LU503344B1

LU503344B1 - Preparation method and application of pesticide adjuvant lipopeptide and derivative

Info

Publication number: LU503344B1
Application number: LU503344A
Authority: LU
Inventors: Tieyong Sheng; Yulan Zhang; Hongtu Xie; Caixia Sun; Junyu Wang
Original assignee: Univ Northeastern; Shenyang Zhongke New Fertilizer Co Ltd; The Inst Of Applied Ecology Chinese Academy Of Sciences
Priority date: 2023-01-12
Filing date: 2023-01-12
Publication date: 2023-07-13

Abstract

The present invention provides a preparation method of a pesticide adjuvant lipopeptide and a derivative. The lipopeptide comprises a peptide chain and an aliphatic chain; the peptide chain and the aliphatic chain are connected by a peptide bond; and the lipopeptide has molecular weight of 2848Da and is linear. The lipopeptide or the lipopeptide derivative may prevent pesticide particles from agglomerating and expanding. The lipopeptide or the lipopeptide derivative may increase the wettability, adhesivity or permeability of the particles and prevent the decomposition of the active component.

Description

PREPARATION METHOD AND APPLICATION OF PESTICIDE 7005068

ADJUVANT LIPOPEPTIDE AND DERIVATIVE

Technical Filed

The present invention relates to the application of a biosurfactant as an adjuvant in the technical field of pesticide, in particular to a preparation method and application of pesticide adjuvant lipopeptide and derivative.

Background

Almost all active compounds for pesticides cannot be used directly, and it is necessary to add various adjuvants to process them into pesticide formulations with practical use value. Pesticide adjuvants are any compounds or substances that are intentionally added to pesticide products to enhance and improve, or contribute to enhancing and improving the physical and chemical properties of the products, to maximize the efficacy of the drug or contribute to the safe application of the drug.

Although the pesticide adjuvants themselves do not have killing toxicity to target organisms, it does not indicate that they are safe for human health and the environment. Surfactants in pesticides play the roles of dispersion, penetration, wetting and absorption promotion, to help to evenly disperse the active compounds into formulations, so as to prevent particles from agglomerating and expanding, increase the wettability, adhesivity or permeability of the particles, and prevent the decomposition of the active ingredients. The commonly used chemical surfactants themselves are often toxic, and destroy the ecosystem and pollute the surface groundwater because of the difficulty in being degraded by soil microorganisms. Most of the adjuvants belong to persistent organic pollution. For example, the concentration of N-methyl-2-pyrrolidone in pollen is as high as 69.3 mg/L for 7 d, which has adverse effects on bee reproduction and development. Adjuvants (cosolvent) are sprayed or washed by rain directly into soil and water, which seriously pollutes farmland, surface water and groundwater. Volatile adjuvants may also increase VOCs in the air in the process of pesticide spraying, especially in the wide application of

UAVs. The control effect of conventional chemical pesticides is not good, and the dosage is increased year by year, often resulting in excessive pesticide residues, and generating serious biological and environmental toxicity. Therefore, the development of surfactant type pesticide adjuvants with high efficiency, safety and environmental protection has very important significance. Biosurfactants have strong surface and interfacial activity, low toxicity and biodegradability. They are widely used in oil exploitation, environmental protection, pharmaceutical and chemical industry, food and agriculture, and has wide potential applications. 0503344

The biosurfactants are secondary metabolites, for self-regulation of biological microenvironment, produced by microorganisms to adapt to the growth of hydrophobic organic compounds in the process of metabolism. The biosurfactant have the common properties of surfactants, have lipophilic and hydrophilic amphipathic molecular structure, and can be well connected with interfaces between solid and liquid phases and between gas and liquid phases to reduce their surface tension and interfacial tension and increase the contact surface between hydrophobic organic compounds and liquid phase, thereby increasing the water content of the hydrophobic organic compounds and improving the utilization of the hydrophobic organic compounds by microorganisms. The biosurfactants have wide bioadaptability, and are not preferred degradation substances of microorganisms in a compost system, but as an organic substance, the biosurfactants finally can be degraded by 100% by microorganisms, and have no residue in the soil. Compared with synthetic surfactants, the biosurfactants have various molecular structure types, some of which have many special functional groups, high surface activity and strong emulsification ability. Good emulsification performance and unique plant cell affinity of the biosurfactants will increase the efficiency of pesticides and reduce the consumption of pesticides, so the biosurfactant is a novel fungicide with ecological and biological safety, increases yield and efficiency and is in line with the development of green ecological agriculture. High lipophilicity of the biosurfactants is conducive to carrying nutritional ions into the roots of crops and conducive to crop growth. The biosurfactants also have anti-bacterial, anti-fungal and antiviral biological pharmacological actions, can control crop diseases, can reduce the crop diseases caused by crop pathogens, and assist and improve the overall quality of products.

