WO2008063011A1 - Method for enhancing the thermal tolerance of entomopathogenic fungal spores, blastospores and enzymes - Google Patents
Method for enhancing the thermal tolerance of entomopathogenic fungal spores, blastospores and enzymes Download PDFInfo
- Publication number
- WO2008063011A1 WO2008063011A1 PCT/KR2007/005886 KR2007005886W WO2008063011A1 WO 2008063011 A1 WO2008063011 A1 WO 2008063011A1 KR 2007005886 W KR2007005886 W KR 2007005886W WO 2008063011 A1 WO2008063011 A1 WO 2008063011A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- spores
- enzymes
- enhancing
- entomopathogenic fungi
- entomopathogenic
- Prior art date
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 52
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 52
- 230000000967 entomopathogenic effect Effects 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000002708 enhancing effect Effects 0.000 title claims abstract description 19
- 230000002538 fungal effect Effects 0.000 title claims abstract description 14
- 241000233866 Fungi Species 0.000 claims abstract description 35
- 241000827797 Beauveria sp. Species 0.000 claims abstract description 19
- 239000000843 powder Substances 0.000 claims abstract description 19
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 239000003463 adsorbent Substances 0.000 claims abstract description 10
- 235000015112 vegetable and seed oil Nutrition 0.000 claims abstract description 6
- 239000008158 vegetable oil Substances 0.000 claims abstract description 6
- 102000012286 Chitinases Human genes 0.000 claims description 21
- 108010022172 Chitinases Proteins 0.000 claims description 21
- 239000004006 olive oil Substances 0.000 claims description 11
- 235000008390 olive oil Nutrition 0.000 claims description 11
- 239000008188 pellet Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 5
- 235000005687 corn oil Nutrition 0.000 claims description 4
- 239000002285 corn oil Substances 0.000 claims description 4
- 239000002385 cottonseed oil Substances 0.000 claims description 4
- 235000012343 cottonseed oil Nutrition 0.000 claims description 4
- 239000003549 soybean oil Substances 0.000 claims description 4
- 235000012424 soybean oil Nutrition 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- -1 pyrophillite Chemical compound 0.000 claims description 3
- 239000000440 bentonite Substances 0.000 claims description 2
- 229910000278 bentonite Inorganic materials 0.000 claims description 2
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 238000004362 fungal culture Methods 0.000 claims 1
- 230000000361 pesticidal effect Effects 0.000 claims 1
- 229960001866 silicon dioxide Drugs 0.000 claims 1
- 239000000304 virulence factor Substances 0.000 claims 1
- 230000007923 virulence factor Effects 0.000 claims 1
- 241000894007 species Species 0.000 abstract description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract description 2
- 239000011707 mineral Substances 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 11
- 239000003921 oil Substances 0.000 description 11
- 235000019198 oils Nutrition 0.000 description 11
- 238000012360 testing method Methods 0.000 description 9
- 238000010438 heat treatment Methods 0.000 description 7
- 238000009630 liquid culture Methods 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000000853 biopesticidal effect Effects 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000004763 spore germination Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000008646 thermal stress Effects 0.000 description 2
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- OMRLTNCLYHKQCK-DHGKCCLASA-N 4-nitrophenyl N-acetyl-beta-D-glucosaminide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 OMRLTNCLYHKQCK-DHGKCCLASA-N 0.000 description 1
- 241000238876 Acari Species 0.000 description 1
- 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 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- QYDYPVFESGNLHU-UHFFFAOYSA-N elaidic acid methyl ester Natural products CCCCCCCCC=CCCCCCCCC(=O)OC QYDYPVFESGNLHU-UHFFFAOYSA-N 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 description 1
- 229940073769 methyl oleate Drugs 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 230000003032 phytopathogenic effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Definitions
- the present invention relates to a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, which can be advantageously used as a material for preparing biopesticides.
