CN102304501A - Complex enzyme preparation and application and method thereof for extracting plant polysaccharides by using same - Google Patents

Complex enzyme preparation and application and method thereof for extracting plant polysaccharides by using same Download PDF

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Publication number
CN102304501A
CN102304501A CN201110255909A CN201110255909A CN102304501A CN 102304501 A CN102304501 A CN 102304501A CN 201110255909 A CN201110255909 A CN 201110255909A CN 201110255909 A CN201110255909 A CN 201110255909A CN 102304501 A CN102304501 A CN 102304501A
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water
compound enzymic
polysaccharides
extract
ppm
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刘敏尧
刘宇
刘靓
刘海庆
刘晓津
董晓芳
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Tianjin Binhai Noao Enzymes Science & Technology Development Co Ltd
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Tianjin Binhai Noao Enzymes Science & Technology Development Co Ltd
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Abstract

The invention provides a complex enzyme preparation and application and a method for extracting plant polysaccharides by using same, belonging to the application field of enzyme preparations. According to the technical scheme of the invention, the complex enzyme preparation comprises the following components in parts by weight: 1-5 parts of cellulase, 1-5 parts of pectase and 2-12 parts of prolease. The invention further provides the application of the complex enzyme preparation for extracting the plant polysaccharides, particularly polysaccharide, from mulberry leaves, and also provides the specific operation steps, and the effect that the extraction rate of the plant polysaccharides is above 60mg/g is finally achieved. The invention has the beneficial effects that the reaction conditions are mild, the conformation of a natural product can be maintained, the stereostructure and the biological activity of the natural product can not be damaged, and the maintaining of the original efficacy of active ingredients can be facilitated; the impurities in a system can be removed, the clarity of extract liquid is improved and the extraction rate of the active ingredients of traditional Chinese medicine is improved; and the reaction conditions are combined in an optimized manner and thus the maximization of the extraction yield is further realized.

