CN102719363B - Preparation method of antibacterial fermentation liquid of Solidago canadesis endophytic fungi - Google Patents
Preparation method of antibacterial fermentation liquid of Solidago canadesis endophytic fungi Download PDFInfo
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- 239000007788 liquid Substances 0.000 title claims abstract description 42
- 241000607059 Solidago Species 0.000 title claims abstract description 37
- 241000233866 Fungi Species 0.000 title claims abstract description 35
- 238000000855 fermentation Methods 0.000 title claims abstract description 19
- 230000004151 fermentation Effects 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 230000000844 anti-bacterial effect Effects 0.000 title abstract description 10
- 241001515917 Chaetomium globosum Species 0.000 claims abstract description 13
- 238000012360 testing method Methods 0.000 claims abstract description 7
- 238000011218 seed culture Methods 0.000 claims abstract description 5
- 239000002609 medium Substances 0.000 claims description 16
- 230000003115 biocidal effect Effects 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 9
- 239000008188 pellet Substances 0.000 claims description 9
- 239000001965 potato dextrose agar Substances 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
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- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000011790 ferrous sulphate Substances 0.000 claims description 3
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 229940001516 sodium nitrate Drugs 0.000 claims description 3
- 235000010344 sodium nitrate Nutrition 0.000 claims description 3
- 239000004317 sodium nitrate Substances 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 244000000004 fungal plant pathogen Species 0.000 abstract description 5
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 16
- 241000193738 Bacillus anthracis Species 0.000 description 6
- 241000223195 Fusarium graminearum Species 0.000 description 6
- 241000233616 Phytophthora capsici Species 0.000 description 6
- 241000221955 Chaetomium Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 229930000044 secondary metabolite Natural products 0.000 description 4
- 241001529387 Colletotrichum gloeosporioides Species 0.000 description 3
- 241000235819 Cytospora Species 0.000 description 3
- 241001270517 Cytospora mali Species 0.000 description 3
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- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 3
- 240000003768 Solanum lycopersicum Species 0.000 description 3
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- 230000000845 anti-microbial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
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- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000299507 Gossypium hirsutum Species 0.000 description 1
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- 244000098338 Triticum aestivum Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
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- 235000012735 amaranth Nutrition 0.000 description 1
- 239000004178 amaranth Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 229930003944 flavone Natural products 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
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- 238000010257 thawing Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
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Abstract
The invention relates to a preparation method of an antibacterial fermentation liquid of Solidago canadesis endophytic fungi, which comprises the following operation steps: 1. activating strain; 2. carrying out first-stage seed culture; 3. carrying out second-stage seed culture; and 4. carrying out fermentation culture to obtain brown Solidago canadesis endophytic fungus fermentation liquid, filtering the Solidago canadesis endophytic fungus fermentation liquid in aseptic condition to remove mycelia, and then passing a 0.22 mu m filter to obtain the antibacterial fermentation liquid of Solidago canadesis endophytic fungi. According to the invention, the preparation method of antibacterial fermentation liquid of Chaetomium globosum yt03 with broad spectrum antibacterial activity is determined; and antibacterial test finds that the Chaetomium globosum yt03 antibacterial fermentation liquid has relatively significant hypha growth inhibiting action on six tested plant pathogenic fungi.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of fermentation culture method, be specifically related to the preparation method of the antibiotic fermented liquid of solidago canadesis endogenetic fungus.
Background technology
Along with the raising of expanding economy and living standards of the people, people more and more concern for the environment pollute the problems such as the eubiosis and human health.Especially require also more and more higher to Food Quality.Pollution-free vegetable and green food have obtained generally acknowledging of the whole society.The development and utilization biological pesticide is carried out biological control, and is imperative.Current, microbial pesticide will be exhibited one's skill to the full in carrying out biological control.Because it has obvious advantage: environmentally safe, nuisanceless, noresidue is difficult for producing resistance, and production cost is low, and zymotechnique is simple, to non-target organism safety.
In for a long time interaction of host plant, coevolution, mutually in the adaptive process, in plant materials, kept the microecosystem balance as the plant endogenesis epiphyte (Endophytic fungi) in one of microbial pesticide source.Simultaneously, in order to keep this balance, plant endogenesis epiphyte may produce the secondary metabolite of or same function similar to its surrounding enviroment (being host plant).Therefore, can think the meta-bolites that the secondary metabolite of plant endogenesis epiphyte can the alternate host plant, and then as the potential or alternate resources of new compound, novel drugs screening, thereby can effectively reduce the R﹠D costs of novel drugs or other purposes active substance.
