concentration
6Bacteria suspension 100-200 μ L coat on this flat board, stainless steel tubule (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm with sterilization, the two ends of pipe are smooth) be placed on the substratum, gently the pressurization, make it contact tight with substratum, treat several minutes after, in each tubule, drip the enterococcus faecalis ANSE228 activated spawn bacterium liquid of some amount respectively, do not make it excessive, cultivate 18-36h, measure antibacterial circle diameter then for 35 ℃-40 ℃.Each tests three repetitions, averages.
Wherein, described MRS flat board is with the MRS substratum behind 121 ℃ of high-temperature sterilizations, pours the flat board after the sterilization into, and the cooling back forms the thick MRS flat board of ganoid 4mm.The prescription of described MRS substratum is: peptone 10g, extractum carnis 10g, yeast extract 5g, K
2HPO
42g, diammonium hydrogen citrate 2g, glucose 20g, sodium acetate 5g, Tween-801mL, MgSO
47H
2O0.58g, MnSO
44H
2O 0.25g, agar 16g, distilled water 1000ml, pH=7.0.
The method of inspection of enterococcus faecalis resistance of the present invention is as follows:
1, high temperature tolerance test: get 5mL enterococcus faecalis ANSE228 viable bacteria bacterium liquid and be injected in the centrifuge tube 1,, select suitable dilution diluent, coating on MRS flat board 1 with ten times of stepwise dilutions of stroke-physiological saline solution.Other gets 5mL enterococcus faecalis ANSE228 viable bacteria bacterium liquid and is injected in the centrifuge tube 2, place 75-90 ℃ of water-bath to heat 10-20min, the enterococcus faecalis ANSE228 viable bacteria bacterium liquid of getting after the heating carries out ten times of stepwise dilutions with stroke-physiological saline solution, select dilution as hereinbefore diluent, coating on MRS flat board 2.Flat board 1 and dull and stereotyped 2 is cultivated 18-36h under 30-45 ℃ of condition, calculate the quantity of enterococcus faecalis heating front and back.
2, the resistance test of simulated gastric fluid: stomach en-is dissolved in the 0.5%-0.85% physiological saline, and making its final concentration is 3g/L, and adjusts the pH value to 2.0-4.0 with the NaOH of concentrated hydrochloric acid or 10%.Get 0.5mL enterococcus faecalis ANSE228 bacteria suspension and join in the simulated gastric fluid of 4.5mL (i.e. ten times of stepwise dilutions), and fully mixed on vibrator rapidly, place 30-45 ℃ to leave standstill and cultivate 2-4h then.In 2h, 4h, take out nutrient solution respectively and count remaining viable count immediately, compare with former viable count.
3, the resistance test of simulation cholate:
Make the solution of 1g/L-1.5g/L with pancreatin, and add 0.3% pig cholate in solution, it is 7.0-9.0 that the NaOH with 10% adjusts pH, uses 0.45 μ m micro-filtrate membrane filtration and degerming then.0.5ml enterococcus faecalis ANSE228 viable bacteria bacterium liquid is inoculated in the 4.5ml simulation cholate, obtains nutrient solution behind the cultivation 24h, count the viable count of remaining enterococcus faecalis ANSE228.With bacterium liquid ten times of stepwise dilutions in sterile saline, and carry out the dull and stereotyped coating of MRS, place 30-45 ℃ to leave standstill cultivation 18-36h (enterococcus faecalis generally will be cultivated just can grow up to obvious single bacterium colony more than the 18h) then.
Method of counting in simulated gastric fluid and the simulation cholate resistance test:
Sample selects suitable extent of dilution to be used for counting with ten times of gradient dilutions of stroke-physiological saline solution.Adopt the MRS nutrient agar, get the 0.5mL diluent and be coated on the flat board, cultivate 24h at 37 ℃.
The present invention also provides a kind of probiotics, and it contains above-mentioned enterococcus faecalis ANSE228.Described probiotics is the enterococcus faecalis ANSE228 that strong, the beneficial natural disposition of biological activity is remarkable and anti-adversity is good that obtains by technologies such as separation repeatedly, purifying, rejuvenation, obtain fermented liquid through production technique such as liquid submerged fermentations, make after drying then.
