CN109824757A - A kind of ring (different bright-different bright) dipeptides, preparation method and its application - Google Patents
A kind of ring (different bright-different bright) dipeptides, preparation method and its application Download PDFInfo
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Abstract
The invention discloses a kind of ring (different bright-different bright) dipeptides, preparation method is fermented with isolated one plant of paecilomyces varioti bacterium SJ1 from wild sea-buckthorn, separation is obtained.Ring (different bright-different bright) dipeptides is additionally provided to the facilitation of budding, improvement crop salt-resistance, induction crop flowers, anti-cucumber downy mildew, the application for promoting crop frost resistance.The bacterial strain is easy culture, can obtain CYCLIC DIPEPTIDES compounds by separating-purifying from fermentation liquid with simpler method, there are no pollution to the environment, and with short production cycle, active superelevation, production and use cost are extremely low, and are easily formed large-scale serial production.The similar cyclic dipeptide compound has apparent facilitation to budding；Obvious effect in terms of salt resistance, induced flowering, anti-cucumber downy mildew, freeze proof, volume increase, raising.With good Development volue, application prospect is good.
Description
Technical field
The present invention relates to field of biotechnology, specifically for a kind of ring (different bright-different bright) dipeptides, preparation method and its answer
With the method for specifically isolating and purifying obtained ring (different bright-different bright) dipeptides from paecilomyces varioti bacterium also relates to such ring two
Peptide compounds answering in terms of growth-promoting, salt resistance, induced flowering, anti-cucumber downy mildew, freeze proof, volume increase, raising
With.
Background technique
Cyclic dipeptides, also known as 2,5- diketopiperazine compound are that formed by 2 amino acid by peptide bond cyclization one is steady
Fixed six-membered ring structure is the smallest cyclic peptide being widely present in nature and natural products.So far, numerous researchers have been
Many kinds of, Various Functions Cyclic dipeptides are isolated from people, other biological, and find it in pesticide, new-type fertilizer, regulator
Application aspect have biggish potentiality to be exploited.
China is a large agricultural country, for a long time the unreasonable applications of pesticide side such as generally existing blindness, excessive application
Formula causes soil universal hardened and is acidified, and soil fertility constantly declines, and quality of agricultural product constantly reduces, and these problems are serious
Hinder the sound development of modern agriculture industry.Crop yield does not increase, and quality persistently reduces, these be all agricultural production into
The performance for entering bottleneck period becomes the main problem of puzzlement agricultural development and peasant's increasing both production and income.In recent years, in supply side structure
Property reform overall background under, the Ministry of Agriculture carry out chemical fertilizer, Pesticide use amount zero growth rate action, by take physics prevention and control, ecology adjust
Control, biological prevention and control and accuracy pesticide applying combined technology, actively push forward pesticide reduction pest controlling, promote pesticide reduction synergy, achieve
Significant results.Meanwhile the exploitation and development of the green products such as biological pesticide, new-type fertilizer have started traditional agrochemical fertilizer and have produced
Industry change tide, wherein CYCLIC DIPEPTIDES compounds research is then the research hotspot of numerous researchers.
Summary of the invention
The object of the present invention is to provide a kind of ring (different bright-different bright) dipeptides, preparation method and applications, from wild sea-buckthorn
(bacterial strain is preserved in Chinese microorganism strain preservation on December 8th, 2014 to one plant of isolated paecilomyces varioti bacterium SJ1
Administration committee's common micro-organisms center, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number are
CGMCCNO.10114), fermented, isolated natural Cyclic dipeptides, Structural Identification are Cyclo (Ile-Ile), have superelevation
The features such as active, multiple functional, green and pollution-free, application prospect is extensive.
