Patent Application
20190327961
This invention relates to a stable composition based on a natural or synthetic aromatic compound as well as to its various uses, in particular in the agricultural field.
In the face of a steadily-increasing population, worldwide agriculture is confronted with multiple challenges, in particular the increased demand for foodstuffs. However, the plant and animal agricultural field is subject to multiple biotic problems, namely the illnesses caused by bacteria, viruses, fungi, nematodes, and all other insects and pests. To overcome these problems, most farmers and breeders use chemical products (pesticides and disinfectants) that are very harmful to human health and to the environment.
It is therefore urgent and necessary to find an alternative to these products that is both effective against the biotic attacks of crops and safe for human health and for the environment.
Multiple alternatives have recently been proposed for replacing the pesticides and the chemical disinfectants, in particular:
Among the biopesticides based on plant extracts, several studies have focused on essential oils and their majority components, such as thymol, carvacrol, eugenol in particular (Koul, O. et al. (2008). Essential Oils as Green Pesticides: Potential and Constraints. Biopestic. Int. 4(1): 63-84; Isman, M. B. and Machial, C. M. (2006). Pesticides Based on Plant Essential Oils: From Traditional Practice to Commercialization. In M. Rai and M. C. Carpinella (Eds.), Naturally Occurring Bioactive Compounds, Elsevier, BV, pp. 29-44). These compounds actually have very promising properties. However, they present problems linked to their volatility, their poor solubility in water, and their capacity for being oxidized quickly (Moretti, M. D. L. et al. (2002). Essential Oil Formulations Useful as a New Tool for Insect Pest Control. AAPS Pharm. Sci. Tech. 3 (2): 13). The volatility of these compounds is at the origin of their short service life in solution. This negatively affects the quality of the formulation since the manufacturers have to use large amounts of it or have to use sophisticated and expensive stabilization methods such as micro- and nano-encapsulation (WO2007063267A1), which makes these products non-competitive in relation to chemical pesticides.
The object of this invention is therefore to find a solution to these problems and to propose in particular a stable formulation comprising aromatic compounds of a volatile nature having an antimicrobial activity (antibacterial, antifungal, antiviral, and antiparasitic) and/or anti-insect.
To respond to this, the invention has as its object a composition in a form that is solid, stable, and dispersible in water, comprising:
Advantageously, such a composition:
The aromatic alcohols have numerous properties depending on their chemical formula, and the composition according to the invention can advantageously be used in particular as a phytosanitary product or fertilizer, but the composition according to the invention can also be used for other applications, such as, for example, an anti-insect application or as an antimicrobial application for sanitizing ambient air, hands, buildings, reusable medical devices, etc.
Other characteristics and advantages of the invention will emerge from the description of the invention, in details, which will follow, and test results produced with the invention demonstrating its effectiveness in numerous applications, results illustrated in particular by the accompanying figures that show:
In terms of the invention, “emulsifying agent” is defined as any compound that can improve the suspension and the dispersion of a composition in water.
When the term emulsifying agent is used in the singular in this application, one or more emulsifying agent(s) should be understood, with the composition according to the invention being able to comprise one or more emulsifying agent(s).
In terms of the invention, “texturing agent” is defined as any compound that has the functions of stabilizing (improves the stability), thickening (increases the viscosity), and/or emulsifying (improves the suspension).
When the term texturing agent is used in the singular in this application, one or more texturing agent(s) should be understood, with the composition according to the invention being able to comprise one or more texturing agent(s).
In terms of the invention, “aromatic alcohol” is defined as an aromatic molecule, having a hydroxyl group OH that is attached to a carbon of a benzene cycle. The aromatic alcohol is also called a phenol.
The term “aromatic alcohol” can be used interchangeably in the singular or in the plural in this application. Whether it is used in the singular or in the plural, at least one aromatic alcohol, i.e., one or more aromatic alcohol(s), should always be understood, with the composition according to the invention being able to comprise one or more aromatic alcohol(s).
In terms of the invention, “anti-insect” is defined as any inhibiting and/or destructive action of insects and pests.
In terms of the invention, “antimicrobial” is defined as any inhibiting and/or destructive action of germs that are bacterial, fungal, parasitic, and viral.
In terms of the invention, “compound” is defined as a molecule or a mixture of molecules.
In terms of the invention, “effervescent acid-base pair” is defined as the association, the combination, of an acid and a base that can produce an effervescent phenomenon when said pair is put into solution.
When the term effervescent acid-base pair is used in the singular in this application, one or more acid-base pair(s) should be understood, with the composition according to the invention being able to comprise one or more acid-base pair(s).
In terms of the invention, “dispersible in water” is defined as being able to disperse, to solubilize in water without forming non-dispersible masses, in particular in irrigation water for spraying plants or watering animals.
In terms of the invention, “essential oil” is defined as any extract that is obtained from one or more aromatic plant(s), preferably the concentrated and hydrophobic liquid of volatile aromatic (fragrant) compounds of a plant. An essential oil can be obtained in particular by mechanical extraction and vapor entrainment or by dry distillation. “Essential oil” is also defined as products that are identical to those described above but obtained by chemical synthesis.
In terms of the invention, “mixture containing an aromatic alcohol” is defined as any mixture of molecules comprising at least one aromatic alcohol. It may preferably involve one or more essential oil(s) or one or more mixture(s) of molecules contained in one or more essential oil(s) or a mixture of one or more essential oil(s) with one or more mixture(s) of molecules contained in one or more essential oil(s).
