CN106282072A - A kind of compound lactobacillus microbial ecological agent and preparation method and application - Google Patents
A kind of compound lactobacillus microbial ecological agent and preparation method and application Download PDFInfo
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- CN106282072A CN106282072A CN201610963539.6A CN201610963539A CN106282072A CN 106282072 A CN106282072 A CN 106282072A CN 201610963539 A CN201610963539 A CN 201610963539A CN 106282072 A CN106282072 A CN 106282072A
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- 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
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- 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
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The present invention relates to a kind of compound lactobacillus microbial ecological agent and preparation method and application, described compound lactobacillus microbial ecological agent has stronger resistance, it is resistant to simulate gastric acid, cholate and hot environment, and higher Viable detection can be kept, its stability is preferable, is more suitable for the requirement of feed industry and livestock breeding industry.Flora in described compound lactobacillus microbial ecological agent scalable animal intestinal, improves animal disease resistant ability, reduces diarrhea rate and mortality rate, also can improve animal feed intake, reduces feedstuff-meat ratio, thus improves breeding performonce fo animals, has good application prospect.
Description
Technical field
The invention belongs to field of fodder, particularly to a kind of compound lactobacillus microbial ecological agent and preparation method thereof with should
With.
Background technology
The abuse of antibiotic brings serious threat to the health of animal husbandry and the mankind.The alternative antibiotic of probiotic bacteria is applied
In breeding production.Lactobacillus plantarum (Lactobacillus plantarum) is the normal flora of humans and animals intestinal, is pressing down
Growth of pathogenic bacteria processed is bred, and promotes that nutrient digestion is absorbed by body, improves the aspects such as immunity of organisms and has important life
Thing function, has wide practical use in husbandry sector.But the big multi-probiotics height to the granulating process in feed manufacturing
The toleration of temperature, transport and gastro-intestinal Fluid and bile is poor, and the kind and the quantity that enter the living plant lactobacillus of intestinal are few.Excrement intestinal
Coccus (Enterococcus faecalis) and enterococcus faecalis (Enterococcus faecium) belong to lactic acid bacteria, and growth is fast,
Adhesion is strong, can produce lactic acid and antibiotic substance, be widely used in the production of broiler, growing and fattening pigs, piglet, milch cow etc..Commonly
Enterococcus faecalis and enterococcus faecalis because of its characteristic such as acidproof, high temperature resistant poor, fail extensively to apply in feed additive.
At present, probiotics preparation is to be widely used in breeding production, but remains in terms of technology of preparing in problems:
(1) product standard disunity, manufacturing enterprise sets up standard voluntarily, lacks scientific basis, probiotic bacteria too high levels or too low, impact
Using effect；(2) producing strain source not clear, most enterprises production strain mutuallys transfer, not through isolation and selection, and bacterium
Planting and degenerate seriously, physiologically active reduces；(3) strain compatibility lack scientific basis, single bacterial strain, double bacterial strains, the most bacterial strains with
Meaning proportioning, it is impossible to the reasonability of compatibility between bacterial strain is described, does not reaches bacterial strain merit, complementary by reasonable compatibility, collaborative
Property is poor；(4) production technology is chaotic, it is impossible to distinguishing by the different physiological propertys of strain and cultivate, many manufacturing enterprises use multi-cultur es
Mixed culture, without fermentation tank, notably uses and heaps fermentation, cause growth of probiotics few, and miscellaneous bacteria grows thickly.
Summary of the invention
In order to make up the deficiencies in the prior art, by relations such as Mutualism in the middle of microorganisms, antagonism and parasitisms, this
Invention provides a kind of compound lactobacillus microbial ecological agent and preparation method and application.
A kind of compound lactobacillus microbial ecological agent that the present invention provides, its active bacteria includes Lactobacillus plantarum
(Lactobacillus plantarum) HEW-A490, its deposit number is CGMCC NO.12554；Enterococcus faecalis
(Enterococcus faecalis) and enterococcus faecalis (Enterococcus faecium).
Preferably, in described compound lactobacillus microbial ecological agent, the living bacteria count of Lactobacillus plantarum HEW-A490 is 2.5
×1010-5.0×1010CFU/g, more preferably 4.0 × 1010CFU/g。
Preferably, in described compound lactobacillus microbial ecological agent, the living bacteria count of enterococcus faecalis is 1.5 × 1010-3.0×
1010CFU/g, more preferably 2.0 × 1010CFU/g。
Preferably, in described compound lactobacillus microbial ecological agent, the living bacteria count of enterococcus faecalis is 2.0 × 1010-3.0×
1010CFU/g, more preferably 2.0 × 1010CFU/g。
Preferably, Lactobacillus plantarum HEW-A490, enterococcus faecalis, enterococcus faecalis in described compound lactobacillus microbial ecological agent
Living bacteria count ratio be 2.5-5:1.5-3:2-3, more preferably 2:1:1.
Preferably, in described compound lactobacillus microbial ecological agent, total living bacteria count is 6.0 × 1010-1.1×1011CFU/
G, more preferably 8.0 × 1010CFU/g。
Preferably, described enterococcus faecalis is enterococcus faecalis HEW-A131, and its deposit number is CGMCC NO.9353, exists
Disclosed in Chinese patent CN104293696A；This bacterium is preserved in Chinese microorganism strain preservation management on June 17th, 2014
Committee's common micro-organisms center.
Preferably, described enterococcus faecalis is enterococcus faecalis HEW-A588, and its deposit number is CGMCC No.10547, exists
Disclosed in Chinese patent CN104862254A, this bacterium is preserved in Chinese microorganism strain preservation management on February 9th, 2015 and entrusts
Member can common micro-organisms center.
Described compound lactobacillus microbial ecological agent also includes that protective agent, described protective agent include the component of following weight portion:
1-10 part trehalose, 1-3 part polyvidon, 2-5 part mannitol, 1-8 part lactose, 0.1-2 part VC, 0.1-2 part VD、0.1-2
Part VE, 0.5-5 part sodium thiosulfate, 1-5 part hydroxymethyl cellulose, 1-10 part arabic gum, 2-8 part fat-free comminuted whey, 2-10
Part glycerol, 0.5-5 part Tween 80,20-40 part corn starch, 20-40 part water.It is further preferred that described protective agent include with
The component of lower weight portion: 5.5 parts of trehaloses, 2 parts of polyvidon, 2.4 parts of mannitol, 3 parts of lactose, 0.5 part of VC, 0.8 part
VD, 0.9 part of VE, 2 parts of sodium thiosulfate, 1.5 parts of hydroxymethyl cellulose, 3 parts of arabic gums, 5 parts of fat-free comminuted wheys, 3 parts of glycerol,
1.8 parts of Tween 80s, 35 parts of corn starchs, 35 parts of water.
Preferably, in described compound lactobacillus microbial ecological agent, protectant weight percentage is 20-70%, further
It is preferably 35%.
The present invention also provides for the preparation method of above-mentioned compound lactobacillus microbial ecological agent, comprises the following steps:
1) by Lactobacillus plantarum (Lactobacillus plantarum) HEW-A490, enterococcus faecalis (Enterococcus
Faecalis) and enterococcus faecalis (Enterococcus faecium) carries out first order seed fermentation respectively, first order seed training is obtained
Nutrient solution；
2) the first order seed culture fluid of Lactobacillus plantarum HEW-A490, enterococcus faecalis and enterococcus faecalis is inoculated in two grades send out
In ferment tank, fermentation culture, it is thus achieved that second order fermentation culture fluid；Or further second order fermentation culture fluid is inoculated in three grade fermemtation tank
In, fermentation culture, it is thus achieved that three grade fermemtation culture fluid；
3) described second order fermentation culture fluid or three grade fermemtation medium centrifugal are obtained compound lactobacillus active bacteria mud；By institute
State active bacteria mud to be mixed in proportion with protective agent, pelletize, be coated, prepare described compound lactobacillus microbial ecological agent.