A kind of ring (different bright-different bright) dipeptides provided by the invention, structural formula are as follows:
Specific step is as follows for a kind of preparation method of ring (different bright-different bright) dipeptides provided by the invention:
The first step, paecilomyces varioti bacterium (Paecilomyces variotii) fermentation:
Paecilomyces varioti bacterium is inoculated in PDA solid medium, 25 DEG C constant temperature incubation 5-7 days.By activated strain
Block, which is dug, with punch agar is inoculated in the triangular flask equipped with 200ml seed culture medium (PDA liquid medium), in 25 DEG C, 180r/
It is used as level-one kind liquid within shaken cultivation 5 days on min shaking table；Level-one kind liquid is accessed in 5L seeding tank, the fermentation of secondary seed tank is carried out
Culture；By determining most suitable culture solution liquid loaded on 121 DEG C of sterilizing 20min in fermentor, first order seed, inoculum concentration are inoculated with after cooling
For 2-10%, 24-30 DEG C of temperature, initial pH6.8-7.6, speed of agitator 140-220r/min, culture obtains fermentation liquid in 5-7 days.
Cultivate formula of liquid are as follows: potato extracting solution 1.0L, yeast extract 1.0g, peptone 3.0g, glucose 15.0g；
Second step, fermentation liquid carry out centrifugal filtration, obtain mycelium and filtering fermentating liquid；
Third step is dried at 60 DEG C after washing mycelium, is weighed, is then crushed through high speed disintegrator, with same volume
Alcohol steep for 24 hours, is extracted 3 times, is mixed with magnetic stirring apparatus, ultrasonic oscillation 1h, is filtered by vacuum, and collects filtrate, as
Extracting solution.
The preparation of 4th step, crude extract: by after the revolving instrument concentration of said extracted liquid with isometric containing 5% acetone
Ethyl acetate is extracted, and is extracted 3 times；Then supernatant is distilled to obtain crude extract with Rotary Evaporators, is dried up under nitrogen evaporator
For use.
5th step takes crude extract through the decompression silica gel column chromatography separation of 200-300 mesh, with methylene chloride, methylene chloride: methanol
(70:1), methylene chloride: methanol (30:1), methylene chloride: methanol (8:1), methylene chloride: methanol (2:1), acetone, methanol 7
Polar system quickly elutes, and collects each flow point, then washes repeatedly through ODS pressurization chromatography, methanol/water (12:88~100) gradient
De-, TLC prepares purifying through HPLC after merging and NMR is analyzed, and determines the Cyclic dipeptides compound are as follows: cyclo (Ile-Ile), knot
Structure are as follows:
The abbreviation of formula Chinese and English indicates: cyclo ring, Ile isoleucine.
Preferably, in the first step, paecilomyces varioti bacterium is inoculated in PDA solid medium, 25 DEG C of constant temperature incubation 5-7
It.By activated strain with punch agar dig block be inoculated in equipped with 200ml seed culture medium (PDA liquid medium) three
Angle bottle was used as level-one kind liquid in shaken cultivation 5 days on 25 DEG C, 180r/min shaking table；Level-one kind liquid is accessed in 5L seeding tank, into
Row secondary seed tank fermented and cultured；By determining most suitable culture solution liquid loaded on 121 DEG C of sterilizing 20min in fermentor, cooling is followed by
Kind first order seed, inoculum concentration 5-10%, 25-28 DEG C of temperature, initial pH 7.2, speed of agitator 160r/min, culture 5-7 days
To fermentation liquid.Cultivate formula of liquid are as follows: potato extracting solution 1.0L, yeast extract 1.0g, peptone 3.0g, glucose 15.0g.
It is furthermore preferred that specific step is as follows for the first step: paecilomyces varioti bacterium being inoculated in PDA solid medium, 25 DEG C
Constant temperature incubation 5-7 days.Activated strain is dug block with punch agar to be inoculated in equipped with 200ml seed culture medium (PDA liquid
Culture medium) triangular flask, on 25 DEG C, 180r/min shaking table shaken cultivation 5 days be used as level-one kind liquid；Level-one kind liquid is accessed into 5L
In seeding tank, secondary seed tank fermented and cultured is carried out；By determining most suitable culture solution liquid loaded on 121 DEG C of sterilizings in fermentor
20min, is inoculated with first order seed after cooling, inoculum concentration 6%, is trained by 28 DEG C of temperature, initial pH 7.2, speed of agitator 160r/min
It supports 5-7 days and obtains fermentation liquid.
The present invention also provides the applications of the ring (different bright-different bright) dipeptides, make including the promotion to budding
With, improve crop salt-resistance, induction crop flowers, anti-cucumber downy mildew, promoted crop frost resistance.
