CN111019930A - Enzyme protective agent, composite enzyme liquid for pesticide degradation, preparation method of composite enzyme liquid and washing combined liquid - Google Patents
Enzyme protective agent, composite enzyme liquid for pesticide degradation, preparation method of composite enzyme liquid and washing combined liquid Download PDFInfo
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- CN111019930A CN111019930A CN201911396609.4A CN201911396609A CN111019930A CN 111019930 A CN111019930 A CN 111019930A CN 201911396609 A CN201911396609 A CN 201911396609A CN 111019930 A CN111019930 A CN 111019930A
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/88—Ampholytes; Electroneutral compounds
- C11D1/94—Mixtures with anionic, cationic or non-ionic compounds
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/02—Inorganic compounds ; Elemental compounds
- C11D3/04—Water-soluble compounds
- C11D3/042—Acids
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38663—Stabilised liquid enzyme compositions
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/50—Perfumes
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/02—Anionic compounds
- C11D1/36—Anionic compounds of unknown constitution, e.g. natural products
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/66—Non-ionic compounds
- C11D1/74—Carboxylates or sulfonates esters of polyoxyalkylene glycols
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
- C11D1/88—Ampholytes; Electroneutral compounds
- C11D1/92—Sulfobetaines ; Sulfitobetaines
Abstract
The invention belongs to the technical field of biological detergents, and particularly relates to an enzyme protective agent, a compound enzyme liquid for pesticide degradation, a preparation method of the compound enzyme liquid and a washing composition liquid, wherein the enzyme protective agent comprises polyol and trehalose, the weight ratio of the polyol to the trehalose is 2: 3-6: 1, the polyol is at least one of glycerol, sorbitol and propylene glycol, the enzyme protective agent can simultaneously stabilize the compound enzyme liquid for pesticide degradation formed by compounding organophosphorus hydrolase and pyrethroid degrading enzyme, and the washing composition liquid can be used for cleaning residual organophosphorus, pyrethroid and other pesticides of fruits, vegetables, tea leaves and the like, and has a good effect.
Description
Technical Field
The invention belongs to the technical field of biological detergents, and particularly relates to an enzyme protective agent, a compound enzyme liquid for pesticide degradation, a preparation method of the compound enzyme liquid and a washing composition liquid.
Background
The data of the national quality control bureau in 2000 show that 47.5 percent of vegetable pesticide residues in China exceed the standard, and common pesticides mainly comprise organic phosphorus, pyrethroid and the like. Organophosphorus pesticides can cause symptoms such as nerve functional disorder, tremor, confusion, language disorder and the like; toxicity of pyrethroid pesticides cumulative long term low dose exposure can cause chronic diseases, and has moderate neurotoxicity, immune system toxicity, cardiovascular toxicity and genetic toxicity to mammals. The WHO has already made strict regulations on the residual limit of the organic phosphorus and the pyrethroid in agricultural products, and the national food safety standard GB2763-2019 of China also clearly defines the maximum residual limit of the application of nearly 400 organic phosphorus and pyrethroid in food.
The traditional main methods for removing pesticide residues are cleaning, peeling, cooking, storing, ozone methods and the like, and have the defects of low degradation rate, weak universality, large limitation and the like. Zongronfen (a research on the removal rate of pesticide residues by a vegetable and fruit detergent) and the like are tested by soaking green vegetables by using a commercially available kitchen detergent, tap water, edible alkali, 1% edible salt, a kitchen detergent added with edible alkali and the like, and the removal rate of the commercially available kitchen detergent to methamidophos and dimethoate is 50% -68%, the removal rate of 1% edible salt is 69%, and the removal rate of the commercially available kitchen detergent after soaking in tap water is only 46%. In comparison, the enzyme-catalyzed pyrethroid pesticide can be effectively degraded, has the advantages of high efficiency, specificity, safety, low cost and the like, has better development prospect, but has great problems of ensuring that the activity of pesticide degrading enzyme is not obviously reduced in the shelf life and having universality on common pesticides such as organophosphorus and pyrethroid and the like.
Patent 201310657828.X discloses a fruit and vegetable cleaning powder composed of Aspergillus niger enzyme liquid, an enzyme protective agent (glycine/potassium sorbate/glycerin) and an adsorbent (calcium carbonate and white carbon black), and capable of effectively and rapidly removing organophosphorus pesticide residues. Liuyuhuan, etc. (ZL201210412214.0) at Zhongshan university also obtains a pyrethroid degrading enzyme solution with the fermentation titer of 2000-3000U/ml, and also discloses (patent 201710019869.4) that esterase est816 recombinant esterase has strong degradation effect on pyrethroid pesticides (including cyfluthrin, cypermethrin, fenvalerate and deltamethrin), and 80 percent of relative enzyme activity is kept after the temperature is kept for 24 hours at 45 ℃.
