CN104150977B - Preparation method and application of alga oligosaccharide biological fertilizer - Google Patents
Preparation method and application of alga oligosaccharide biological fertilizer Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an alga oligosaccharide biological fertilizer and relates to alga fertilizers. The method for preparing the alga oligosaccharide biological fertilizer comprises the following steps: activating flammeovirga pacifica H2 by using an activating medium flat plate, inoculating in a seed medium, and performing shaking culture to obtain fermentation liquor, wherein a collection number of the flammeovirga pacifica H2 is CCTCC NO:M2012229; (2) inoculating the fermentation liquor obtained in the step (1) into a saccharogenic culture medium, continuously shaking for fermenting and culturing, centrifuging, and collecting the supernatant; (3) filtering the supernatant by using a tubular membrane, and collecting the filtrate; (4) treating the filtrate collected in the step (3) by using protease to obtain the alga oligosaccharide biological fertilizer. The prepared alga oligosaccharide biological fertilizer can be applied to crop growing.
Description
Technical field
The present invention relates to alga fertilizer, more particularly to a kind of preparation method and application of alga oligosaccharides bio-fertilizer.
Background technology
Marine alga is the huge adsorbent of a class, and it can concentrate the nutriment in 440,000 times of oceans, is especially suitable for supplying
Barren soil.In soil substantial amounts of nutriment by rainwater and river be discharged in ocean formed reasonably biology follow
Ring.The nitrogen that about 21,500,000 tons of the whole world and 2,000,000 tons of potassium are flowed into river by rainwater from land, then flow into again
To ocean.Marine alga as agricultural plant growth conditioning agent be exactly those were originally derived from land nutriments pass through marine alga this
" adsorbent " reconfigures and returns in terrestrial plant.
Alga fertilizer is using sea-plant marine alga as primary raw material, the bio-feritlizer being processed into through science, its development
Experienced three phases, be rot marine alga, varec (powder), marine algae extract respectively.Core substance in alga fertilizer is pure sky
So marine algae extract, primary raw material is selected from natural seaweed, processes through special biochemical process, extracts the essence substance in marine alga,
Greatly remain natural activity component, containing the incomparable potassium of substantial amounts of non-nitrogen containing organic matter, land plant, calcium, magnesium, zinc,
Iodine etc. more than 40 plants mineral matter element and abundant vitamin, particularly with algal polysaccharides specific in marine alga, alginic acid, height
Degree unrighted acid and multiple natural plants growth regulator, have very high biologically active, can stimulate non-spy in plant body
The generation of the specific activities factor, adjusts Endogenous hormone balance.
Active principle in alga fertilizer after specially treated, in the activated state being easily absorbed by plants, after a procedure
2-3 hour enter in plant body, and there is Assimilation and conductivity speed quickly.Natural plants growth regulating is contained in alga fertilizer
Agent is such as：Auxin, cytokinin-like substance and gibberellin etc., have very high biologically active, the ratio of various materials and land
In plant, each hormone ratio is close.Alginic acid in alga fertilizer can reduce the surface tension of water, forms one in plant surface
Layer film, increases contact area, makes water-soluble substances be easier to enter plant cell through cauline leaf superficial cell film, makes plant
Most effectively absorb the nutrient content in seaweed extracted liquor, if therefore liquid seaweed manure and insecticide, bactericide and chemistry
Fertilizer is used in mixed way, better, it is possible to decrease sprays expense, has synergistic effect to agricultural chemicals and chemical fertilizer.External report sea
In alginic acid, contained natural antibiotics have antivirus action.Alga fertilizer raw material be selected from natural seaweed, with terrestrial plant have good
Compatibility, environmentally safe nontoxic to people, animal, have the advantages that other any fertilizer are all incomparable.
Alga fertilizer can directly make soil or make soil increase organic matter, the various beneficial micro- life in activation soil by plant
Thing, these biologies can play a part catalyst in the circulation of phytomicroorganism metabolin, so that the biopotency of soil is increased.
The metabolin of plant and edaphon can provide more nutrients for plant.Alga fertilizer is a kind of natural biological preparation, can be with
The plant soil ecosystem harmoniously works.The native compound that it contains such as Sodium Alginate is natural soil conditioner,
The formation of soil aggregate can be promoted, improve soil internal void space, coordinate solid, liquid, gas proportions in soil, recover
Due to soil over-burden and chemical contamination and lose lac balance, increase geobiont vigor, promote available nutrient
Release, be conducive to root growth, improve the resistance of crop.