Preferably, ring (different bright-different bright) dipeptides is small under salt stress to improve to the facilitation of budding
The germination percentage of wheat increases wheat root long under salt stress and increases wheat root quantity under salt stress.
It is furthermore preferred that ring (different bright-different bright) dipeptides is dense to the facilitation action of wheat seed germinating under salt stress
Degree is 20-40ng/ml.
It is furthermore preferred that ring (different bright-different bright) dipeptides is tolerable to the facilitation of wheat seed germinating under salt stress
Salinity is the salt water not higher than 0.8%, and the salt water is sodium chloride solution.
Preferably, the effect of two inducing peptide crop flowers of the ring (different bright-different bright) is induction winter jujube tree and capsicum bud
Differentiation, promotes winter jujube tree and capsicum Blooming.
It is furthermore preferred that the onset of action concentration of two inducing peptide crop flowers of the ring (different bright-different bright) is 20-30ng/ml.
Preferably, it is 5- that ring (different bright-different bright) dipeptides, which reduces the onset of action concentration of the disease incidence of cucumber downy mildew,
10ng/ml。
Preferably, it is to reduce potted tomato by freeze injury that ring (different bright-different bright) dipeptides, which promotes the effect of crop frost resistance,
Degree, the tolerable low temperature range of the potted tomato for spraying root and spraying treatment through Cyclic dipeptides is -10-0 DEG C, and Cyclic dipeptides onset of action is dense
Degree is 1-5ng/ml.
Cyclic dipeptides solution described above is aqueous solution.
Compared with prior art, the beneficial effects of the present invention are:
1, ring (different bright-different bright) dipeptides of new stereoeffect is provided, and utilizes paecilomyces varioti bacterium system for the first time
Standby CYCLIC DIPEPTIDES compounds out；
2, paecilomyces varioti bacterium bacterial strain that the present invention uses is easy culture, can be from fermentation liquid with simpler method
Middle separating-purifying obtains CYCLIC DIPEPTIDES compounds, and there are no pollution to the environment, with short production cycle, active superelevation, production and use at
This is extremely low, and is easily formed large-scale serial production.
3, the similar cyclic dipeptide compound has apparent facilitation to budding；In salt resistance, induced flowering, resist
Cucumber downy mildew, improves utilization rate of fertilizer etc. obvious effect at freeze proof, volume increase.With good Development volue, application prospect
Well.
Detailed description of the invention
The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.
Fig. 1: fermentation mycelium yield (%) result figure under the conditions of different carbon source in embodiment 1；
Fig. 2: fermentation mycelium yield (%) result figure under the conditions of different nitrogen sources in embodiment 1；
Fig. 3: fermentation mycelium yield (%) result figure under the conditions of different vaccination amount in embodiment 1；
Fig. 4: fermentation mycelium yield (%) result figure under condition of different temperatures in embodiment 1；
Fig. 5: fermentation mycelium yield (%) result figure under condition of different pH in embodiment 1；
Fig. 6: fermentation mycelium yield (%) result figure under the conditions of different rotating speeds in embodiment 1；
Fig. 7: fermentation mycelium yield (%) result figure under the conditions of different incubation times in embodiment 1；
Fig. 8: the Cyclic dipeptides product test sample HPLC chromatogram being prepared into embodiment 2；
Fig. 9: the HPLC chromatogram of the reference substance of Cyclic dipeptides is detected in embodiment 2；
Figure 10: Cyclic dipeptides nucleus magnetic hydrogen spectrum (NMR) figure being prepared into embodiment 2；
Figure 11: influence schematic diagram of the various concentration Cyclic dipeptides seed soaking to wheat germination in embodiment 3；
Figure 12: influence schematic diagram of the Cyclic dipeptides to wheat germination under different salinity in embodiment 3；
Figure 13: the influence schematic diagram after Cyclic dipeptides punching is applied in embodiment 4 to winter date flowering season；
Figure 14: the influence schematic diagram after Cyclic dipeptides punching is applied in embodiment 4 to the capsicum florescence；
Figure 15: the influence schematic diagram of various concentration Cyclic dipeptides pouring root, spraying harmful ability freeze proof to tomato.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
Embodiment 1: the fermentation technology optimization of paecilomyces varioti bacterium (Paecilomyces variotii)
1) optimization of fermentation medium
Paecilomyces varioti bacterium after activating in PDA culture medium is inoculated in seed culture medium and is cultivated, by tradition
Zymotechnique carries out fermented and cultured, and plate count is carried out after culture, calculates Fungal biodiversity.The fermentation training of traditional zymotic technique
The formula for supporting base is as shown in table 1 below:
Table 1 is traditional zymotic technique fermentation condition
Corn flour, soluble starch, glucose, lactose are replaced into the sucrose in traditional zymotic culture medium, fermentation medium
In other compositions it is constant, by inoculum concentration be 2%, 28 DEG C of temperature, initial pH 7.0, speed of agitator 180r/min, cultivate 7d after
Fungal biodiversity is recorded respectively, and as can be seen from Figure 1, optimum carbon source is glucose.
