Edible oil-in-water nanoemulsion formulations for pre-harvest treatment and / or post-harvest preservation of fruits or vegetables

Abstract

Edible oil-in-water nanoemulsion formulations for pre-harvest treatment and / or post-harvest preservation of fruits or vegetables are prepared by a high energy process (e.g., by ultrasound) from an oily solution of edible oil (e.g., high oleic sunflower oil) as a solvent, wherein an aqueous solution of melatonin (e.g., 100 [mu] M) and an edible fat-soluble antioxidant (e.g., 100 [mu] M alpha-tocopherol), and an edible water-soluble stabilizer (e.g., 1% w / v dextrin) is contained. These externally applied (e.g. By spraying) formulations are suitable for pre-harvest application and / or post-harvest preservation of fruits or vegetables, in particular stone fruits, in particular sweet cherries. For two varieties of sweet cherries and two varieties of plums, preservation with these formulations is significantly superior to preservation with an aqueous melatonin solution, or with a commercial formulation or without treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of pre-harvest treatment and / or post-harvest preservation of fruits or vegetables by applying chemical preparations. Background Art

[0002] In nature, senescence or decay of fruits and vegetables is an irreversible process involving a series of changes with a decrease in color, flavor and nutrition, and ultimately a shortened shelf life. Known attempts to improve the post-harvest preservation of fruits or vegetables include physical storage methods (e.g., refrigeration, controlled atmosphere storage and ventilation) and treatment with certain chemicals (e.g., nitric oxide, chlorine dioxide, calcium chloride, methyl salicylate, acetylsalicylic acid, 1-methylcyclopropane and β-aminobutyric acid).

[0003] In the food industry, the postharvest preservation of several Prunus fruits (sometimes simply called stone fruits, such as sweet cherries, plums, peaches, nectarines, apricots, etc.) is of great interest. Different edible coatings (applied in liquid form by spraying, dipping, brushing or dripping) have been proposed to improve the postharvest preservation of stone fruits.

[0004] It is well known that the application of exogenous melatonin (a hormone also known as N-acetyl-5-methoxytryptamine) improves the post-harvest preservation of fruits and vegetables (see review, e.g., T. Xu et al.; "Melatonin is a potential target for improving post-harvest preservation of fruits and vegetables"; Frontiers in Plant Science, 2019; doi: 10.3389 / fpls.2019.01388). In particular, soaking in an aqueous solution of melatonin has been found to help improve the preservation of sweet cherries (see, e.g., F. Wang et al.; "Exogenous melatonin delays postharvest fruit senescence and maintains the quality of sweet cherries"; Food Chemistry; 2019; doi.org / 10.1016 / j.foodchem.2019.125311).

[0005] Commercial Products Naturcover (Decco Iberica Post Cosecha, SAU, Paterna, Valencia, Spain) contains sucrose, fatty acid esters (E-473) and other additives and is sold to prevent cherries, plums and other stone fruits from rotting after harvest (see https: / / www.deccoiberica.es / producto / naturcover-cp / ; accessed on January 24, 2022).

[0006] Among stone fruits, cherries, especially sweet cherries (European sweet cherries, Prunus avium), are extremely difficult to handle after harvest and are highly perishable, with a shelf life of 7 to 14 days under refrigeration.

[0007] In agriculture, preharvest treatments of fruits and vegetables are also of interest, not only to increase crop yield, but also to improve quality characteristics at harvest, such as weight, color, firmness, total soluble solids, and titratable acidity. For example, foliar spraying with a 0.3 mM aqueous solution of melatonin is known to be an effective method for improving yield and quality characteristics of sweet cherry crops (see, e.g., A. Carrion-Antoli et al.; "Effects of Melatonin Treatment on Sweet Cherry Tree Yield and Fruit Quality"; Agronomy 2022, vol 12, p. 3. https: / / doi.org / 10.3390 / agronomy12010003; and references therein).