- Biological pesticides comprising microbes can be understood as a biological formulation which is prepared from microbe itself or from other formulations comprising microbes and is used for an efficient control of various kinds of weeds, phytopathogenic fungi, or pests such as insects, mites and nematodes, etc. that are harmful to many crops.
- the present invention is a result of works to solve the problems as described above. Specifically, purpose of the present invention is to provide a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, which can be advantageously used as a material for preparing biopesticides.
- the present invention is accomplished through a series of studies for increasing the stability of entomopathogenic fungal spores by using an oil as a preservation medium so as to prevent exposure of the spores to moisture as much as possible, after confirming the fact that the activity of the spores is significantly lowered by the presence of moisture in general, and for the enzyme (e.g., chitinase), considering a tendency that the enzyme activity is lowered when the enzyme is dissolved in water, the enzyme dissolved in water is first pulverized to reduce its contact with water and then kept in oils to increase their thermal tolerance.
- the present invention provides a method for enhancing the thermal tolerance of spores and enzymes that are produced by entomopathogenic fungi. More specifically, the present invention provides a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, characterized in that spore powder of entomopathogenic fungi or powder to which enzymes originating from entomopathogenic fungi are adsorbed is added to vegetable oils, and then the resulting mixtures are stored until further use.
- the present invention also relates to a novel entomopathogenic fungal species of
- Beauveria sp. DBB2507 (Deposit No.: KCCM 10892P) having a property of efficiently controlling the harmful insects in agriculture by treatment of spores or enzymes that are produced by entomopathogenic fungi.
- the above-described vegetable oils of the present invention can be selected from soybean oil, olive oil, cotton seed oil, or corn oil.
- olive oil can be selected.
- the powder to which enzymes originating from entomopathogenic fungi are adsorbed can be obtained by freeze-drying of the pellets that are formed by adding enzyme adsorbents to a supernatant resulting from centrifuge of culture media comprising entomopathogenic fungi.
- the above-described drying step is not limited to freeze-drying, and it is evident that a skilled person can freely select any of known drying methods depending on his or her needs.
- kaoline can be preferably used for the above-described enzyme adsorbent of the present invention.
- the above-described enzymes originating from entomopathogenic fungi can be chitinase, but are not limited thereto. It can include several kinds of enzymes relating to virulence.
- the novel ent ⁇ nopathogenic fungal species of Beauveria sp. DBB2507 (Deposit No.: KCCM 10892P) was used to determine the claimed method for enhancing the thermal tolerance of the spores and the enzymes produced by entomopathogenic fungi.
- the method for enhancing the thermal stability of entomopathogenic fungal spores, blastospores and enzymes according to the present invention is found to be a useful method to maintain and enhance the activities of the spores, blastospores and the enzymes produced by entomopathogenic fungi, which can be used as a material for producing biopesticides.
- Figure 1 shows the thermal stability of Beauveria sp.DBB2507 spores and blastospores in water, as an embodiment of the present invention (left: spores, right: blastospores).
- Figure 2 shows the stability of Beauveria sp.DBB2507 spores in several kinds of oils between room temperature and heat-treatment condition, as an embodiment of the present invention (left: at room temperature, right: under heat treatment condition).
- Figure 3 shows the thermal stability of Beauveria sp.DBB2507 spores and blastospores in oil at different time of thermal-stress, as an embodiment of the present invention (left: spores, right: blastospores).
- Figure 4 shows the thermal stability of Beauveria sp.DBB2507 chitinase existed as liquid form at different time of thermal-stress, as an embodiment of the present invention.
- Figure 5 shows the amount of chitinase harvested during the process of centrifuge, wherein said chitinase has been produced by Beauveria sp.DBB2507 in liquid culture, as an embodiment of the present invention.
- Figure 6 shows the absorption capacity of enzyme adsorbents for chitinase that has been produced by Beauveria sp.DBB2507 in liquid culture, as an embodiment of the present invention.
- Figure 7 shows the thermal stability of Beauveria sp.DBB2507 chitinase adsorbed onto enzyme adsorbent at several conditions wherein said chitinase has been produced by liquid culture of Beauveria sp.DBB2507, as an embodiment of the present invention.
