CN101732819A - Organic phosphorus degrading enzyme preparation and preparation method thereof - Google Patents
Organic phosphorus degrading enzyme preparation and preparation method thereof Download PDFInfo
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- CN101732819A CN101732819A CN 200810228798 CN200810228798A CN101732819A CN 101732819 A CN101732819 A CN 101732819A CN 200810228798 CN200810228798 CN 200810228798 CN 200810228798 A CN200810228798 A CN 200810228798A CN 101732819 A CN101732819 A CN 101732819A
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Abstract
The invention relates to microbe technology, in particular to a degrading enzyme preparation for degrading residual organic phosphorus on vegetables and fruits and a preparation method thereof. The invention particularly relates to organic phosphorus degrading enzyme preparation obtained by fermenting Methylobacterium. The preparation process comprises the following steps: after fermenting the inoculated strain, centrifugally collecting thalli, and breaking thalli to obtain the organic phosphorus degrading enzyme preparation. The organic phosphorus pesticide degrading enzyme preparation can degrade residual organic phosphorus pesticides on vegetables and fruits.
Description
Technical field
The present invention relates to microbial technique, residual organic phosphorus degrading enzyme preparation and preparation method thereof on a kind of specifically vegetable and fruit that is used to degrade.
Background technology
Organophosphorus pesticide is the primary categories in the agricultural chemicals, has played important function in agricultural production.But it kills and wounds nontarget organism and problem of environment pollution caused can not be ignored equally.The low dosage organophosphor that remains in the food such as vegetables, fruit can produce chronic toxicity to the people, brings out DPN, apoplexy etc.In addition, some organophosphor has very high toxicity to honeybee, fish.The residual toxicity problem of organophosphorus pesticide more and more is subjected to people's attention.The degraded of organophosphor in environment mainly contains present photochemical degradating, chemical degradation, 3 kinds of forms of microbial degradation.Utilize at present microorganism with and metabolite organophosphor is degraded is to be used gradually.Organophosphor contains three phosphide keys usually, and a phosphoric acid ester bond is hydrolyzed and will reduces its toxicity greatly.Opd degradable organophosphorus pesticide molecule destroys the phosphate bond of organophosphor and makes its detoxification.Because various organophosphorus pesticides all have similar structure, be the substituting group difference, the multiple organophosphorus pesticide so a kind of opd often can be degraded.
Summary of the invention
The purpose of this invention is to provide simple organic phosphorus degrading enzyme preparation of a kind of technology and preparation method thereof.
For achieving the above object, the technical solution used in the present invention is:
The organic phosphorus degrading enzyme preparation: the methyl bacillus promptly gets the organic phosphorus degrading enzyme preparation after fermentation.
Described organic phosphorus degrading enzyme preparation also comprises N-oleoyl contract amino acid sodium, sodium acid carbonate and sodium carboxymethylcellulose more, counts by weight and counts 5-20 part organic phosphorus degrading enzyme preparation, 2-8 part N-oleoyl contract amino acid sodium, 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose more by weight.Described methyl bacillus is inoculated in beef-protein medium in 30-35 ℃ through sweat for percentage 5-10% methyl bacillus at first by volume, and shaken cultivation 48-60h is stand-by; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, stand-by; To add the sucrose solution of the EDTA of 1/30-1/40 zymotic fluid and 1/10-1/15 fermentating liquid volume in the zymotic fluid centrifuged deposit at last in the 30-60min ice bath, add the frozen water of 1/30-1/40 fermentating liquid volume again in the centrifugation again at the 30-60min ice bath, centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, precipitation is gained.
The preparation method of organic phosphorus degrading enzyme preparation: at first by volume percentage 5-10% methyl bacillus is inoculated in beef-protein medium in 30-35 ℃, and shaken cultivation 48-60h is stand-by; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, stand-by; To add the sucrose solution of the EDTA of 1/30-1/40 zymotic fluid and 1/10-1/15 fermentating liquid volume in the zymotic fluid centrifuged deposit at last in the 30-60min ice bath, add the frozen water of 1/30-1/40 fermentating liquid volume again in the centrifugation again at the 30-60min ice bath, centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, precipitation is the organic phosphorus degrading enzyme preparation, behind above-mentioned mixing.
The freeze drying of described organic phosphorus degrading enzyme preparation is pulverized and is added water and mixes, and in 15-35 ℃, is made into 20-40 purpose particle in comminutor.Described organic phosphorus degrading enzyme preparation also comprises N-oleoyl contract amino acid sodium, sodium acid carbonate and sodium carboxymethylcellulose more; count by weight and count 5-20 part organic phosphorus degrading enzyme preparation, 2-8 part N-oleoyl contract amino acid sodium, 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose more by weight; by behind the above-mentioned mixing in tablet press machine compressing tablet; be pressed into thickness 1.0-1.5cm; diameter 1.0-1.5cm; the tablet of weight 2.0-4.0g obtains organophosphorus insecticide degradation agent effervescent tablet.Described centrifugal all under 4 ℃, with 6000-8000r/min, centrifugal 30-40min.Tank pressure 0.02-0.05MPa, throughput 1: 1.0-1.5v/v/min, mixing speed are 300 rev/mins during described seed tank culture.
