CN109810929A - A method of utilizing the clay standby active microbial inoculum of cyanobacteria - Google Patents
A method of utilizing the clay standby active microbial inoculum of cyanobacteria Download PDFInfo
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- CN109810929A CN109810929A CN201910237935.4A CN201910237935A CN109810929A CN 109810929 A CN109810929 A CN 109810929A CN 201910237935 A CN201910237935 A CN 201910237935A CN 109810929 A CN109810929 A CN 109810929A
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Abstract
The invention discloses a kind of methods using the clay standby active microbial inoculum of cyanobacteria, belong to the fields such as biotechnology, feed addictive.The method of the present invention includes the following steps: the auxiliary materials such as cyanobacteria mud stalk, wheat bran being uniformly mixed, the solid medium that moisture content is 30%-60% is prepared into, after sterilizing, being cooled to room temperature, bacillus is inoculated with, solid fermentation culture is carried out at 37 DEG C.The present invention carries out resource utilization using cyanobacteria mud as raw material, to cyanobacteria mud, and cyanobacteria solid medium culture bacillus is made after auxiliary material adjusts moisture content, high pressure steam sterilization, can be used for feed addictive and agricultural production.
Description
Technical field
The present invention relates to a kind of methods using the clay standby active microbial inoculum of cyanobacteria, belong to biotechnology, feed addition
The fields such as agent.
Background technique
Freshwater lake and river seriously affect environmental ecology and drinking water source since eutrophy causes cyanobacterial bloom, such as
Taihu Lake cyanobacteria is frequently broken out every year, causes serious crisis to locality.It is mainly carried out at present by salvaging mode for Taihu Lake cyanobacteria
It administers, salvages within such as 2017 nearly 11,790,000 tons, dry matter calculates about 110,000 tons.40~70% are mainly contained in cyanobacteria dry matter
Protein and 18~25% polysaccharide.Place forms cyanobacteria mud to salvaging and blue algae dewatering and carries out financial subsidies, occupies big
Social public resource is measured, therefore especially urgent to the exploitation of the further application technology as the second resource of cyanobacteria mud.
Cyanobacteria mud resource comparative maturity technology includes compost fermentation, biogas fermentation and biological plastics.But blue algae fermentation
Manufactured compost not exclusively agrees with agricultural planting season；Cyanobacteria it is carbon containing it is lower it is unfavorable to biogas fermentation (need external source to supplement, such as with
Pig manure common fermentation), forming a large amount of algae-residues also can not further utilize, and discarding causes secondary pollution；If as biological plastics,
Price depends on wherein protein content, and manufactured plastics have cyanobacteria stink.In addition there are cyanobacteria muddy water solutions to prepare amino acid, uses
In foliar fertilizer or feed addictive, but condition is harsh (high temperature and concentrated acid), secondary pollution easy to form, and cost is also higher.Separately
Outside, also have been reported that cyanobacteria mud is used to prepare single cell protein for culture yeasts, but algae toxin present in it is potential hidden
Suffer from.In addition, cyanobacteria mud, which also has, can be used for Trichoderma fermentation, the biocontrol agent as plant, crops.
Summary of the invention
The present invention makes full use of the abundant enzyme system of cyanobacteria mud abundant nutrition and bacillus, is consolidated by adding stalk
State fermentation, preparation form the microecological microbial agent of no or low algae toxin, can be applied to herding and culture fishery.Meanwhile stalk is straight
It fetches derived from livestock-raising field, agricultural wastes, waste recycling can be carried out.
The first purpose of the invention is to provide a kind of methods for preparing microecological microbial agent, and the method is with cyanobacteria mud and straw
Stalk is raw material, and the microorganism for being inoculated with bacillus carries out solid state fermentation.
In one embodiment of the invention, the specific steps of the method include:
(1) cyanobacteria mud is uniformly mixed with auxiliary material by 1:0.5-2.5, sterilizes, prepares solid medium；
(2) gemma for being 4.5-5.4 by every 100g inoculation 1~5mL OD value in the solid medium obtained to step (1)
Bacillus bacterium solution ferments in 30-37 DEG C, obtains microbial bacterial agent.