Abstract
The present invention relates to a method for enhancing the thermal tolerance of entomopathogenic fungal spores and enzymes. More specifically, the present invention relates to a method for enhancing the thermal tolerance of entomopathogenic fungal spores and enzymes, characterized in that spore powder produced by entomopathogenic fungi and an enzyme(s) originated from the entomopathogenic fungi, which are adsorbed onto mineral adsorbents and the resulting powders are added into vegetable oils to give a mixture which can be preserved for a later use. Furthermore, the present invention relates to a novel entomopathogenic fungal species of Beauveria sp. DBB2507 (Deposit No.: KCCM 10892P) that is used for an efficient preservation of the spores and the enzymes produced by entomopathogenic fungi.
Description
Description
METHOD FOR ENHANCING THE THERMAL TOLERANCE OF ENTOMOPATHOGENIC FUNGAL SPORES, BLASTOSPORES AND ENZYMES
Technical Field
[1] The present invention relates to a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, which can be advantageously used as a material for preparing biopesticides. Background Art
[2] Recently, many problems have been created by an excessive use of chemical pesticides, which causes a high level of resistance for harmful insects in agriculture, a disturbance in ecological system, an adverse effect on human health, etc., and many concerns related thereto have been continuously raised. As an alternative to such chemical pesticides, more attention is being paid to the development of biopesticides which utilize entomopathogenic fungi that are present in nature.
[3] Biological pesticides comprising microbes can be understood as a biological formulation which is prepared from microbe itself or from other formulations comprising microbes and is used for an efficient control of various kinds of weeds, phytopathogenic fungi, or pests such as insects, mites and nematodes, etc. that are harmful to many crops.
[4] Meanwhile, since the biological function of chitinase has been recently determined for fungi, microbes and plants, its important role as a mediator for an ecological interaction among them is being noticed. In this connection, biological control using chitinase is also getting more attention.
[5] Biological pesticides comprising such entomopathogenic fungi have many advantages that they are less toxic and therefore are less harmful to crops compared to synthetic organic pesticides, and they have less impact on ecological system and do not cause a tolerance or resistance to pesticides, etc. However, because activity of enzymes that are produced by fungi themselves cannot be maintained for a long period of time during manufacturing process, the short shelf life has been remained as a problem. Disclosure of Invention Technical Problem
[6] The present invention is a result of works to solve the problems as described above.
Specifically, purpose of the present invention is to provide a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, which can be advantageously used as a material for preparing biopesticides.
[7] The present invention is accomplished through a series of studies for increasing the stability of entomopathogenic fungal spores by using an oil as a preservation medium so as to prevent exposure of the spores to moisture as much as possible, after confirming the fact that the activity of the spores is significantly lowered by the presence of moisture in general, and for the enzyme (e.g., chitinase), considering a tendency that the enzyme activity is lowered when the enzyme is dissolved in water, the enzyme dissolved in water is first pulverized to reduce its contact with water and then kept in oils to increase their thermal tolerance. Technical Solution
[8] In order to accomplish the above-described purpose, the present invention provides a method for enhancing the thermal tolerance of spores and enzymes that are produced by entomopathogenic fungi. More specifically, the present invention provides a method for enhancing the thermal tolerance of spores and enzymes produced by entomopathogenic fungi, characterized in that spore powder of entomopathogenic fungi or powder to which enzymes originating from entomopathogenic fungi are adsorbed is added to vegetable oils, and then the resulting mixtures are stored until further use.
[9] The present invention also relates to a novel entomopathogenic fungal species of
Beauveria sp. DBB2507 (Deposit No.: KCCM 10892P) having a property of efficiently controlling the harmful insects in agriculture by treatment of spores or enzymes that are produced by entomopathogenic fungi.
[10] The above-described vegetable oils of the present invention can be selected from soybean oil, olive oil, cotton seed oil, or corn oil. Preferably, olive oil can be selected.
