CN105647830B - It is a kind of to enhance the composite bacteria agent of animal immunizing power, preparation method and applications - Google Patents
It is a kind of to enhance the composite bacteria agent of animal immunizing power, preparation method and applications Download PDFInfo
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Abstract
The present invention relates to animal-breeding fields, particularly relate to composite bacteria agent, the preparation method and applications of a kind of enhancing animal immunizing power.In the composite bacteria agent, remembered with number of cells, including following components: 1-10 parts of side spore bacillus brevis, 1-10 parts of candida utili, 5-15 parts of bacillus licheniformis, 5-15 parts of aspergillus oryzae.The preparation method of above-mentioned bacteria fermentation powder is also provided.Composite bacteria agent of the present invention, combination matching, and carry out mixed fermentation, it influences each other between each bacterial strain, the various nutritional ingredients in culture medium, after being added in animal feed, the colony balance of adjustable animal intestinal tract can more fully be utilized, strengthen immunity and stress ability, and helminth can be effectively prevented.
Description
Technical field
The present invention relates to animal-breeding fields, particularly relate to hybrid bacterial strain yeast powder, the system of a kind of enhancing animal immunizing power
Preparation Method and its application.
Background technique
The immune function of animal is executed by many immunocytes and immune molecule, immunocyte and immune molecule
Quantity and activity, determine the power of its immunity.The quantity of immune substance and activity are closely related with feed ingredient.Therefore think
Improve animal autoimmune function, it is necessary to pick up from the ingredient of feed.In the feed addictive used in recent years, has and much contain
It is improved the additive of animal immunizing power.The use of feed addictive is to sides such as immunity, the raising growth rates for improving animal
It has great promotion effect in face, and culture efficiency is very significant.But the much things of feed addictive used in current various countries
Learn synthetic drug or hormonal substance.With these additive feeding animals, usually there are various toxic side effects, make cause of disease
Bacterium generates anti-drug resistance, and the Harmful Residue increases in animal product.After people have eaten the animal product for feeding these additives,
The incidence for leading to problems such as deformity, cancer, early ageing, children precocious significantly improves, and seriously endangers human health.Therefore, respectively
Government, state, which has all made laws, limits the use of chemical classes feed addictive, and China has also put into effect a variety of decree regulations.
As degree of concern of the people to health is higher and higher, people increasingly pay attention to the Arrangement Problem of food.It removes
Except requirement basic food safety, health, green also become the selection and pursuit of more people.And the immunity of animal is to close
It is the key factor of animal disease resistant power.Therefore the function of immune system for how exciting animal itself, improves the immunity of animal, is
The emphasis studied during animal-breeding.
Summary of the invention
The invention proposes a kind of hybrid bacterial strain yeast powder for enhancing animal immunizing power, preparation method and applications, can
The intracorporal colony balance of animal is adjusted, the resistance and stress ability of animal are enhanced.
The technical scheme of the present invention is realized as follows:
A kind of composite bacteria agent, is remembered with number of cells, including following components: 1-10 parts of side spore bacillus brevis, Candida utilis
1-10 parts of yeast, 5-15 parts of bacillus licheniformis, 5-15 parts of aspergillus oryzae.
A kind of preparation method for the composite bacteria fermentation powder enhancing animal immune, comprising the following steps:
(1) culture medium needed for preparing composite bacteria fermentation；
(2) above-mentioned bacterial strains are activated respectively and bacterial strain fermentation liquor is made, take bacterial strain fermentation liquor obtained, mixed by formula ratio equal
It after even, is added in above-mentioned mix bacterium agent fermentation culture medium, culture obtains composite bacteria fermentation liquid in incubator, after drying to obtain the final product
Mix bacterium agent yeast powder.
Further, in the step (1), the culture medium includes the component of following parts by weight, 1-10 parts of glucose, sugarcane
1-8 parts sugared, 2-15 parts of wheat bran, 1-5 parts of peptone, 3-7 parts of coconut powder, 5-10 parts of defatted soy flour, 100-400 parts of purified water.
Further, in the fermentation culture medium, in terms of total number of bacteria, total bacterial strain concentration is 10 in composite bacteria agent6-108cfu/
mL。
Further, in the step (2), condition of culture is 35-40 DEG C, is cultivated 45-50 hours.