Asparagus is a kind of Gracilaria marine alga, belongs to red algae sea-plant, and breeding low cost, growth cycle are short, simultaneously
It is also a kind of high dietary-fiber, high protein, low fat, low calorie, and the natural high-quality ocean vegetable rich in minerals and vitamins
Dish.In recent years, coastal various places asparagus aquaculture development is rapid, adds asparagus has that frond is big, strong adaptability, growth are fast etc.
Advantage, various places asparagus resource very abundant.Because asparagus frond is harder and algae fishy smell is denseer, among the people seldom directly eat,
In addition to fraction is as abalone feed, major part is used as the raw material of agar-agar production.But it is in recent years continuous due to agar-agar substitute
Occur and the cause influence such as discharge of a large amount of discarded object is developed using the more large-scale of asparagus agar-agar enterprise, cause to support
Grow overstocking in a large number of asparagus.Therefore, expand the range of application of asparagus, change the single situation of asparagus converted products, meaning
Great.
Chinese patent CN103304304A discloses a kind of preparation method of iodate chitosan oligosaccharide seaweed manure, and step is such as
Under：(1) enzyme digestion reaction of shitosan：After the clipped pulverizing of shitosan, carry out enzyme digestion reaction with non-specific complex enzyme, divided
The sub- degree of polymerization is in the chitosan oligosaccharide solution of 4-8.(2) iodination reaction of chitosan oligosaccharide：To in the chitosan oligosaccharide solution obtaining with certain speed
Degree adds liquor kalii iodide, carries out the iodination reaction of chitosan oligosaccharide, obtains iodate chitosan oligosaccharide solution.(3) chelating of seaweed extracted liquor
Reaction：Take the seaweed extracted liquor of enzyme solution preparation, with iodate chitosan oligosaccharide solution, carry out chelatropic reaction according to certain ratio,
Compound certain density trace element, finally give iodate chitosan oligosaccharide seaweed manure.
Chinese patent CN103304278A discloses a kind of marine alga bacterial manure containing algae leaching liquor, described marine alga bacterial manure be by
Following preparation methods obtain：(1) marine alga extraction；(2) preparation of fermentation medium；(3) inoculate and ferment.Described marine alga bacterial manure
It is a kind of marine alga active bio microbial inoculum fertilizer producing acquisition using pure physics and biological method, have marine alga activity and biological bacteria concurrently
The live body regulatory function of agent, its living bacteria count >=2.0 × 109CFU/ml, it is rich in simultaneously and degraded through biofermentation by algin
The plant growth regulating activity materials such as the alginic acid of conversion, alga oligosaccharides, gibberellin (GA), heteroauxin (IAA).
Chinese patent CN103710281A discloses a kind of bacillusmusilaginosiengineering bacterial strain and its in seaweed manure
Application, contains abundant bacillusmusilaginosiengineering using the marine alga microbial manure of this bacterial strain preparation and breeds the pod membrane that metabolism produces
Polysaccharide (or exocellular polysaccharide) and by the alginic acid through biofermentation Degradation and Transformation for the algae component, alga oligosaccharides, gibberellin (GA), Yin
The plant growth regulating activity materials such as indolylbutyric acid (IAA), living bacteria count >=2.0 × 109CFU/ml.
The applicant provides a kind of asparagus agaropectin oligose and preparation method thereof in Chinese patent CN102827899A
With application, preparation method：1) bacterial strain Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2 is put down with activation medium
After plate is activated, it is inoculated in seed culture medium, shaken cultivation obtains zymotic fluid；Described activation medium flat board is：Contain through 60
～80 mesh sieve after gardon asparagus powder 1%, yeast extract 0.2%, NaCl 3%, agar-agar powder 1.5%, distilled water prepare, through 121
DEG C, use after 20min sterilizing；Described seed culture medium is：Containing CMC-Na2 0.5%～1.5%, yeast extract 0.2%, NaCl
3%, distilled water is prepared, and through 121 DEG C, uses after 20min sterilizing；Described Pacific Ocean heat color bacillus (Flammeovirga
Pacifica) H2 is preserved in China typical culture collection center on June 13rd, 2012, and deposit number is CCTCC NO:
M2012229.
