Fermented organic matter with anti-pathogenic activity of potato endophyte, preparation process and application thereof, and fermented product
阅读说明：本技术 马铃薯内生菌的具有抗病原活性的发酵有机物、其制备工艺、应用以及发酵产物 (Fermented organic matter with anti-pathogenic activity of potato endophyte, preparation process and application thereof, and fermented product ) 是由 冯涛 艾洪莲 何隽 杨会祥 李正辉 刘吉开 于 2020-07-23 设计创作，主要内容包括：本发明涉及发酵技术领域,具体而言,涉及马铃薯内生菌的具有抗病原活性的发酵有机物、其制备工艺、应用以及发酵产物。该发酵有机物选自以下结构式所示化合物中的任意一种：<Image he="372" wi="596" file="DDA0002597764500000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>和<Image he="383" wi="557" file="DDA0002597764500000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>该有机物为首次利用马铃薯内生菌Trichotheciumcrotocinigenum得到的化合物,其为马铃薯内生菌制备得到的新化合物提供新的思路,扩大了马铃薯内生菌制备化合物的类型和范围。且制备该有机物的方法简洁,易于实施,可以大规模工业化生产。(The invention relates to the technical field of fermentation, in particular to a fermented organic matter with anti-pathogenic activity of potato endophyte, a preparation process and application thereof, and a fermented product. The fermentation organic matter is selected from any one of the compounds shown in the following structural formula: and the organic matter is a compound obtained by utilizing potato endophyte Trichothecium crotoconigenium for the first time, provides a new thought for preparing a new compound by the potato endophyte, and enlarges the type and range of the compound prepared by the potato endophyte. And the method for preparing the organic matter is simple and easy to implement, and can be used for large-scale industrial production.)
1. A fermented organic matter of potato endophyte with anti-pathogenic activity, characterized in that the fermented organic matter is selected from any one of the compounds represented by the following structural formula:
2. a process for preparing an anti-pathogenic fermented organic substance from potato endophyte according to claim 1, comprising post-treating the fermented organic substance with a fermentation product of potato endophyte Trichothecium crotoconigenin.
3. The process according to claim 2, wherein the post-treatment comprises: and carrying out liquid-liquid separation on the fermentation product, and then carrying out silica gel column separation, medium-pressure chromatographic separation and high-pressure liquid phase preparation on the organic phase obtained by the liquid-liquid separation in turn.
4. The process according to claim 2, wherein the post-treatment comprises: and extracting the fermentation product by using ethyl acetate, then carrying out silica gel column separation on the extract liquor, then carrying out medium-pressure chromatographic separation on the fourth elution combined solution obtained by silica gel column separation, and then carrying out high-pressure liquid phase on the second elution combined solution obtained by medium-pressure chromatographic separation.
5. The preparation process according to claim 3 or 4, wherein the silica gel column separation is eluted by: eluting chloroform and methanol at concentration gradient of 100:0, 50:1, 30:1, 20:1, 10:1 and 5: 1;
preferably, the elution process for the medium pressure chromatographic separation is: methanol and water are mixed according to concentration gradients of 10:90, 20:80, 30:70, 40:60, 50:50, 60: 40: eluting the fourth elution combined solution obtained by the silica gel column separation at the ratio of 70:30, 80:20, 90:10 and 100: 0;
preferably, the elution process of the high pressure liquid phase separation is: and eluting the second elution combined solution obtained by the medium-pressure chromatographic separation by using acetonitrile and water according to the concentration gradient of 25:75 and 50: 50.
6. The process according to claim 2, wherein the fermentation process of Trichothecium crotocinigenin is as follows: inoculating the Trichothecium crotoconigenium into a culture medium and then fermenting;
preferably, the conditions of the fermentation are: 24-26 ℃, the pH value is 5.9-6.1, the rotating speed is 220-280r/min, and the air inlet amount is 0.8-1.2 vvm.