In above-mentioned preparation method,
The primary-seed medium used during Lactobacillus plantarum HEW-A490 fermentation is: lactose 0.5-3%, sucrose 0.5-
2%, whole milk powder 01-1.%, bean cake powder 0.5-2%, yeast leaching powder 0.5-3.%, NaH2PO40.1-0.7%, KH2PO4
0.1-0.7%, magnesium chloride 0.01-0.1%, ferric ammonium citrate 0.01-0.1%, MnSO40.01-0.1%, calcium chloride 0.05-
1%, surplus is water, pH5.8-7.2；Preferably lactose 1.25%, sucrose 0.8%, whole milk powder 0.5%, bean cake powder 1.5%,
Yeast leaching powder 0.85%, NaH2PO40.4%, KH2PO40.6%, magnesium chloride 0.05%, ferric ammonium citrate 0.04%, MnSO4
0.04%, calcium chloride 0.1%, surplus is water, pH6.4 ± 0.2.
The primary-seed medium used during Enterococcus faecalis fermentation is: sucrose 1-5%, soy peptone 0.5-2.5%, ferment
Female extract 0.1-1.0%, MgSO40.05-0.2%, MnSO40.01-1.0%, NaCl0.1-2.0%, dibasic ammonium citrate
0.1-0.5%, CaCO30.1-1.0%, surplus is water, pH7.0 ± 0.2；It is preferably: sucrose 2.5%, soy peptone
1.8%, yeast extract 0.4%, MgSO40.2%, MnSO40.045%, NaCl0.2%, dibasic ammonium citrate 0.2%,
CaCO30.06%, surplus is water, pH7.0 ± 0.2.
The primary-seed medium used during enterococcus faecalis fermentation is: sucrose 1-5%, glucose 0.5-1.5%, peptone
1-3%, yeast extract 0.5-2.0%, MgSO40.1-0.5%, MnSO40.01-0.05%, NaCl 0.5-2.0%, citric acid
Diammonium 0.1-0.5%, CaCO30.05-1.0%, surplus is water, pH6.8 ± 0.2；It is preferably: sucrose 1.5%, glucose
0.5%, peptone 1.6%, yeast extract 0.8%, MgSO40.3%, MnSO40.02%, NaCl0.5%, dibasic ammonium citrate
0.1%, CaCO30.05%, surplus is water, pH6.8 ± 0.2.
Fermentation medium (including second order fermentation culture medium and three grade fermemtation culture medium) is made up of following component: molasses 1-
3%, brown sugar 0.5-2%, Semen Maydis pulp 1-3%, yeast extract 0.5-2%, ammonium sulfate 0.1-0.5%, citric acid hydrogen diamine 0.01-
0.5%, K2HPO40.1-0.5%, calcium carbonate 0.2-1.0%, NaCl0.2-1%, MnSO40.01-0.1%, surplus is water,
pH6.0-7.0；It is preferably molasses 1.5%, brown sugar 0.85%, Semen Maydis pulp 1.2%, yeast extract 0.7%, ammonium sulfate 0.2%, Fructus Citri Limoniae
Acid diammonium hydrogen 0.03%, K2HPO40.1%, calcium carbonate 0.4%, NaCl0.5%, MnSO40.031%, surplus is water, pH6.6
±0.2。
Fermentation condition (including that second order fermentation is cultivated and three grade fermemtation is cultivated) including: cultivation temperature 28-45 DEG C, speed of agitator
80-200r/m, fermentation time is 10-15h；It is preferably: cultivation temperature 35 DEG C, speed of agitator 110r/m, fermentation time is 15h.
Preferably, the inoculum concentration of the first order seed culture fluid of Lactobacillus plantarum HEW-A490, enterococcus faecalis, enterococcus faecalis is divided
It is not 2%, 1.5% and 1%.
Preferably, in described second order fermentation culture fluid, Lactobacillus plantarum HEW-A490, enterococcus faecalis, the having of enterococcus faecalis
Effect viable count is respectively 3.5 × 109-1.0×1010CFU/mL、2.5×109-5.0×109CFU/mL、2.0×109-4.5×
109CFU/mL, more preferably 9.0 × 109CFU/mL、4.0×109CFU/mL、4.0×109CFU/mL。
Preferably, in described three grade fermemtation culture fluid, Lactobacillus plantarum HEW-A490, enterococcus faecalis, the having of enterococcus faecalis
Effect viable count is respectively 1.0 × 1010-5.0×1010CFU/mL、1.0×1010-3.0×1010CFU/mL、1.0×1010-3.0
×1010CFU/mL, more preferably 4.5 × 1010CFU/mL、1.8×1010CFU/mL、1.8×1010CFU/mL。
Preferably, in described second order fermentation tank, three grade fermemtation tank, the liquid amount of culture medium is 30-70%, the most excellent
Elect 60% as.
Preferably, when three grade fermemtation is cultivated, after fermentation a period of time, such as, after fermentation 4-15h, regulated by Feeding ammonia water
Three grade fermemtation liquid is to pH5.5, when pH no longer declines, fermentation ends.
Described protective agent includes in terms of parts by weight: 1-10 part trehalose, 1-3 part polyvidon, 2-5 part manna
Alcohol, 1-8 part lactose, 0.1-2 part VC, 0.1-2 part VD, 0.1-2 part VE, 0.5-5 part sodium thiosulfate, 1-5 part hydroxylmethyl cellulose
Element, 1-10 part arabic gum, 2-8 part fat-free comminuted whey, 2-10 part glycerol, 0.5-5 part Tween 80,20-40 part corn starch,
20-40 part water；Preferably include: 5.5 parts of trehaloses, 2 parts of polyvidon, 2.4 parts of mannitol, 3 parts of lactose, 0.5 part of VC、
0.8 part of VD, 0.9 part of VE, 2 parts of sodium thiosulfate, 1.5 parts of hydroxymethyl cellulose, 3 parts of arabic gums, 5 parts of fat-free comminuted wheys, 3 parts
Glycerol, 1.8 parts of Tween 80s, 35 parts of corn starchs, 35 parts of water.
Preferably, described bacterium mud and protectant part by weight are 0.5-2:0.5-4, more preferably 1.5:1.
Preferably, in described bacterium mud, living bacteria count is 5.0 × 1011-1.50×1011CFU/g, more preferably 8.0
×1011CFU/g。
The described liquid that is coated includes by weight percentage: starch 1-10%, bovine serum albumin 1-5%, lecithin 1-
3%, citric acid 0.5-4%, gelatin 0.5-5%, sodium carboxymethyl cellulose 0.1-5%, maltodextrin 1-10%, oligofructose 2-
8%, glucosan 1-10%, surplus is water；Preferably include: starch 8.5%, bovine serum albumin 2%, lecithin 1.5%, Fructus Citri Limoniae
Acid 1.2%, gelatin 2.4%, sodium carboxymethyl cellulose 1.5%, maltodextrin 4%, oligofructose 3.5%, glucosan 6%, remaining
Amount is water.
The present invention also provides for above-mentioned compound lactobacillus microbial ecological agent and is preparing animal feed or preparing in feed additive
Application, or the application in livestock-raising.
Above-mentioned application in livestock-raising includes improving breeding performonce fo animals, reproductive performance, promotes animal growth.
Application of the present invention does not the most include therapeutic purposes.
The present invention also provides for the animal feed containing above-mentioned compound lactobacillus microbial ecological agent or feed additive.
The addition suggestion in animal feed of above-mentioned compound lactobacillus microbial ecological agent is 0.05-0.15% (weight percent
Than).
Compound lactobacillus microbial ecological agent of the present invention has stronger resistance, it is possible to tolerance simulation gastric acid, cholate and
Hot environment, and higher Viable detection can be kept, its Viable detection can reach more than 90%, and its stability is preferable,
It is more suitable for the requirement of feed industry and livestock breeding industry, has good application prospect.
The method have the benefit that
1, the flora in compound lactobacillus microbial ecological agent scalable animal intestinal of the present invention, improves animal disease resistant ability,
Reduce diarrhea rate and mortality rate, also can improve animal feed intake, reduce feedstuff-meat ratio, thus improve breeding performonce fo animals.
2, the microbial ecological agent consumption of the present invention is less, such as can play notable work when being only 0.05% in feedstuff
With, it is in particular in:
1) compound lactobacillus microbial ecological agent can effectively suppress the infection to chicken of the chicken pathogenic escherichia coli.
2) feed compound lactobacillus microbial ecological agent and be remarkably improved sow total yield coefficient, young number of living, reduce weak young number, dead
Tire number；Compared with matched group, piglet birth weight improves 6%, and weigth at birth significantly improves 15.60g, weaned piglet incubation rate
Significantly improving 9.5%, sow early productivity significantly reduces 10%, and sow prolonged labor rate significantly reduces 20%, sow sickness rate
Significantly reduce 33%.Compound lactobacillus microbial ecological agent can be effectively improved the reproductive performance of sow, improves sow growth performance,
Reducing sow sickness rate, the growth for suckling pig provides guarantee.