At present, few reports are reported on the research on the combination of degrading enzymes of two types of pesticides which are in the front of the pesticide market, and the activity change of the two types of enzymes after combination and the universality on the degradation of organic phosphorus and pyrethrins are considered, so the research on the combination and the application of the two types of degrading enzymes of the pesticides has important significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and can simultaneously stabilize the organophosphorus hydrolase and the pyrethroid degrading enzyme.
The invention is realized by the following steps:
the invention provides an enzyme protective agent which comprises polyhydric alcohol and trehalose, wherein the weight ratio of the polyhydric alcohol to the trehalose is 2: 3-6: 1, and the polyhydric alcohol is at least one of glycerol, sorbitol and propylene glycol.
The enzyme protective agent is used for protecting at least one of cellulase, lipase, esterase and protease.
The invention also provides a compound enzyme liquid for degrading pesticides, which comprises an enzyme protective agent, a pyrethroid hydrolase liquid, an organophosphorus hydrolase liquid, a preservative and xanthan gum.
The compound enzyme liquid for degrading the pesticide comprises the following components in percentage by weight: 25-35 parts of pyrethrin hydrolase liquid, 25-35 parts of organophosphorus hydrolase liquid, 15-45 parts of enzyme protective agent, 0.05-0.1 part of preservative and 0.01-0.1 part of xanthan gum; the preservative is at least one of methylisothiazolinone, methylchloroisothiazolinone, sorbic acid and benzoic acid; the chrysanthemum ester hydrolase is microbial lipase C244A; the organophosphorus hydrolase is esterase MPH.
The invention also provides a preparation method of the compound enzyme liquid for degrading the pesticide, which comprises the following steps:
(1) measuring liquid chrysanthemum ester hydrolase C244A and preservative, and stirring uniformly;
(2) uniformly mixing xanthan gum and trehalose of which the amount is ten times that of the xanthan gum, slowly pouring the mixture into the step (1), and stirring to fully dissolve the mixture;
(3) measuring liquid organophosphorus hydrolase MPH, and pouring into the step (1);
(4) pouring the rest of trehalose and polyol into the step (1), and continuously stirring to obtain the product.
The invention also provides a washing combined liquid, which comprises complex enzyme liquid for degrading pesticides, glycolipid, acid-base regulator and essence, wherein the glycolipid is selected from at least one of rhamnolipid and sophorolipid, and the pH of the washing combined liquid is regulated to be between 5.0 and 8.0 by the acid-base regulator.
The washing combined liquid also comprises a surfactant, wherein the surfactant is at least one of polysorbate 80, alkyl glycoside and hydroxysultaine.
The washing combined liquid comprises the following components in percentage by weight: 70-95 parts of compound enzyme liquid for degrading pesticide, 2-15 parts of glycolipid, 0.5-10 parts of surfactant and 0.05-0.1 part of essence.
The washing composition liquid is used for cleaning residual pesticides such as organophosphorus, pyrethroid and the like of fruits, vegetables, tea leaves and the like.
The use method of the washing combined liquid comprises the following steps: firstly, adding the composition into water according to the proportion of 0.1-1% to form a cleaning solution, soaking a substrate to be cleaned in the cleaning solution for 3-8min, and then taking out and cleaning, wherein the temperature of the cleaning solution is lower than 40 ℃.
The invention has the following beneficial effects:
1. trehalose can nonspecifically protect liquid enzyme protein from invariance inactivation, polyhydric alcohol can also provide a better environment for the stability of hydrolase, the two can synergistically enhance the protection of the liquid enzyme protein, and the universality is wide.
2. The xanthan gum in the composite enzyme liquid and the washing combined liquid provided by the invention can be dissolved into a highly viscous solution in water, so that the pesticide degrading enzyme can be kept as a single entity in the solution, and the stability of the pesticide degrading enzyme is enhanced.
3. The complex enzyme liquid and the washing combined liquid provided by the invention are combined with the chrysanthemum ester hydrolase C244A and the organophosphorus hydrolase MPH according to a specific proportion, so that the thermal stability of the pesticide degradation complex enzyme liquid is enhanced, and the practical requirement that the degradation amount is several times of the maximum pesticide residue limit of the national standard GB2763-2019 is fully met.
