CN105566510A - Glucooligosaccharide and preparation method and application thereof - Google Patents
Glucooligosaccharide and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses glucooligosaccharide with succinic acid and pyruvoyl modified groups and a de-modified derivative of the glucooligosaccharide. A main chain of the glucooligosaccharide is composed of eight glycosyls and is free of branched chain connection, and the glycosyls of the main chain are connected by one alpha-1, 3-glycosidic bond and six beta-1, 3 glycosidic bonds. The glucooligosaccharide is obtained by directly separating from fermentation supernate after passing liquid mixed fermentation Agrobacterium ZX09 and Paenibacillus S09, and a glucooligosaccharide dry powder product is obtained after subjecting the fermentation supernate to alcohol precipitation, centrifuging and drying. The preparation method is quick, effective, simple in process, high in yield, low in cost, free of pollution and suitable for industrialized production. The glucooligosaccharide prepared by the method is high in water solubility, uniform in molecular weight and structure, easy to prepare and purify, high in bioactivity and capable of effectively activating resistant, to pathogenic fungi, of plants, can serve as a plant induced resistance agent and a plant protective agent, has resistance inducing effect on crops remarkably better than that of kelp oligosaccharide and has wide application value in the aspects of prevention and control of plant diseases and pests.
Description
Technical field
The invention belongs to agriculture applied technology field, be specifically related to the few carbohydrates and their derivative in the obtained Portugal of a kind of microorganism mixed fermentation, and the preparation method of Portugal's oligosaccharides and plant inducer resist with plant protection in application.
Background technology
Oligosaccharides (Oligosaccharide), also known as oligosaccharide or oligose, is generally the glucide with straight or branched be formed by connecting by glycosidic link by 3 ~ 10 monose.Can by body metabolism and absorption and with or without biological function according to it, oligosaccharides can be divided into common oligosaccharides and functional oligosaccharide.Common oligosaccharides such as sucrose, maltose, lactose and trehalose etc. can be digested, are energy matters, without special physiologic function.Functional oligosaccharide generally has low heat value, taste is sweet, viscosity is large, water absorbability by force, not by physicochemical properties such as pipe intestinal digestings, and it is soluble in water, have no side effect, no antigen, body storage effect is more weak, there is different physiological roles, such as improve intestinal microflora, improve food utilization efficiency, reduce serum cholesterol and neutral fat content, improve immunity of organisms, antiviral, anti-inflammatory and plant inducer anti-etc.The defense mechanism of wherein β-Portugal's oligosaccharide kind exciton energy activated plant self, the expression of the transduction of inducing plant resistance signal and disease-resistant gene of being correlated with, and be called as " plant immunization vaccine ", there are great Application and Development potentiality.
The mass-producing application of current oligosaccharides still has difficulties, and its bottleneck factor is mainly the source of oligosaccharides, preparation method and cost etc.The preparation method of oligosaccharides comprises chemosynthesis, enzymic synthesis and polysaccharide edman degradation Edman.As Chinese patent CN1373134A (β-1; 3-glucooligosan and application) β-1 that obtained by chemical synthesis; 3-Portugal oligosaccharides can be used as the degerming agent of farm crop for field of biological pesticide; but the method relates to hydroxyl protection, the step such as deprotection and chromatographic separation and purification of multistep; synthesis cycle is long; difficulty is comparatively large, and productive rate is lower.The approach of current macromolecular polysaccharide degraded mainly contains chemical acid hydrolysis, microorganism enzymolysis, thermal destruction, mechano-degradation, radiation degradation etc., and the most frequently used is acid hydrolysis and enzyme hydrolysis method.As the Chinese patent CN102312021A preparation method of Derain oligosaccharides (a kind of can) have employed acid hydrolyzation preparation can Derain oligosaccharides.The product polymerization degree heterogeneity that acid hydrolyzation obtains, molecular weight distribution is wider, is difficult to separation and purification, and yield is lower.Enzymolysis process has specificity usually, and product structure is comparatively homogeneous, does not destroy glycosidic structure, and production technique cleans, and by product is few, free from environmental pollution, is the desirable route preparing oligosaccharides.But existing enzymolysis process is subject to the restriction of enzyme source and kind, the shortcoming such as the ubiquity enzymolysis cycle is long, efficiency is low and production cost is high.