[0008] Thus, despite the extensive research and development that has been done, there is still room for improvement in the art of pre-harvest processing and / or post-harvest preservation of fruits or vegetables. Summary of the invention

[0009] One aspect of the present invention relates to providing an edible oil-in-water (o / w) nanoemulsion formulation, which is prepared by a high-energy method involving the use of a mechanical device from: a certain amount of an oily solution, the oily solution comprising one or more edible oils as a solvent, melatonin and one or more edible fat-soluble antioxidants; and a certain amount of an aqueous solution, the aqueous solution comprising water as a solvent and one or more edible water-soluble stabilizers; wherein the amount of the oily solution is 0.10:100 to 5:100 in a v / v ratio to the amount of the aqueous solution. In a specific embodiment, the amount of the oily solution is 1.0:100 to 2.5:100 in a v / v ratio to the amount of the aqueous solution.

[0010] In a specific embodiment, the amount of melatonin and edible fat-soluble antioxidant in the oil solution independently produces a concentration of 10 μM to 5.0 mM in the final formulation. In a specific embodiment, the amount of melatonin and edible fat-soluble antioxidant independently produces a concentration of 50 μM to 500 μM in the final formulation.

[0011] The preparation of the edible o / w nanoemulsion formulation of the present invention can be carried out by an oily solution containing one or more edible oils as solvents. In a specific embodiment, the edible oil is sunflower oil, which is mainly composed of oleic acid, linoleic acid, palmitic acid and stearic acid, and the w / w percentages of these four acids are different. In a specific embodiment, the edible oil is high oleic sunflower oil, which is stable and has a neutral taste characteristic, and wherein the w / w percentage of oleic acid is in the range of 80% to 90%.

[0012] E numbers ("E" stands for "Europe") are codes used within the European Union and the European Free Trade Association for substances used as food additives. They usually appear on food labels and their safety assessment and approval is the responsibility of the European Food Safety Authority. Some food additives with E codes are of natural origin; some are artificially produced; and some are both.

[0013] In a specific embodiment, the edible fat-soluble antioxidant in the oily solution used in the preparation of the edible o / w nanoemulsion formulation is a vitamin E vitamin or a mixture thereof. In a specific embodiment, the vitamin E vitamin is tocopherol, whose food additive code is between E306 and E309. In a specific embodiment, the tocopherol is alpha-tocopherol, whose food additive code is E307.

[0014] The edible o / w nanoemulsion formulations of the present invention have oily droplets (typically <200 nm in diameter) dispersed in an aqueous phase. There is no apparent aggregation of droplets in the o / w nanoemulsion formulations, in part due to the presence of edible water-soluble stabilizers in the aqueous solution used to prepare the formulation. In a particular embodiment, the w / v concentration of the edible water-soluble stabilizers in the aqueous solution used to prepare the formulation is in the range of 0.50% to 5.0%. In a particular embodiment, their w / v concentration is in the range of 1.0% to 2.0%.

[0015] In a particular embodiment, the water-soluble stabilizers in the aqueous solution are food additives, in particular: polyvinyl pyrrolidone (E1201), poly(vinyl alcohol) (E1203), polydextrose (E1200), starch derivatives (E1401) or mixtures thereof. In a particular embodiment, they are starch derivatives (E1401), in particular dextrins (E1400). The term "dextrin" is understood here as any edible ingredient from the group of low molecular weight carbohydrates produced by the hydrolysis of starch or glycogen or a mixture thereof.

[0016] In a particular embodiment, the mechanical device involved in the high energy preparation method is an ultrasonic generator, a high pressure valve homogenizer or a microfluidizer. In a particular embodiment, the mechanical device is an ultrasonic generator.

[0017] Another aspect of the invention relates to the use of the edible o / w nanoemulsion formulations disclosed above for pre-harvest treatment and / or post-harvest preservation of fruits or vegetables, the purpose of which is to increase crop yield and / or preservation, respectively. This aspect is closely related to methods for treating fruits or vegetables before or after harvest by applying one or more edible o / w nanoemulsion formulations disclosed above, the purpose of which is to increase crop yield and / or preservation. The application of the formulation can be by spraying, dipping, brushing or dripping. In a specific embodiment, the application is by spraying. In a specific embodiment, the fruit can be a drupe. In a specific embodiment, the drupe is a sweet cherry.

