CN106305721A - Green alga oligosaccharide plant vaccine and preparation method thereof - Google Patents
Green alga oligosaccharide plant vaccine and preparation method thereof Download PDFInfo
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- CN106305721A CN106305721A CN201610691254.1A CN201610691254A CN106305721A CN 106305721 A CN106305721 A CN 106305721A CN 201610691254 A CN201610691254 A CN 201610691254A CN 106305721 A CN106305721 A CN 106305721A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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Abstract
The invention provides a green alga oligosaccharide plant vaccine and a preparation method thereof, and belongs to the field of plant vaccines. The plant vaccine consists of enteromorpha oligosaccharide and laminine, wherein enteromorpha oligosaccharide is prepared by accurately extracting natural green alga enteromorpha serving as a raw material; and oligosaccharide with different sizes is produced by controlling the reaction conditions and can induce expression of genes of some disease resistance and stress resistance related molecules of the plant, so that the stress resistance of the plant is improved. Two components of the enteromorpha oligosaccharide vaccine can significantly stimulate immune system response of the plant and have significant effects in application to resistance of all the wheatrust and wheat sharp eyespot; moreover, the synergistic effect of the two components is significant.
Description
Technical field
The invention belongs to plant vaccine field, be specifically related to a kind of chlorella oligosaccharide plant vaccine and preparation method thereof.
Background technology
Plant vaccine refers to induce plant to produce resistance, thus resists pathogen invasion, and suppression pathogenic microorganism is raw
Long, make plant exempt from disease or alleviate that a class of disease is biological or abiotic material, mainly have weak strain epidemic disease malicious, nontoxic
Seedling, live bacterial vaccines, protide vaccine, oligosaccharide vaccine, little molecule and other metabolite vaccine, Transgenic Plant Vaccines.
Applying on producing at present more has amino-oligosaccharide, activator protein, bacillus subtilis, peak, Chongqing 99 plant protection etc..
Several, world agro-chemical companies have developed the plant disease resistance inductor of commercialization the most in succession at present.U.S. Eden
The Messenger effective ingredient of Bioscienes company comes from the harpin egg in epidemic caused by fire pathogen pathogenic bacteria (Erwinia amylovory)
In vain, it is used for preventing and treating the powdery mildews such as mandarin orange, Fructus Piperis, Fructus Lycopersici esculenti, Fructus Cucumidis sativi.The plant disease resistance inductor of Syngenta Co., Ltd of Switzerland in recent years
Actigard mixed with antibacterial can prevent and treat watermelon crystal mildew, it is possible to significantly improves the effect of antibacterial, is substantially reduced chemistry agriculture
The usage amount of medicine.The Chitosan of Xu Cheng chemical company of Korea S derives from the biological shells such as shrimp Eriocheir sinensis, is used for processing seed, has
Disease-preventing and yield-increasing effect.In recent years, plant immunization lures antiradiation drug to also obtain in China to develop rapidly.As many merits that a class is novel
Energy biological product, the administration section the most at home such as oligosaccharide, abscisic acid registers, and obtains large-area popularization and application.But
With chlorella Entermorpha as raw material, the product of exploitation oligosaccharide plant vaccine have not been reported.
CN103449892A discloses a kind of plant vaccine, its formula combination, powder: astragalus polysaccharides 1-2 part, oligochitosan
0.5-1 part, barbaloin 1-2 part, SILICA FUME 5-20 part, photosynthetic induction element 0.3-0.7 part, tea saponin 0.1-0.3 part.Water preparation: be
Above formula combination being got up, dissolves in the ethanol of purity 25%-50%, drug ratios accounts for 2%-3%, bottled i.e. can be planted
Thing vaccine.Plant can be made to produce induction of antibodies, the invasion of opposing pathogen, the growth of suppression sex pheromone, make plant exempt from raw
Disease or alleviate disease." utilizing plant vaccine and growth regulator mepiquat chloride to control cotton verticillium wilt " have studied plant vaccine Chongqing
Peak 99 plant protection, activator protein, amino-oligosaccharide are used alone and are used in mixed way cotton yellow with plant growth regulator mepiquat chloride
The prevention effect of disease of withering, and select activator protein and mepiquat chloride mixing to use to have studied different spraying time and spraying times to cotton
The prevention effect of flower verticillium wilt.Result shows: Chongqing peak 99 plant protection, activator protein, amino-oligosaccharide and mepiquat chloride are used alone, with
Mepiquat chloride prevention effect is best (average preventive effect 41.3%), next to that activator protein (average preventive effect 36.7%).3 kinds of plant vaccines and
Mepiquat chloride is used in mixed way and all can improve prevention effect to some extent, and change peak 99 plant protection+mepiquat chloride, activator protein+mepiquat chloride, ammonia
The average preventive effect of base oligosaccharide+mepiquat chloride is respectively 52.9%, 52.2%, 47.9%.