3) compared with matched group, add compound lactobacillus preparation and be remarkably improved piglet daily gain, reduce feedstuff-meat ratio, diarrhoea
Rate and mortality rate；Also can substantially reduce the harmful bacteria quantity such as escherichia coli in intestinal, and can keep and improve lactic acid bacteria in intestinal
Deng probiotics content, thus the invasion of Competitive assays pathogenic bacterium, field planting and breeding, thus improve piglet survival rate.
3, the preparation method of compound lactobacillus microbial ecological agent of the present invention not only solve the energy consumption of single bacterium fermenting and producing high,
The problem such as easily pollute, technique is loaded down with trivial details, and each strain bio activity can also be improved, and solve the problems such as spawn degeneration.
Accompanying drawing explanation
Fig. 1 is Lactobacillus plantarum HEW-A490 colonial morphology figure in MRS culture medium；
Fig. 2 is Lactobacillus plantarum HEW-A490 bacterial strain Gram’s staining figure.
Detailed description of the invention
Explaining the present invention below in conjunction with embodiment, case study on implementation is merely to illustrate the present invention.Unless stated otherwise, the present invention
Technological means used by is method known in those skilled in the art.It addition, embodiment is interpreted as illustrative,
And unrestricted the scope of the present invention, the spirit and scope of the invention are limited only by the claims that follow.For people in the art
For Yuan, on the premise of without departing substantially from spirit and scope of the present invention, the material component in these embodiments and consumption are carried out
Various changes or change fall within protection scope of the present invention.
Lactobacillus plantarum (Lactobacillus plantarum) HEW-A490 used in following example is in 2016
(being called for short CGMCC, address is: north to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 27, in
North Star West Road, Jing Shi Chaoyang District 1 No. 3 Institute of Microorganism, Academia Sinica of institute, postcode: 100101), deposit number is
CGMCC NO.12554.Enterococcus faecalis is enterococcus faecalis HEW-A131, is preserved in China Microbiological bacterium on June 17th, 2014
Planting preservation administration committee common micro-organisms center, deposit number is CGMCC NO.9353, at Chinese patent
Disclosed in CN104293696A.Enterococcus faecalis is enterococcus faecalis HEW-A588, is preserved in Chinese micro-life on February 9th, 2015
Thing culture presevation administration committee's common micro-organisms center, deposit number CGMCC No.10547, at Chinese patent
Disclosed in CN104862254A.
Embodiment 1 Lactobacillus plantarum HEW-A490, enterococcus faecalis HEW-A131, the activation culture of enterococcus faecalis HEW-A588
And the preparation of compound lactobacillus microbial ecological agent
1) taking the Lactobacillus plantarum HEW-A490 of glycerol pipe preservation, isolated and purified on MRS flat board, picking growing way is preferable
Bacterial strain is streak culture 20h on MRS inclined-plane, is inoculated in 100mL MRS liquid with inoculating loop picking one ring bacterium in aseptic operating platform
In body culture medium, 20h is cultivated in concussion, then according to 2% inoculum concentration is inoculated in primary-seed medium, cultivation temperature is 30-
40 DEG C, rotating speed is 120-220r/m, and incubation time is 10-30h, is prepared into Lactobacillus plantarum first order seed culture fluid (viable count
≥2.0×109CFU/mL).Its fermentation condition is preferably 37 DEG C, 160r/m, cultivates 15h.
Described primary-seed medium is: lactose 1.25%, sucrose 0.8%, whole milk powder 0.5%, bean cake powder 1.5%,
Yeast leaching powder 0.85%, NaH2PO40.4%, KH2PO40.6%, magnesium chloride 0.05%, ferric ammonium citrate 0.04%, MnSO4
0.04%, calcium chloride 0.1%, surplus is water, pH6.4 ± 0.2.
2) the enterococcus faecalis HEW-A131 of glycerol pipe preservation is taken, isolated and purified on MRS flat board, the preferable bacterium of picking growing way
Strain is streak culture 20h on MRS inclined-plane, is inoculated in 100mL MRS liquid with inoculating loop picking one ring bacterium in aseptic operating platform
In culture medium, 15h, then according to 1% inoculum concentration is inoculated in primary-seed medium, 35 DEG C are cultivated in concussion, and 180r/m shakes
Cultivate 15h, obtain enterococcus faecalis first order seed culture fluid (viable count >=1.0 × 109CFU/mL)。
Described primary-seed medium is: sucrose 2.5%, soy peptone 1.8%, yeast extract 0.4%, MgSO4
0.2%, MnSO40.045%, NaCl 0.2%, dibasic ammonium citrate 0.2%, CaCO30.06%, surplus is water, pH7.0 ±
0.2。
3) the enterococcus faecalis HEW-A588 of glycerol pipe preservation is taken, isolated and purified on MRS flat board, the preferable bacterium of picking growing way
Strain is streak culture 20h on MRS inclined-plane, is inoculated in 100mL MRS liquid with inoculating loop picking one ring bacterium in aseptic operating platform
In culture medium, 20h is cultivated in concussion, then according to 1% inoculum concentration is inoculated in primary-seed medium, 37 DEG C, 150r/min shakes
Swing cultivation 15h, obtain enterococcus faecalis first order seed culture fluid (viable count >=1.0 × 109CFU/mL)。
Described primary-seed medium is: sucrose 1.5%, glucose 0.5%, peptone 1.6%, yeast extract 0.8%,
MgSO40.3%, MnSO40.02%, NaCl 0.5%, dibasic ammonium citrate 0.1%, CaCO30.05%, surplus is water,
pH6.8±0.2。
4) by the first order seed culture fluid of Lactobacillus plantarum, enterococcus faecalis and enterococcus faecalis respectively according to 2%, 1.5% and
1% inoculum concentration is inoculated in 500L fermentation tank simultaneously, and liquid amount is 350L, cultivation temperature 35 DEG C, speed of agitator 110r/m, fermentation
Time is 10-15h, it is thus achieved that second order fermentation culture fluid, and in fermentation liquid, Lactobacillus plantarum living bacteria count reaches 9.0 × 109CFU/mL,
Enterococcus faecalis living bacteria count reaches 4.0 × 109CFU/mL, enterococcus faecalis living bacteria count reaches 4.0 × 109CFU/mL。
In described 500L fermentation tank, medium component is: molasses 1.5%, brown sugar 0.85%, Semen Maydis pulp 1.2%, yeast extract
0.7%, ammonium sulfate 0.2%, citric acid hydrogen diamine 0.03%, K2HPO40.1%, calcium carbonate 0.4%, NaCl0.5%, MnSO4
0.031%, surplus is water, pH6.6 ± 0.2.
350L second order fermentation culture fluid is transferred in 20T fermentation tank, carries out three grades of amplification culture.By fermentation temperature control
At 35 DEG C, liquid amount 15T, after fermentation a period of time, regulate three grade fermemtation liquid pH5.5 by Feeding ammonia water, when pH no longer declines
Time, fermentation ends, in fermentation liquid, Lactobacillus plantarum living bacteria count reaches 4.5 × 1010CFU/mL, enterococcus faecalis living bacteria count reaches
1.8×1010CFU/mL, enterococcus faecalis living bacteria count reaches 1.8 × 1010CFU/mL, total viable count reaches 8.1 × 1010CFU/mL。
Described three grade fermemtation liquid culture medium composition is identical with second order fermentation liquid culture medium composition.