Description

A kind of compound enzymic preparation and be used to extract the application and the method for vegetable polysaccharides
Technical field
The present invention relates to a kind of compound enzymic preparation and be used to extract the application and the method for vegetable polysaccharides, particularly relate to a kind of method of from the Chinese medicine mulberry leaf, extracting polysaccharides of Folium Mori.
Background technology
Natural polysaccharide is one type of important biological material, has many-sided biological activity and nourishing function, can be used as the effective constituent of various medicines and healthcare products, comprises that for prevention and treatment the multiple disease of cancer all has great potential.Common vegetable polysaccharides has tremella polysaccharide, bitter melon polysaccharide, astragalus polysaccharides, TPS, lentinan, ganoderan, polysaccharides from ginkgo biloba, capsule of weeping forsythia polysaccharide etc.
Because the plant medicine composition is very complicated and a lot of valuable active constituent contents are very low; Be trace even trace; Therefore, how fast and effeciently extraction separation and purifying low levels composition from Chinese medicinal materials, and keep higher activity to become the critical process in the tcm development.But there are problems such as effective component extraction rate is not high, the contaminant removal rate is low in traditional extraction and separation method (like decocting method, pickling process, percolation process, circumfluence method etc.), and these root problems are restricting the process of tcm development.Therefore, need to seek a kind of have the atopic height, fast, efficient, reaction conditions is gentle and the method for the extraction vegetable polysaccharides that is easy to control.
Mulberry leaf different name Herba adianti myriosori is the leaf of Moraceae Morus Morus alba L..The Morus plant has multiple biological activitys such as hypoglycemic, hypotensive, antibiotic and antiviral.Modern pharmacological research proof mulberry leaf can suppress blood sugar increasing, have the effect of prevention and treatment mellitus.In addition, mulberry leaf also have the effect of expelling wind and clearing away heat, cooling blood for improving eyesight, cure mainly rheumatism heating, headache, hot eyes, thirsty, cough due to lung-heat, wandering arthritis etc.Have the gentle characteristics such as have no side effect of the property of medicine, formally be included into the row of " be food be again medicine " by health ministry.Polysaccharides of Folium Mori is as one of effective constituent in the mulberry leaf; Show that through modern pharmacological research it has hypoglycemic activity; Rat tetraoxypyrimidine type mellitus are had therapeutic action, thus extraction, separation, purifying polysaccharides of Folium Mori composition to process the prospect of various Chinese patent medicines very wide.
Summary of the invention
The object of the present invention is to provide a kind of compound enzymic preparation; And it is applied in the extraction vegetable polysaccharides, especially in the polysaccharides of Folium Mori, further provided concrete working method; This method can be carried out highly selective to effective ingredients in plant and transformed under the condition of gentleness; Not only can overcome in the industry in the pure water extracting method commonly used problems such as effective component extraction rate is low, complex procedures, improve extraction system clarity, change the medicinal material quality, can also in extraction, change the structure of original natural component; Increase the physiologically active of extract, have the potential industrial application value.
For realizing this purpose, the present invention adopts following technical scheme:
A kind of compound enzymic preparation is characterized in that: the component that comprises following weight part:
Cellulase 1-5 part
Polygalacturonase 1-5 part
Proteolytic enzyme 2-12 part
Said proteolytic enzyme is selected at least a in papoid, Sumizyme MP, the neutral protease for use.
Above-mentioned compound enzymic preparation is used to extract the application of vegetable polysaccharides.
Above-mentioned compound enzymic preparation is used to extract the method for vegetable polysaccharides, it is characterized in that may further comprise the steps:
(1) get vegetable material, pulverize, add zero(ppm) water, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation and add suitable zero(ppm) water, placed in the water-bath under the 40-60 ℃ of condition activation 10-30 minute;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 4.0-6.0, under 40-60 ℃ of condition enzymolysis 0.5-2.0 hour;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution at little state that boils;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains vegetable polysaccharides medicinal extract.
Said plant is under the condition of mulberry leaf, uses above-mentioned zymin extraction polysaccharides of Folium Mori and may further comprise the steps:
(1) get mulberry tree through air-dry mulberry leaf, pulverize, add zero(ppm) water, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation and add suitable zero(ppm) water, placed in the water-bath under the 40-60 ℃ of condition activation 10-30 minute;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 4.0-5.5, under 40-55 ℃ of condition enzymolysis 0.5-2.0 hour;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution at little state that boils;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains polysaccharides of Folium Mori medicinal extract.
In the method for extraction vegetable polysaccharides above-mentioned and further extraction polysaccharides of Folium Mori, said plant is crushed to the 40-100 order, and the mass ratio of the zero(ppm) water of vegetable material and adding is 1: 10-30.
The mass ratio of described compound enzymic preparation and zero(ppm) water is 1: 5-15.
Compound enzymic preparation addition described in the step (2) is the 1-4% of step (1) vegetable material addition.
Extraction time described in the step (4) is 1-2 hour.
The present invention selects for use the principle of this kind zymin to be: cellulase: can decompose plant cellulose; Polygalacturonase: can destroy plant cell wall; Proteolytic enzyme: can remove the protein in the extract, improve the purity of extract.Because plant class cellularstructure is identical, three kinds of combinations are applicable to the extraction of the interior polysaccharide of cell of plant, have wider range of application.
Compared with prior art, the present invention has the following advantages:
(1) the present invention adopts combined-enzyme method to extract vegetable polysaccharides, and reaction conditions is gentle, can keep the conformation of natural product, does not destroy its three-dimensional arrangement and biological activity, and the original drug effect of composition helps remaining valid.
(2) the present invention selects for use prozyme not only can remove impurity in the system, improves the clarity of extracting solution, has improved the Chinese medicine extraction ratio of effective constituents simultaneously again.
(3) the present invention has carried out optimum combination to proportioning, extraction time, extraction temperature, enzyme concentration, the pH value of prozyme, has realized the maximization that vegetable polysaccharides extracts yield.