The invention has the advantages that:
1. preparation of the present invention has broad spectrum activity degraded, can degrade DDVP, phoxim, acephatemet, parathion etc. to organophosphorus insecticide.
2. preparation of the present invention soaks with common employing clear water, washing agent or cleans and compare, and effect is better, can degrade more than 75% to organophosphorus insecticide residual on the vegetables and fruits.
The specific embodiment
Embodiment 1
1) seed liquor fermentation: beef-protein medium, the methyl bacillus is inserted with the inoculum concentration of percent by volume 10% in the sterilization back, and 30 ℃, 180r/min cultivates 60h.
2) ferment tank: by volume the inoculum concentration of percentage 8% moves into seeding tank with seed liquor, carries out fermented and cultured, and condition is: jar 30 ℃ of temperature, tank pressure 0.05MPa, throughput 1: 1.5v/v/min, 300 rev/mins of mixing speeds, fermentation time 60 hours.
3) with zymotic fluid in 4 ℃, 7000r/min. centrifugal 30min, abandon supernatant, in bacterium mud, add the pH7.5 of 1/40 fermentating liquid volume, the sucrose solution of the EDTA of 0.05mol/L and 1/15 fermentating liquid volume, shake up gently, thalline is fully suspended, ice bath 60min, 4 ℃, the centrifugal 30min of 7000r/min, abandon high sepage, in bacterium mud, add the frozen water of 1/40 fermentating liquid volume, shake up gently, ice bath 60min, 4 ℃, the centrifugal 30min of 7000r/min gets supernatant, adding fine ground ammonium sulfate to saturation degree while stirring and be 100%, 4 ℃ saltouts and spends the night; Centrifugal, collect albumen precipitation, be the organic phosphorus degrading enzyme preparation.
The organic phosphorus degrading enzyme preparation of 8 parts of parts by weight of above-mentioned gained is added water mix, in comminutor, be made into 20 purpose particles, stand-by;
Meter by weight then, 20 parts of sodium acid carbonates, 5 parts of N-oleoyls amino acid sodium that contracts more, 0.5 part of sodium carboxymethylcellulose sodium acid carbonate mixes, and compressing tablet in tablet press machine is pressed into thickness 1.0cm, diameter 1.2cm, the tablet of weight 3.0g.
Above-mentioned stand-by organic phosphorus degrading enzyme preparation is mixed with above-mentioned tablet, compressing tablet in tablet press machine, 32 ℃ of compressing tablet temperature are pressed into thickness 1.0cm, diameter 1.2cm, the tablet of weight 3.0g obtains organophosphorus insecticide digestive enzyme effervescent tablet.
With the clean clear and clear water of washing of above-mentioned organic phosphorus degrading enzyme preparation and organophosphorus insecticide digestive enzyme effervescent tablet and same concentration to phoxim residual on the rape test (referring to table 1) of degrading.
The agent of table 1 organic phosphorus degrading enzyme is to the degradation effect of phoxim residual on the rape
Embodiment 2
Difference from Example 1 is:
1) seed liquor fermentation: beef-protein medium, the methyl bacillus is inserted with the inoculum concentration of percent by volume 5% in the sterilization back, and 35 ℃, 180r/min cultivates 48h.
2) ferment tank: by volume the inoculum concentration of percentage 5% moves into seeding tank with seed liquor, carries out fermented and cultured, and condition is: jar 25 ℃ of temperature, tank pressure 0.02MPa, throughput 1: 1.0v/v/min, 300 rev/mins of mixing speeds, fermentation time 48 hours.
3) with zymotic fluid in 4 ℃, 6000r/min. centrifugal 40min. abandons supernatant, the pH7.5 that in bacterium mud, adds 1/30 fermentating liquid volume, 0.05mol/L EDTA and the sucrose solution of 1/10 fermentating liquid volume, shake up gently, thalline is fully suspended, ice bath 30min, 4 ℃, the centrifugal 40min of 6000r/min abandons high sepage, the frozen water that in bacterium mud, adds 1/30 fermentating liquid volume, shake up ice bath 30min, 4 ℃ gently, the centrifugal 40min of 6000r/min, supernatant, add fine ground ammonium sulfate to saturation degree while stirring and be 100%, 4 ℃ and saltout and spend the night; Centrifugal, collect albumen precipitation, be the organic phosphorus degrading enzyme preparation.