Summary of the invention
The present invention provides a kind of chlorella oligosaccharide plant vaccine and preparation method thereof, and described plant vaccine is by natural green-alga
Entermorpha is that raw material enteromorpha oligosaccharide and laminine through precisely extracting preparation forms, and by controlling reaction condition, produces different big
Little oligosaccharide, it can be induced the expression of some gene with disease-resistant degeneration-resistant correlation molecule of plant, thus improve the degeneration-resistant of plant
Property.Enteromorpha oligosaccharide vaccine can the immune system response of significant stimulation plant, in the full rust of anti-Semen Tritici aestivi and anti-wheat sharp eyespot
Application in effect notable.
The present invention is achieved by the following technical solutions:
A kind of chlorella oligosaccharide plant vaccine, its active component is made up of enteromorpha oligosaccharide and laminine, wherein enteromorpha oligosaccharide
Preparation method specifically includes following steps:
1) pretreatment: dry Entermorpha powder is broken to below 20 mesh, adds the tap water of 10 times of weight, puts in extraction pot and extracts；
2) degraded: add 2～10% potassium hydroxide of Entermorpha weight ratio, 0～2% potassium permanganate, 0～2%EDTA-in extraction pot
2Na, and it is gradually heating to 90～100 DEG C of extractions 0.5～4h；
3) neutralize: degradation solution acid is neutralized to 6.0～7.0；
4) purification: being filtered by degradation solution or centrifugal, it is 2000-that the filtrate obtained or centrifugal liquid pass sequentially through molecular cut off
The NF membrane of 3500Da and 300-500Da, intercepts the part of molecular weight 300-2000Da, obtains nanofiltration concentrated solution；
5) it is dried: it is 25-35% that nanofiltration concentrated solution is concentrated into solid content further, is spray-dried, i.e. can get Entermorpha few
Sugar.
Preferably, in described enteromorpha oligosaccharide and laminine, ratio of weight and number is 3-5:1.It is further preferred that it is described
Enteromorpha oligosaccharide and laminine in ratio of weight and number be 4:1.
Preferably, described step 2) in potassium hydroxide addition be 5%, potassium permanganate addition is 1%, EDTA-2Na adds
Amount is 1%；Described degraded optimum condition is degradation temperature 95 DEG C, degradation time 3h.
Preferably, in described step 3), optimum pH is 6.5.
Preferably, in the filter operation in described step 4), the mesh number of filter cloth is 600-1000 mesh；In described step 4)
In centrifugally operated, centrifugal rotational speed is 3500 revs/min, and centrifugation time is 10min.
The preparation method of the present invention is conducted in-depth research by applicant according to the combination of above-mentioned condition, it has been found that
In the most preferred embodiment of the present invention, the preparation method of described enteromorpha oligosaccharide specifically comprises the following steps that
1) pretreatment: dry Entermorpha powder is broken to below 20 mesh, adds the tap water of 10 times of weight, puts in extraction pot and carries
Take；
2) degraded: addition 5% potassium hydroxide of Entermorpha weight ratio, 1% potassium permanganate, 1%EDTA-2Na in extraction pot, and gradually
It is warming up to 95 DEG C and extracts 3h；
3) neutralize: degradation solution acid is neutralized to 6.5；
4) purification: being filtered by degradation solution or centrifugal, it is 2000-that the filtrate obtained or centrifugal liquid pass sequentially through molecular cut off
The NF membrane of 3500Da and 300-500Da, intercepts the part of molecular weight 300-2000Da, obtains nanofiltration concentrated solution；
5) it is dried: it is 25-35% that nanofiltration concentrated solution is concentrated into solid content further, is spray-dried, i.e. can get Entermorpha few
Sugar.