Further, in the step (2), the drying mode of composite bacteria agent is spray drying or freeze-drying.
Further, before spray drying, porous-starch is added in composite bacteria fermentation liquid, is uniformly mixed.
Application of the above-mentioned composite bacteria agent or composite bacteria fermentation powder on the preparation of preparation enhancing animal immunizing power.
Further, the animal is domestic animal.
Further, the animal is poultry.
Heretofore described bacterial strain is purchased from Chinese industrial Microbiological Culture Collection administrative center, and identified is respectively side
Spore bacillus brevis (Brevibacillus laterosporus), number CICC21185；Candida utili (Candida
Utilis), number CICC32211；Bacillus licheniformis (Bacillus licheniformis), number CICC10037；
Aspergillus oryzae (Aspergillus oryzae), number CICC2195.
Beneficial effects of the present invention:
Side spore bacillus brevis is the pathogen of non-spinal animal, has research to confirm there is nematicide effect.
Candida utili is called Torula utilis Henneb or edible torula.The content of its protein and vitamin B all compares beer
Brewer yeast is high, it can be using urea and nitric acid as nitrogen source, and not needing any growth factor is added in the medium can grow.It
Pentose and hexose can be utilized, nitrate can be assimilated, moreover it is possible to which it is edible to produce people and animals using molasses, wood hydrolysis liquid etc.
Protein.
Bacillus licheniformis, which can be adjusted, is in microecological balance between microorganism, between microorganism and host in enteron aisle,
Simultaneously participate in metabolism in enteron aisle, strengthen immunity and anti-stress ability.
Aspergillus oryzae is a kind of bacterial strain for producing complex enzyme, in addition to producing protease, can also produce amylase, carbohydrase, cellulase,
Phytase etc. can decompose the macromolecular substances in culture, be degraded, for the metabolism of other bacterial strains and breeding institute
It needs, is conducive to the efficiency for improving fermentation, improves the quantity of beneficial bacterium in bacterium powder；Porous-starch is added before spray drying, it can be with
The survival rate of bacterial strain in the drying process is protected, the vigor of bacterial strain in bacterium powder is improved.
Compound strain and compound strain yeast powder of the present invention by combination matching, and carries out mixed fermentation, each bacterium
It influences each other between strain, can be more fully using the various nutritional ingredients in culture medium, fermentation efficiency greatly improves, is added to dynamic
After in object feed, the colony balance of adjustable animal intestinal tract, strengthen immunity and stress ability reduce the disease incidence of animal,
The digestive system function for adjusting animal, greatly improves the utilization rate of feed, daily gain dramatically increases.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical solution in the present invention is clearly and completely described, it is clear that
Described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the implementation in the present invention
Example, every other embodiment obtained by those of ordinary skill in the art without making creative efforts belong to
The scope of protection of the invention.
Embodiment 1
A kind of composite bacteria agent, with bacterium number scale, including following components: 1 part of side spore bacillus brevis, candida utili
1 part, 5 parts of bacillus licheniformis, 5 parts of aspergillus oryzae.
A kind of preparation method for the composite bacteria fermentation powder enhancing animal immune, comprising the following steps:
(1) glucose 1g, sucrose 1g, wheat bran 2g, peptone 1g, coconut powder 3g, 5g parts of defatted soy flour mixings are taken, are added
Purified water 100g is uniformly mixed, culture medium needed for composite bacteria fermentation is made；
(2) above-mentioned bacterial strains are activated respectively and bacterial strain fermentation liquor is made, take bacterial strain fermentation liquor obtained, after mixing, bacterium
After strain fermentation liquid mixing, with bacterium number scale, including following components: 1 part of side spore bacillus brevis, 1 part of candida utili, ground
5 parts of clothing bacillus, 5 parts of aspergillus oryzae；It is added in above-mentioned mix bacterium agent fermentation culture medium；In terms of total number of bacteria, fermented and cultured
Total bacterial strain concentration is 10 in composite bacteria agent in object6cfu/mL.It is cultivated 45 hours in 35 DEG C of incubator and obtains composite bacteria fermentation
Porous-starch is added in liquid, is uniformly mixed, up to mix bacterium agent yeast powder after spray drying.