When the term mixture containing an aromatic alcohol is used in the singular in this application, one or more mixture(s) containing an aromatic alcohol should be understood, with the composition according to the invention being able to comprise one or more mixture(s) containing an aromatic alcohol.
In terms of the invention, “stable” product is defined as a product whose molecular composition as well as the texture, color, and effectiveness are constant over time.
In terms of the invention, “solid” is defined as a preferably uniform non-liquid form: powder, pellet, or tablet.
The invention therefore has as its object a composition in a form that is solid, stable, and dispersible in water.
The composition according to the invention can in particular come in the form of powder, pellets, or tablets.
The composition according to the invention comprises at least one aromatic alcohol or at least one mixture containing at least one aromatic alcohol.
Said at least one aromatic alcohol is a natural or synthetic aromatic alcohol.
Preferably, said at least one aromatic alcohol is mono-, di- or sesquiterpenic. It can be selected in particular from among thymol, menthol, eugenol, carvacrol, and cinnamaldehyde.
According to a particularly suitable embodiment, the composition comprises at least one aromatic alcohol that is contained in an essential oil, and preferably the composition according to the invention comprises at least one essential oil, such as, for example, the essential oil of thyme, oregano, clove, mint.
Said at least one aromatic alcohol and/or said at least one mixture comprising at least one aromatic alcohol, present in the composition according to the invention, play(s) an active ingredient role. They have effects and effectiveness that vary depending on their nature, but they preferably have an antibacterial and/or anti-insect activity, as is the case in particular of aromatic alcohols selected from among thymol, menthol, eugenol, carvacrol, and cinnamaldehyde.
In a preferred way, the aromatic alcohol or the mixture comprising at least one aromatic alcohol represents between 0.1 and 25% by weight of the composition.
In addition to the aromatic alcohol (or the mixture containing it), the composition according to the invention also comprises at least one emulsifying agent and/or one oil. The emulsifying agent and/or oil is/are preferably selected from among vegetable oils, mineral oils, soy lecithin, cellulose, pectin, and glycerol.
Said at least one emulsifying agent and/or oil makes it possible in particular to liquefy and/or to keep in suspension the aromatic alcohol when it is put into solution. Actually, the emulsifiers and/or oils interact both with the aromatic alcohol molecules and with the water molecules, which prevents the aromatic alcohol molecules from floating to the surface of the water.
In a preferred way, the emulsifying agent and/or the oil represent(s) between 0.01 and 2% by weight of the composition.
The composition according to the invention, in addition to these first two components, also comprises at least one texturing agent. It is preferably selected from among locust bean gum, guar gum, and cassia gum.
Said at least one texturing agent makes it possible in particular to improve the stabilization of the aromatic alcohols present in the composition both during storage and during the solubilization. Actually, the texturing agent makes it possible, during manufacturing, to fix the aromatic alcohols and the emulsifiers and/or oils that are liquid and consequently to obtain a sievable powder. During storage, the volatile aromatic alcohols remain fixed on the texturing agents and do not evaporate. At the time of the solubilization, the texturing agents create a viscosity that promotes the dispersion of aromatic alcohols and prevent them from floating.
In a preferred way, said at least one texturing agent represents between 5 and 12% by weight of the composition. The ratio of the gum does not exceed 12%, preferably 10%, in the final composition, if not once in solution, the texturing agent runs the risk of forming a paste that is difficult to disperse.
Finally, the composition according to the invention also comprises at least one effervescent acid-base pair. It is preferably selected from among the following acid-base pairs: sodium bicarbonate-citric acid, calcium bicarbonate-citric acid, potassium bicarbonate-citric acid, sodium bicarbonate-tartaric acid, calcium bicarbonate-tartaric acid, potassium bicarbonate-tartaric acid, sodium bicarbonate-maleic acid, potassium bicarbonate-maleic acid, sodium bicarbonate-ascorbic acid, calcium bicarbonate-ascorbic acid, and potassium bicarbonate-ascorbic acid. Preferably, the acid from the effervescent pair represents between 20 and 25% by weight of the composition, and the base of the effervescent pair represents between 60 and 74.89% by weight of the composition.
Said at least one effervescent acid-base pair that is present in the composition makes it possible in particular to improve the dissolution of the aromatic alcohol once in solution.
In a preferred way, said at least one effervescent acid-base pair represents between 50 and 94.89% by weight of the composition.
According to a variant, the composition according to the invention consists exclusively of:
One composition according to this variant can consist in particular as follows:
According to another variant, in addition to said at least one aromatic alcohol (or at least one mixture containing at least one aromatic alcohol), said at least one emulsifying agent and/or oil, said at least one texturing agent, and said at least one effervescent acid-base pair, the composition according to the invention comprises at least one other compound, such as, for example, at least one compound that is selected from among the vitamins, minerals, hormones, dyes, etc.
The composition according to the invention can be obtained by any suitable method that makes it possible to obtain a composition in solid form with the above-mentioned components.
It can involve in particular a method comprising the following steps:
This first step makes it possible to obtain good dissolution of the aromatic alcohols, without any recrystallization problem.