[11] Meanwhile, the powder to which enzymes originating from entomopathogenic fungi are adsorbed can be obtained by freeze-drying of the pellets that are formed by adding enzyme adsorbents to a supernatant resulting from centrifuge of culture media comprising entomopathogenic fungi. The above-described drying step is not limited to freeze-drying, and it is evident that a skilled person can freely select any of known drying methods depending on his or her needs. In addition, for the above-described enzyme adsorbent of the present invention, kaoline can be preferably used.
[12] The above-described enzymes originating from entomopathogenic fungi can be chitinase, but are not limited thereto. It can include several kinds of enzymes relating to virulence.
[13] Meanwhile, according to the present invention the novel entαnopathogenic fungal species of Beauveria sp. DBB2507 (Deposit No.: KCCM 10892P) was used to determine the claimed method for enhancing the thermal tolerance of the spores and the enzymes produced by entomopathogenic fungi.
Advantageous Effects
[14] As it has been described above, the method for enhancing the thermal stability of entomopathogenic fungal spores, blastospores and enzymes according to the present invention is found to be a useful method to maintain and enhance the activities of the spores, blastospores and the enzymes produced by entomopathogenic fungi, which can be used as a material for producing biopesticides.
[15] Furthermore, it is expected that the method for enhancing the thermal stability of entomopathogenic fungal spores, blastospores and enzymes according to the present invention can be advantageously used for the formulation and mass production of biopesticides having a high level of controlling activity. Brief Description of the Drawings
[16] Figure 1 shows the thermal stability of Beauveria sp.DBB2507 spores and blastospores in water, as an embodiment of the present invention (left: spores, right: blastospores).
[17] Figure 2 shows the stability of Beauveria sp.DBB2507 spores in several kinds of oils between room temperature and heat-treatment condition, as an embodiment of the present invention (left: at room temperature, right: under heat treatment condition).
[18] Figure 3 shows the thermal stability of Beauveria sp.DBB2507 spores and blastospores in oil at different time of thermal-stress, as an embodiment of the present invention (left: spores, right: blastospores).
[19] Figure 4 shows the thermal stability of Beauveria sp.DBB2507 chitinase existed as liquid form at different time of thermal-stress, as an embodiment of the present invention.
[20] Figure 5 shows the amount of chitinase harvested during the process of centrifuge, wherein said chitinase has been produced by Beauveria sp.DBB2507 in liquid culture, as an embodiment of the present invention.
[21] Figure 6 shows the absorption capacity of enzyme adsorbents for chitinase that has been produced by Beauveria sp.DBB2507 in liquid culture, as an embodiment of the present invention.
[22] Figure 7 shows the thermal stability of Beauveria sp.DBB2507 chitinase adsorbed
onto enzyme adsorbent at several conditions wherein said chitinase has been produced by liquid culture of Beauveria sp.DBB2507, as an embodiment of the present invention.
Mode for the Invention
[23] The present invention will now be described in greater detail with reference to the following preferred examples. However, it is only to specifically exemplify the present invention and in no case the scope of the present invention is limited by these examples. It is construed that the scope of the present invention is limited only by the attached claims of the invention.
[24]
[25] <Example 1> Method of enhancing the thermal stability of the spores and blastospores that are produced by entomopathogenic fungi
[26] 1-1. Test for determining a thermal stability of entomopathogenic fungal spores and blastospores
[27] Beauveria sp.DBB2507 strain (Deposit No.; KCCM 10892P) was subjected to a plate culture using SDA+Y agar (Saubraud dextrose culture media + 0.5 wt% of yeast extract) and also to a liquid culture using SDA+Y broth to obtain spores and blastospores, respectively, which were then subjected to a freeze-drying for thermal stability test. Specifically, suspensions comprising the spores or blastospores were kept in an incubator at 5O0C for 60 min, or 120 min. \QμJl of the resulting suspensions were then added dropwise to SDA+Y agar plate. After culturing at 280C for 12 hours, number of the germinated spores per one hundred spores was counted to obtain the spore germination ratio.