[0018] One of the parameters directly related to fruit senescence or decay is the decrease in firmness index (FI) over storage time (the higher the FI, the better the market acceptance). As shown in the attached comparative tests using sweet cherries and plums (two varieties each), treatment with the edible o / w nanoemulsion formulations of the present invention is significantly better than treatment with an aqueous solution of melatonin having the same hormone concentration in order to maintain a high FI of the fruit during storage; they are also significantly better than treatment with commercial formulations used to prevent post-harvest decay of cherries, plums and other stone fruits; and they are significantly better than no treatment (control).

[0019] When fruits and vegetables are infected by pathogens (e.g. fungi), the pathogen infection (PI) does not increase with storage time and is related to preservation (the lower the PI, the better the market acceptance). As shown in the attached comparative tests using sweet cherries and plums (two varieties each), in order to maintain a low PI, especially when the initial pathogen load is high, treatment with the edible o / w nanoemulsion formulation of the present invention is significantly better than treatment with an aqueous solution of melatonin with the same hormone concentration, and is significantly better than no treatment (control).

[0020] Other parameters relevant to fruit preservation are: total soluble solids (TSS) content, titratable acidity (TA) and pH. As shown in the attached comparative tests using sweet cherries and plums (two varieties each), treatment with the edible o / w nanoemulsion formulation of the present invention does not significantly change these three parameters compared to treatment with an aqueous solution of melatonin having the same hormone concentration, treatment with a commercial formulation for fruit preservation and no treatment (control).

[0021] In conclusion, applying the edible o / w nanoemulsion formulation of the present invention to fruits or vegetables improves preservation without compromising their quality.

[0022] Throughout the specification and claims, the word "comprise" and its variations are not intended to exclude other technical features, additives, components or steps. In addition, the word "comprise" covers the situation of "consisting of..." The following detailed embodiments / examples are provided by way of illustration, but are not intended to limit the present invention. Additional objects, advantages and features of the present invention will become apparent to those skilled in the art after reviewing the specification, or may be learned by practicing the present invention. In addition, the present invention covers all possible combinations of the specific embodiments described herein. DETAILED DESCRIPTION

[0023] Preparation of the formulation according to the invention

[0024] The materials used were as follows: α-tocopherol (E-307, Sigma Aldrich, St. Louis, MO, USA), melatonin (Fluka Honeywell Research Chemicals, Seltzer, Germany); high oleic sunflower oil (GM Food, Vilamaria, Spain); dextrin (E-1400, Quimics Dalmau, Barcelona, ​​Spain); and deionized and purified water (MiliQ).

[0025] Dextrin was mixed with water under slow stirring, first at 45°C for 30 min and then at room temperature until the dextrin was completely dissolved, thereby preparing a 1.5% w / v aqueous solution of dextrin. Oily solutions of α-tocopherol and melatonin were prepared separately in high oleic sunflower oil, with a concentration of 667 μM for each product. Then 15 mL of the oily solution was poured into 1000 mL of the aqueous solution of dextrin; thus, the concentrations of α-tocopherol and melatonin in the final o / w nanoemulsion formulation were both about 100 μM. The pouring was done slowly and stirred. The entire mixture was kept under slow stirring for 10 min; then ultrasound was performed at 4°C for 30 min; finally, it was rotated steadily in a stirrer (to avoid the formation of bubbles), and when the o / w nanoemulsion was transparent or slightly translucent (i.e., when the droplets were too small to be visible to the naked eye), it was considered complete. This o / w nanoemulsion formulation is represented here by FINV (from Formulation INVention).

[0026] Two formulations were prepared for comparison

[0027] For comparison purposes, Naturcover was used, obtained by dissolving the commercial product in water according to the manufacturer's recommendations. (Decco Iberica Post Cosecha, SAU, Paterna, Valencia, Spain), here called CNAT (according to Comparative NATurcover). Naturcover Sold to prevent post-harvest rot in cherries, plums, and other stone fruits.

[0028] For comparative purposes, a 100 μM aqueous solution of melatonin was also used as a formulation, as this solution is believed to delay post-harvest decay of sweet cherries (see, e.g., F. Wang et al.; supra, p. 2). This formulation is referred to herein as CMEL (for Comparative MELatonin).