(4) compound enzymic preparation of the present invention is used for extracting the method for vegetable polysaccharides, and step (4) has the inactivator preparation, has carried out the effect that water is put forward simultaneously again, carries with water at the employing enzyme and carrying under the bonded condition, has improved the yield of activeconstituents.
(5) adopt the present invention from the medicinal plant mulberry leaf, to extract polysaccharides of Folium Mori, the polysaccharides of Folium Mori extraction yield is greater than 70mg/g, and general water extracting alcohol formulation, extraction yield generally is merely 20-30mg/g, so the enzyme formulation is extracted the yield height.
(6) simple, easy to operate, the less investment of technology of the present invention, can not produce noise and pollution, adapt to explained hereafter and use.
Embodiment
Embodiment 1
(1) get mulberry tree through air-dry mulberry leaf, be crushed to 100 orders, get this powder 9g, add zero(ppm) water 180ml, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation: cellulase 0.02g, polygalacturonase 0.02g, papoid 0.05g adds zero(ppm) water 0.9g, 40 ℃ of water-baths 30 minutes;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 5.0, and enzymolysis is 1 hour under 50 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains polysaccharides of Folium Mori medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the polysaccharides of Folium Mori extraction yield is 71.63mg/g.
Embodiment 2
(1) get mulberry tree through air-dry mulberry leaf, be crushed to 100 orders, get this powder 10g, add zero(ppm) water 200ml, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation: cellulase 0.04g, polygalacturonase 0.04g, papoid 0.1g adds zero(ppm) water 0.9g, 40 ℃ of water-baths 30 minutes;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 5.0, and enzymolysis is 30 minutes under 50 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains polysaccharides of Folium Mori medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the polysaccharides of Folium Mori extraction yield is 70.53mg/g.
Embodiment 3:
(1) get mulberry tree through air-dry mulberry leaf, be crushed to 100 orders, get this powder 20g, add zero(ppm) water 400ml, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation 0.2g, wherein cellulase: polygalacturonase: papoid=2: 2: 5 adds zero(ppm) water 2g, 40 ℃ of water-baths 30 minutes;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 5.0, and enzymolysis is 1 hour under 45 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains polysaccharides of Folium Mori medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the polysaccharides of Folium Mori extraction yield is 75.54mg/g.
Embodiment 4
(1) get mulberry tree through air-dry mulberry leaf, be crushed to 60 orders, get this powder 10g, add zero(ppm) water 300ml, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation: cellulase 0.09g, polygalacturonase 0.09g, neutral protease 0.18g adds zero(ppm) water 1.8g, 60 ℃ of water-baths 10 minutes;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 5.5, and enzymolysis is 1 hour under 60 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 1 hour;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains polysaccharides of Folium Mori medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the polysaccharides of Folium Mori extraction yield is 66.67mg/g.
Embodiment 5: capsule of weeping forsythia polysaccharide
(1) get air-dry Folium Forsythiae, be crushed to 100 orders, get this powder 10g, add zero(ppm) water 100ml, it is subsequent use to boil postcooling;
(2) (cellulase: polygalacturonase: 0.4g papoid=1: 2: 5) adds zero(ppm) water 4g, 40 ℃ of water-baths 20 minutes to get compound enzymic preparation;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 4.0, and enzymolysis is 1 hour under 40 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains capsule of weeping forsythia polysaccharide medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, capsule of weeping forsythia polysaccharide extract rate is 64.32mg/g.
Embodiment 6: polysaccharides from ginkgo biloba
(1) get air-dry Ginkgo Leaf, be crushed to 40 orders, get this powder 10g, add zero(ppm) water 200ml, it is subsequent use to boil postcooling;
(2) (cellulase: polygalacturonase: 0.4g Sumizyme MP=2: 3: 5) adds zero(ppm) water 6g, 45 ℃ of water-baths 30 minutes to get compound enzymic preparation;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 4.5, and enzymolysis is 0.5 hour under 45 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains capsule of weeping forsythia polysaccharide medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the polysaccharides from ginkgo biloba extraction yield is 65.01mg/g.
Embodiment 7: tremella polysaccharide
(1) get the exsiccant white fungus, be crushed to 100 orders, get this powder 10g, add zero(ppm) water 200ml, it is subsequent use to boil postcooling;
(2) (cellulase: polygalacturonase: 0.16g papoid=1: 5: 2) adds zero(ppm) water 1.6g, 45 ℃ of water-baths 30 minutes to get compound enzymic preparation;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 4.5, and enzymolysis is 0.5 hour under 45 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains tremella polysaccharide medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the tremella polysaccharide extraction yield is 60.37mg/g.
Embodiment 8: bitter melon polysaccharide
(1) get the exsiccant balsam pear, be crushed to 80 orders, get this powder 10g, add zero(ppm) water 300ml, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation (cellulase 0.04g, polygalacturonase 0.04g, neutral protease 0.06g, Sumizyme MP 0.04g adds zero(ppm) water 1.8g, 50 ℃ of water-baths 30 minutes;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 6.0, and enzymolysis is 1.5 hours under 50 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains bitter melon polysaccharide medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the bitter melon polysaccharide extraction yield is 63.33mg/g.
Embodiment 9: astragalus polysaccharides
(1) get the Chinese medicinal materials Radix Astragali, be crushed to 100 orders, get this powder 10g, add zero(ppm) water 150ml, it is subsequent use to boil postcooling;
(2) (cellulase: polygalacturonase: 0.22g Sumizyme MP=5: 5: 12) adds zero(ppm) water 3.3g, 60 ℃ of water-baths 30 minutes to get compound enzymic preparation;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 5.0, and enzymolysis is 1.5 hours under 60 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains astragalus polysaccharides medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the astragalus polysaccharides extraction yield is 68.91mg/g.
Embodiment 10: lentinan
(1) get the exsiccant mushroom, be crushed to 100 orders, get this powder 10g, add zero(ppm) water 250ml, it is subsequent use to boil postcooling;
(2) (cellulase: polygalacturonase: 0.34g papoid=1: 5: 11) adds zero(ppm) water 3.4g, 45 ℃ of water-baths 20 minutes to get compound enzymic preparation;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 5.0, and enzymolysis is 2 hours under 45 ℃ of conditions;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution little state that boils, 2 hours;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains lentinan medicinal extract.
Adopt the phenol sulfuric acid process to measure polysaccharide content, the lentinan extraction yield is 70.06mg/g.