4. When the complex enzyme solution and the washing combined solution provided by the invention are used for cleaning fruits and vegetables, the complex enzyme solution and the washing combined solution have the effects of keeping the fruits and the vegetables fresh and brightening the fruits and the vegetables due to the specific addition of the trehalose, and have excellent antibacterial and anticorrosive effects due to the glycolipid and the specific preservative component.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides an enzyme protective agent, which comprises polyalcohol and trehalose, wherein the weight ratio of the polyalcohol to the trehalose is 2: 3-6: 1, the trehalose can nonspecifically protect liquid enzyme protein from invariable inactivation, the polyalcohol can also provide a better environment for the stability of hydrolase, the polyalcohol and the trehalose can synergistically enhance the protection of the liquid enzyme protein, and the enzyme protective agent is used for protecting at least one of cellulase, lipase, esterase and protease.
The invention also provides a compound enzyme liquid for degrading pesticide, which comprises the following components in percentage by weight: 25-35 parts of pyrethrin hydrolase liquid, 25-35 parts of organophosphorus hydrolase liquid, 15-45 parts of enzyme protective agent, 0.05-0.1 part of preservative and 0.01-0.1 part of xanthan gum; the preservative is at least one of methylisothiazolinone, methylchloroisothiazolinone, sorbic acid and benzoic acid; the chrysanthemum ester hydrolase is microbial lipase C244A; the organophosphorus hydrolase is esterase MPH.
The preparation method of the compound enzyme liquid for pesticide degradation comprises the following steps:
(1) measuring liquid chrysanthemum ester hydrolase C244A and preservative, and stirring uniformly;
(2) uniformly mixing xanthan gum and trehalose of which the amount is ten times that of the xanthan gum, slowly pouring the mixture into the step (1), and stirring to fully dissolve the mixture;
(3) measuring liquid organophosphorus hydrolase MPH, and pouring into the step (1);
(4) pouring the rest of trehalose and polyol into the step (1), and continuously stirring to obtain the product.
The invention also provides a washing combined liquid, which comprises the following components in proportion: 70-95 parts of composite enzyme liquid for degrading pesticides, 2-15 parts of glycolipid, 0.5-10 parts of surfactant, 0.05-0.1 part of essence and an acid-base regulator, wherein the glycolipid is at least one of rhamnolipid and sophorolipid, and the pH of the washing composition liquid is regulated by the acid-base regulator to be between 5.0 and 8.0.
The use method of the washing combined liquid comprises the following steps: firstly, adding the composition into water according to the proportion of 0.1-1% to form a cleaning solution, soaking a substrate to be cleaned in the cleaning solution for 3-8min, and then taking out and cleaning, wherein the temperature of the cleaning solution is lower than 40 ℃.
Enzyme activity determination of organophosphorus hydrolase and chrysanthemum ester hydrolase
The method for measuring the enzyme activity of the organophosphorus hydrolase is disclosed in the patent CN108998289A [0129] line [0131] line.
The method for measuring the production rate of generating p-nitrophenol by using p-nitrophenylpalmitate (marked as 'pNPP') for the enzyme activity of the chrysanthemum ester hydrolase comprises the following steps:
preparing a 10mL glass test tube, sucking 5900 mu L of 0.05mmol/L pH7.6 Tris-hydrochloric acid buffer solution 75 mu L of 10mmol/L p-nitrophenylpalmitate isopropanol solution, mixing uniformly, carrying out water bath at 25 ℃ for 3min, adding 120uL of pyrethrin degrading enzyme sample water diluent, timing from 0s, mixing uniformly, adjusting the zero point of a spectrophotometer by taking a blank sample as a control, measuring the light absorption value of reaction liquid in the test tube at 400nm, and recording the measured value at 30s as A1(for measuring the dilution degree, should be controlled between 0.15-0.25), the value measured at the 90s is recorded as A2. Reading absorbance values at ambient temperatures as close as possible to 25 ℃; the relative error of two parallel measurement values of the same sample is not more than 10.0 percent, and the average value of the two measurement values is the final enzyme activity measurement value.