A kind of method preparing above-mentioned chlorella oligosaccharide plant vaccine, it specifically includes following steps: by enteromorpha oligosaccharide and elder brother
It is dissolved in the ethanol water of 35-45% after the mixing of cloth propylhomoserin, shakes up and get final product.
The present invention is also claimed above-mentioned chlorella oligosaccharide plant vaccine in the full rust of anti-Semen Tritici aestivi and anti-wheat sharp eyespot
Application.
The present invention compared with prior art has a following technical advantage:
1) enteromorpha oligosaccharide can induce the expression of some gene with disease-resistant degeneration-resistant correlation molecule of plant, thus improves the anti-of plant
Inverse property, it can be greatly lowered the infection probability of gaeumannomyce bacterium and Rhizoctonia cerealis.And after it combines with laminine,
The infection probability of gaeumannomyce bacterium and Rhizoctonia cerealis is greatly lowered especially relative to being used alone enteromorpha oligosaccharide, and individually makes
With laminine then without this effect, this shows that two kinds of active components produce anti-gaeumannomyce bacterium and Rhizoctonia cerealis induction plant
Aspect has synergism.Therefore, plant vaccine of the present invention has the significant full rust of anti-Semen Tritici aestivi and wheat sharp eyespot
Effect.
2) yield of enteromorpha oligosaccharide is high, thus production cost is greatly lowered.Effect example of the present invention shows, according to this
The yield of the enteromorpha oligosaccharide of invention preparation is high, and its yield is between 38%-43%, few far above the Entermorpha prepared by prior art
Sugar, and the purity of enteromorpha oligosaccharide that the present invention prepares is high, is not less than the enteromorpha oligosaccharide that prior art is obtained.
Detailed description of the invention
Below by specific embodiment, the present invention is further elaborated:
Embodiment 1:
Dry Entermorpha pulverizer is pulverized, and crosses 20 mesh sieves, takes 10kg and puts in extraction pot, adds 100kg tap water, put into extraction pot
In extract；It is subsequently adding 0.5kg potassium hydroxide, 0.1kg potassium permanganate, 0.1KgEDTA-2Na, and is gradually heating to 95 DEG C
Extract 3h；It is neutralized to 6.5 with acid；Degradation solution plate-and-frame filtration (750 mesh filter cloth), obtains 100L filtrate, and passes through molecular cut off
The NF membrane of 3500Da, the effluent 73.5L NF membrane by molecular cut off 500DA, it is concentrated into 5L to concentrated solution volume, dense
Contracting liquid is concentrated into solid content 30% more further with Three-effect concentration device, is spray-dried, obtains 4.37Kg enteromorpha oligosaccharide.
Embodiment 2:
Dry Entermorpha pulverizer is pulverized, and crosses 20 mesh sieves, takes 15kg and puts in extraction pot, adds 150kg tap water, put into extraction pot
In extract；In extraction pot, add 0.3Kg potassium hydroxide, and be gradually heating to 90 DEG C of extraction 4h；It is neutralized to 7.0 with acid；
Degradation solution plate-and-frame filtration (750 mesh filter cloth), obtains 150L filtrate and by the NF membrane of molecular cut off 3500Da, effluent
The 137L NF membrane by molecular cut off 500DA, is concentrated into 60L to concentrated solution volume, and concentrated solution enters with Three-effect concentration device again
One step is concentrated into solid content 30%, is spray-dried, obtains 6.1kg enteromorpha oligosaccharide.
Embodiment 3-embodiment 8
Preparing enteromorpha oligosaccharide according to parameter as described in Table 1, in addition to parameters described below difference, embodiment 3-embodiment 5 preparation method is same
Embodiment 1, embodiment 6-embodiment 8 is with embodiment 2.