5) three grade fermemtation liquid step 4 obtained is through 15000r/m centrifuge 30-40min, it is thus achieved that compound lactobacillus
Active bacteria mud.And active bacteria mud is stirred according to 1.5:1 weight ratio with protective agent in agitator tank, by the regulation that adds water
Its humidity is maintained at 30-45%, obtains microcapsule wet-milling, puts in granulator and pelletizes, is subsequently adding and is coated liquid so that it is formed
Coatings, then be dried, making moisture≤6%, cross 20 mesh sieves and obtain compound lactobacillus microbial ecological agent, its viable count reaches
8.0×1010CFU/g.Lactobacillus plantarum HEW-A490 effective active is 4.0 × 1010CFU/g, enterococcus faecalis HEW-A131 has
Effect activity is 2.0 × 1010CFU/g, enterococcus faecalis HEW-A588 effective active are 2.0 × 1010CFU/g。
Described protective agent includes in terms of parts by weight: 5.5 portions of trehaloses, 2 parts of polyvidon, 2.4 portions of mannitol, 3
Part lactose, 0.5 part of VC, 0.8 part of VD, 0.9 part of VE, 2 parts of sodium thiosulfate, 1.5 parts of hydroxymethyl cellulose, 3 parts of arabic gums, 5 parts
Fat-free comminuted whey, 3 parts of glycerol, 1.8 parts of Tween 80s, 35 parts of corn starchs, 35 parts of water.
The described liquid that is coated includes by weight percentage: starch 8.5%, bovine serum albumin 2%, lecithin 1.5%, lemon
Lemon acid 1.2%, gelatin 2.4%, sodium carboxymethyl cellulose 1.5%, maltodextrin 4%, oligofructose 3.5%, glucosan 6%,
Surplus is water.
The preparation of embodiment 2 compound lactobacillus microbial ecological agent
By the identical method of embodiment 1 respectively by Lactobacillus plantarum HEW-A490, enterococcus faecalis HEW-A131, enterococcus faecalis
This three strains bacterium of HEW-A588 carries out seed culture, two grades, three grade fermemtation cultivation respectively, is centrifuged respectively obtaining this three strains bacterium
Active bacteria mud, by Lactobacillus plantarum HEW-A490, enterococcus faecalis HEW-A131, the active bacteria mud of enterococcus faecalis HEW-A588 and guarantor
Protecting agent to stir in agitator tank according to 0.4:0.8:1.0:1.5 weight ratio, by adding water, its humidity of regulation is maintained at 30-
45%, obtain microcapsule wet-milling, put in granulator and pelletize, be subsequently adding and be coated liquid so that it is form coatings, then do
Dry, make moisture≤6%, cross 20 mesh sieves and obtain compound lactobacillus microbial ecological agent, its viable count reaches 1.0 × 1011CFU/g.Plant
Thing lactobacillus HEW-A490 effective active is 2.0 × 1010CFU/g, enterococcus faecalis HEW-A131 effective active is 4.0 ×
1010CFU/g, enterococcus faecalis HEW-A588 effective active are 4.0 × 1010CFU/g。
The preparation of embodiment 3 compound lactobacillus microbial ecological agent
With embodiment 1 differ only in three grade fermemtation liquid case study on implementation 1 step 4 obtained through 15000r/m centrifuge
Centrifugal 30-40min, it is thus achieved that the active bacteria mud of compound lactobacillus.And active bacteria mud is being stirred according to 2:0.8 weight ratio with protective agent
Mixing in tank and stir, by adding water, its humidity of regulation is maintained at 30-45%, obtains microcapsule wet-milling, puts in granulator and carry out
Pelletizing, be subsequently adding and be coated liquid so that it is form coatings, then be dried, make moisture≤6%, crossing 20 mesh sieves must be combined
Lactobacillus micro-ecological preparation, its viable count reaches 2.0 × 1011CFU/g.Lactobacillus plantarum HEW-A490 effective active is 1.0 ×
1011CFU/g, enterococcus faecalis HEW-A131 effective active is 5.0 × 1010CFU/g, enterococcus faecalis HEW-A588 effective active are
5.0×1010CFU/g。
The preparation of embodiment 4 compound lactobacillus microbial ecological agent
With embodiment 1 differ only in three grade fermemtation liquid case study on implementation 1 step 4 obtained through 15000r/m centrifuge
Centrifugal 30-40min, it is thus achieved that the active bacteria mud of compound lactobacillus.And active bacteria mud is being stirred according to 1:1 weight ratio with protective agent
Stirring in tank, by adding water, its humidity of regulation is maintained at 30-45%, obtains microcapsule wet-milling, puts in granulator and make
Grain, is subsequently adding and is coated liquid so that it is forms coatings, then is dried, and makes moisture≤6%, crosses 20 mesh sieves and obtains Composite Milk
Acid bacterium microbial ecological agent, its viable count reaches 5.0 × 1010CFU/g.Lactobacillus plantarum HEW-A490 effective active is 2.4 ×
1010CFU/g, enterococcus faecalis HEW-A131 effective active is 1.3 × 1010CFU/g, enterococcus faecalis HEW-A588 effective active are
1.3×1010CFU/g。
The preparation of embodiment 5 compound lactobacillus microbial ecological agent
Include (in terms of parts by weight) with the protective agent that differs only in of embodiment 1: 3 parts of trehaloses, 1.5 parts of polyethylene pyrroles
Cough up ketone, 1 part of mannitol, 3 parts of lactose, 0.3 part of VC, 0.5 part of VD, 0.6 part of VE, 2 parts of sodium thiosulfate, 1.5 parts of hydroxylmethyl cellulose
Element, 1.5 parts of arabic gums, 3 parts of fat-free comminuted wheys, 2 parts of glycerol, 2 parts of Tween 80s, 40 parts of corn starchs, 40 parts of water.Obtain answers
Closing lactobacillus micro-ecological preparation, its viable count reaches 6.0 × 1010CFU/g.Lactobacillus plantarum HEW-A490 effective active is 3 ×
1010CFU/g, enterococcus faecalis HEW-A131 effective active is 1.5 × 1010CFU/g, enterococcus faecalis HEW-A588 effective active are
1.5×1010CFU/g。
The preparation of embodiment 6 compound lactobacillus microbial ecological agent
With embodiment 1 differ only in three grade fermemtation liquid case study on implementation 1 step 4 obtained through 15000r/m centrifuge
Centrifugal 30-40min, it is thus achieved that the active bacteria mud of compound lactobacillus.And active bacteria mud is being stirred according to 0.2:1 weight ratio with protective agent
Mixing in tank and stir, by adding water, its humidity of regulation is maintained at 30-45%, obtains microcapsule wet-milling, puts in granulator and carry out
Pelletizing, be subsequently adding and be coated liquid so that it is form coatings, then be dried, make moisture≤6%, crossing 20 mesh sieves must be combined
Lactobacillus micro-ecological preparation, its viable count reaches 1.0 × 1010CFU/g.Lactobacillus plantarum HEW-A490 effective active is 6 ×
109CFU/g, enterococcus faecalis HEW-A131 effective active is 2 × 109CFU/g, enterococcus faecalis HEW-A588 effective active be 2 ×
109CFU/g。
The prebiotic performance of experimental example 1 compound lactobacillus microbial ecological agent and Detection of Stability
Laboratory sample: the compound lactobacillus microbial ecological agent of embodiment 1 preparation.
1. acid resistance measures
Weighing compound lactobacillus microbial ecological agent 1g in 9mL standard intraocular's gastric juice, after processing 1h, 2h and 3h, detection is lived
Bacterium number, respectively with process before the contrast of bacterial strain viable count, before treatment compound lactobacillus microbial ecological agent viable count be 8.0 ×
1010CFU/g, after processing 1h, viable count is 7.9 × 1010CFU/g；After processing 2h, viable count is 7.7 × 1010CFU/g；Process
After 3h, viable count is 7.5 × 1010CFU/g.After standard intraocular's gastric juice of pH1.5 processes 3h, compound lactobacillus microbial ecological agent
Survival rate reach 93.75%, show that microbial ecological agent has the highest acid resistance, gastric acid can be tolerated, arrive smoothly intestinal and play
Its effect.
2. bile tolerance performance measurement
Its concentration is made to be 1%, 2% and 3% by phosphate buffer adds Fel Sus domestica salt, standby after sterilizing, take 1g Composite Milk
Acid bacterium microbial ecological agent is in cholate-phosphate buffer, after processing 1h, 2h and 3h, detects viable count, respectively viable bacteria front with process
Number contrast, calculates survival rate, it is judged that the bile tolerance ability of bacterial strain.Result is as shown in table 1, and compound lactobacillus microbial ecological agent exists
3% cholate-MRS culture medium processes 3h survival rate and reaches 99.59%, show that this bacterial strain has higher bile tolerance performance, Ke Yi
Survive under intestinal height cholate environment, thus play a role.