Moreover, the biosurfactants are non-toxic, used less and easy to be degraded by microorganisms, and have good environmental friendliness. The biosurfactants contain carboxyl in structure, and have the functions of neutralizing and improving alkaline soil. Their degradation process is also a metabolic process of soil microorganisms, which not only can enhance soil biological vitality, but also can play an important role in improving soil compaction. The biosurfactants are suitable for environments with extreme temperature, pH value and salinity. These compounds do not harm the microorganisms, and can promote the improvement of the microenvironment to increase the degradation efficiency of organic matter by the microorganisms. The microorganisms in the soil form a symbiotic proliferation relationship, inhibit the growth and reproduction of harmful pathogens and transform the pathogens into beneficial bacteria which interact and promote each other to play a LUS03344 synergistic effect of the population, enhance stress resistance and disease resistance of crops, reduce the disease index of continuous cropping crops, and greatly alleviate continuous cropping obstacles after application of successive years. According to structural features and microbial sources, the biosurfactants which are studied extensively currently can be divided into glycolipids, lipopeptides, lipoproteins, fatty acids, phospholipids, neutral lipids and polymer surfactants, wherein lipopeptides, sophorolipids, trehalose lipids and lipopeptides are widely studied and applied. The biosurfactants are fermented generally by taking all kinds of vegetable oil or n-hexane as carbon sources, and have diversified components, long fermentation cycle with the change of specific culture conditions, easy generation of foam in the fermentation process, and high production cost. However, such surfactants are largely required in the actual production of pesticides.

The lipopeptide is composed of two parts of a hydrophilic peptide chain and a lipophilic fatty acid chain, that is, about 10 polypeptides and fatty acid chains form a circular or linear lipopeptide. The lipopeptides are mainly derived from secondary metabolites of microorganisms and exhibit multiple biological activities. In 1970,

Bernheimer and Avigard used the lipopeptide as a pesticide due to its insect resistant characteristic (Zhai Ying, Lv Shuang * Study on Antibacterial Activity of Novel

Lipopeptides. Chinese Journal of Urban and Rural Industrial Hygiene. 2006, 4(114): 19-21).

Summary

The present invention discloses a lipopeptide and a derivative thereof, and a preparation method and application thereof.

The lipopeptide is extracted from the fermentation broth of Staphylococcus epidermidis, can significantly induce the expression of defensin in skin keratinocytes and has a good effect on inhibiting Staphylococcus aureus infection. À first invention purpose of the present invention is to provide a lipopeptide. The lipopeptide comprises a peptide chain and an aliphatic chain; the peptide chain and the aliphatic chain are connected by a peptide bond; a structure is shown as a formula which is linear and has molecular weight of 2848Da.

A lipopeptide derivative, characterized in that the lipopeptide derivative comprises a peptide chain and an aliphatic chain; the peptide chain and the aliphatic chain are connected by a peptide bond;

The lipopeptide is extracted from Staphylococcus epidermidis, and the

Staphylococcus epidermidis comprises skin symbiotic bacteria Staphylococcus epidermidis 1457 or Staphylococcus epidermidis 12228 or Staphylococcus LUS03344 epidermidis RP62A.

The Staphylococcus epidermidis is fermented; the supernatant of acid-precipitated Staphylococcus epidermidis fermentation broth is collected and extracted by methanol to obtain a lipopeptide extract; and the lipopeptide extract is purified by HPLC to obtain the lipopeptide.

The preparation method comprises the steps: (1) fermenting Staphylococcus epidermidis at a refrigerator of -80°C; taking out the preserved Staphylococcus epidermidis 1457 or 12228 or RP62A; activating; and then transferring to 200ml of

TSB liquid medium for fermentation at 37°C and 220rpm for 16h; (2) conducting acid precipitation on the fermentation broth for centrifuging the Staphylococcus epidermidis fermentation broth; adjusting the supernatant of the fermentation broth with 6M hydrochloric acid to a pH of 2.0; and standing at 4°C overnight; (3) centrifuging the supernatant of the acid-precipitated Staphylococcus epidermidis fermentation broth; extracting precipitates in methanol; centrifuging the methanol solution extracted overnight, and collecting the supernatant; drying the methanol extraction solution by distillation on a rotary evaporator; and finally extracting with an oil pump to obtain the lipopeptide extract; (4) dissolving the lipopeptide extract, and then conducting gradient elution using a separated high pressure liquid chromatograph Agilentl200 and C18 column by taking water containing 0.1% TFA and acetonitrile containing 0.1% TFA as a mobile phase; collecting each elution peak; and identifying the molecular weight of each elution peak by primary mass spectrometry to obtain a target product.

Fatty acids are connected to the peptide chain by butyl octyl phthalyl and

N-methylmorpholine, and the peptide chain is synthesized by a solid phase synthesis method.

It is conducive to dispersing the active compound evenly in a formulation.

The lipopeptide or the lipopeptide derivative may prevent pesticide particles from agglomerating and expanding.

The lipopeptide or the lipopeptide derivative may increase the wettability, adhesivity or permeability of the particles and prevent the decomposition of the active component.