The solution is then added to a powder containing at least one texturing agent and an effervescent acid-base pair, preferably under the following conditions:
The texturing agent makes it possible to stabilize the volatile molecules by absorption in the final composition. The effervescent acid-base pair improves the solubilization of the composition that can be carried out without necessarily having recourse to manual or mechanical stirring.
During the stirring in the mechanical mixing mechanism, the initial powder that consists of the texturing agent and the acid-base pair gradually absorbs the liquid that consists of the aromatic alcohol and the emulsifying agent and/or oil until a powder sometimes containing small lumps is obtained. These small lumps are converted into powder by the action of the calibrator; the calibration is done after the powder exits from the mixing mechanism. (The final powder obtained after calibration is preferably stored in hermetically-sealed bags protected from heat and humidity at ambient temperature.) Depending on the aromatic alcohols, and/or mixtures containing them, present in the composition, the composition according to the invention can be used for various applications. Before use, the solid composition according to the invention is preferably put into solution. In a preferred way, the composition/water ratio is between 100 g per 1,000 liters of water up to 25 kg per 1,000 liters of water.
In particular, the aromatic alcohols have antibacterial and/or antifungal and/or antiviral and/or anti-insect properties.
The object of the invention is therefore in particular the use of a composition according to the invention as a phytosanitary product, in particular for preventing and/or combatting the plant illnesses (some or all of the plants) caused by fungi, bacteria, viruses, nematodes and/or pests.
For its use by application on the plants (some or all of the plants), including on fruits, vegetables and/or flowers post-harvest, the composition according to the invention can be used as irrigation water or in leaf spraying.
The plant illnesses caused by bacteria for which the composition according to the invention is particularly useful for prevention and/or treatment are preferably selected from among the soft rot caused by the Erwinia species, the bacterial canker caused by the Pseudomonas species, or the crown gall caused by the Agrobacterium species.
The plant illnesses caused by fungi for which the composition according to the invention is particularly useful for prevention and/or treatment are preferably selected from among the Fusarium wilt caused by the Fusarium species, the mildew caused by the Phytophtora species, the powdery mildew caused by the Podosphaera and Oidium species, the early blight caused by the Alternaria species, the sooty mold caused by the Alternaria and Cladosporium species, or the gray rot caused by the Botrytis species.
The pests causing plant illnesses against which the composition according to the invention is particularly useful for prevention and/or treatment are selected from among aphids, gnats, soil mites, or budworms.
The composition according to the invention can be used specifically for preventing and/or combatting the illnesses of fruits, vegetables, and/or flowers post-harvest caused by fungi, bacteria, viruses, nematodes, and/or pests. In particular, the composition according to the invention can be used for preventing and/or combatting the rot of citrus fruit caused by the Penicillium and Geotrichum species, or for preventing and/or combatting the fungal deterioration of dates caused by the Aspergillus species.
The composition according to the invention can also be used for an antifungal treatment during the coating of seeds, in particular seeds selected from among wheat, barley, lentils, chickpeas, and beans.
According to another aspect, the composition according to the invention can be used as a phytosanitary product for:
The composition according to the invention can also advantageously be used to stimulate the growth of plants, in particular to stimulate the rhizogenesis in plants.
Independently of its use in application on the vegetables, either as a phytosanitary product or for stimulating their growth, the composition according to the invention can be used directly on objects, soils, walls, etc., in particular for:
The composition according to the invention can also be used as a bath for decontaminating animal carcasses in slaughterhouses or for decontaminating poultry eggs before placing them in incubators.
According to another aspect, the composition according to the invention is particularly effective and can be used to disinfect reusable medical devices, in particular fibroscopes, coloscopes, bronchoscopes, and sinuscopes.
The composition can also be used:
The composition according to the invention can therefore be used for numerous applications. It offers the advantage of coming in solid and stable form, which makes possible a long-term storage of at least two years and an easy and economical transfer. In addition, the composition is very easily dispersible in water, which makes possible an easy and economical use in solution.
The invention is now illustrated by non-limiting examples of compositions according to the invention, of uses and test results demonstrating their effectiveness.
The composition of Example 1 consists of:
This composition is obtained by implementing the following steps:
15 g of thymol is preheated to 50° C. and mixed with 0.2 ml of soy lecithin, and then the mixture is added to the combination of 10 g of locust bean gum, 50.54 g of sodium bicarbonate, and 24.26 g of citric acid. All of it is suspended in 10 l of water.
The composition of Example 2 consists of:
This composition is obtained by implementing the following steps:
15 g of menthol is preheated to 50° C. and mixed with 0.2 ml of mineral oil, and then the mixture is added to the combination of 10 g of guar gum, 50.54 g of potassium bicarbonate, and 24.26 g of tartaric acid. All of it is suspended in 10 l of water.
The composition of Example 3 consists of:
This composition is obtained by implementing the following steps:
15 ml of carvacrol is preheated to 50° C. and mixed with 0.2 ml of glycerol, and then the mixture is added to the combination of 10 g of xanthan gum, 50.54 g of potassium bicarbonate, and 24.26 g of maleic acid. All of it is suspended in 10 l of water.
The composition of Example 4 consists of:
This composition is obtained by implementing the following steps:
15 ml of menthol is preheated to 50° C. and mixed with 0.2 ml of soy lecithin, and then the mixture is added to the combination of 10 g of locust bean gum, 50.54 g of sodium bicarbonate, and 24.26 g of tartaric acid. All of it is suspended in 10 l of water.
