OA21103A - Process for manufacturing a biopesticide by nano formulation based on essential oils of clove (Syzygium aromaticum) and ginger (Zingiber officinalis). - Google Patents

Abstract

In the perpetual fight against microbial agents associated with the degradation of foodstuffs, chemical synthesis products are greatly requested by approximately 94% of farmers in black Africa. Despite their effectiveness, there is a resurgence of microbial resistance phenomena, the accumulation of chemical residues in food can lead to food poisoning in humans, contamination of groundwater and environmental pollution. Given the growing consumer demand for natural foodstuffs called "organic", these foods treated with chemical pesticides are for the vast majority banned from sale in certain markets due to non-compliance with requirements related to agricultural practices. The manufacturing process of a biopesticide by nano formulation based on essential oils of Syzygium aromaticum and Zingiber officinalis, the subject of the present invention, remedies this situation. The Nano biopesticide resulting from it has several advantages, namely: it leads to a biopesticide rich and diversified in bioactive compounds from the two combined essential oils of tropical flora plants. The constituent molecules of the Nano bio pesticide have a very fine size of 20-200 nanometers and can therefore easily penetrate between plant tissues to protect them from phytopathogenic attacks. This Nano biopesticide is more effective compared to other biopesticides found on the market. It has no impact on human health and the environment. It is a reliable alternative compared to chemically synthesized phytosanitary products with a view to improving yields and agricultural practices. /-

Description

I. INTRODUCTION

The present invention relates to a process for manufacturing a biopesticide by nano formulation based on essential oils of clove (Syzygium aromaticum) and ginger (Zingiber officinalis). This is a new generation of bio-pesticide, combining the bioactive molecules of two essential oils with very effective antimicrobial potential, a broad spectrum of action and respectful of human health and the environment.

Pesticides are a set of chemical and biological products intended to ensure the destruction or prevent the action of animals, plants or microorganisms harmful to agriculture. They include insecticides, fungicides, herbicides and parasiticides designed to have a biocidal action.

Although having demonstrated a certain effectiveness, the preponderant use of chemically synthesized pesticides has revealed an increase in microbial resistance phenomena, environmental pollution, the destruction of biodiversity, the contamination of groundwater and the accumulation chemical residues in foods that can lead to food poisoning in humans and the occurrence of numerous chronic diseases.

The side effects of chemical synthesis products have pushed farmers to question this farming practice. It therefore proved necessary to look for another alternative that would take over in the antimicrobial fight in order to guarantee food safety and security. It is this fact that has piqued the interest in developing this manufacturing process by nanoformulation of a bio-pesticide based on essential oils of Syzygium aromaticum (Clove) and Zingiber officinalis (Ginger).

A Bio pesticide is a synthetic product formulated from natural substances derived from animals, microorganisms or plants and used to combat organisms considered harmful to agricultural products. In a current geosphere where organic is intended to be a luxury for the certification and marketing of foodstuffs in the international market, the biological properties of plants serve as a basis for the development of this formula, because the raw material used is mainly made up of water and essential oils from plants, notably clove (Syzygium aromaticum) and ginger (Zingiber officinale).

These essential oils which are concentrates of secondary metabolites including eugenol and gingerol which are powerful antimicrobial compounds therefore their effectiveness in crop protection no longer needs to be demonstrated. Clove and ginger are two plants of tropical flora, available and abundantly cultivated in the Littoral regions, more precisely in the locality of Njombé-Penja and West Cameroon.

One of the major advantages of this so-called nanoformulation process is the reduction in the size of bioactive molecules, their encapsulation in micelles and their biotransformation into very small particles (nanometer). All this makes it possible to improve their stability, reduce their photosensitivity, optimize their effectiveness and potentiate their antimicrobial powers against pathogens in agricultural products.

The small size of the bioactive molecules obtained gives them a greater ability to diffuse between the tissues of the plant material to easily reach the plant pathogen and destroy it. This is the curative method of treating plant matter. In addition, in a preventive manner, biotransformed secondary metabolites have the capacity to induce a form of protection in plants. This consists of the synthesis of numerous other antimicrobial compounds within the plant which can enable it to cope with a possible biotic or abiotic attack (preventive mode).

II. STATE OF THE ART

Plant protection products are varied and are found in three forms: liquid, solid and powder. They can be used by spraying, depositing or even fumigation. Bio pesticides are phytosanitary products manufactured to this day in the traditional way by farmers from dried Neem, Thyme and Chili plant powders. The manufacturing process for these products uses simplified equipment and production methods, namely: drying of plants, grinding, addition to water and homogenization. Except that this method remains limited due to the lack of control of the doses applicable by the populations, a reported effectiveness of less than 50% because the majority of the active compounds remain trapped in the tissues of the plant. This very often pushes farmers to resort again to chemically synthesized pesticides.