In one embodiment of the invention, the microorganism of the bacillus includes but is not limited to bacillus subtilis
Bacterium, bacillus licheniformis or bacillus megaterium.
In one embodiment of the invention, the cyanobacteria is Microcystis aeruginosa, anabena, synnema algae, pseudo- anabena, quasi- column
One or more of born of the same parents algae, the algae that quivers, floating silk algae, sheath silk algae, section ball algae, Shu Qiuzao.
Further, the moisture content of the cyanobacteria mud is 85-95%.
Further, the cyanobacteria mud is the fresh cyanobacteria mud that algae moisture place leaving from station is salvaged, or protecting after salvaging in low temperature
The cyanobacteria mud deposited, or moisture content after dehydration are the blue alga cake of 50-80%.
Further, in step (1), the auxiliary material is stalk, wheat bran or combinations thereof.
Further, in step (1), the sterilising conditions are 121 DEG C of high pressure steam sterilization 30min, are gone out in steam
It is applicable under bacterium or other sterilising conditions under the conditions of boiling etc..
Further, in step (1), the cyanobacteria solid medium contains: 30-60 parts of cyanobacteria mud, stalk, wheat bran
Or combinations thereof 40-70 parts.
Further, in step (1), the cyanobacteria solid medium also contains glucose and NaCl, KH2PO3、
K2HPO3、MgSO4、FeSO4、GaCl2At least one of equal inorganic salts.
Further, in step (1), the moisture content of the cyanobacteria solid medium is 30%-60%.
Further, in step (2), the fermented incubation time 24-72h.
In one embodiment of the invention, the microbial bacterial agent that the method also obtains step (2) is dried.
In one embodiment of the invention, it is also smashed after drying, obtains solid powder microbial inoculum.
A second object of the present invention is to provide the microbial bacterial agents prepared using any of the above-described method.
Application of the microbial bacterial agent in terms of preparing animal feed is also claimed in the present invention.
In one embodiment of the invention, the application is that animal feed is added in the microbial inoculum according to a certain percentage
In, stirring feeds livestock.
In one embodiment of the invention, the livestock includes but is not limited to ox, pig, sheep.
The utility model has the advantages that the present invention carries out resource utilization for bloom blue algae is salvaged, pass through the cyanobacteria that will be salvaged and auxiliary material
Solid state fermentation is carried out after sterilizing is simply mixed, takes full advantage of and contains the nutrition group such as a large amount of albumen (40-60%), polysaccharide in cyanobacteria
At feature, simple raw material is cooperated to carry out solid state fermentation using bacillus, free amino acid total amount reaches in the product after making fermentation
1000mg/100g or more, hydrolysis amino acid total amount utilize the abundant enzyme system of bacillus to cyanobacteria mud simultaneously up to 15.16g/100g
Middle algae toxin MC degrades, and after the 60h that ferments, can't detect algae toxin in culture medium.
Detailed description of the invention
Fig. 1 is algae toxin mark product chromatogram；
Fig. 2 is algae toxin standard quality spectrogram；
Fig. 3 is the chromatogram of fermentation 0h sample (a) and algae toxin standard specimen (b)；
Fig. 4 is the chromatogram of fermentation sample (a) and algae toxin standard specimen (b) for 24 hours；
Fig. 5 is the chromatogram of fermentation 48h sample (a) and algae toxin standard specimen (b)；
Fig. 6 is the chromatogram of fermentation 60h sample (a) and algae toxin standard specimen (b).
Specific embodiment
In conjunction with specific embodiments, a specific embodiment of the invention is furtherd elucidate, following embodiment is for illustrating
The present invention, but be not intended to limit the scope of the invention.
The bacillus that the present invention uses includes:
1. bacillus subtilis is commercialized bacterial strain, number ATCC 14579；
2. bacillus licheniformis, number CICIM B0109 is purchased from Southern Yangtze University's Chinese Universities ' industrial microorganism platform.