The aromatic alcohols do not disperse easily in water because of their hydrophobic nature. The prior art proposes a method for dispersion of these compounds by the addition of a surfactant and a solvent (WO2009124392A1), but this method is not suitable because it is known that the surfactants and the solvents reduce the activity of the phenolic compounds (Remmal, A., Bouchikhi, T., Tantaoui-Elaraki, A., Ettayebi, M. (1993). Inhibition of Antibacterial Activity of Essential Oils by Tween 80 and Ethanol in Liquid Medium. J. Pharm. Belg. 48: 352-356). In addition, certain aromatic alcohols, such as thymol or menthol, are in crystallized form at ambient temperature, and once in contact with water, if an attempt is made to make them liquid by heating them, they recrystallize.
The composition according to the invention combining the aromatic alcohols with a texturing agent, an emulsifying agent (and/or one oil), and an effervescent acid-base pair makes it possible at the same time to prevent the recrystallization of aromatic alcohols in crystallized form, to ensure the stability of aromatic alcohols and of the composition, its homogeneity, and to facilitate its dispersion during its solubilization.
The form and the dispersion in water of the composition of Example 1 was evaluated in comparison to other compositions comprising just an aromatic alcohol or with only one or more component(s) of the composition according to the invention.
The composition C1 consists of 15% thymol only.
This composition is obtained by the suspension of 15 g of thymol in 10 l of water.
The composition C2 that consists of:
This composition is obtained by implementing the following steps:
The composition C3 that consists of:
This composition is obtained by implementing the following steps:
The composition C4 that consists of:
This composition is obtained by implementing the following steps:
The results are presented in Table 1 and in the figures.
These results reveal that only the combination of the components of the composition according to the invention makes it possible to obtain a powder containing an aromatic alcohol that is stable, homogeneous, and easily dispersible.
For the purpose of evaluating the effectiveness of the composition according to the invention (Example 1): it was tested in vitro on several strains of bacteria, yeasts, and molds causing illnesses and damage in plants.
The 15 strains tested in this study were isolated, purified, and identified: 11 strains of fungal origin, including 5 of the genus Fusarium, one of the genus Penicillium, one of the genus Geotrichum, one of the genus Alternaria, one of the genus Sclerotonia, and the last of the genus Helminthosporium. Two strains of yeast origin, one of the genus Candida, and the other of the genus Saccharomyces. The two strains that remain are of bacterial origin.
The culture media used are:
The testing of the antifungal activity of the composition according to the invention was carried out as follows:
The testing of the antibacterial activity of the composition according to the invention was carried out by following the same approach as the one produced in the testing of the antifungal activity except that the culture medium used for this test is Mueller-Hinton (agar and broth); the inoculated bacterial inoculum is on the order of 107 bacteria/ml, and the incubation temperature is on the order of 37° C.
The values of CMI and CMF obtained with the composition according to the invention are summarized in Table 2.
Fusarium oxysporum spdianthi
Fusarium oxysporum spalbedinis
Fusarium oxysporum spgladioli
Fusarium oxysporum spcubense
Fusarium nival
Penicillium digitatum
Geotrichum candidum
Alternaria alternata
Sclerotinia homoeocarpa
Helminthosporium
Cladosporium
Candida albicans
Saccharomyces cerevisiae
According to these results, the composition has proven very effective against the fungal species used in this testing.
In addition, the CMI and CMB values obtained with the composition of Example 1 in the testing of the antifungal activity on the species of bacteria are presented in Table 3.
Escherichia coli
Staphylococcus aureus
These results reveal a very significant antibacterial activity of the composition according to the invention.
For the purpose of evaluating the effectiveness of the composition according to the invention of Example 1, it was tested in vitro or in vivo on several insects, parasites, and nematodes.
The adults of the Macrosiphum rosae species (rosebush aphid) were sampled using a fine brush and brought into contact with various doses of the composition according to the invention.
In the Petri dishes, we deposit an absorbent paper disk saturated with solutions containing increasing doses of the composition according to the invention (1.67; 3.33; 6.67, and 13.34 g/l). Petri dishes were used as controls containing regular water. 20 adults were put into each dish; the experiment was set up in triplicate.
Likewise, the in vivo effectiveness of the composition according to the invention (Example 1) was tested on the Rhizoglyphus callae species (soil mite) in their usual environment, which is the soil. To do this, flower pots (freesia) containing soil naturally infested with these creatures (brought in from a farm producing cut flowers) were irrigated with solutions containing increasing doses of the composition according to the invention (1.67; 3.33; 6.67, and 13.34 g/l). Pots being used as controls received only water as an irrigation solution; the experiment was repeated three times.
After several hours of contact with the irrigation solution, several grams of soil from each pot were sampled and suspended in water; observation and counting under the binocular magnifying glass were carried out for the purpose of determining the percentage of mortality for each dose used in this testing.
In addition, the composition according to the invention was also tested in vitro on nematodes and more specifically the Heterodera spp species starting from infected soil (previously treated with various concentrations of the composition of Example 1). The method used is that of Baerrman. It is a method that consists in separating the nematodes from soil particles depending on their sizes and their weights according to the following steps:
The dilutions that are used are:
1.67 g of the composition in 1 l of water
3.33 g of the composition in 1 l of water
6.67 g of the composition in 1 l of water
13.34 g of the composition in 1 l of water.