Major parameter in table 1 embodiment 3-embodiment 8
Measure the yield according to the preparation-obtained enteromorpha oligosaccharide of embodiment of the present invention 1-embodiment 8 respectively, many in enteromorpha oligosaccharide
The remaining rate of sugar, its measurement result is as shown in table 2.Yield=the enteromorpha oligosaccharide of enteromorpha oligosaccharide/dry Entermorpha × 100%.The mensuration of polysaccharide
Method measures according to method disclosed in prior art, its remaining rate be in enteromorpha oligosaccharide polyoses content divided by dry waterside before extracting
Polyoses content in tongue.Measurement result is as shown in table 2.
Enteromorpha oligosaccharide that the different preparation method of table 2 obtains and impurity content
| Enteromorpha oligosaccharide yield (%) | Impurity content (%) in oligosaccharide | |
| Embodiment 1 | 43.72 | 0.45 |
| Embodiment 2 | 40.67 | 0.59 |
| Embodiment 3 | 43.62 | 0.24 |
| Embodiment 4 | 42.61 | 0.43 |
| Embodiment 5 | 40.18 | 0.51 |
| Embodiment 6 | 41.92 | 0.49 |
| Embodiment 7 | 43.10 | 0.43 |
| Embodiment 8 | 39.95 | 0.43 |
As can be seen from Table 2, according to the yield height of enteromorpha oligosaccharide prepared by the present invention, and in enteromorpha oligosaccharide, polysaccharide residual volume is few,
The purity of product is high.Wherein the yield with the product of the preparation of the method described in embodiment 5 is the highest, for the optimal enforcement of the present invention
Example.
Embodiment 9 uses enteromorpha oligosaccharide vaccine that the present invention prepares to banded sclerotial blight and the infection rate of full rust pathogenic bacterium
Impact.
Using the enteromorpha oligosaccharide of above-described embodiment 5 preparation as object of study, study it with laminine compatibility to pathogenic bacterium
The impact of infection rate.In experiment in vitro, enteromorpha oligosaccharide and laminine all do not embody gaeumannomyce bacterium and Rhizoctonia cerealis
Directly inhibition.
Immunization method: start to spray plant vaccine of the present invention every 5 days from wheat during jointing stage, spray 6 times continuously
After, use gaeumannomyce bacterium and Rhizoctonia cerealis to remove infection experiment Semen Tritici aestivi, observe prevalence.Wherein enteromorpha oligosaccharide is at plant vaccine
In concentration be 0.5%.
Table 3 uses the infection rate of the pathogenic bacterium after plant vaccine
| Enteromorpha oligosaccharide and laminine weight ratio in vaccine | Gaeumannomyce bacterium infection rate (%) | Rhizoctonia cerealis infection rate (%) | |
| Vaccine 1 | Enteromorpha oligosaccharide 0.5% | 25.6 | 31.2 |
| Vaccine 2 | 3:1 | 14.6 | 9.6 |
| Vaccine 3 | 4:1 | 2.1 | 1.2 |
| Vaccine 4 | 5:1 | 5.6 | 3.9 |
| Vaccine 5 | Laminine 0.5% | 98.8 | 99.2 |
| Matched group | Do not use vaccine | 100 | 100 |
As can be seen from Table 3, enteromorpha oligosaccharide can induce the expression of some gene with disease-resistant degeneration-resistant correlation molecule of plant, thus
Improving the resistance of plant, it can be greatly lowered the infection probability of gaeumannomyce bacterium and Rhizoctonia cerealis.And work as itself and elder brother
After the combination of cloth propylhomoserin, the infection probability of gaeumannomyce bacterium and Rhizoctonia cerealis is relative to being used alone enteromorpha oligosaccharide the most significantly
Reducing, be used alone laminine then without this effect, this shows that two kinds of active components produce anti-gaeumannomyce induction plant
Bacterium and Rhizoctonia cerealis aspect have synergism.Therefore, plant vaccine of the present invention has significant anti-Semen Tritici aestivi and entirely becomes rusty
The sick effect with wheat sharp eyespot.
