CN107603911A - A kind of preparation method of Composite microbe inocula for aquatic product culture - Google Patents
A kind of preparation method of Composite microbe inocula for aquatic product culture Download PDFInfo
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Abstract
The present invention relates to field of biological product, and in particular to a kind of preparation method of Composite microbe inocula for aquatic product culture.The present invention includes bacillus subtilis, three floras of candida utili and Bifidobacterium, can be in water body in the presence of nutriment after application water body, quick breeding, form advantageous flora, oxygen demand is relatively low during use, can be with pollutants such as the residual baits in fast decoupled breeding water body, improve water bottom environment, the fermentate that the composite bacteria liquid matrix of centrifugal drying extraction is obtained by fermenting, add mixed trace elements and vermiculite is mixed, obtained complex micro organism fungicide by the suction-operated of vermiculite can rapid subsidence to water bottom, directly act on water bottom environment, with good stability and high efficiency, it is simultaneously micro- soluble in water in complex micro organism fungicide, it is easy to volatilize, trace element required for can supplementing in aquatic products growth course, improve the yield and quality of aquatic products.
Description
Technical field
The present invention relates to field of biological product, and in particular to a kind of preparation side of Composite microbe inocula for aquatic product culture
Method.
Background technology
Water body is that the existence of the aquatic livestocks such as fishes and shrimps grows had to rely on environment, and the quality of water quality is that aquaculture is special
It is the key of intensive culture success or failure.With developing rapidly for culture fishery, water pollution is increasingly severe.Breeding water body is dirty
Dye refers to the activity due to the mankind（Production, life etc.）, cultivation object itself secretion and excreta, bait is superfluous, medicine
The factors such as residual cause water body to destroy, the phenomenon of water quality factor dysequilibrium.In recent years, China's culture fishery is quickly grown,
Cultivation scale constantly expands.But the aquaculture majority in China is using the intensive aquaculture model of high density, seedling at present
Kind put excess in a suitable place to breed, heavy dressing bait throwing in, make residual bait, aquatile excreta and corpse, breeding water body basic sediment, each
The eutrophy factor one water body of coexistence such as kind organic and inorganic fertilizer, and then irregular change occurs, generation harmful substance such as ammonia nitrogen,
Nitrite, hydrogen sulfide etc., cause aquiculture animal morbidity even dead.And the discharge of trade effluent and sanitary sewage is undoubtedly
The pollution of breeding water body has been aggravated, has seriously constrained the sustainable development of China's culture fishery.
Due to often carrying out excessive fertilising bait throwing in breeding process so that a large amount of residual baits, fertilizer for fish feed material, cultivated animals row
Letting out ammonia nitrogen, nitrite, sulfide etc. caused by thing and biological remains turns into major pollutants, hence in so that breeding water body
Self-purification capacity degradation so that the trace element of aquatic products seriously lacks.
There are the restorative procedures such as physics, chemistry, biology applied to water treatment at present.Micro- life is mainly used in the present invention
Thing reparation, its be it is a kind of absorbed to environmental pollutants using microorganism, the technology of metabolic degradation.
Complex micro organism fungicide microorganism formulation made of two or more and the mutual not microorganism fungus kind of antagonism.
Such microbial inoculum typically has the good characteristics such as species complete, compatibility reasonable, functional, economic benefit height.
Complex micro organism fungicide is divided into bacterium microbial inoculum, unwrapping wire bacteria agent (as resisted by specific microbe species in its product
Raw mushroom), fungal inoculant (such as mycorrhizal fungi class), Azotica microbial inoculum；It is divided into root nodule mushroom microbial inoculum by its mechanism of action, consolidates
Nitrogen mushroom microbial inoculum, phosphorus decomposing mushroom microbial inoculum, potassium decomposing mushroom microbial inoculum；It can be divided into simple microbial bacterial agent by its product inclusion and answer
Close (or compound) microbial bacterial agent etc..
Generally speaking, composite microbic bacterial fertilizer can promote soil fertility, manufacture and assist crops to absorb nutrition, enhancing
Plant disease-resistant and drought-resistant ability.
Specifically, the middle water bottom of aquaculture at present is seriously polluted, can although carrying out processing using chemical agent
To reach preferable effect, but it is also easy to produce secondary pollution.
Therefore, invent a kind of composite microbic bacterial fertilizer used for aquiculture of function admirable, China's aquatic products is adapted to this
The demand of aquaculture, this will promote the development of China's aquaculture significantly.
The content of the invention
The technical problems to be solved by the invention：The technical problems to be solved by the invention：For current aquaculture
Middle water bottom is seriously polluted, although preferable effect can be reached by carrying out processing using chemical agent, is also easy to produce secondary dirt
A kind of the problem of dye, there is provided preparation method of Composite microbe inocula for aquatic product culture.