[0029] The results corresponding to untreated fruit as negative control are indicated here as CNEG (according to Control NEGative).

[0030] Plant material and treatment

[0031] Two varieties (Prime Giant and Skeena) (Prunus avium L.) of sweet cherry (European sweet cherry, Prunus avium L.) and two varieties (Angeleno and Rose) of plum (Prunus salicina) were sampled. The fruit was randomly distributed in boxes, and each of the control group (CNEG) and three formulation groups (FINV, CNAT and CMEL) had 5 boxes (n=5). Sweet cherries were randomly distributed in the laboratory and kept in stable conditions of 23°C and 50% relative humidity (RH). Plums were randomly distributed in the laboratory and kept in stable conditions of 19°C and 82% RH.

[0032] Each box was sprayed with 20 mL of each of the three formulations until all fruit surfaces were completely covered. Sampling and quality parameter determinations were performed on days 0, 3 and 5 for sweet cherries and on days 0, 2, 4 and 8 for plums as described below. The values ​​on day 0 correspond to the values ​​determined before treatment. The differences in sampling days are due to the fruit survival period. Pathogen infection (PI) was determined daily for all fruits from day 0 until the last day of the corresponding analysis period.

[0033] On each sampling day, the firmness index (FI) was first determined, and then the fruits were frozen in liquid nitrogen and stored at -80°C to determine TSS content, TA and pH (see below). All data were statistically processed by two-way analysis of variance (ANOVA) using SPSS25.0 statistical software package; and two-way multiple comparisons were performed for the control group and the four preparations.

[0034] Determination of hardness index (FI)

[0035] The FI of the fruit was determined by a fruit penetration hardness tester PCE-PTR200 (PCE Iberica, Albacete, Spain), which measures the force (in Newtons, N) required to push a punch. The FI was obtained by making a small incision on the surface of the fruit and inserting the punch into the flesh until the punch left a mark. The punch was varied according to the type of fruit.

[0036] Determination of pathogen infection (PI)

[0037] PI was determined on each day of the experiment. It is expressed as the percentage of infected fruit in the box compared to the total number of fruit in the box. Fruit was considered infected when visible symptoms of infection appeared on the surface of the fruit. PI was considered to be mainly due to fungi.

[0038] Determination of total soluble solids (TSS) content

[0039] TSS content was determined as described (N. Teribia et al.; New Biotechnology; 2016; vol. 33; pp. 824-833) with slight modifications. The frozen fruits were ground until a fine powder was obtained. A 5.0 g sample of the powder, kept frozen in liquid nitrogen, was suspended in 50 mL of water. The TSS content (expressed in Brix) was determined on 1.0 mL of the final sample using a refractometer (Hanna Instruments, Padova, Italy).

[0040] Determination of Titratable Acidity (TA)

[0041] TA was determined as described (N.Teribia et al.; 2016; supra) with slight modifications. 10 mL of the final sample was diluted with 100 mL of water and then titrated with 0.10 M NaOH using 1% phenolphthalein as an indicator. TA values ​​were expressed as grams of malic acid per gram of fruit (gg -1 ) to indicate.

[0042] Determination of pH

[0043] The pH of the juice obtained from the frozen material was measured using a pH meter.

[0044] Results of TSS content, TA and pH: Statistical analysis

[0045] For the four fruit varieties studied (Prime Giant sweet cherry, Skeena sweet cherry, Angeleno plum and Rose plum), the results obtained for the three quality parameters in the title (not shown) were not statistically significantly different, either when compared with time or with treatments (CNEG, CNAT, CMEL and FINV).

[0046] Hardness Index (FI): Results and Conclusions

[0047] Table 1 (Prime Giant) and Table 2 (Skeena) show the obtained sweet cherry (Prunus avium L.) FI results as mean ± SD (n = 5). Statistically significant differences (P < 0.05) between FINV and CNEG, CNAT or CMEL are marked as a, b or c, respectively.