[28] It was found that the suspension of the spores and the blastospores that had been produced by entomopathogenic fungi had a very low level of germination ratio (i.e., only about 10% under the condition of 5O0C heat treatment for an hour), indicating that the spores and the blastospores have a very low thermal stability (see Figure 1).
[29] 1-2. Gbmparative test to determine the ambient temperature stability and the thermal stability of the entomopathogenic fungal spores prepared in different kinds of oils
[30] Beauveria sp.DBB2507strain was subjected to a solid culture for three weeks using
SDA+Y agar plate. Spores were harvested with a brush and then freeze-dried to produce spore powder that is used for a next test. Specifically, thus-obtained spore powder was added to several kinds of oils including soybean oil, olive oil, cotton seed oil, corn oil, mineral oil, and methyl oleate, etc. After mixing the spore powder with oils, the mixture was kept at ambient temperature (250C) or heat-treated (5O0C
incubator) for 120 min. Thereafter, lOβfl of the spore suspension was added dropwise to SDA+Y agar plate. After culturing at 280C for 12 hours, number of the germinated spores per one hundred spores was counted to obtain the spore germination ratio.
[31] As a result, it was found that the ambient temperature stability of the spores present in oils such as soybean oil, olive oil, cotton seed oil and corn oil is similar to that of the non-treated spores (i.e., almost 90% of the spores have germinated; see Figure 2). In addition, under 5O0C heat treatment condition, the spores present in olive oil showed spore germination ratio similar to that of the non-treated spores (see Figure X). Therefore, it was confirmed that when spores are stored in an olive oil, their thermal tolerance is improved compared to those stored in water.
[32] 1-3. Test for determining a thermal stability of entomopathogenic fungal spores with different period of heat treatment time
[33] Beauveria sp.DBB2507strain was subjected to a solid culture for three weeks using
SDA+Y agar plate. Spores were harvested with a brush and then freeze-dried to prepare spore powder that is used for a next test. Specifically, thus-obtained spore powder was added to olive oil. After mixing the powder with the oil, the mixture was subjected to a heat treatment (5O0C incubator) for 60 min and 120 min. Thereafter, \QμJl of the spore suspension was added dropwise to SDA+Y agar plate. After culturing at 280C for 12 hours, number of the germinated spores per one hundred spores was counted to obtain the spore germination ratio.
[34] As a result, as it is shown in Figure 3, it was found that not only the spores but also the blastospores of the entomopathogenic fungi are heat stable when they were kept in olive oil (see Figure 3).
[35]
[36] <Example 2> Method for enhancing the thermal stability of enzymes produced by entomopathogenic fungi.
[37] 2-1. Test for determining the thermal stability of an enzyme
[38] Beauveria sp. DBB2507 strain was subjected to a liquid culture for three days using
SDA+Y broth. After centrifuging the culture broth at 15,000 rpm for 5 min, the supernatant was prepared and kept in 5O0C incubator for 60 min and 120 min. Enzyme activity of chitinase was then measured. Specifically, the enzyme solution (100/i2), p- nitrophenyl β-D-N-acetylglucosaminide (PNG; 100 //#) as a substrate, and 0.1M citrate-phosphate buffer, pH 6.2 (3OO/i6) were mixed and then the enzyme reaction was carried out at 370C for one hour. Then the adsorbance of p-nitrophenol, the reaction product, was measured at 405 nm.
[39] As a result, it was found that the activity of chitinase existed as solution that has been produced by entomopathogenic fungi ( Beauveria sp.DBB2507) was reduced from 4.2 unit/hr at the beginning to 0.34 unit/hr after two hours of heat treatment at 5O0C (see Figure 4).