The preparation of 1 bacillus seed liquor of embodiment
Culture medium involved in the present embodiment:
LB liquid medium formula: the addition 5g yeast extract into 1L distilled water, 10g tryptone, 10gNaCl,
NaOH tune pH7.0 or so, 121 DEG C of high pressure steam sterilization 20-30min；
LB solid culture based formulas: the addition 15-20g agar powder into 1L LB liquid medium, NaOH tune pH7.0 or so,
121 DEG C of high pressure steam sterilization 20-30min.
Withered grass or bacillus licheniformis are seeded on LB solid medium and activated, after 37 DEG C of plate culture 16-24h, is chosen
Take single colonie to be inoculated into LB liquid medium, 37 DEG C, 200rpm shaking table grow to the logarithm middle and later periods, obtain OD value in 4.5-
5.4 bacillus seed liquor.
The solid state fermentation culture of 2 bacillus subtilis of embodiment
Cyanobacteria solid medium used in the present embodiment: 53 parts, 47 parts stalk mixing of cyanobacteria mud (moisture content 93%) are equal
It is even, it is dispensed into triangular flask, 121 DEG C of high pressure steam sterilization 30min, obtains the cyanobacteria culture medium that moisture content is 50%.
The bacillus subtilis seed liquor that embodiment 1 is obtained is inoculated on cyanobacteria culture medium by volume mass than 1%,
After mixing evenly, be placed in 37 DEG C of incubators and cultivate 48h, interval is stirred, final gemma viable count up to 2.6 ×
109cfu/g。
The solid state fermentation culture of 3 bacillus licheniformis of embodiment
Cyanobacteria solid medium used in the present embodiment: 53 parts, 47 parts stalk mixing of cyanobacteria mud (moisture content 93%) are equal
It is even, it is dispensed into triangular flask, 121 DEG C of high pressure steam sterilization 30min, obtains the cyanobacteria culture medium that moisture content is 50%.
The bacillus licheniformis seed liquor that embodiment 1 is obtained is inoculated on cyanobacteria culture medium by volume mass than 1%,
After mixing evenly, be placed in 37 DEG C of incubators and cultivate 48h, interval is stirred, final gemma viable count up to 2 ×
109cfu/g。
The solid state fermentation culture of 4 bacillus subtilis of embodiment
Cyanobacteria solid medium used in the present embodiment: 53 parts, 47 parts wheat bran mixing of cyanobacteria mud (moisture content 93%) are equal
It is even, it is dispensed into triangular flask, 121 DEG C of high pressure steam sterilization 30min, obtains the cyanobacteria culture medium that moisture content is 50%.
The bacillus subtilis seed liquor that embodiment 1 is obtained is inoculated on cyanobacteria culture medium by volume mass than 1%,
After mixing evenly, be placed in 37 DEG C of incubators and cultivate 48h, interval is stirred, final gemma viable count up to 2.3 ×
1010cfu/g。
Nutritional ingredient in tunning is detected, wherein free amino acid total amount reaches 1002mg/100g, hydrolyzes ammonia
Base acid total amount reaches 17.31g/100g.
The solid state fermentation culture of 5 bacillus licheniformis of embodiment
Cyanobacteria solid medium used in the present embodiment: 53 parts, 47 parts wheat bran mixing of cyanobacteria mud (moisture content 93%) are equal
It is even, it is dispensed into triangular flask, 121 DEG C of high pressure steam sterilization 30min, obtains the cyanobacteria culture medium that moisture content is 50%.
The bacillus licheniformis seed liquor that embodiment 1 is obtained is inoculated on cyanobacteria culture medium by volume mass than 1%,
After mixing evenly, be placed in 37 DEG C of incubators and cultivate 48h, interval is stirred, final gemma viable count up to 2.8 ×
1010cfu/g。
Kjeldahl determination is carried out to tunning, as the result is shown: protein is from the 18.8% to fermentation 48h of fermentation 0h
25%, increase 33%.
Nutritional ingredient in tunning is detected, wherein free amino acid total amount reaches 1450mg/100g, hydrolyzes ammonia
Base acid total amount reaches 14.47g/100g.
The solid state fermentation culture of 6 bacillus licheniformis of embodiment
Cyanobacteria solid medium used in the present embodiment: 63 parts, 37 parts wheat bran mixing of cyanobacteria mud (moisture content 93%) are equal
It is even, it is dispensed into triangular flask, 121 DEG C of high pressure steam sterilization 30min, obtains the cyanobacteria culture medium that moisture content is 60%.