[0048] Table 3 (Angeleno) and Table 4 (Rose) show the resulting FI data for plum (Prunus salicina) as mean ± SD (n = 5). Statistically significant differences between FINV and CNEG, CNAT or CMEL are marked as a, b or c, respectively.

[0049] Post-harvest FI measurements of sweet cherry and plum fruit showed statistically significant differences related to species and variety. In all cases, the fruit treated with the nanoemulsion formulation according to the invention (FINV) maintained higher firmness and for a longer period of time than the fruit treated with the control / comparative conditions (CNEG, CNAT, CMEL).

[0050] For Prime Giant sweet cherries, after 3 days of post-harvest storage, the fruit treated with FINV was 22% firmer than the untreated (CNEG) fruit. Similarly, at the end of post-harvest storage, 5 days after the treatment, the Prime Giant sweet cherries treated with FINV had lost 20% of their initial firmness, but they were still significantly firmer (35%) than the fruit treated with A similar situation was found for Skeena sweet cherries after 5 days of storage after harvest: the firmness of Skeena sweet cherries treated with FINV was significantly higher than that of their counterparts from CNEG, CNAT and CMEL (they were 30%, 27% and 23% lower, respectively, see Table 2). In fact, Skeena sweet cherries treated with FINV had a constant FI, while in the other three cases the FI gradually decreased over time.

[0051] The FI progression of plums was different from that of cherries. Angelo plums showed a transient decrease and a final increase (see Table 3), while Rose plums showed a gradual increase regardless of treatment (see Table 4). These results may be related to differences in size and dehydration progression in plums compared to sweet cherries. Nevertheless, at the end of the experiment, 8 days after the start of the treatment, the FI of Angeleno plums treated with FINV was superior to that of Angeleno plums treated with CNEG and CNAT (25% and 30% better, respectively). At the end of the experiment, the FI of Rose plums treated with FINV was slightly superior to that of Rose plums treated with CNEG (only 15% better).

[0052] Table 1. Relationship between the firmness index (FI, in Newtons (N)) and time (t, in days (d)) of Prime Giant sweet cherries after three treatments and the control (untreated)

[0053]

[0054] Table 2. Relationship between firmness index (FI, in Newton (N)) and time (t, in days (d)) of Skeena sweet cherries under four conditions

[0055]

[0056] Table 3. Relationship between firmness index (FI, in Newtons (N)) and time (t, in days (d)) of Angeleno plums after three treatments and control (untreated)

[0057]

[0058] Table 4. Relationship between the firmness index (FI, in Newton (N)) and time (t, in days (d)) of Rose plums after three treatments and the control (untreated)

[0059]

[0060] Pathogen infection (PI): Results and conclusions

[0061] Table 5 (Prime Giant) and Table 6 (Skeena) show the resulting pathogen infection (PI) data for sweet cherry (Prunus avium L.) as mean ± SD (n = 5). Statistically significant differences (P < 0.05) between FINV and CNEG, CNAT or CMEL are marked as a, b or c, respectively.

[0062] Table 7 (Angeleno) and Table 8 (Rose) show the resulting PI data for plum (Prunus salicina) as mean ± SD (n = 5). Statistically significant differences between FINV and CNEG, CNAT or CMEL are marked as a, b or c, respectively.

[0063] Prime Giant sweet cherries showed the highest PI on all days studied (for the three treatments as well as no treatment) compared to the other three fruit varieties studied.

[0064] Untreated fruit, Prime Giant sweet cherry, showed a steady increase in PI over time, from 0% on day 1 to 85% on day 5 (see Table 5, CNEG). For this variety, no statistically significant differences were found between the PI values ​​corresponding to the CNEG, CNAT and CMEL treatments within a given day. However, on day 5, the PI values ​​corresponding to FINV were 25%, 16% and 15% lower than those corresponding to CNEG, CNAT and CMEL, respectively (see Table 5, t=5d).

[0065] Overall, Skeena sweet cherries showed lower PIs than Prime Giant sweet cherries, which may be due to differences in pathogen loads in the orchards. The PI of Skeena sweet cherries was clearly visible on day 2 and increased steadily over time (see Table 6). On the last day of the study (day 5), the PI corresponding to FINV (25%) was not significantly different from the PI corresponding to CNAT, but was significantly less than the PI corresponding to CNEG and CMEL (about 37%).