The activity of the pesticide degrading enzyme in the sample is calculated according to the following formula:
X＝Vt*(A2-A1)*N/14.7*1.0*Vs*t＝3.455*(A2-A1)*N
in the formula: the activity of the pesticide degrading enzyme in the X-liquid (solid) sample is u/mL (or u/g), and the result is an integer; Vt-Total volume of reaction (6095. mu.L); vs — sample volume (120 μ Ι _); 1.0-optical path (cm); n-dilution multiple; t-time interval, 1 min; 14.7-p-nitrophenol molar extinction coefficient (cm) of 400nm2/umol)。
1. Influence of enzyme protective agent on stability of organophosphorus hydrolase or pyrethrin hydrolysate
Example 1
The embodiment provides an enzyme protective agent, and the enzyme protective agent is used for a chrysanthemum ester hydrolase C244A, and the specific mixture ratio is as follows: 26 parts of sorbitol solution, 7 parts of trehalose, 66.9 parts of chrysanthemum ester hydrolase C244A and 0.1 part of methylchloroisothiazolinone.
Example 2
The embodiment provides an enzyme protective agent, and the enzyme protective agent is used for a chrysanthemum ester hydrolase C244A, and the specific mixture ratio is as follows: 30 parts of sorbitol solution, 3 parts of trehalose, 66.9 parts of chrysanthemum ester hydrolase C244A and 0.1 part of methylchloroisothiazolinone.
Example 3
The embodiment provides an enzyme protective agent, and the enzyme protective agent is used for a chrysanthemum ester hydrolase C244A, and the specific mixture ratio is as follows: 11 parts of sorbitol solution, 22 parts of trehalose, 66.9 parts of chrysanthemum ester hydrolase C244A and 0.1 part of methylchloroisothiazolinone.
Example 4
The embodiment provides an enzyme protective agent, and the enzyme protective agent is used for organophosphorus hydrolase MPH, and the mixture ratio is as follows: 25 parts of glycerol, 14 parts of trehalose, 60.9 parts of organophosphorus hydrolase MPH and 0.1 part of methylisothiazolinone.
The enzyme protectant was replaced with water for control experiments:
comparative example 1: 66.9 parts of chrysanthemum ester hydrolase C244A liquid, 33 parts of water and 0.1 part of methylisothiazolinone.
Comparative example 2: 60.9 parts of organophosphorus hydrolase MPH liquid, 39 parts of water and 0.1 part of methylisothiazolinone.
The instant enzyme activity of each group of samples and the enzyme activity after standing for 30 days at 40 ℃ are determined, and the specific results are shown in table 1.
Table 1 stability Effect of enzyme protecting Agents on Single enzymes
As shown in Table 1, test results show that the comparative ratio of the embodiment after the enzyme protective agent is added is obviously stable, the enzyme protective agent in the embodiment 1 is better in enzyme stability protection than the enzyme protective agents in the embodiments 2 and 3, namely the enzyme protective agent has certain influence on the enzyme stability, and the enzyme protective agent has better enzyme stability effect when the weight ratio of the polyalcohol to the trehalose is 2: 3-6: 1.
2. Influence of enzyme protective agent on stability of compound enzyme liquid and washing combined liquid
Example 5
The embodiment provides a compound enzyme solution, which comprises the following specific mixture ratio: 45 parts of enzyme protective agent (2/1 parts of glycerol/trehalose), 27 parts of organophosphorus hydrolase MPH, 27.9 parts of pyrethroid hydrolase C244A, 0.04 part of xanthan gum and 0.06 part of methylisothiazolinone.
Example 6
The embodiment provides a compound enzyme solution, which comprises the following specific mixture ratio: 15 parts of enzyme protective agent (4/1 parts of propylene glycol/trehalose), 42 parts of organophosphorus hydrolase MPH, 42.88 parts of pyrethrin hydrolase C244A, 0.04 part of xanthan gum and 0.08 part of methyl chloroisothiazolinone.
Example 7
The embodiment provides a compound enzyme solution, which comprises the following specific mixture ratio: 30 parts of an enzyme protective agent (glycerol/trehalose ═ 2/1), 34 parts of organophosphorus hydrolase MPH, 35.86 parts of pyrethroid hydrolase C244A, 0.04 part of xanthan gum and 0.1 part of methylisothiazolinone.
The specific preparation method of the compound enzyme solution comprises the following steps:
(1) accurately weighing 35.86 parts of chrysanthemum ester hydrolase C244A and 0.1 part of methylisothiazolinone, and uniformly stirring;
(2) uniformly mixing 0.04 part of xanthan gum and 0.4 part of trehalose, slowly pouring into the mixture in the step (1), and stirring to fully dissolve the xanthan gum and the trehalose;
(3) accurately measuring 34 parts of organophosphorus hydrolase MPH, and pouring into the reactor (1);
(4) pouring the rest of trehalose and 20 parts of glycerol into the mixture (1), and continuously stirring to obtain the finished product.