Yeast extract, bean cake powder, peptone, beef extract are added in traditional zymotic culture medium, and fully intermeshing is carried out to 4 kinds of nitrogen sources
Combination, forms 10 kinds of nitrogen source formulas, records Fungal biodiversity respectively after fermented and cultured, as can be seen from Figure 2, optimum nitrogen source is yeast
Cream+peptone.
Combined sorting is optimized according to Orthogonal Experiment and Design, obtains Optimal compositions of fermentation medium formula are as follows: potato is extracted
Liquid 1.0L, yeast extract 1.0g, peptone 3.0g, glucose 15.0g.
(2) optimization of fermentation condition
Using the culture medium after screening as Optimal compositions of fermentation medium, inoculum concentration is respectively 2%, 4%, 6%, 8%, 10%, into
Row shake flask fermentation, records Fungal biodiversity respectively, as shown in figure 3, filtering out most suitable inoculum concentration is 6%.
Using the culture medium after screening as Optimal compositions of fermentation medium, respectively at 24 DEG C, 25 DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 30 DEG C
Lower carry out shake flask fermentation, records Fungal biodiversity respectively, as shown in figure 4, filtering out optimum temperature is 28 DEG C.
Using the culture medium after screening as Optimal compositions of fermentation medium, different pH gradients 6.8,7.0,7.2,7.4,7.6 is set
Shake flask fermentation is carried out, records Fungal biodiversity respectively, as shown in figure 5, filtering out most suitable initial pH is 7.2.
Using the culture medium after screening as Optimal compositions of fermentation medium, respectively in revolving speed 140r/min, 160r/min, 180r/
Shake flask fermentation is carried out under min, 200r/min, 220r/min, records Fungal biodiversity respectively, as shown in fig. 6, filtering out most suitable turn
Speed is 160r/min.
Using the culture medium after screening as Optimal compositions of fermentation medium, shake flask fermentation 4d, 5d, 6d, 7d, 8d, record respectively respectively
Fungal biodiversity, as shown in fig. 7, filtering out most suitable incubation time is 5d.
The embodiment shows the culture medium after optimization are as follows: potato extracting solution 1.0L, yeast extract 1.0g, peptone 3.0g,
Glucose 15.0g；Optimal conditions of fermentation are as follows: inoculum concentration 6%, 28 DEG C of temperature, initial pH 7.2, speed of agitator 160r/min,
Cultivate 5d.
Compared with traditional fermentation process, use cost is low, application effect is preferable for zymotechnique after optimization, thallus biology
Amount increases 10%.
Embodiment 2: the fermentation of paecilomyces varioti bacterium (Paecilomyces variotii) and the preparation of Cyclic dipeptides compound
1) paecilomyces varioti bacterium is inoculated in PDA solid medium, 25 DEG C constant temperature incubation 5 days；By activated strain
Block, which is dug, with punch agar is inoculated in the triangular flask equipped with 200ml seed culture medium (PDA liquid medium), in 25 DEG C, 180r/
Shaken cultivation 5-7 days on min shaking table, as level-one kind liquid；Level-one kind liquid is accessed in 5L seeding tank, secondary seed tank hair is carried out
Ferment culture；By determining most suitable culture solution loaded on 121 DEG C of sterilizing 20min in fermentor, first order seed, inoculum concentration are inoculated with after cooling
It is 6%, 25.8 DEG C of temperature, initial pH7.2, speed of agitator 160r/min, cultivates 5d, daily sample detection is final that target produces
Object.Fermentative medium formula are as follows: potato extracting solution 1.0L, yeast extract 1.0g, peptone 3.0g, glucose 15.0g.