In order to solve the above technical problems, the present invention is using technical scheme as described below：
A kind of preparation method of Composite microbe inocula for aquatic product culture, the preparation method comprise the following steps：
（1）The primary nutrient solution of mixing bacterium powder addition is activated and is mixed, obtains mixed bacteria liquid, then mixed bacteria liquid is added
Expand culture in two level nutrient solution to exponential phase, adjust OD600Value, obtains mixed bacteria liquid to be dried；
（2）Above-mentioned mixed bacteria liquid to be dried is put into centrifuge and centrifuged, supernatant is removed, is precipitated, by precipitation, water and jelly
Dry protective agent is well mixed, and obtains bacteria suspension, and obtained bacteria suspension is moved into vacuum freeze drier and is freeze-dried, obtains drying
Thing；
（3）By above-mentioned dried object and the distilled water of 4 times of dried object quality, centrifuged after mixing, remove supernatant liquor, collected lower floor and sink
Form sediment, and by after lower sediment drying constant weight, cooling, obtain the matrix of composite bacteria agent；
（4）The matrix addition fermented nutritive liquid of above-mentioned composite bacteria agent is fermented, after the completion of fermentation, fermentate is collected, will send out
Ferment thing, mixed trace elements additive, the vermiculite of 100 mesh are well mixed, and are stood, and freeze-drying, are pulverized and sieved, and collect sieving
Grain, obtains Composite microbe inocula for aquatic product culture.
The step（1）Middle mixing bacterium powder includes bacillus subtilis, candida utili and Bifidobacterium, withered grass bud
The mass ratio of spore bacillus, candida utili and Bifidobacterium is 2：1：1, primary nutrient solution formula number meter, take ox
1.2 ~ 2 parts of meat extract, 2.5 ~ 5 parts of trypticase, 8 ~ 10 parts of potato full-powder, 12 ~ 15 parts of agar, 1.5 ~ 2.5 parts of glucose, NaCl 3
~ 5 parts, KH2PO41.2 ~ 2.5 parts, 0.6 ~ 1 part of Tween-80,0.2 ~ 0.8 part of NAC, 1000 parts of water, pH value 7.0 ±
0.2,121 DEG C of sterilizing 30min, obtains primary nutrient solution.
The step（1）The middle condition for expanding culture：Temperature is 30 ~ 35 DEG C, 18 ~ 24h of incubation time；OD600Regulation
It is worth for 1.5, if OD600＞ 1.5, OD is diluted to two level nutrient solution600=1.5；If OD600＜ 1.5 can first be centrifuged and be removed part afterwards
Supernatant, then adjust to OD600=1.5, the formula number meter of two level nutrient solution, take 12 ~ 17 parts of trypticase, peptone 1
~ 5 parts, 0.2 ~ 0.5 part of beef extract, 20 ~ 40 parts of maltose, 10 ~ 15 parts of glucose, 1 ~ 3 part of NaCl, 15 ~ 20 parts of agar,
KH2PO41.2 ~ 2.5 parts, MgSO4·7H20.8 ~ 1.6 part of O, 5 ~ 10 parts of vitamin, 1000 parts of water, pH value 7.2 ± 0.1,
121 DEG C of sterilizing 20min, obtain two level nutrient solution.
The step（2）Middle freeze drying protectant is that mass fraction is 40% glycerite, and precipitation, water and freeze drying protectant obtain
Mass ratio is 1：6：4.
The step（4）The formula number meter of middle fermented nutritive liquid, takes 10 ~ 15 parts of wheat bran, peptone 10 ~ 15
Part, 16 ~ 20 parts of millet powder, Na2CO31.2 ~ 2 parts, MgSO4·7H20.1 ~ 0.2 part of O, FeSO4·7H20.01 ~ 0.03 part of O,
2 ~ 6 parts of NaCl, 1000 parts of water, stir, obtain fermented nutritive liquid；The fermentation condition is 20 ~ 35 DEG C, and the time is 18 ~ 24h.
The step（4）Middle fermentate, mixed trace elements additive, 100 mesh vermiculite mass ratio be 2：1：6, it is micro-
The component of secondary element additive includes, and according to the mass fraction, takes FeSO4·7H20.03 ~ 0.05 part of O, ZnSO4·7H2O 0.01
~ 0.05 part, Na2SeO3 0.03 ~ 0.09 part, MgSO4·7H20.05 ~ 0.2 part of O, Ca (IO3)2 0.02 ~ 0.09 part.