Table 1 compound lactobacillus microbial ecological agent survival rate in variable concentrations cholate-MRS culture medium
3. resistance to elevated temperatures measures
Taking 1g compound lactobacillus microbial ecological agent respectively, 90 DEG C, 100 DEG C, 110 DEG C process 1min, answering after detection process
Close lactobacillus micro-ecological preparation viable count, contrast before processing, calculate survival rate.Result shows compound lactobacillus microbial ecological agent
90 DEG C, 100 DEG C, 110 DEG C process after survival rate be respectively 100%, 100%, 99.95%, show compound lactobacillus Tiny ecosystem
Preparation can tolerate high temperature, such that it is able to the high temperature of tolerance feed manufacturing, keeps activity.
The most resistance to high humidity performance measurement
Weighing 1g compound lactobacillus microbial ecological agent, 85 DEG C of steam process 1min, and the compound lactobacillus after detection processes is micro-
Ecological agent viable count, contrasts before processing, calculates survival rate.Result shows that the compound lactobacillus microbial ecological agent after processing is deposited
Motility rate reaches 92%, shows that compound lactobacillus microbial ecological agent can tolerate high humidity environment, can keep activity in Feed Manufacturing.
5. Detection of Stability
Compound lactobacillus microbial ecological agent product is sealed respectively the most at normal temperatures preserve January, 3 months, 6 months, 9 months,
After 12 months, detection Lactobacillus plantarum HEW-A490, enterococcus faecalis HEW-A131 and the work of tri-kinds of bacterium of enterococcus faecalis HEW-A588
Bacterium sum, measures viable bacteria retention ratio compared with initial viable count, and observes product characteristics, the results are shown in Table 2.
Table 2 room temperature preserves the impact on compound lactobacillus microbial ecological agent survival rate
As shown in Table 2, pig preserves after January, 3 months, 6 months, 9 months, 12 months sealing with probiotics, product
Shape is good, and its bacterium survival rate is all >=99.5%, shows that compound lactobacillus microbial ecological agent stability is the best.
Experimental example 2 compound lactobacillus microbial ecological agent attacks bacterium protection test
Laboratory sample: the compound lactobacillus microbial ecological agent of embodiment 1 preparation.
Taking the yellow-feather broiler 200 that 30 close ages in days of body weight are in a good state of health, I group is blank group, normally raises
Feed basal diet；II group is prevention group, after feeding basal diet+0.1% compound lactobacillus microbial ecological agent continuous one week, and abdominal cavity
Injection chicken pathogenic escherichia coli bacteria suspension；III group is treatment group, first lumbar injection chicken pathogenic escherichia coli bacteria suspension, then raises
Feed basal diet+0.1% compound lactobacillus microbial ecological agent to off-test；IV group is positive controls, through lumbar injection chicken
After Escherichia coli bacteria suspension, normally feed basal diet.Basal diet, all without antibiotic, is attacked bacterium and is observed after one week
And recording the health status of broiler, death condition, the mental status, appetite, feces etc., result is as shown in table 3.
Bacterium protection test result attacked by table 3
During test, the chicken of II group occurs the situation of lethargy after attacking bacterium, but a period of time back recovers normal.Ⅳ
Group chicken occurs that after attacking bacterium lassitude, feed intake and amount of drinking water decline, the situation of loose and watery stool, starts death occur, deposit after 24h
Motility rate only 56%.III group of chicken is after attacking bacterium compared with IV group, feeds compound lactobacillus microbial ecological agent, decreases the dead shape of chicken
Condition, and improve chicken diet and drinking-water situation.Show that compound lactobacillus microbial ecological agent can effectively suppress chicken pathogenic escherichia coli
Infection to chicken.
The impact on sow growth performance of the experimental example 3 compound lactobacillus microbial ecological agent
Laboratory sample: the compound lactobacillus microbial ecological agent of embodiment 1 preparation.
Select the sow 120 of the latter half of gestation that health, body condition, body weight and parity are close, be randomly divided into 4 groups, often organize 30
Head, often group sets 3 repetitions, each repetition 10.Preliminary experiment enters formal test after 1 week, test period I group feeds basal diet
For blank group；II feeds basal diet+0.05% compound lactobacillus microbial ecological agent；III group feeds basal diet+0.1%
Compound lactobacillus microbial ecological agent；IV feeds basal diet+0.15% compound lactobacillus microbial ecological agent.Experimental period is 80 days,
Other feedings and managements carry out according to conventional program and keep consistent.Record during test the total yield coefficient of every sow, young number of living,
Weak young number, birth piglet weight, sow actual band son's number etc., observe sow body condition simultaneously, record sow premature labor number, prolonged labor number, three
(mastitis, metritis, agalactia syndrome) morbidity number etc. levied by connection.Result is as shown in table 4, it is known that feed compound lactobacillus Tiny ecosystem
Preparation is remarkably improved sow total yield coefficient, young number of living, and reduces weak young number, stillborn fetus number, compared with matched group, adds various dose
Microbial ecological agent can improve piglet birth weight and piglet litter weight in various degree, come into being than matched group piglet respectively for II, III, IV group
Heavily improve 6%, 10%, 8.67%, weigth at birth significantly improves 15.60,19.24%, 21.33% (P < 0.05) respectively,
Weaned piglet incubation rate significantly improves 9.5%, 11.38%, 11.7% (P < 0.05) respectively, the most notable fall of sow early productivity
Low 10%, 16.67%, 16.67% (P < 0.05), sow prolonged labor rate significantly reduces 20%, 23.33%, 26.67% respectively
(P < 0.05), sow sickness rate significantly reduces 33%, 33.33%, 33.33% (P < 0.05).Sow body condition feed compound
After lactobacillus micro-ecological preparation, the most there is not situation about becoming thin in sow.In a word, compound lactobacillus microbial ecological agent can be effective
Improving the reproductive performance of sow, improve sow growth performance, reduce sow sickness rate, the growth for suckling pig provides guarantor
Barrier.
The impact on sow growth performance of the table 4 compound lactobacillus microbial ecological agent
Note: same column numerically marking-up parent phase is with person or unmarked person, and difference is not notable (P ＞ 0.05), and different person's difference shows
Write (P < 0.05).
Experimental example 4 compound lactobacillus microbial ecological agent is on piglet growth performance and the impact of enteric microorganism
Laboratory sample: the compound lactobacillus microbial ecological agent of embodiment 1 preparation.
Select the 40 age in days ablactational baby pig 120 that body weight is close, be randomly divided into 4 groups, often 3 repetitions of group, each repetition 10,
I group feeds basal diet is blank group；II feeds basal diet+0.05% compound lactobacillus microbial ecological agent；Raise for III group
Feed basal diet+0.1% compound lactobacillus microbial ecological agent；IV feeds basal diet+0.15% compound lactobacillus Tiny ecosystem system
Agent.All feeding piglets are in same pig house, and each group separately, is thrown something and fed 3 times every day, free choice feeding and drinking-water, every day clear excrement once,
Sterilization and immune programme for children manage routinely and carry out.Raise 42 days, personal management during test, every day entry piglet empty body weight,
Feed intake, diarrhoea and death condition.After off-test, often group 3 piglets of random choose, detection piglet ileum, caecum, colon and
Lactic acid bacteria in rectum section and colibacillary content, result is as shown in table 5.Compared with matched group, add compound lactobacillus system
Agent is remarkably improved piglet and in a few days increases weight, and reduces feedstuff-meat ratio, diarrhea rate and mortality rate, just can reach when addition is 0.05%
Remarkable result, shows that this microbial ecological agent can improve weaned piglets.Each intestinal segment E. CoIi content is along with compound lactobacillus
Microbial ecological agent increases, and all has reduction in various degree, especially colon with E. CoIi content in caecum compared with matched group,
Significantly reduce；Each intestinal segment content of lactic acid bacteria increases along with compound lactobacillus microbial ecological agent, all has and increases in various degree, especially
Caecum, colon, significantly improve with E. CoIi content in caecum compared with matched group.The micro-life of compound lactobacillus is added in feedstuff
State can substantially reduce the harmful bacteria quantity such as escherichia coli in intestinal, and can keep and improve the probiotics such as lactic acid bacteria in intestinal and contain
Amount, thus the invasion of Competitive assays pathogenic bacterium, field planting and breeding, thus improve piglet survival rate.
