Device for stored products protection and uses thereof

Abstract

The present disclosure relates to a method for protecting stored agricultural dry products during storage, consisting of a controlled release system of essential oils and a shell for allowing oil diffusion in vapour phase towards the outer atmosphere without direct contact of the support material with stored products. This system is characterized by being suitable for use within containers for protecting stored dried agricultural plant products for a long period of time, in particular more than 4 weeks, more preferably more than 8 or 12 weeks.

Claims

1. A method of enhancing long-term protection of an agricultural product, comprising: storing the agricultural product in a dried form; and exposing the stored agricultural product to an essential oil, wherein the essential oil includes both eugenol and pulegone, wherein the agricultural product is stored for at least four weeks.

2. The method of claim 1, wherein the agricultural product is stored for at least eight weeks.

3. The method of claim 1, wherein the combination of the eugenol and the pulegone have a concentration of at least 40 weight percent of the essential oil.

4. The method of claim 1, wherein the stored dried agricultural product comprises at least one of a cereal, a pulse, and a nut.

5. The method of claim 1, wherein the stored dried agricultural product comprises at least one of rice, a bean, maize, rye, whey, wheat, and a nut.

6. The method of claim 1, wherein the essential oil includes at least one of a stabilizer, an antioxidant, and a food dye.

7. The method of claim 6, wherein an antioxidant is added to the essential oil and the antioxidant added includes at least one of butylated hydroxytoluene, ascorbyl palmitate, calcium disodium ethylenediamine-tetraacetate, sorbic acid, 4-Hydroxymethyl-2,6-di-tert-butylphenol, monoglyceride citrate, 2,4,5 trihydroxybutyrophenone; tertiary butylhydroquinone, and 2,4,5-Trihydroxybutyrophenone.

8. The method of claim 1, wherein the essential oil is derived from a plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of disclosure.

(2) FIG. 1Schematic representation of containers with the device now disclosed, wherein 1 represents a 65 L high density polyethylene container, in particular with 63 cm height and 36.5 cm in diameter; 2 represents a probe in the case of fungal and insecticidal study, in particular with a 3 cm diameter and 13 cm length, and the sampling point in the case of the study of the propagation profiles, in particular 9 mm in diameter and 16 cm length; 3 represents a protection device, in particular with 7 cm diameter and 3.3 cm height; 3a represents a brass metal structure of the outer shell and 3b represents a metallic net stainless-steel outer shell.

(3) FIG. 2Represents the effect of 3.5 months treatments on grain maize germination: (A) in vitro after 4 days and (B) in vivo 17 days after sowing (DAS).

(4) FIG. 3Represents the effects of treatments on some physiological parameters: Anet photosynthesis, Btotal chlorophyll, Cstomatal conductance and Dtranspiration rate.

(5) FIG. 4Represents the effects of treatments with essential oils on some plant growth parameters.

(6) FIG. 5Represents the time course of germination capacity and grain moisture during 50, 93 and 162 days of storage in the 2 containers with 1 (C1) and 3 (C3) essential oils released devices.

(7) FIG. 6Represents the maize mass variation towards the main active components of essential oil (MAC)eugenol and pulegone, or their mixtureand essential oils vapours along 35 days of exposure.

DETAILED DESCRIPTION

(8) The present disclosure relates to a method for controlling insects and microorganisms in stored products consisting of a controlled release system of plant essential oils combining the encapsulation of essential oils in biocompatible microstructures, a physical support/biocompatible screen for fixing/impregnating the microparticles containing the essential oils and a device for containment the structure impregnated with the essential oils. This system is characterized by being suitable for use in storage containers and under confined environment. This system is suitable for long time period storage of stored products.

(9) The present disclosure relates to a device, in particular a delivery device to be placed within a container for stored products. This device explores the insecticidal and fungicidal properties of essential oils using sustained release devices in a confined environment.

(10) This disclosure is based on the propagation property of essential oils vapours inside storage containers for stored products in an amount sufficient to reach vapour stabilization, ensure sterility of the environment, without altering the quality of stored products throughout the storage period.