Compared with other method, advantageous effects are the present invention：
（1）The present invention includes three bacillus subtilis, candida utili and Bifidobacterium floras, can be in water after application water body
In body in the presence of nutriment, quick breeding, advantageous flora is formed, oxygen demand is relatively low during use, can be quick
Residual bait, medicament residue, macromolecular organic pollution in decomposition breeding water body etc., degradation of ammonia nitrogen, phosphorus, nitrite, hydrogen sulfide
Deng material, water bottom environment is improved；
（2）The fermentate that the composite bacteria liquid matrix of centrifugal drying extraction of the present invention is obtained by fermenting, adds mixed trace elements
Mixed with vermiculite, obtained complex micro organism fungicide by the suction-operated of vermiculite can rapid subsidence to water bottom,
Water bottom environment is directly acted on, there is good stability and high efficiency, while the micro member in complex micro organism fungicide
Element is soluble in water, is easy to volatilize, the trace element required for can supplementing in aquatic products growth course, improves the yield of aquatic products
And quality.
Embodiment
Mix the preparation of bacterium powder：Bacillus subtilis, candida utili and Bifidobacterium in mass ratio 2:1:1 mixing is equal
It is even.
The preparation of primary nutrient solution：Primary nutrient solution formula number meter, takes 1.2 ~ 2 parts of beef extract, trypticase 2.5
~ 5 parts, 8 ~ 10 parts of potato full-powder, 12 ~ 15 parts of agar, 1.5 ~ 2.5 parts of glucose, 3 ~ 5 parts of NaCl, KH2PO41.2 ~ 2.5 parts,
0.6 ~ 1 part of Tween-80,0.2 ~ 0.8 part of NAC, 1000 parts of water, 7.0 ± 0.2,121 DEG C of sterilizing 30min of pH value, are obtained
Primary nutrient solution.
The preparation of two level nutrient solution liquid：The formula number meter of two level nutrient solution, takes 12 ~ 17 parts of trypticase, albumen
1 ~ 5 part of peptone, 0.2 ~ 0.5 part of beef extract, 20 ~ 40 parts of maltose, 10 ~ 15 parts of glucose, 1 ~ 3 part of NaCl, 15 ~ 20 parts of agar,
KH2PO41.2 ~ 2.5 parts, MgSO4·7H20.8 ~ 1.6 part of O, 5 ~ 10 parts of vitamin, 1000 parts of water, pH value 7.2 ± 0.1,
121 DEG C of sterilizing 20min, obtain two level nutrient solution.
The preparation of fermented nutritive liquid：The formula number meter of fermented nutritive liquid, takes 10 ~ 15 parts of wheat bran, and peptone 10 ~
15 parts, 16 ~ 20 parts of millet powder, Na2CO31.2 ~ 2 parts, MgSO4·7H20.1 ~ 0.2 part of O, FeSO4·7H2O 0.01~0.03
Part, 2 ~ 6 parts of NaCl, 1000 parts of water, stir, obtain fermented nutritive liquid；The fermentation condition be 20 ~ 35 DEG C, the time be 18 ~
24h。
The preparation of mixed trace elements additive：Trace mineral supplement is counted in parts by weight, takes FeSO4·7H2O 0.03
~ 0.05 part, ZnSO4·7H20.01 ~ 0.05 part of O, Na2SeO3 0.03 ~ 0.09 part, MgSO4·7H20.05 ~ 0.2 part of O, Ca
(IO3)2 0.02 ~ 0.09 part, obtain mixed trace elements additive.
A kind of preparation method of Composite microbe inocula for aquatic product culture of the present invention, comprises the following steps：
（1）Mixing bacterium powder is added in primary nutrient solution and is activated and is mixed, mixing bacterium powder and primary nutrient solution
Mass ratio is 1:9, mixed bacteria liquid is obtained, then mixed bacteria liquid is added in two level nutrient solution, the addition of mixed bacteria liquid is trained for two level
The 40% of nutrient solution volume, under conditions of 30 ~ 35 DEG C, expand culture to exponential phase, adjust OD600Value, if OD600＞ 1.5,
OD is diluted to two level nutrient solution600=1.5, if OD600＜ 1.5 can first be centrifuged and be removed part supernatant afterwards, then be adjusted to OD600=
1.5, obtain mixed bacteria liquid to be dried；
（2）Above-mentioned mixed bacteria liquid to be dried is put into centrifuge, 5000 r/min centrifuge 15 min, remove supernatant, obtain
Precipitation, precipitation, water and freeze drying protectant are well mixed, freeze drying protectant is that mass fraction is 40% glycerite, precipitation, water
And freeze drying protectant obtains mass ratio as 1:6:4, bacteria suspension is obtained, obtained bacteria suspension is moved into freeze in vacuum freeze drier and done
It is dry, -70 DEG C of pre-freeze 2h, obtain dried object, dried object water content≤3%；
（3）By above-mentioned dried object and the distilled water of 4 times of dried object quality, 4 DEG C of 5000 r/min centrifuges 10 min after mixing, moves
Supernatant is removed, is precipitated, repeats aforesaid operations twice, the component collection of nutrient solution in precipitation is removed, by centrifuge washing
Thalline, thalline is kept into 80 DEG C of freeze-day with constant temperature, 105 DEG C of holding more than 3h constant weights, dried object is cooled down, obtains the base of composite bacteria agent
Body；
（4）The matrix of above-mentioned composite bacteria agent addition fermented nutritive liquid is fermented, composite bacteria agent matrix and fermented nutritive liquid
Mass ratio is 2:6, fermentation condition is 20 ~ 35 DEG C, and the time is 18 ~ 24h, after the completion of fermentation, collects fermentate, obtains viable count
200000000000 ~ 400,000,000,000 cfu/g fermentate, add fermentate, mixed trace elements additive, 100 mesh vermiculite in mass ratio
2:1:6 is well mixed, stands, and freeze-drying, is pulverized and sieved with the sieve of 200 mesh, collects sieving particle, obtains used for aquiculture multiple
Close microbial bacterial agent.