Summary of the invention
The object of the present invention is to provide a kind of Portugal oligosaccharides and remove modificationization derivative, and the preparation method of these compounds.The present invention above-mentioned Portugal oligosaccharides is also provided and go modificationization derivative plant inducer resist and in protection in application.
To achieve these goals, technical scheme of the present invention is as follows:
A kind of Portugal oligosaccharides, its structural formula is such as formula shown in I:
Above-mentioned Portugal oligosaccharides is the linear molecule be formed by connecting by 6 β-1,3-glycosidic links and 1 α-1,3-glycosidic link by glucose unit, not containing side chain, carries a succinyl-modification group and a pyruvic acid modification group.
Above-mentioned Portugal oligosaccharides removes a modificationization derivative by macromolecule alkali for hydrolysis removal succinyl-modification group, and its structural formula is such as formula shown in II:
Prepare a method of removing modificationization derivative for the Portugal's oligosaccharides shown in above-mentioned formula II, by macromolecule alkali for hydrolysis, alkali used is NaOH or KOH.
What above-mentioned Portugal oligosaccharides removed succinyl-and pyruvic acid modification group by acid hydrolytic reaction simultaneously removes a modificationization derivative, and its structural formula is as shown in formula III:
Prepare a method of removing modificationization derivative for the Portugal's oligosaccharides shown in above-mentioned formula III, by acid hydrolytic reaction, acid used is hydrochloric acid, sulfuric acid, trifluoroacetic acid or acetic acid.
The present invention also provides the preparation method of above-mentioned Portugal oligosaccharides, comprise the following steps: first by edaphic bacillus ZX09 and series bacillus S09 respectively through solid medium rejuvenation, cultivate in picking list bacterium colony to seed liquor and obtain seed culture fluid, then the seed liquor of ZX09 and S09 bacterium is seeded in mixed fermentive culture medium successively, shaking culture at 28 ~ 37 DEG C, afterwards after collected by centrifugation supernatant concentration, add alcohol settling and obtain Portugal's oligosaccharides crude product, obtain Portugal's oligosaccharides crude product after Portugal's oligosaccharides crude product freeze-drying, obtain the Portugal's oligosaccharides refined finally by Gel filtration.
Edaphic bacillus ZX09 described in the present invention is on January 21st, 2010 in China typical culture collection center (CCTCC) preservation, and deposit number is CCTCCNO:M2010020.Series bacillus S09 described in the present invention is preserved in China typical culture collection center (CCTCC) on May 30th, 2012, and preservation registration number is CCTCCM2012196.
Edaphic bacillus ZX09 preferentially utilizes sucrose substrate to grow and synthesis of oligose precursor, carries out secondary metabolism and processing for series bacillus S09.Described edaphic bacillus ZX09 and the seed liquor volume ratio of series bacillus S09 are 1 ~ 5:1, and it is 0 ~ 36h that edaphic bacillus ZX09 is seeded to the inoculation time of mixed fermentive culture medium poor prior to series bacillus S09.
Consisting of of described mixed fermentive culture medium: sucrose 20 ~ 40g/L, urea 0.7 ~ 1.0g/L, potassium primary phosphate 0.5 ~ 1.0g/L, Calcium Chloride Powder Anhydrous 0.1 ~ 0.3g/L, bitter salt 0.1 ~ 0.3g/L, ferrous sulfate 0.01 ~ 0.03g/L, zinc chloride 0.002 ~ 0.005g/L, manganous sulfate 0.001 ~ 0.003g/L, pH value is 5 ~ 10.
The time of described shaking culture is 48 ~ 72h.
Further, the invention provides the few carbohydrates and their derivative in above-mentioned Portugal to resist and the application in plant protection at plant inducer.The few carbohydrates and their derivative in above-mentioned Portugal can produce disease-resistant factor by inducing plant, and Promoting plant growth, suppress disease to occur, can be used for preparing plant inducer and resist and plant protection series products.