[40] 2-2. Test for determining the amount of enzyme harvested during centrifuge process
[41] Beauveria sp.DBB2507 strain was subjected to a liquid culture for three days in
SDA+ Y broth. After centrifuge at 15,000 rpm for 5 min, supernatant and pellet were separated. Resulting pellet comprising blastospores and mycelium was adjusted to the concentration before the centrifuge by using 0.1M citrate-phosphate buffer, pH 6.2. The chitinase activity was measured for the supernatant and the pellet suspension obtained above, respectively.
[42] It was also found that the harvest efficiency for chitinase by general centrifugal precipitation of the enzyme which is present in an aqueous solution is rather low (4.5%; 0.3/6.7, see Figure 5) although above process is an essential step.
[43] 2-3. Test for selecting adsorbents that can be used for improving the harvesting efficiency of enzyme
[44] Beauveria sp.DBB2507 strain was subjected to a liquid culture for three days in
SDA+ Y broth. After centrifuge at 15,000 rpm for 5 min, supernatant was obtained. To the resulting supernatant, several kinds of enzyme adsorbents including silica gel, cellulose, pyrophillite, skim milk, bentonite, celite, kaoline, and polyvinyl alcohol, etc. were added to the concentration of 0.5% (w/v). The mixture was ketp at ambient temperature for 30 min with shaking, and then centrifuged at 15,000 rpm for 5 min to obtain the pellet to which the enzyme was adsorbed. The chitinase activity was determined for the supernatant obtained above and the pellet to which the enzyme was adsorbed to find out the absorption ratio for each of the above-described adsorbents.
[45] In order to improve the harvesting efficiency of chitinase, mineral substances and polysaccharides were further used and the absorption ratio was determined therefor. As a result, it was found that among various candidate substances, the pellet treated with kaoline exhibited the highest chitinase activity of 11.1 unit/hr. At the same time, the lowest chitinase activity of 0.2 unit/hr was observed for the remaining solution. The absorption ratio for kaoline was about 90.2% (see Figure 6).
[46] 2-4. Test for determining thermal stability of the enzyme-adsorbed powder in oil
[47] The above-described pellet to which the enzyme has been adsorbed was subjected to a freeze-drying. The resulting enzyme- adsorbed powder was added to a vegetable oil, i.e., olive oil, and mixed. The mixture was then heat-treated in 5O0C incubator for 60
min and 120 min. By comparing with a sample which was prepared by adding said enzyme-adsorbed powder into water, the thermal stability of chitinase in oil was determined. [48] As a result, it was found that when chitinase was adsorbed first to kaoline and the resulting powder was dispersed in an olive oil, thermal stability thereof was about 8 to 10 times higher than the enzyme present in liquid form. In addition, compared to the bare enzyme present in powder form, it had a thermal stability that is about 1.25 times higher (see Figure 7).
Claims
[1] A method for enhancing the thermal stability of spores, blastospores and enzymes produced by entomopathogenic fungi, characterized in that powder of the spores produced by entomopathogenic fungi or powder adsorbed with the enzymes originating from entomopathogenic fungi are added to vegetable oils to give a mixture and the resulting mixtures are preserved.
[2] The method for enhancing the thermal stability of spores and enzymes produced by entomopathogenic fungi according to Claim 1, characterized in that said vegetable oils are at least one selected from the group consisting of soybean oil, olive oil, cotton seed oil, and corn oil.
[3] The method for enhancing the thermal stability of spores and enzymes produced by entomopathogenic fungi according to Claim 1, characterized in that said enzymes originating from entomopathogenic fungi are chitinase, other known pesticidal enzymes or other virulence factors.
[4] The method for enhancing the thermal stability of spores and enzymes produced by entomopathogenic fungi according to Claim 1, characterized in that said powder that is adsorbed with the enzymes originating from entomopathogenic fungi is obtained from freeze-drying of pellets that are formed by adding enzyme-adsorbents to a supernatant obtained from the centrifuge of the fungal culture broth.