Solidago canadesis (
Solidgo canadensis) as a kind of alien species, reproductivity is extremely strong, velocity of propagation is fast, and growth vigor is obvious, and ecological suitability is wide, strives sunlight with surrounding plants, strives fertilizer, until other plant is dead, thereby species diversity is constituted a serious threat.Solidago canadesis can be done and view and admire cut-flower, feed, nectar source, can be used for simultaneously the production of natural pigment and refines essential oil.At present, have been found that the flavones ingredient that has stronger anti-oxidant, free radical eliminate activity and multiple bacteriostatic activity in the solidago canadesis body.The plant vat liquor of reporting in addition in addition solidago canadesis has significant allelopathy to seed germination and the growth of seedling of many weeds (such as bare headed barnyard grass and Herba Setariae viridis) and various vegetables (alfalfa, red three-coloured amaranth, wheat, soybean and cotton).According to the plant endogenesis epiphyte endosymbiotic theory, solidago canadesis probably exists special endogenetic fungus, by producing the special secondary metabolite with anti-microbial activity.At present, also there is not solidago canadesis endogenetic fungus secondary metabolite both at home and abroad to the research report of phytopathogen bacteriostatic activity.
Summary of the invention
The object of the present invention is to provide a kind of preparation method with antibiotic fermented liquid of solidago canadesis endogenetic fungus of stronger inhibition plant pathogenic fungi activity.
Solidago canadesis endogenetic fungus called after chaetomium globosum yt03(of the present invention
Chaetomium globosumYt03), now be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, No. 1 institute in Beichen West Rd., Beijing, preservation date on July 20th, 2011, deposit number is CGMCC No.5088.
Solidago canadesis endogenetic fungus described in the invention be therefrom state Hefei City, Anhui Province solidago canadesis (
Solidgo canadensis) separate in the plant living body and to obtain.
Solidago canadesis endogenetic fungus called after chaetomium globosum yt03(
Chaetomium globosumYt03), be deposited in Chinese Typical Representative culture collection center, preservation date on July 20th, 2011, deposit number is CGMCC No.5088;
The concrete fermentation preparation manipulation step of the antibiotic fermented liquid of solidago canadesis endogenetic fungus of the present invention is as follows:
(1) actication of culture
Under aseptic condition, with a little solidago canadesis endogenetic fungus test tube slant preservation of bacteria strain of transfering loop picking, transfer in sterilized potato dextrose agar solid medium flat board, put and cultivate 28 ℃ ± 1 ℃ of the temperature inside the box, activation culture 5~7 days obtains activated spawn;
(2) seed culture
Activated spawn being broken under aseptic condition with aseptic punch tool (internal diameter 5mm) is 5mm bacterium dish directly, inoculates 5 ferfas dish in the triangle shaking flask that contains the 50ml liquid nutrient medium, and constant-temperature table is cultivated, 160 rev/mins of shaking speed, 28 ℃ ± 2 ℃ of temperature were cultivated 2~3 days, got the shaking table seed;
(3) fermentation culture
The shaking table seed is inoculated in the 250ml triangle shaking flask that the 50ml liquid nutrient medium is housed, inoculum size 5%, 160 rev/mins of shaking speed, 28 ℃ ± 2 ℃ of temperature were cultivated 5~7 days, obtained solidago canadesis endogenetic fungus fermented liquid; Solidago canadesis endogenetic fungus fermented liquid removes by filter mycelium under aseptic condition, cross 0.22 micron filter, namely obtains the antibiotic fermented liquid of solidago canadesis endogenetic fungus;
The fermentation culture feature: cultivated the 1st day, thalline is the powder of a small amount of grey; Cultivated the 3rd day, the fine powder of pale pink reduces, and the little mycelium pellet of black occurs; Cultivated the 5th day, the black mycelium pellet continues to increase, and the volume change is large, and fermented liquid is tawny; Cultivated the 7th day, and the mycelium pellet of a large amount of black occurred, volume also becomes greatly, and fermented liquid is brown;
Described potato dextrose agar solid medium: 1 liter of potato 200 gram, glucose 20 grams, agar 18 grams, distilled water;
Described liquid nutrient medium: peptone 15 grams, glucose 20 grams, calcium chloride 0.5 gram, dipotassium hydrogen phosphate 0.1 gram, ferrous sulfate
0.01 gram, SODIUMNITRATE 1 gram, 1000 milliliters in water, pH nature;
Before using with above-mentioned two kinds of substratum 30 min that under 121 ℃ of temperature, pressure 0.1MPa, sterilize.
The present invention finds by the antibacterial tests to the antibiotic fermented liquid of chaetomium globosum yt03, this bacterium to for examination six kind of plant pathogenic fungies (be the Phytophthora capsici germ (
Phytophthora capsici), the tomato wilt bacterium (
Fusarium oxysporum), Valsa mali
(Cytospora mandshurica),Apple anthrax bacteria
(Colletotrichum gloeosporioides),The jujube anthrax bacteria
(Gloelsporium fructigenum),Fusarium graminearum (
Fusarium graminearum)) have and comparatively significantly suppress the mycelial growth effect.The present invention utilizes endophytic fungus resources of plants, in the hope of obtaining anti-plant pathogenic fungi disease novel agricultural antibiotics natural component.