Composite bacteria agent or composite bacteria fermentation liquid obtained above can be used for preparing the preparation for improving immunity of poultry
Embodiment 2
A kind of composite bacteria agent, with bacterium number scale, including following components: 10 parts of side spore bacillus brevis, Candida utilis ferment
It is 10 parts, 15 parts of bacillus licheniformis, 15 parts of aspergillus oryzae female.
A kind of preparation method for the composite bacteria fermentation powder enhancing animal immune, comprising the following steps:
(1) glucose 10g, sucrose 8g, wheat bran 15g, peptone 5g, coconut powder 7g, defatted soy flour 10g are taken, mixes, adds
Enter purified water 400g, is uniformly mixed, culture medium needed for composite bacteria fermentation is made；
(2) above-mentioned bacterial strains are activated respectively and bacterial strain fermentation liquor is made, take bacterial strain fermentation liquor obtained, after mixing, bacterium
After strain fermentation liquid mixing, with bacterium number scale, including following components: 10 parts of side spore bacillus brevis, 10 parts of candida utili,
15 parts of bacillus licheniformis, 15 parts of aspergillus oryzae；It is added in above-mentioned mix bacterium agent fermentation culture medium；In terms of total number of bacteria, fermentation
Total bacterial strain concentration is 10 in composite bacteria agent in culture8cfu/mL；It is cultivated 50 hours in 40 DEG C of incubator and obtains composite bacteria agent
Fermentation liquid, up to mix bacterium agent yeast powder after freeze-drying.
Composite bacteria agent or composite bacteria fermentation liquid obtained above can be used for preparing the preparation for improving domestic animal immunity.
Embodiment 3
A kind of composite bacteria agent, with bacterium number scale, including following components: 5 parts of side spore bacillus brevis, candida utili
5 parts, 10 parts of bacillus licheniformis, 10 parts of aspergillus oryzae.
A kind of preparation method for the composite bacteria fermentation powder enhancing animal immune, comprising the following steps:
(1) glucose 5g, sucrose 5g, wheat bran 10g, peptone 3g, coconut powder 5g, defatted soy flour 7g are taken, is mixed, is added
Purified water 200g is uniformly mixed, culture medium needed for composite bacteria fermentation is made；
(2) above-mentioned bacterial strains are activated respectively and bacterial strain fermentation liquor is made, take bacterial strain fermentation liquor obtained, after mixing, bacterium
After strain fermentation liquid mixing, with bacterium number scale, including following components: 10 parts of side spore bacillus brevis, 10 parts of candida utili,
15 parts of bacillus licheniformis, 15 parts of aspergillus oryzae；It is added in above-mentioned mix bacterium agent fermentation culture medium, in terms of total number of bacteria, fermentation
Total bacterial strain concentration is 10 in composite bacteria agent in culture7cfu/mL；It is cultivated 48 hours in 38 DEG C of incubator and obtains composite bacteria agent
Porous-starch is added in fermentation liquid, is uniformly mixed, up to mix bacterium agent yeast powder after spray drying.
Composite bacteria agent or composite bacteria fermentation liquid obtained above, which can be used for preparing, improves poultry or domestic animal immunity
Preparation.
Influence of the mix bacterium agent yeast powder of the present invention to experimental animal immune power
Cleaning grade Kunming mouse 80, half male and half female, weight (20 ± 2) g is randomly divided into 8 groups, every group 10.Experiment
Group prepares mix bacterium agent yeast powder according to the method that 3 embodiments provide.Remove spore short brood cell in side in the composite bacteria agent of control group 1
Bacillus；Remove side spore bacillus brevis in the composite bacteria agent of control group 2；Remove lichens brood cell's bar in the composite bacteria agent of control group 3
Bacterium；Remove aspergillus oryzae in the composite bacteria agent of control group 4；Other operations are same as Example 3.Using weight as standard, experimental group is pressed
The administration standard of 0.04g/kg, gastric infusion；Using the physiological saline of peer accumulated amount as blank control group, continuous gavage 10
It.Influence by following metrics evaluation mix bacterium agent yeast powder to mouse immunity.Its result is shown in Table 1-3 respectively.