Table 5 compound lactobacillus microbial ecological agent is on piglet growth performance and the impact of fecal microorganism
Note: colleague's data different letter representation significant difference (P ＜ 0.05) of shoulder mark, takes on mark same letter or represents without shoulder mark
Difference is not notable (P ＞ 0.05)
Hereinafter Lactobacillus plantarum HEW-A490 is further described.
Lactobacillus plantarum (Lactobacillus plantarum) HEW-A490 is to separate from healthy chicken intestinal contents
Lactic acid bacillus mycopremna, determine that it is plant breast bar by colony morphological observation, physiological and biochemical property, molecular biology identification etc.
Bacterium.This bacterial strain has acid and alkali-resistance, resistance to elevated temperatures, strong stress resistance, and putrefaction bacteria is had inhibitory action etc..
Lactobacillus plantarum (Lactobacillus plantarum) HEW-A490 is in being preserved on May 27th, 2016
(being called for short CGMCC, address is: Chaoyang District, Beijing City North Star west at state's Microbiological Culture Collection administration committee common micro-organisms center
Road 1 No. 3 Institute of Microorganism, Academia Sinica of institute, postcode: 100101), deposit number is CGMCC NO.12554, point
The named Lactobacillus plantarum of class (Lactobacillus plantarum).
Lactobacillus plantarum (Lactobacillus plantarum) HEW-A490 has following microbial characteristic: at MRS
The raw bacterium colony of culture medium is creamy white, circular and regular edges, moistening and smooth, easily provoke, colony diameter is 2～3mm, bacterium colony shape
State is shown in Fig. 1；Microscopic morphology is shown in Fig. 2；Its thalline is rod-short, atrichia, without spore, Gram-positive；Lactobacillus plantarum HEW-
A490 facultative anaerobe, growth Suitable ranges: 4 DEG C-65 DEG C, optimum growth temperature: 25 DEG C-45 DEG C；Growth is suitable
PH2.5-10, optimum pH is 5-8；Some physiological-biochemical characteristics is shown in Table S1.
Table S1 Lactobacillus plantarum HEW-A490 some physiological-biochemical characteristics
Note: "+" represent reacting positive；"-" represents reaction negative.
The sequence of the 16S rDNA of bacterial strain HEW-A490 is carried out Blast comparison, from data with known array in GenBank
Storehouse obtains the 16S rDNA of relevant kind, phylogenetic tree construction, and by the cellular morphology of strain, physiological and biochemical property
Comprehensively determine that this bacterial strain belongs to Lactobacillus with experimental datas such as 16S rDNA sequences, be ultimately determined to Lactobacillus plantarum
(Lactobacillus plantarum)。
Lactobacillus plantarum HEW-A490 has significant bacteriostasis property, can effectively suppress Salmonella enteritidis, Pullorum Disease husky
The growth and breeding of the putrefaction bacteria such as door Salmonella, staphylococcus aureus, Pseudomonas aeruginosa, campylobacter jejuni and escherichia coli.
Lactobacillus plantarum HEW-A490 has stronger resistance, can simulated gastric fluid high temperature resistant, resistance to, bile tolerance, and
The survival rate of more than 90% can be kept.
The separation screening of Lactobacillus plantarum HEW-A490, qualification and preservation
1, Lactobacillus plantarum is isolated and purified
The aseptic healthy chicken intestinal contents that takes carries out enrichment culture in 100mL MRS fluid medium, draws 0.5mL rich
Collect culture fluid in 4.5mL sterile saline, carry out 10 times of gradient dilutions successively, select suitable dilution factor to take 0.1mL dilute
Releasing liquid on the culture medium flat plate of MRS+ calcium carbonate, coating uniformly, is inverted for 37 DEG C and is cultivated 24-48h, and picking produces molten calcium circle not
Streak inoculation pure culture is carried out with the bacterium colony of form, standby in 4 DEG C of preservations.
2, the observation of colonial morphology
Bacterium colony is creamy white on MRS flat board, circular, neat in edge, shows moistening smooth, easily provokes.
3, the screening of good plant lactobacillus
Microscopy chooses Gram-positive bacillus, and totally 58 strain of asporulate Lactobacillus plantarum, numbers respectively, by following side
Method is screened.
Hereinafter being only described the performance of bacterial strain HEW-A490, remaining vegetable lactobacillus performance is all not as bacterial strain HEW-
A490.1) prepared by bacterial strain HEW-A490 fermentation liquid
Bacterial strain HEW-A490 is rule on MRS inclined-plane, cultivates 20h, inclined-plane is added 2mL sterile saline system for 37 DEG C
Become bacteria suspension, bacteria suspension is transferred in 300mL seed culture medium, carries out shake flask fermentation cultivation, 37 DEG C, 180r/min concussion
15h, makes HEW-A490 fermentation liquid.Detection fermentation liquid viable count is 5.4 × 109CFU/mL, fermentation liquid is saved in refrigerator in 4 DEG C
Interior standby.
Wherein, seed culture medium is made up of following component: glucose 0.5%, sucrose 1.2%, yeast extract 0.8%, pancreas egg
White peptone 1.0%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate 0.05%, sodium chloride 0.25%, calcium carbonate 0.17%, surplus is water,
pH6.8±0.2.The condition of shake flask fermentation is: fermentation temperature 37 DEG C, and rotating speed is 180r/m, fermentation time 15h.
Second order fermentation is cultivated as a example by 50L fermentor cultivation, and 50L fermentation tank pilot scale culture medium is sucrose 1.5%, and Semen Maydis forms sediment
Powder 2.0%, Dried Corn Steep Liquor Powder 1.2%, yeast extract 1.4%, dipotassium hydrogen phosphate 0.08%, magnesium sulfate 0.05%, sodium chloride
0.2%, calcium carbonate 0.15%, manganese sulfate 0.03%, surplus is water, pH6.8 ± 0.2.
50L fermentation tank pilot scale fermentation condition is: liquid amount is 30L culture medium, and inoculum concentration is 300mL, and fermentation temperature is 37
DEG C, speed of agitator 120r/min, fermentation time 10h.
Three grade fermemtation is cultivated as a example by 5T fermentor cultivation, and 5T fermentation tank culture medium is identical with second order fermentation culture medium.5T
Ferment tank condition is: liquid amount is 3.5T culture medium, and inoculum concentration is 30L, and fermentation temperature is 37 DEG C, speed of agitator 110r/
Min, fermentation time 20h.Viable count is up to 2.5 × 1010CFU/mL。
2) acid resistance measures
Take the HEW-A490 fermentation liquid 10mL of preparation in 90mL standard intraocular's gastric juice, after processing 1h, 2h and 3h respectively, inspection
Surveying viable count, respectively bacterial strain viable count contrast front with process, Lactobacillus plantarum HEW-A490 fermentation liquid viable count is before treatment
5.4×109CFU/mL, after processing 1h, viable count is 5.2 × 109CFU/mL；After processing 2h, viable count is 5.0 × 109CFU/
mL；After processing 3h, viable count is 4.9 × 109CFU/mL.After standard intraocular's gastric juice of pH1.5 processes 3h, bacterial strain HEW-A490
Survival rate reach 90.74%, show that this bacterial strain has the highest acid resistance, gastric acid can be tolerated, arrive smoothly intestinal and play it and make
With.
3) bile tolerance performance measurement
Make its concentration be respectively 0.1%, 0.2% and 0.3% by MRS fluid medium adds Fel Sus domestica salt, make MRS-
Bile salt culture-medium, standby after sterilizing, take 10mL HEW-A490 fermentation liquid in MRS-bile salt culture-medium, process respectively 1h, 2h and
After 3h, detecting viable count, viable count contrast front with process, calculates survival rate, it is judged that the bile tolerance ability of bacterial strain respectively.Result is such as
Shown in table S2, bacterial strain HEW-A490 processes 3h survival rate in 3% cholate-MRS culture medium and reaches 97.69%, shows that this bacterial strain has
Higher bile tolerance performance, can survive under intestinal height cholate environment, thus play a role.
Table S2 bacterial strain HEW-A490 survival rate in variable concentrations cholate-MRS culture medium
4) resistance to elevated temperatures measures
Take 10mL bacterial strain HEW-A490 fermentation liquid in test tube, 85 DEG C of water-baths heat 15min, the bacterium after detection heating
Strain HEW-A490 fermentation liquid viable count, contrasts before processing, calculates survival rate.Result shows that the survival rate of bacterial strain HEW-A490 reaches
90%, show this bacterial strain can tolerate high temperature, such that it is able to the high temperature of tolerance feed manufacturing, keeps activity.