Detailed Embodiments

Embodiment 1 1. Adjuvant of avermectin water emulsion

Avermectin is a macrolide antibiotic insecticide and acaricide, and has high 0503344 biological activity, the effects of contact and stomach toxicity and a wide insecticidal spectrum. At present, most of the domestic products are emulsified oil with very low content of the active compound, and a large amount of toluene and xylene organic 5 solvent adjuvants are used, which are highly volatile, pollutes the environment and are harmful to human health. The solvent may be not used or the amount of the solvent can be reduced by preparing the emulsified oil into water emulsion.

In the present invention, 20% avermectin crude oil (i.e, xylene solution of avermectin), edible oil with 3% volume fraction, a large amount of water and lipopeptide (2g/L) are mixed under stirring to form a uniform 1.8% avermectin water emulsion which is lacteous. The appearance is a milky white solution, and emulsification dispersibility and stability tests are conducted according to GB/1603.

The water emulsion can be quickly and evenly dispersed in water after entering the water, and has no oil slick on top and no precipitate on bottom. The stability of the emulsion is qualified. The formulation uses the water as the main solvent, and organic solvent content is not more than 8%. Compared with the similar avermectin emulsified oil, the formulation has the advantages of being non-combustible, non-explosive and mild to people, livestock and the environment and is a preferred drug for the production of pollution-free fruits and vegetables. 2. Adjuvant of atrazine water suspension agent as herbicide

The bacterial fermentation broth containing 45 g/L lipopeptide, as a biosurfactant adjuvant, is added to the atrazine active compound. When the lipopeptide content is 1 g/L, 48% atrazine water suspension agent under uniform suspension can be prepared, and the suspension agent has no obviously settled atrazine water suspension agent within 30 min. 2 g/L methylcellulose is further added. The suspension is better; no bottom deposit appears within 45 min; and the suspension agent has good thermal storage stability and cold storage stability. The above prepared atrazine water suspension agent is used for weeding corn fields, and it is found that the weeding effect is better than that of the commercially available atrazine water suspension agent and a similar weeding effect can be achieved with a 30% reduction in the dosage.

Claims

CLAIMS LU503344

1. À lipopeptide, characterized in that the lipopeptide comprises a peptide chain and an aliphatic chain; the peptide chain and the aliphatic chain are connected by a peptide bond; a structure is shown as a formula (I) which is linear and has molecular weight of 2848Da.

2. A lipopeptide derivative, characterized in that the lipopeptide derivative comprises a peptide chain and an aliphatic chain; and the peptide chain and the aliphatic chain are connected by a peptide bond.

3. The lipopeptide derivative according to claim 2, characterized in that the lipopeptide is extracted from Staphylococcus epidermidis, and the Staphylococcus epidermidis comprises skin symbiotic bacteria Staphylococcus epidermidis 1457 or Staphylococcus epidermidis 12228 or Staphylococcus epidermidis RP62A.

4. The lipopeptide derivative according to claim 3, characterized in that the Staphylococcus epidermidis is fermented; the supernatant of acid-precipitated Staphylococcus epidermidis fermentation broth is collected and extracted by methanol to obtain a lipopeptide extract; and the lipopeptide extract is purified by HPLC to obtain the lipopeptide.

5. A preparation method of the lipopeptide derivative of claim 4, characterized by comprising the steps: (1) fermenting Staphylococcus epidermidis at a refrigerator of -80°C; taking out the preserved Staphylococcus epidermidis 1457 or 12228 or RP62A; activating; and then transferring to 200ml of TSB liquid medium for fermentation at 37°C and 220rpm for 16h; (2) conducting acid precipitation on the fermentation broth for centrifuging the Staphylococcus epidermidis fermentation broth; adjusting the supernatant of the fermentation broth with 6M hydrochloric acid to a pH of 2.0; and standing at 4°C overnight; (3) centrifuging the supernatant of the acid-precipitated Staphylococcus epidermidis fermentation broth; extracting precipitates in methanol; centrifuging the methanol solution extracted overnight, and collecting the supernatant; drying the methanol extraction solution by distillation on a rotary evaporator, and finally extracting with an oil pump to obtain the lipopeptide extract; (4) dissolving the lipopeptide extract, and then conducting gradient elution using a separated high pressure liquid chromatograph Agilentl200 and C18 column by taking water containing 0.1% TFA and acetonitrile containing 0.1% TFA as a mobile phase; collecting each elution peak; and identifying the molecular weight of each elution peak by primary mass spectrometry to obtain a target product.

6. The preparation method of the lipopeptide derivative according to claim 5,

characterized in that fatty acids are connected to the peptide chain by butyl octyl LUS03344 phthalyl and N-methylmorpholine, and the peptide chain is synthesized by a solid phase synthesis method.

7. An application of the lipopeptide or the lipopeptide derivative of claim 1 or 2, characterized in that the lipopeptide or the lipopeptide derivative can be used for preparing pesticides.