The bacillus licheniformis seed liquor that embodiment 1 is obtained is inoculated on cyanobacteria culture medium by volume mass than 1%,
After mixing evenly, culture, interval in 37 DEG C of incubators is placed in be stirred, fermentation 48h gemma viable count up to 3 ×
1010cfu/g。
To tunning carry out kjeldahl determination, as the result is shown: protein from fermentation 0h 20% to fermentation 48h 26%,
Increase 30%.
Nutritional ingredient in tunning is detected, wherein free amino acid total amount reaches 2153mg/100g, hydrolyzes ammonia
Base acid total amount reaches 15.16g/100g.
The algae toxin content of fermentation process is detected, as a result as shown in figs. 1 to 6, the property material of algae toxin exists
The retention time of HPLC is respectively 3.30min, 4.47min, 5.05min, and ferment 0h, i.e., measurement algae toxin contains after inoculation seed liquor
Amount, the results show that detecting the algae toxin property material of retention time 3.38min and 5.03min；Fermentation for 24 hours and 48h, is only examined
Measure retention time be 5.03min algae toxin property material, ferment 60h when, without algae toxin property material detect, it is seen that hair
Algae toxin is completely degraded after ferment 60h.
Comparative example 1:
With embodiment 5, difference is specific embodiment, and the water content of cyanobacteria mud culture medium is adjusted to 30%.As a result it shows
Show bacillus licheniformis viable count up to 7.7 × 109Cfu/g, viable bacteria amount is opposite to be reduced.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for preparing microecological microbial agent, which is characterized in that using cyanobacteria mud as raw material, be inoculated with micro- life of bacillus
Object carries out solid state fermentation；The specific steps of the solid state fermentation include:
(1) cyanobacteria mud is uniformly mixed with auxiliary material by 1:0.5-2.5, sterilizes, prepares solid medium；
(2) the gemma bar for being 4.5-5.4 by every 100g inoculation 1~5mL OD value in the solid medium obtained to step (1)
Bacterium bacterium solution ferments in 30-37 DEG C, obtains microbial bacterial agent.
2. the method according to claim 1, wherein the microorganism of the bacillus is including but not limited to withered
Careless bacillus, bacillus licheniformis or bacillus megaterium.
3. method according to claim 1 or 2, which is characterized in that the cyanobacteria is Microcystis aeruginosa, anabena, synnema algae, puppet
One or more of anabena, quasi- column born of the same parents algae, the algae that quivers, floating silk algae, sheath silk algae, section ball algae, Shu Qiuzao.
4. according to the method described in claim 3, it is characterized in that, the moisture content of the cyanobacteria mud is 90-95%.
5. the method according to claim 1, wherein in step (1), the auxiliary material be stalk, wheat bran or its
Combination.
6. the method according to claim 1, wherein in step (2), the fermented incubation time 24-
72h。
7. the method according to claim 1, wherein the microbial bacterial agent also obtained to step (2) is dried
And/or it smashes.
8. the microbial bacterial agent prepared using any the method for claim 1~7.
9. application of the microbial bacterial agent according to any one of claims 8 in terms of preparing animal feed.
10. the feed containing microbial bacterial agent described in claim 8.
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| CN110205135A (en) * | 2019-07-08 | 2019-09-06 | 山东多芬农业有限公司 | A method of soil and fixed nitrogen are administered using cyanobacteria |
| CN110810286A (en) * | 2019-12-06 | 2020-02-21 | 怀化学院 | Method for ecologically breeding loaches by mixing bacteria and algae |
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| CN101555489A (en) * | 2009-05-19 | 2009-10-14 | 江苏省农业科学院 | Biological preparation of EMB bacteria, preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110205135A (en) * | 2019-07-08 | 2019-09-06 | 山东多芬农业有限公司 | A method of soil and fixed nitrogen are administered using cyanobacteria |
| CN110810286A (en) * | 2019-12-06 | 2020-02-21 | 怀化学院 | Method for ecologically breeding loaches by mixing bacteria and algae |
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