The Application Mode by Spraying
The mortality percentage of the insects used in this testing was calculated according to the following formula:
where Nt and Nm respectively represent the total number of insects and the number of dead insects.
The results are presented in Table 4.
Macrosiphum
rosae
Rhizoglyphus
callae
Heterodera spp
These results clearly reveal that the composition according to the invention generates a very powerful insecticide effect. The 6.67 g/l dose has proven mortal for aphids and nematodes while the 3.33 g/l dose is effective against soil mites, after several hours of contact only.
The composition according to the invention (Example 1) was tested in vivo on the vascular Fusarium wilt in carnations caused by Fusarium oxysporum sp dianthi. Eight seedlings per batch of the Martina variety were used in this testing.
The fusarium spores (106 spores/ml) were inoculated into the siliceous sand near the roots of each plant.
Five batches were set up:
PNINT: Untreated, non-infected plants.
PINT: Untreated, infected plants.
PIT 3.33 g/l: Infected plants treated with 3.3 g/l of the composition according to the invention.
PIT 6.67 g/l: Infected plants treated with 6.67 g/l of the composition according to the invention.
PIT 13.34 g/l: Infected plants treated with 13.34 g/l of the composition according to the invention.
To evaluate the effectiveness of the composition, the measurements of multiple agromorphological types were compared, in particular the number of shoots, the distance between nodes, the number of nodes, the length of the stem, the diameter of the stem, and the number of flower buds.
The dilutions used are:
3.33 g of the composition in 1 l of water.
6.67 g of the composition in 1 l of water.
13.34 g of the composition in 1 l of water.
The application mode is an application by irrigation.
The results that are obtained are presented in Table 6.
These results clearly reveal that the plants that are infected and treated by the composition according to the invention do much better than the non-infected plants; the dose that provided the better results in terms of agromophological types is 3.3 g/l.
Furthermore, the dose 13.34 g/l has proven to be too high for the carnations, since it generated a phytotoxic effect on the aerial part and even on the root system of the plant.
The importance of these results comes from the fact that the fungal load of the soil is a decisive factor for the health of the plant; actually, the higher this load is, the more the plant is stressed by the bacteria and the fungi that enter into competition with the plant on the essential nutrients for its growth.
The use of the composition in the irrigation water therefore makes it possible to reduce the fungal load of the soil. This will have a positive impact on the health and well-being of the young plant that develops.
The stimulating effect of the growth of the composition according to the invention (Example 1) was also tested on seedlings of carnations of West Diamond variety.
To do this, five batches were set up:
PNT: Non-treated plants.
PT 1 g/l: Plants treated with 1 g/l of the composition according to the invention.
PT 1.65 g/l: Plants treated with 1.65 g/l of the composition according to the invention.
PT 3.33 g/l: Plants treated with 3.33 g/l of the composition according to the invention.
PT 6.67 g/l: Treated with 6.67 g/l of the composition according to the invention.
The stimulating effect of the composition according to the invention (Example 1) was quantified by taking measurements of the agromorphological types (the number of shoots, the distance between nodes, the number of nodes, the length of the stem, the diameter of the stem, and the number of flower buds) after three months of treatment at a rate of three applications per week of irrigation water.
The dilutions used are:
1 g of the composition in 1 l of water.
1.67 g of the composition in 1 l of water.
3.3 g of the composition in 1 l of water.
6.67 g of the composition in 1 l of water.
The application method is by irrigation.
The results that are obtained are presented in Table 7.
These results reveal a convincing effect of the composition according to the invention; the dose 3.3 g/l has proven most effective since the plants that have received this dose of irrigation water have very high-performing agromorphological natures compared to the control plants.
The stimulating effect of the composition according to the invention (Example 1) was tested on date palm seedlings obtained from the germination of the stones of dates of the Medjool variety. Seedlings in leaf stage received different doses of the composition of Example 1 according to the invention (0; 1.65; 3.33; 6.67 g/l). After 4 months of treatment (at a rate of twice per week), the seedlings were harvested, and the wet and dry weights of the root portion of each seedling were determined.
The dilutions used are:
1.65 g of the composition in 1 l of water
3.3 g of the composition in 1 l of water
6.67 g of the composition in 1 l of water
The application mode is by irrigation.
The results are presented in Table 8.
These results reveal the stimulating effect of the rhizogenesis of the composition according to the invention in date palm seedlings; the two doses 1.65 and 3.33 g/l have proven the most effective, with the dose 1.65 g/l being superior compared to the control seedlings that have received only water throughout the experiment. Further, the dose 6.67 g/l has proven phytotoxic in the cells of the root system of the young seedlings.
So as to verify the feasibility of the use of the composition according to the invention on the post-harvest preservation of dates, testing was carried out. This testing consisted in soaking the dates for 10 minutes in solutions containing various concentrations of the composition of Example 1 (0; 3.33, and 6.6 g/l). Next, the dates were rinsed with water, and then dried before being stored in hermetically-sealed plastic boxes. For the purpose of evaluating the bacterial load (FMAT) and the fungal load of the treated dates, sampling by swabbing eight dates from each box was carried out. The sampling surface is 1 cm2 per date.
The dilutions used are:
3.33 g of the composition in 1 l of water
6.6 g of the composition in 1 l of water
The application mode is by soaking.
The results obtained are presented in Tables 9 and 10.