[5] The method for enhancing the thermal stability of spores and enzymes produced by entomopathogenic fungi according to Claim 4, characterized in that said enzyme adsorbents are at least one selected from the group consisting of kaoline, silicagel, pyrophillite, and bentonite.
[6] A novel fungal strain of Beauveria sp. DBB2507 (Deposit No.; KCCM 10892P), which can be used for enhancing the thermal stability of spores and enzymes produced by entomopathogenic fungi.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2006-0114935 | 2006-11-21 | ||
| KR20060114935A KR100835689B1 (en) | 2006-11-21 | 2006-11-21 | Method for Enhancing the Thermal Tolerance of Entomopathogenic Fungal Spores, blastospores and enzymes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2008063011A1 true WO2008063011A1 (en) | 2008-05-29 |
Family
ID=39429901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2007/005886 WO2008063011A1 (en) | 2006-11-21 | 2007-11-21 | Method for enhancing the thermal tolerance of entomopathogenic fungal spores, blastospores and enzymes |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR100835689B1 (en) |
| WO (1) | WO2008063011A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013096383A2 (en) | 2011-12-19 | 2013-06-27 | Novozymes Biologicals Holdings A/S | Bio-pestcide methods and compositions |
| WO2015069708A1 (en) | 2013-11-08 | 2015-05-14 | Novozymes Bioag A/S | Compositions and methods for treating pests |
| WO2015077278A1 (en) | 2013-11-20 | 2015-05-28 | Novozymes Bioag A/S | Compositions and methods comprising chromobacterium for controlling plant nematode pests and plant insect pests |
| CN106993801A (en) * | 2016-01-22 | 2017-08-01 | 许昌世纪香生物科技有限公司 | The preparation method of broken wall Hericium erinaceus conidia powder |
| WO2018228966A1 (en) * | 2017-06-15 | 2018-12-20 | Dsm Ip Assets B.V. | Frozen enzyme pellets |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101661566B1 (en) * | 2015-07-13 | 2016-10-04 | 한국생명공학연구원 | Manufacturing method of Biopesticide using Paecilomyces sp |
| CN106810351A (en) * | 2016-12-23 | 2017-06-09 | 浙江海洋大学 | A kind of method that hydrolyzed aquatic products accessory substance prepares amino acid foliage fertilizer |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5360607A (en) * | 1991-01-10 | 1994-11-01 | W. R. Grace & Co.-Conn. | Method for production and use of pathogenic fungal preparation for pest control |
| US5730973A (en) * | 1994-02-15 | 1998-03-24 | Hoechst Schering Agrevo Gmbh | Water-dispersible granules of spores or live Beauveria bassiana |
| KR20010036693A (en) * | 1999-10-11 | 2001-05-07 | 우종일 | Microorganism insect composition comprising BT |
| US20060110366A1 (en) * | 2001-01-30 | 2006-05-25 | Yufera Eduardo P | Entomopathogenic microorganism spores carrier and method for controlling harmful insects |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100191896B1 (en) * | 1996-10-12 | 1999-06-15 | 박원훈 | An insectcidal composition which contains beauveria sp. and its producing method |
-
2006
- 2006-11-21 KR KR20060114935A patent/KR100835689B1/en active IP Right Grant
-
2007
- 2007-11-21 WO PCT/KR2007/005886 patent/WO2008063011A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5360607A (en) * | 1991-01-10 | 1994-11-01 | W. R. Grace & Co.-Conn. | Method for production and use of pathogenic fungal preparation for pest control |
| US5730973A (en) * | 1994-02-15 | 1998-03-24 | Hoechst Schering Agrevo Gmbh | Water-dispersible granules of spores or live Beauveria bassiana |
| KR20010036693A (en) * | 1999-10-11 | 2001-05-07 | 우종일 | Microorganism insect composition comprising BT |
| US20060110366A1 (en) * | 2001-01-30 | 2006-05-25 | Yufera Eduardo P | Entomopathogenic microorganism spores carrier and method for controlling harmful insects |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013096383A2 (en) | 2011-12-19 | 2013-06-27 | Novozymes Biologicals Holdings A/S | Bio-pestcide methods and compositions |