(11) Therefore, this disclosure relates to the design of a protection system for long period stored products, preferably dried foods, more preferably grains, the most preferably dried cereal grains and pulses.

(12) In an embodiment, this protective device involves the use of plant essential oils evidencing protection against insets and fungi.

(13) In an embodiment, the protection system of the present disclosure allows the use of one, two, or even more essential oils combined in order to improve the efficacy of the protection system or to control one or more undesired pests.

(14) In an embodiment, this disclosure also includes the design of the delivering device for the sustained release of the essential oils in closed containers of medium volume with a dimension superior to 60 L, preferably from 60 to 300 L, more preferably from 60 to 150 L, even more preferably 60 to 100 L of capacity.

(15) In an embodiment, the device described herein supports the material containing the oil or oil mixture in its interior and allows the subsequent propagation of the oil inside the container by diffusion in the gaseous atmosphere present in the interstitial spaces of the stored product. The oil concentration in the gaseous atmosphere increases till reaching the saturation point correspondent to pressure and temperature conditions inside the container.

(16) In an embodiment of the present disclosure, essential oils from aromatic plants containing high contents of eugenol and/or pulegone may be used.

(17) In an embodiment, the essential oils of Syzygium aromaticum (dove) and Mentha pulegium (pennyroyal) are preferred (Table 1). Still preferably the compounds eugenol and pulegone can also be used.

(18) In an embodiment, dove and pennyroyal essential oils were selected based on their pesticide potential and their environmental safeness.

(19) TABLE-US-00001 TABLE 1 Chemical composition (%) of Pennyroyal (Mentha pulegium) and Clove (Syzygium aromaticum) obtained by GC-MS % Components Components Mentha pulegium Syzygium aromaticum 3-Methyl cyclohexanone 0.3 -Pinene 0.5 1,8-Cineole 0.1 Sabinene 0.1 -Pinene 0.4 3-Octanol 0.8 Limonene 0.9 Terpinolene 0.1 Menthone 0.3 p-Menth-3-en-8-ol* 0.8 Isomenthone 0.2 Cis-Isopulegone 2.5 Pulegone 86 Piperitenone 2.2 B-Caryophyllene 0.7 13.4 -Humulene 1.1 1.5 -Caryophyllene oxide 0.1 0.4 Humulene epoxide* 0.2 0.1 Methyl salicylate 0.1 Eugenol 78.1 -Cubebene 0.1 -Copaene 0.3 Eugenol acetate 5.2 trans,trans--Farnesene 0.1 trans-Calamenene 0.1 -Cadinene 0.1 *Identification based on Mass Spectra only

(20) In an embodiment, additional essential oils from other plants can be used, in particular provided that in its composition more than 40% (w/w, mass of active component mass of essential oil) of pulegone and/or eugenol are present.

(21) In an embodiment, pulegone and eugenol commercially available can be used.

(22) Tables 1 and Table 2, summarize the richness in pulegone and eugenol, respectively, of some plant essential oils (Essential Oil DataBase of National Institute of Plant Genome Research http://nipgr.res.in/Essoildb/index.html), that can be use in the present disclosure.

(23) TABLE-US-00002 TABLE 2 Plant essential oil rich in pulegone. Content of pulegone (%) Plant species Plant part (w.sub.pulegone/w.sub.total oil composition) Acinos suaveolens aerial part 67.70 Calamintha nepeta ssp. aerial part 75.50 var. subisodonda Calamintha nepeta aerial part 41.00 Cunila angustifolia leaf 56.50-72.30 Hedeoma mandoniana aerial part 43.20 Hedeoma multiflorum aerial part 66.00 Hedeoma multiflorum aerial part 62.10 Micromeria thymifolia aerial part 50.40 Minthostachys leaf and 63.00 verticillata stem Mintosthachys mollis leaf 42.80 Satureja abyssinica aerial part 43.50 Satureja brownei flower 54.63 Satureja odora leaf and 41.60 flower Ziziphora clinopodioides aerial part 45.80 ssp. rigida