7. The preparation process of claim 6, wherein the culture medium comprises, in mass percent, 0.45-0.55 wt% of yeast powder, 0.12-0.18 wt% of pork peptone, 4.8-5.2 wt% of glucose, 0.045-0.055 wt% of potassium dihydrogen phosphate, and 0.045-0.055 wt% of magnesium sulfate.
8. The process according to claim 6, wherein the amount of the inoculum of Trichothecium crotocinigenin is 6-12 vt%.
9. A fermentation product comprising the fermented organic of claim 1.
10. Use of a fermented organic matter of potato endophyte according to claim 1, a fermented organic matter produced by a process for producing an anti-pathogenic fermented organic matter of potato endophyte according to any one of claims 2 to 8, or a fermentation product according to claim 9 for inhibiting phytopathogens or for producing a phytocide;
preferably, the phytopathogen comprises any one of Phytophthorainfestans, alternariolani, rhizoctonialani and fusarium oxysporum.
Technical Field
The invention relates to the technical field of fermentation, in particular to a fermented organic matter with anti-pathogenic activity of potato endophyte, a preparation process and application thereof, and a fermented product.
Background
Endophytes are bacteria that live in the intercellular spaces or cells of various tissues and organs of healthy plants at some or all stages of their life history, and their endogenesis can be demonstrated by histological methods or by methods of isolation from severely surface-sterilized plant tissues or by direct production of amplified microbial DNA from within plant tissues. In the prior art, different endophytes are generally obtained by separating potato endophytes, and then the influence of the endophytes on the growth of plants or the action of the endophytes is detected or evaluated, but the research on fermentation products or fermentation organic matters of the endophytes is less.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a fermented organic matter with anti-pathogenic activity of potato endophyte, a preparation process and an application thereof, and a fermented product. The fermentation organic matter is a new compound, provides a new idea for preparing the new compound by the potato endophyte, and enlarges the type and range of the compound prepared by the potato endophyte.
The invention is realized by the following steps:
in a first aspect, embodiments of the present invention provide a fermented organic matter with anti-pathogenic activity of potato endophyte, wherein the fermented organic matter is selected from any one of compounds represented by the following structural formula:
In a second aspect, embodiments of the present invention provide a process for preparing an anti-pathogenic fermented organic substance from potato endophyte, which comprises performing post-treatment on a fermentation product of potato endophyte trichothecin, thereby obtaining a fermented organic substance.
In a preferred embodiment, the post-processing comprises: liquid-liquid separation is carried out on the fermentation product, and then silica gel column separation, medium-pressure chromatographic separation and high-pressure liquid phase preparation are carried out on the organic phase obtained by the liquid-liquid separation in turn. The liquid-liquid separation can be extraction or other means that can be used for separation.
In a preferred embodiment, the post-processing comprises: and extracting the fermentation product by using ethyl acetate, then carrying out silica gel column separation on the extract liquor, then carrying out medium-pressure chromatographic separation on the fourth elution combined solution obtained by silica gel column separation, and then carrying out high-pressure liquid phase on the second elution combined solution obtained by medium-pressure chromatographic separation.
In a preferred embodiment, the elution process for the silica gel column separation is: eluting chloroform and methanol at concentration gradient of 100:0, 50:1, 30:1, 20:1, 10:1 and 5:1, and collecting eluate obtained from each elution to obtain 6 kinds of elution composition solutions.
In the gradient elution with chloroform and methanol, chloroform and methanol are mixed in the volume ratio of 100:0, 50:1, 30:1, 20:1, 10:1 and 5:1 respectively to form eluates, and then the eluates are eluted, and the eluates with corresponding concentrations are collected after the elution. The method comprises the steps of mixing chloroform and methanol according to the volume ratio of 100:0 to form eluent, then eluting, collecting the eluent as a first elution combined solution, mixing chloroform and methanol according to the volume ratio of 50:1 to form eluent, then eluting, collecting the eluent as a second elution combined solution, mixing chloroform and methanol according to different volumes, and then eluting to obtain 6 elution combined solutions.
The following gradient elution according to concentration is the same as the gradient elution of chloroform and methanol, and is to mix solvents according to volume ratio to form eluent, then elute and collect the eluent combination liquid with different components.