2) fermentation liquid carries out centrifugal filtration, obtains mycelium and filtering fermentating liquid；It is dried at 60 DEG C after mycelium is washed,
Weighing, then crushes through high speed disintegrator, for 24 hours with the alcohol steep of same volume, extracts 3 times, is mixed with magnetic stirring apparatus,
Ultrasonic oscillation 1h, vacuum filtration collect filtrate, as extracting solution；It will be contained after extracting solution revolving instrument concentration with isometric
There is the ethyl acetate of 5% acetone to be extracted, extracts 3 times；Then supernatant is distilled to obtain crude extract, nitrogen with Rotary Evaporators
It blows and is dried up for use under instrument；Take crude extract through the decompression silica gel column chromatography separation of 200-300 mesh, with methylene chloride, methylene chloride: methanol
(70:1), methylene chloride: methanol (30:1), methylene chloride: methanol (8:1), methylene chloride: methanol (2:1), acetone, methanol 7
Polar system quickly elutes, and collects each flow point, then repeatedly through ODS pressurization chromatography, methanol/water 12:88~100 (by 12% first
Alcohol solution becomes pure water) gradient elution, through HPLC (RP chromatography after TLC merging；Mobile phase: MeOH/H2O=50%~
100%, 40min, 1.0ml/min；Chromatographic column: C18) preparation is purified and nucleus magnetic hydrogen spectrum NMR analysis, it is (different bright-different to finally obtain ring
It is bright) dipeptide compound aqueous solution, concentration 5-15mg/ml.Cyclic dipeptides reference substance HPLC chromatogram is as shown in Figure 9, it is seen that ring two
Peptide retention time about 60 minutes, the Cyclic dipeptides product test sample HPLC chromatogram being prepared into was as shown in Figure 8.The Cyclic dipeptides of generation
It is as shown in Figure 10 that nucleus magnetic hydrogen spectrum (NMR) analyzes chromatogram.Its structural formula known to measurement are as follows:
Embodiment 3: influence of the Cyclic dipeptides compound to wheat germination under salt stress
(1) wheat seed (wheat breed: tobacco grower 15) is impregnated into 2h (seed soaking with the Cyclic dipeptides solution of clear water and various concentration
Cyclic dipeptides concentration is respectively 20,30,40ng/ml): 1. common germination condition: takes 50, the seed soaked seed to be placed on respectively and be lined with
In the big culture dish of filter paper, add clear water, adding water height is the 50% of seed height, pays attention to keeping the skin wet in germination process.It is placed in
It germinates under 15-25 DEG C of dark temperature match curing conditions, counts the extreme length and quantity of fibrous root；2. simulating salt resistance condition: by above-mentioned examination
Testing the clear water being added in culture medium and changing concentration into is 0.6%NaCl salt water, 0.8%NaCl salt water, while being compareed with clear water,
Other conditions are constant, count the extreme length and quantity of fibrous root.
Calculation method: being calculated by formula each concentration for the treatment of percentage of seedgermination, and unit is percentage (%).
In formula:
I-germination percentage；
D0-germination number；
Dt-seed sum.
The result shows that (being shown in Table 2,3, Figure 11, Figure 12), with the germination percentage for increasing wheat seed of Cyclic dipeptides seed soaking concentration
Obviously increase, longest root long is consequently increased, and also has certain facilitation for fibrous root sum.Salt resistance test result table
Bright, under 0.6% salinity, Cyclic dipeptides can also show to improve wheat seed germinating rate and promote the work of root long and quantity
With.