The organic phosphorus degrading enzyme preparation of 5 parts of parts by weight of above-mentioned gained is added water mix, in comminutor, be made into 30 purpose particles in 15 ℃, stand-by;
Meter by weight then, 50 parts of sodium acid carbonates, 2 parts of N-oleoyls amino acid sodium that contracts more, 1 part of sodium carboxymethylcellulose sodium acid carbonate mixes, and compressing tablet in tablet press machine is pressed into thickness 1.5cm, diameter 1.5cm, the tablet of weight 2.0g.
Above-mentioned stand-by organic phosphorus degrading enzyme preparation is mixed with above-mentioned tablet, compressing tablet in tablet press machine, 25 ℃ of compressing tablet temperature are pressed into thickness 1.5cm, diameter 1.5cm, the tablet of weight 2.0g obtains organophosphorus insecticide digestive enzyme effervescent tablet.
With the clean clear and clear water of washing of above-mentioned organic phosphorus degrading enzyme preparation and organophosphorus insecticide digestive enzyme effervescent tablet and same concentration to phoxim residual on the rape test (referring to table 2) of degrading.
The agent of table 2 organic phosphorus degrading enzyme is to the degradation effect of phoxim residual on the rape
Embodiment 3
Difference from Example 1 is:
1) seed liquor fermentation: beef-protein medium, the methyl bacillus is inserted with the inoculum concentration of percent by volume 5% in the sterilization back, and 32 ℃, 180r/min cultivates 52h.
2) ferment tank: by volume the inoculum concentration of percentage 5% moves into seeding tank with seed liquor, carries out fermented and cultured, and condition is: jar 28 ℃ of temperature, tank pressure 0.04MPa, throughput 1: 1.2v/v/min, 300 rev/mins of mixing speeds, fermentation time 52 hours.
3) with zymotic fluid in 4 ℃, 8000r/min. centrifugal 35min. abandons supernatant, the pH7.5 that in bacterium mud, adds 1/30 fermentating liquid volume, 0.05mol/L EDTA and the sucrose solution of 1/10 fermentating liquid volume, shake up gently, thalline is fully suspended, ice bath 50min, 4 ℃, the centrifugal 35min of 8000r/min abandons high sepage, the frozen water that in bacterium mud, adds 1/30 fermentating liquid volume, shake up ice bath 50min, 4 ℃ gently, the centrifugal 35min of 8000r/min, supernatant, add fine ground ammonium sulfate to saturation degree while stirring and be 100%, 4 ℃ and saltout and spend the night; Centrifugal, collect albumen precipitation, be the organic phosphorus degrading enzyme preparation.
The organic phosphorus degrading enzyme preparation of 15 parts of parts by weight is added water mix, in comminutor, be made into 35 purpose particles, stand-by.
Meter by weight then, 20 parts of sodium acid carbonates, 5 parts of N-oleoyls contract amino acid sodium and 1.0 parts of sodium carboxymethylcellulose sodium acid carbonates mix more, and compressing tablet in tablet press machine is pressed into thickness 1.2cm, diameter 1.4cm, the tablet of weight 3.0g.
Above-mentioned stand-by organic phosphorus degrading enzyme preparation is mixed with above-mentioned tablet, compressing tablet in tablet press machine, 32 ℃ of compressing tablet temperature are pressed into thickness 1.2cm, diameter 1.4cm, the tablet of weight 4.0g obtains the special-purpose organophosphorus insecticide degradation agent of vegetables and fruits effervescent tablet.
With the clean clear and clear water of washing of above-mentioned organophosphorus insecticide digestive enzyme effervescent tablet and same concentration to DDVP residual on the rape test (referring to table 3) of degrading.
The agent of table 3 organic phosphorus degrading enzyme is to the degradation effect of DDVP residual on the rape
Claims (8)
1. organic phosphorus degrading enzyme preparation is characterized in that: the methyl bacillus promptly gets the organic phosphorus degrading enzyme preparation after fermentation.
2. by the described organic phosphorus degrading enzyme preparation of claim 1; it is characterized in that: described organic phosphorus degrading enzyme preparation also comprises N-oleoyl contract amino acid sodium, sodium acid carbonate and sodium carboxymethylcellulose more, counts 5-20 part organic phosphorus degrading enzyme preparation, 2-8 part N-oleoyl contract amino acid sodium, 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose by weight more.
3. by claim 1 or 2 described organic phosphorus degrading enzyme preparations, it is characterized in that: described methyl bacillus is inoculated in beef-protein medium in 30-35 ℃ through sweat for percentage 5-10% methyl bacillus at first by volume, shaken cultivation 48-60h, stand-by; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, stand-by; To add the sucrose solution of the EDTA of 1/30-1/40 zymotic fluid and 1/10-1/15 fermentating liquid volume in the zymotic fluid centrifuged deposit at last in the 30-60min ice bath, add the frozen water of 1/30-1/40 fermentating liquid volume again in the centrifugation again at the 30-60min ice bath, centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, precipitation is gained.