Tables 9 and 10 respectively present the results of the bacterial load and the fungal load contained in the treated dates and those that are not treated. The dose 6.67 g/1l has proven the most effective since the soaked dates at this dose are free of bacteria and fungi that can interfere with their preservation for a month and at ambient temperature.
The composition according to the invention (Example 1) was also tested on the vase life of cut flowers and more specifically of carnations. To do this, the carnation flowers were cut to equal lengths of 55 cm before being put to soak in solutions containing two concentrations of the composition according to the invention (1.6 and 3.3 g/l) immediately after harvest.
The measurements taken into consideration for evaluating the effect of the composition according to the invention are:
The dilutions used are:
1.6 g of the composition in 1 l of water
3.3 g of the composition in 1 l of water
The application mode is as follows: putting into a vase.
The results obtained are presented in Tables 11 and 12.
These results reveal that the composition according to the invention improves the vase life of the cut flowers.
The effectiveness of the composition according to the invention (Example 1) was tested on the microbial load (bacteria, yeasts and molds, parasites) of the water used in watering poultry. The sample is moved to the laboratory and preserved wet for the in vitro test.
The culture media used are:
The testing of the antibacterial activity of the composition according to the invention was carried out as follows:
The testing of the antifungal activity of the composition according to the invention was carried out by following the same protocol as that produced in the testing of the antibacterial activity except that the incubation temperature is on the order of 27° C., and the incubation time is from 3 to 5 days.
Regarding the testing of the antiparasitic activity, a volume of 10 μl of a water sample was placed between a thin glass strip and the Malassez cell. The number of cells in 10 rectangles was then counted.
The results obtained are presented in Table 13.
Salmonella
Staphylococci
These results reveal that the treatment of the surface water by the composition brings about a significant reduction in the microbial load. This reduction increases with the concentration of the composition.
The composition according to the invention was also tested in vivo on chicks. Twelve chicks per batch were used in this testing. During the entire period of the testing, the temperature of the animal house was adjusted to 28° C., and the animals had free access to water and to the feed that was continuously available.
The water used in this testing is the surface water sampled from the reservoir for distribution of watering water that was used in the in vitro testing.
The objectives of this testing are:
The distribution of the batches is as follows:
To evaluate the effectiveness of the composition, the measurements of multiple zootechnical types during the following three breeding phases were compared:
Mean live weight (g)=weight of all of the chicks of one batch/number of chicks of this batch
Increase in Live Weight (g)=P2−P1
The results obtained are presented in Table 14.
These results reveal that the animals treated by the composition according to the invention do better than the untreated animals; the dose that provided the best results in terms of zootechnical types is 2 g/1.
The effect of the composition according to the invention (Example 1) was tested on the reduction of the intestinal load (revivable aerobic bacteria and the parasite load) of the broiler chicks for 35 days. Every 7 days, the samples of droppings from various batches were sampled and solubilized in physiological water (1 g of droppings in 9 ml of physiological water).
Dilutions are then prepared from the stock solution.
The culture medium that is used is:
Regarding the parasite load, a volume of 10 μl of the sample is placed between a thin glass strip and the Malassez cell. The number of cells in 10 rectangles is then counted. The results are presented in Tables 16 and 17.
The results that are obtained clearly reveal that the animals that are treated by the composition according to the invention have a lower intestinal load in relation to untreated animals; the dose that provided the best results in terms of the reduction of the intestinal load is 2 g/l.
Comparison Study Between the Antibacterial Action of the Composition According to the Invention and Glutaraldehyde Against E. coli In Vitro.
In microplates with 96 wells each containing 130 μl of sterile Muiller-Hinton stock (autoclaving for 15 minutes at 110° C.), a variable volume of the stock solution of glutaraldehyde [1/10 (100 mg/ml), 1/100 (10 mg/ml), 1/250 (4 mg/ml), 1/500 (2 mg/ml), 1/1,000 (1 mg/ml), 1/2,000 (0.5 mg/ml), 1/4,000 (0.25 mg/ml), 1/8,000 (0.125 mg/ml), and 1/16,000 (0.062 mg/ml)] and of the composition according to the invention of Example 1 [1/10 (133.4 mg/ml), 1/100 (66.7 mg/ml), 1/250 (26.68 mg/ml), 1/500 (13.34 mg/ml), 1/1,000 (6.67 mg/ml), 1/2,000 (3.33 mg/ml), 1/4,000 (1.66 mg/ml), 1/8,000 (0.83 mg/ml), and 1/16,000 (0.42 mg/ml)] was added so as to obtain the final concentrations for each disinfectant. After, 20 μl of the inoculum was added into each well. Positive and negative controls were also prepared. The plates are then incubated at 37° C. for 24 hours. The bacterial growth was tracked by the optical density using the spectrophotometer with a 600-nm wavelength.
The results are presented in Table 18.
These results present the effect of glutaraldehyde and the preparation of the HE at various concentrations on an E. coli suspension on the order of 108 UFC/ml. They reveal that the composition according to the invention is more effective than the disinfectant (glutaraldehyde). The inhibiting minimal concentration (CMI) is 1/100 for the composition according to the invention, whereas for the glutaraldehyde, the CMI is obtained only with the pure disinfectant. A more significant partial inhibition is noted for the 1/250 to 1/1,000 concentrations of the composition according to the invention in relation to glutaraldehyde.