(1) Immune Organs Index: de- neck puts to death mouse, takes mouse thymus and spleen, after weighing, calculates thymus index and spleen
Dirty index.Thymus index=thymic weight/weight；Index and spleen index=spleen weight/weight
(2) immune function detects
The experiment of mouse carbonic clearance: it by 100mL/kg weight from 4 times of dilution of the injection of mouse tail vein clump of india ink, injects
After prepared Chinese ink respectively in 2min, 10min from intraocular 20 μ L of corner of the eyes venous blood sampling, and be added to 0.1% Na2CO3In solution 2mL,
600nm wavelength densitometric value (OD), with 0.1% Na2CO3Solution cleans up index K as blank control, calculating.
K=(lgOD1-lg OD2)/(t1-t2)
Delayed allergy measurement: mouse web portion is lost hair or feathers with vulcanized sodium, with the 2,4-dinitrofluorobenzene (letter of 10mg/mL
Claim DNFB) solution 40 μ L smear sensitization.Auris dextra two sides is applied to 10 μ L of the DNFB solution of same concentration after 5 days to be attacked.
It takes off neck afterwards for 24 hours and puts to death mouse, left and right auricular concha takes diameter 10mm auricle with punch, weighs, and evaluates delayed with the difference of two auricles
Allergy value.
(3) serum IL-2 measures
Eye socket takes blood 1.5mL, stands 30min, and 2500r/min is centrifuged 15min, takes supernatant, measures IL-2 in serum and contains
Amount.
1 mouse thymus of table and spleen organ index compare
As can be seen from Table 1, compared with blank control group, the thymus gland and spleen internal organs of 3 embodiment groups and control group 1-4
Index dramatically increases, and 3 embodiment group organ indexs are apparently higher than control group 1-4.Illustrate stomach-filling institute of the present invention
The composite bacteria fermentation powder stated can significantly improve the thymus gland and spleen organ index of mouse, remove any one component, effect
It is substantially reduced.The change of the organ index of thymus gland and spleen can react the state of body's immunity.Therefore, of the present invention
Composite bacteria fermentation powder can improve the immune function of mouse.
2 mouse carbonic clearance ability of table compares
As can be seen from Table 2,3 embodiment groups and control group 1-4 are more certain than blank control group mouse carbonic clearance index
The raising of degree；And the raising degree of 3 embodiment groups becomes apparent, compared with control group 1-4, significant difference.Illustrate this
The invention composite bacteria fermentation powder can significantly improve the non-specific row immune function of mouse, and effect is substantially better than control group
1-4。
3 Mice Auricle weight difference of table compares
By finding out in table 3, each embodiment group and control group 1-4 mouse or so auricle weight difference are obviously reduced, and 3 realities
Example group weight gain amplitude is applied significantly lower than control group 1-4, significant difference.Illustrate that composite bacteria agent of the present invention promotes delaying type to become
The effect of state reaction is substantially better than other groups, beneficial effect and its significant.
4 mice serum IL-2 comparision contents of table
It is obtained by table 4, compared with blank control group, 3 embodiment groups and control group 1-4 mice serum IL-2 obviously increase
Add, and 3 embodiment group increasing degrees are apparently higher than control group 1-4, there are significant differences.Illustrate of the present invention compound
Bacteria fermentation powder can significantly improve body soluble Interleukin-2, enhance body's immunity.Remove any of them portion, makees
It is substantially reduced with effect, difference is very significant.
In summary experimental data, illustrate composite bacteria fermentation powder of the present invention can increase immune organ thymus gland and
The weight of spleen improves body nonspecific immunity function, improves soluble Interleukin-2, and composite bacteria agent of the present invention is each
It influences each other between component, plays the role of collaboration and be added, the function and effect for improving mouse immunity are apparently higher than other groups, go
Fall any one component, effect has different degrees of decline, and there are notable differences.
Influence of the composite bacteria agent of the present invention to domestic animal immunity
Domestic animal 48 are selected similar in age in days respectively, is allocated as 8 groups at random, and 3 groups are experimental group, and 5 groups are control group, each place
Single column is managed to feed.Experimental group feeds the feed for being added to composite bacteria agent or composite bacteria fermentation powder of the present invention；Control
Group 1-4 prepares composite bacteria agent according to above method, and the ratio being added in feed is identical as experimental group, and blank control group is only
Feeding feed stuff.All other method for breeding of processing are all the same.Continuous feeding 3 months.Pig that domestic animal selects daily raising more and
Sheep.Continuous feeding 3 months, investigates influence of the composite bacteria agent of the present invention to domestic animal immunity.The result is shown in tables 5 and table 6.