(24) TABLE-US-00003 TABLE 3 Plant essential oil rich in eugenol. Content of eugenol (%) Plant species Plant part (w.sub.eugenol/w.sub.total oil composition) |Hyptis recurvata leaf 68.80 Marrubium vulgare aerial part 2.10-50.10 Ocimum basilicum leaf 9.49-41.20 Ocimum basilicum herb 2.90-43.20 Ocimum gratissimum leaf 75.40 chemovar. l Ocimum gratissimum apical part 52.80-63.60 of branches Ocimum gratissimum leaf 68.81 Ocimum micranthum leaf 46.55 Ocimum sanctum leaf 53.40 Origanum micranthum leaf 72.90-90.40 Pimento dioica leaf 45.40-83.68

(25) Several bioassays were conducted. The biological assays, both in vitro and in vivo, were performed in order to confirm the fungicidal and insecticidal potential of the essential oils obtained from clove and pennyroyal and that of their major compounds eugenol and pulegone, respectively.

(26) Limonene, used as an insecticide and insect repellent, found in many oils and fruits including orange, lemon, grapefruit, berry, leaf, caraway, dill, bergamot, peppermint and spearmint oils was also included in the present disclosure just as reference. The Federal and Drug Administration determined that limonene products, labelled and used as specified in this Registration Eligibility Decision, will not pose unreasonable risks or adverse effects to humans or the environment.

(27) The fungi studied were Aspergillus candidus, A. niger, Fusarium culmorum and Penicillium islandicum. They were obtained from the IICT Mycology laboratory.

(28) The insects studied were Sitophilus zeamais and Acanthoscelides obtectus, obtained from the IICT Entomological laboratory.

(29) Maize and bean samples were not sprayed with any pesticide.

Example 1Antifungal Activity Tests

(30) The screening of S. aromaticum and M. pulegium essential oils and their major components, eugenol and pulegone, respectively, was carried out using a direct plating technique in order to evaluate their ability to inhibit the growth of Aspergillus candidus, A. niger, Penicillium islandicum and Fusarium culmorum.

(31) In vitro assays. For the determination of direct plating effect of essential oil on the growth of the fungi on different concentrations 0.5, 1.0, 2.5 and 5.0 L/mL of essential oil were put on the surface of a Petri dish with 20 mL of potato dextrose agar medium (PDA). These Petri dishes were inoculated with a 5 mm diameter disk of fungi grown on PDA medium for 8 days at 28 C. This disk was placed on the agar surface and the dish sealed immediately with parafilm to prevent loss of essential oil to the atmosphere and incubated at 28 C. Inhibition concentrations of essential oil were determined by periodic evaluation of fungi growth during 25 weeks. For each concentration, four replicate dishes were used.

(32) For the determination of volatile phase effect of essential oil on the growth of the fungi different concentrations 1.0, 2.5 and 5.0 L/mL of essential oil were added to sterile filter papers (10 mm diameter, Whatman No. 1 and placed adherent to the inner surface of each Petri dishes lid (90 mm)). Petri dishes were inoculated with a 5 mm diameter disk of fungi grown on PDA medium for 8 days at 28 C. This disk was placed on the agar surface and the dish sealed immediately with parafilm and incubated at 28 C. Inhibition concentrations of volatile phase of essential oils were determined by a periodic evaluation the fungi growth over 25 weeks. In the control, equal amounts of sterilized water was added to filter papers and placed onto the lid of Petri dishes. For each concentration, four replicate Petri dishes were used. The absence of growth was recorded during 25 weeks. The results are show in table 4.