So as to compare the antibacterial action of the composition according to the invention and of glutaraldehyde, the following steps were carried out:
The results obtained are presented in Table 19.
The results obtained revealed the presence of a layer of bacteria before disinfection; the same result was obtained after the disinfection by glutaraldehyde, whereas after the disinfection by the composition according to the invention, the bacterial load was only 50 UFC/ml with the 1/1,000 concentration and zero with the 1/500 concentration.
Further, these results reveal a significant reduction of the microbial load of the fibroscope after the disinfection by the composition according to the invention. This shows the advantage of using the composition according to the invention as an alternative treatment to glutaraldehyde.
Drenching and Waxing Treatment of Clementines (“Afourar” Variety). Fruits Experimentally Infected by Injection of Spores
24 hours after the treatment, each piece of fruit of the six batches was infected by injection of a volume of 100 μl of a suspension of spores of the Penicillium digitatum strain (106 spores/ml), using a syringe equipped with a very fine needle (30-gauge), inclined tangential to the albedo surface. The injection point is marked by a circle traced with an indelible marker. The fruits were then incubated at a temperature of 20° C.
Daily monitoring of the six batches was carried out, and the appearance as well as the development of rot at the injection points was noted for the fruits of each batch.
The results obtained reveal that rot begins starting from the third day after infection in the batch washed with the chemical fungicides, whereas in the batch washed with the composition according to the invention, rot begins only after four days. The results also reveal that the rot in the batch washed with the chemical fungicides develops in a faster way with a diameter of 3.2 cm±0.7 after 7 days, in relation to the batch washed with the composition according to the invention where the diameter of the rot reaches only 2 cm±0.6 after 7 days. The results also reveal a delay in the appearance of spores in the batch treated with the composition according to the invention in comparison with the batch treated with chemical fungicides.
For the batches 3 and 4, rot begins starting from the fourth day after infection in the batch treated with the mixture of the wax plus the composition of the invention and the batch treated with the mixture of the wax plus imazalil. However, a faster development of rot was noted in the batch treated with the mixture of wax and imazalil (2.55 cm±0.3 in diameter after 7 days), in relation to the development of rot in the batch treated with the mixture of wax and the composition according to the invention (1.66 cm±0.3 in diameter after 7 days).
For the batches 5 and 6: Rot in the batch that is washed and waxed with the preparation of the invention (1.2 cm±0.4 in diameter after 7 days) develops sparingly in relation to the batch that is washed and waxed with chemical fungicides (2.07 cm±0.5 in diameter after 7 days). Similar results were obtained for the “Maroc Late” variety.
Therefore, the preparation of the invention makes possible a better protection than the chemical fungicides when it is used for washing and waxing fruits that are experimentally infected with an extreme number of spores located in the injection point.
Drenching and Waxing Treatment of Clementines (“Afourar” Variety). Non-Infected, Experimentally Damaged Fruits
Three batches of 10 fruits of the “Afourar” variety were prepared:
Two equidistant holes (diameter 1 mm+depth 2 mm) that pass through the flavedo and the albedo and that extend to the pulp were made on each piece of fruit. Next, these fruits were preserved in hermetically-sealed boxes and saturated with moisture. The boxes were incubated at a temperature of 27° C.
The percentage of the surface of rotted fruits at the holes was noted for the fruits of each batch.
The results are presented in Table 20.
The results reveal a very significant reduction in the percentage of rotten surface in fruits treated with the preparation of the invention in relation to the control fruits treated with water and just wax. This reduction is also noteworthy in relation to the fruits treated with chemical fungicides.
These results reveal that the composition according to the invention makes possible a preservation of the fruits for 12 days without rot whereas for the batch treated with chemical fungicides, rot appeared on the 6th day. For the batch without treatment, rot appeared on the 4th day.
Treatment by Soaking Clementines, Directly on Site at a Citrus Fruit Farm
Field tests were carried out at a citrus fruit farm. In these tests, were used a batch of four boxes of fruits of the “Afourar” variety that were soaked immediately after being picked up in the water containing the preparation of the invention at the dose of 1 g per ton, another batch of 4 boxes of fruit soaked immediately after being picked up in just water. 30 fruits were sampled from each batch, and the fungal load of each piece of fruit was evaluated by the number of spores (units forming a colony) per square centimeter of the surface of the fruit.
The results obtained reveal that the fungal load of the fruits soaked in a solution with the composition according to the invention is on the order of 105 spores per cm2, smaller than the fungal load of the fruits soaked in the water itself that reaches 108 spores per cm2. These results reveal that the fruits soaked in the preparation of the invention will arrive at the packing station with a more reduced initial load (1,000 times smaller) in relation to that of the fruits soaked in just water.
Drenching and Waxing on Infected Clementines, Tests in a Packing Station
Field tests were carried out in a station for packing citrus fruit. In these tests, a batch of 6 tons of fruits of the “Afourar” variety was washed in the drench chamber with a paste that contains water and the preparation of the invention at a dose of 3 kg per ton; next, in the waxing step, this same batch was waxed with a mixture of wax and the preparation of the invention at a dose of 5 kg per ton. Another batch of 6 tons of fruit of the “Afourar” variety was washed in the drenching step with a paste consisting of chemical fungicide (imazalil at a dose of 500 cc/hl and orthophenylphenol at a dose of 750 c/hl), and next waxed with the mixture of the wax and the imazalil at a dose of 3,000 ppm. 30 fruits from each batch were sampled after the drenching step and 30 fruits after the waxing step so as to evaluate their fungal load.