The few carbohydrates and their derivative in above-mentioned Portugal resists and the application in plant protection at plant inducer, and its concrete application method is: the Portugal's oligosaccharides or derivatives thereof aqueous solution by concentration being 0.2 ~ 1.0g/L, is evenly sprayed on plant leaf surface.
Preferably, the concentration of described Portugal's oligosaccharides or derivatives thereof aqueous solution is 0.5g/L.
Portugal of the present invention oligosaccharides is a kind of natural sugar chain molecule, and be white or micro-yellow powder, sugared content reaches 85-95%, protein content 3%-12%, very easily water-soluble, has no side effect, has biodegradability, free from environmental pollution.The preparation technology of the few carbohydrates and their derivative in above-mentioned Portugal is simple, and with low cost, productive rate is high, and product is separation and purification very easily, is beneficial to industrialization scale operation.In addition, Portugal's oligosaccharides that this method obtains has no side effect, biological activity is higher, effectively can produce disease-resistant factor by inducing plant, strengthen plant disease-resistant ability, compared with the laminari-oligo saccharide of same dose, oligosaccharides of the present invention more can reduce the sickness rate of the crop such as potato, cucumber effectively, and Induced resistant effect is more remarkable.
Accompanying drawing explanation
Fig. 1 is the mass spectroscopy spectrogram of Portugal oligosaccharides I of the present invention.
Fig. 2 is the two-dimensional nucleus magnetic analysis spectrogram of Portugal oligosaccharides I of the present invention.
Fig. 3 is the effect contrast figure that Portugal oligosaccharides I of the present invention alleviates that Botrytis cinerea infects Arabidopsis leaf.
Fig. 4 is after Portugal oligosaccharides I of the present invention process, H in Arabidopsis leaf cell
2o
2variation diagram horizontal in Whitfield's ointment.
Fig. 5 is after Portugal oligosaccharides I process 36h of the present invention, pathogenesis-related proteins (PR1, resistance indicator protein in born of the same parents; PR2, dextranase; PR3, chitinase; PR5, class monellin) and Pdf1.2 (methyl jasmonate approach resistance protein) gene transcription level variation diagram.
Fig. 6 is after Portugal oligosaccharides I process 48h of the present invention, born of the same parents' endo-dextranase and chitinase variation diagram more flat than enzyme running water.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail.
Embodiment 1
The present embodiment illustrates and edaphic bacillus ZX09 and series bacillus S09 is carried out the method that Hybrid NC machine tool fermentation obtains Portugal's oligosaccharides.
A. the preparation of seed liquor: by single for ZX09 bacterium colony inoculation in inorganic salt-liquid sucrose substratum, in 30 DEG C of shaking culture 24h, substratum consists of: sucrose 20g, SODIUMNITRATE 0.5g, potassium primary phosphate 0.5g, Calcium Chloride Powder Anhydrous 0.1g, bitter salt 0.3g, ferrous sulfate 0.02g, zinc chloride 0.003g, manganous sulfate 0.001g, water 1000mL, pH7.0.121 DEG C of autoclaving 20min.By single for S09 bacterium colony inoculation in LBO liquid nutrient medium, in 35 DEG C of shaking culture 24h, substratum consists of sodium-chlor 0.5g, yeast extract paste 0.5g, peptone 1.0g, oatmeal 0.5g, 121 DEG C of high pressure steam sterilization 20min.
B. Hybrid NC machine tool: the seed liquor of cultured ZX09 bacterium and S09 bacterium is seeded in mixed fermentive culture medium in the ratio of 3:1, in 30 DEG C of shaking culture 48h, consisting of of mixed fermentive culture medium: sucrose 20g, urea 0.8g, potassium primary phosphate 0.75g, Calcium Chloride Powder Anhydrous 0.15g, bitter salt 0.15g, ferrous sulfate 0.02g, zinc chloride 0.003g, manganous sulfate 0.0015g, water 1000mL, pH7.0.121 DEG C of high pressure steam sterilization 20min.