Pig serum immunoglobulin is measured using following methods: randomly choosing 2 the 15th day and the 30th day early morning
Jugular vein blood collection, 37 DEG C of standings 2h, 4 DEG C of 4h, centrifugation, 3500rpm are centrifuged 8min, and serum, -20 DEG C of preservations are precipitated.Measure serum
Middle IgA, IgG and IgM concentration.
The changes of contents of 5 pig serum immunoglobulin of table
Table 5 the result shows that, blank photo group is compared, 3 embodiment groups and control group 1-4, immune globulin in Swine serum
Bai Hanliang has different degrees of raising, and the raising of 3 embodiment groups is more more significant than control group 1-4.Meanwhile the hair of pig
Sick rate also significantly decreases.Illustrate that composite bacteria agent and composite bacteria fermentation powder of the present invention can significantly improve exempting from for pig
Epidemic disease power, improves pig disease resistance, and function and effect are substantially better than other processing groups.
It has also been found that the weight gain of 3 embodiment group pigs is fast in experimentation, the state of mind is substantially better than other processing groups, pig manure
Just color burn illustrates that composite bacteria agent of the present invention can be improved the digestion power of pig, improves efficiency of feed utilization.
Influence of the composite bacteria agent of the present invention of table 6 to sheep disease incidence
Table 6 the result shows that, compared with other processing groups, add the resulting composite bacteria agent of the embodiment of the present invention, the hair of sheep
Sick rate is substantially reduced, and difference is very significant.Illustrate that composite bacteria agent and composite bacteria fermentation powder of the present invention can enhance sheep
Immunity, function and effect are substantially better than other processing groups.
In addition, by the administering transgenic of other domestic animals such as ox, horse, it was also found that adding composite bacteria agent of the present invention, family
Poultry disease incidence has different degrees of reduction, reduces amplitude generally 15% or so, illustrates composite bacteria agent of the present invention
The immunity of other domestic animals can be enhanced, there is significant beneficial effect.
Influence of the composite bacteria agent of the present invention to immunity of poultry
Test site, Ji Lan, wire mesh cage are unified into disinfection treatment before the test.It chooses and is good for similar in 328 1 age in days weight
Health chick, half male and half female.It is allocated as 8 groups at random, every group 45,3 groups are experimental group, and 5 groups are control group, and the single column of each processing is fed
It supports.Experimental group feeds the feed for being added to composite bacteria agent or composite bacteria fermentation powder of the present invention；Control group 1-4 according to
Above method prepares composite bacteria agent, and the ratio being added in feed is identical as experimental group, blank control group feeding feed stuff.Institute
There are other method for breeding of processing all the same.Each component component component group respectively randomly selects 10 in 20,30,45,65 ages in days respectively,
It cuts open and wins the bursa of farbricius, spleen after killing, after sucking surface moisture with filter paper, weighing calculates bursal index and spleen index, as a result sees
Table 7 and table 8.
Bursal index=bursa of farbricius weight in wet base (mg)/weight (g)
Spleen index=spleen weight in wet base (mg)/weight (g)
Influence of 7 composite bacteria agent of table to chicken bursa index
As can be seen from Table 7, with the increase of chick age in days, downward trend after first rising is presented in bursal index.Than
Compared with the same Japanese instar chickling of different disposal, the bursal index of 3 embodiment groups and control group 1-4 chick is above blank control
Group, and 3 embodiment group bursal index are apparently higher than control group 1-4, and difference is fairly obvious.Illustrate of the present invention multiple
Combined bacteria agent can increase the bursal index for improving chick, enhance the immunity of chicken.