| WO2013096383A3 (en) * | 2011-12-19 | 2013-10-10 | Novozymes Biologicals Holdings A/S | Bio-pestcide methods and compositions |
| EP3172966A1 (en) | 2011-12-19 | 2017-05-31 | Novozymes Bioag A/S | Biopesticide method and compositions |
| WO2015069708A1 (en) | 2013-11-08 | 2015-05-14 | Novozymes Bioag A/S | Compositions and methods for treating pests |
| WO2015077278A1 (en) | 2013-11-20 | 2015-05-28 | Novozymes Bioag A/S | Compositions and methods comprising chromobacterium for controlling plant nematode pests and plant insect pests |
| CN106993801A (en) * | 2016-01-22 | 2017-08-01 | 许昌世纪香生物科技有限公司 | The preparation method of broken wall Hericium erinaceus conidia powder |
| WO2018228966A1 (en) * | 2017-06-15 | 2018-12-20 | Dsm Ip Assets B.V. | Frozen enzyme pellets |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100835689B1 (en) | 2008-06-09 |
| KR20080045812A (en) | 2008-05-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2736382C1 (en) | Compositions and methods for fusarium disease control | |
| US9090884B2 (en) | Formulations of viable microorganisms and their methods of production and use | |
| KR101126060B1 (en) | Microorganism capable of controlling plant diseases and plant disease-controlling agent using the microorganism | |
| US10993443B2 (en) | Methods and compositions for improving soybean yield | |
| WO2008063011A1 (en) | Method for enhancing the thermal tolerance of entomopathogenic fungal spores, blastospores and enzymes | |
| CN108135180A (en) | For the microbial composite and method of biological protection | |
| BR0017570B1 (en) | COMPOSITION AND METHOD TO PROTECT OR TREAT A PLANT, ROOT OR FRUIT | |
| CN1154715C (en) | Microbial agricultural chemical | |
| JPH078769B2 (en) | Method for controlling soil nematode and nematode control composition used therefor | |
| CN111394284B (en) | Serratia marcescens MB21 and application thereof | |
| He et al. | Effective biocontrol of soybean root rot by a novel bacterial strain Bacillus siamensis HT1 | |
| AU2003267883A1 (en) | Grass endophytes | |
| CN106244480A (en) | One strain vacation Grignon anthropi and the application of preventing and treating plant nematode thereof | |
| JP4310466B2 (en) | Composition and method for biological control of soybean black root rot | |
| KR102207732B1 (en) | Microbial agent for control of Plutella xylostella larva using Metarhizium anisopliae FT319 and its culture media | |
| Cheng et al. | Biological control of Qinghai plateau terrestrial weeds with the A. alternata HL-1 | |
| KR100251230B1 (en) | Novel strain belonging to exserohilum monoceras and use of the same | |
| Banu et al. | Mass multiplication and formulation of a nematophagous fungus, Paecilomyces lilacinus | |
| HUE028607T2 (en) | Glomus iranicum var. tenuihypharum var. nov. strain and its use as bio-nematicide | |
| KR102145620B1 (en) | Microbial agent for control of Plutella xylostella larva using Isaria fumosorosea FT337 and its culture media | |
| Roswanjaya et al. | Conidia Production of Beauveria Bassiana in Solid Substrate Fermentation Using a Biphasic System | |
| KR20120110375A (en) | Mass production method of paecilomyces lilacinus hy-4 | |
| Ahonsi et al. | Selection of non-pathogenic ethylene-producing rhizobacteria for accelerated depletion of Striga hermonthica seed bank | |
| KR101256179B1 (en) | Culture Media for Microoganism Comprising Natural Material | |
| Kumar et al. | Role of different substrates in mass production and pathogenicity of Alternaria macrospora for the management of parthenium weed |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 07834192 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 07834192 Country of ref document: EP Kind code of ref document: A1 |