Content of the invention
Present invention aims to the not extensive present situation of existing asparagus range of application, provide and can be directly applied to
Soil, expands a kind of preparation method and application of alga oligosaccharides bio-fertilizer of asparagus range of application.
The preparation method of described alga oligosaccharides bio-fertilizer, comprises the following steps：
1) Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2 is activated with activation medium flat board
Afterwards, it is inoculated in seed culture medium, shaken cultivation obtains zymotic fluid；Described Pacific Ocean heat color bacillus (Flammeovirga
Pacifica) the Pacific Ocean heat color bacillus that H2 and the applicant provide in Chinese patent CN102827899A
(Flammeovirga Pacifica) H2 is identical, and this bacterial strain is preserved in China typical culture collection on June 13rd, 2012
Center, deposit number is CCTCC NO:M2012229, address is Wuhan, China university (postcode 430072)；
2) by step 1) in the zymotic fluid that obtains inoculate in product sugar culture-medium, be centrifuged after continuing oscillation and fermentation cultivation, receive
Collection supernatant；
3) supernatant is filtered with tubular membrane, collected filter liquor；
4) by step 3) the filter liquor Protease Treatment collected, obtain final product alga oligosaccharides bio-fertilizer.
In step 1) in, described activation medium flat board can be：Containing the gardon asparagus powder after sieving through 60～80 mesh
1.5%, yeast extract 0.2%, NaCl 3.5%, agar-agar powder 1%, distilled water is prepared, and through 121 DEG C, uses after 20min sterilizing；Institute
Stating seed culture medium can be：Containing CMC-Na 1%～2%, yeast extract 0.2%, NaCl 3.5%, distilled water is prepared, through 121 DEG C,
Use after 20min sterilizing；The condition of described shaken cultivation can be：Under the conditions of 25～37 DEG C, 250r/min shaken cultivation is extremely
OD600=1.0～1.5.
In step 2) in, described product sugar culture-medium can be：Containing the gardon asparagus powder 1%～5% sieved through 60～80 mesh,
MgSO40.1%～0.5%, KCl 0.05～0.2%, distilled water is prepared, and through 121 DEG C, uses after 20min sterilizing；Zymotic fluid
Inoculation by mass percentage can be for producing the 5% of sugar culture-medium；The condition of described oscillation and fermentation cultivation can be：In 25～37 DEG C of conditions
Under, 300r/min oscillation and fermentation cultivation 24～72h；The condition of described centrifugation can be：11000rpm is centrifuged 15min.
In step 3) in, described filtration can adopt membrane filter system, by supernatant successively with 0.8 μm, 0.45 μm and 0.22 μ
M tubular membrane is filtered；Described tubular membrane can adopt rolling organic film.
In step 4) in, described protease can for example give birth to work bioengineering (Shanghai) stock using commercially available papain
The papain (P16J12-100g) that part Co., Ltd produces；Described process can process 3h in 37 DEG C.
Prepared alga oligosaccharides bio-fertilizer can be applied in arable farming.
The method of described application can be：With water, alga oligosaccharides bio-fertilizer is diluted to containing final concentration of 25～500mg/L's
Oligosaccharides, once, the application dosage of first 4 times is 50mL/ time to every 7～10d pouring root, and application dosage is 100mL/ time later, effectively carries
High Soil Phosphorus, protein, organic content, especially improve the content of potassium, thus increasing soil fertility.
The present invention adopts the method that bacterial strain is co-cultured with asparagus, and the enzyme system produced using Pacific Ocean heat color bacillus H2 is direct
Degraded asparagus, discharges various plant active factors, after filtration sterilization, adds papain, algae PD is
Polypeptide and amino acid, provide the nitrogenous fertilizer of high-quality simultaneously for plant.
The present invention inoculates Pacific Ocean heat color bacillus H2 in the culture medium containing asparagus, the enzyme system produced using bacterial strain
The algal polysaccharides containing in asparagus are degraded to alga oligosaccharides, remove through membrane filtration after thalline and the gardon asparagus powder of residual,
Add papain to be digested, obtain final product described bio-fertilizer.The present invention gives the application mode of described bio-fertilizer,
Germination period and seedling initial stage carry out pouring root 4 times by the amount of 25mg, after be promoted to the amount pouring root of 50mg, effectively increase in soil
Phosphorus, protein, organic content, thus increase soil fertility.