Preferably, the elution process for the medium pressure chromatographic separation is: methanol and water are mixed according to concentration gradients of 10:90, 20:80, 30:70, 40:60, 50:50, 60: 40: eluting the fourth elution combined solution obtained by the silica gel column separation at the ratio of 70:30, 80:20, 90:10 and 100: 0; that is to say, the medium pressure chromatography gives a total of 10 elution combinations.
Preferably, the elution process of the high pressure liquid phase separation is: and eluting the second elution combined solution obtained by the medium-pressure chromatographic separation by using acetonitrile and water according to the concentration gradient of 25:75 and 50:50 to obtain 2 elution combined solutions, wherein each elution combined solution corresponds to one of the compounds shown in the structural formula.
In a preferred embodiment, the fermentation process for Trichothecium crotoconigenin is: inoculating Trichothecium crotoconigenium into a culture medium and then fermenting;
preferably, the conditions of the fermentation are: 24-26 ℃, the pH value is 5.9-6.1, the rotating speed is 220-280r/min, and the air inlet amount is 0.8-1.2 vvm.
In a preferred embodiment, the culture medium comprises, in mass percent, 0.45-0.55 wt% of yeast powder, 0.12-0.18 wt% of pork peptone, 4.8-5.2 wt% of glucose, 0.045-0.055 wt% of potassium dihydrogen phosphate and 0.045-0.055 wt% of magnesium sulfate.
In a preferred embodiment, the amount of inoculum of Trichotechnium crotocinigenin is 6-12 vt%.
In a third aspect, embodiments of the present invention provide a fermentation product comprising the above-described fermented organic matter.
In a fourth aspect, embodiments of the present invention provide a fermented organic matter of potato endophyte having anti-pathogenic activity, a fermented organic matter prepared by the preparation process of the fermented organic matter of potato endophyte having anti-pathogenic activity, or an application of the fermented product in inhibiting plant pathogenic bacteria or preparing a plant bactericide;
preferably, the phytopathogen comprises any one of Phytophthorainfestans, alternariolani, rhizoctonialani and fusarium oxysporum (blast).
The invention has the following beneficial effects: the embodiment of the invention provides a novel fermentation compound of Trichothecium crotoconigenin, which is a novel compound obtained from potato endophyte Trichothecium crotoconigenin for the first time, provides a novel idea for preparing the novel compound from the potato endophyte, and expands the type and range of the compound prepared from the potato endophyte. And the method for preparing the organic matter is simple and easy to implement, and can be used for large-scale industrial production.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment of the invention provides a fermented organic matter with anti-pathogenic activity for potato endophyte, which is selected from any one of the compounds shown in the following structural formula:
The embodiment of the invention also provides a preparation process of the fermented organic matter, which comprises the following steps:
first, trichothecium crocinum is a conventionally known potato endophyte, which may be purchased from the market or obtained by self-culture using potato as a raw material. The procedure for obtaining Trichotechnium crocinigenin by potato culture was as follows: the potato is disinfected and cut into pieces, then the root pieces, the stem pieces and the leaf pieces of the potato are respectively inoculated on a solid culture medium containing mixed liquid of penicillin and streptomycin, 1 to 2 pieces are put on each plate, and the potato is cultured in a constant temperature incubator at the temperature of 22 to 25 ℃. And (4) observing regularly every day, and when macroscopic colonies grow to the periphery of the culture medium in different tissues of the potatoes, selecting single colonies with obvious differences for purification culture according to the colony morphology, the color and the growth time.
The single colonies with obvious differences include, but are not limited to, the following characteristics: the hyphae were white, and the center of the colony was white, sparse and growing radially.
The conditions for the purification culture were as follows: picking white hyphae at the front edge of a colony to a fresh culture plate, repeating for 3 times, observing the form of the hyphae under a common optical microscope, and if the form is uniform, successfully purifying.
The purified cultured Trichothecium crotoconigenium has the following physiological and biochemical characteristics: the colony grown on PDA medium had white radial hyphae in the center and white hyphae in the periphery.