Example 1
Mixing bacterium powder is added to 10h is activated and be mixed in primary nutrient solution, mixing bacterium powder and primary nutrient solution
Mass ratio is 1:9, mixed bacteria liquid is obtained, then mixed bacteria liquid is added in two level nutrient solution, the addition of mixed bacteria liquid is trained for two level
The 40% of nutrient solution volume, under conditions of 30 DEG C, expand culture to exponential phase, adjust OD600Value, if OD600＞ 1.5, use
Two level nutrient solution is diluted to OD600=1.5, if OD600＜ 1.5 can first be centrifuged and be removed part supernatant afterwards, then be adjusted to OD600=
1.5, obtain mixed bacteria liquid to be dried.
Using Drug sensitivity test, three kinds of bacterium of detection whether there is inhibitory action between each other.
The specific growing state of mixed bacteria liquid such as table 1
Table 1
| Strain | Mixed bacteria liquid growing state |
| Bacillus subtilis | + |
| Candida utili | + |
| Bifidobacterium | + |
| Bacillus subtilis, Bifidobacterium | + + |
| Bacillus subtilis, candida utili | + + |
| Candida utili, Bifidobacterium | + + |
| Bacillus subtilis, candida utili, Bifidobacterium | + + + |
Test result indicates that mutually without the antagonism relationship between three kinds of function bacteriums.
Embodiment 2
Mix the preparation of bacterium powder：Bacillus subtilis, candida utili and Bifidobacterium in mass ratio 2:1:1 is well mixed.
The preparation of primary nutrient solution：Primary nutrient solution formula number meter, takes 1.2 parts of beef extract, trypticase 2.5
Part, 8 parts of potato full-powder, 12 parts of agar, 1.5 parts of glucose, 3 parts of NaCl, KH2PO41.2 parts, 0.6 part of Tween-80, half Guang
0.2 part of amino acid, 1000 parts of water, 7.0 ± 0.2,121 DEG C of sterilizing 30min of pH value, obtain primary nutrient solution.
The preparation of two level nutrient solution liquid：The formula number meter of two level nutrient solution, takes 12 parts of trypticase, peptone 1
Part, 0.2 part of beef extract, 20 parts of maltose, 10 parts of glucose, 1 part of NaCl, 15 parts of agar, KH2PO41.2 parts, MgSO4·
7H20.8 part of O, 5 parts of vitamin, 1000 parts of water, 7.2 ± 0.1,121 DEG C of sterilizing 20min of pH value, obtain two level nutrient solution.
The preparation of fermented nutritive liquid：The formula number meter of fermented nutritive liquid, takes 10 parts of wheat bran, 10 parts of peptone,
16 parts of millet powder, Na2CO31.2 parts, MgSO4·7H20.1 part of O, FeSO4·7H20.01 part of O, 2 parts of NaCl, 1000 parts of water,
Stir, obtain fermented nutritive liquid；The fermentation condition is 20 DEG C, time 18h.
The preparation of mixed trace elements additive：Trace mineral supplement is counted in parts by weight, takes FeSO4·7H2O 0.03
Part, ZnSO4·7H20.01 part of O, Na2SeO3 0.03 part, MgSO4·7H20.05 part of O, Ca (IO3)2 0.02 part, it must mix
Trace mineral supplement.