The antibacterial tests of the antibiotic fermented liquid of solidago canadesis endogenetic fungus of the present invention is as follows:
1. plant pathogenic fungi:
The Phytophthora capsici germ (
Phytophthora capsici), the tomato wilt bacterium (
Fusarium oxysporum), Valsa mali (
Cytospora mandshurica), apple anthrax bacteria (
Colletotrichum gloeosporioides), the jujube anthrax bacteria
(Gloelsporium fructigenum), fusarium graminearum (
Fusarium graminearum);
2. the cultivation of pathogenic micro-organism
The slant culture of getting pathogenic bacteria accesses respectively the dull and stereotyped activation of potato dextrose agar solid medium, cultivates 5 days in (28 ± 2) ℃ thermostat container;
3. suppress the mycelial growth rate method and measure anti-microbial activity
The antibiotic fermented liquid of the solidago canadesis endogenetic fungus of 6 milliliters of the present invention's preparations of adding and the substratum of 54 milliliters of aseptic thawings of potato dextrose agar in 100 milliliters aseptic triangular flask, shake up, respectively getting respectively 10 milliliters, to place diameter be to make flat board in 9 centimetres of sterile petri dish, put into 1 for examination pathogenic bacteria bacterium cake (diameter is 6 millimeters) on each substratum plane after the cooling, the mycelia face of bacterium cake is attached to media surface, places 28 ± 1 ℃ to cultivate 96 hours flat board.Adopt the right-angled intersection method to measure colony growth diameter, calculate inhibiting rate with following formula:
Table 1: the fermented liquid of chaetomium globosum yt03 of the present invention is to the anti-bacterial result of six kind of plant pathogenic micro-organisms
By as seen from Table 1 upper, the present invention prepares resulting solidago canadesis endogenetic fungus endogenetic fungus chaetomium globosum yt03 tunning the test plant pathogenic fungi is had broad spectrum antibiotic activity.Chaetomium globosum yt03 fermenation raw liquid to the Phytophthora capsici germ (
Phytophthora capsici), the jujube anthrax bacteria
(Gloelsporium fructigenum)And fusarium graminearum (
Fusarium graminearum) mycelial growth inhibition rate surpass 50%.To the tomato wilt bacterium (
Fusarium oxysporum), Valsa mali
(Cytospora mandshurica)And apple anthrax bacteria
(Colletotrichum gloeosporioides)Mycelial growth stronger restraining effect is also arranged.The present invention utilizes endophytic fungus resources of plants, by the natural component of fermentation preparation in the hope of acquisition anti-plant pathogenic fungi disease novel agricultural activity.
Embodiment
Embodiment:
The used solidago canadesis endogenetic fungus of present embodiment called after chaetomium globosum yt03(
Chaetomium globosumYt03), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on July 20th, 2011, deposit number is CGMCC No.5088;
Chaetomium globosum yt03(
Chaetomium globosumYt03) bacterial strain is the natural microbial medicine source that fermentation obtains anti-Plant diseases novel agricultural antibiotics activeconstituents.
The concrete preparation manipulation step of the antibiotic fermented liquid of solidago canadesis endogenetic fungus is as follows:
(1) actication of culture
Under aseptic condition (Bechtop), with a little test tube slant preservation of bacteria strain chaetomium globosum yt03(of transfering loop picking
Chaetomium globosumYt03), transfer in sterilized potato dextrose agar solid medium flat board, put and cultivated 28 ℃ of activation culture of the temperature inside the box 5 days, obtain activated spawn;
(2) seed culture
With aseptic punch tool (internal diameter 5mm) (Bechtop) under aseptic condition activated spawn being broken into directly is 5mm bacterium dish, 5 ferfas dish are inoculated in the triangle shaking flask that contains the 50ml liquid nutrient medium, constant-temperature table is cultivated, 160 rev/mins of shaking speed, 28 ℃ of temperature, cultivated 2 days, and got the shaking table seed;
(4) fermentation culture: the shaking table seed is inoculated in the 250ml triangle shaking flask that the 50ml liquid nutrient medium is housed, inoculum size 5%, 160 rev/mins of shaking speed, 28 ℃ of temperature were cultivated 7 days, obtained solidago canadesis endogenetic fungus fermented liquid; Solidago canadesis endogenetic fungus fermented liquid removes by filter mycelium under aseptic condition, cross 0.22 micron filter, namely obtains the antibiotic fermented liquid of solidago canadesis endogenetic fungus;
The fermentation culture feature: cultivated the 1st day, thalline is the powder of a small amount of grey; Cultivated the 3rd day, the fine powder of pale pink reduces, and the little mycelium pellet of black occurs; Cultivated the 5th day, the black mycelium pellet continues to increase, and the volume change is large, and fermented liquid is tawny; Cultivated the 7th day, and the mycelium pellet of a large amount of black occurred, volume also becomes greatly, and fermented liquid is brown;
Potato dextrose agar solid medium: 1 liter of potato 200 gram, glucose 20 grams, agar 18 grams, distilled water; Liquid nutrient medium: peptone 15 grams, glucose 20 grams, calcium chloride 0.5 gram, dipotassium hydrogen phosphate 0.1 gram, ferrous sulfate
0.01 gram, SODIUMNITRATE 1 gram, 1000 milliliters in water, pH nature.Above two kinds of substratum 30 min that under 121 ℃ of temperature, pressure 0.1MPa, sterilize before using.