Above example is only in order to illustrate technical scheme, rather than is limited；Although with reference to aforementioned reality
Execute example the present invention has been described in detail, for the person of ordinary skill of the art, still can be to aforementioned enforcement
Technical scheme described in example is modified, or wherein portion of techniques feature is carried out equivalent；And these are revised or replace
Change, do not make the essence of appropriate technical solution depart from the spirit and scope of claimed technical solution of the invention.
Claims (9)
1. a chlorella oligosaccharide plant vaccine, its active component is made up of enteromorpha oligosaccharide and laminine, wherein enteromorpha oligosaccharide
Preparation method specifically include following steps:
1) pretreatment: dry Entermorpha powder is broken to below 20 mesh, adds the tap water of 10 times of weight, puts in extraction pot and carries
Take；
2) degraded: add 2～10% potassium hydroxide of Entermorpha weight ratio, 0～2% potassium permanganate, 0～2%EDTA-in extraction pot
2Na, and it is gradually heating to 90～100 DEG C of extractions 0.5～4h；
3) neutralize: degradation solution acid is neutralized to 6.0～7.0；
4) purification: being filtered by degradation solution or centrifugal, it is 2000-that the filtrate obtained or centrifugal liquid pass sequentially through molecular cut off
The NF membrane of 3500Da and 300-500Da, intercepts the part of molecular weight 300-2000Da, obtains nanofiltration concentrated solution；
5) it is dried: it is 25-35% that nanofiltration concentrated solution is concentrated into solid content further, is spray-dried, i.e. can get Entermorpha few
Sugar.
Chlorella oligosaccharide plant vaccine the most according to claim 1, it is characterised in that described enteromorpha oligosaccharide and laminine
Middle ratio of weight and number is 3-5:1.
Chlorella oligosaccharide plant vaccine the most according to claim 1, it is characterised in that described enteromorpha oligosaccharide and laminine
Middle ratio of weight and number is 4:1.
Chlorella oligosaccharide plant vaccine the most according to claim 1, it is characterised in that described step 2) in potassium hydroxide add
Amount is 5%, and potassium permanganate addition is 1%, EDTA-2Na addition is 1%；Described degraded optimum condition is degradation temperature 95 DEG C,
Degradation time 3h.
Chlorella oligosaccharide plant vaccine the most according to claim 1, it is characterised in that in described step 3), optimum pH is
6.5。
Chlorella oligosaccharide plant vaccine the most according to claim 1, it is characterised in that the filter operation in described step 4)
The mesh number of middle filter cloth is 600-1000 mesh；In centrifugally operated in described step 4), centrifugal rotational speed is 3500 revs/min, centrifugation time
For 10min.
Chlorella oligosaccharide plant vaccine the most according to claim 1, it is characterised in that the preparation method of described enteromorpha oligosaccharide
Specifically comprise the following steps that
1) pretreatment: dry Entermorpha powder is broken to below 20 mesh, adds the tap water of 10 times of weight, puts in extraction pot and carries
Take；
2) degraded: addition 5% potassium hydroxide of Entermorpha weight ratio, 1% potassium permanganate, 1%EDTA-2Na in extraction pot, and gradually
It is warming up to 95 DEG C and extracts 3h；
3) neutralize: degradation solution acid is neutralized to 6.5；
4) purification: being filtered by degradation solution or centrifugal, it is 2000-that the filtrate obtained or centrifugal liquid pass sequentially through molecular cut off
The NF membrane of 3500Da and 300-500Da, intercepts the part of molecular weight 300-2000Da, obtains nanofiltration concentrated solution；
5) it is dried: it is 25-35% that nanofiltration concentrated solution is concentrated into solid content further, is spray-dried, i.e. can get Entermorpha few
Sugar.
8. preparing a method for chlorella oligosaccharide plant vaccine described in claim 1, it specifically includes following steps: Entermorpha is few
Sugar and laminine are dissolved in the ethanol water of 35-45% after mixing, shake up and get final product.
9. chlorella oligosaccharide plant vaccine application in the full rust of anti-Semen Tritici aestivi and anti-wheat sharp eyespot described in claim 1.
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| CN112655709A (en) * | 2020-12-24 | 2021-04-16 | 南京农业大学 | Application of 2-amino-3-methyl caproic acid as plant immunity inducer |
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