A kind of preparation method of Composite microbe inocula for aquatic product culture of the present invention, comprises the following steps：
（1）Mixing bacterium powder is added to 10h is activated and be mixed in primary nutrient solution, mixing bacterium powder and primary nutrient solution
Mass ratio be 1:9, mixed bacteria liquid is obtained, then mixed bacteria liquid is added in two level nutrient solution, the addition of mixed bacteria liquid is two level
The 40% of nutrient solution volume, under conditions of 30 DEG C, expand culture to exponential phase, adjust OD600Value, if OD600＞ 1.5,
OD is diluted to two level nutrient solution600=1.5, if OD600＜ 1.5 can first be centrifuged and be removed part supernatant afterwards, then be adjusted to OD600=
1.5 obtain mixed bacteria liquid to be dried；
（2）Above-mentioned mixed bacteria liquid to be dried is put into centrifuge, 5000 r/min centrifuge 15 min, remove supernatant, obtain
Precipitation, precipitation, water and freeze drying protectant are well mixed, freeze drying protectant is that mass fraction is 40% glycerite, precipitation, water
And freeze drying protectant obtains mass ratio as 1:6:4, bacteria suspension is obtained, obtained bacteria suspension is moved into freeze in vacuum freeze drier and done
It is dry, -70 DEG C of pre-freeze 2h, obtain dried object, dried object water content≤3%；
（3）By above-mentioned dried object and the distilled water of 4 times of dried object quality, 4 DEG C of 5000 r/min centrifuges 10 min after mixing, moves
Supernatant is removed, is precipitated, repeats aforesaid operations twice, the component collection of nutrient solution in precipitation is removed, by centrifuge washing
Thalline, thalline is kept into 80 DEG C of freeze-day with constant temperature, 105 DEG C of holding more than 3h constant weights, dried object is cooled down, obtains the base of composite bacteria agent
Body；
（4）The matrix of above-mentioned composite bacteria agent addition fermented nutritive liquid is fermented, composite bacteria agent matrix and fermented nutritive liquid
Mass ratio is 2:6, fermentation condition is 20 DEG C, time 18h, after the completion of fermentation, collects fermentate, obtains viable count 200,000,000,000
Cfu/g fermentate, add fermentate, mixed trace elements additive, the vermiculite in mass ratio 2 of 100 mesh:1:6 is well mixed,
Stand, freeze-drying, pulverized and sieved with the sieve of 200 mesh, collect sieving particle, obtain Composite microbe inocula for aquatic product culture.
Embodiment 3
Mix the preparation of bacterium powder：Bacillus subtilis, candida utili and Bifidobacterium in mass ratio 2:1:1 is well mixed.
The preparation of primary nutrient solution：Primary nutrient solution formula number meter, takes 1.6 parts of beef extract, trypticase 3.5
Part, 9 parts of potato full-powder, 13 parts of agar, 2.0 parts of glucose, 4 parts of NaCl, KH2PO41.8 parts, 0.8 part of Tween-80, half Guang
0.6 part of amino acid, 1000 parts of water, 7.0 ± 0.2,121 DEG C of sterilizing 30min of pH value, obtain primary nutrient solution.
The preparation of two level nutrient solution liquid：The formula number meter of two level nutrient solution, takes 15 parts of trypticase, peptone 4
Part, 0.3 part of beef extract, 35 parts of maltose, 13 parts of glucose, 2 parts of NaCl, 17 parts of agar, KH2PO41.8 parts, MgSO4·
7H21.2 parts of O, 7 parts of vitamin, 1000 parts of water, 7.2 ± 0.1,121 DEG C of sterilizing 20min of pH value, obtain two level nutrient solution.
The preparation of fermented nutritive liquid：The formula number meter of fermented nutritive liquid, takes 13 parts of wheat bran, 13 parts of peptone,
18 parts of millet powder, Na2CO31.6 parts, MgSO4·7H20.1 part of O, FeSO4·7H20.02 part of O, 4 parts of NaCl, 1000 parts of water,
Stir, obtain fermented nutritive liquid；The fermentation condition is 28 DEG C, time 21h.
The preparation of mixed trace elements additive：Trace mineral supplement is counted in parts by weight, takes FeSO4·7H2O 0.04
Part, ZnSO4·7H20.04 part of O, Na2SeO3 0.07 part, MgSO4·7H20.10 part of O, Ca (IO3)2 0.06 part, it must mix
Trace mineral supplement.