(33) TABLE-US-00004 TABLE 4 Increased period of storage without any fungal contamination using two techniques of essential oils application: direct plating and in vitro under saturated atmospheres (vapour phase) Time (weeks) Conc. Aspergillus Aspergillus Fusarium Penicillium EO/MAC L/mL) candidus niger culmorum islandicum Syzygium Direct plating Control 1 1 1 1 aromaticum 0.5 25 25 25 25 1.0 25 25 25 25 2.5 25 25 25 25 5.0 25 25 25 25 Vapour phase Control 1 1 1 1 0.5 23 24 25 25 1.0 25 25 25 25 2.5 25 25 25 25 5.0 25 25 25 25 Mentha Direct plating Control 1 1 1 1 pulegium 1.0 10 11 25 25 2.5 25 15 25 25 5.0 25 25 25 25 Vapour phase Control 1 1 1 1 1.0 7 6 25 25 2.5 15 13 25 25 5.0 25 25 25 25 Eugenol Direct plating Control 1 0.5 25 25 25 25 1.0 25 25 25 25 2.5 25 25 25 25 5.0 25 25 25 25 Vapour phase Control 1 1 1 1 0.5 21 23 25 25 1.0 25 25 25 25 2.5 25 25 25 25 5.0 25 25 25 25

(34) 1.2 In vivo tests. The efficacy screening of the vapour of clove and pennyroyal essential oils (OE) and their main active components (MAC) eugenol, pulegone or their mixture was, carried out using a modified technique describe in Bluma, R., Landa, M. F., Etcheverry, M., 2009. Impact of volatile compounds generated by essential oils on Aspergillus section Flavi growth parameters and aflatoxin accumulation. J. Sci. Food Agric. 89 (9), 1473-1480. For the determination of volatile phase effect of essential oil on fungi growth different concentrations of essential oil were added to sterile filter papers (10 mm diameter, Whatman No. 1), namely, 0.5 L/mL, 1.0 L/mL, 2.5 L/mL and 5.0 L/mL and placed adherent to the inner surface of each Petri dishes lid (90 mm).

(35) The grains were disinfected at surface as describe by Pitt & Hocking (Pitt, J. I., Hocking, A. D., 2009. Fungi and food spoilage. Springer, New York).

(36) Ten dried grains were placed on Petri dishes with 20 mL of PDA medium with chloramphenicol (1%). These dishes were sealed immediately with parafilm and incubated at 28 C. Inhibition concentrations of volatile phase of essential oils were determined by periodic observation of fungi growth over 25 weeks. Control with no treated grains was also performed. For each concentration ten replicates were done.

(37) TABLE-US-00005 TABLE 5 Increased period of storage without any fungal contamination tested in vivo under satured atmospheres (vapour phase). Time (weeks) Conc. Syzigium Mentha (L/mL) aromaticum Eugenol pulegium Pulegone Eugenol:Pulegone Vapour phase Control 1 1 1 1 0.5 2 2 2 2 2.5 5 25 20 20 5.0 25 25 25 25 Control 1 0.25:0.25 1 0.50:0.50 10 0.25:0.75 3 0.75:0.25 25

Example 2Insecticidal Activity Tests

(38) Maize grain with an average moisture content of 140.5% and ten unsexed adult insects aged from 1 to 4 days were used. The stock cultures of insects and the biological tests were carried out in a single incubator at 27 C. and 755% relative humidity.

(39) Assay Procedures

(40) Different concentrations of essential oil were added to sterile filter papers, for the determination of volatile phase effect of essential oil on insect growth (20 mm diameter, Whatman No. 1), 18.8 L/mL of S. aromaticum and pulegone (for maize) and 7.5 L/mL of eugenol, pulegone, limonene and 5.6:5.6 L/mL for the mixtures eugenol:pulegone and eugenol:limonene (for beans), and placed adherent to the inner surface of each plastic jars of 25 mL; 10 g of stored product (maize or bean) and 10 unsexed adult insects aged from 1 to 4 days old were introduced in the plastic jars, which were hermetically sealed with parafilm. Control with non-treated samples was also performed. Ten replicates per treatment and control were set up. All the replicates were kept in an incubator at 27 C. and 75%5% relative humidity.