Claims (9)

1. compound enzymic preparation is characterized in that: the component that comprises following weight part:
Cellulase 1-5 part
Polygalacturonase 1-5 part
Proteolytic enzyme 2-12 part
2. the described a kind of compound enzymic preparation of claim 1 is characterized in that: said proteolytic enzyme is selected at least a in papoid, Sumizyme MP, the neutral protease for use.
3. the described compound enzymic preparation of claim 1 application that is used to extract vegetable polysaccharides.
4. the described compound enzymic preparation of claim 1 is used to extract the method for vegetable polysaccharides, it is characterized in that may further comprise the steps:
(1) get vegetable material, pulverize, add zero(ppm) water, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation and add suitable zero(ppm) water, placed in the water-bath under the 40-60 ℃ of condition activation 10-30 minute;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 4.0-6.0, under 40-60 ℃ of condition enzymolysis 0.5-2.0 hour;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution at little state that boils;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains vegetable polysaccharides medicinal extract.
5. the described compound enzymic preparation of claim 1 is used to extract the method for vegetable polysaccharides, it is characterized in that: said plant is mulberry leaf, may further comprise the steps:
(1) get mulberry tree through air-dry mulberry leaf, pulverize, add zero(ppm) water, it is subsequent use to boil postcooling;
(2) get compound enzymic preparation and add suitable zero(ppm) water, placed in the water-bath under the 40-60 ℃ of condition activation 10-30 minute;
(3) solution that step (2) and step (1) is obtained mixes, and regulates pH to 4.0-5.5, under 40-55 ℃ of condition enzymolysis 0.5-2.0 hour;
(4) decoct enzymolysis solution, the control Heating temperature keeps solution at little state that boils;
(5) centrifugal, get supernatant liquid filtering, will filtrate concentrates, and obtains polysaccharides of Folium Mori medicinal extract.
6. claim 4 or 5 described compound enzymic preparations are used to extract the method for vegetable polysaccharides, it is characterized in that the plant described in the step (1) is crushed to the 40-100 order, and the mass ratio of the zero(ppm) water of vegetable material and adding is 1: 10-30.
7. claim 4 or 5 described compound enzymic preparations are used to extract the method for vegetable polysaccharides, it is characterized in that: the mass ratio of compound enzymic preparation described in the step (2) and zero(ppm) water is 1: 5-15.
8. claim 4 or 5 described compound enzymic preparations are used to extract the method for vegetable polysaccharides, it is characterized in that: the compound enzymic preparation addition described in the step (2) is the 1-4% of step (1) vegetable material addition.
9. claim 4 or 5 described compound enzymic preparations are used to extract the method for vegetable polysaccharides, it is characterized in that: the extraction time described in the step (4) is 1-2 hour.
CN201110255909A 2011-09-01 2011-09-01 Complex enzyme preparation and application and method thereof for extracting plant polysaccharides by using same Pending CN102304501A (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102603914A (en) * 2012-04-20 2012-07-25 河南中医学院 Preparation method of Caulis Lonicerae polysaccharide
CN105542026A (en) * 2016-01-19 2016-05-04 济南大学 Method for efficiently extracting okra polysaccharide
CN107875196A (en) * 2017-11-17 2018-04-06 成都赛诺联创生物科技有限公司 Complex enzyme, extract solution system, medicament, extract and preparation method thereof, application
CN107880142A (en) * 2016-09-30 2018-04-06 华南农业大学 A kind of preparation method and applications of moringa root Thick many candies
CN109627283A (en) * 2018-12-05 2019-04-16 湖南希尔天然药业有限公司 Method that is a kind of while extracting protein of folium mori and mulberry leaf polysaccharide
CN112142866A (en) * 2020-08-28 2020-12-29 盐城工学院 Extraction method of froggrass polysaccharide with blood sugar reducing effect
CN113249359A (en) * 2021-05-24 2021-08-13 山西农业大学 Method for extracting mulberry leaf functional components by using enzyme
CN113498792A (en) * 2021-06-09 2021-10-15 云南星耀生物制品有限公司 Application of bacillus subtilis preparation in preventing and treating cruciferae clubroot