C. the abstraction and purification of Portugal's oligosaccharides: collect mixed fungus fermentation supernatant liquor, after concentrated by Rotary Evaporators, adopt the Portugal's oligosaccharide compositions in alcohol settling fermented liquid, obtains Portugal's oligosaccharides crude product after freeze-drying.Chong Rong Portugal oligosaccharides crude product, after being made into 20% Portugal's oligosaccharide solution, crosses SephadexG25 gel chromatography column, remove the impurity such as high molecular weight protein and small molecules sucrose, what utilize DNS method monitoring reducing sugar goes out peak position, collects elutriant, after freeze-drying, obtain purifying Portugal oligosaccharides dry powder.
Mass spectrum and nucleus magnetic resonance is utilized to resolve the information such as the molecular weight of Portugal's oligosaccharides and structure respectively.The molecular weight showing this Portugal's oligosaccharides from mass spectrometry results is (741*2+2)=1484Da, with two negative charges, shows the existence (Fig. 1) of two modification groups.By two-dimensional nucleus magnetic spectrum elucidation, (Fig. 2, a are to obtain the backbone structure of Portugal's oligosaccharides, modification group and position thereof further
1h-
1h chemical shift is correlated with COSY spectrum, and b is that heteronuclear Multiple-quantum is correlated with HMQC spectrum, and c is that heteronuclear list quantum is correlated with hsqc spectrum).
Embodiment 2
The preparation of Portugal's oligosaccharide derivative.
Portugal's oligosaccharides after purifying is mixed with the aqueous solution of 10mg/mL, add final concentration be 0.2M NaOH react 2-5h, with in HCl and after, cross SephadexG25 carry out desalination, collect oligosaccharides eluted peak, Portugal sample, obtain the Portugal oligosaccharide derivative II removing succinyl-modification group.Portugal's oligosaccharides after purifying is mixed with the aqueous solution of 10mg/mL, add final concentration be 0.5M HCl react 5h, with in NaOH and after, cross SephadexG25 carry out desalination, collect oligosaccharides eluted peak, Portugal sample, obtain the Portugal oligosaccharide derivative III simultaneously removing succinyl-and pyruvic acid modification.
Embodiment 3
Portugal's oligosaccharides infects the control of Arabidopis thaliana to Botrytis cinerea.
Portugal's oligosaccharides aqueous solution of preparation 0.2-1.0g/L, is evenly sprayed to and grows on the arabidopsis thaliana blade of suitable size, within one day, sprays once, to spray clear water in contrast.The Botrytis cinerea of cultivation two days later, solid medium punches by pre-treatment Portugal oligosaccharides, is labelled to by mold agar block in Arabidopsis leaf and carries out infecting cultivation.Within 1-3 days, observe the gradient of infection of control group and Portugal's oligosaccharides group plant leaf afterwards.
Result shows, spray Portugal's oligosaccharide solution in advance and can reduce arabidopsis thaliana leaves infected rate, germ patch obviously diminishes.Figure 3 shows that spraying clear water and shifting to an earlier date spray concentration is arabidopsis thaliana blade figure after Portugal's oligosaccharide solution of 0.5g/L.
Embodiment 4
Portugal's oligosaccharides is to the induction of Arabidopis thaliana resistance.
Portugal's oligosaccharides aqueous solution of preparation 0.2-1.0g/L, be evenly sprayed to and grow on the arabidopsis thaliana blade of suitable size, after different time, clip blade is placed in rapidly liquid nitrogen after weighing frozen.
The mensuration of blade cell hydrogen peroxide level: take 100mg blade, adds acetone high-speed homogenization, and centrifuging and taking supernatant 200 μ L, adds 600 μ L dipotassium hydrogen phosphate damping fluid (50mM, pH8.2), vibration mixing.Add after 1mL chloroform after concuss, centrifuging and taking 500 μ L upper strata aqueous phase, add tiron mixing, then add 200 μ L strong aquas vibrations, centrifugal collecting precipitation.After the 2M sulphuric acid soln dissolution precipitation of 500 μ L, under 415nm wavelength, measure absorbancy.