4. preparation method by the described organic phosphorus degrading enzyme preparation of claim 1, it is characterized in that: at first by volume percentage 5-10% methyl bacillus is inoculated in beef-protein medium in 30-35 ℃, and shaken cultivation 48-60h is stand-by; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, stand-by; To add the sucrose solution of the EDTA of 1/30-1/40 zymotic fluid and 1/10-1/15 fermentating liquid volume in the zymotic fluid centrifuged deposit at last in the 30-60min ice bath, add the frozen water of 1/30-1/40 fermentating liquid volume again in the centrifugation again at the 30-60min ice bath, centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, precipitation is the organic phosphorus degrading enzyme preparation.
5. by the preparation method of the described organic phosphorus degrading enzyme preparation of claim 4, it is characterized in that: the freeze drying of described organic phosphorus degrading enzyme preparation, pulverize and add water and mix, in comminutor,, be made into 20-40 purpose particle in 15-35 ℃.
6. press the preparation method of the described organic phosphorus degrading enzyme preparation of claim 4; it is characterized in that: described organic phosphorus degrading enzyme preparation also comprises the N-oleoyl amino acid sodium that contracts more; sodium acid carbonate and sodium carboxymethylcellulose; count by weight and count 5-20 part organic phosphorus degrading enzyme preparation by weight; 2-8 part N-oleoyl amino acid sodium that contracts more; 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose; by behind the above-mentioned mixing in tablet press machine compressing tablet; be pressed into thickness 1.0-1.5cm; diameter 1.0-1.5cm; the tablet of weight 2.0-4.0g obtains organophosphorus insecticide degradation agent effervescent tablet.
7. by the preparation method of the described organic phosphorus degrading enzyme preparation of claim 4, it is characterized in that: described centrifugal all under 4 ℃, with 6000-8000r/min, centrifugal 30-40min.
8. by the preparation method of the described organic phosphorus degrading enzyme preparation of claim 4, it is characterized in that: tank pressure 0.02-0.05MPa, throughput 1: 1.0-1.5v/v/min, mixing speed are 300 rev/mins during described seed tank culture.
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| CN 200810228798 CN101732819B (en) | 2008-11-14 | 2008-11-14 | Organic phosphorus degrading enzyme preparation and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993839A (en) * | 2010-07-23 | 2011-03-30 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
| CN102199550A (en) * | 2011-03-24 | 2011-09-28 | 深圳西德赛科技有限公司 | Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent |
| CN103146658A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院沈阳应用生态研究所 | Organophosphorus pesticide degrading enzyme transformed through mutation and encoding gene thereof |
| CN103834595A (en) * | 2014-03-07 | 2014-06-04 | 江苏农林职业技术学院 | Methylobacterium and application thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103146664B (en) * | 2013-02-04 | 2014-07-02 | 辽宁中科生物工程有限公司 | Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof |
| CN103146665B (en) * | 2013-02-04 | 2014-05-14 | 中国科学院沈阳应用生态研究所 | Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059668A (en) * | 1991-10-12 | 1992-03-25 | 徐谦 | Eliminating agent for chemical left over from vegetable |
| US5453375A (en) * | 1993-08-18 | 1995-09-26 | Lockheed Idaho Technologies Company | Method and compositions for the degradation of tributyl phosphate in chemical waste mixtures |
| US6599733B1 (en) * | 1998-12-07 | 2003-07-29 | The United States Of America As Represented By The Secretary Of The Army | Microbial biodegradation of phosphonates |
| CN101293131B (en) * | 2007-04-29 | 2014-10-29 | 上海优你生物科技股份有限公司 | Complex enzyme preparation for eliminating residual pesticides and application thereof |
-
2008
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993839A (en) * | 2010-07-23 | 2011-03-30 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
| CN101993839B (en) * | 2010-07-23 | 2012-06-27 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
| CN102199550A (en) * | 2011-03-24 | 2011-09-28 | 深圳西德赛科技有限公司 | Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent |
| CN103146658A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院沈阳应用生态研究所 | Organophosphorus pesticide degrading enzyme transformed through mutation and encoding gene thereof |
| CN103834595A (en) * | 2014-03-07 | 2014-06-04 | 江苏农林职业技术学院 | Methylobacterium and application thereof |
| CN103834595B (en) * | 2014-03-07 | 2015-10-28 | 江苏农林职业技术学院 | A kind of Methylobacterium and application thereof |
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| CN101732819B (en) | 2012-07-25 |
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