A kind of preparation method of Composite microbe inocula for aquatic product culture of the present invention, comprises the following steps：
（1）Mixing bacterium powder is added to 10h is activated and be mixed in primary nutrient solution, mixing bacterium powder and primary nutrient solution
Mass ratio be 1:9, mixed bacteria liquid is obtained, then mixed bacteria liquid is added in two level nutrient solution, the addition of mixed bacteria liquid is two level
The 40% of nutrient solution volume, under conditions of 32 DEG C, expand culture to exponential phase, adjust OD600Value, if OD600＞ 1.5,
OD is diluted to two level nutrient solution600=1.5, if OD600＜ 1.5 can first be centrifuged and be removed part supernatant afterwards, then be adjusted to OD600=
1.5 obtain mixed bacteria liquid to be dried；
（2）Above-mentioned mixed bacteria liquid to be dried is put into centrifuge, 5000 r/min centrifuge 15 min, remove supernatant, obtain
Precipitation, precipitation, water and freeze drying protectant are well mixed, freeze drying protectant is that mass fraction is 40% glycerite, precipitation, water
And freeze drying protectant obtains mass ratio as 1:6:4, bacteria suspension is obtained, obtained bacteria suspension is moved into freeze in vacuum freeze drier and done
It is dry, -70 DEG C of pre-freeze 2h, obtain dried object, dried object water content≤3%；
（3）By above-mentioned dried object and the distilled water of 4 times of dried object quality, 4 DEG C of 5000 r/min centrifuges 10 min after mixing, moves
Supernatant is removed, is precipitated, repeats aforesaid operations twice, the component collection of nutrient solution in precipitation is removed, by centrifuge washing
Thalline, thalline is kept into 80 DEG C of freeze-day with constant temperature, 105 DEG C of holding more than 3h constant weights, dried object is cooled down, obtains the base of composite bacteria agent
Body；
（4）The matrix of above-mentioned composite bacteria agent addition fermented nutritive liquid is fermented, composite bacteria agent matrix and fermented nutritive liquid
Mass ratio is 2:6, fermentation condition is 27 DEG C, time 22h, after the completion of fermentation, collects fermentate, obtains viable count 300,000,000,000
Cfu/g fermentate, add fermentate, mixed trace elements additive, the vermiculite in mass ratio 2 of 100 mesh:1:6 is well mixed,
Stand, freeze-drying, pulverized and sieved with the sieve of 200 mesh, collect sieving particle, obtain Composite microbe inocula for aquatic product culture.
Embodiment 4
Mix the preparation of bacterium powder：Bacillus subtilis, candida utili and Bifidobacterium in mass ratio 2:1:1 is well mixed.
The preparation of primary nutrient solution：Primary nutrient solution formula number meter, take 2 parts of beef extract, 5 parts of trypticase, horse
10 parts of the full powder of bell potato, 15 parts of agar, 2.5 parts of glucose, 5 parts of NaCl, KH2PO42.5 parts, 1 part of Tween-80, NAC
0.8 part, 1000 parts of water, 7.0 ± 0.2,121 DEG C of sterilizing 30min of pH value, obtain primary nutrient solution.
The preparation of two level nutrient solution liquid：The formula number meter of two level nutrient solution, takes 17 parts of trypticase, peptone 5
Part, 0.5 part of beef extract, 40 parts of maltose, 15 parts of glucose, 3 parts of NaCl, 20 parts of agar, KH2PO42.5 parts, MgSO4·
7H2O1.6 parts, 10 parts of vitamin, 1000 parts of water, 7.2 ± 0.1,121 DEG C of sterilizing 20min of pH value, obtain two level nutrient solution.
The preparation of fermented nutritive liquid：The formula number meter of fermented nutritive liquid, takes 15 parts of wheat bran, 15 parts of peptone,
20 parts of millet powder, Na2CO32 parts, MgSO4·7H2O0.2 parts, FeSO4·7H20.03 part of O, 6 parts of NaCl, 1000 parts of water, are stirred
Mix uniformly, obtain fermented nutritive liquid；The fermentation condition is 35 DEG C, time 24h.
The preparation of mixed trace elements additive：Trace mineral supplement is counted in parts by weight, takes FeSO4·7H2O 0.05
Part, ZnSO4·7H20.05 part of O, Na2SeO3 0.09 part, MgSO4·7H20.2 part of O, Ca (IO3)2 0.09 part, it must mix micro-
Secondary element additive.