The results are presented in Table 21.
The results obtained reveal that there is a significant difference in the fungal load of fruits treated with the composition according to the invention, which is 1,000 times smaller than that of the fruits treated with the chemical fungicides after the drenching step and after the waxing step.
Tracking of the Rot Rate of Clementines in a Packing Station
Field tests were performed in a citrus fruit packing station. In these tests, a first batch of 6 tons (imazalil batch) of fruits of the “Afourar” variety was washed with a paste consisting of chemical fungicides (imazalil at a dose of 500 cc/hl and orthophenylphenol at a dose of 750 cc/hl) in the drenching step, and waxed with a mixture of the wax and imazalil at a dose of 300 ppm in the waxing step. A second batch of 6 tons of fruits of the “Afourar” variety (composition batch) was washed with a paste containing water and the composition according to the invention at the dose of 3 kg per ton, and waxed with a mixture of wax and the composition according to the invention at the dose of 5 kg per ton. After being put into boxes, two sub-batches of 15 boxes were sampled from each batch and distributed as follows:
The sub-batches were monitored once per week for a month. A count of rotten fruits in each batch makes it possible to detect the change in the rate of rotting depending on the storage temperature and the treatment used.
The results are presented in Table 22.
The results obtained reveal that the rate of rotting in the sub-batches preserved at a temperature of 25° C. is higher in comparison with the sub-batches preserved at a temperature of 4° C. The results also reveal that the fruits treated with the composition according to the invention have a rate of rotting of between 0.5% and 2.82% at a temperature of 4° C., and between 4.5% and 6.91% at 25° C.; this rate is lower in relation to the rate of rotting in fruits treated with the chemical fungicides, which varies between 1.35% and 4.67% at a storage temperature of 4° C., and between 4.62% and 9.68% at a temperature of 25° C.
Treatment of Ambient Air in a Clementine Packing Station
Tests were carried out in a citrus fruit packing station. In these tests, the ambient air from various zones of the station was treated by spraying the preparation of the invention at a dose of 10 kg per ton of water twice per day; between 12 hours and 13 hours and at 18 hours. Air samples (Petri dish with open PDA medium for 5 minutes) were taken so as to evaluate the fungal load of the ambient air of each zone of the station before and after treatment and this four times per day:
The results obtained are presented in Table 23.
The results reveal that the fungal load of the air from the various zones was very low at the beginning of the day due to the treatment of the air with the preparation of the invention carried out at the end of the preceding day. During the day, this load gradually increases up until noon or a spraying of the preparation of the invention causes a significant reduction of this load. From the afternoon until the end of the day, a slight increase in the fungal load of the ambient air from various zones was noted which will be decreased by a second spraying of the preparation of the invention. This approach makes it possible to pack the fruits without the risk of contamination by the microflora of the air.
Evaluation of the Effect of the Composition According to the Invention in the Laboratory
A strain of penicillium digitatum, a strain of penicillium italicum, and a strain of geotrichum candidum were isolated starting from rotten oranges, and then purified and identified.
One drop of 10 μl of a stock suspension of 106 spores/ml of each of 3 strains was cultivated at the surface of the Petri dishes of the Sabouraud medium with chloramphenicol containing various concentrations of the composition according to the invention. The rate of growth of the three strains is tracked daily by measuring the diameter of the colonies.
The results obtained are presented in Table 24.
These results reveal that the composition according to the invention exerts a very strong partial inhibition on the three strains studied with the 0.75 g/l concentration that corresponds to a treatment with 0.75 kg per ton of the composition. Starting from the 1.25 concentration, total inhibition is noted on the three strains studied.
These results clearly reveal that the composition according to the invention has a very high level of effectiveness against the primary fungi responsible for the rotting of citrus fruit post-harvest.
Evaluation of the Effect of the Composition According to the Invention on Cherry Tomatoes Post-Harvest
Two batches of 120 cherry tomatoes of the Angel D1A1 variety were treated with two different preparations:
After washing, each batch was divided into two sub-batches of 60 cherry tomatoes:
The tomatoes of each sub-batch were tracked daily, and the percentage of the dehydrated tomatoes was noted.
The results are presented in Table 25.
The results obtained reveal that the percentage of dehydrated tomatoes in sub-batches preserved at a temperature of 25° C. is higher in comparison with the sub-batches preserved at a temperature of 8° C. The results also reveal that the batch treated with the composition according to the invention has a smaller percentage of dehydrated tomatoes than the number of dehydrated tomatoes in the batch treated with chlorine.
Another test was carried out. Four batches of 20 cherry tomatoes of the Angel D1A1 variety were treated with three different preparations:
After treatment, the tomatoes from each batch were damaged at the stem with a fine syringe (22 G×1/4″) and preserved at a temperature of 27° C.
The tomatoes were monitored daily, and the percentage of tomatoes that have stem rot was noted.
The results are presented in Table 26.
The results reveal a very significant reduction in the percentage of rotten tomatoes in the fruits washed with the composition of the invention in relation to the control fruits washed with water. This reduction is also noteworthy in relation to the fruits washed with chlorine.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16/70691 | Nov 2016 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/057198 | 11/17/2017 | WO | 00 |