5) bacteriostasis property measures
By Salmonella enteritidis, S. pullonum, staphylococcus aureus, Pseudomonas aeruginosa, jejunum campylobacter bar
Bacterium and escherichia coli, as antibacterial indicator bacteria, prepare bacteria suspension respectively so that it is concentration is 109CFU/mL, is coated on Carnis Bovis seu Bubali cream egg
On white peptone culture medium flat plate, then the Oxford cup after sterilizing is placed on the flat board containing indicator bacteria, respectively to each Oxford cup
Middle addition 0.2mL concentration is 109CFU/mL bacterial strain HEW-A490 fermentation liquid.Do not make it excessive, cultivate 24-48h, then survey for 37 DEG C
Amount antibacterial circle diameter.Result is such as shown in Table S3, it is known that bacterial strain HEW-A490 is to Salmonella enteritidis, S. pullonum, golden yellow
This six pathogen strains bacterium of color staphylococcus, Pseudomonas aeruginosa, campylobacter jejuni and escherichia coli has relatively high inhibition effect, and it presses down
Bacterium performance is strong.
The table S3 bacterial strain HEW-A490 fungistatic effect to pathogen
6) adhesion property detection
The bacterial strain HEW-A490 PBS solution activated is cleaned 2-3 time, is prepared as 108The bacteria suspension of CFU/mL, will training
The HT-29 cell dissociation supported, the RPMI-1640 cell culture fluid using Gibco company of the U.S. to produce makes cell culture fluid
(5×104Individual/mL), addition 2mL cell suspension in the six every holes of orifice plate the most placing coverslip, 37 DEG C, CO2In incubator
Cultivate.After cell is adherent, aseptic PBS rinses 2 times, and every hole adds 1mL HEW-A490 bacterium solution (mycetome 1 × 108Individual/mL) with
The mixed liquor of 1mL1640 cell culture fluid, 37 DEG C, CO2Incubator is cultivated 2h.Aseptic PBS rinses, and 10% formaldehyde is fixed
0.5h, Gram’s staining, microscopy, 20 visuals field of random choose, calculate the bacterial population adhered on 50 cells.Result shows this bacterium
Strain reaches 27 in the adhesion number of Intestinal epithelium cell HT-29 cell, illustrates that the adhesive capacity of this bacterial strain is stronger.Probiotic bacteria is permissible
By adhering to enterocyte receptor, occupy-place field planting also stops pathogen to adhere to cell receptor, suppresses pathogen, regulates intestinal
Road colony balance.
Consider the acid-fast ability of each Lactobacillus plantarum, bile tolerance ability, heat-resisting ability, bacteriostatic activity and adhesiveness
Can, final acquisition one strain bacterium HEW-A490, its acid-fast ability, bile tolerance ability, heat-resisting ability and adhesion property are strong, normal to chicken
See pathogenic bacterium Salmonella enteritidis, S. pullonum, staphylococcus aureus, Pseudomonas aeruginosa, jejunum campylobacter
Bacillus and colibacillary good antimicrobial effect.
4, the qualification that bacterial strain belongs to
Bacterial strain HEW-A490 has a following microbial characteristic: be creamy white at the raw bacterium colony of MRS culture medium, circular and edge
Regular, moistening and smooth, easily to provoke, colony diameter is 2～3mm, and colonial morphology is shown in Fig. 1；Microscopic morphology is shown in Fig. 2；Its thalline in
Rod-short, atrichia, without spore, Gram-positive；Lactobacillus plantarum HEW-A490 facultative anaerobe, grows preference temperature model
Enclose: 4 DEG C-65 DEG C, optimum growth temperature: 25 DEG C-45 DEG C；Growth appropriate pH 2.5-10, optimum pH is 5-8.
Bacterial strain HEW-A490 is dyeed respectively, microscopy, choosing nonspore-bearing Gram positive bacillus, to carry out physiology raw
Change and test (catalase, gelatin liquefaction, nitrate reduction and sugar alcohols fermentation etc.), reference " common bacteria system identification handbook ",
" lactobacillus bacteria taxonomic identification and experimental technique " identifies the kind of bacterial strain.Result display bacterial strain HEW-A490 is plant breast bar
Bacterium.The some physiological-biochemical characteristics of this bacterium is shown in Table S1.
5,16S rDNA sequencing
Modified CTAB method is used to extract the genomic DNA of bacterial strain HEW-A490.Utilize universal primer to bacterial strain HEW-A490
16S rDNA genetic fragment carry out PCR amplification, PCR amplification system (25 μ L) includes primers F and each 1.0 μ L of R, and TaqDNA is polymerized
Enzyme 1.5U, 10 × Taq enzyme buffer 2.5 μ L, Mg2+(25mmoL/L) 1.5 μ L, strain gene group DNA 50ng.Reaction condition is:
94 DEG C of denaturations 5min；94 DEG C of degeneration 40s, 53 DEG C of annealing 1min, 72 DEG C extend 1min30s, totally 30 circulations；72 DEG C of temperature baths
10min.Reaction takes 3 μ L PCR primer after terminating and carries out electrophoresis detection on 1% agarose gel.Gained PCR primer reclaims pure
Change, send biological engineering (Shanghai) Co., Ltd. to carry out the mensuration of DNA sequence.By measured sequence and the 16S in GenBank
RDNA sequence carries out Blast analyses and comparison, and results strain HEW-A490 reaches 99.96% with the homology of lactobacillus.Pass through bacterium
The morphological characteristic of strain HEW-A490, physiological and biochemical property, 16S rDNA feature, determine that bacterial strain HEW-A490 is Lactobacillus plantarum
(Lactobacillus plantarum).The 16S rDNA sequence of bacterial strain HEW-A490 is as shown in sequence table.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Sequence table
<110>Beijing Hao Shiwo Bioisystech Co., Ltd
<120>a kind of compound lactobacillus microbial ecological agent and preparation method and application
<130> KHP161116615.4
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1428
<212> DNA
<213>Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
ggttcctaaa aggttacccc accgactttg ggtgttacaa actctcatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga ttactagcga 120
ttccgacttc atgtaggcga gttgcagcct acaatccgaa ctgagaatgg ctttaagaga 180
ttagcttact ctcgcgagtt cgcaactcgt tgtaccatcc attgtagcac gtgtgtagcc 240
caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg 300
cagtctcacc agagtgccca acttaatgct ggcaactgat aataagggtt gcgctcgttg 360
cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc acctgtatcc 420
atgtccccga agggaacgtc taatctctta gatttgcata gtatgtcaag acctggtaag 480
gttcttcgcg tagcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa 540
ttcctttgag tttcagcctt gcggccgtac tccccaggcg gaatgcttaa tgcgttagct 600
gcagcactga agggcggaaa ccctccaaca cttagcattc atcgtttacg gtatggacta 660
ccagggtatc taatcctgtt tgctacccat actttcgagc ctcagcgtca gttacagacc 720
agacagccgc cttcgccact ggtgttcttc catatatcta cgcatttcac cgctacacat 780
ggagttccac tgtcctcttc tgcactcaag tttcccagtt tccgatgcac ttcttcggtt 840
gagccgaagg ctttcacatc agacttaaaa aaccgcctgc gctcgcttta cgcccaataa 900
atccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg 960
ctttctggtt aaataccgtc aatacctgaa cagttactct cagatatgtt cttctttaac 1020
aacagagttt tacgagccga aacccttctt cactcacgcg gcgttgctcc atcagacttt 1080
cgtccattgt ggaagattcc ctactgctgc ctcccgtagg agtttgggcc gtgtctcagt 1140
cccaatgtgg ccgattaccc tctcaggtcg gctacgtatc attgccatgg tgagccgtta 1200
ccccaccatc tagctaatac gccgcgggac catccaaaag tgatagccaa agccatcttt 1260
caagctcgga ccatgcggtc caagttgtta tgcggtatta gcatctgttt ccaggtgtta 1320
tcccccgctt ctgggcaggt ttcccacgtg ttactcacca gttcgccact cactcaaatg 1380
taaatcatga tgcaagcacc aatcaatacc agagttcgtt cgacttgc 1428
Claims (10)
1. a compound lactobacillus microbial ecological agent, it is characterised in that its active bacteria includes Lactobacillus plantarum
(Lactobacillus plantarum) HEW-A490, its deposit number is CGMCC NO.12554；Enterococcus faecalis
(Enterococcus faecalis) and enterococcus faecalis (Enterococcus faecium).