The nano biopesticide based on combinations of essential oils resulting from this formulation process has the advantage of making all the bioactive molecules of the two plants available at a nanometric size. This makes it possible to optimize the effectiveness to more than 95% and broaden the spectrum of action against phytopathogenic microorganisms such as Fusarium solani, Colletotrichum gloeosporoides, Lasiodiploidia theobromae, Rhizopus stolonifer and Aspergillus niger.

III. DESCRIPTION OF THE INVENTION

In the present invention, nanobiopesticide means the combination of low volume of essential oils of clove or ginger, very small quantity of surfactant (Tween 80, Glycerol, Propylene glycol monostearate) and large volume of water. The whole is subjected to the shearing and molecular encapsulation process. The nano biopesticide in the form of a milky solution is manufactured under rigorous hygienic conditions and in compliance with scientific formulation requirements.

III.1. Detailed description of the manufacturing process

The process for manufacturing a biopesticide by nano formulation based on combinations of essential oils of Syzygium aromaticum (Clove) and Zingiber officinalis (Ginger) is carried out in three main stages: (1) extraction of essential oils clove flower buds and ginger rhizomes, (2) shearing and low temperature molecular encapsulation, (3) stabilization testing and packaging.

III.l.l. Extraction of essential oils from plants

The essential oils of clove (Syzygium aromaticum) and ginger (Zingiber officinale) are extracted by hydrodistillation using Clevenger-type equipment according to the process in Figure 1 below. This process is divided into the following 8 subsidiary steps:

- Supply: dry clove flower buds and fresh ginger rhizomes were collected from peasant plantations on the Littoral;

Sorting and cleaning: this consists of washing the plants with running water in order to eliminate solid debris, sand and mud;

Cutting: the ginger rhizomes are cut into thin strips with a scalpel;

Maceration: One kilogram of each plant is introduced separately into a container to which two (02) liters of water are added and the whole is covered for 24 hours, with the aim of softening the tissues of the plant material in order to prepare it for distillation;

Hydrodistillation: the macerated plants are introduced into a heating tank with an additional liter of water. Hydrodistillation consists of subjecting macerated plant matter to high boiling temperatures of water (+100°C) for 10 hours for cloves and 6 hours for ginger. This is to induce the weakening of plant cells and the release of volatile bioactive compounds which will be carried by water vapor towards a refrigerant where they will be condensed in the form of droplets. These droplets are made up of both floral water (hydrophilic phase) and essential oil (hydrophobic phase);

Purification of the essential oil: the hydrophobic or oily phase which floats is recovered and dried using anhydrous sodium sulfate (chemical desiccant which has the capacity to capture traces of water present in a mixture);

Storage: the essential oils obtained are placed in dark/opaque bottles and stored in the refrigerator at -4°C. This is to protect the compounds in the oils against oxidation in the light and the volatility of the molecules.

The analysis of the chemical composition of essential oils is done by

Gas chromatography coupled with mass spectrometry.

Clean water

Tinted water-matter

Droplets and plant traces of water

Figure l: Process for extracting essential oils by Hydrodistillation

IIL1.2. Low temperature molecular shearing and encapsulation

The pure essential oils obtained were subjected to high pressure and rotation speed of a magnetic bar under the effect of a magnetic stirrer at low temperature with the addition of surfactant (Tween 80), cosurfactants (Glycerol, Propylene monostearate glycol) and water. The mixture is made to ensure the hydrophilic lipophilic balance (HLB) between the four components in Figure 2 (Clove essential oil, Ginger essential oil, Tween 80, Glycerol and Propylene glycerol monosterate). Thus, the oil particles become very tiny and disperse in the water to form stable micelles. This second step has 5 sub-steps:

Choice of components

The components used in the formulation of the Nanobiopesticide which is the subject of the present invention are in particular the essential oils (EO) of plants and water. Tween 80 (HLB=15.01) was used as a surfactant in the mixture; Propylene glycol monostearate (HLB=1.8) and glycerol (HLB=3.8) were used as cosurfactants.

Table: ingredients for the formulation of 100 ml of Nanobiopesticide

Materials Quantity 1 Clove essential oil 7 mL 2 Ginger essential oil 3 mL 3 Tween 80 3 mL 4 Propylene glycol monostearate (MPG) 1 mL 5 Glycerol (Gly) f 1 mL 6 H ₂ O 85

Source: our care

Combination of essential oils

The two essential oils obtained from clove and ginger are introduced into a beaker and stirred in a closed Erlenmeyer flask using a magnetic stirrer at 400 rpm for a period of 30 minutes for a final volume of 10 mL of combined essential oil (HECO).