Brief description
Fig. 1 is content of soil available phosphor after variable concentrations oligosaccharides pouring root.
Fig. 2 is Soil Available potassium content after variable concentrations oligosaccharides pouring root.
Fig. 3 is Soil available nitrogen content after variable concentrations oligosaccharides pouring root.
Fig. 4 is soil organic matter content after variable concentrations oligosaccharides pouring root.
Fig. 5 is Flavobacterium (Flavobacterium) proportion of different disposal group.
In Fig. 1～5, respectively it is labeled as：1 represents control group, i.e. 0mg/L；2 represent experimental group, are 25mg/L；3 represent in fact
Test group, be 50mg/L；4 represent experimental group, are 100mg/L；5 represent experimental group, are 500mg/L.
Specific embodiment
In order to understand the present invention, below the present invention is further illustrated with embodiment, but the present invention be not limited solely to following
Embodiment.
The preparation of embodiment 1 alga oligosaccharides bio-fertilizer
(1) by Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2 activation medium flat board (containing through 60 mesh
Gardon asparagus powder 1.5% after sieving, yeast extract 0.2%, NaCl 3.5%, agar-agar powder 1%, distilled water is prepared, through 121 DEG C,
20min sterilizing after using) activated after, be inoculated in seed culture medium (containing CMC-Na 1.5%, yeast extract 0.2%, NaCl
3.5%, distilled water prepare, through 121 DEG C, 20min sterilizing after using) in, under the conditions of 25 DEG C, 250r/min shaken cultivation is extremely
OD600=1.2, obtain zymotic fluid；
(2) zymotic fluid obtaining in step (1) is inoculated into product sugar culture-medium (containing the gardon asparagus powder sieved through 60 mesh
1%, MgSO40.1%, KCl 0.2%, distilled water prepare, through 121 DEG C, 20min sterilizing after using) in, under the conditions of 25 DEG C,
300r/min oscillation and fermentation cultivation 72h, rear 11000rpm is centrifuged 15min, collects supernatant；
(3) adopt membrane filter system, by supernatant successively with 0.8 μm, 0.45 μm and 0.22 μm of rolling organic film were carried out
Filter, collects filter liquor；
(4) (the P16J12- of Sangon Biotech (Shanghai) Co., Ltd. is added to the filter liquor in step (3)
100g) papain (every liter of addition 8U), processes 3h, as alga oligosaccharides bio-fertilizer in 37 DEG C.
The prepared application process of alga oligosaccharides bio-fertilizer given below and effect：
Take the alga oligosaccharides preparing, be diluted with water into final concentration of 25mg/L, 50mg/L, 100mg/L, 500mg/ respectively
L, and with clear water for comparison.It is applied in the root system of cherry tomato.
Frequency of administration is that every 7～10d applies once, and the application dosage of first 4 times is 50mL, is for latter 4 times 100mL.
The soil constituent of analysis different experiments group, and the composition of rhizosphere microbial flora, find available nitrogen, rapid available phosphorus with
And the content of the soil organism has different degrees of raising, and the content of available potassium then improves more than 3 times.Concrete change is shown in
Fig. 1～4.
Flavobacterium (Flavobacterium) is the main phosphorus decomposing Pseudomonas of a class in soil, biological applying alga oligosaccharides
In soil after fertilizer, the content of Flavobacterium is also improved, as shown in Figure 5.
Embodiment 2
Similar to Example 1, its difference is that cultivation temperature described in step (1) is 37 DEG C, shaken cultivation to OD600
=1.5, the described content producing gardon asparagus powder in sugar culture-medium of step (2) is 5%, MgSO40.3%.
Embodiment 3
Similar to Example 1, its difference is that the described content producing gardon asparagus powder in sugar culture-medium of step (2) is 3%,
KCl0.05%.Described oscillation and fermentation cultivation temperature is 37 DEG C, and incubation time is 24h.
Embodiment 4
Similar to Example 1, its difference is that the described incubation time of step (2) is 48h.
Embodiment 5
Similar to Example 4, its difference is that the described cultivation temperature of step (2) is 30 DEG C.