Compound lactobacillus microbial ecological agent the most according to claim 1, it is characterised in that wherein Lactobacillus plantarum HEW-
The living bacteria count of A490 is 2.5 × 1010-5.0×1010CFU/g, the living bacteria count of enterococcus faecalis is 1.5 × 1010-3.0
×1010CFU/g, the living bacteria count of enterococcus faecalis is 2.0 × 1010-3.0×1010CFU/g；
Preferably, wherein the living bacteria count of Lactobacillus plantarum HEW-A490 is 4.0 × 1010CFU/g, the effective of enterococcus faecalis lives
Bacterium number is 2.0 × 1010CFU/g, the living bacteria count of enterococcus faecalis is 2.0 × 1010CFU/g。
Compound lactobacillus microbial ecological agent the most according to claim 1 and 2, it is characterised in that Lactobacillus plantarum HEW-
A490, enterococcus faecalis, the living bacteria count ratio of enterococcus faecalis are 2.5-5:1.5-3:2-3, preferably 2:1:1.
Compound lactobacillus microbial ecological agent the most according to claim 1 and 2, it is characterised in that described compound lactobacillus is micro-
In ecological agent, total living bacteria count is 6.0 × 1010-1.1×1011CFU/g, preferably 8.0 × 1010CFU/g。
Compound lactobacillus microbial ecological agent the most according to claim 1 and 2, it is characterised in that also include protective agent, described
Protective agent includes the component of following weight portion: 1-10 part trehalose, 1-3 part polyvidon, 2-5 part mannitol, 1-8 part breast
Sugar, 0.1-2 part VC, 0.1-2 part VD, 0.1-2 part VE, 0.5-5 part sodium thiosulfate, 1-5 part hydroxymethyl cellulose, 1-10 part Ah
Draw primary glue, 2-8 part fat-free comminuted whey, 2-10 part glycerol, 0.5-5 part Tween 80,20-40 part corn starch；Preferably, described guarantor
Protect agent and include the component of following weight portion: 5.5 portions of trehaloses, 2 parts of polyvidon, 2.4 portions of mannitol, 3 parts of lactose, 0.5
Part VC, 0.8 part of VD, 0.9 part of VE, 2 parts of sodium thiosulfate, 1.5 parts of hydroxymethyl cellulose, 3 parts of arabic gums, 5 parts of skimmed wheys
Powder, 3 parts of glycerol, 1.8 parts of Tween 80s, 35 parts of corn starchs, 35 parts of water；
And/or, in described compound lactobacillus microbial ecological agent, protectant weight percentage is 20-70%, preferably 35%.
6. according to the compound lactobacillus microbial ecological agent described in any one of claim 1-5, it is characterised in that described enterococcus faecalis
For enterococcus faecalis HEW-A131, its deposit number is CGMCC NO.9353；And/or, described enterococcus faecalis is enterococcus faecalis HEW-
A588, its deposit number is CGMCC No.10547.
7. the preparation method of compound lactobacillus microbial ecological agent described in any one of claim 1-6, it is characterised in that include following
Step:
1) Lactobacillus plantarum HEW-A490, enterococcus faecalis and enterococcus faecalis are carried out respectively first order seed fermentation, obtain one-level kind
Sub-culture fluid；
2) the first order seed culture fluid of Lactobacillus plantarum HEW-A490, enterococcus faecalis and enterococcus faecalis is inoculated in second order fermentation tank
In, fermentation culture, it is thus achieved that second order fermentation culture fluid；Or further second order fermentation culture fluid is inoculated in three grade fermemtation tank,
Fermentation culture, it is thus achieved that three grade fermemtation culture fluid；
3) described second order fermentation culture fluid or three grade fermemtation medium centrifugal are obtained compound lactobacillus active bacteria mud；By described work
Property bacterium mud be mixed in proportion with protective agent, pelletize, be coated, prepare described compound lactobacillus microbial ecological agent；
Wherein, Lactobacillus plantarum HEW-A490 primary-seed medium is: lactose 0.5-3%, sucrose 0.5-2%, whole milk powder
01-1.%, bean cake powder 0.5-2%, yeast leaching powder 0.5-3.%, NaH2PO40.1-0.7%, KH2PO40.1-0.7%, chlorination
Magnesium 0.01-0.1%, ferric ammonium citrate 0.01-0.1%, MnSO40.01-0.1%, calcium chloride 0.05-1%, surplus is water,
pH5.8-7.2；It is preferably lactose 1.25%, sucrose 0.8%, whole milk powder 0.5%, bean cake powder 1.5%, yeast leaching powder
0.85%, NaH2PO40.4%, KH2PO40.6%, magnesium chloride 0.05%, ferric ammonium citrate 0.04%, MnSO40.04%, chlorine
Changing calcium 0.1%, surplus is water, pH6.4 ± 0.2；
Enterococcus faecalis fermentation primary-seed medium is: sucrose 1-5%, soy peptone 0.5-2.5%, yeast extract 0.1-
1.0%, MgSO40.05-0.2%, MnSO40.01-1.0%, NaCl0.1-2.0%, dibasic ammonium citrate 0.1-0.5%,
CaCO30.1-1.0%, surplus is water, pH7.0 ± 0.2；Being preferably: sucrose 2.5%, soy peptone 1.8%, yeast extracts
Thing 0.4%, MgSO40.2%, MnSO40.045%, NaCl0.2%, dibasic ammonium citrate 0.2%, CaCO30.06%, surplus
For water, pH7.0 ± 0.2；
Enterococcus faecalis primary-seed medium is: sucrose 1-5%, glucose 0.5-1.5%, peptone 1-3%, yeast extract 0.5-
2.0%, MgSO40.1-0.5%, MnSO40.01-0.05%, NaCl0.5-2.0%, dibasic ammonium citrate 0.1-0.5%,
CaCO30.05-1.0%, surplus is water, pH6.8 ± 0.2；It is preferably: sucrose 1.5%, glucose 0.5%, peptone
1.6%, yeast extract 0.8%, MgSO40.3%, MnSO40.02%, NaCl 0.5%, dibasic ammonium citrate 0.1%, CaCO3
0.05%, surplus is water, pH6.8 ± 0.2.
8. the preparation method described in claim 7, it is characterised in that fermentation medium is made up of following component: molasses 1-3%,
Brown sugar 0.5-2%, Semen Maydis pulp 1-3%, yeast extract 0.5-2%, ammonium sulfate 0.1-0.5%, citric acid hydrogen diamine 0.01-0.5%,
K2HPO40.1-0.5%, calcium carbonate 0.2-1.0%, NaCl 0.2-1%, MnSO40.01-0.1%, surplus is water, pH6.0-
7.0；It is preferably: molasses 1.5%, brown sugar 0.85%, Semen Maydis pulp 1.2%, yeast extract 0.7%, ammonium sulfate 0.2%, hydrogen citrate
Diamidogen 0.03%, K2HPO40.1%, calcium carbonate 0.4%, NaCl 0.5%, MnSO40.031%, surplus is water, pH6.6 ±
0.2；
And/or, fermentation condition includes: cultivation temperature 28-45 DEG C, speed of agitator 80-200r/m, and fermentation time is 10-15h；Excellent
Electing as: cultivation temperature 35 DEG C, speed of agitator 110r/m, fermentation time is 15h.
9. method system described in compound lactobacillus microbial ecological agent described in any one of claim 1-6 or any one of claim 7-8
Standby compound lactobacillus microbial ecological agent is preparing animal feed or the application preparing in feed additive, or in livestock-raising
The application of non-treatment purpose.
10. contain compound lactobacillus microbial ecological agent described in any one of claim 1-6 or containing any one of claim 7-8 institute
The animal feed of compound lactobacillus microbial ecological agent prepared by the method for stating or feed additive.
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Denomination of invention: Compound lactic acid bacteria microecological preparation as well as preparation method and application thereof Effective date of registration: 20200628 Granted publication date: 20191220 Pledgee: Beijing ustron Tongsheng financing Company limited by guarantee Pledgor: BEIJING HESWOF BIOTECHNOLOGY Co.,Ltd. Registration number: Y2020990000669 |
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