The mensuration of Whitfield's ointment (SA) level in blade cell: get blade 100mg, adds centrifuging and taking supernatant after methyl alcohol 500 μ L homogenate, dries up with Nitrogen evaporator; Add 300 μ L5% trichoroacetic acid(TCA) dissolution precipitations; Add 550 μ L Ethyl acetate-cyclohexane (1:1) solution extractions, centrifugation aqueous phase and organic phase, in transfer upper organic phase to clean centrifuge tube.Repeat to merge upper layer of extraction liquid after this operates 2-3 time, dry up rear dissolve with methanol with Nitrogen evaporator, detect free state SA content with HPLC.Add 200 μ L8M hydrochloric acid solns in lower floor's aqueous phase, 80 DEG C of water-bath 1h make combined SA become free state SA, detect combined SA content with HPLC.
The mensuration of PR gene transcriptional level in born of the same parents: take 100mg blade, with Trizol test kit extracting blade total serum IgE, after RNA reverse transcription being become cDNA with the Reverse Transcriptase kit of Invitrogen, with poly ubiquitin enzyme UBQ5 gene for internal reference, carry out real-time fluorescence quantitative PCR reaction (QRT-PCR), analyze the expression of pathogenesis-related proteins in born of the same parents from transcriptional level.
Born of the same parents' endo-dextranase and chitinase activity measure: after appropriate blade is ground fragmentation in liquid nitrogen, dissolve mixing 1h with the phosphoric acid buffer (pH6.5,0.1M) containing protease inhibitor cocktail; Collected by centrifugation supernatant liquor is crude enzyme liquid.At 37 DEG C, hatch 1h with the laminarin of 1% and the chitin solution of 1% for substrate respectively and carry out enzyme reaction.With the growing amount of reducing sugar in DNS method assaying reaction liquid.An enzyme lives unit definition for discharging the enzyme amount required for 1 μm of ol glucose in 1h from substrate.
Result shows, and after Portugal's oligosaccharide solution process Arabidopis thaliana, can significantly improve H in blade cell
2o
2with Whitfield's ointment level, strengthen the transcriptional level of PR gene and Pdf1.2, and the synthesis of born of the same parents' endo-dextranase and chitinase can be promoted.Fig. 4, Fig. 5 and Fig. 6 are be result figure after Portugal's oligosaccharide solution process Arabidopis thaliana of 0.5g/L by concentration.Be after Portugal's oligosaccharide solution process Arabidopis thaliana of 0.5g/L by concentration, H in blade cell can be significantly improved
2o
2with Whitfield's ointment level (Fig. 4); Strengthen PR gene (PR1, resistance indicator protein gene; PR2, glucanase gene; PR3, chitinase gene; PR5, class sweet protein gene) and the transcriptional level (Fig. 5) of Pdf1.2 (methyl jasmonate approach resistance protein gene), and the synthesis (Fig. 6) of born of the same parents' endo-dextranase and chitinase can be promoted.To sum up result illustrates, microorganism Portugal of the present invention oligosaccharides can excite a series of metabolic regulation systems in plant materials fast and effectively, inducing plant disease resistance, strengthens plant disease-resistant and anti-adversity ability, has the application potential as plant immunization resistance inductor and plant protection agent.
Embodiment 5
The field test that Portugal's few carbohydrates and their derivative prevention crop pest occurs.
Portugal's oligosaccharides or derivatives thereof is mixed with the mother liquor of 10%, by 200 times of dilutions during use.Spray test is carried out to different crops (potato, cucumber, tobacco, tomato).Using clear water process as negative control, with the enzymolysis product laminari-oligo saccharide of laminarin for positive control, every day sprays once, and it is good for soaking No drip type with blade.Severity Scaling standard is: 0 grade, without scab; 1 grade, lesion area accounts for whole leaf area less than 5%; 3 grades, lesion area accounts for whole leaf area 6% ~ 10%; 5 grades, lesion area accounts for whole leaf area 11% ~ 20%; 7 grades, lesion area accounts for whole leaf area 21% ~ 50%; 9 grades, lesion area accounts for whole leaf area more than 50%.
Sickness rate=morbidity strain number/investigation strain number × 100%
Disease index=Σ (the sick number of sheets × relative level numerical value at different levels)/(investigating total number of sheets × highest typical value) × 100
Disease resisting effect=(contrast disease index-process disease index)/contrast disease index × 100%
The few carbohydrates and their derivative in Portugal to the Induced resistant effect of Different Crop in table 1, Portugal oligosaccharides I and laminari-oligo saccharide to the Induced resistant effect of Different Crop in table 2.