(41) The progeny (F1) was evaluated monthly by common procedures after removing the parent adults. Insect mortality was assessed during five months (0.25, 1, 8, 134 and 149 days). The development index and the life cycle were also evaluated.

(42) TABLE-US-00006 TABLE 6 Increased the period of storage without any insect contamination tested in vitro under saturated atmospheres (vapour phase). Days Stored Conc. 0.25 1 8 134 149 Insect product (L/mL) Insect Mortality (%) Sitophilus Maize Control 0 0 0 0 0 zeamais Syzigium aromaticum 18.8 23 28 92 100 100 Pulegone 18.8 46 100 100 100 100 Acanthoscelides Bean Control 0 29 100 100 100 obtectus Eugenol 7.5 0 70 100 100 100 Pulegone 7.5 0 100 100 100 100 Limonene 7.5 0 100 100 100 100 Eugenol:Pulegone 5.6:5.6 0 20 100 100 100 Eugenol:Limonene 5.6:5.6 0 100 100 100 100

(43) Pulegone efficacy was very high for S. zeamais and A. obtectus, after one day, 100% mortality for concentrations of 18.8 e 7.5. L/mL, respectively.

Example 3Effects of Treatments on the Germination and the Seedling of the Reference Stored Products

Example 3.1In Vitro Germination

(44) In an embodiment, for the in vitro germination assay were used 10 seeds and 5 ml of sterile water per petri dish and the incubation temperature was 25 C. The treatments related to methods of the present disclosure do not induce any significant change on seed germination capacity. In the other hand, when limonene was used a significant decrease on germination was observed (FIG. 2A).

Example 3.2In Vivo Effects

(45) In an embodiment, the assay was performed in pots in a greenhouse with environmental monitoring (temperature, humidity and PAR intensity) and controlled irrigation. For each treatment, 6 pots with 2 plants each were used. Plants produced from treated seeds did not show significant differences on the life cycle in relation to the control without any treatment. No relevant differences in phenotypic (germination, chlorophyll and height) and physiological parameters (photosynthesis, stomatal conductance and transpiration (FIG. 3) were found.

(46) Treatment with eugenol showed a similar result on germination. All the other modalities induced a slight decrease, except limonene, used as reference that really affected the germination (49.1%).

(47) Plants obtained from grains treated with limonene were the only ones with a small decrease (1.9%) in total chlorophyll content related to the control. All plants from the seeds submitted to treatments with the bioactive products here proposed, showed good growth parameters and good usefulness for the object of present disclosure.

(48) In what concerns to the height of plants (FIG. 4A), only the plants from seeds treated with limonene showed a small variation relatively to the control. The number of cobs (4B) obtained shows that the plants productivity was not affected by any treatment.

Example 4Real Scale Tests

(49) In an embodiment, the mechanism of action and efficiency behind the disclosure was supported by conducting three studies in simultaneously: evaluation of the fungicidal and insecticidal activity performance; evaluation of the treated seed germination; main components vapour propagation profile measurement and monitoring in an ecosystem/full scale storage situation.

(50) The ecosystem considered for this demonstration reproduced the maize storage during 4/5 months at 25 C. in a high-density polyethylene container with 65 L. The device (s) was packaged into the grains bed during container filling with the dried product stored.

Example 4.1Fungicidal and Insecticidal Activity

(51) In an embodiment, for the evaluation of essential oils insecticidal and fungicidal activity in stored grain protection and germination 2 hypotheses were considered, using 1 or 3 release devices in each container (FIG. 1) plus one container without device (control).

(52) The device is composed by a metal shell with a porous stainless steel mesh supported in a cylindrical brass structure (FIG. 1). Two cotton semicircles were impregnated individually with 12 mL of clove oil and the other with 12 mL of mint-pennyroyal oil and placed in the internal shell.