The present invention also provides the application of above-mentioned probiotics in breed, its be with probiotics as additive, add in the drinking-water or feed of cultivated animals.Wherein, the addition of described enterococcus faecalis ANSE228 in drinking-water is 10
8-10
11CFU/L drinking-water; The addition of described enterococcus faecalis ANSE228 in feed is 10
7-10
10The CFU/kg feed.Cultivated animals described in the present invention comprises fryer, laying hen and pig, also comprises fish, duck or ruminating animal, as ox or sheep.
Enterococcus faecalis ANSE228 of the present invention is less at its consumption, for example only is 10 in feed
7Can significantly improve the production performance of animal during CFU/kg, make that the fryer feedstuff-meat ratio reduces by 33.14%, the piglet death rate is reduced to 0%, the laying hen feedstuff-egg ratio reduces by 19.8%, has significantly improved the production performance of animal, has reduced production cost.
Beneficial effect of the present invention is:
1. enterococcus faecalis ANSE228 of the present invention has significant beneficial natural disposition, can significantly suppress the growth and breeding of pathogenic bacterium such as white dysentery Salmonellas, intestinal bacteria and streptococcus aureus.
2. enterococcus faecalis ANSE228 of the present invention has stronger resistance, can tolerate simulation hydrochloric acid in gastric juice, simulation cholate and hot environment, and can keep higher viable bacteria survival rate, and its viable bacteria survival rate can reach more than 80%.Because temperature is higher in the process that feed processing is granulated, and reaches 75-90 ℃, so bacterial strain need tolerate that this temperature range could guarantee that animal has is viable bacteria, and viable bacteria just has beneficial natural disposition.Tolerance hydrochloric acid in gastric juice and cholate are in order to make enterococcus faecalis can bring into play its effect in animal digestive tract.
3. the present invention has excavated the new purposes of known enterococcus faecalis, has opened up a new Application Areas.
4. after the probiotics that enterococcus faecalis ANSE228 makes among the present invention entered animal intestinal, powerful enterococcus faecalis ANSE228 activated rapidly, breeds, and formed useful dominant microflora simultaneously in the intestinal microecology environment.
5. enterococcus faecalis ANSE228 of the present invention, has significant beneficial natural disposition and resistance, not only can improve the food utilization efficiency of animal, save cost, and improved in the animal body stable state of environment in the digestive tube, and promote absorbing of nutrition, promote growth of animal, effect such as increase economic efficiency is indicating good prospects for application.
Description of drawings
Fig. 1 is the inhibition zone of enterococcus faecalis ANSE228 to the white dysentery Salmonellas.
Fig. 2 is that enterococcus faecalis ANSE228 is to colibacillary inhibition zone.
Fig. 3 is the inhibition zone of enterococcus faecalis ANSE228 to golden yellow Salmonellas.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 enterococcus faecalis ANSE228 preparation of fermentation liquid
(deposit number is CGMCC No.4082, and viable bacteria concentration is 10 to get enterococcus faecalis ANSE228
9CFU/ml), be inoculated in and carry out shake flask fermentation in the 50ml substratum and cultivate, leavening temperature is 37 ℃, pH value 7.0, leaves standstill cultivation, fermentation time 24h.
Wherein, the shake flask fermentation substratum is made up of following component: Semen Maydis powder 15g, dregs of beans 20g, glucose 5g, dipotassium hydrogen phosphate 0.15g, potassium primary phosphate 1.5g, manganese sulfate monohydrate 1.0g, magnesium sulfate heptahydrate 1.5g, distilled water 1000mL, the pH value is 7.0.
Shake flask fermentation carries out the fermentor tank pilot plant test after finishing, and gets 50ml shake flask fermentation seed liquor and is inoculated in the 10L fermentor tank, and liquid amount is that 5L, leavening temperature are 37 ℃, pH value 7.0, mixing speed 300r/min, fermentation time 36h.Fermentor tank pilot scale substratum is formed same shake flask fermentation.
After the fermentation ends, be kept in 4 ℃ of refrigerators fermented liquid standby.
The checking of embodiment 2 enterococcus faecalis ANSE228 benefit natural disposition
Preparation thickness is the MRS flat board about 4mm, is 10 with pathogenic bacterium (intestinal bacteria, streptococcus aureus and Salmonellas) concentration
6Bacteria suspension 100 μ L coat on this flat board, with stainless steel tubule (the circular tubule of internal diameter 6mm, external diameter 8mm, high 10mm of aseptic technique with sterilization, the two ends of pipe are smooth), be placed on the substratum, gently pressurization, make it contact tight with substratum, after treating several minutes, the fermented liquid (activated spawn bacterium liquid) that the embodiment 1 of dropping 200-300 microlitre prepares in each tubule does not make it excessive respectively, cultivate 24h under 37 ℃ of temperature, measure antibacterial circle diameter then.Each tests three repetitions, averages.The result as Figure 1-3.