Example 8
The embodiment provides a compound enzyme solution, which comprises the following specific mixture ratio: 30 parts of an enzyme protective agent (glycerol/trehalose ═ 9/1), 34 parts of organophosphorus hydrolase MPH, 35.86 parts of pyrethroid hydrolase C244A, 0.04 part of xanthan gum and 0.1 part of methylisothiazolinone.
Comparative example 3
The enzyme protective agent is replaced by water as a contrast, and the specific mixture ratio is as follows: 34 parts of organophosphorus hydrolase MPH, 35.86 parts of pyrethrin hydrolase C244A, 30 parts of water, 0.04 part of xanthan gum and 0.1 part of methylisothiazolinone
Example 9
The embodiment provides a washing composition liquid, which comprises the following specific components in percentage by weight: 70 parts of complex enzyme solution (example 7), 5 parts of rhamnolipid, 2 parts of tween 80, 3 parts of hydroxysultaine and 0.07 part of lemon essence, and the pH is adjusted to 5.8 with dilute hydrochloric acid.
Example 10
The embodiment provides a washing composition liquid, which comprises the following specific components in percentage by weight: 95 parts of complex enzyme solution (example 7), 3 parts of sophorolipid, 1 part of tween 80, 0.9 part of hydroxysultaine, 0.07 part of lemon essence, and the pH is adjusted to 6.0 with dilute hydrochloric acid.
The instant enzyme activity of each group of samples and the enzyme activity after standing for 30 days at 40 ℃ are determined, and the specific results are shown in Table 2.
Table 2 Effect of enzyme protecting Agents on the stability of Complex enzyme solutions and Wash compositions
As shown in Table 2, the test results show that the examples 5, 6, 7 and 8 added with the enzyme protective agent are obviously stable to the comparison example 3, the enzyme protective agent in the examples 5, 6 and 7 is better in enzyme stability protection than the enzyme protective agent in the example 8, namely the enzyme protective agent has certain influence on the enzyme stability, the enzyme protective agent has better enzyme stability effect when the weight ratio of the polyalcohol to the trehalose is 2: 3-6: 1, and the examples 9 and 10 show that the chrysanthemum ester hydrolase C244A and the organophosphorus hydrolase MPH in the washing combined liquid are also good in stability.
3. Removing effect of compound enzyme liquid and washing composition liquid on common pesticides
The qualitative test of the color development method of the organophosphorus pesticide quick test card shows that the color development difference of the lemon peel is obvious before and after the soaking in example 9, the lemon peel is nearly white before the soaking, blue after the soaking and close to a pure water negative control group, and the organophosphorus residue of the lemon peel is lower than the detection limit of the quick test card after the soaking.
The test of the special quick test card color developing box for the pyrethrin shows that the color developing degree of the green tea and the black tea which are positive before after being soaked in the sample of the example 9 is close to that of the negative control group. In addition, the cleaning effect of the composite enzyme liquid in the embodiment 7 and the enzyme-containing cleaning liquid in the embodiment 9 on common pyrethrin is shown in table 3, and the degradation efficiency on the pyrethrin can reach more than 80%, which shows that the pyrethroid degradation effect is good.
Table 3 HPLC method for detecting the removing effect of the compound enzyme liquid and the washing composition liquid on the common pyrethrin
After the compound enzyme liquid and the washing combined liquid are placed for 30 days, the chrysanthemum ester hydrolase C244A and the organophosphorus hydrolase MPH also have high enzyme activity, and can be used for cleaning residual organophosphorus, pyrethroid and other pesticides of fruits, vegetables, tea leaves and the like.
The complex enzyme liquid and the washing combined liquid provided by the invention are combined with the chrysanthemum ester hydrolase C244A and the organophosphorus hydrolase MPH according to a specific proportion, so that the thermal stability of the pesticide degradation complex enzyme liquid is enhanced, and the practical requirement that the degradation amount is several times of the maximum pesticide residue limit of the national standard GB2763-2019 is fully met.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The enzyme protective agent is characterized by comprising polyhydric alcohol and trehalose, wherein the weight ratio of the polyhydric alcohol to the trehalose is 2: 3-6: 1, and the polyhydric alcohol is at least one of glycerol, sorbitol and propylene glycol.