A kind of preparation method of Composite microbe inocula for aquatic product culture of the present invention, comprises the following steps：
（1）Mixing bacterium powder is added to 10h is activated and be mixed in primary nutrient solution, mixing bacterium powder and primary nutrient solution
Mass ratio be 1:9, mixed bacteria liquid is obtained, then mixed bacteria liquid is added in two level nutrient solution, the addition of mixed bacteria liquid is two level
The 40% of nutrient solution volume, under conditions of 35 DEG C, expand culture to exponential phase, adjust OD600Value, if OD600＞ 1.5,
OD is diluted to two level nutrient solution600=1.5, if OD600＜ 1.5 can first be centrifuged and be removed part supernatant afterwards, then be adjusted to OD600=
1.5, obtain mixed bacteria liquid to be dried；
（2）Above-mentioned mixed bacteria liquid to be dried is put into centrifuge, 5000 r/min centrifuge 15 min, remove supernatant, obtain
Precipitation, precipitation, water and freeze drying protectant are well mixed, freeze drying protectant is that mass fraction is 40% glycerite, precipitation, water
And freeze drying protectant obtains mass ratio as 1:6:4, bacteria suspension is obtained, obtained bacteria suspension is moved into freeze in vacuum freeze drier and done
It is dry, -70 DEG C of pre-freeze 2h, obtain dried object, dried object water content≤3%；
（3）By above-mentioned dried object and the distilled water of 4 times of dried object quality, 4 DEG C of 5000 r/min centrifuges 10 min after mixing, moves
Supernatant is removed, is precipitated, repeats aforesaid operations twice, the component collection of nutrient solution in precipitation is removed, by centrifuge washing
Thalline, thalline is kept into 80 DEG C of freeze-day with constant temperature, 105 DEG C of holding more than 3h constant weights, dried object is cooled down, obtains the base of composite bacteria agent
Body；
（4）The matrix of above-mentioned composite bacteria agent addition fermented nutritive liquid is fermented, composite bacteria agent matrix and fermented nutritive liquid
Mass ratio is 2:6, fermentation condition be 35 DEG C, time 24h, after the completion of fermentation, collect fermentate, obtain viable count 200,000,000,000 ~
400000000000 cfu/g fermentate, add fermentate, mixed trace elements additive, the vermiculite in mass ratio 2 of 100 mesh:1:6 is mixed
Close uniformly, stand, freeze-drying, pulverized and sieved with the sieve of 200 mesh, collect sieving particle, obtain composite microbial used for aquiculture
Thing microbial inoculum.
Comparative example：The microbial bacterial agent used for aquiculture of Shandong bio tech ltd production
Method：4 parts of aquaculture system 2L are taken, it is each to add embodiment 2,3 and 4 and the microbial bacterial agent 2ml prepared by comparative example,
Add microbial inoculum once within every three days, continuous dosing is after three weeks, measurement result.
Each component content table of water body such as table 2 before experiment
Table 2
| COD（mg/L） | NH3-N(mg/L） | Total nitrogen (mg/L） | Total phosphorus (mg/L） |
| 265 | 16.8 | 31.53 | 2.82 |
Water body detects concrete condition such as table 3 after adding microbial bacterial agent
Table 3
| Detection project | Embodiment 2 | Embodiment 3 | Embodiment 4 | Comparative example |
| COD（mg/L） | 35.9 | 27.6 | 15.3 | 48.65 |
| NH3-N(mg/L） | 1.77 | 1.32 | 0.69 | 3.59 |
| Total nitrogen (mg/L） | 3.29 | 2.68 | 0.97 | 6.88 |
| Total phosphorus (mg/L） | 1.21 | 0.95 | 0.23 | 1.97 |
From the foregoing, it will be observed that the microbial bacterial agent that the present invention is produced, the middle water bottom for effectively solving aquaculture is with serious pollution
Problem, and secondary pollution is not likely to produce, it is a kind of safe and efficient Composite microbe inocula for aquatic product culture, is worthy to be popularized and makes
With.
Claims (6)
1. a kind of preparation method of Composite microbe inocula for aquatic product culture, it is characterised in that the preparation method includes following step
Suddenly：
（1）The primary nutrient solution of mixing bacterium powder addition is activated and is mixed, obtains mixed bacteria liquid, then mixed bacteria liquid is added
Expand culture in two level nutrient solution to exponential phase, adjust OD600Value, obtains mixed bacteria liquid to be dried；
（2）Above-mentioned mixed bacteria liquid to be dried is put into centrifuge and centrifuged, supernatant is removed, is precipitated, by precipitation, water and jelly
Dry protective agent is well mixed, and obtains bacteria suspension, and obtained bacteria suspension is moved into vacuum freeze drier and is freeze-dried, obtains drying
Thing；
（3）By above-mentioned dried object and the distilled water of 4 times of dried object quality, centrifuged after mixing, remove supernatant liquor, collected lower floor and sink
Form sediment, and by after lower sediment drying constant weight, cooling, obtain the matrix of composite bacteria agent；
（4）The matrix addition fermented nutritive liquid of above-mentioned composite bacteria agent is fermented, after the completion of fermentation, fermentate is collected, will send out
Ferment thing, mixed trace elements additive, the vermiculite of 100 mesh are well mixed, and are stood, and freeze-drying, are pulverized and sieved, and collect sieving
Grain, obtains Composite microbe inocula for aquatic product culture.