Homogenization of Tween 80 with Propylene Glycol Monostearate and Glycerol

A mixture of 3 mL of surfactant (tween 80) and 2 mL (v/v) of cosurfactant (MPG + Gly) are stirred in an Erlenmeyer flask using a magnetic stirrer at 400 rpm for a period of 30 minutes to homogenize the surfactants and form 5 mL of surfactant.

Dispatching of combined oil in surfactant

This step consists of adding the 10 mL of essential oil combined with the 5 mL of surfactant. Everything is stirred under magnetic stirring (800 rpm) in a closed vacuum for 1 hour. Then, the mixture is introduced into a Bain Marie Shaker at 45°C for 30 minutes. Then, this mixture is again subjected to rotary stirring at 800 rpm for 30 minutes. During this stage, there is molecular shearing and reduction in the size of the bioactive molecules of essential oils.

Encapsulation of molecules

Using a graduated burette, 85 mL of distilled water is poured at a constant flow rate of 2 mL/min into the HECO-Surfactant mixture while stirring. After adding water, the HECO-Surfactant-Water mixture is kept under magnetic stirring at 800 rpm for 4 hours. The formulated mixture is stored for 72 hours in the refrigerator at + 4 °C and subjected again to magnetic stirring at 400 rpm for 1 hour. Here, very small bioactive molecules are encapsulated in water micelles, which reduces their photosensitivity and volatility. At the end of this step we obtain the Biopesticide which is subsequently subjected to a particle size analysis.

Particle size analysis

The biopesticide thus formulated is subjected to a particle size measurement using a Mastersizer Hydro 2000 Laser Granulometer in order to determine the size of the particles present in the mixture. For a nanoformulation, the particle size of the mixture must be between 20-200 nanometer. It is this step which confirms the presence of essential oil nanoparticles in this biopesticide and gives it the name Nanobiopesticide.

400 rpm 30 minutes

.Magnetic stirring

HE: Essential oil

HECO: Combined essential oils

Figure 2; Shearing and molecular encapsulation process

IIL1.3. Stabilization test and conditioning

After 72 hours of storage, the Nanobiopesticide is subjected to centrifugation testing, washability and rheological tests in order to determine its stability over time.

- Centrifugation test: It consists of subjecting 3 mL of Nanobiopesticide to a high rotation speed (3000 rpm) for 10 minutes and observing its behavior. The aim is to determine whether the Nanobiopesticide remains uniphasic after centrifugation.

Washability test: It consists of adding 10 mL of distilled water to 2 mL of Nanobiopesticide and observing whether the solution obtained remains homogeneous.

Rheological tests: This consists of storing the Nanobiopesticide at room temperature for 180 days and observing daily whether or not there is the appearance of flocculation, coalescence, creaming and sedimentation phenomena. A good nanoformulation should not allow any of these phenomena to appear.

Nanobiopesticide is packaged in translucent plastic bottles. For optimal effectiveness, 100 mL of this Nanobiopesticide must be added to 100 L of water before application and/or spraying on crops in meadows or post-harvest.

Claims (1)

i. Process for manufacturing a liquid Nanobiopesticide based on essential oils of clove and ginger, characterized in that it comprises the following steps: • Extraction by hydrodistillation of essential oils from clove and ginger through cleaning, cutting, maceration, distillation and purification, • Formulation of Nanobiopesticide in liquid form by shearing and molecular encapsulation of essential oils in water micelles, • Stabilization of the Nanobiopesticide through centrifugation, washability and rheological phenomena tests. ii. Process for manufacturing a liquid Nanobiopesticide based on essential oils of clove and ginger according to claim 1, characterized in that the active compounds of clove oil and ginger are optimized with a view to improve their spectrum of action against plant pathogens; iii. Process for manufacturing a liquid Nanobiopesticide based on essential oils of clove and ginger according to claims 1 and 2, characterized in that the nanoformulation process used results in a biopesticide therefore the molecules have a very small size which oscillates between 20-200 nanometers and therefore easily penetrates the tissues of the plant in order to protect it against attacks by phytopathogens. iv. Nanobiopesticide according to the preceding claims, characterized in that it can be used as a phytosanitary product in the sanitation of agricultural plantations and places where crops are stored as a microbicide, insecticide and insect repellent against phytopathogenic agents such as bacteria, fungi, viruses, fleas and insects. v. Liquid nanobiopesticide based on essential oils of clove and ginger obtained according to the process of claim 1.