2 Cyclic dipeptides of table are to measurement result (3 days) in the common growing floor of tobacco grower 15
| CK | 20ng/ml | 30ng/ml | 40ng/ml | |
| Germination percentage (%) | 32 | 42 | 50 | 66 |
| Longest root average (mm) | 24.59 | 26.46 | 27.75 | 28.88 |
| 3 or more fibrous root ratios (%) | 0 | 1 | 2 | 3 |
Note: CK refers to that clear water is soaked seed in upper table.
3 Cyclic dipeptides of table are to 15 salt tolerance indoor measurement result (7 days) of tobacco grower
Embodiment 4: influence of the Cyclic dipeptides compound to capsicum, winter jujube florescence
With concentration be 20-30ng/ml Cyclic dipeptides compound water solution carried out before winter jujube tree, capsicum bloom punching apply, capsicum
Foliar spray, jujube tree pouring root find advance flowering period.As shown in Figure 13 and Figure 14, winter jujube tree amount relatively compares big, and capsicum relatively compares the florescence
5-6 days in advance.Thus it is speculated that Cyclic dipeptides compound can induce bud differentiation, promote plant Blooming.
Embodiment 5: inductive effect of the Cyclic dipeptides to cucumber downy mildew
It is " the green 21-10 of saliva " for examination cucumber seeds.(per acre with the water-soluble liquid irrigating root of 5-10ng/ml Cyclic dipeptides by plastic tent cucumber
5 liters of Cyclic dipeptides solution of pouring root) after processing, investigation cucumber downy mildew disease index, sick leaf rate.
Cucumber downy mildew grade scale: 0 grade: disease-free spot；1 grade: lesion area accounts for 5% or less entire leaf area；3 grades:
Lesion area accounts for the 6%-10% of entire leaf area；5 grades: lesion area accounts for the 11%-25% of entire leaf area；7 grades: scab face
Product accounts for the 26%-50% of entire leaf area；9 grades: lesion area accounts for 50% or more of entire leaf area.
As shown in table 4, the results showed that, it can significantly reduce the disease incidence of cucumber downy mildew, and the state of an illness after being handled with Cyclic dipeptides
Index is substantially reduced.
Inductive effect of 4 Cyclic dipeptides of table to cucumber downy mildew
Embodiment 6: the influence of Cyclic dipeptides harmful ability freeze proof to tomato
Potted tomato is filled after diluting 3000-6000 times (Cyclic dipeptides concentration is 1-5ng/ml) with Cyclic dipeptides fermentation liquid
Root and spraying and low-temperature treatment (- 10 DEG C), while control treatment is set, as shown in figure 15, from left to right successively are as follows: conventional treatment
CK, chemical Water soluble fertilizer, Cyclic dipeptides fermentation liquid dilute 3000 times of pourings, Cyclic dipeptides fermentation liquid dilutes 6000 times of spraying, Cyclic dipeptides hairs
Zymotic fluid dilutes 6000 times of pourings, 6000 times of pourings of Cyclic dipeptides fermentation liquid dilution+spraying, observes after 10 days.The result shows that through low temperature
There is freeze injury phenomenon in the tomato of processing, but tomato is more right by the degree of freeze injury after the irrigation of Cyclic dipeptides fermentation liquid, spraying treatment
According to mitigation.Thus infer, Cyclic dipeptides substance may participate in Genes For Plant Tolerance Low temperature regulation.
Embodiment 7: influence of the Cyclic dipeptides to great Jiang yield
It is sowed after being dressed seed with Cyclic dipeptides aqueous solution to kind of Jiang Jinhang, and carries out pouring root punching in cauline leaf and root growth peak period
It applies, while setting Routine control.As a result, it has been found that being 40.64 jin through Cyclic dipeptides treated great Jiang per mu yield, control per mu yield is
30.24 jin.It can be seen that Cyclic dipeptides can effectively improve the yield of great Jiang.