The table 1 Portugal Induced resistant effect of few carbohydrates and their derivative to Different Crop compares
As can be seen from Table 1, after this several crop applying oligosaccharides or derivatives thereof of potato, cucumber, tobacco and tomato, sickness rate and disease index all obviously decline, and disease resistance obviously increases.Wherein, the Induced resistant effect outline of oligosaccharides I removes modificationization derivative I I and III higher than it.During using oligosaccharides, without adverse side effect, occur without bad poisoning phenomenon.
Table 2 Portugal oligosaccharides I compares with the Induced resistant effect of laminari-oligo saccharide to Different Crop
As can be seen from Table 2, Portugal's oligosaccharides I and laminari-oligo saccharide all can reduce sickness rate and the disease index of this several crop of potato, cucumber, tobacco and tomato.Under same dose, the Induced resistant effect of Portugal oligosaccharides I obviously will be better than laminari-oligo saccharide.Compared with laminari-oligo saccharide, Portugal oligosaccharides preparation technology of the present invention is simple, and production cost is low, and product structure is homogeneous, more remarkable to the Induced resistant effect of crop, has the application potential as plant immunization resistance inductor and plant protection agent.
Claims (10)
1. Portugal's oligosaccharides, its structural formula is such as formula shown in I:
Described Portugal oligosaccharides is the linear molecule be formed by connecting by 6 β-1,3-glycosidic links and 1 α-1,3-glycosidic link by glucose unit, not containing side chain, carries a succinyl-modification group and a pyruvic acid modification group.
2. the derivative of Portugal as claimed in claim 1 oligosaccharides, its structural formula is such as formula shown in II or formula III:
3. the preparation method of the derivative of Portugal as claimed in claim 2 oligosaccharides, it is characterized in that, obtained the derivative of the Portugal's oligosaccharides shown in formula II by macromolecule alkali for hydrolysis, alkali used is NaOH or KOH; Obtained the derivative of the Portugal's oligosaccharides shown in formula III by acid hydrolytic reaction, acid used is hydrochloric acid, sulfuric acid, trifluoroacetic acid or acetic acid.
4. the preparation method of Portugal as claimed in claim 1 oligosaccharides, it is characterized in that, comprise the following steps: first by edaphic bacillus ZX09 and series bacillus S09 respectively through solid medium rejuvenation, cultivate in picking list bacterium colony to seed liquor and obtain seed culture fluid, then the seed liquor of ZX09 and S09 bacterium is seeded in mixed fermentive culture medium successively, shaking culture at 28 ~ 37 DEG C, afterwards after collected by centrifugation supernatant concentration, add alcohol settling and obtain Portugal's oligosaccharides crude product, Portugal's oligosaccharides crude product is obtained after Portugal's oligosaccharides crude product freeze-drying, the Portugal's oligosaccharides refined is obtained finally by gel permeation chromatography.
5. the preparation method of Portugal as claimed in claim 4 oligosaccharides, it is characterized in that, described edaphic bacillus ZX09 and the seed liquor volume ratio of series bacillus S09 are 1 ~ 5:1, and it is 0 ~ 36h that edaphic bacillus ZX09 is seeded to the inoculation time of mixed fermentive culture medium poor prior to series bacillus S09.
6. the preparation method of Portugal as claimed in claim 4 oligosaccharides, it is characterized in that, consisting of of described mixed fermentive culture medium: sucrose 20 ~ 40g/L, urea 0.7 ~ 1.0g/L, potassium primary phosphate 0.5 ~ 1.0g/L, Calcium Chloride Powder Anhydrous 0.1 ~ 0.3g/L, bitter salt 0.1 ~ 0.3g/L, ferrous sulfate 0.01 ~ 0.03g/L, zinc chloride 0.002 ~ 0.005g/L, manganous sulfate 0.001 ~ 0.003g/L, pH value is 5 ~ 10.
7. the preparation method of Portugal as claimed in claim 4 oligosaccharides, it is characterized in that, the time of described shaking culture is 48 ~ 72h.