(53) Each container had 6 side probes (distributed according to FIG. 1), used to evaluate the essential oils insecticidal activity in the evolution of insects S. zeamais life cycle. 36 days after the grain storage with the release devices containing OE, insects, 10 unsexed adults aged from 1 to 4 days old, were placed in these probes together with 10 g of maize. Independently the number of release devices present in the containers (1 or 3), an insect average mortality of 30% was observed after 50 days after the initial maize storage, in opposite to 0% of mortality in the control assay. Comparing the containers with 1 and 3 devices, the mortality % increased after 30 days, nearly doubled in the container with 3 devices (47% and 63%, respectively). Concerning the infestation level, the container with one central device reduced by 23% the progeny number (F1) when compared with the control assay (only 167 to 217), while with 3 devices was observed an increase to 33% (only 145 to 217).

(54) After 30 days of assays, the container with 3 devices showed an insect average mortality of 63%, while the container with 1 device showed 47% of mortality.

(55) The F1 insect mortality shows a dependence on the number of devices placed in the container. After 30 days of assay, the container with 1 device showed 47% of mortality and the container with 3 devices exhibited 63% of mortality. The mortality observed in the control assay is related to natural mortality

(56) The fungicidal effect of essential oils vapour was evaluated on maize samples withdrawn from the container interior with a vertical probe, which allows sampling at any depth of the grain bed.

(57) After 36, 50 and 90 days of cereal storage, no fungi development was observed inside containers. After grains observation under laboratory conditions and appropriated culture media (PDA) the fungistatic effect of essential oils was confirmed.

Example 4.2Treated Seeds Germination Capacity

(58) In an embodiment, after 50 days of storage, in the 2 containers with the EOs release devices, the grains showed the same germination rate than the observed in the control assay (between 83-90%). This trend is still found after 93 days of storage (80%) and after 5 months (70%), which indicates that these types of treatments allowing the maintenance of the germination capacity during storage.

Example 4.3Container Atmosphere Composition

(59) To study the evolution of the spatial and temporal pulegone concentration, two containers with 1 and 2 release devices were used, similar to those described in 4.1. In each container, 24 ml of pulegone were impregnated in cotton.

(60) The spatial concentration profiles along time were outlined by collecting over time samples of 500 L from the container atmosphere, through the 6 sampling ports (detailed presentation in FIG. 1) using a 500 L gas-tight Hamilton syringe.

(61) The composition of the samples was measured in a Dani Master GC-PID, equipped with a Varian CP-8944 Column (30 m0.25 mm I.D.) according to the following operating conditions: injector temperature at 100 C. with a 1:15 split ratio, detector temperature at 200 C.; initial column temperature of 60 C., subsequently increased to 120 C. at a rate of 10 C./min, and held isothermal for 10 minutes, then increased to 190 C. at a heating rate of 25 C./min, held isothermal for 5 minutes. Nitrogen was used as carrier gas at a constant 2 mL flow rate.

(62) After 20 days of storage time, the spatial concentration profiles of pulegone indicated a complete stabilization in the entire container atmosphere at a concentration level corresponding to 10% of the standard pulegone saturation value, either using one or two devices. These values were maintained during all the storage period (4/5 months).

(63) Considering the results from the insecticide assays (section 4.1), it may be assumed that these concentration levels granted an effective protection against the insect development. This explains why all containers exhibited the same average mortality 50 days after the beginning of the experiment (15 days after placing the insects in containers probes).

Example 5Determination of Essential Oils Retention Level in Maize

(64) In an embodiment, the measurement of the oils vapour retention coefficient on the maize grains was performed using around 30 g maize packed in perforated Petri dishes. These dishes were placed inside a sealable recipient at a 25 C., which contained, at the bottom, a vessel filled with essential oils liquid. This arrangement provided the ideal conditions for the evaporation and saturation of the atmosphere of the recipient promoting the contact between the essential oils vapour and the grains. This procedure was reproduced for both essential oils and their main active components (eugenol and pulegone, or their mixture). The mass retention of the essential oils in the grains was monitored by weighting the grains over time (FIG. 6).

(65) The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable. The following claims further set out particular embodiments of the disclosure.