Embodiment 8: influence of the Cyclic dipeptides to urea nitrogen use efficiency
Using wheat as test material, different fertilization: urea, urea+Cyclic dipeptides, controlled release urine is carried out in its breeding time
Element, control release urea+Cyclic dipeptides bury bag nutrients release method using indoor incubator hydrostatic nutrient extraction (25 DEG C) and soil and measure
For trying the Nutrient Release Characteristics of coated fertilizer；Content of inorganic nitrogen in soil (air-dried pedotheque) is extracted with the CaCl2 of 0.01mol/L
(Tu Shui is than 1: 10), in leaching liquor NO3--N and NH4+-N content using continuous flow injection analyzer (Bran+Luebbe,
AA3, Germany) measurement, total nitrogen content is disappeared using H2SO4-H2O2 boils, Kjeldahl nitrogen determination.As a result, it has been found that control release urea
Utilization rate of nitrogen fertilizer dramatically increase 9.6 percentage points than urea；The utilization rate of nitrogen fertilizer of " urea+Cyclic dipeptides " processing compares Urea treatment
Dramatically increase 10.1 percentage points；The utilization rate of nitrogen fertilizer of " control release urea+Cyclic dipeptides " processing is dramatically increased than control release urea processing
12.4 percentage points, 11.9 percentage points are dramatically increased than " urea+Cyclic dipeptides " processing.Thus it can be extrapolated that control release urea is with applying
Cyclic dipeptides can effectively improve utilization rate of nitrogen fertilizer.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above, the skill of the art
Variation, addition or the replacement that art personnel are made in knowledge of the invention, also should belong to protection scope of the present invention.
Claims (10)
1. a kind of ring (different bright-different bright) dipeptides, which is characterized in that its structural formula are as follows:
2. a kind of preparation method of ring (different bright-different bright) dipeptides according to claim 1, which is characterized in that the ring
(bacterial strain is in 2014 12 with isolated one plant of paecilomyces varioti bacterium SJ1 from wild sea-buckthorn for (different bright-different bright) dipeptides
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on 8th are as follows: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, deposit number CGMCCNO.10114) it is fermented, separation be made, the preparation method includes the following steps:
The first step, the fermentation of paecilomyces varioti bacterium:
Paecilomyces varioti bacterium is inoculated in PDA solid medium, 25 DEG C constant temperature incubation 5-7 days；Activated strain is used and is beaten
Hole device agar digs block and is inoculated in the triangular flask equipped with 200ml seed culture medium, in shaken cultivation 5 on 25 DEG C, 180r/min shaking table
It is as level-one kind liquid；Level-one kind liquid is accessed in 5L seeding tank, secondary seed tank fermented and cultured is carried out；By determining most suitable training
Nutrient solution liquid is inoculated with first order seed, inoculum concentration 2-10%, temperature 24-30 after cooling loaded on 121 DEG C of sterilizing 20min in fermentor
DEG C, initial pH6.8-7.6, speed of agitator 140-220r/min, culture obtain fermentation liquid in 4-8 days；Cultivate formula of liquid are as follows: potato
Extracting solution 1.0L, yeast extract 1.0g, peptone 3.0g, glucose 15.0g；
Second step, fermentation liquid carry out centrifugal filtration, obtain mycelium and filtering fermentating liquid；
Third step is dried at 60 DEG C after washing mycelium, is weighed, is then crushed through high speed disintegrator, with the ethyl alcohol of same volume
Extraction for 24 hours, is extracted 3 times, is mixed with magnetic stirring apparatus, ultrasonic oscillation 1h, is filtered by vacuum, and is collected filtrate, is as extracted
Liquid；
The preparation of 4th step, crude extract: isometric acetic acid containing 5% acetone will be used after the revolving instrument concentration of said extracted liquid
Ethyl ester is extracted, and is extracted 3 times；Then supernatant is distilled to obtain crude extract with Rotary Evaporators, is dried up under nitrogen evaporator stand-by；
5th step takes crude extract through the decompression silica gel column chromatography separation of 200-300 mesh, with methylene chloride, methylene chloride: methanol (70:
1), methylene chloride: methanol (30:1), methylene chloride: methanol (8:1), methylene chloride: 7 methanol (2:1), acetone, methanol polarity
System quickly elutes, and collects each flow point, then repeatedly through ODS pressurization chromatography, methanol/water (12:88~100) gradient elution, TLC
Purifying is prepared through HPLC after merging and NMR is analyzed.