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
夏平,等: "复合酶法提取桑叶中多糖的工艺条件优化", 《安徽农业科学》 *
张桂征,等: "桑叶多糖提取方法的研究进展", 《广西农业科学》 *
张艳艳: "桑叶多糖的研究进展", 《内江科技》 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102603914A (en) * 2012-04-20 2012-07-25 河南中医学院 Preparation method of Caulis Lonicerae polysaccharide
CN105542026A (en) * 2016-01-19 2016-05-04 济南大学 Method for efficiently extracting okra polysaccharide
CN107880142A (en) * 2016-09-30 2018-04-06 华南农业大学 A kind of preparation method and applications of moringa root Thick many candies
CN107875196A (en) * 2017-11-17 2018-04-06 成都赛诺联创生物科技有限公司 Complex enzyme, extract solution system, medicament, extract and preparation method thereof, application
CN109627283A (en) * 2018-12-05 2019-04-16 湖南希尔天然药业有限公司 Method that is a kind of while extracting protein of folium mori and mulberry leaf polysaccharide
CN112142866A (en) * 2020-08-28 2020-12-29 盐城工学院 Extraction method of froggrass polysaccharide with blood sugar reducing effect
CN113249359A (en) * 2021-05-24 2021-08-13 山西农业大学 Method for extracting mulberry leaf functional components by using enzyme
CN113249359B (en) * 2021-05-24 2023-05-30 山西农业大学 Method for extracting mulberry leaf functional ingredient by utilizing enzyme
CN113498792A (en) * 2021-06-09 2021-10-15 云南星耀生物制品有限公司 Application of bacillus subtilis preparation in preventing and treating cruciferae clubroot
CN113498792B (en) * 2021-06-09 2022-07-01 云南星耀生物制品有限公司 Application of bacillus subtilis preparation in preventing and treating clubroot of cruciferae

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Application publication date: 20120104