Claims (1)
1. the preparation method of the antibiotic fermented liquid of solidago canadesis endogenetic fungus, it is characterized in that: described solidago canadesis endogenetic fungus called after chaetomium globosum yt03 (Chaetomium globosum yt03), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3, No. 1 institute in Beichen West Rd., Beijing, preservation date on July 20th, 2011, deposit number is CGMCC No.5088;
Concrete fermentation preparation manipulation step is as follows:
(1) actication of culture
Under aseptic condition, with a little solidago canadesis endogenetic fungus test tube slant preservation of bacteria strain of transfering loop picking, transfer in sterilized potato dextrose agar solid medium flat board, put and cultivate 28 ℃ ± 1 ℃ of the temperature inside the box, activation culture 5~7 days obtains activated spawn;
(2) seed culture
Under aseptic condition activated spawn being broken into diameter with aseptic punch tool is 5mm bacterium dish, inoculates 5 ferfas dish in the triangle shaking flask that contains the 50ml liquid nutrient medium, and constant-temperature table is cultivated, 160 rev/mins of shaking speed, 28 ± 2 ℃ of temperature were cultivated 2~3 days, got the shaking table seed;
(3) fermentation culture
The shaking table seed is inoculated in the 250ml triangle shaking flask that the 50ml liquid nutrient medium is housed, inoculum size 5%, 160 rev/mins of shaking speed, 28 ℃ ± 2 ℃ of temperature were cultivated 5~7 days, obtained solidago canadesis endogenetic fungus fermented liquid; Solidago canadesis endogenetic fungus fermented liquid removes by filter mycelium under aseptic condition, cross 0.22 micron filter, namely obtains the antibiotic fermented liquid of solidago canadesis endogenetic fungus;
The fermentation culture feature: cultivated the 1st day, thalline is the powder of a small amount of grey; Cultivated the 3rd day, the fine powder of pale pink reduces, and the little mycelium pellet of black occurs; Cultivated the 5th day, the black mycelium pellet continues to increase, and the volume change is large, and fermented liquid is tawny; Cultivated the 7th day, and the mycelium pellet of a large amount of black occurred, volume also becomes greatly, and fermented liquid is brown;
Described potato dextrose agar solid medium: 1 liter of potato 200 gram, glucose 20 grams, agar 18 grams, distilled water;
Described liquid nutrient medium: peptone 15 grams, glucose 20 grams, calcium chloride 0.5 gram, dipotassium hydrogen phosphate 0.1 gram, ferrous sulfate 0.01 gram, SODIUMNITRATE 1 gram, 1000 milliliters in water, pH nature;
Above-mentioned two kinds of substratum 30min that under 121 ℃ of temperature, pressure 0.1MPa, sterilizes before using.
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CN103194490A (en) * | 2013-03-26 | 2013-07-10 | 安徽农业大学 | Method for preparing phytopathogen-resisting ginkgo biloba L. endophytic-fungus fermentation broth |
CN103255065A (en) * | 2013-04-22 | 2013-08-21 | 安徽农业大学 | Method for preparing anti-phytopathogen fermented liquid for camptotheca acuminate endophytic fungi |
CN107164233B (en) * | 2016-12-28 | 2020-08-25 | 贵州省植物保护研究所 | Chaetomium globosum LJ-S2L1 strain and application thereof |
CN109706083B (en) * | 2018-12-10 | 2023-05-30 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | Eucommia ulmoides endophytic fungus with remarkably increased antibacterial activity after metabolic regulation |
CN114774287B (en) * | 2022-04-08 | 2023-06-27 | 西北农林科技大学 | Endophytic chaetomium globosum MG2 and application thereof in preventing and treating apple tree rot |
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