[0066] For Angeleno plums and Rose plums, no statistically significant differences in PI values ​​were observed between the three treatments and the control (see Tables 7 and 8). It is noteworthy that Angeleno plums exhibited very little PI over the 8 days studied, reaching only a maximum of about 15% on day 8. Overall, Rose plums exhibited a higher PI than Angeleno plums, similar to that of Skeena sweet cherries, with the notable difference that they did not increase steadily but suddenly on day 4 and remained almost constant until day 8.

[0067] Therefore, the overall conclusion of this study is that for the prevention of PI in sweet cherry, especially under conditions of high pathogen load, FINV treatment is more useful than the other two treatments (CNAT, CMEL) and no treatment (CNEG).

[0068] Table 5. Relationship between pathogen infection (PI, in %) and time (t, in days (d)) of Prime Giant sweet cherry after three treatments and control (untreated)

[0069]

[0070] Table 6. Relationship between pathogen infection (PI, in %) and time (t, in days (d)) of Skeena sweet cherry after three treatments and control (untreated)

[0071]

[0072] Table 7. Relationship between pathogen infection (PI, in %) and time (t, in days (d)) of Angeleno plum after three treatments and control (untreated)

[0073]

[0074] Table 8. Relationship between pathogen infection (PI, in %) and time (t, in days (d)) of Rose plums after three treatments and control (untreated)

[0075]

Claims

1. An edible oil-in-water nanoemulsion formulation prepared by a high energy process involving the use of a mechanical device from: A quantity of an oily solution comprising: one or more edible oils as solvent; Melatonin; and one or more edible fat-soluble antioxidants; as well as A certain amount of aqueous solution, comprising: Water as solvent; and one or more edible water-soluble stabilizers; The amount of the oily solution to the amount of the aqueous solution is in a v / v ratio of 0.10:100 to 5:

100.

2. The preparation according to claim 1, wherein The amount of the oily solution to the amount of the aqueous solution is in a v / v ratio of 1.0:100 to 2.5:

100.

3. The preparation according to any one of claims 1 to 2, wherein The amounts of melatonin and the edible fat-soluble antioxidant in the oil solution independently produce a concentration of 10 μM to 5.0 mM in the final formulation.

4. The preparation according to any one of claims 1 to 3, wherein The amounts of melatonin and the edible fat-soluble antioxidant in the oil solution independently produce a concentration of 50 μM to 500 mM in the final formulation.

5. The preparation according to any one of claims 1 to 4, wherein The edible oil used as a solvent in the oily solution is high oleic sunflower oil.

6. The preparation according to any one of claims 1 to 5, wherein The edible fat-soluble antioxidant in the oily solution is vitamin E or a mixture thereof.

7. The preparation according to claim 6, wherein The vitamin E vitamin is alpha-tocopherol.

8. The preparation according to any one of claims 1 to 7, wherein The w / v concentration of the edible water-soluble stabilizer in the aqueous solution is in total 0.50% to 5.0%.

9. The preparation according to any one of claims 1 to 8, wherein The w / v concentration of the edible water-soluble stabilizer in the aqueous solution is in total 1.0% to 2.0%.

10. The preparation according to any one of claims 1 to 9, wherein The water-soluble stabilizer in the aqueous solution is selected from polyvinyl pyrrolidone, poly(vinyl alcohol), polydextrose, starch derivatives and mixtures thereof.

11. The preparation according to claim 10, wherein The edible water-soluble stabilizer is a starch derivative.

12. The preparation according to claim 11, wherein The starch derivative is dextrin.

13. The preparation according to any one of claims 1 to 12, wherein The mechanical device involved in the high energy method is selected from the group consisting of: an ultrasonic generator, a high pressure valve homogenizer and a microfluidizer.

14. Use of the preparation according to any one of claims 1 to 13 for pre-harvest treatment and / or post-harvest storage of fruits or vegetables.

15. The use according to claim 14, wherein The fruit is sweet cherry.

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