8. the few carbohydrates and their derivative in Portugal as claimed in claim 1 or 2 resists and the application in plant protection at plant inducer as plant induced resistance agent and plant protection product.
9. the few carbohydrates and their derivative in Portugal as claimed in claim 8 resists and the application in plant protection at plant inducer as plant induced resistance agent and plant protection product; it is characterized in that; concrete application method is: the aqueous solution by concentration being Portugal's oligosaccharides or derivatives thereof of 0.2 ~ 1.0g/L, is evenly sprayed on plant leaf surface.
10. the few carbohydrates and their derivative in Portugal as claimed in claim 9 resists and the application in plant protection at plant inducer as plant induced resistance agent and plant protection product, and it is characterized in that, the concentration of the aqueous solution of described Portugal's oligosaccharides or derivatives thereof is 0.5g/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114304146A (en) * | 2021-12-07 | 2022-04-12 | 西北农林科技大学 | Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof |
| CN114605564A (en) * | 2020-12-09 | 2022-06-10 | 南京理工大学 | Hetero-poly-oligosaccharide and application thereof in improving disease resistance of plants |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100141A (en) * | 1993-07-05 | 1995-03-15 | 武田药品工业株式会社 | Method of producing beta-1,3-glucan |
| CN101935623A (en) * | 2010-04-14 | 2011-01-05 | 南京理工大学 | Agrobacterium ZX09, water-soluble beta-glucan prepared from Agrobacterium ZX09 and preparation method thereof and application on reducing blood sugar |
| CN102312021A (en) * | 2010-06-30 | 2012-01-11 | 中国科学院大连化学物理研究所 | Preparation method of Curdlan oligomers |
| CN102827791A (en) * | 2012-08-09 | 2012-12-19 | 广西大学 | Agrobacterium radiobacter mutant strain and method for preparing beta-1, 3 glucan as fish and shrimp disease-resistant factor feed additive through utilizing agrobacterium radiobacter mutant strain |
| CN103059154A (en) * | 2012-11-29 | 2013-04-24 | 滨州学院 | Fungal glucan oligomer chrome complex and preparation method thereof |
| CN104745656A (en) * | 2015-03-20 | 2015-07-01 | 江南大学 | Method for directly producing beta-1,3-glucooligosaccharides by virtue of thermal gel fermentation liquor |
-
2015
- 2015-12-18 CN CN201510957864.7A patent/CN105566510B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1100141A (en) * | 1993-07-05 | 1995-03-15 | 武田药品工业株式会社 | Method of producing beta-1,3-glucan |
| CN101935623A (en) * | 2010-04-14 | 2011-01-05 | 南京理工大学 | Agrobacterium ZX09, water-soluble beta-glucan prepared from Agrobacterium ZX09 and preparation method thereof and application on reducing blood sugar |
| CN102312021A (en) * | 2010-06-30 | 2012-01-11 | 中国科学院大连化学物理研究所 | Preparation method of Curdlan oligomers |
| CN102827791A (en) * | 2012-08-09 | 2012-12-19 | 广西大学 | Agrobacterium radiobacter mutant strain and method for preparing beta-1, 3 glucan as fish and shrimp disease-resistant factor feed additive through utilizing agrobacterium radiobacter mutant strain |
| CN103059154A (en) * | 2012-11-29 | 2013-04-24 | 滨州学院 | Fungal glucan oligomer chrome complex and preparation method thereof |
| CN104745656A (en) * | 2015-03-20 | 2015-07-01 | 江南大学 | Method for directly producing beta-1,3-glucooligosaccharides by virtue of thermal gel fermentation liquor |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114605564A (en) * | 2020-12-09 | 2022-06-10 | 南京理工大学 | Hetero-poly-oligosaccharide and application thereof in improving disease resistance of plants |
| WO2022121962A1 (en) * | 2020-12-09 | 2022-06-16 | 南京理工大学 | Heteropoly oligosaccharide and application thereof in improving plant disease resistance |
| CN114304146A (en) * | 2021-12-07 | 2022-04-12 | 西北农林科技大学 | Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof |
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