3. a kind of preparation method of ring (different bright-different bright) dipeptides according to claim 2, which is characterized in that as if the first step
In family name's paecilomycerol fermentation process, fermentation condition are as follows: first order seed inoculum concentration is 6%, 28 DEG C of temperature, initial pH 7.2, is stirred
Revolving speed 160r/min is cultivated 5 days.
4. a kind of application of ring (different bright-different bright) dipeptides according to claim 1, which is characterized in that including to Crop Species
The facilitation of son germination improves crop salt-resistance, induction crop flowers, anti-cucumber downy mildew, promotes crop frost resistance.
5. a kind of application of ring (different bright-different bright) dipeptides according to claim 4, which is characterized in that the ring (it is different it is bright-
It is different bright) dipeptides to the facilitation of budding be improve salt stress under wheat germination percentage, increase salt stress under wheat
Wheat root quantity under root long and increase salt stress, ring (different bright-different bright) dipeptides is to wheat seed germinating under salt stress
Facilitation action concentration is 20-40ng/ml.
6. a kind of application of ring (different bright-different bright) dipeptides according to claim 5, which is characterized in that the ring (it is different it is bright-
It is different bright) to can tolerate salinity to the facilitation of wheat seed germinating under salt stress be the salt water not higher than 0.8% to dipeptides, it is described
Salt water is sodium chloride solution.
7. a kind of application of ring (different bright-different bright) dipeptides according to claim 4, which is characterized in that the ring (it is different it is bright-
It is different bright) effects of two inducing peptide crop flowers is induction winter jujube tree and capsicum bud differentiation, promote winter jujube tree and capsicum to open in advance
Flower.
8. a kind of application of ring (different bright-different bright) dipeptides according to claim 7, which is characterized in that the ring (it is different it is bright-
It is different bright) the onset of action concentration of two inducing peptide crop flowers is 20-30ng/ml.
9. a kind of application of ring (different bright-different bright) dipeptides according to claim 4, which is characterized in that the ring (it is different it is bright-
It is different bright) dipeptides reduce cucumber downy mildew disease incidence onset of action concentration be 5-10ng/ml.
10. a kind of application of ring (different bright-different bright) dipeptides according to claim 9, which is characterized in that the ring (it is different it is bright-
It is different bright) to promote the effect of crop frost resistance be to reduce potted tomato by freeze injury degree to dipeptides, root and spraying treatment are sprayed through Cyclic dipeptides
Potted tomato can tolerate low temperature range be -10-0 DEG C, Cyclic dipeptides onset of action concentration be 1-5ng/ml.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007122446A1 (en) * | 2006-04-26 | 2007-11-01 | Commissariat A L'energie Atomique | Cyclodipeptide synthetases and their use for synthesis of cyclo(leu-leu) cyclodipeptide |
| CN105307514A (en) * | 2013-06-10 | 2016-02-03 | 三得利控股株式会社 | Plant extract containing diketopiperazine and method for producing same |
| CN106456633A (en) * | 2014-06-20 | 2017-02-22 | 三得利控股株式会社 | Glucose metabolism ameliorating agent |
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2019
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Patent Citations (3)
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|---|---|---|---|---|
| WO2007122446A1 (en) * | 2006-04-26 | 2007-11-01 | Commissariat A L'energie Atomique | Cyclodipeptide synthetases and their use for synthesis of cyclo(leu-leu) cyclodipeptide |
| CN105307514A (en) * | 2013-06-10 | 2016-02-03 | 三得利控股株式会社 | Plant extract containing diketopiperazine and method for producing same |
| CN106456633A (en) * | 2014-06-20 | 2017-02-22 | 三得利控股株式会社 | Glucose metabolism ameliorating agent |
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| LI ZHENG等: "Hymeniacidon perleve Associated Bioactive Bacterium Pseudomonas sp.NJ6-3-1", 《BIOCHEMISTRY AND MICROBIOLOGY》 * |
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Denomination of invention: A cyclic (isoleucine isoleucine) dipeptide, its preparation method and Application Effective date of registration: 20210610 Granted publication date: 20201225 Pledgee: Tai'an financing Company limited by guarantee Pledgor: SHANDONG PENGBO BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021980004604 |
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