Claims (10)
1. a kind of preparation method of alga oligosaccharides bio-fertilizer is it is characterised in that comprise the following steps：
1), after Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2 being activated with activation medium flat board, connect
Plant in seed culture medium, shaken cultivation obtains zymotic fluid；Described Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2
Deposit number is CCTCC NO:M2012229；Described activation medium flat board is：Containing the dragon vegetable powder after sieving through 60～80 mesh
End 1.5%, yeast extract 0.2%, NaCl 3.5%, agar-agar powder 1%, distilled water is prepared, and through 121 DEG C, uses after 20min sterilizing；
Described seed culture medium is：Containing CMC-Na 1%～2%, yeast extract 0.2%, NaCl 3.5%, distilled water is prepared, through 121 DEG C,
Use after 20min sterilizing；
2) by step 1) in the zymotic fluid that obtains inoculate in product sugar culture-medium, be centrifuged, in collection after continuing oscillation and fermentation cultivation
Clear liquid；Described product sugar culture-medium be：Containing the gardon asparagus powder 1%～5% sieved through 60～80 mesh, MgSO40.1%～
0.5%, KCl 0.05～0.2%, distilled water is prepared, and through 121 DEG C, uses after 20min sterilizing；
3) supernatant is filtered with tubular membrane, collected filter liquor；
4) by step 3) the filter liquor Protease Treatment collected, obtain final product alga oligosaccharides bio-fertilizer；Described protease adopts commercially available
Papain.
2. as claimed in claim 1 a kind of preparation method of alga oligosaccharides bio-fertilizer it is characterised in that in step 1) in, described shake
The condition swinging culture is：Under the conditions of 25～37 DEG C, 250r/min shaken cultivation to OD600=1.0～1.5.
3. as claimed in claim 1 a kind of preparation method of alga oligosaccharides bio-fertilizer it is characterised in that in step 2) in, zymotic fluid
Inoculation is produce sugar culture-medium 5% by mass percentage.
4. as claimed in claim 1 a kind of preparation method of alga oligosaccharides bio-fertilizer it is characterised in that in step 2) in, described shake
The condition swinging fermented and cultured is：Under the conditions of 25～37 DEG C, 300r/min oscillation and fermentation cultivation 24～72h；The bar of described centrifugation
Part is：11000rpm is centrifuged 15min.
5. as claimed in claim 1 a kind of preparation method of alga oligosaccharides bio-fertilizer it is characterised in that in step 3) in, described mistake
Filter adopts membrane filter system, and by supernatant successively with 0.8 μm, 0.45 μm and 0.22 μm of tubular membrane are filtered.
6. as claimed in claim 5 a kind of preparation method of alga oligosaccharides bio-fertilizer it is characterised in that described tubular membrane using volume
Formula organic film.
7. as claimed in claim 1 a kind of preparation method of alga oligosaccharides bio-fertilizer it is characterised in that described commercially available pawpaw egg
White enzyme adopts the papain P16J12-100g that Sangon Biotech (Shanghai) Co., Ltd. produces.
8. as claimed in claim 1 a kind of preparation method of alga oligosaccharides bio-fertilizer it is characterised in that in step 4) in, described place
Reason is to process 3h in 37 DEG C.
9. the alga oligosaccharides life as prepared by described a kind of preparation method of alga oligosaccharides bio-fertilizer arbitrary in claim 1～8
The fertile application in arable farming of thing.
10. application as claimed in claim 9 is it is characterised in that the method for application is：With water, alga oligosaccharides bio-fertilizer is diluted to
Oligosaccharides containing final concentration of 25～500mg/L, once, the application dosage of first 4 times is 50mL/ time to every 7～10d pouring root, Yi Houshi
It it is 100mL/ time with dosage.
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| CN105794860A (en) * | 2016-04-07 | 2016-07-27 | 山东省农业科学院农业质量标准与检测技术研究所 | Microbial agent for controlling quarantine fungus diseases of apples and promoting yield increase |
| CN113739845A (en) * | 2021-08-12 | 2021-12-03 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Application method of sargassum fusiforme oligosaccharide |
| CN114573394A (en) * | 2022-03-18 | 2022-06-03 | 新洋丰农业科技股份有限公司 | Fertilizer biological synergistic additive for promoting photosynthesis and preventing physiological yellowing and preparation method thereof |
| CN115745674B (en) * | 2022-11-04 | 2024-06-18 | 江西农业大学 | Microbial oligosaccharide chelated trace element water-soluble fertilizer and preparation method thereof |
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