Then fermenting the Trichothecium crotoconigenium, inoculating the Trichothecium crotoconigenium into a culture medium according to the inoculation amount of 6-12 vt%, and then fermenting. Specifically, the amount of inoculation may be 6 vt%, 7 vt%, 8 vt%, 9 vt%, 10 vt%, 11 vt%, or 12 vt%, and may be any value between the above-mentioned values of the various amounts of inoculation, such as 8.5 vt%, 9.5 vt%, 10.5 vt%, or 11.5 vt%. In some preferred embodiments, the amount of inoculum of Trichothecium crotoconigenium is 9 vt%. The inoculation amount can further ensure the fermentation of the Trichothecium crotoconigenium, and is beneficial to obtaining the required fermented organic matters through subsequent separation.
Further, the fermentation conditions were: 24-26 ℃, the pH value is 5.9-6.1, the rotating speed is 220-280r/min, and the air inlet amount is 0.8-1.2 vvm. In some preferred embodiments, the fermentation conditions are: at 25 deg.C, pH 6.0 and rotation speed 160 r/min. And vvm represents the aeration ratio, namely the ratio of the aeration quantity per minute to the actual feed liquid volume of the tank body. By adopting the fermentation conditions, the fermentation effect can be further ensured, and the fermented organic matters can be obtained.
Further, the culture medium comprises, by mass, 0.45-0.55 wt% of yeast powder, 0.12-0.18 wt% of pork peptone, 4.8-5.2 wt% of glucose, 0.045-0.055 wt% of potassium dihydrogen phosphate and 0.045-0.055 wt% of magnesium sulfate. The culture medium can further ensure the culture effect and is beneficial to separating and obtaining the required organic matters.
And performing post-treatment on the fermentation product, wherein the post-treatment comprises liquid-liquid separation, specifically, extracting the fermentation product by using ethyl acetate, collecting an organic phase, and then, purifying and separating the organic phase by using silica gel column separation, and specifically, the elution process of the silica gel column separation is as follows: eluting chloroform and methanol at concentration gradient of 100:0, 50:1, 30:1, 20:1, 10:1 and 5:1, and collecting eluate of each time to obtain 6 kinds of elution composition solutions.
And then separating the fourth elution combined solution by using medium pressure chromatography, specifically, separating methanol and water according to the concentration gradient of 10:90, 20:80, 30:70, 40:60, 50:50, 60: 40: eluting the fourth elution composition from the silica gel column at a ratio of 70:30, 80:20, 90:10 and 100: 0.
And then carrying out high-pressure liquid-phase separation on the second elution combined solution obtained by the medium-pressure chromatographic separation, specifically, eluting the second elution combined solution obtained by the medium-pressure chromatographic separation by using acetonitrile and water according to the concentration gradient of 25:75 and 50:50 to obtain 2 elution combined solutions, wherein each elution combined solution corresponds to one of the compounds shown in the structural formula. Specifically, 2 eluate combinations obtained by HPLC were dried separately, i.e., LYBR115 and LYBR117 were obtained.
The specific operations of silica gel column separation, medium-pressure chromatography separation, high-pressure liquid phase separation and the like are the same as those in the prior art, and the detailed description of the embodiments of the present invention is omitted.
By adopting the mode for separation and purification, impurities in the fermentation product can be effectively separated to obtain the required compound.
The embodiment of the invention provides a fermentation product, which comprises the fermentation organic matter. The fermentation product may also be a fermentation product obtained after fermentation of the aforementioned Trichothecium crotoconigenin, which necessarily includes the aforementioned fermentation organic.
The embodiment of the invention provides the fermented organic matter, the fermented organic matter prepared by the preparation process of the fermented organic matter or the application of the fermented product in inhibiting plant pathogenic bacteria or preparing plant bactericide; and the phytopathogen includes any one of Phytophthorainfestans, alternariolani, rhizoctonialani and fusarium oxysporum (blast).
The features and properties of the present invention are described in further detail below with reference to examples.
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