2. the preparation method of complex micro organism fungicide according to claim 1, it is characterised in that the step（1）In mix
Closing bacterium powder includes bacillus subtilis, candida utili and Bifidobacterium, bacillus subtilis, candida utili and double
The mass ratio of discrimination bacillus is 2：1：1, primary nutrient solution formula number meter, take 1.2 ~ 2 parts of beef extract, trypticase 2.5 ~ 5
Part, 8 ~ 10 parts of potato full-powder, 12 ~ 15 parts of agar, 1.5 ~ 2.5 parts of glucose, 3 ~ 5 parts of NaCl, KH2PO41.2 ~ 2.5 parts, tell
- 80 0.6 ~ 1 parts of temperature, 0.2 ~ 0.8 part of NAC, 1000 parts of water, 7.0 ± 0.2,121 DEG C of sterilizing 30min of pH value, are obtained
Primary nutrient solution.
3. the preparation method of complex micro organism fungicide according to claim 1, it is characterised in that the step（1）Middle expansion
The condition cultivated greatly：Temperature is 30 ~ 35 DEG C, 18 ~ 24h of incubation time；OD600Regulated value be 1.5, if OD600＞ 1.5, with two
Level nutrient solution is diluted to OD600=1.5；If OD600＜ 1.5 can first be centrifuged and be removed part supernatant afterwards, then be adjusted to OD600=
1.5, the formula number meter of two level nutrient solution, take 12 ~ 17 parts of trypticase, 1 ~ 5 part of peptone, 0.2 ~ 0.5 part of beef extract,
20 ~ 40 parts of maltose, 10 ~ 15 parts of glucose, 1 ~ 3 part of NaCl, 15 ~ 20 parts of agar, KH2PO41.2 ~ 2.5 parts, MgSO4·
7H20.8 ~ 1.6 part of O, 5 ~ 10 parts of vitamin, 1000 parts of water, 7.2 ± 0.1,121 DEG C of sterilizing 20min of pH value, obtain two level
Nutrient solution.
4. the preparation method of complex micro organism fungicide according to claim 1, it is characterised in that the step（2）Middle jelly
Dry protective agent is that mass fraction is 40% glycerite, and precipitation, water and freeze drying protectant obtain mass ratio as 1：6：4.
5. the preparation method of complex micro organism fungicide according to claim 1, it is characterised in that the step（4）Middle hair
The formula number meter of ferment nutrient solution, take 10 ~ 15 parts of wheat bran, 10 ~ 15 parts of peptone, 16 ~ 20 parts of millet powder, Na2CO3
1.2 ~ 2 parts, MgSO4·7H20.1 ~ 0.2 part of O, FeSO4·7H20.01 ~ 0.03 part of O, 2 ~ 6 parts of NaCl, 1000 parts of water, stirring
Uniformly, fermented nutritive liquid is obtained；The fermentation condition is 20 ~ 35 DEG C, and the time is 18 ~ 24h.
6. the preparation method of complex micro organism fungicide according to claim 1, it is characterised in that the step（4）Middle hair
Ferment thing, mixed trace elements additive, 100 mesh vermiculite mass ratio be 2：1：6, the component of trace mineral supplement includes,
According to the mass fraction, FeSO is taken4·7H20.03 ~ 0.05 part of O, ZnSO4·7H20.01 ~ 0.05 part of O, Na2SeO3 0.03~
0.09 part, MgSO4·7H20.05 ~ 0.2 part of O, Ca (IO3)2 0.02 ~ 0.09 part.
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Cited By (3)
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| CN108813395A (en) * | 2018-04-24 | 2018-11-16 | 贵州梵净山生态农业股份有限公司 | A kind of fermented beef fabricated product of low nitrite content |
| CN108813396A (en) * | 2018-04-24 | 2018-11-16 | 贵州梵净山生态农业股份有限公司 | A kind of application of multi-strain fermentation agent in beef processing |
| CN111808764A (en) * | 2019-04-11 | 2020-10-23 | 河北维尔利动物药业集团有限公司 | Preparation method of bacillus subtilis composite liquid microbial inoculum for preventing and treating epidemic diseases of livestock and poultry |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108813395A (en) * | 2018-04-24 | 2018-11-16 | 贵州梵净山生态农业股份有限公司 | A kind of fermented beef fabricated product of low nitrite content |
| CN108813396A (en) * | 2018-04-24 | 2018-11-16 | 贵州梵净山生态农业股份有限公司 | A kind of application of multi-strain fermentation agent in beef processing |
| CN111808764A (en) * | 2019-04-11 | 2020-10-23 | 河北维尔利动物药业集团有限公司 | Preparation method of bacillus subtilis composite liquid microbial inoculum for preventing and treating epidemic diseases of livestock and poultry |
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Application publication date: 20180119 |