2. The enzyme protectant of claim 1, wherein: used for protecting at least one of cellulase, lipase, esterase and protease.
3. A complex enzyme liquid for degradation of agricultural chemicals comprising the enzyme protecting agent according to claim 1 or 2, characterized in that: also comprises pyrethrin hydrolase liquid, organophosphorus hydrolase liquid, preservative and xanthan gum.
4. The complex enzyme liquid for pesticide degradation according to claim 3, characterized in that the composition ratio of each component is as follows: 25-35 parts of pyrethrin hydrolase liquid, 25-35 parts of organophosphorus hydrolase liquid, 15-45 parts of enzyme protective agent, 0.05-0.1 part of preservative and 0.01-0.1 part of xanthan gum; the preservative is at least one of methylisothiazolinone, methylchloroisothiazolinone, sorbic acid and benzoic acid; the chrysanthemum ester hydrolase is microbial lipase C244A; the organophosphorus hydrolase is esterase MPH.
5. The method for preparing a complex enzyme liquid for degradation of agricultural chemicals according to claim 4, characterized in that: the method comprises the following steps:
(1) measuring liquid chrysanthemum ester hydrolase C244A and preservative, and stirring uniformly;
(2) uniformly mixing xanthan gum and trehalose of which the amount is ten times that of the xanthan gum, slowly pouring the mixture into the step (1), and stirring to fully dissolve the mixture;
(3) measuring liquid organophosphorus hydrolase MPH, and pouring into the step (1);
(4) pouring the rest of trehalose and polyol into the step (1), and continuously stirring to obtain the product.
6. A washing composition liquid comprising the complex enzyme liquid for degradation of agricultural chemicals according to claims 3 to 4, characterized in that: the washing liquid also comprises glycolipid, pH regulator and essence, wherein the glycolipid is at least one of rhamnolipid and sophorolipid, and the pH of the washing liquid is regulated to be between 5.0 and 8.0 by the pH regulator.
7. The washing composition of claim 6, wherein: the surfactant is at least one of polysorbate-80, alkyl glycoside and hydroxysultaine.
8. The washing composition of claim 7, wherein the components are in the following proportions: 70-95 parts of compound enzyme liquid for degrading pesticide, 2-15 parts of glycolipid, 0.5-10 parts of surfactant and 0.05-0.1 part of essence.
9. The washing composition as claimed in any one of claims 6 to 8, wherein: the cleaning agent is used for cleaning residual pesticides such as organophosphorus and pyrethroid and the like of fruits, vegetables, tea leaves and the like.
10. A method of using a washing composition as claimed in any one of claims 6 to 9, wherein: adding the combined solution into water according to the proportion of 0.1-1% to form a cleaning solution, soaking a substrate to be cleaned in the cleaning solution for 3-8min, and then taking out and cleaning, wherein the temperature of the cleaning solution is lower than 40 ℃.
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| CN201911396609.4A CN111019930A (en) | 2019-12-30 | 2019-12-30 | Enzyme protective agent, composite enzyme liquid for pesticide degradation, preparation method of composite enzyme liquid and washing combined liquid |
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| CN201911396609.4A CN111019930A (en) | 2019-12-30 | 2019-12-30 | Enzyme protective agent, composite enzyme liquid for pesticide degradation, preparation method of composite enzyme liquid and washing combined liquid |
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| CN111019930A true CN111019930A (en) | 2020-04-17 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114794158A (en) * | 2022-05-24 | 2022-07-29 | 武汉新华扬生物股份有限公司 | Composition for preventing germs and removing pesticide residues of tea leaves and preparation method and application thereof |
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| CN108998289A (en) * | 2018-09-26 | 2018-12-14 | 武汉新华扬生物股份有限公司 | Enzyme stabilizers, Recompounded pesticide hydrolysis enzyme solution and preparation method thereof and fruits and vegetables cleaning solution and preparation method thereof |
| CN111117982A (en) * | 2019-12-25 | 2020-05-08 | 武汉新华扬生物股份有限公司 | Pyrethroid degrading enzyme, encoding gene thereof, recombinant strain and application |
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| CN101293131A (en) * | 2007-04-29 | 2008-10-29 | 上海普天欣生物技术有限公司 | Complex enzyme preparation for eliminating residual pesticides and application thereof |
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| CN114794158A (en) * | 2022-05-24 | 2022-07-29 | 武汉新华扬生物股份有限公司 | Composition for preventing germs and removing pesticide residues of tea leaves and preparation method and application thereof |
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