Influence of 8 composite bacteria agent of table to spleen index
As can be seen from Table 8, compared with blank control group, the bursal index of 3 embodiment groups and control group 1-4 chick
It is significantly increased.Meanwhile the spleen index of 3 embodiments is apparently higher than control group 1-4.Illustrate to add 3 embodiments of the invention
Composite bacteria agent the spleen index of chicken can be improved, and by mixed fermentation between each bacterial strain of composite bacteria agent of the present invention, mutually
It mutually influences, generates synergistic effect, remove any one bacterial strain, function and effect significantly reduce.In experimentation also
It was found that the disease incidence of 3 embodiment group chickens is significantly lower than other groups, the feed intake of chicken increases, and speed of weight increment is apparently higher than other
Processing group, the abnormal excrement such as yellow excrement, green excrement significantly reduce, and chicken digestion power is substantially better than other processing groups.
In addition, being found by the administering transgenic to other poultry such as duck, geese, composite bacteria agent of the present invention can also be mentioned
The immune function of other high poultry, reduces the disease incidence of poultry, improves its digestion power, speed of weight increment also has different degrees of increasing
Add.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of composite bacteria agent, which is characterized in that in the composite bacteria agent, remembered with number of cells, including following components: side spore
1-10 parts of bacillus brevis, 1-10 parts of candida utili, 5-15 parts of bacillus licheniformis, 5-15 parts of aspergillus oryzae.
2. a kind of preparation method for the composite bacteria fermentation powder for enhancing animal immune, which comprises the following steps:
Composite bacteria agent described in claim 1 is added to culture in fermentation medium and obtains composite bacteria fermentation liquid, after dry
Up to mix bacterium agent yeast powder.
3. a kind of preparation method of composite bacteria fermentation powder for enhancing animal immune as claimed in claim 2, it is characterised in that:
Each bacterial strain is added to before fermentation medium in the composite bacteria agent, activates bacterial strain fermentation liquor is made respectively；The fermented and cultured
Base includes the component of following parts by weight, 1-10 parts of glucose, 1-8 parts of sucrose, 2-15 parts of wheat bran, 1-5 parts of peptone, coconut powder
3-7 parts, 5-10 parts of defatted soy flour, 100-400 parts of purified water.
4. a kind of preparation method of composite bacteria fermentation powder for enhancing animal immune as claimed in claim 2, it is characterised in that:
In the fermentation medium, in terms of total number of bacteria, total bacterial strain concentration is 10 in composite bacteria agent6-108cfu/mL。
5. a kind of preparation method of composite bacteria fermentation powder for enhancing animal immune as claimed in claim 2, it is characterised in that:
The condition of culture of the composite bacteria agent is 35-40 DEG C, is cultivated 45-50 hours.
6. a kind of preparation method of composite bacteria fermentation powder for enhancing animal immune as claimed in claim 2, which is characterized in that
The drying mode of the composite bacteria fermentation liquid is spray drying or freeze-drying.
7. a kind of preparation method of composite bacteria fermentation powder for enhancing animal immune as claimed in claim 6, it is characterised in that:
Before spray drying, porous-starch is added in composite bacteria fermentation liquid, is uniformly mixed.
8. composite bacteria agent as described in claim 1 or composite bacteria fermentation powder as claimed in claim 3 enhance in preparation
Application on the preparation of animal immunizing power.
9. as claim 8 application, it is characterised in that: the animal be domestic animal.
10. as claim 8 application, it is characterised in that: the animal be poultry.
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| CN102273548B (en) * | 2011-08-17 | 2012-10-31 | 湖南泰谷生物科技有限责任公司 | Feeding microecological preparation and preparation method thereof |
| CN103190532A (en) * | 2012-01-10 | 2013-07-10 | 康建国 | Poultry micro-ecological preparation |
| CN103224886B (en) * | 2013-04-24 | 2015-03-04 | 武汉市天辰生物科技有限公司 | Composite microbe agent for aquatic product culture and application thereof |
| CN103689218B (en) * | 2013-12-05 | 2015-12-02 | 中国农业大学 | A kind ofly improve the composite bacteria agent capable and application thereof that feed fibre degradation rate and positive regulation rumen fermentation propionic acid generate |
| CN103981129B (en) * | 2014-05-06 | 2016-07-27 | 武汉科前动物生物制品有限责任公司 | A kind of complex microorganism preparations and its preparation method and application |
| CN104126734B (en) * | 2014-08-22 | 2016-08-24 | 成都合成生物科技有限公司 | A kind of microbial ecological agent for animals and preparation method thereof |
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2016
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