Wherein, described MRS flat board is with the MRS substratum behind 121 ℃ of high-temperature sterilizations, pours the flat board after the sterilization into, and the cooling back forms the thick MRS flat board of ganoid 4mm.The prescription of described MRS substratum is: peptone 10g, extractum carnis 10g, yeast extract 5g, K
2HPO
42g, diammonium hydrogen citrate 2g, glucose 20g, sodium acetate 5g, Tween-801mL, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, agar 16g, distilled water 1000ml, pH=7.0.
Enterococcus faecalis ANSE228 is 1.20cm to the antibacterial circle diameter of white dysentery Salmonellas, and the antibacterial circle diameter of intestinal bacteria and streptococcus aureus is respectively 1.16cm and 1.12cm.
The checking of embodiment 3 enterococcus faecalis ANSE228 resistance
1, the fermented liquid that the 5mL embodiment 1 that keeps prepares of going bail for is injected in the centrifuge tube 1, adopts ten times of stepwise dilutions, coating on MRS flat board 1.The centrifuge tube 2 that the fermented liquid that 5mL embodiment 1 prepares will be housed again places 80 ℃ of water-baths to heat 15min, and the enterococcus faecalis bacterium liquid of getting after the heating carries out ten times of stepwise dilutions, coating on MRS flat board 2.Before will heating at last and the flat board after the heating all under 37 ℃ of conditions, cultivate 24h, calculate the quantity before and after the enterococcus faecalis ANSE228 heating.
The result shows that the viable bacteria survival rate has reached 85%.
2, the resistance test of simulated gastric fluid: stomach en-is dissolved in 0.5% physiological saline, and making its final concentration is 3g/L, and adjusts pH value to 2.0 with the NaOH of concentrated hydrochloric acid or 10%.Get 0.5mL enterococcus faecalis ANSE228 bacteria suspension and join in the simulated gastric fluid of 4.5mL (i.e. ten times of dilutions), and fully mixed on vibrator rapidly, place 37 ℃ to leave standstill and cultivate 2h then.After handling 2h, take out nutrient solution and count remaining viable count immediately, compare with former viable count.The result shows that the viable bacteria survival rate is 96%.
3, the resistance test of simulation cholate: make the solution of 1g/L with pancreatin, and add 0.3% pig cholate in solution, it is 8.0 that the NaOH with 10% adjusts pH, uses 0.45 μ m micro-filtrate membrane filtration and degerming then.0.5ml enterococcus faecalis ANSE228 viable bacteria bacterium liquid is inoculated in the 4.5ml simulation cholate, obtains nutrient solution behind the cultivation 24h, count the viable count of remaining enterococcus faecalis ANSE228.With bacterium liquid ten times of stepwise dilutions in sterile saline, and carry out the dull and stereotyped coating of MRS, place 37 ℃ to leave standstill cultivation 24h then.The result shows that the viable bacteria survival rate is 96%.
Embodiment 4 growth of meat chicken performance tests
Select 120 the 30 healthy fryer of age in days for use, weigh, be divided into 4 treatment group at random, 5 repetitions are established in each processing, and each repeats 6 chickens, and male and female respectively accounts for 1/2.Test drinking-water is respectively: (1) common drinking-water (control group); (2) enterococcus faecalis ANSE228 viable bacteria concentration is 10 in the drinking-water
10CFU/L; (3) enterococcus faecalis ANSE228 viable bacteria concentration is 10 in the drinking-water
9CFU/L; (4) enterococcus faecalis ANSE228 viable bacteria concentration is 10 in the drinking-water
8CFU/L.Test chicken free choice feeding and drinking-water, feeding and management and immune programme for children are with reference to the feeding of broiler administrative manual.
Table 1 enterococcus faecalis ANSE228 is to the growth of meat chicken Effect on Performance
Annotate: do not contain same letter person with line data shoulder mark and represent significant difference (P＜0.05) (down together)
This test-results shows that enterococcus faecalis ANSE228 has significantly improved fryer average daily gain (P=0.0123) and feed efficiency (P=0.0006).Simultaneously, enterococcus faecalis ANSE228 viable bacteria concentration is 10 in the drinking-water
10During CFU/L, the broiler fodder transformation efficiency is the highest.
The test of embodiment 5 laying hen production performances:
Test designs for single-factor, and 576 brown commodity of Luo Man are divided into 3 processing immediately for laying hen, and each handles 6 repetitions, and each repeats 8 cages, 4 chickens of every cage.Handle 1 and be negative contrast, the basal diet of feeding does not contain any medicated feed additive; Handling 2 is over against photograph, adds the brown kangdisu sulfas 4mg/kg of Zinc-bacitracin 20mg/kg on the basal diet basis; Handle 3 and on the basal diet basis, add 3 * 10
9The enterococcus faecalis ANSE228 probiotics of CFU/kg.Test chicken free choice feeding and drinking-water, feeding and management and immune programme for children are with reference to the layer breeding administrative manual.
Table 2 enterococcus faecalis ANSE228 is to the influence of laying hen production performance
The result of this test shows that enterococcus faecalis ANSE228 can significantly improve feedstuff-egg ratio of laying hen and the egg productivity (p＜0.05) that improves laying hen, and can reduce production costs, improve the production performance of laying hen, increase economic efficiency, effectively substitute Antibiotic Additive, be fit to large-scale promotion and use.
The test of embodiment 6 piglet growth performances
This test selects for use 90 of ternary (Du * length * big) the hybridization weanling pigs of 35 ± 1 ages in days, mean body weight 7.86 ± 0.06kg health to distribute to advance three processing immediately, each handles 5 repetitions, each repeats 6 piglets (male and female half and half), initial body weight no significant difference (p＞0.05) between each repeating groups.
Three processing are respectively: blank group, microbiotic control group (75mg/kg duomycin) and test group (add 4 * 10
9CFU/kg enterococcus faecalis ANSE228 probiotics).Test pig free choice feeding and drinking-water, feeding and management and immune programme for children are with reference to the feeding piglet administrative manual.
Table 3 enterococcus faecalis ANSE228 is to the influence of piglet growth performance
This test-results shows that enterococcus faecalis ANSE228 can significantly improve the average daily gain (p＜0.05) of piglet, improves feedstuff-meat ratio, and has significantly reduced diarrhea rate and the death rate (p＜0.05) of piglet.Improved production performance and the economic benefit of piglet, can effectively substitute Antibiotic Additive, be fit to large-scale promotion and use.
Though, above used general explanation, specific embodiments and experiment, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. an enterococcus faecalis (Enterococcus faecium) ANSE228, its deposit number is CGMCC No.4082.
2. a method of cultivating the described enterococcus faecalis ANSE228 of claim 1 is characterized in that, gets enterococcus faecalis ANSE228 seed liquor 1-5ml, is inoculated into to carry out the shake flask fermentation cultivation in the 50-100ml substratum.
3. cultural method according to claim 2, it is characterized in that, described substratum is made up of following component: Semen Maydis powder 10-25g/L, dregs of beans 10-30g/L, glucose 0-15g/L, dipotassium hydrogen phosphate 0.1-0.2g, potassium primary phosphate 1.0-2.0g, manganese sulfate monohydrate 0.5-2.0g, magnesium sulfate heptahydrate 0.5-2.0g, distilled water 800-1200mL, the pH value is 6.5-7.5.
4. cultural method according to claim 2 is characterized in that, the condition of described shake flask fermentation is: leavening temperature 25-45 ℃, fermentation time 18-36h, pH value 6.5-7.5, leave standstill cultivation.
5. the application of the described enterococcus faecalis ANSE228 of claim 1 in suppressing white dysentery Salmonellas and/or intestinal bacteria and/or streptococcus aureus.
6. the application of the described enterococcus faecalis ANSE228 of claim 1 in the hot environment of simulated gastric fluid and/or simulation cholate and/or 75-90 ℃.
7. a probiotics is characterized in that, it contains the described enterococcus faecalis ANSE228 of claim 1.
8. the application of the described probiotics of claim 7 in breed is characterized in that, probiotics as additive, is added in the drinking-water or feed of cultivated animals.
9. application according to claim 8 is characterized in that, the addition of described enterococcus faecalis ANSE228 in drinking-water is 10
8-10
11CFU/L drinking-water; The addition of described enterococcus faecalis ANSE228 in feed is 10
7-10
10The CFU/kg feed.
10. according to claim 5 or 6 described application, it is characterized in that described cultivated animals comprises fryer, laying hen and pig, also comprise fish, duck, ox or sheep.
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